Kasai S, Mito M
Second Department of Surgery, Asahikawa Medical College, Hokkaido, Japan.
Cryobiology. 1993 Feb;30(1):1-11. doi: 10.1006/cryo.1993.1001.
This study aimed to develop methods for the large-scale preparation of hepatocytes from large animal livers and for the mass cryopreservation of isolated hepatocytes. Isolated hepatocytes were obtained from Beagle dogs weighing around 10 kg by collagenase digestion plus preperfusion with the Ca2+ chelator ethylene glycol bis N,N'-tetraacetic acid. The cell yield was 2.1 +/- 0.45 x 10(10)/liver with 90% viability by the trypan blue dye exclusion test, and the estimated cell yield was 72 +/- 13%. The optimal conditions were large-scale cryopreservation of dog hepatocytes were (i) a freezing rate of 1-10 degrees C/min, (ii) a dimethyl sulfoxide (Me2SO) concentration of 20% (final concentration: 10%), (iii) a cell density that did not exceed 10(8)/ml, and (iv) rapid thawing in a 37 degrees C water bath. The viability of preserved hepatocytes was 75 +/- 3.0% and the estimated recovery rate was approximately 50%. Preserved hepatocytes showed 20-50% of the metabolic activity of fresh cells, as assessed by ammonia and fructose loading tests, ATP content, and [14C]leucine uptake. Following culture after thawing, the morphological normalization of preserved cells was confirmed by light and electron microscopy.
本研究旨在开发从大型动物肝脏大规模制备肝细胞以及对分离的肝细胞进行大规模冷冻保存的方法。通过胶原酶消化加用Ca2+螯合剂乙二醇双N，N'-四乙酸预灌注，从体重约10 kg的比格犬获取分离的肝细胞。通过台盼蓝染料排除试验，细胞产量为2.1±0.45×10(10)/肝脏，活力为90%，估计细胞产量为72±13%。犬肝细胞大规模冷冻保存的最佳条件为：(i)冷冻速率为1-10℃/分钟；(ii)二甲基亚砜(Me2SO)浓度为20%（最终浓度：10%）；(iii)细胞密度不超过10(8)/ml；(iv)在37℃水浴中快速解冻。保存的肝细胞活力为75±3.0%，估计回收率约为50%。通过氨和果糖负荷试验、ATP含量以及[14C]亮氨酸摄取评估，保存的肝细胞显示出新鲜细胞20-50%的代谢活性。解冻后培养，通过光学显微镜和电子显微镜确认了保存细胞的形态正常化。
