Growth Of Hepatocytes In Culture Research Articles

Pleiotrophin/Heparin-Binding Growth-Associated Molecule as a Mitogen of Rat Hepatocytes and Its Role in Regeneration and Development of Liver

Previously pleiotrophin (PTN) was identified among proteins secreted by Swiss 3T3 cells as a mitogen for cultured adult rat hepatocytes. The present study showed that the growth of rat hepatocytes was enhanced when cultured with rat hepatic stellate cells (HSCs). HSCs expressed PTN mRNA and secreted its protein in the co-cultures. Recombinant PTN enhanced the growth of hepatocytes in culture, suggesting that HSCs stimulate the growth of hepatocytes through the action of PTN. To know the biological role of PTN in the growth of hepatocytes in vivo, we examined the expression of PTN in four regeneration models of adult liver and embryonic liver of rat. The expression of PTN mRNA in the liver was markedly up-regulated by the treatment with D-galactosamine (GalN) or with acetylaminofluorene followed by partial hepatectomy. HSCs expressed PTN mRNA in response to GalN treatment and its protein was found on hepatocytes. The mRNA expression of N-syndecan, a PTN receptor, was up-regulated in GalN-treated hepatocytes. The mesenchymal cells in the septum transversum enclosing the embryonic liver, but not embryonic HSCs, expressed PTN mRNA. We suggest that PTN is secreted from activated adult HSCs and embryonic mesenchymal cells as a mitogen of parenchymal cells in adult and embryonic liver, respectively.

Mechanisms controlling growth of hepatocytes in primary culture

Mature hepatocytes in primary culture express most of the functions and hormonal responsiveness seen in normal liver studied in vivo. The growth of hepatocytes in culture is regulated by various growth factors. We have identified a hepatocyte growth factor that is isolated from rat platelets. We found that rat platelets also contain a growth inhibitor, transforming growth factor-beta which is secreted as a latent molecule. Its latency is due to its binding with a masking protein. Growth of hepatocytes is also suppressed by interleukin-1 (IL-1) and IL-6. Moreover, the growth and functions of liver cells in culture are regulated reciprocally by cell density: at higher cell density liver-specific functions are expressed and growth is suppressed, whereas the opposite situation is observed at lower cell density. In contrast, neonatal hepatocytes in culture grow autonomously without a requirement for added hormones. This autonomous growth is due to an autocrine mechanism in which the cells secrete one or more growth factors into the culture medium. However, this autonomous growth ceases one week after birth at a time when the cells begin to express differentiated characteristics. Based upon these data, the mechanisms of liver regeneration, differentiation, and hepatocarcinogenesis are discussed.

A simple medium for the study of hepatocyte growth in culture under defined conditions.

The combination (1:1) of Dulbecco's modified Eagle's medium and Waymouth's medium MAB 87/3 was found to provide favorable conditions for serum-free culture and growth of adult rat hepatocytes. In this simple medium, a majority of hepatocytes stimulated by epidermal growth factor plus insulin entered S phase and divided, with a normal (13 h) interval between DNA synthesis and cell division. The proliferative response did not require extra substratum or the presence of serum, even during cell isolation and plating.

L-Proline is an essential amino acid for hepatocyte growth in culture

For improvement of the culture conditions of adult rat hepatocytes in primary culture in collagen coated dishes, effects of various commercial culture media on the induction of replicative DNA synthesis of the cells stimulated by insulin plus epidermal growth factor were studied. Proline-deficient media, such as Leibovitz's L-15, Eagle's minimal essential medium and Dulbecco's modified minimal essential medium, did not induce DNA synthesis in hepatocytes, whereas proline-rich media, such as Williams medium E, McCoy's 5A and Ham's F-12, induced markedly hepatocyte proliferation. Moreover, when the proline-deficient media were supplemented with L-proline, the cells synthesized DNA in response to the two hormones. Cis-4-hydroxyl-L-proline strongly inhibited the induction of DNA synthesis, without affecting protein synthesis of the cells or showing any cytotoxicity. This inhibition was recovered completely by adding excess proline to the medium. Addition of other amino acids not present in the medium did not promote DNA synthesis. These findings indicate that L-proline is essential for induction of hepatocyte proliferation in culture, through its affect on synthesis of intracellular collagen.