Basic Research
Copyright ©2005 Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Nov 28, 2005; 11(44): 6968-6974
Published online Nov 28, 2005. doi: 10.3748/wjg.v11.i44.6968
Induction of pancreatic duct cells of neonatal rats into insulin-producing cells with fetal bovine serum: A natural protocol and its use for patch clamp experiments
San-Hua Leng, Fu-Er Lu
San-Hua Leng, Fu-Er Lu, Institute of Integrative Traditional Chinese and Western Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China
Author contributions: All authors contributed equally to the work.
Supported by the National Natural Science Foundation of China, No. 30472254
Correspondence to: Professor Fu-Er Lu, Institute of Integrative Traditional Chinese and Western Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China. felu@tjh.tjmu.edu.cn
Telephone: +86-27-83662220 Fax: +86-27-83646605
Received: December 28, 2004
Revised: March 23, 2005
Accepted: March 24, 2005
Published online: November 28, 2005
Abstract

AIM: To induce the pancreatic duct cells into endocrine cells with a new natural protocol for electrophysiological study.

METHODS: The pancreatic duct cells of neonatal rats were isolated, cultured and induced into endocrine cells with 15% fetal bovine serum for a period of 20 d. During this period, insulin secretion, MTT value, and morphological change of neonatal and adult pancreatic islet cells were comparatively investigated. Pancreatic β-cells were identified by morphological and electrophysiological characteristics, while ATP sensitive potassium channels (KATP), voltage-dependent potassium channels (KV), and voltage-dependent calcium channels (KCA) in β-cells were identified by patch clamp technique.

RESULTS: After incubation with fetal bovine serum, the neonatal duct cells budded out, changed from duct-like cells into islet clusters. In the first 4 d, MTT value and insulin secretion increased slowly (MTT value from 0.024±0.003 to 0.028±0.003, insulin secretion from 2.6±0.6 to 3.1±0.8 mIU/L). Then MTT value and insulin secretion increased quickly from d 5 to d 10 (MTT value from 0.028±0.003 to 0.052±0.008, insulin secretion from 3.1±0.8 to 18.3±2.6 mIU/L), then reached high plateau (MTT value >0.052±0.008, insulin secretion >18.3±2.6 mIU/L). In contrast, for the isolated adult pancreatic islet cells, both insulin release and MTT value were stable in the first 4 d (MTT value from 0.029±0.01 to 0.031±0.011, insulin secretion from 13.9±3.1 to 14.3±3.3 mIU/L), but afterwards they reduced gradually (MTT value <0.031±0.011, insulin secretion <8.2±1.5 mIU/L), and the pancreatic islet cells became dispersed, broken or atrophied correspondingly. The differentiated neonatal cells were identified as pancreatic islet cells by dithizone staining method, and pancreatic β-cells were further identified by both morphological features and electrophysiological characteristics, i.e. the existence of recording currents from KATP, KV, and KCA.

CONCLUSION: Islet cells differentiated from neonatal pancreatic duct cells with the new natural protocol are more advantageous in performing patch clamp study over the isolated adult pancreatic islet cells.

Keywords: Pancreatic duct cells; Pancreatic precursor cells; Insulin-producing cells; Patch clamp; Experimental protocol; ATP sensitive potassium channels; Voltage-dependent potassium channels; Voltage-dependent calcium channels