Development of a High Throughput Method for Vitrification

摘要： 为了减少使用动物进行毒性测试，公司已经开发出体外生物工程。 皮肤模型和其他人体组织结构，用于预测性毒性测试，以取代传统的测试，如 作为Draize皮肤刺激试验，也可用作临床前动物模型。目前，组织或皮肤 构造是按订单生产的，需要几周的准备时间才能使用。质量控制 检查是在装运后进行的，这给制造商带来了严重的召回责任。有能力 低温保存组织模型将解决关键的后勤问题，创建可以按需存储的组织库 进行预筛选，以确保质量控制。这将创造制造规模经济，并减少 制造商和最终用户的成本。我们已经开发了一种专利的无冰玻璃化方法 生物工程皮肤构造的超低温保存。使用这一专利工艺，我们一贯 证明了几种类型的人类生物工程上皮构造的80%的生存能力。这些生存能力 结果在复温后持续了几天，生存能力和功能评估都符合 制造商在毒理学测试中使用的验收标准。然而，保护的一个限制因素是 更大的规模是一次玻璃化多个结构的能力。我们目前的方法允许处理 一次最多6-12个构造。超过12个的处理会由于细胞毒性而导致存活率降低 暴露在冷冻保护剂中的时间太长。在此阶段I中，我们计划测试使用 特别设计的盒式磁带，一次可容纳多达24个构件。盒式磁带将允许保存 多个构建件简化了玻璃化冷冻过程，同时消除了构建件之间的差异 操作员错误。结果将通过评估组织活性和所需的功能分析来衡量。 由厂家进行质量控制。这项提议的成功将符合我们公司的目标 将我们的玻璃化过程商业化，储存按需的人类生物工程组织，以便分发到 药物或化学筛选和研究应用的客户。在论证了阶段可行性之后 I，我们将提交一份第二阶段提案，主要目标是定制开发一款全自动 用于构建低温保存和在使用前移除低温保护剂的机器人样品处理系统 预测性毒性测试。

ABSTRACT: In an effort to reduce the use of animals for toxicity testing, companies have developed bioengineered in vitro skin models and other human tissue constructs for predictive toxicity testing to replace traditional assays such as the Draize skin irritation test and also for use as preclinical animal models. Presently, tissues or skin constructs are made-to-order and require a lead time of several weeks before they can be used. Quality control checks are conducted post shipment creating serious recall liabilities for the manufacturer. The ability to cryopreserve tissue models would solve key logistical issues creating “on-demand” banks of tissues which can be prescreened to ensure quality control. This would create economies of manufacturing scale and reduce costs to manufacturers and end users. We have developed a patented ice-free vitrification method for cryopreservation of bioengineered skin constructs. Using this patented process, we have consistently demonstrated >80% viability of several types of human bioengineered epithelial constructs. These viability results persisted for several days post-rewarming and both viability and functional assessments meet the manufacturer's acceptance criteria for use in toxicology tests. However, a limiting factor for preservation at larger scale is the ability to vitrify multiple constructs at one time. Our current method allows for processing of up to 6-12 constructs at one time. Processing of more than 12 results in reduce viability due to cytotoxicity from exposure to cryoprotectants for too long. In this Phase I, proposal we plan to test the feasibility of using a specially designed cassette that can hold up to 24 constructs at once. The cassette would allow preservation of multiple constructs streamlining the vitrification process while removing variation between constructs due to operator error. The outcome will be measured by assessment of tissue viability and functional assays required by manufacturers for quality control. The success of this proposal will be inline with our company goal of commercializing our vitrification process by storing on-demand human bioengineered tissues for distribution to customers for drug or chemical screening and research applications. After demonstration of feasibility in Phase I, we will submit a Phase II proposal with the primary objective of custom development of a fully automated robotic sample handling system for construct cryopreservation and removal of cryoprotectants prior to use for predictive toxicity testing.