Abstract Cells are routinely cryopreserved for various applications. Successful cryopreservation requires the loading of cryoprotective agents (CPAs) to prevent cells from cryoinjury during freezing and thawing, as well as the unloading of CPAs before use since CPAs are often toxic. Moreover, the CPA loading/unloading processes are usually effortful and error-prone. In this study, a membrane-based microfluidic device is introduced to aid the CPA loading and unloading processes. The device consists of two helical microfluidic channels with a sandwiched layer of microfiltration membrane. CPA exchange across the membrane via diffusion and filtration is realized when cell suspension and replacement fluid flow counter-currently in the channels. CPA concentration in the cell suspension can be regulated in a well-controlled, progressive manner. A theoretical model has been developed to better characterize the dynamic change and distribution of the CPA concentration, as well as the cell volume response, with respect to various mass transfer parameters within the device during CPA loading and unloading steps. Physical experiments have been conducted to validate the proposed model. Furthermore, operating conditions of the device have been optimized under the guidance of the established model and different devices with various sizes have been fabricated to evaluate their performance characteristics with respect to different levels of throughput. The optimized condition enables the loading of DMSO (from 0% to 10%, v/v) at a rate of 180 ml/h and unloading (from 10% to 1%, v/v) at 5 ml/h with a single pass through the initial prototype. A second enlarged device further improves the DMSO unloading rate to 180 ml/h with the washing solution consumption rate at 225 ml/h. The higher throughput and reduced washing solution requirement demonstrate the significant advancement in efficiency with the presented approach. Lastly, the system is easy to operate and the process is robust, thus provides a reliable solution for loading/unloading of CPAs for various sized cell suspensions.

中文翻译：

---

基于膜的微流体加载和卸载冷冻保护剂的理论和实验研究

摘要 细胞通常被冷冻保存用于各种应用。成功的冷冻保存需要加载冷冻保护剂 (CPA) 以防止细胞在冷冻和解冻过程中受到冷冻损伤，以及在使用前卸载 CPA，因为 CPA 通常是有毒的。此外，CPA 加载/卸载过程通常既费力又容易出错。在这项研究中，引入了一种基于膜的微流体装置来帮助 CPA 加载和卸载过程。该装置由两个螺旋微流体通道组成，中间夹着一层微滤膜。当细胞悬浮液和置换液在通道中逆流流动时，通过扩散和过滤实现跨膜的 CPA 交换。细胞悬浮液中的 CPA 浓度可以以可控的、渐进的方式进行调节。已经开发了一个理论模型，以更好地表征 CPA 浓度的动态变化和分布，以及细胞体积响应，与 CPA 加载和卸载步骤期间设备内的各种传质参数有关。已经进行了物理实验以验证所提出的模型。此外，在建立的模型的指导下优化了设备的运行条件，并制造了各种尺寸的不同设备，以评估它们在不同吞吐量水平下的性能特征。优化的条件使 DMSO（从 0% 到 10%，v/v）以 180 ml/h 的速率加载，并以 5 ml/h 的速率卸载（从 10% 到 1%，v/v），使用单个通过初始原型。第二个放大装置进一步将 DMSO 卸载率提高到 180 毫升/小时，洗涤液消耗率为 225 毫升/小时。更高的吞吐量和减少的洗涤溶液需求证明了所提出的方法在效率方面的显着进步。最后，该系统易于操作且过程稳健，从而为加载/卸载各种大小的细胞悬液的 CPA 提供了可靠的解决方案。