Picoliter Droplets for Single Cell Cryopreservation

描述(申请人提供)：低温保存是一种保存生物细胞和组织的技术，它依赖于这样一个事实，即分子扩散，从而大大减少了细胞在低温下的损伤。在传统的冷冻保存中，选择的冷却速率介于引起溶液效应的速率(慢速率)和导致细胞内结冰的速率(快速率)之间。对于许多细胞类型，这些范围是重叠的，因此有必要添加为每种细胞类型量身定做的冷冻保护剂(CPA)。 一种有希望的替代方法是玻璃化，即冷却时形成非晶态玻璃状固体。从理论上讲，玻璃化冷冻可以避免冰形成造成的所有损害，并可以为细胞保存提供一种简单有效的方法，而不受细胞类型的影响。不幸的是，它需要极端的冷却速度，再加上高的且通常是有毒的CPA浓度，以防止冰晶以实际可实现的冷却速度形成。其中一个原因是，细胞外的冰通常在自发(均匀)成核发生之前很久就从颗粒物质(异质成核)开始。这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玻璃化状态在低浓度和无毒的冷冻保护剂浓度下实现。这是因为人们认识到，将溶液分成多个液滴，不仅大大减少了特定液滴含有冰核的可能性，而且还减少了可能发生均匀成核的特征时间。 为了验证这一假设，第一个目标是开发一种微流控装置，用于系统研究 在反(油包水)乳状液中超低温保存。这将允许创建单分散的水相CPA液滴，并将其快速冷却到低温，无论是在芯片上，在骤降冷却的石英微毛细管内，和/或在传统的低温阶段内。接下来，我们将研究液滴尺寸和冷却速度对玻璃化临界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，这些信息将被用于在肝细胞上测试各种指示的方案，在有和没有共培养成纤维细胞的情况下，存活率、增殖能力和肝特异性功能将衡量成功。相关性：通过将单个细胞包裹在微小的水滴中而获得的玻璃化所需的冷冻保护剂水平的降低，可以使不同种类的细胞悬浮液得以保存。

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.