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均涉及用于收集，扩展，修饰和重新植入活细胞和组织的技术。制备结合活细胞的治疗产物的过程对于TH产品的稳定性和效力至关重要，但可能对组件细胞固有地有害。例如，在组织中广泛实践的胶原酶消化技术，从胰腺中获得诸如胰岛或肝脏肝细胞的孤立细胞，伴有有害的副作用和其他相关问题。这个广泛实践的程序已经识别出主要是由于控制消化过程而产生可行数量的最佳数量的困难。此外，该过程是苛刻甚至有毒的，导致了一些有价值的细胞的必然损失。此外，依赖最纯粹形式的酶的过程非常昂贵，并且可能会受到批处理变化，这导致了令人沮丧的变异性和不一致性，以优化和标准化这些过程。这里提出了一种全新的方法，该方法可以最大程度地减少并有可能消除组织酶消化的需求。取而代之的是，提出的过程依赖于已知的细胞敏感性冻结损伤，从而通过促进的差异频率和冷冻保存技术来影响不同细胞类型的分离。已经证明了这种新型方法的可行性用于隔离猪胰岛，这是通过胰岛移植对I型糖尿病治疗治疗的广泛接受的模型。为了获得基于细胞的疗法的胰岛，胰岛移植的TE田完全依赖于酶消化过程，这些过程破坏了供体组织的细胞外基质，释放了被夹住的小岛以进一步加工和纯化。相比之下，我们建议通过对内分泌组织和外分泌组织的差异灌注来预处理胰腺，旨在与保留胰岛同时造成外分泌组织的破坏。 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腺泡组织在保存内分泌部分的同时，凭借冷冻保护剂浸润。然后，固体冷冻的胰腺可以使无限期的存储和生物群体和随后的加工进行粉碎和裂缝，并将腺体碎裂成包含冷冻胰岛的微小片段。最后，将冻结的组织解冻以释放功能性胰岛并破坏腺泡组织。在完成了这种创新新方法的最初概念概念证明之后，这一阶段I研究提议开发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冷冻生物学科学的最新进展相结合，以系统地优化基线技术，同时开发了一种通过热机械应力来促进组织破裂的方法，从而提高了差异冻结干扰和可行的胰岛隔离的有效性。这项研究汇集了在第二阶段研究中，将这种新颖概念从可行性到常规练习以及随后在人体组织中验证的新颖概念所必需的冰冻生物学专业知识和热机械工程的独特组合。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，这些疗法都涉及生物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。一个典型的例子是，通过移植来制备用于I型糖尿病的潜在治疗的分离胰岛。这项研究的重点是开发一种新的新型替代技术来通过依靠胰腺的差异冻结破坏来释放在原位有选择性地冷冻保存的胰岛来开发酶消化。