Non-Enzymatic Cryogenic Isolation of Therapeutic Cells

描述（由申请人提供）：在现代医学中，细胞疗法、再生医学和组织工程都涉及用于收获、扩增、修饰和重新植入活细胞和组织的技术。制备掺入活细胞的治疗产品的方法对于产品的稳定性和效力是至关重要的，但可能固有地对组分细胞有害。例如，广泛实践的胶原酶消化组织以获得分离的细胞如来自胰腺的胰岛或来自肝脏的肝细胞的技术充满了有害的副作用和其他相关问题。这一广泛应用的方法存在缺陷，主要是由于难以控制消化过程以产生最佳数量的活细胞。此外，这个过程是苛刻的，甚至是有毒的，会导致一些不可避免的有价值的细胞损失。此外，依赖于最纯形式的酶的方法非常昂贵，并且可能会受到批次变化的影响，这导致在试图优化和标准化这些方法时令人沮丧的可变性和不一致性。这里提出了一种全新的方法，该方法最大限度地减少并可能消除对组织的酶消化的需要。相反，所提出的方法依赖于已知的细胞对冷冻损伤的敏感性，以通过促进的差异冷冻和冷冻保存技术来影响不同细胞类型的分离。这种新方法的可行性已被证明用于分离猪胰岛，这是一种广泛接受的模型，用于研究通过胰岛移植治疗I型糖尿病。为了获得用于基于细胞的治疗的胰岛，胰岛移植领域完全依赖于酶消化过程，该酶消化过程破坏供体组织的细胞外基质，释放截留的胰岛用于进一步加工和纯化。相比之下，我们建议通过内分泌和外分泌组织的差异灌注来预处理胰腺，其设计的方式是在保留胰岛的同时最大限度地破坏外分泌组织。更具体地说，这种新的冷冻隔离方法包括通过血管通路用冷冻保护剂初始灌注内分泌组织（胰岛），并且仅在胰岛充分平衡后，外分泌成分（腺泡）注入纯水溶液（蒸馏水或生理盐水）然后在促进冰形成和破坏腺泡组织的条件下冷却整个胰腺，同时通过由于冷冻保护剂渗透。然后，固体冷冻胰腺可以无限期储存和生物库，并进行后续加工，将腺体粉碎并破碎成含有冷冻保存的胰岛的微小碎片。最后，解冻冷冻破坏的组织以释放功能性胰岛和破坏的腺泡组织。在完成了这种创新新方法的初步概念验证后，这项第一阶段研究建议开发一种用于低温隔离的设备原型，并评估其性能以建立基线协议。该方法将基础研究工具与低温生物学科学的最新进展相结合，以系统地优化基线技术，同时开发一种通过热机械应力促进组织破裂的方法，从而提高差异冷冻破坏和可行胰岛分离的有效性。该研究汇集了低温生物学和热机械工程专业知识的独特组合，将这一新概念从可行性转化为常规实践，并随后在第二阶段研究中在人体组织中进行验证。 公共卫生相关性：再生医学和组织工程中的基于细胞的疗法都涉及活细胞的获取和再植入过程，目前依赖于昂贵、不一致甚至有毒的酶消化过程。一个主要的例子是制备分离的胰岛，用于通过移植治疗I型糖尿病。这项研究的重点是开发一种新的和新颖的替代技术，以酶消化，而不是依赖于差异冷冻破坏胰腺释放胰岛，选择性冷冻保存在原位。

DESCRIPTION (provided by applicant): In modern day medicine, cellular therapies, regenerative medicine and tissue engineering all involve technologies for harvesting, expanding, modifying and re-implanting live viable cells and tissues. Processes for preparing the therapeutic products that incorporate living cells are critical for the stability and potency of th products but may be inherently injurious to the component cells. For example, the widely practiced technique of collagenase digestion of tissues to obtain isolated cells such as pancreatic islets from pancreata, or hepatocytes from liver is fraught with detrimental side-effects and other associated problems. This widely practiced procedure has recognized pitfalls due principally to the difficulty of controlling the digestive process to yield an optimum quantityof viable cells. Moreover, the process is harsh and even toxic, causing some inevitable loss of valuable cells. Furthermore, the process relying upon the purest forms of the enzymes are very expensive and may be subject to batch variations that have led to frustrating variability and inconsistency in attempts to optimize and standardize these processes. A totally new approach is proposed here that minimizes and potentially eliminates the need for enzymatic digestion of the tissue. Instead, the proposed process relies upon known susceptibilities of cells to freezing injury, to affect the separation of different cell types by virtue of a facilitated differential frezing and cryopreservation techniques. Feasibility for this novel approach has been demonstrated for isolating porcine pancreatic islets, which is a widely accepted model for research into the treatment of type I diabetes by islet transplantation. To obtain islets for cell-based therapies, te field of islet transplantation relies totally upon enzymatic digestion processes that destroy the extracellular matrix of the donor tissue releasing the entrapped islets for further processing and purification. In contrast, we propose to pre-treat the pancreas by differential perfusion of the endocrine and exocrine tissue in a way designed to maximize the destruction of the exocrine tissue at the same time as preserving the islets. More specifically, this new cryo-isolation approach involves an initial perfusion of the endocrine tissue (islets) with cryoprotective agents via a vascular access and after adequate equilibration of the islets only, the exocrine component (acini) is infused with a purely aqueous solution (distilled water or saline) via the ductal system The entire pancreas is then cooled under conditions that promote ice formation and destruction of the acinar tissue while preserving the endocrine portion by virtue of the cryoprotectant infiltration. The solid frozen pancreas is then amenable to indefinite storage and biobanking and subsequent processing to pulverize and fracture the gland into tiny fragments containing the cryopreserved islets. Finally, the freeze-disrupted tissue is thawed to release functional islets and destroyed acinar tissue. Having completed the initial proof-of-concept of this innovative new approach, this Phase I study proposes to develop a device prototype for cryo-isolation and evaluate its performance to establish baseline protocols. The approach combines basic research tools with recent advances in cryobiology science to systematically optimize the baseline technique, while developing a method to promote tissue fracturing by means of thermo-mechanical stresses, thereby increasing the effectiveness of differential freeze disruption and viable islet isolation. The study brings together a unique combination of expertise in cryobiology and thermo-mechanical engineering necessary to take this novel concept from feasibility to routine practice and subsequently validation in human tissue in a Phase II study. PUBLIC HEALTH RELEVANCE: Cell-based therapies in regenerative medicine and tissue engineering, which all involve processes for procurement and re-implantation of living cells, currently rely upon expensive, inconsistent and even toxic enzyme-digestion processes. A prime example is the preparation of isolated pancreatic islets for the potential treatment of Type I diabetes by transplantation. This research is focused on the development of a new and novel alternative technique to enzymatic digestion by relying instead on differential freeze destruction of the pancreas to release islets that are selectively cryopreserved in situ.