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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(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采集、扩增、修改和重新植入活细胞和组织的技术。含有活细胞的治疗产品的制备过程对TH产品的稳定性和有效性至关重要，但可能固有地对组成细胞造成伤害。例如，广泛使用的胶原酶消化组织以获得分离细胞的技术，如从胰腺获得胰岛，或从肝脏获得肝细胞，都充满了有害的副作用和其他相关的问题。这一广泛应用的程序已经认识到缺陷，主要是由于控制消化过程以产生最佳数量的活细胞的困难。此外，这一过程是严酷的，甚至是有毒的，导致一些有价值的细胞不可避免地损失。此外，依赖最纯形式的酶的过程非常昂贵，可能会受到批次变化的影响，这导致在尝试优化和标准化这些过程时存在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，最大限度地减少并潜在地消除了对组织的酶消化的需要。相反，建议的过程依赖于已知的细胞对冷冻损伤的敏感性，通过促进差异化加热和冷冻保存技术来影响不同类型细胞的分离。这一新方法在分离猪胰岛方面的可行性已被证明，这是一种被广泛接受的研究模型，用于胰岛移植治疗I型糖尿病的研究。为了获得胰岛用于细胞治疗，胰岛移植领域完全依赖于酶消化过程，破坏供体组织的细胞外基质，释放包裹的胰岛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不同灌流来对胰腺进行预治疗，这种方法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包括通过血管通路向内分泌组织(胰岛)初始灌流冷冻保护剂，在仅对胰岛进行充分平衡后，通过导管系统向外分泌成分(腺泡)注入纯水溶液(蒸馏水或生理盐水)，然后在促进结冰和腺泡组织破坏的条件下冷却整个胰腺，同时通过冷冻保护剂渗透保留内分泌部分。然后，固体冷冻的胰腺可以无限期地储存和生物库，以及随后的处理，以将腺体粉碎和断裂成包含低温保存的胰岛的微小碎片。最后，冷冻破坏的组织解冻以释放功能性胰岛和破坏的腺泡组织。在完成了这一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个低温隔离设备原型，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，系统地优化了基线技术，同时开发了一种通过热机械应力促进组织破裂的方法，从而提高了差别化冷冻破坏和可行的胰岛隔离的有效性。这项研究汇集了低温生物学和热机械工程方面的独特专业知识，这是将这一新概念从可行性转化为常规实践以及随后在第二阶段研究中在人体组织中进行验证所必需的。与公共卫生相关：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采购和重新植入过程，目前依赖于昂贵、不一致甚至有毒的酶消化过程。一个最好的例子是准备分离的胰岛，用于通过移植治疗I型糖尿病的可能性。这项研究的重点是开发一种新的替代酶消化的新技术，即依赖于胰腺的差异冷冻破坏来释放选择性地在原位冷冻保存的胰岛。