Extended primary culture of human hepatocytes in a collagen gel sandwich system

Abstract

To develop a strategy for extended primary culture of human hepatocytes, we placed human hepatocytes between two layers of collagen gel, called a "collagen gel sandwich." Maintenance of hepatocellular functions in this system was compared with that of identical hepatocyte preparations cultured on dry-collagen coated dishes or cocultured with rat liver epithelial cells. Human hepatocytes in a collagen gel sandwich (five separate cultures) survived for more than 4 wk, with the longest period of culture being 78 d. They maintained polygonal morphology with bile canaliculuslike structures and high levels of albumin secretion throughout the period of culture. In contrast, hepatocytes on dry-collagen became feature-less, and albumin secretion could not be detected after 14 d of culture. This loss of albumin secretion was partially recovered by overlaying one layer of collagen gel. Ethoxyresorufin O-deethylase activity, associated with cytochrome P450 1A2, was detected basally up to 29 d in collagen gel sandwich culture. These activities were induced four- to eightfold after induction with dibenz(a,h)anthracene. Cocultures also maintained basal activity up to 29 d. However, their inducibility was lower than that of hepatocytes in collagen gel sandwich. No ethoxyresorufin O-deethylase activity was detected in hepatocytes cultured on dry-collagen at 7 d. Thus, the collagen gel sandwich system preserves differentiated morphology and functions of human hepatocytes in primary culture for a prolonged period of time. This system is a promising model for studying human hepatocellular function, including protein synthesis and drug metabolism in vitro.

Primary porcine hepatocytes with portal vein serum cultured on microcarriers or in spheroidal aggregates

“…Our proceeding studies had prove d that Cytodex-3 was the best microcarrier for primary hepatocytes [3] . Hepatocytes on Cytodex-3 were readily to shape the microstructure similar to th at of liver so they could grow and function well.…”

“…Then it was proposed that syntheses of albumin and urea could be promoted by adding some nutritional factors and hormones into the monolayer culture system. By now some improvements has been achieved in searching for a new effective system to cultivate primary hepatocytes in vitro by the follo wing methods: adding hormonal agents such as insulin, glucagon, transferrin an d hydrocortisone into the basal culture medium [1] ; adding extracellular biological matrix into the culture system [2][3][4] ; enriching the cultu re system with hepatocytes growth stimulating factors such as hepatocytes growth factor and epithelial cell growth factor etc [5,6] ; co-culturing with other endodermal and mesodermal cells; three-dimensional cultivation such as microcapsular and macrocapsular culture, spheroid aggregate, microcarrier culture and hollow fiber culture [7,8] ; adding several amino acids and nutritional substances into the culture system; and adding penetrating-inducer such as pentobarbiturate to the culture system [9] . The above measures all aimed to make a circumstance mimic to the in vivo enviroment for the hepatocytes.…”

Cryopreservation and gel collagen culture of porcine hepatocytes

“…For up to 10 days' cultures, the typical cellular polygonal morphological characteristics of culture hepatocytes could be observed, and the bound between hepatocytes appear brightly and clearly; the leakage of aspartate aminotransferase was less and the level of urea nitrogen synthesized by hepatocytes was higher. Through sandwich culture can hold hepatocytes synthesizing and metabolizing abilities, the cultivated hepatocytes was also at monolayer attaching state, and do not have normal three-dimension construction in vivo, so it needs to be further ameliorated [26,29,43] . In conclusion, through the study of many important factors in hypothermic preservation, we can optimize the hypothermic preservation protocols or cultivated conditions, and established a hepatocytes storage room.…”

“…When the mixed collagen matrix was fixed in an hour, the gel was washed slowly and medium was added, and hepatocytes were incubated at 37 with 100 mL/L CO 2 900 mL/L humidity. The methods of culture and observation were the same to the control group [26,27] . When thawed hepatocytes were cultivated in sandwich configuration, the collagen solution was prepared just as before, then T-flasks were coated with it and incubated one hour at 37 .…”

Hepatic Differentiation of Human Embryonic and Induced Pluripotent Stem Cells for Regenerative Medicine

“…Although the superiority of the culture conditions in the 3D dynamic culture system has been clearly demonstrated, it is difficult to determine which parameters are critical for this advantage in hepatic maturation. Using matrigel or collagen gel, it has been shown that static 3D culture conditions prolong hepatic function of primary hepatocytes (Kono et al 1997). It has been proposed that the 3D structure allows more physiological cellto-cell interactions, and induces polarity in hepatocytes (Haouzi et al 2005).…”