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Chowdhury S, Beeton K, Wallace Z, Moore M, Bidwell GL, Janorkar AV. Extending the Three-Dimensional Culture of Adipocytes Through Surface Coatings. Bioengineering (Basel) 2025; 12:266. [PMID: 40150730 PMCID: PMC11939550 DOI: 10.3390/bioengineering12030266] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2025] [Revised: 02/28/2025] [Accepted: 03/05/2025] [Indexed: 03/29/2025] Open
Abstract
To mimic the important features of progressing adiposity, in vitro adipose cell culture models must allow gradual intracellular fat accumulation in the three-dimensional (3D) arrangement of adipose-derived stem cells (ASCs) over a long-term culture period. Previously, elastin-like polypeptide (ELP) and polyethyleneimine (PEI) have been used to culture human adipose-derived stem cells (hASCs) as 3D spheroids and to differentiate them to adipocytes over a relatively long culture period of up to 5 weeks. In this study, to further enhance the spheroid adhesion properties, ELP was fused with Arginine-Glycine-Aspartic Acid (RGD) residues, known for their role as cell-attachment sites. This study aimed to assess whether the addition of RGD to the C-or N-terminus of ELP would impact the spheroid-forming ability of ELP-PEI coatings. ELP-RGD conjugates were produced using genetically modified Escherichia coli to express ELP-(RGD)3 and (RGD)3-ELP, followed by chemical conjugation with PEI. SDS gel electrophoresis, FTIR spectroscopy, and turbidimetry analyses revealed that ELP was conjugated with RGD without much alteration in the molecular weight, functional groups present, and transition temperature of ELP. The addition of RGD to ELP also did not affect the chemical conjugation capacity of ELP to PEI. We observed that the ELP-PEI coating formed slightly larger spheroids (61.8 ± 3.2 µm) compared to the ELP-(RGD)3-PEI and (RGD)3-ELP-PEI coatings (56.6 ± 3.0 and 53.4 ± 2.4 µm, respectively). Despite the size difference, ELP-(RGD)3-PEI coatings exhibited superior spheroid retention during media changes, with minimal spheroid loss. DNA assay results confirmed a significant decrease in the DNA concentration (p < 0.05) after the 20 media changes for spheroids cultured on the ELP-PEI coating, indicating spheroid loss. However, there was no significant difference in DNA concentration before and after 20 media changes for spheroids cultured on the ELP-(RGD)3-PEI and (RGD)3-ELP-PEI coatings (p > 0.05). These findings suggest that RGD incorporation does not hinder the initial spheroid formation ability of the ELP-PEI coating and enhances spheroid retention under dynamic culture conditions.
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Affiliation(s)
- Sheetal Chowdhury
- Department of Biomedical Materials Science, University of Mississippi Medical Center, 2500 N State Street, Jackson, MS 39216, USA
| | - Komal Beeton
- Department of Cell and Molecular Biology, University of Mississippi Medical Center, 2500 N State Street, Jackson, MS 39216, USA
| | - Zacchaeus Wallace
- Department of Biomedical Materials Science, University of Mississippi Medical Center, 2500 N State Street, Jackson, MS 39216, USA
| | - Maggie Moore
- Department of Biomedical Materials Science, University of Mississippi Medical Center, 2500 N State Street, Jackson, MS 39216, USA
| | - Gene L. Bidwell
- Department of Neurology, University of Mississippi Medical Center, 2500 N State Street, Jackson, MS 39216, USA
| | - Amol V. Janorkar
- Department of Biomedical Materials Science, University of Mississippi Medical Center, 2500 N State Street, Jackson, MS 39216, USA
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Kartasheva-Ebertz D, Gaston J, Lair-Mehiri L, Massault PP, Scatton O, Vaillant JC, Morozov VA, Pol S, Lagaye S. Adult human liver slice cultures: Modelling of liver fibrosis and evaluation of new anti-fibrotic drugs. World J Hepatol 2021; 13:187-217. [PMID: 33708350 PMCID: PMC7934011 DOI: 10.4254/wjh.v13.i2.187] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/27/2020] [Revised: 11/04/2020] [Accepted: 12/30/2020] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND Liver fibrosis can result in end-stage liver failure and death.
AIM To examine human liver fibrogenesis and anti-fibrotic therapies, we evaluated the three dimensional ex vivo liver slice (LS) model.
METHODS Fibrotic liver samples (F0 to F4 fibrosis stage according to the METAVIR score) were collected from patients after liver resection. Human liver slices (HLS) were cultivated for up to 21 days. Hepatitis C virus (HCV) infection, alcohol (ethanol stimulation) and steatosis (palmitate stimulation) were examined in fibrotic (F2 to F4) liver slices infected (or not) with HCV. F0-F1 HLS were used as controls. At day 0, either ursodeoxycholic acid (choleretic and hepatoprotective properties) and/or α-tocopherol (antioxidant properties) were added to standard of care on HLS and fibrotic liver slices, infected (or not) with HCV. Expression of the biomarkers of fibrosis and the triglyceride production were checked by quantitative reverse transcription polymerase chain reaction and/or enzyme-linked immunosorbent assay.
RESULTS The cultures were viable in vitro for 21 days allowing to study fibrosis inducers and to estimate the effect of anti-fibrotic drugs. Expression of the biomarkers of fibrosis and the progression to steatosis (estimated by triglycerides production) was increased with the addition of HCV and /or ethanol or palmitate. From day 15 of the follow-up studies, a significant decrease of both transforming growth factor β-1 and Procol1A1 expression and triglycerides production was observed when a combined anti-fibrotic treatment was applied on HCV infected F2-F4 LS cultures.
CONCLUSION These results show that the human three dimensional ex vivo model effectively reflects the in vivo processes in damaged human liver (viral, alcoholic, nonalcoholic steatohepatitis liver diseases) and provides the proof of concept that the LS examined model permits a rapid evaluation of new anti-fibrotic therapies when used alone or in combination.
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Affiliation(s)
- Daria Kartasheva-Ebertz
- Institut Pasteur, Immunobiologie des Cellules Dendritiques, INSERM U1223, Paris 75015, France
- BioSPC, Université de Paris, Paris 75005, France
| | - Jesintha Gaston
- Institut Pasteur, Immunobiologie des Cellules Dendritiques, INSERM U1223, Paris 75015, France
- BioSPC, Université de Paris, Paris 75005, France
| | - Loriane Lair-Mehiri
- Institut Pasteur, Immunobiologie des Cellules Dendritiques, INSERM U1223, Paris 75015, France
- BioSPC, Université de Paris, Paris 75005, France
| | - Pierre-Philippe Massault
- Service de Chirurgie digestive, Hépato-biliaire et Endocrinienne, AP-HP, Groupe Hospitalier Cochin, Paris 75014, France
| | - Olivier Scatton
- Service de Chirurgie digestive et Hépato bilio pancréatique, AP-HP, Groupe Hospitalier La Pitié-Salpétrière, Medecine Sorbonne Université, Paris 75013, France
| | - Jean-Christophe Vaillant
- Service de Chirurgie digestive et Hépato bilio pancréatique, AP-HP, Groupe Hospitalier La Pitié-Salpétrière, Medecine Sorbonne Université, Paris 75013, France
| | - Vladimir Alexei Morozov
- Center for HIV and Retrovirology, Department of Infectious Diseases, Robert Koch Institute, Berlin 13353, Germany
| | - Stanislas Pol
- Institut Pasteur, Immunobiologie des Cellules Dendritiques, INSERM U1223, Paris 75015, France
- Département d'Hépatologie, AP-HP, Groupe Hospitalier Cochin, Université de Paris, Paris 75014, France
| | - Sylvie Lagaye
- Institut Pasteur, Immunobiologie des Cellules Dendritiques, INSERM U1223, Paris 75015, France
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Costa EC, de Melo-Diogo D, Moreira AF, Carvalho MP, Correia IJ. Spheroids Formation on Non-Adhesive Surfaces by Liquid Overlay Technique: Considerations and Practical Approaches. Biotechnol J 2017; 13. [DOI: 10.1002/biot.201700417] [Citation(s) in RCA: 73] [Impact Index Per Article: 9.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2017] [Revised: 10/03/2017] [Accepted: 10/16/2017] [Indexed: 12/11/2022]
Affiliation(s)
- Elisabete C. Costa
- CICS-UBI − Health Sciences Research Centre; Universidade da Beira Interior; 6200-506 Covilhã Portugal
| | - Duarte de Melo-Diogo
- CICS-UBI − Health Sciences Research Centre; Universidade da Beira Interior; 6200-506 Covilhã Portugal
| | - André F. Moreira
- CICS-UBI − Health Sciences Research Centre; Universidade da Beira Interior; 6200-506 Covilhã Portugal
| | - Marco P. Carvalho
- CICS-UBI − Health Sciences Research Centre; Universidade da Beira Interior; 6200-506 Covilhã Portugal
| | - Ilídio J. Correia
- CICS-UBI − Health Sciences Research Centre; Universidade da Beira Interior; 6200-506 Covilhã Portugal
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Abstract
Primary hepatocytes form spheroids under some culture conditions. These spheroids exhibit many tissuelike ultrastructures and retain many liver-specific functions over a long period of time. They are attractive for many applications employing liver cells. The ability to maintain their viability and functions at a reduced temperature to allow for transportation to the site of their application will facilitate their use. Furthermore, with their structural and functional similarity, they could possibly be used as a model system for studying various liver ischemias. The effect of hypothermic treatment was assessed by oxygen consumption rate, ATP, H2O2, and caspase 8 content, as well as albumin and urea synthesis, during and posttreatment. No single outcome variable gives a superlative quantification of hypothermic damage. Taken together, the hypothermic treatment can be seen as increasingly damaging as the temperature decreases from 21°C to 15°C and 4°C. The addition of the chemical protectants glutathione, N-acetyl-L-cystein (NAC), and tauroursodeoxycholic acid (TUDCA) decreased the damaging effect of hypothermic treatment. This protection effect was even more profound when spheroids were preincubated with the protectant for 24 h, and was most prominent at 4°C. The viability of the hypothermically treated hepatocyte spheroids was confirmed by laser scanning confocal microscopy. The method reported provides a means of maintaining spheroids' viability and may allow for their distribution to application sites at a distance.
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Affiliation(s)
- Pamela H Lai
- Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, MN 55455-0132, USA
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Liu Y, Hu K, Wang Y. Primary Hepatocytes Cultured on a Fiber-Embedded PDMS Chip to Study Drug Metabolism. Polymers (Basel) 2017; 9:E215. [PMID: 30970894 PMCID: PMC6431835 DOI: 10.3390/polym9060215] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2017] [Revised: 05/25/2017] [Accepted: 06/07/2017] [Indexed: 11/26/2022] Open
Abstract
In vitro drug screening using reliable and predictable liver models remains a challenge. The identification of an ideal biological substrate is essential to maintain hepatocyte functions during in vitro culture. Here, we developed a fiber-embedded polydimethylsiloxane (PDMS) chip to culture hepatocytes. Hepatocyte spheroids formed in this device were subjected to different flow rates, of which a flow rate of 50 μL/min provided the optimal microenvironment for spheroid formation, maintained significantly higher rates of albumin and urea synthesis, yielded higher CYP3A1 (cytochrome P450 3A1) and CYP2C11 (cytochrome P450 2C11) enzyme activities for metabolism, and demonstrated higher expression levels of liver-specific genes. In vitro metabolism tests on tolbutamide and testosterone by hepatocytes indicated predicted clearance rates of 1.98 ± 0.43 and 40.80 ± 10.13 mL/min/kg, respectively, which showed a good in vitro⁻in vivo correspondence. These results indicate that this system provides a strategy for the construction of functional engineered liver tissue that can be used to study drug metabolism.
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Affiliation(s)
- Yaowen Liu
- College of Food Science, Sichuan Agricultural University, Yaan 625014, China.
- School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, China.
| | - Ke Hu
- College of Food Science, Sichuan Agricultural University, Yaan 625014, China.
| | - Yihao Wang
- College of Food Science, Sichuan Agricultural University, Yaan 625014, China.
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Weeks CA, Aden B, Zhang J, Singh A, Hickey RD, Kilbey SM, Nyberg SL, Janorkar AV. Effect of amine content and chemistry on long-term, three-dimensional hepatocyte spheroid culture atop aminated elastin-like polypeptide coatings. J Biomed Mater Res A 2016; 105:377-388. [PMID: 27648820 DOI: 10.1002/jbm.a.35910] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2016] [Revised: 09/12/2016] [Accepted: 09/16/2016] [Indexed: 01/22/2023]
Abstract
Culture conditions that induce hepatic spheroidal aggregates sustain liver cells with metabolism that mimics in vivo hepatocytes. Here we present an array of elastin-like polypeptide conjugate coating materials (Aminated-ELPs) that are biocompatible, have spheroid-forming capacity, can be coated atop traditional culture surfaces, and maintain structural integrity while ensuring adherence of spheroids over long culture period. The Aminated-ELPs were synthesized either by direct conjugation of ELP and various polyelectrolytes or by conjugating both ELP and various small electrolytes to the reactive polymer poly(2-vinyl-4,4-dimethyl azlactone) (PVDMA). Spheroid morphology, cellular metabolic function, and liver-specific gene expression over the long-term, 20-day culture period were assessed through optical microscopy, measurement of total protein content and albumin and urea production, and quantitative real-time (qRT) PCR. We found that the amine content of the Aminated-ELP coatings dictated the initial hepatocyte attachment, but not the subsequent hepatocyte spheroid formation and their continued attachment. A lower amine content was generally found to sustain higher albumin production by the spheroids. Out of the 19 Aminated-ELP coatings tested, we found that the lysine-containing substrates comprising ELP-polylysine or ELP-PVDMA-butanediamine proved to consistently culture productive spheroidal hepatocytes. We suggest that the incorporation of lysine functional groups in Aminated-ELP rendered more biocompatible surfaces, increasing spheroid attachment and leading to increased liver-specific function. Taken together, the Aminated-ELP array presented here has the potential to create in vitro hepatocyte culture models that mimic in vivo liver functionality and thus, lead to better understanding of liver pathophysiology and superior screening methods for drug efficacy and toxicity. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 377-388, 2017.
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Affiliation(s)
- C Andrew Weeks
- Department of Biomedical Materials Science, School of Dentistry, University of Mississippi Medical Center, 2500 North State Street, Jackson, Mississippi, 39216
| | - Bethany Aden
- Departments of Chemistry & Chemical and Biomolecular Engineering, University of Tennessee, 322 Buehler Hall, 1420 Circle Drive, Knoxville, Tennessee, 37996
| | - Junlin Zhang
- Department of Surgery, School of Medicine, University of Mississippi Medical Center, 2500 North State Street, Jackson, Mississippi, 39216
| | - Anisha Singh
- Department of Surgery, Mayo Clinic, 200 1st St SW, Rochester, Minnesota, 55905
| | - Raymond D Hickey
- Department of Surgery, Mayo Clinic, 200 1st St SW, Rochester, Minnesota, 55905
| | - S Michael Kilbey
- Departments of Chemistry & Chemical and Biomolecular Engineering, University of Tennessee, 322 Buehler Hall, 1420 Circle Drive, Knoxville, Tennessee, 37996
| | - Scott L Nyberg
- Department of Surgery, Mayo Clinic, 200 1st St SW, Rochester, Minnesota, 55905
| | - Amol V Janorkar
- Department of Biomedical Materials Science, School of Dentistry, University of Mississippi Medical Center, 2500 North State Street, Jackson, Mississippi, 39216
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Liu Y, Wei J, Lu J, Lei D, Yan S, Li X. Micropatterned coculture of hepatocytes on electrospun fibers as a potential in vitro model for predictive drug metabolism. MATERIALS SCIENCE & ENGINEERING. C, MATERIALS FOR BIOLOGICAL APPLICATIONS 2016; 63:475-84. [PMID: 27040241 DOI: 10.1016/j.msec.2016.03.025] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/19/2016] [Revised: 02/14/2016] [Accepted: 03/07/2016] [Indexed: 12/26/2022]
Abstract
The liver is the major organ of importance to determine drug dispositions in the body, thus the development of hepatocyte culture systems is of great scientific and practical interests to provide reliable and predictable models for in vitro drug screening. In the current study, to address the challenges of a rapid function loss of primary hepatocytes, the coculture of hepatocytes with fibroblasts and endothelial cells (Hep-Fib-EC) was established on micropatterned fibrous scaffolds. Liver-specific functions, such as the albumin secretion and urea synthesis, were well maintained in the coculture system, accompanied by a rapid formation of multicellular hepatocyte spheroids. The activities of phase I (CYP3A11 and CYP2C9) and phase II enzymes indicated a gradual increase for cocultured hepatocytes, and a maximum level was achieved after 5 days and maintained throughout 15 days of culture. The metabolism testing on model drugs indicated that the scaled clearance rates for hepatocytes in the Hep-Fib-EC coculture system were significantly higher than those of other culture methods, and a linear regression analysis indicated good correlations between the observed data of rats and in vitro predicted values during 15 days of culture. In addition, the enzyme activities and drug clearance rates of hepatocytes in the Hep-Fib-EC coculture model experienced sensitive responsiveness to the inducers and inhibitors of metabolizing enzymes. These results demonstrated the feasibility of micropatterned coculture of hepatocytes as a potential in vitro testing model for the prediction of in vivo drug metabolism.
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Affiliation(s)
- Yaowen Liu
- Key Laboratory of Advanced Technologies of Materials, Ministry of Education, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, PR China; College of Food Science, Sichuan Agricultural University, Yaan 625014, PR China
| | - Jiaojun Wei
- Key Laboratory of Advanced Technologies of Materials, Ministry of Education, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, PR China
| | - Jinfu Lu
- Key Laboratory of Advanced Technologies of Materials, Ministry of Education, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, PR China
| | - Dongmei Lei
- Key Laboratory of Advanced Technologies of Materials, Ministry of Education, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, PR China
| | - Shili Yan
- Key Laboratory of Advanced Technologies of Materials, Ministry of Education, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, PR China
| | - Xiaohong Li
- Key Laboratory of Advanced Technologies of Materials, Ministry of Education, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, PR China.
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Hepatocyte spheroid culture on fibrous scaffolds with grafted functional ligands as an in vitro model for predicting drug metabolism and hepatotoxicity. Acta Biomater 2015; 28:138-148. [PMID: 26409440 DOI: 10.1016/j.actbio.2015.09.027] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2015] [Revised: 08/18/2015] [Accepted: 09/22/2015] [Indexed: 12/25/2022]
Abstract
The identification of a biologic substrate for maintaining hepatocyte functions is essential to provide reliable and predictable models for in vitro drug screening. In the current study, a three-dimensional culture of hepatocytes was established on highly porous fibrous scaffolds with grafted galactose and RGD to afford extensive cell-cell and cell-scaffold interactions spatially. The pore size and ligand densities indicated significant effects on the formation of hepatocyte spheroids in balancing the cell retention, adhesion, and migration on fibrous scaffolds. Fibrous scaffolds with an average pore size of 60 μm and surface grafting densities of galactose at 5.9 nmol/cm(2) and RGD at 6.9 pmol/cm(2) provided optimal microenvironments for hepatocyte infiltration and multicellular spheroid formation. Significant promotions were also demonstrated in the syntheses of albumin and urea and the activities of phase I (CYP 3A11 and CYP 2C9) and phase II enzymes. The in vitro metabolism tests on testosterone and acetaminophen by hepatocytes on the optimal scaffolds indicated the predicated clearance rates of 50.7 and 22.6 ml/min/kg, respectively, which were comparable to the in vivo values of rats. The in vitro hepatotoxicity tests on amiodarone hydrochloride and acetaminophen predicted the half maximal effective concentrations (EC50) to reflect the in vivo toxic plasma concentrations in human. In addition, the enzyme activities, predicted clearance rates and hepatotoxicity values of hepatocytes on the optimal scaffolds experienced sensitive responsiveness to specific inducers or inhibitors of CYP 3A11 and phase II enzymes, exhibiting in vivo-in vitro correlations to a certain extent. These results demonstrate the feasibility of hepatocyte spheroid culture on fibrous scaffolds as an potential in vitro testing model to predict the in vivo drug metabolism, hepatotoxicity, and drug-drug interactions.
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9
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Turner PA, Weeks CA, McMurphy AJ, Janorkar AV. Spheroid organization kinetics of H35 rat hepatoma model cell system on elastin-like polypeptide-polyethyleneimine copolymer substrates. J Biomed Mater Res A 2013; 102:852-61. [PMID: 23564487 DOI: 10.1002/jbm.a.34743] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2013] [Revised: 03/15/2013] [Accepted: 04/02/2013] [Indexed: 01/28/2023]
Abstract
Though two-dimensional systems have yielded some success in deriving morphological and functional markers of hepatocyte culture, they largely fail to capture the three-dimensional organization, long-term viability, and functionality of the hepatic tissue. We have engineered a system for inducing self-assembly of model H35 rat hepatoma spheroids using a copolymer comprised of biocompatible elastin-like polypeptide (ELP) chemically conjugated to positively charged polyethyleneimine (PEI). We have achieved a conjugation ratio of 30 mol %, though our studies analyzing spheroid organization kinetics indicate conjugate ratios of 5 mol % and greater to be optimal for cell culture based on least variability in spheroid sizes and minimum incidence of overgrown aggregates. Furthermore, our ELP-PEI system indicated the potential for influencing ultimate spheroid dimensions, with spheroid size inversely related to polyelectrolyte conjugation. Overall, this study provides a good starting point to investigate functional correlations between spheroid size and functional markers and their future use as an in vitro diagnostic or tissue engineering tool.
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Affiliation(s)
- Paul A Turner
- Department of Biomedical Materials Science, School of Dentistry, University of Mississippi Medical Center, Jackson, Mississippi
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Acikgöz A, Giri S, Cho MG, Bader A. Morphological and Functional Analysis of Hepatocyte Spheroids Generated on Poly-HEMA-Treated Surfaces under the Influence of Fetal Calf Serum and Nonparenchymal Cells. Biomolecules 2013; 3:242-69. [PMID: 24970167 PMCID: PMC4030890 DOI: 10.3390/biom3010242] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2012] [Revised: 02/07/2013] [Accepted: 02/11/2013] [Indexed: 01/16/2023] Open
Abstract
Poly (2-hydroxyethyl methacrylate) (HEMA) has been used as a clinical material, in the form of a soft hydrogel, for various surgical procedures, including endovascular surgery of liver. It is a clear liquid compound and, as a soft, flexible, water-absorbing material, has been used to make soft contact lenses from small, concave, spinning molds. Primary rat hepatocyte spheroids were created on a poly-HEMA-coated surface with the intention of inducing hepatic tissue formation and improving liver functions. We investigated spheroid formation of primary adult rat hepatocyte cells and characterized hepatic-specific functions under the special influence of fetal calf serum (FCS) and nonparencymal cells (NPC) up to six days in different culture systems (e.g., hepatocytes + FCS, hepatocytes – FCS, NPC + FCS, NPC – FCS, co-culture + FCS, co-culture – FCS) in both the spheroid model and sandwich model. Immunohistologically, we detected gap junctions, Ito cell/Kupffer cells, sinusoidal endothelial cells and an extracellular matrix in the spheroid model. FCS has no positive effect in the sandwich model, but has a negative effect in the spheroid model on albumin production, and no influence in urea production in either model. We found more cell viability in smaller diameter spheroids than larger ones by using the apoptosis test. Furthermore, there is no positive influence of the serum or NPC on spheroid formation, suggesting that it may only depend on the physical condition of the culture system. Since the sandwich culture has been considered a “gold standard” in vitro culture model, the hepatocyte spheroids generated on the poly-HEMA-coated surface were compared with those in the sandwich model. Major liver-specific functions, such as albumin secretion and urea synthesis, were evaluated in both the spheroid and sandwich model. The synthesis performance in the spheroid compared to the sandwich culture increases approximately by a factor of 1.5. Disintegration of plasma membranes in both models was measured by lactate dehydrogenase (LDH) release in both models. Additionally, diazepam was used as a substrate in drug metabolism studies to characterize the differences in the biotransformation potential with metabolite profiles in both models. It showed that the diazepam metabolism activities in the spheroid model is about 10-fold lower than the sandwich model. The poly-HEMA-based hepatocyte spheroid is a promising new platform towards hepatic tissue engineering leading to in vitro hepatic tissue formation.
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Affiliation(s)
- Ali Acikgöz
- Department of Cell Techniques and Applied Stem Cell Biology, Center for Biotechnology and Biomedicine (BBZ), University of Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany.
| | - Shibashish Giri
- Department of Cell Techniques and Applied Stem Cell Biology, Center for Biotechnology and Biomedicine (BBZ), University of Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany.
| | - Man-Gi Cho
- Department of Bio-Chemical Engineering, Graduate School, Dongseo University, Busan 617-716, Republic of Korea.
| | - Augustinus Bader
- Department of Cell Techniques and Applied Stem Cell Biology, Center for Biotechnology and Biomedicine (BBZ), University of Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany.
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Wu FJ, Friend JR, Lazar A, Mann HJ, Remmel RP, Cerra FB, Hu WS. Hollow fiber bioartificial liver utilizing collagen-entrapped porcine hepatocyte spheroids. Biotechnol Bioeng 2012; 52:34-44. [PMID: 18629850 DOI: 10.1002/(sici)1097-0290(19961005)52:1<34::aid-bit4>3.0.co;2-#] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
A xenogeneic hollow fiber bioreactor utilizing collagen-entrapped dispersed hepatocytes has been developed as an extracorporeal bioartificial liver (BAL) for potential treatment of acute human fulminant hepatitis. Prolonged viability, enhanced liver-specific functions, and differentiated state have been observed in primary porcine hepatocytes cultivated as spheroids compared to dispersed hepatocytes plated on a monolayer. Entrapment of spheroids into the BAL can potentially improve performance over the existing device. Therefore, studies were conducted to evaluate the feasibility of utilizing spheroids as the functionally active component of our hybrid device. Confocal microscopy indicated high viability of spheroids entrapped into cylindrical collagen gel. Entrapment of spheroids alone into collagen gel showed reduced ability to contract collagen gel. By mixing spheroids with dispersed cells, the extent of collagen gel contraction was increased. Hepatocyte spheroids collagen-entrapped into BAL devices were maintained for over 9 days. Assessment of albumin synthesis and ureagenesis within a spheroid-entrapment BAL indicated higher or at least as high activity on a per-cell basis compared to a dispersed hepatocyte-entrapment BAL device. Clearance of 4-methylumbelliferone to its glucuronide was detected throughout the culture period as a marker of phase II conjugation activity. A spheroid-entrapment bioartificial liver warrants further studies for potential human therapy. (c) 1996 John Wiley & Sons, Inc.
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Affiliation(s)
- F J Wu
- Department of Chemical Engineering and Materials Science, University of Minnesota, 421 Washington Ave. S.E., Minneapolis, Minnesota 55455
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Powers MJ, Rodriguez RE, Griffith LG. Cell-substratum adhesion strength as a determinant of hepatocyte aggregate morphology. Biotechnol Bioeng 2012; 53:415-26. [PMID: 18634032 DOI: 10.1002/(sici)1097-0290(19970220)53:4<415::aid-bit10>3.0.co;2-f] [Citation(s) in RCA: 73] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
Cultured hepatocytes typically form multicellular aggregates which are either monolayered or spheroidal in morphology. We propose that the aggregate morphology resulting from a particular cell-substratum interaction has a biophysical basis: when cell contractile forces are greater than cell-substratum adhesion forces, spheroidal aggregates form; when cell contractile forces are weaker than cell-substratum adhesion forces, cells remain essentially spread and form monolayered aggregates. We tested this hypothesis by systematically varying the morphology of hepatocellular aggregates formed on substrata coated with a series of different concentrations of Matrigel, and correlating aggregate morphology with the cell-substratum adhesion strength measured in a shear flow detachment assay. Aggregate morphology was binary-spheroidal aggregates formed at low Matrigel concentrations and monolayered aggregates formed at high Matrigel concentrations. Cell-substratum adhesion strength was similarly binary, with low adhesion strengths correlated with spheroidal aggregates and high adhesion strengths correlated with formation of monolayered aggregates. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 415-426, 1997.
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Affiliation(s)
- M J Powers
- Department of Chemical Engineering, Massachusetts Institute of Technology, 66-556, Cambridge, Massachusetts 02139
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Abstract
Aggregation of hepatocytes in culture is an important phenomenon to control in tissue engineering applications. Aggregation generally enhances maintenance of differentiated functions but inhibits cell growth. At present there exists insufficient information for rational design of substrata that control aggregation. Indeed, the cellular mechanism(s) underlying the aggregation process is poorly understood, although cell motility is generally considered to be an essential phenomenon. In this article we provide the first study investigating the relationship between hepatocyte aggregation and motility behavior on various extracellular matrix substrata, including Matrigel, laminin, and fibronectin. We find that the extent of aggregation depends on the concentration of the extracellular matrix proteins, as well as on the type. Furthermore, we find that the extent of aggregation appears to be independent of classical single-cell locomotion. In fact, under conditions giving rise to substantial aggregation, the fraction of cells exhibiting classical locomotion is essentially negligible. Instead, aggregation appears to involve intracellular contacts accomplished via a different form of cell motility: active cell membrane extensions followed by adhesive cell-cell interactions. An implication of these findings is that aggregation may be largely governed by relative strengths of cell-cell versus cell-substratum interactions. These observations could be helpful for improved design of cell transplantation devices and cell culture substrata. (c) 1996 by John Wiley & Sons, Inc.
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Affiliation(s)
- M J Powers
- Department of Chemical Engineering, Massachusetts Institute of Technology, Room 66-556, Cambridge, Massachusetts 02139
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14
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Bao J, Fisher JE, Lillegard JB, Wang W, Amiot B, Yu Y, Dietz AB, Nahmias Y, Nyberg SL. Serum-free medium and mesenchymal stromal cells enhance functionality and stabilize integrity of rat hepatocyte spheroids. Cell Transplant 2012; 22:299-308. [PMID: 23006214 DOI: 10.3727/096368912x656054] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
Long-term culture of hepatocyte spheroids with high ammonia clearance is valuable for therapeutic applications, especially the bioartificial liver. However, the optimal conditions are not well studied. We hypothesized that liver urea cycle enzymes can be induced by high protein diet and maintain on a higher expression level in rat hepatocyte spheroids by serum-free medium (SFM) culture and coculture with mesenchymal stromal cells (MSCs). Rats were feed normal protein diet (NPD) or high protein diet (HPD) for 7 days before liver digestion and isolation of hepatocytes. Hepatocyte spheroids were formed and maintained in a rocked suspension culture with or without MSCs in SFM or 10% serum-containing medium (SCM). Spheroid viability, kinetics of spheroid formation, hepatic functions, gene expression, and biochemical activities of rat hepatocyte spheroids were tested over 14 days of culture. We observed that urea cycle enzymes of hepatocyte spheroids can be induced by high protein diet. SFM and MSCs enhanced ammonia clearance and ureagenesis and stabilized integrity of hepatocyte spheroids compared to control conditions over 14 days. Hepatocytes from high protein diet-fed rats formed spheroids and maintained a high level of ammonia detoxification for over 14 days in a novel SFM. Hepatic functionality and spheroid integrity were further stabilized by coculture of hepatocytes with MSCs in the spheroid microenvironment. These findings have direct application to development of the spheroid reservoir bioartificial liver.
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Affiliation(s)
- Ji Bao
- Department of Pathology, West China Hospital, Chengdu, Sichuan, People's Republic of China
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15
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Abu-Absi SF, Hansen LK, Hu WS. Three-dimensional co-culture of hepatocytes and stellate cells. Cytotechnology 2011; 45:125-40. [PMID: 19003250 DOI: 10.1007/s10616-004-7996-6] [Citation(s) in RCA: 56] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2004] [Accepted: 12/21/2004] [Indexed: 10/25/2022] Open
Abstract
Hepatocytes self-assemble in culture to form compacted spherical aggregates, or spheroids, that mimic the structure of the liver by forming tight junctions and bile canalicular channels. Hepatocyte spheroids thus resemble the liver to a great extent. However, liver tissue contains other cell types and has bile ducts and sinusoids formed by endothelial cells. Reproducing 3-D co-culture in vitro could provide a means to develop a more complex tissue-like structure. Stellate cells participate in revascularization after liver injury by excreting between hepatocytes a laminin trail that endothelial cells follow to form sinusoids. In this study we investigated co-culture of rat hepatocytes and a rat hepatic stellate cell line, HSC-T6. HSC-T6, which does not grow in serum-free spheroid medium, was able to grow under co-culture conditions. Using a three-dimensional cell tracking technique, the interactions of HSC-T6 and hepatocyte spheroids were visualized. The two cell types formed heterospheroids in culture, and HSC-T6 cell invasion into hepatocyte spheroids and subsequent retraction was observed. RT-PCR revealed that albumin and cytochrome P450 2B1/2 expression were better maintained in co-culture conditions. These three-dimensional heterospheroids provide an attractive system for in vitro studies of hepatocyte-stellate cell interactions.
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Affiliation(s)
- Susan Fugett Abu-Absi
- Departments of Chemical Engineering and Materials Science, University of Minnesota, 55455-0132, Minneapolis, MN, USA
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16
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Evenou F, Fujii T, Sakai Y. Spontaneous formation of stably-attached and 3D-organized hepatocyte aggregates on oxygen-permeable polydimethylsiloxane membranes having 3D microstructures. Biomed Microdevices 2010; 12:465-75. [DOI: 10.1007/s10544-010-9403-8] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
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17
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A Mathematical Model of Liver Cell Aggregation In Vitro. Bull Math Biol 2008; 71:906-30. [DOI: 10.1007/s11538-008-9387-1] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2008] [Accepted: 12/03/2008] [Indexed: 12/11/2022]
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18
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Gerlach JC, Zeilinger K, Patzer II JF. Bioartificial liver systems: why, what, whither? Regen Med 2008; 3:575-95. [DOI: 10.2217/17460751.3.4.575] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023] Open
Abstract
Acute liver disease is a life-threatening condition for which liver transplantation is the only recognized effective therapy. While etiology varies considerably, the clinical course of acute liver failure is common among the etiologies: encephalopathy progressing toward coma and multiple organ failure. Detoxification processes, such as molecular adsorbent recirculating system (MARS®) and Prometheus, have had limited success in altering blood chemistries positively in clinical evaluations, but have not been shown to be clinically effective with regard to patient survival or other clinical outcomes in any Phase III prospective, randomized trial. Bioartificial liver systems, which use liver cells (hepatocytes) to provide metabolic support as well as detoxification, have shown promising results in early clinical evaluations, but again have not demonstrated clinical significance in any Phase III prospective, randomized trial. Cell transplantation therapy has had limited success but is not practicable for wide use owing to a lack of cells (whole-organ transplantation has priority). New approaches in regenerative medicine for treatment of liver disease need to be directed toward providing a functional cell source, expandable in large quantities, for use in various applications. To this end, a novel bioreactor design is described that closely mimics the native liver cell environment and is easily scaled from microscopic (<1 ml cells) to clinical (∼600 ml cells) size, while maintaining the same local cell environment throughout the bioreactor. The bioreactor is used for study of primary liver cell isolates, liver-derived cell lines and stem/progenitor cells.
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Affiliation(s)
- Jörg C Gerlach
- Departments of Surgery & Bioengineering, McGowan Institute for Regenerative Medicine, Bridgeside Point Bldg., 100 Technology Drive, Suite 225, Pittsburgh, PA 15219-3130, USA
- Charite - Campus Virchow, Humboldt University Berlin, Germany
| | | | - John F Patzer II
- Departments of Bioengineering, Chemical Engineering & Surgery, McGowan Institute for Regenerative Medicine, University of Pittsburgh, PA, USA
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19
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Abstract
In this study, we prepared polystyrene (PS) nanofibers as hepatocytes culture substrates
by electrospinning method and subsequently coated with specific ligand (poly(N--vinylbenzyl--
β-D-galactopyranosyl-(14)-D-gluconamide)(PVLA) for hepatocytes attachment. Rat hepatocytes’
behavior on the PVLA-coated and non-coated PS nanofibrous matrices have been investigated.
Electrospun PS fiber structures revealed randomly aligned fibers with average diameter of 500 nm.
Fabricated PS nanofibers had no bonding points like cotton fibers. Analyses by ATR/FTIR and
ESCA revealed that PVLA was successfully coated to the surfaces of PS nanofibers. More
hepatocytes were attached on the surface of PS nanofibers coated with PVLA than that on noncoated
PS nanofibers. PS nanofibrous matrix could incorporate many cells into the interior of the
matrix probably due to the suitable pore size. Cell viabilities cultured on PVLA-coated PS
nanofibrous mats were maintained for 2 weeks, while it was decreased rapidly on PVLA-coated PS
dishes. High hepatic function especially albumin secretion was maintained for 2 weeks on
nanofibrous mats but rapidly decreased on flat PS dishes. These results indicate that nanofibrous
structure enabled spheroid-like culture results in providing cell-cell communication and subsequent
long-term maintenance of specific cell function.
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20
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Meng Q, Wu D, Zhang G, Qiu H. Direct self-assembly of hepatocytes spheroids within hollow fibers in presence of collagen. Biotechnol Lett 2006; 28:279-84. [PMID: 16555013 DOI: 10.1007/s10529-005-5531-2] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2005] [Revised: 11/25/2005] [Accepted: 11/25/2005] [Indexed: 10/24/2022]
Abstract
Opposite to the established view that collagen is an extracellular substratum for only dispersed hepatocyte culture, hepatocyte spheroids were directly formed within hollow fibers by addition of moderate concentrations of soluble collagen. Morphologically, these spheroids indicated a close relationship with their in vivo structure of liver. The albumin and urea synthetic profiles confirmed that those spheroids maintained liver-specific functions for at least 8 days. Spheroid formation by addition of collagen not only presents a potential methodology for clinical use of spheroids in bioartificial liver device but also indicates a likely function of collagen for self-assembly of primary cells in tissue engineering.
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Affiliation(s)
- Qin Meng
- College of Materials Science and Chemical Engineering, Zhejiang University, Zhejiang 310027, China.
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21
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Török E, Vogel C, Lütgehetmann M, Ma PX, Dandri M, Petersen J, Burda MR, Siebert K, Düllmann J, Rogiers X, Pollok JM. Morphological and functional analysis of rat hepatocyte spheroids generated on poly(L-lactic acid) polymer in a pulsatile flow bioreactor. ACTA ACUST UNITED AC 2006; 12:1881-90. [PMID: 16889518 DOI: 10.1089/ten.2006.12.1881] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/30/2023]
Abstract
Liver neo-tissue suitable for transplantation has not been established. Primary rat hepatocytes were cultured on three-dimensional biodegradable polymer matrices in a pulsatile flow bioreactor with the intention of inducing tissue formation and improving cell survival. Functional and structural analysis of the hepatocytes forming liver neo-tissue was performed. Biodegradable poly(L-lactic acid) (PLLA) polymer discs were seeded with 4 x 10(6) primary rat hepatocytes each, were exposed to a pulsatile medium flow of 24 mL/min for 1, 2, 4, or 6 days and were investigated for monoethylglycinexylidine (MEGX) formation, ammonia detoxification, Cytokeratin 18 (CK18) expression, and preserved glycogen storage. Fine structural details were obtained using scanning and transmission electron microscopy. Spheroids of viable hepatocytes were formed. MEGX-specific production was maintained and ammonia removal capacity remained high during the entire flow-culture period of 6 days. CK18 distribution was normal. Periodic-acid- Schiff reaction demonstrated homogenous glycogen storage. The hepatocytes reassembled to form intercellular junctions and bile canaliculi. Functional and morphological analysis of rat hepatocytes forming spheroids in a pulsatile flow bioreactor indicated preserved and intact hepatocyte morphology and specific function. Pulsatile flow culture on PLLA scaffolds is a promising new method of hepatic tissue engineering leading to liver neo-tissue formation.
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Affiliation(s)
- Eva Török
- Department of Hepatobiliary Surgery and Visceral Transplantation, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
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22
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Ulloa-Montoya F, Verfaillie CM, Hu WS. Culture systems for pluripotent stem cells. J Biosci Bioeng 2005; 100:12-27. [PMID: 16233846 DOI: 10.1263/jbb.100.12] [Citation(s) in RCA: 95] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2005] [Accepted: 05/12/2005] [Indexed: 02/07/2023]
Abstract
Pluripotent stem cells have the capacity to self renew and to differentiate to cells of the three somatic germ layers that comprise an organism. Embryonic stem cells are the most studied pluripotent stem cells. Pluripotent stem cells have also been derived from adult tissues. Both embryonic and adult stem cells represent valuable sources of cells for applications in cell therapy, drug screening and tissue engineering. While expanding stem cells in culture, it is critical to maintain their self-renewal and differentiation capacity. In generating particular cell types for specific applications, it is important to direct their differentiation to the desired lineage. Challenges in expansion of undifferentiated stem cells for clinical applications include the removal of feeder layers and non-defined components in the culture medium. Our limited basic knowledge on the requirements for maintaining pluripotency of adult pluripotent stem cells and the lack of appropriate markers associated with pluripotency hinders the progress toward their wide spread application. In vitro differentiation of stem cells usually produces a mixed population of different cell lineages with the desired cell type present only at a small proportion. Use of growth factors that promote differentiation, expansion or survival of specific cell types is key in controlling the differentiation towards specific cell lineages. A variety of bioreactors for cell cultivation exist and can be readily adapted for stem cell cultivation and differentiation. They provide a well-controlled environment for studying the process of stem cell propagation and differentiation. Their wide use will facilitate the development of processes for stem cell application.
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Affiliation(s)
- Fernando Ulloa-Montoya
- Department of Chemical Engineering and Materials Science, University of Minnesota, 421 Washington Avenue SE, Minneapolis, MN 55455-0132, USA
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Abu-Absi SF, Hu WS, Hansen LK. Dexamethasone effects on rat hepatocyte spheroid formation and function. ACTA ACUST UNITED AC 2005; 11:415-26. [PMID: 15869420 DOI: 10.1089/ten.2005.11.415] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
Hepatocytes cultured on moderately adhesive surfaces or in spinner flasks spontaneously self-assemble into spherical tissue-like aggregates (spheroids). These spheroids have smooth surfaces and tissue-like polarized cell morphology, including bile canalicular-like channels, and maintain high viability and liver-specific functions for extended culture periods. Dexamethasone (DEX), a synthetic glucocorticoid, is known to elicit various responses in gene expression, and is often added to hepatocyte culture medium. The morphology and liver-specific protein production of hepatocyte spheroids were assessed under DEX concentrations ranging from 50 nM to 10 microM. DEX altered the kinetics of spheroid formation in a concentration-dependent fashion, with increasing concentrations inhibiting aggregation and promoting aggregate disassembly on culture dishes. DEX addition to spinner cultures resulted in smaller, more irregularly shaped spheroids and a higher incidence of aggregate clumping. Albumin and urea production were also higher in DEX cultures, but this effect was not as sensitive to concentration and occurred irrespective of the state of aggregation. RTPCR was utilized to assess the mRNA levels of extracellular matrix proteins, E-cadherin, and cytochrome P-450 enzymes. Results indicated a slight increase in fibronectin and collagen III mRNA early in the cultures, possibly contributing to the changes in morphology observed.
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Affiliation(s)
- Susan Fugett Abu-Absi
- Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, USA
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24
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Ma M, Xu J, Purcell WM. Biochemical and functional changes of rat liver spheroids during spheroid formation and maintenance in culture: I. morphological maturation and kinetic changes of energy metabolism, albumin synthesis, and activities of some enzymes. J Cell Biochem 2004; 90:1166-75. [PMID: 14635190 DOI: 10.1002/jcb.10730] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
In the process of isolated single liver cells coming together to form three-dimensional spheroids, cells undergo dramatic environmental changes. How liver cells respond to these changes has not been well studied before. This study characterized the functional and biochemical changes during liver spheroid formation and maintenance. Spheroids were prepared in 6-well plates from freshly isolated liver cells from male Sprague rats by a gyrotatory-mediated method. Morphological formation, and functional and biochemical parameters of liver spheroids were evaluated over a period of 21 days in culture. Liver spheroid formation was divided into two stages, immature (1-5 days) and mature (>5 days), according to their size and shape, and changes in their functionality. Galactose and pyruvate consumption was maintained at a relatively stable level throughout the period of observation. However, glucose secretion and cellular GPT and GOT activities were higher in immature spheroids, decreased upto day 5 and remained stable thereafter. Cellular gamma-glutamyltransferase (gamma-GT) and lactate dehydrogenase (LDH) activities were initially undetectable or low and increased as spheroids matured. Albumin secretion decreased rapidly within the first 2 days and increased as spheroids matured. It is concluded that cells undergo functional and biochemical changes during spheroid formation following isolation of liver cells from intact tissue. Functionality and biochemical properties recovered and were maintained in mature spheroids. A relatively stable period (6-15 days) of functionality in mature spheroids was identified and is recommended for applications of the model.
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Affiliation(s)
- Mingwen Ma
- Centre for Research in Biomedicine, Faculty of Applied Sciences, University of the West of England, Bristol, United Kingdom
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25
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Enmon RM, O'Connor KC, Song H, Lacks DJ, Schwartz DK. Aggregation kinetics of well and poorly differentiated human prostate cancer cells. Biotechnol Bioeng 2002; 80:580-8. [PMID: 12355469 DOI: 10.1002/bit.10394] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Aggregation of attachment-dependent animal cells represents a series of motility, collision, and adhesion events applicable to such diverse fields as tissue engineering, bioseparations, and drug testing. Aggregation of human prostate cancer cells in liquid-overlay culture was modeled using Smoluchowski's collision theory. Using well (LNCaP) and poorly differentiated (DU 145 and PC 3) cell lines, the biological relevance of the model was assessed by comparing aggregation rates with diffusive and adhesive properties. Diffusion coefficients ranged from 5 to 90 microm(2)/min for single LNCaP and PC 3 cells, respectively. Similar diffusivities were predicted by the persistent random walk model and Einstein relation, indicating random motion. LNCaP cells were the most adhesive in our study with reduced cell shedding, 100% adhesion probability, and enhanced expression of E-cadherin. There was an increase in DU 145 cells staining positive for E-cadherin from nearly 20% of single cells to uniform staining across the surface of all aggregates; under 30% of PC 3 aggregates stained positive. Aggregation rates were more consistent with adhesive properties than with motilities, suggesting that aggregation in our study was reaction-controlled. Relative to other assays employed here, aggregation rates were more sensitive to phenotypic differences in cell lines and described size-dependent changes in aggregation at a finer resolution. In particular, model results suggest similar aggregation rates for two-dimensional DU 145 and PC 3 aggregates and upwards of 4-fold higher rates for larger three-dimensional DU 145 spheroids, consistent with expression of E-cadherin. The kinetic model has application to spheroid production, to cell flocculation and as an adhesion assay.
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Affiliation(s)
- Richard M Enmon
- Department of Chemical Engineering, Tulane University, Lindy Boggs Center, Suite 300, New Orleans, Louisiana 70118, USA
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26
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Powers MJ, Janigian DM, Wack KE, Baker CS, Beer Stolz D, Griffith LG. Functional behavior of primary rat liver cells in a three-dimensional perfused microarray bioreactor. TISSUE ENGINEERING 2002; 8:499-513. [PMID: 12167234 DOI: 10.1089/107632702760184745] [Citation(s) in RCA: 190] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
We have previously described the design and operation of a microfabricated bioreactor that supports perfused 3D culture of liver cells and facilitates evolution of tissue-like morphological structures. Here, we describe the functional viability of cells maintained in this microarray bioreactor and examine the influence of different seeding protocols on the evolution of structure and function in comparison with static culture. Primary rat hepatocytes were seeded into the perfusion reactors either as single-cell suspensions immediately after isolation or as spheroidal aggregates formed over a 2- to 3-day period. Initial studies in which cells were cultured for 7 days postisolation revealed significantly greater functional activity and morphological stability of cells that were preaggregated for up to 3 days before seeding in the reactor, compared with direct seeding of single cells. Total albumin secretion and urea genesis rates in single-cell reactor cultures declined significantly during this initial culture period while remaining constant in preaggregated reactor cultures. Longer term studies indicate that rates of albumin secretion and urea genesis are maintained at constant levels through 15 days postisolation. These metabolic rates are an order of magnitude higher than observed for the same preaggregated structures cultured statically with comparable medium ratio and exchange conditions. The metabolic function data are supported by light microscopy images showing viable tissue structures, and electron microscopy images that reveal tight junctions, glycogen storage, and bile canaliculi.
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Affiliation(s)
- Mark J Powers
- Division of Biological Engineering, Biotechnology Process Engineering Center, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA
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27
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Schwartz RE, Reyes M, Koodie L, Jiang Y, Blackstad M, Lund T, Lenvik T, Johnson S, Hu WS, Verfaillie CM. Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells. J Clin Invest 2002. [PMID: 12021244 DOI: 10.1172/jci0215182] [Citation(s) in RCA: 748] [Impact Index Per Article: 32.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
We have derived from normal human, mouse, and rat postnatal bone marrow primitive, multipotent adult progenitor cells (MAPCs) that can differentiate into most mesodermal cells and neuroectodermal cells in vitro and into all embryonic lineages in vivo. Here, we show that MAPCs can also differentiate into hepatocyte-like cells in vitro. Human, mouse, and rat MAPCs, cultured on Matrigel with FGF-4 and HGF, differentiated into epithelioid cells that expressed hepatocyte nuclear factor-3beta (HNF-3beta), GATA4, cytokeratin 19 (CK19), transthyretin, and alpha-fetoprotein by day 7, and expressed CK18, HNF-4, and HNF-1alpha on days 14-28. Virtually all human, as well as a majority of rodent cells stained positive for albumin and CK18 on day 21; 5% (rodent) to 25% (human) cells were binucleated by day 21. These cells also acquired functional characteristics of hepatocytes: they secreted urea and albumin, had phenobarbital-inducible cytochrome p450, could take up LDL, and stored glycogen. MAPCs, which can be expanded in vitro and maintained in an undifferentiated state for more than 100 population doublings, can thus differentiate into cells with morphological, phenotypic, and functional characteristics of hepatocytes. MAPCs may therefore be an ideal cell for in vivo therapies for liver disorders or for use in bioartificial liver devices.
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Affiliation(s)
- Robert E Schwartz
- Stem Cell Institute, University of Minnesota, Minneapolis 55455, USA
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28
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Abu-Absi SF, Friend JR, Hansen LK, Hu WS. Structural polarity and functional bile canaliculi in rat hepatocyte spheroids. Exp Cell Res 2002; 274:56-67. [PMID: 11855857 DOI: 10.1006/excr.2001.5467] [Citation(s) in RCA: 178] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Primary hepatocytes self-assemble into spheroids that possess tight junctions and microvilli-lined channels. We hypothesized that polarity develops gradually and that the channels structurally and functionally resemble bile canaliculi. Immunofluorescence labeling of apical and basolateral proteins demonstrated reorganization of the membrane proteins into a polarized distribution during spheroid culture. By means of fluorescent dextran diffusion and confocal microscopy, an extensive network of channels was revealed in the interior of the spheroids. These channels connected over several planes and opened to pores on the surface. To examine the content of apical proteins in the channel membranes, the bile canalicular enzyme dipeptidyl peptidase IV (DPPIV) was localized using a fluorogenic substrate, Ala-Pro-cresyl violet. The results show that DPPIV activity is heterogeneously distributed in spheroids and localized in part to channels. Bile acid excretion was then investigated to demonstrate functional polarity. A fluorescent bile acid analogue, fluorescein isothiocyanate-labeled glycocholate, was taken up into the spheroids and excreted into bile canalicular channels. Due to the structural polarity of spheroids and their ability to excrete bile into channels, they are a unique three-dimensional model of in vitro liver tissue self-assembly. (Videoanimations of some results are available at http://hugroup.cems.umn.edu/research_movies).
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Affiliation(s)
- Susan Fugett Abu-Absi
- Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota 55455, USA
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29
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Tzanakakis ES, Hsiao CC, Matsushita T, Remmel RP, Hu WS. Probing enhanced cytochrome P450 2B1/2 activity in rat hepatocyte spheroids through confocal laser scanning microscopy. Cell Transplant 2002; 10:329-42. [PMID: 11437078 DOI: 10.3727/000000001783986783] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
Cytochrome P450 (CYP450) enzymes are essential for xenobiotic metabolism. Although CYP450s are found in many tissues, CYP2B1/2 are primarily expressed in the rat liver. The constitutive expression in vivo of CYP2B1/2 is low but it is induced in the presence of various drugs such as phenobarbital (PB). In this study, CYP2B1/2 activity in cultured hepatocytes was assessed in situ with the introduction of a fluorogenic substrate, pentoxyresorufin. The product of 7-pentoxyresorufin-O-dealkylation (PROD), which is catalyzed specifically by CYP2B1/2, was detected using confocal laser scanning microscopy (CLSM). Primary hepatocytes cultured as monolayers on collagen-coated surfaces exhibited background PROD activity and minimal PB inducibility after 4 days in culture. In contrast, rat hepatocytes organized in compacted aggregates, or spheroids, exhibited higher levels of PROD activity and retained their ability for PB induction. The results from the CLSM analysis were verified by RT-PCR and Western immunoblotting analysis. Furthermore, CLSM in conjunction with image processing techniques and three-dimensional reconstruction revealed the localization of enhanced PROD activity in the center of spheroids. The results support the use of CLSM as a powerful tool for investigating CYP2B1/2 activity in cultured rat hepatocytes.
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Affiliation(s)
- E S Tzanakakis
- Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis 55455-0132, USA
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30
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Abstract
Recent studies performed in our laboratory have shown that a brief period of preculture prior to cryopreservation improves the postthaw viability of hepatocytes. The purpose of this investigation is to characterize specific metabolic and biochemical characteristics of the hepatocytes (both frozen and nonfrozen) to help elucidate the role of preculture on the postthaw viability. Fresh and thawed hepatocytes were cultured in a bioartificial liver (BAL) to determine albumin secretion as a function of time in culture. In addition, cell extracts were analyzed using nuclear magnetic resonance (NMR) spectroscopy to quantify changes in cell membrane composition and energetics as a function of time in culture prefreeze and postthaw. The results of these studies showed an increase in albumin concentration in the culture medium with time in culture for the period tested for both fresh and frozen and thawed hepatocytes. NMR spectroscopy of lipid extracts indicates that in vitro culture of hepatocytes results in an increase in cholesterol relative to membrane phospholipid. Moreover, the NMR results also indicate phospholipid interconversion, via specific lipases in cultured hepatocytes, and these changes are consistent with water permeability measurements performed previously. Significant changes in phosphoenergetics were also observed, with the net energy charge for the cells increasing significantly with time in culture. In addition, NMR spectra show increased levels of 6-phosphogluconate, another indicator of the cellular response to the stresses of isolation and ex vivo culture. These results suggest that energetic considerations may be a significant factor in the ability of hepatocytes to survive the stresses of freezing and thawing. Significant shifts in membrane phospholipids may also influence membrane permeability and postthaw survival.
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Affiliation(s)
- A Hubel
- Department of Laboratory Medicine and Pathology, University of Minnesota, Mayo Mail Code 609, Minneapolis, Minnesota 55455, USA.
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31
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Tzanakakis ES, Hansen LK, Hu WS. The role of actin filaments and microtubules in hepatocyte spheroid self-assembly. CELL MOTILITY AND THE CYTOSKELETON 2001; 48:175-89. [PMID: 11223949 DOI: 10.1002/1097-0169(200103)48:3<175::aid-cm1007>3.0.co;2-2] [Citation(s) in RCA: 83] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/11/2023]
Abstract
Cultured rat hepatocytes self-assemble into three-dimensional structures or spheroids that exhibit ultrastructural characteristics of native hepatic tissue and enhanced liver-specific functions. The spheroid formation process involves cell translocation and changes in cell shape, indicative of the reorganization of the cytoskeletal elements. To elucidate the function of the cytoskeleton, hepatocytes undergoing spheroid formation were treated with drugs that disrupt the different cytoskeletal components. Cytochalasin D, which targets the actin filaments, caused inhibition of spheroid formation. The role of microtubules in this process was assessed by incubating the cells with taxol or nocodazole. Perturbation of microtubules had minimal effects on spheroid assembly. Scanning electron micrographs showed no morphological differences between spheroids formed in control cultures and those formed in the presence of taxol or nocodazole. In addition, the effects of those agents on hepatocyte functions were investigated. Albumin secretion and cytochrome P450 2B1/2 activities of hepatocytes were comparable in spheroids formed in the presence of taxol or nocodazole to those formed in control cultures. The levels of these liver-specific activities were lower in cytochalasin D--treated cultures where only dispersed cells or cell clumps were found but spheroids had not found. Thus, hepatocytes require an intact actin network to self-assemble efficiently into functional tissue-like structures. Perturbation of the microtubule lattice does not impair the formation process. Events that transpire during hepatocyte spheroid self-assembly exhibit striking similarities to processes commonly observed in tissue morphogenesis. The results provide insight into the mechanisms that cells employ to organize into tissues and can contribute to our understanding of how to control the cellular assembly in tissue engineering and clinical applications.
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MESH Headings
- Actins/physiology
- Albumins/biosynthesis
- Angiogenesis Inhibitors/pharmacology
- Animals
- Antineoplastic Agents/pharmacology
- Aryl Hydrocarbon Hydroxylases
- Cells, Cultured
- Cytochalasin D/pharmacology
- Cytochrome P-450 CYP2B1/biosynthesis
- Cytochrome P-450 Enzyme System/biosynthesis
- Cytoskeleton/drug effects
- Cytoskeleton/metabolism
- Cytoskeleton/ultrastructure
- Dose-Response Relationship, Drug
- Hepatocytes/drug effects
- Hepatocytes/metabolism
- Hepatocytes/ultrastructure
- Liver/metabolism
- Male
- Microscopy, Confocal
- Microscopy, Electron, Scanning
- Microtubules/drug effects
- Microtubules/physiology
- Microtubules/ultrastructure
- Models, Biological
- Movement
- Nocodazole/pharmacology
- Nucleic Acid Synthesis Inhibitors/pharmacology
- Oxazines/metabolism
- Paclitaxel/pharmacology
- Rats
- Rats, Sprague-Dawley
- Regeneration
- Spheroids, Cellular/drug effects
- Spheroids, Cellular/metabolism
- Spheroids, Cellular/ultrastructure
- Steroid Hydroxylases/biosynthesis
- Time Factors
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Affiliation(s)
- E S Tzanakakis
- Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, 55455, USA
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32
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Fujii Y, Nakazawa K, Funatsu K. Intensive promotion of spheroid formation by soluble factors in a hepatocyte-conditioned medium. JOURNAL OF BIOMATERIALS SCIENCE. POLYMER EDITION 2001; 11:731-45. [PMID: 11011770 DOI: 10.1163/156856200743986] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
We developed a hybrid artificial liver and a drug metabolism simulator using polyurethane foam (PUF) in which primary hepatocytes spontaneously form functional spheroids. Gel filtration liquid chromatography analysis of a hepatocyte-conditioned medium during spheroid formation showed that some substances secreted by primary rat hepatocytes accumulated advantageously inside the pores of PUF compared with outside. Similar substances were detected in a hepatocyte-conditioned medium from a positively-charged surface by concentrating the substances using an ultrafiltration membrane of a molecular weight-cutoff of 50 kD. These substances were shown to act as soluble factors on freshly isolated primary rat hepatocytes to promote spontaneous and rapid spheroid formation, depending on their concentration by preventing them from initially attaching and spreading on a positively-charged surface. In particular, using 50-fold concentrated substances, about 80% of total hepatocytes formed the floating spheroids within 72 h of culture. The resulting spheroids had a diameter distribution mainly ranging from 40 to 70 microm and expressed high-level liver-specific functions compared with a conventional monolayer.
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Affiliation(s)
- Y Fujii
- Department of Chemical Systems and Engineering, Graduate School of Engineering, Kyushu University, Fukuoka, Japan
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33
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Abstract
Hepatocytes are being studied for a wide variety of applications, including drug metabolism studies, gene therapy, and use in liver-assist devices for temporary liver support. The ability to cryopreserve isolated hepatocytes would permit the pooling of cells to reach the required therapeutic coordination of the cell supply with patient care regimes and the completion of safety and quality-control testing. The objective of this investigation was to develop a method of cryopreserving isolated hepatocytes that will retain high levels of function and facilitate the use of the cells in different applications. Freshly isolated hepatocytes were cultured in a spinner flask for different periods of time, up to 48 h. The cells were cryopreserved by use of a range of solution concentrations and cooling rates. For fresh, nonfrozen hepatocytes precultured for 24 h prior to being plated on collagen, the albumin secretion rate was 0.88 +/- 0.62 mg/ml/h. When the cells were precultured for 24 h, frozen in a solution containing 10% Me2SO with a cooling rate of 1 degrees C/min, thawed, plated on collagen, and cultured, the albumin secretion rate was 0.21 +/- 0.24 microg/ml/h. In contrast, freshly isolated hepatocytes cryopreserved without preculture and cultured on collagen had an albumin secretion rate of 0.07 +/- 0.08 mg/ml/h. The influences of different solution compositions and cooling rates on postthaw function of precultured hepatocytes were also determined. These results indicate that the use of a preliminary culture step prior to cryopreservation can enhance the postthaw function of hepatocytes.
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Affiliation(s)
- T B Darr
- Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis 55455, USA
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34
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Semler EJ, Ranucci CS, Moghe PV. Mechanochemical manipulation of hepatocyte aggregation can selectively induce or repress liver-specific function. Biotechnol Bioeng 2000; 69:359-69. [PMID: 10862674 DOI: 10.1002/1097-0290(20000820)69:4<359::aid-bit2>3.0.co;2-q] [Citation(s) in RCA: 105] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023]
Abstract
Controlled activation of hepatocyte aggregation is critical to three-dimensional (3D) multicellular morphogenesis during native regeneration of liver as well as tissue reconstruction therapies. In this work, we quantify the stimulatory effects of two model hepatotrophic activators, epidermal growth factor (EGF) and hepatocyte growth factor (HGF), on the aggregation kinetics and liver-specific function of hepatocytes cultured on organotypic substrates with differing mechanical resistivity. Substrate-specific morphogenesis of cultured hepatocytes is induced on a tissue basement membrane extract, Matrigel, formulated at two distinct levels of mechanical compliance (storage modulus G', at oscillatory shear rate 1 rad/s, was 34 Pa for basal Matrigel and 118 Pa for crosslinked Matrigel). Overall, we report that growth factor stimulation selectively promotes the kinetics of aggregation in the form of two-dimensional corded aggregates on basal Matrigel and three-dimensional spheroidal aggregates on crosslinked Matrigel. Our analysis also indicates that costimulation with EGF and HGF (20 ng/mL each) cooperatively maximizes the kinetics of aggregation in a substrate-specific manner. In addition, we show that the role of growth factor stimulation on hepatocyte function is sensitively governed by the mechanical compliance of the substrate. In particular, on matrices with high compliance, costimulatory aggregation is shown to elicit a marked increase in albumin secretion rate, whereas on matrices with low compliance aggregation results in effective functional repression to basal, unstimulated levels. Thus, our studies highlight a novel interplay of physicochemical parameters of the culture microenvironment, leading to selective enhancement or repression of differentiated functions of hepatocytes, in concert with the activation of cellular morphogenesis.
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Affiliation(s)
- E J Semler
- Department of Chemical and Biochemical Engineering, Rutgers University, 98 Brett Road, Piscataway, New Jersey 08854, USA
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35
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Brenner J, Hülser DF. Production of tissue plasminogen activator (tPA) in two and three dimensionally growing cultures of Bowes melanoma cells. Biotechnol Bioeng 2000; 51:422-33. [DOI: 10.1002/(sici)1097-0290(19960820)51:4<422::aid-bit5>3.0.co;2-k] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
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36
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Abstract
A potential approach to facilitate the performance of implanted hepatocytes is to enable their aggregation and re-expression of their differentiated function prior to implantation. Here we examined the behavior of freshly isolated rat adult hepatocytes seeded within a novel three-dimensional (3-D) scaffold based on alginate. The attractive features of this scaffold include a highly porous structure (sponge-like) with interconnecting pores, and pore sizes with diameters of 100-150 microm. Due to their hydrophilic nature, seeding hepatocytes onto the alginate sponges was efficient. DNA measurements showed that the total cell number within the sponges did not change over 2 weeks, indicating that hepatocytes do not proliferate under these culture conditions. Nearly all seeded cells maintained viability, according to the MTT assay. Within 24 h post-seeding, small clusters of viable cells, were seen scattered within the sponge. More than 90% of the seeded cells participated in the aggregation; the high efficiency is attributed to the non-adherent nature of alginate. The spheroids had smooth boundaries and by day 4 in culture reached an average diameter of 100 microm, which is at the same magnitude of the sponge pore size. The cells appeared to synthesize fibronectin which was deposited on the spheroids. No laminin or collagen type IV were detected in the deposit. The 3-D arrangement of hepatocytes within the alginate sponges promoted their functional expression; within a week the cells secreted the maximal albumin secretion rate of 60 microg albumin/10(6) cells/day. Urea secretion rate did not depend on cell aggregation and was similar to that obtained when hepatocytes were cultured on collagen type I coated dishes (100 microg/10(6) cells/day). Our studies show that alginate sponges can provide a conducive environment to facilitate the performance of cultured hepatocytes by enhancing their aggregation.
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Affiliation(s)
- R Glicklis
- Unit Biotechnology, Faculty of Engineering Sciences, Beer-Sheva, Israel
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37
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Hansen LK, Albrecht JH. Regulation of the hepatocyte cell cycle by type I collagen matrix: role of cyclin D1. J Cell Sci 1999; 112 ( Pt 17):2971-81. [PMID: 10444391 DOI: 10.1242/jcs.112.17.2971] [Citation(s) in RCA: 59] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
Rat hepatocytes adherent to a rigid film of type I collagen will spread and enter S phase, while those attached to collagen gel or a dried collagen substrate remain round and quiescent. The current studies were initiated to determine the mechanism by which these different substrates differentially influence cell cycle progression. Cyclin D1 mRNA and protein expression and associated kinase activity was low on dried collagen relative to collagen film. In contrast, cyclin E and cdk2 protein levels were similar on the two substrates. Although cyclin E and cdk2 were present, cells on dried collagen lacked cdk2 kinase activity. p27 protein levels did not differ between dried collagen and film, but more p27 was associated with cdk2 in cells on dried collagen than those on collagen film. Cyclin D1 expression on collagen film was inhibited by cytochalasin D and exoenzyme C3, suggesting a role for the GTP-binding protein, Rho, in regulating cyclin D1 expression. Cyclin D1 over-expression induced hepatocytes into S phase in the absence of cell shape change on dried collagen or collagen gel. These results demonstrate a novel, substrate-dependent mechanism for cyclin D1 expression in hepatocytes, and also demonstrate that cyclin D1 over-expression allows shape-independent S phase entry.
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Affiliation(s)
- L K Hansen
- Department of Laboratory Medicine and Pathology, and Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA.
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38
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Hsiao CC, Wu JR, Wu FJ, Ko WJ, Remmel RP, Hu WS. Receding cytochrome P450 activity in disassembling hepatocyte spheroids. TISSUE ENGINEERING 1999; 5:207-21. [PMID: 10434069 DOI: 10.1089/ten.1999.5.207] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
Primary rat hepatocytes can self-assemble to form multicellular spheroids when plated onto Primaria petri dishes or suspended in stirred vessels. These spheroids exhibit prolonged viability, enhanced liver-specific functions and differentiated ultrastructure compared to monolayer cultures. Upon transfer to collagen coated surface, or upon the addition of fetal bovine serum (FBS) to the culture, these spheroids began to disassemble and spread on the surface. The dynamics of cytochrome P450 CYP1A1/2 activity in the course of spheroid disassembly was examined in situ by detection of the fluorescent product, resorufin, of ethoxyresorufin O-dealkylation. Optical sectioning of the disassembling spheroids by confocal microscopy demonstrated that hepatocytes that reverted to monolayer exhibited markedly lower CYP1A1/2 activity than those that remained in a multilayered structure. This occurred whether the disassembly was caused by incubation with FBS-containing medium or by cultivation on a collagen-coated surface. When spheroids were cultured on the surface of agar, the disassembly process was retarded even in the presence of FBS. However, even in those intact spheroids, the exposure to FBS markedly decreased CYP1A1/2 activity. The decreased CYP1A1/2 activity was correlated to a diminished smooth endoplasmic reticulum as seen in the transmission electron micrograph. The results clearly demonstrate that the disassembly of hepatocyte spheroids led to decreased CYP1A1/2 activity. Furthermore, FBS contained a factor that caused CYP1A1/2 to decrease even in intact spheroids.
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Affiliation(s)
- C C Hsiao
- Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, MN 55455-0132, USA
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39
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Wu FJ, Friend JR, Remmel RP, Cerra FB, Hu WS. Enhanced cytochrome P450 IA1 activity of self-assembled rat hepatocyte spheroids. Cell Transplant 1999; 8:233-46. [PMID: 10442736 DOI: 10.1177/096368979900800304] [Citation(s) in RCA: 68] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
Primary rat hepatocytes can self-assemble to form multicellular spheroids when plated onto Primaria petri dishes. Spheroids have been observed to exhibit enhanced liver-specific functions and differentiated ultrastructure compared to monolayer cultures on dry collagen. With confocal scanning laser microscopy, CYP1A1 activity was evaluated in situ by detecting resorufin. This highly fluorescent molecule is the P450-mediated product of 7-ethoxyresorufin O-dealkylation (EROD). Significantly higher P450 activity was observed in spheroids compared to monolayers on collagen upon induction with 50 microM beta-naphthoflavone (BNF), a CYP1A inducer. This was confirmed by measuring microsomal EROD activity. The distribution of CYP1A1 activity within spheroids was heterogeneous, with higher activity localized to the hepatocytes in the interior. During the process of spheroid formation, cells were initially seen to attach and spread out as a monolayer. This stage was associated with relatively low CYP1A1 activity. As cells formed multicellular structures and aggregated into spheroids, the level of CYP1A1 activity increased over time. At least a fivefold higher fluorescence intensity was observed in spheroids compared to that of monolayers maintained on collagen. The higher P450 activity within spheroids may be associated with their ability to maintain a greater degree of differentiation compared to monolayers. These studies demonstrate the potential of hepatocyte spheroids as a model system for investigating drug metabolism, tissue engineering, and tissue self-assembly.
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Affiliation(s)
- F J Wu
- Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis 55455-0132, USA
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40
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Abstract
The ability to understand and control the morphogenesis of mammalian cells is a fundamental objective of cell and developmental biology and tissue engineering research. Numerous processes, both biochemical and biophysical in nature, have been studied in an attempt to elucidate the mechanisms underlying this behavior. We focus here on the contributions of biophysical phenomena to the morphogenetic behavior of pure and mixed cell populations on solid surfaces in vitro. These principles are illustrated using characteristic liver tissue cells as a model system. The studies discussed demonstrate that cell-substratum and cell-cell adhesive forces are critical determinants of the ultimate morphology, cytoarchitecture, and organization achieved by these cells in vitro.
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Affiliation(s)
- M J Powers
- Department of Chemical Engineering and Center for Biomedical Engineering, Massachusetts Institute of Technology, Cambridge 02139, USA
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41
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Abstract
Spheroids are multicellular aggregates that exhibit a more tissue-like morphology and function when compared to monolayer cultures of the same cells. Hepatocyte spheroids are presently under investigation for use of an artificial liver. The ability to cryopreserve hepatocyte spheroids is essential for their clinical and commercial application. A multicompartment model was formulated to predict water content as a function of temperature during freezing. The theoretical predictions of water transport indicate that there will be spatial differences in water content of the spheroid during freezing and that due to the rapid decrease in water transport with decreasing temperature, the undercooling of the intracellular solution during freezing will increase steadily. These results indicate that conventional freezing of hepatocyte spheroids will be difficult to accomplish due to transport limitations in the spheroids.
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Affiliation(s)
- B Korniski
- Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis 55455, USA
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42
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Hansen LK, Hsiao CC, Friend JR, Wu FJ, Bridge GA, Remmel RP, Cerra FB, Hu WS. Enhanced Morphology and Function in Hepatocyte Spheroids: A Model of Tissue Self-Assembly. ACTA ACUST UNITED AC 1998. [DOI: 10.1089/ten.1998.4.65] [Citation(s) in RCA: 37] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Affiliation(s)
- Linda K. Hansen
- Department of Laboratory Medicine and Pathology, and Biomedical Engineering Institute, University of Minnesota, Minneapolis, Minnesota 55455
| | - Chang-Chun Hsiao
- Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota 55455
| | - Julie R. Friend
- Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota 55455
| | - Florence J. Wu
- Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota 55455
- Present address: Advanced Tissue Sciences, La Jolla, California 92037
| | - Gary A. Bridge
- Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota 55455
| | - Rory P. Remmel
- Department of Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota 55455
| | - Frank B. Cerra
- Biomedical Engineering Institute, University of Minnesota, Minneapolis, Minnesota 55455
- Department of Surgery, University of Minnesota, Minneapolis, Minnesota 55455
| | - Wei-Shou Hu
- Biomedical Engineering Institute, University of Minnesota, Minneapolis, Minnesota 55455
- Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota 55455
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43
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Kamihira M, Yamada K, Hamamoto R, Iijima S. Spheroid formation of hepatocytes using synthetic polymer. Ann N Y Acad Sci 1997; 831:398-407. [PMID: 9616730 DOI: 10.1111/j.1749-6632.1997.tb52213.x] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
Abstract
It is known that hepatocytes show the different morphology depending on the surface condition of cultural substratum. The cells form a floating cell-aggregate called spheroid on proteoglycan-coated or positively charged dishes. The liver cell functions are generally high and maintained for the long-term in the spheroid culture. Since spheroid morphology is preferable for the construction of a bioartificial liver, it is important to develop an effective method of preparing spheroids. In this regard, we examined a preparation method of functional spheroid-like cell-aggregates, in which a synthetic polymer, Eudragit was added to culture medium for inducing liver cell-aggregation. The cell-cell attachment of the aggregate was loose at the beginning of the culture, but it became tight and spheroids were formed 2-3 days after inoculation. When 0.1% Eudragit was added to the medium, the liver functions such as albumin secretion, ammonia removal and urea synthesis were enhanced compared with monolayer and conventional spheroid cultures. The spheroid formation was also performed with suspension culture in a spinner flask. Approximately 80% of the cells inoculated formed spheroids by the addition of the polymer. Moreover, the polymer showed a protective effect from cell damage by agitation. Since this procedure does not require surface for cell attachment, a large amount of spheroids can be prepared in suspension culture.
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Affiliation(s)
- M Kamihira
- Department of Biotechnology, Graduate School of Engineering, Nagoya University, Japan.
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44
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Wu FJ, Friend JR, Hsiao CC, Zilliox MJ, Ko WJ, Cerra FB, Hu WS. Efficient assembly of rat hepatocyte spheroids for tissue engineering applications. Biotechnol Bioeng 1996; 50:404-15. [DOI: 10.1002/(sici)1097-0290(19960520)50:4<404::aid-bit7>3.0.co;2-p] [Citation(s) in RCA: 117] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022]
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45
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Peshwa MV, Wu FJ, Sharp HL, Cerra FB, Hu WS. Mechanistics of formation and ultrastructural evaluation of hepatocyte spheroids. In Vitro Cell Dev Biol Anim 1996; 32:197-203. [PMID: 8727043 DOI: 10.1007/bf02722946] [Citation(s) in RCA: 82] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/01/2023]
Abstract
Freshly harvested rat hepatocytes form spheroids on uncoated positively charged polystyrene surfaces. Time lapse microscopy revealed that cell movement and reorganization were involved in spheroid formation. Ultrastructural evaluation using scanning and transmission electron microscopy indicated polarized cellular morphology and extensive cell-cell communication within spheroids. Bile canalicular structures were observed to surround each individual hepatocyte, forming an intricate three-dimensional continuous network of channels that appeared to end as pores/holes on the surface of the spheroid. The maintenance of differentiated cellular morphology coincided with preservation of hepatocyte viability and enhanced levels of tissue specific functions in spheroids.
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Affiliation(s)
- M V Peshwa
- Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis 55455-0132, USA
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46
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Wu FJ, Peshwa MV, Cerra FB, Hu WS. Entrapment of Hepatocyte Spheroids in a Hollow Fiber Bioreactor as a Potential Bioartificial Liver. ACTA ACUST UNITED AC 1995; 1:29-40. [DOI: 10.1089/ten.1995.1.29] [Citation(s) in RCA: 45] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Affiliation(s)
- Florence J. Wu
- Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota 55455
| | - Madhusudan V. Peshwa
- Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota 55455
| | - Frank B. Cerra
- Department of Surgery, University of Minnesota, Minneapolis, Minnesota 55455
| | - Wei-Shou Hu
- Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota 55455
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