Collagen sandwich culture affects intracellular polyamine levels of human hepatocytes

Abstract

Extracellular matrices, like collagen layers, play an important role in preventing dedifferentiation of hepatocytes in long-term culture experiments. It has also been shown that polyamines are crucial for cell growth and liver differentiation - regeneration. Primary cultured hepatocytes with their low mitotic activity might be a valuable tool in studying the role of polyamines in differentiation. Here, our goal was to investigate whether an extracellular cell culture matrix can influence intracellular polyamine levels in human hepatocytes during long-term culture. Primary human hepatocytes were isolated from surgical tissue resections and were maintained either in single collagen (SG) or double collagen gel (DG) layer (sandwich) culture systems. Cell viability and function were examined and intracellular polyamine levels were measured using a highly sensitive high performance liquid chromatography (HPLC) method. Hepatocytes showed high viability in both culture systems used, but albumin secretion was diminished in SG cultured hepatocytes after 14 days. In general, total intracellular polyamine levels of hepatocytes decreased markedly in both SG and DG within the first days of culture, but remained constant until day 21 with a SG/DG ratio of about 1.4. Individual polyamines levels were dependent on the culture time and system, where spermine decreased and putrescine increased in both SG and DG over time (day 14), but spermidine increased only in DG. Our results suggest that polyamine levels, in particular putrescine, might be important regulators of hepatocyte specific function in vitro and therefore serve as a marker of differentiation for cultivated human hepatocytes.

GLUT1 Expression Is Increased in Hepatocellular Carcinoma and Promotes Tumorigenesis

“…20 Primary human hepatocytes (PHHs) were isolated and cultured as previously described. 21 Human liver tissue for cell isolation was obtained according to the guidelines of the charitable state-controlled foundation Human Tissue and Cell Research with the patient's informed consent. Hypoxia was induced by incubation with 2,2Ј-dipyridyl (DP) (100 mol/L; Sigma Aldrich, Deisenhofen, Germany) or exposure to 1% O 2 for the indicated periods of time.…”

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.…”

“…One million cells were cultivated with L-15 medium. Nonattached hepatocytes were washed after one hour; second layer of collagen solution was spread to sandwich the hepatocytes after 24 h. Thirty minutes later, sandwich configuration hepatocytes were formed [28][29][30] , and the methods of culture and observation were just performed as before.…”

The Role of PI3K p110gamma in chronic liver injury

“…Mice were fed standard chow (Ssniff® R/M-H Cat.# V1534-0) or an NASH inducing diet (NASH model) which was also prepared by Ssniff (Soest, Germany) and contains 17% fat, supplemented with 1.25% cholesterol and 0.5% cholate, according to Matsuzawa et al (Matsuzawa et al, 2007 Surgery, University of Regensburg, Germany) using a modified two-step EGTA/ collagenase perfusion procedure (Hellerbrand et al, 2008,Ryan et al, 1993,Weiss et al, 2002. Further, HSC were isolated on a regular basis within our working group from 8-12…”