Picoliter Droplets for Single Cell Cryopreservation

用于单细胞冷冻保存的皮升液滴

基本信息

批准号：
7493520
负责人：
Jon F Edd
金额：
$ 4.62万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-09-01 至 2009-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7493520
关键词：
Analysis of Variance Biological Biological Preservation Blood capillaries Cell Death Cell Membrane Permeability Cell Nucleus Cell Size Cell Therapy Cells Characteristics Chemicals Classification Coculture Techniques Condition Confined Spaces Coupled Cryopreservation Crystal Formation Crystallization Data Dehydration Depth Development Devices Differential Scanning Calorimetry Diffusion Doctor of Philosophy Emulsions Event Excision Fibroblasts Fluorescence Freezing Generations Glass Goals Hand Hepatocyte Ice Individual Liquid substance Liver Failure Mammalian Cell Measures Methods Metric Microfluidic Microchips Microfluidics Microscopic Microscopy Modeling Molecular Monitor Neuro-Oncological Ventral Antigen 2 Nitrogen Oils Optics Particulate Particulate Matter Phase Probability Protocols documentation Publishing Quartz Range Rate Recovery Reporting Research Solid Solutions Spectroscopy, Fourier Transform Infrared Staging Standards of Weights and Measures Suspension substance Suspensions Techniques Temperature Testing Time Tissues Travel Water Work X ray diffraction analysis X-Ray Diffraction aqueous capillary cell injury cell type cell water chemical reaction cryogenics experience extracellular interest novel novel strategies particle prevent radius bone structure research study size success theories

项目摘要

DESCRIPTION (provided by applicant): Cryopreservation is a technique for preserving biological cells and tissues that relies on the fact that molecular diffusion, and thus cellular injury, are vastly reduced at cryogenic temperatures. In conventional cryopreservation, cooling rate is chosen to lie between rates that cause solution effects (slow rates) and those that cause intracellular ice formation (fast rates). For many cell types; however, these ranges overlap so that the addition of cryoprotectant chemicals (CPAs), tailored to each cell type, is necessary. One promising alternative is vitrification, or the formation of a non-crystalline glass-like solid on cooling. In theory, vitrification should avoid all damages from ice formation and could provide a simple and effective method for cell preservation independent of cell type. Unfortunately, it requires extreme cooling rates coupled with high and typically toxic CPA concentrations to prevent ice crystal formation at practically realizable cooling rates. One reason for this is that extracellular ice often initiates from particulate matter (heterogeneous nucleation) long before spontaneous (homogeneous) nucleation would occur. Both forms of ice nucleation are stochastic, which leads to the hypothesis of this work: cryopreservation of biological cells captured within microscopic aqueous droplets will enable the vitrified state to be achieved at low and nontoxic cryoprotectant concentrations. This follows from the realizations that dividing a solution into numerous droplets dramatically decreases not only the probability that a particular droplet contains an ice nucleator, but also the characteristic time in which homogeneous nucleation is likely to occur. To test this hypothesis, the first aim is to develop a microfluidic device for the systematic study of cryopreservation in an inverse (water-in-oil) emulsion. This will allow the creation of monodisperse aqueous-CPA droplets and their rapid cooling to cryogenic temperatures, either on-chip, within a plunge-cooled quartz micro-capillary, and/or inside a conventional cryostage. Next, the effects of droplet size and cooling rate on the critical CPA concentration for vitrification will be studied. This will entail the development of a physicochemical model of droplet vitrification which will guide the search for conditions enabling low-CPA vitrification. Vitrification will be assessed with methods such as X-ray diffraction, FTIR, and/or DSC. Finally, this information will be used to test a variety of indicated protocols on hepatocytes, with and without cocultured fibroblasts, where viability, proliferative ability and hepatospecific function will measure success. Relevance: The reduction in cryoprotectant levels required for vitrification gained by encapsulation of single cells in microscopic droplets of water could enable the preservation of heterogeneous cell suspensions.

描述（由申请人提供）：冷冻保存是一种用于保存生物细胞和组织的技术，依赖于以下事实：在低温温度下，分子扩散以及因此细胞损伤大大降低。在常规的冷冻保存中，选择冷却速率位于引起解决方案效应的速率（缓慢速率）和导致细胞内冰形成的速率（快速速率）。对于许多细胞类型；但是，这些范围重叠，因此必须添加针对每种细胞类型量身定制的冷冻保护剂化学物质（CPA）。一种有希望的替代方法是玻璃化，或在冷却时形成非晶体类似玻璃的固体。从理论上讲，玻璃化应避免形成冰的所有损害，并可以为独立于细胞类型的细胞保存提供一种简单有效的方法。不幸的是，它需要极端的冷却速率，加上高且通常有毒的CPA浓度，以防止以实际上可实现的冷却速率形成冰晶。这样做的一个原因是，细胞外冰通常在自发（同质）成核之前很久以颗粒物（异质成核）的形式引发。两种形式的冰核都是随机的，这导致了这项工作的假设：在微观水滴中捕获的生物细胞的冷冻保存将使在低和无毒的冷冻保护剂浓度下实现玻璃化状态。这是从将溶液划分为众多液滴的实现不仅降低了特定液滴包含冰核的概率，而且还可能发生同质成核的特征时间。为了检验这一假设，第一个目的是开发一种微流体设备，以进行系统研究在反向（油中）乳液中的冷冻保存。这将使单分散水溶液液滴以及在凹陷冷却的石英微毛细管内和/或传统的低温危险中产生片上的低温温度。接下来，将研究液滴尺寸和冷却速率对玻璃化临界CPA浓度的影响。这将需要开发液滴玻璃化的物理化学模型，该模型将指导寻找能够实现低CPA玻璃化的条件。玻璃化将通过X射线衍射，FTIR和/或DSC等方法进行评估。最后，这些信息将用于测试有或没有共培养成纤维细胞的肝细胞上的各种指示方案，在这里，生存力，增殖能力和肝特异性功能将衡量成功。相关性：通过在水的微观液滴中封装单个细胞获得的玻璃化所需的冷冻保护剂水平的降低，可以保留异质细胞悬浮液。