Novel cryopreservation method for stabilization of manufactured therapeutic cells

一种基于细胞兼容封装的新型玻璃化冷冻方法 自然杀伤细胞免疫疗法 摘要：使用自然杀伤(NK)细胞作为治疗血液系统和实体肿瘤的免疫疗法已经 在过去的几年里得到了很大的关注。NK细胞的关键优势包括没有输血风险- 相关的移植物抗宿主疾病和降低肿瘤逃逸的可能性。事实上，NK细胞 在不需要MHC的情况下识别目标，I类允许使用同种异体产品。在密歇根大学 明尼苏达州，米勒博士和他的NK细胞计划团队已经用捐赠者NK治疗了400多名癌症患者 细胞和单次给药是充分测试其抗癌活性的主要限制。为了允许第三方、场外- 货架大规模制造需要多剂量策略，我们必须能够冷冻和解冻NK细胞 随需应变，生存能力和功能损失最小。然而，目前的冷冻保存实践严重依赖于 关于使用有毒的冷冻保护剂(CPA)，如二甲基亚砜(DMSO)和冻融协议 不是针对NK细胞进行优化的。结果是：1)自然杀伤细胞解冻后活性显著下降 B)丧失NK细胞的细胞毒作用和细胞因子的产生功能。 Pi Aksan教授的实验室最近开发了一种基于细胞兼容封装的玻璃化方法，该方法 不需要细胞毒性和致突变的CPA。这项建议的主要目标是发展和 这一新方法适用于玻璃化冷冻和保存扩张的血液和诱导的多能干细胞 (IPSC)来源的NK细胞。我们的方法避免了在冷却过程中有害的细胞内/细胞外冰结晶， 并在升温过程中重新结晶，因此消除了冻结/解冻和低温储存导致的损失 存活率和解冻后功能。为实现主要目标，我们将实现以下具体目标： 具体目标1：设计胶囊凝胶并确定细胞相容的CPA组合物 这将抑制细胞内外冰的结晶和生长、玻璃化和共晶融化， 这是有害的。 具体目标2：使用优化的冷冻-解冻使解冻后的NK细胞活性和功能最大化 协议。 具体目标3：验证NK细胞在-80oC和液体中的短期和中期储存稳定性 氮气。

Novel Cytocompatible Encapsulation-based Vitrification Method for Natural Killer Cell Immunotherapy Summary: Use of natural killer (NK) cells as immunotherapy against hematologic and solid tumor cancers has gained much attention in the last few years. Critical advantages of NK cells include lack of risk for transfusion- associated graft-versus-host disease and decreased likelihood of tumor escape. The fact that NK cells recognize targets without needing MHC class I allows for the use of allogeneic products. At the University of Minnesota, co-I Dr. Miller and his NK cell Program team have treated over 400 cancer patients with donor NK cells and single dosing is a major limitation to fully test their anti-cancer activity. To allow for third party, off-the- shelf large- scale manufacturing needed for multidose strategies, we must be able to freeze and thaw NK cells on demand with minimal loss of viability and function. However, current cryopreservation practice heavily relies on the use of toxic cryoprotectant agents (CPAs) such as dimethyl sulfoxide (DMSO) and freeze/thaw protocols that are not optimized for NK cells. As a result, there is a) a significant decrease in post-thaw viability of the NK cells, and b) loss of NK cell cytotoxicity and cytokine production function. PI Prof. Aksan's lab has recently developed a cytocompatible encapsulation-based vitrification method that does not require cytotoxic and mutagenic CPAs. The main goal of this proposal is development and adaptation of this novel method to vitrify and preserve expanded blood and induced pluripotent stem cell (iPSC) derived NK cells. Our method avoids detrimental intra-/extracellular ice crystallization during cooling, and re-crystallization during warming, therefore eliminating freeze/thaw and cryogenic storage-induced loss of viability and post-thaw function. To reach our main goal, we will pursue the following specific aims: Specific Aim 1: Engineer the encapsulation gel and determine the cytocompatible CPA compositions that will inhibit intra-/extracellular ice crystallization and growth, devitrification, and eutectic melting, which are detrimental. Specific Aim 2: Maximize post-thaw NK cell viability and function using optimized freeze-thaw protocols. Specific Aim 3: Verify short and medium-term storage stability of NK cells at -80oC, and in liquid nitrogen.