Development of a High Throughput Method for Vitrification

摘要： 为了减少使用动物进行毒性测试，公司开发了体外生物工程 皮肤模型和其它人体组织构建物，用于预测毒性测试， 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 这些结构是按订单制造的，在使用之前需要几个星期的准备时间。质量控制 检查是在装运后进行的，这对制造商造成了严重的召回责任。的能力 冷冻保存组织模型将解决关键的后勤问题，创造“按需”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， 制造商和最终用户的成本。我们开发了一种专利的无冰玻璃化方法， 冷冻保存生物工程皮肤结构。使用这种专利工艺，我们始终 证实了几种类型的人生物工程化上皮构建体的>80%活力。这些可行性 结果在复温后持续了几天，存活力和功能评估均符合 用于毒理学试验的制造商验收标准。然而，保存的一个限制因素是， 更大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个构建体。处理超过12个导致由于细胞毒性导致活力降低， 暴露于冷冻保护剂太长时间。在第一阶段，我们计划测试使用 一次最多可容纳24个构建体的特殊设计的盒。磁带可以保存 多个构建体简化了玻璃化过程，同时消除了构建体之间的变化， 操作员错误。将通过评估组织活力和所需的功能测定来测量结果 制造商进行质量控制。这项建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，储存按需人类生物工程组织， 用于药物或化学品筛选和研究应用。在阶段论证可行性后 我，我们将提交一个第二阶段的建议与自定义开发的全自动化的主要目标 用于构建体冷冻保存和在用于以下用途之前去除冷冻保护剂的机器人样品处理系统 预测毒性试验。

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.