Non-Enzymatic Cryogenic Isolation of Therapeutic Cells

治疗细胞的非酶低温分离

基本信息

批准号：
8394427
负责人：
MICHAEL John TAYLOR
金额：
$ 26.86万
依托单位：
CELL AND TISSUE SYSTEMS, INC.
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-15 至 2013-12-14
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8394427
关键词：
Acinus organ component Address Adverse effects Affect Basic Science Blood Vessels Cell Therapy Cells Cellular Structures Coupled Cryopreservation Cryoprotective Agents Cryosurgery Data Dependency Development Device or Instrument Development Devices Digestion Ductal Effectiveness Endocrine Engineering Enzymes Extracellular Matrix Family suidae Fracture Freezing Future Gland Goals Harvest Hepatocyte Ice Implant In Situ Infiltration Injury Insulin-Dependent Diabetes Mellitus Islet Cell Islets of Langerhans Islets of Langerhans Transplantation Legal patent Life Liver Measures Mechanical Stress Mechanics Medicine Methods Modeling Operative Surgical Procedures Pancreas Performance Perfusion Phase Positioning Attribute Predisposition Preparation Procedures Process Protocols documentation Recording of previous events Regenerative Medicine Research Research Proposals Rewarming Saline Scheme Science Small Business Innovation Research Grant Solid Solutions Specimen Structure System Systems Integration Techniques Technology Temperature Therapeutic Time Tissue Donors Tissue Engineering Tissues Transplantation Validation Variant Water aqueous base biobank cell preparation cell type collagenase cost effective cryobiology cryogenics design experience human tissue implantation innovation instrumentation islet knowledge base multidisciplinary novel novel strategies operation phase 1 study phase 2 study prototype routine practice tool transplantation medicine

项目摘要

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.

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过程对于产品的稳定性和效力至关重要，但可能对成分细胞本身有害。例如，广泛应用的胶原酶消化组织以获得分离的细胞（例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。这种广泛实施的程序已认识到其缺陷，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，这个过程非常严酷，甚至有毒，不可避免地会造成一些有价值的细胞损失。此外，依赖于最纯净形式的酶的过程非常昂贵，并且可能会受到批次变化的影响，这导致在优化和标准化这些过程的尝试中出现令人沮丧的变化和不一致。这里提出了一种全新的方法，可以最大限度地减少甚至可能消除组织酶消化的需要。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，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。这种新方法分离猪胰岛的可行性已得到证实，猪胰岛是胰岛移植治疗 I 型糖尿病研究中广泛接受的模型。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，该过程破坏供体组织的细胞外基质，释放捕获的胰岛以进行进一步处理和纯化。相反，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，其方式旨在最大限度地破坏外分泌组织，同时保留胰岛。更具体地说，这种新的冷冻隔离方法涉及通过血管通路用冷冻保护剂对内分泌组织（胰岛）进行初始灌注，仅在胰岛充分平衡后，将纯水溶液注入外分泌成分（腺泡）。然后，整个胰腺在促进冰形成和腺泡组织破坏的条件下冷却，同时通过导管系统保留内分泌部分冷冻保护剂渗透。然后，固体冷冻胰腺可以无限期储存和生物库，以及随后的加工，将腺体粉碎并破碎成含有冷冻保存胰岛的微小碎片。最后，冷冻破坏的组织被解冻以释放功能性胰岛和被破坏的腺泡组织。在完成了这种创新新方法的初步概念验证后，该第一阶段研究建议开发一种用于冷冻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，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，将这一新颖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。公共健康相关性：再生医学和组织工程中的细胞疗法都涉及活细胞的获取和重新植入过程，目前依赖于昂贵、不一致甚至有毒的酶消化过程。一个典型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酶消化替代技术，依靠不同的冷冻破坏胰腺来释放选择性原位冷冻保存的胰岛。