Mass Preparation of Primary Porcine Hepatocytes and the Design of a Hybrid Artificial Liver Module using Spheroid Culture for a Clinical Trial

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Abstract
To isolate a large number of porcine hepatocytes, we originally developed a mass preparation method that combined the usual collagenase perfusion method of a whole liver with a collagenase redigestion method of tissue fragments after liver perfusion. Using a pig of 10kg, collagenase perfusion only resulted in a yield of 63 ± 78 x 108 total cells with a viability of 69.2 ± 25.3 %, but our combined method had a yield of 167 ± 31 x 108 total cells with a viability of 87.9 ± 4.4 % (mean ± SD). Also, the combined method was applied to two pigs of 10kg body weight at the same time, and isolated 387 ± 89 x 108 hepatocytes with a viability of 87.1 ± 6.9 % and a purity of 93.6 ± 2.8 % in 11 experiments. We designed a large multi-capillary polyurethane foam (MC-PUF) packed-bed module containing 1 x 1010 porcine hepatocytes on a clinical trial scale. The porcine hepatocytes in the module formed spherical multicellular aggregates (spheroids) of 200 – 500 μm diameter. Most hepatocytes forming spheroids were viable judged by fluorescein diacetate and ethidium bromide staining. The activities of ammonia removal, albumin secretion and oxygen consumption of the large MC-PUF module were the same as for a small MC-PUF module containing 2 x 108 porcine hepatocytes, and were maintained for at least 9 days of culture. These results show that a large MC-PUF module is successfully scaled up 50 times. In conclusion, we succeeded in developing a mass preparation method of porcine hepatocytes and a large hybrid artificial liver module on a clinical trial scale.