Culture with Growth Factor Supplements Improves the Viability and Function of Rat Hepatocytes

Hyung Na Gun; Goo Kim Dong; Sun Jung Eun

doi:10.4285/jkstn.2015.29.3.139

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Gun, Dong, and Eun: Culture with Growth Factor Supplements Improves the Viability and Function of Rat Hepatocytes

Original Article

J Korean Soc Transplant 2015;29(3):139-147.

Published online: 10 September 2015

DOI: https://doi.org/10.4285/jkstn.2015.29.3.139

Culture with Growth Factor Supplements Improves the Viability and Function of Rat Hepatocytes

Hyung Na Gun, M.D.¹, Goo Kim Dong, Ph.D.¹, Sun Jung Eun, Ph.D.²

¹Departments of Surgery

²Pathology, Seoul St. Mary’ s Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea

Corresponding author: Dong Goo Kim Department of Surgery, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul 06591, Korea Tel: 82-2-2258-6096, Fax: 82-2-595-2822 E-mail: kimdg@catholic.ac.kr

Received 7 June 2015 Revised 7 June 2015 Accepted 11 August 2015

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

To identify the optimum culture conditions by investigating isolated rat hepatocytes cultured in medium containing different growth factors.

Methods

Hepatocytes were isolated from rats using a two-step perfusion technique and divided into the following four groups cultured in medium containing different growth factors: control, epidermal growth factor (EGF), insulin, and EGF+insulin. The viability of the cultured rat hepatocytes and liver function parameters, including albumin, ammonia, and urea in the culture medium, were measured. Hepatocyte morphology was examined by staining with hematoxylin and eosin, and albumin receptor expression was confirmed by immunofluorescence.

Results

Slightly higher viability was observed in the growth factor groups than in the control group, although without significance (P=0.073). The levels of albumin (P=0.001), ammonia (P＜0.001), and urea (P=0.041) differed significantly among the four groups. The functional parameters in the growth factor groups, particularly the EGF+insulin group, were significantly superior to those in the control group. The morphology of the hepatocytes in all growth factor groups was well maintained at 10 days. However, the control group showed deterioration in cell morphology by day 7.

Conclusions

Morphological and functional assessment indicated that the presence of growth factors, particularly EGF+insulin, provided culture conditions superior to those of non-supplemented medium.

Keywords: Culture, Epidermal growth factor, Hepatocytes, Insulin, Rats

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Fig. 1.

(A) Operative view of a cannulation in portal vein and (B) an infusion of collagenase solution with whitish discoloration of liver.

Fig. 2.

Evaluation of cell viability by trypan blue exclusion test (×200). Viable cells with an intact plasma membrane exclude dyes such as trypan blue, whereas damaged cells (arrows) become stained, particularly intensively in the nucleus. The viability of isolated hepatocytes in this photograph was about 85%.

Fig. 3.

The change of viability according to growth factor supplement in culture medium. Data are average value±SEM. Abbreviation: EGF, epidermal growth factor. ^a P＜0.05 vs. control on each day.

Fig. 4.

The change of liver function test including (A) albumin, (B) ammonia, and (C) urea according to growth factor supplement in culture medium. Data are average value±SEM. Abbreviation: EGF, epidermal growth factor. ^a P<0.05 versus control on each day.

Fig. 5.

Histologic feature of cultured rat hepatocytes (HE stain, ×400): (A) 7 days, (B) 10 days, and (C) 14 days after culture. Abbreviation: EGF, epidermal growth factor.

Fig. 6.

Histologic feature of cultured rat hepatocytes (immunofluorescent staining, ×100): (A) 3 days and (B) 7 days after culture. Abbreviation: EGF, epidermal growth factor.

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