Maintenance of periportal and pericentral oxygen tensions in primary rat hepatocyte cultures: Influence on cellular DNA and protein content monitored by flow cytometry

✍ Scribed by Claudia Holzer; Peter Maier

• Publisher: John Wiley and Sons
• Year: 1987
• Tongue: English
• Weight: 656 KB
• Volume: 133
• Category: Article
• ISSN: 0021-9541
• DOI: 10.1002/jcp.1041330213

✦ Synopsis

Primary hepatocytes were cultured at oxygen tensions similar to those reported to be present in periportal (13% 02) and pericentral (4% 0,) regions of the liver lobules. Cellular DNA and protein content of individual hepatocytes were determined simultaneously by two-parameter (DNAlprotein) flow cytometry after 1, 4, and 7 days in culture. p 0 2 tensions monitored on line in conventional plastic culture dishes revealed that the depletion of the p 0 2 in the culture medium depended on the number of hepatocytes plated. When cultured as monolayer after 4-7 days at periportal (13% 0 2 ) and more pronounced at pericentral oxygen concentration (4% O,), u p to 90% of the hepatocytes showed degenerated nuclei but normal protein content. By using culture dishes with teflon membrane bottoms the oxygen tension in the culture medium was accurately maintained by the incubator atmosphere. At pericentral oxygen tension the fraction of 2 N cells increased by about 20%. That of the 4N cell was not affected, and the contribution of 8N hepatocytes dropped to 70% compared to cultures at periportal oxygen tension. Concomitantly, in the 4% 0, hepatocyte cultures the protein content of the 2 N and the 4N cells was better preserved and increased by up to 10%. These results suggest that in vitro at pericentral oxygen conditions (4% 0,) ageing of hepatocytes is delayed, regenerating processes are better maintained, and, furthermore, freshly isolated 4N hepatocytes have the potency to adapt their metabolism in vitro to periportal as well as to perivenous oxygen tensions.

PP and PC oxygen tension on protein and DNA content was then compared in 7-day cultures.