The mixed coculture effect of primary rat hepatocytes and bone marrow cells is caused by soluble factors derived from bone marrow cells
Section snippets
Cells
Primary rat hepatocytes and BMCs were simultaneously isolated from 6–7-week-old male Wistar rats (Kyudo, Tosu). Hepatocytes were prepared using a two-step collagenase perfusion method (7), and their viability was confirmed to be >95% using Trypan blue exclusion. BMCs were obtained from the femurs by flushing with culture medium containing 20% heat-inactivated fetal bovine serum (HI-FBS) and dispersed by passing through a 22-gauge needle. The BMCs were subsequently filtered through a stainless
Optimization of coculture conditions for hepatocytes and BMCs
Heat-inactivated serum is usually used in BMC cultures, but serum-free medium (rat [10], dog [8]) or untreated FBS-supplemented medium (porcine) (11) have been used in spheroid cultures of primary hepatocytes. Therefore, we examined the effect of the heat-inactivated treatment of FBS in cocultures of hepatocytes and BMCs. On the seventh day of culture, the albumin production activity of the cocultured hepatocytes in the 20%-HI-FBS-supplemented culture medium (9.31±0.52 μg/well/d) was slightly
Discussion
Apoptosis and cell functions were previously reported to be affected by the composition of the culture medium (12). Furthermore, it has become clear that soluble factors secreted by BMCs participate in the coculture effects of hepatocytes and BMCs (6). Therefore, we hypothesized that the coculture effect of hepatocytes and BMCs might be the result of an anti-apoptotic effect. However, no anti-apoptotic effect of coculturing was detected (Fig. 5). Furthermore, there was no improvement in the
Acknowledgments
This work was supported in part by Grant-in-Aids for Young Scientists (A): 15686035 and for Scientific Research (B): 18360399 from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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