Non-enzymatic methods for isolation of stromal vascular fraction and adipose-derived stem cells: A systematic review

Table 1 Summary of the included studies in the systematic review

Ref.	Method of separation	No. of samples	Open/closed	Time taken	Cell counts	Significance
Raposio et al[31]	Vibrating shaker at 6000 rpm for 6 min followed by centrifugation at 1600 rpm for 6 min	-	Open	15 min	125000 nucleated cells per cc of lipoaspirate, but only about 5% of these were found to be progenitor cells	A pellet is formed at the end
Cohen et al[32]	LipocubeNano decanted for 3 min in a syringe: First, Port 1 is used to pass the fat graft once, resulting in 1 mm parcel sizes. After that, the fat is transferred 10 times back and forth between Ports 2 and 3, smoothing and homogenizing the fat tissue. Finally, to produce the final product, Nanofat, the fat was transferred once from Port 3 to Port 4 via a 500-micron single filter. Tulip NanoTransfer Kit. After decantation for 3 min, transfer millifat from Port 1 to 2. Flush the fat between Ports 2 and 3 for 10 times. Collect the final product by transferring fat from Ports 3 to 4 in a single stroke	10 patients	Closed	Not mentioned (Approximately less than 10 min for both methods)	LipocubeNano resulted in relatively high cell counts (2.24 × 106/mL) and cell viability (96.75%), whereas Tulip’s NanoTransfer method resulted in a lower cell count of 1.44 × 106/mL and cell viability of 96.75%	-
Tonnard et al[2]	Lipoaspirate is washed and rinsed followed by 30 passes done between two 10 mL syringes connected by leur lock and the resultant whitish fluid is filtered over a sterile nylon cloth	67 cases	Open	Not mentioned (Approximately less than 10 min)	1975000 cells per 100 mL of lipoaspirate	Introduced the concept of nanofat
Sesé et al[33]	Enzymatic dissociation using GID stromal vascular fraction protocol. Mechanical dissociation using Tulip. NanoTransfer Protocol	20 for enzymatic and 6 for mechanical	Closed	Not available	Enzymatically dissociated stromal vascular fraction resulted in 0.68 million cells/g lipoaspirate, whereas mechanically disaggregated nanofat resulted in 6.63 million cells/g lipoaspirate	-
Bianchi et al[34]	Lipogems	-	Closed	Less than 20 min	The significantly higher percentage of mature pericytes and MSCs, and lower number of hematopoietic elements, than enzymatically digested lipoaspirates	-
Bright et al[35]	Centrifugation for 2 min at 200 g of lipoaspirate. Followed by an ultrasonic cavitation device probe using Hielschler UP200s set at 50% amplitude and cycle of 0.4 for 1 min with the probe lowered and 30 s at the top of the tube. The resultant fluid is subjected to centrifugation for 5 min at 300 g with a temperature not rising above 43 degrees and preferably not over 37 degrees	-	Open	-	169 million cells were injected intraarticularly for a patient with anterior cruciate tear mentioned but not specified the volume	Described as a patented procedure. Different modifications of this technique also have been described based on the indication and site of therapeutic application
Gentile et al[36]	Mystem system – washing and filtration. Fastem- Filtration and centrifugation for 10 min at 1700 rpm	10 for Fastem and 10 for Mystem	Closed for both systems	Not available	Cell yield achieved with Mystem is less than that with Fastem and Cytori (enzymatic isolation technique)	Improved contour seen after breast reconstruction with fat grafts enriched with ADSCs from Fastem (equal to Cytori) greater than Mystem
van Dongen et al[22]	Centrifuged at 300 rpm for 2.5 min followed by non-enzymatic dissociation performed by pushing the lipoaspirate to and through a fractionator 30 times. The resultant fluid is centrifuged for 2.5 min at 3000 rpm	-	Open	10 min	2.7 × 106/mL	-
Chaput et al[37]	Vortexing and centrifuging. Vibrating shaker for 6 min at 3200 rpm followed by centrifugation for 6 min at 558 g followed by 100 micrometre sieves followed by centrifugation for 10 min	21	Open	22 min	The percentage of ADSCs in SVF extracted by vortexing and centrifugation, dissociation by inter-syringe process, and enzymatic isolation techniques are 5.81 ± 1.3, 38.11 ± 5.1, and 21.45 ± 2.52, respectively	Final pellet for vortexing and centrifuging
	Dissociation by inter-syringe processing 30 passes through leur lock connected syringes passed through 100-micrometre sieve followed by centrifugation for 10 min		Open	11 mins		Final pellet for dissociation by inter-syringe processing
Copcu et al[38]	Centrifuged at 500 g for 2 min. Adinizing was first performed with a 4000-micron Adinizer; after approximately 25 passes, the cutting process was continued with the next-smaller diameter disk followed by centrifugation for 6 min at 1600 g	24 patients	Open	Not mentioned	93% mean viability and cell counts of 28.66 to 88.88 × 106 from 100 mL of condensed fat	Volumes ranging from 3-12 mL can be produced depending on the indication
Rose et al[39]	Sedimentation for 1 h, centrifugation at 3000 rpm for 3 min or washing with normal saline combined with 3 min of centrifugation at 3000 rpm	24 fat samples	Open	-	The mean cell count per high-powered field of histologically intact adipocytes was 27.1 for specimens processed by sedimentation, 14.2 for centrifuging, and 11.8 for washing	-
Amirkhani et al[40]	Dissected for 10 s using a blender mixer followed by sonic cavitation for 2 min at 18 MHz followed by centrifugation for 10 min at 900 g followed by suspension with 150 mM ammonium chloride for 5 min and centrifugation for 5 min at 400 g. The pellets are then resuspended in DMEM supplemented with 10% FBS and then seeded into a T25 culture flask. After 24 h, the adherent cells were used for further confirmation tests. The SVFs harvested by both methods were suspended in PBS and then incubated for 30 min at 4 °C with the antibodies conjugated with FITC against CD34, CD44, CD73, CD90, and CD105 biomarkers	-	Open	Less than 30 min	Viable cells 2.6 × 105 cells from 1 mL of fat tissue	-
Victor[41]	Ultrasonic cavitation performed using a 200 W generator (SONIC 200) for a range of 10 to 20 min at a frequency of about 20-30 kHz	-	Open	-	From 2 million up to 22 million stromal vascular cells per mL of adipose tissue	Described as a patented procedure
Domenis et al[42]	Fastem - automated system performing filtration and centrifugation for 10 min at 1700 rpm	6	Closed	Not available	Only mentioned that cell yield from Fastem was less than Lipokit and less than Cytori	Enriched grafting has greater subcutaneous thickness
Condé-Green et al[43]	Centrifugation followed by vortexing for 3 min. Centrifugation followed by RBC lysis	9	Open	Not available	cSVF of 1.2 × 104 per mL for the first method and 2.3 × 104 per mL for the second method	Mechanical methods have greater cells positive for CD14 than with enzymatic process which is a marker for monocytes and macrophages
Markarian et al[44]	The first method involved RBC lysis of lipoaspirate and then centrifugation for 10 min at 600 g. The second and third techniques each included an additional initial stage of centrifugation at 800 g and 1280 g for 15 min, respectively	10	Open	Not available	The cell yield obtained from collagenase was greater than that of mechanical and trypsin. The second and third methods produced viable cells that had not proliferated even after 14 d	-
Shah et al[45]	Rigorous washing in PBS with handshaking followed by centrifugation for 15 min at 900 g	13	Open	1 h for mechanical and almost 3 h for isolation with collagenase	The mechanical method produced 19 times fewer cells compared to the enzymatic extraction technique	-
Condé-Green et al[46]	Lipoaspirate is subjected to RBC lysis followed by 15 min of centrifugation at 900 g	10	Open	Not available	The highest concentration of ADSCs was found in the pellet found at the bottom after centrifugation	-
Baptista et al[47]	The procedure followed in the same sequence. RBC lysis, centrifugation for 15 min at 900 g, resuspension in fetal bovine serum plus dimethyl sulfoxide, cryopreservation at –196 degrees centigrade	13	Open	Mechanical processing required less time	Cell yield was less with mechanical compared to enzymatic processing	Adherent cells were positive for CD44, CD90, CD105, and CD34 and negative for CD45 and CD73

MSCs: Mesenchymal stem cells; ADSCs: Adipose-derived stem cells; SVF: Stromal vascular fraction; FBS: Foetal bovine serum; RBC: Red blood cell; cSVF: Cellular stromal vascular fraction; PBS: Phosphate buffered saline.

Table 2 Risk of bias in the included studies based on office of health assessment and translation criteria

Ref.	Did the selection of study participants appropriate?	Did the study account for confounding and modifying variables?	Were the outcome data complete without attrition bias?	Can we be confident in exposure characterization?	Can we be confident in outcome assessment?	Were all measured outcomes reported?
Raposio et al[31]	++	+	++	++	++	++
Chaput et al[37]	++	++	+	+	++	+
Cohen et al[32]	++	+	+	- (NR)	+	+
Copcu et al[38]	++	++	++	+	+	++
Tonnard et al[2]	++	+	+	+	+	+
Sesé et al[33]	++	+	+	+	+	+
Rose et al[39]	++	+	++	++	++	++
Bianchi et al[34]	++	++	++	++	++	++
van Dongen et al[22]	++	+	+	- (NR)	+	+
Amirkhani et al[40]	++	++	++	++	++	++
Victor[41]	++	++	++	++	++	++
Bright et al[35]	++	-	+	+	+	+
Domenis et al[42]	++	++	+	- (NR)	+	+
Gentile et al[36]	++	+	+	+	+	+
Condé-Green et al[43]	++	++	++	++	++	++
Markarian et al.[44]	++	++	+	++	++	++
Shah et al[45]	++	++	++	++	++	++
Condé-Green et al[46]	++	++	++	- (NR)	+	+
Baptista et al[47]	++	+	+	++	++	++

NR: Not reported; ++: Reflects a low risk of bias; +: Reflects probably a low risk of bias; -: Reflects probably a high risk of bias.