Ann Surg Treat Res.  2014 Aug;87(2):53-60. 10.4174/astr.2014.87.2.53.

Effects of glucose concentration in the medium on rat hepatocyte culture

• Na GH
• Kim DG
• Kim YH
• Han JH
• Jung ES

• KMID: 1882831
• DOI: http://doi.org/10.4174/astr.2014.87.2.53

Abstract

PURPOSE
To determine the optimum culture conditions by investigating isolated rat hepatocytes cultured in medium containing different glucose concentrations.
METHODS
Hepatocytes were isolated from rats using a two-step perfusion technique and divided into the following two groups cultured in medium containing different glucose concentrations: (1) low-glucose group and (2) high-glucose group. Total cell count and viability of cultured rat hepatocytes and liver function parameters (i.e., concentrations of albumin, ammonia, and urea in the culture medium) were measured. The morphology of cultured rat hepatocytes was examined by staining with hematoxylin and eosin, and albumin receptor expression was confirmed by immunofluorescence.
RESULTS
Total cell count and viability showed smaller increases in the low-glucose group than the high-glucose group, although the difference was not statistically significant (P = 0.112 and P = 0.147, respectively). The levels of albumin (P = 0.943), ammonia (P = 0.744), and urea (P = 0.709) were not significantly different between the two groups. In both groups, the function of cultured hepatocytes decreased significantly over time. The morphology of hepatocytes was well maintained in both groups at 3 days. On day 7, the cytoplasm was transformed into a spindle shape. On day 10, these changes were exaggerated, and were more prominent in the high-glucose group.
CONCLUSION
Morphological assessment indicated that low-glucose culture medium is better than high-glucose culture medium for culturing of hepatocytes, although there was not significantly different in functional assessment. The cultured hepatocytes with low-glucose culture medium could be maintained for 7 days.

Keyword

Culture; Glucose; Hepatocytes; Cell transplantation; Rats

MeSH Terms

Ammonia
Animals
Cell Count
Cell Transplantation
Cytoplasm
Eosine Yellowish-(YS)
Fluorescent Antibody Technique
Glucose*
Hematoxylin
Hepatocytes*
Liver
Perfusion
Rats*
Receptors, Albumin
Urea
Ammonia
Eosine Yellowish-(YS)
Glucose
Hematoxylin
Receptors, Albumin
Urea

Figure

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  Fig. 1 Operative view of cannulation in the portal vein (A) and infusion of collagenase solution with whitish discoloration of the liver (B).

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  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, with particularly strong staining in the nucleus. The viability of isolated hepatocytes in this photograph was about 85%.

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  Fig. 3 The changes in total cell count (A) and viability (B) according to glucose concentration in the culture medium. Data are means ± standard error of the mean. Asterisks indicate significant differences between time points (*P < 0.05).

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  Fig. 4 Changes in liver function, including albumin (A), ammonia (B), and urea (C) levels, according to the glucose concentration in the culture medium. Data are means ± standard error of the mean. Asterisks indicate significant differences between time points (*P < 0.05).

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  Fig. 5 Histological features of cultured rat hepatocytes (H&E, ×400). (A) Low-glucose group. (B) High-glucose group.

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  Fig. 6 Histological features of cultured rat hepatocytes (immunofluorescence staining, ×200). (A) Low-glucose group. (B) High-glucose group.

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