Taylor M J, Baicu S
Cell and Tissue Systems, North Charleston, South Carolina 29406, USA.
Transplant Proc. 2011 Nov;43(9):3181-3. doi: 10.1016/j.transproceed.2011.10.001.
A critical component of treating type I diabetes by transplantation is the availability of sufficient high-quality islets. Currently, islets can be obtained only by reliance on an expensive, inconsistent, and toxic enzyme digestion process. As an alternative, we hypothesize that cryobiologic techniques can be used for differential freeze destruction of the pancreas to release islets that are selectively cryopreserved in situ.
Pancreases were procured from juvenile pigs with the use of approved procedures. The concept of cryo-isolation is based on differential processing of the pancreas in 5 stages: 1) infiltrating islets in situ preferentially with a cryoprotectant (CPA) cocktail via antegrade perfusion of the major arteries; 2) retrograde ductal infusion of water (or saline solution) to fully distend the gland; 3) freezing the entire pancreas to -160°C, and stored in liquid nitrogen; 4) mechanically crushing and pulverizing the frozen pancreas into small fragments; and 5) thawing, filtering and washing the frozen fragments with RPMI 1640 culture medium to remove the CPA. Finally, the filtered effluent (cryo-isolate) was stained with dithizone for identification of intact islets, and samples were taken for static glucose-stimulated insullin release assessment.
As predicted the cryo-isolated contained small fragments of residual tissue comprising an amorphous mass of acinar tissue with largely intact embedded islets. The degree of cleavage of the cryoprotected islets from the freeze-destroyed exocrine cells, was variable. Islets were typically larger than their counterparts isolated from juvenile pigs with conventional enzyme-digestion techniques. Functionally, the islets from replicate cryo-isolates responded to a glucose challenge with a mean stimulation index = 3.3 ± 0.7 (n = 3).
An enzyme-free method of islet isolation relying on in situ cryopreservation of islets with simultaneous freeze-destruction of acinar tissue is feasible and proposed as a novel method that avoids the problems associated with conventional collagenase digestion methods.
通过移植治疗I型糖尿病的一个关键要素是要有足够数量的高质量胰岛。目前，胰岛只能通过依赖一种昂贵、不稳定且有毒的酶消化过程来获取。作为一种替代方法，我们推测低温生物学技术可用于对胰腺进行差异性冷冻破坏，以释放原位选择性冷冻保存的胰岛。
使用批准的程序从幼年猪获取胰腺。冷冻分离的概念基于胰腺的五个阶段的差异性处理：1）通过主要动脉的顺行灌注，使原位胰岛优先浸润一种冷冻保护剂（CPA）混合物；2）通过逆行导管注入水（或盐溶液）以使腺体充分扩张；3）将整个胰腺冷冻至-160°C，并储存在液氮中；4）将冷冻的胰腺机械粉碎成小碎片；5）用RPMI 1640培养基对冷冻碎片进行解冻、过滤和洗涤以去除CPA。最后，将过滤后的流出物（冷冻分离物）用双硫腙染色以鉴定完整的胰岛，并采集样本进行静态葡萄糖刺激胰岛素释放评估。
如预期的那样，冷冻分离物包含残留组织的小碎片，由无定形的腺泡组织块和大部分完整嵌入的胰岛组成。冷冻保护的胰岛与冷冻破坏的外分泌细胞的分离程度各不相同。胰岛通常比用传统酶消化技术从幼年猪分离的胰岛更大。在功能上，重复的冷冻分离物中的胰岛对葡萄糖刺激的反应平均刺激指数 = 3.3 ± 0.7（n = 3）。
一种依赖于胰岛原位冷冻保存同时对腺泡组织进行冷冻破坏的无酶胰岛分离方法是可行的，并被提议作为一种避免与传统胶原酶消化方法相关问题的新方法。
