Dawidson I, Simonsen R, Aggarwal S, Coorpender L, Diller K, Rajotte R, Raskin P, Redman H, Rosenstock J
Department of Surgery, University of Texas Health Science Center, Dallas 75235.
Cryobiology. 1988 Apr;25(2):83-93. doi: 10.1016/0011-2240(88)90001-6.
Human fetal pancreata (HFP) were obtained from dilatation and extraction aborted fetuses of 11-18 weeks' gestation. The pancreas was excised under sterile conditions and kept in culture medium at 4 degrees C, prior to stepwise digestion into 50- to 150-micron fragments. The fragmented pieces were allowed to sediment by gravity, then transferred to tissue culture for 24-48 h, and cryopreserved. The freeze-thaw protocol used stepwise equilibration with dimethyl sulfoxide, nucleation of the sample at -10 degrees C, and a slow cooling rate of 0.25 degrees C/min to -40 degrees C, followed by submersion in liquid nitrogen (-196 degrees C). Rapid thawing at 300 degrees C/min from -196 degrees C was employed. Both fresh and frozen-thawed HFP fragments appeared viable as judged by light and electron microscopy, and secreted insulin in a perifusion system upon stimulation with glucose (28 mM) and theophylline (10 mM) or glucose (2.8 mM) and theophylline (10 mM). Six patients with Type I insulin-dependent diabetes mellitus, already requiring immunosuppression for a kidney transplant, had intraportal injection of 20 cryopreserved-thawed and pooled HFP fragments. Up to the 1-year post-transplant follow-up, there has been no evidence of in vivo insulin or C-peptide production. The usefulness of cryopreserved human fetal pancreata as a source of insulin-producing tissue for diabetic patients, therefore, remains to be demonstrated.
人胎儿胰腺（HFP）取自妊娠11 - 18周经扩张刮宫流产的胎儿。在无菌条件下切除胰腺，于4℃保存在培养基中，然后逐步消化成50至150微米的片段。将片段通过重力沉淀，然后转移至组织培养24 - 48小时，再进行冷冻保存。冷冻 - 解冻方案采用与二甲基亚砜逐步平衡、在 - 10℃使样品成核，以及以0.25℃/分钟的缓慢冷却速率降温至 - 40℃，随后浸入液氮（ - 196℃）。采用从 - 196℃以300℃/分钟的速率快速解冻。通过光学显微镜和电子显微镜判断，新鲜的和冷冻 - 解冻后的HFP片段均显示有活力，并且在葡萄糖（28 mM）和茶碱（10 mM）或葡萄糖（2.8 mM）和茶碱（10 mM）刺激下，在灌流系统中分泌胰岛素。6例I型胰岛素依赖型糖尿病患者，因已需要免疫抑制进行肾移植，接受了门静脉注射20个冷冻 - 解冻并汇集的HFP片段。直至移植后1年的随访期，尚无体内胰岛素或C肽产生的证据。因此，冷冻保存的人胎儿胰腺作为糖尿病患者胰岛素产生组织来源的实用性仍有待证实。
