Introduction

While hydrogel encapsulation of cells has been developed to treat multiple diseases, methods to cryopreserve and maintain the composite function of therapeutic encapsulated cell products are still needed to facilitate their storage and distribution. While methods to preserve encapsulated cells, and post-synthesis have received recent attention, effective preservation mediums have not been fully defined.

Methods

We employed a two-tiered screen of an initial library of 32 different cryopreservation agent (CPA) formulations composed of different cell-permeable and impermeable agents. Formulations were assayed using dark field microscopy to evaluate alginate hydrogel matrix integrity, followed by cell viability analyses and measurements of functional secretion activity.

Results

The structural integrity of large > 1 mm alginate capsules were highly sensitive to freezing and thawing in media alone but could be recovered by a number of CPA formulations containing different cell-permeable and impermeable agents. Subsequent viability screens identified two top-performing CPA formulations that maximized capsule integrity and cell viability after storage at − 80 °C. The top formulation (10% Dimethyl sulfoxide (DMSO) and 0.3 M glucose) was demonstrated to preserve hydrogel integrity and retain cell viability beyond a critical USA FDA set 70% viability threshold while maintaining protein secretion and resultant cell potency.

Conclusions

This prioritized screen identified a cryopreservation solution that maintains the integrity of large alginate capsules and yields high viabilities and potency. Importantly, this formulation is serum-free, non-toxic, and can support the development of clinically translatable encapsulated cell-based therapeutics.

中文翻译：

---

开发用于水凝胶封装的细胞疗法冷冻保存的无血清培养基

介绍

虽然细胞的水凝胶封装已被开发用于治疗多种疾病，但仍然需要冷冻保存和维持治疗性封装细胞产品的复合功能的方法，以促进其储存和分配。虽然保存封装细胞和合成后的方法最近受到关注，但有效的保存介质尚未完全确定。

方法

我们对由不同细胞渗透剂和不可渗透剂组成的 32 种不同冷冻保存剂 (CPA) 制剂的初始库进行了两层筛选。使用暗视野显微镜对制剂进行分析，以评估藻酸盐水凝胶基质的完整性，然后进行细胞活力分析和功能性分泌活性测量。

结果

> 1 mm 的大藻酸盐胶囊的结构完整性对单独在培养基中冷冻和解冻高度敏感，但可以通过许多含有不同细胞渗透性和不可渗透性试剂的 CPA 配方来恢复。随后的活力筛选确定了两种性能最佳的 CPA 配方，可在 - 80 °C 储存后最大限度地提高胶囊的完整性和细胞活力。顶级配方（10% 二甲基亚砜 (DMSO) 和 0.3 M 葡萄糖）被证明可以保持水凝胶的完整性，并保持细胞活力超过美国 FDA 设定的 70% 活力阈值，同时保持蛋白质分泌和由此产生的细胞效力。

结论

这种优先筛选确定了一种冷冻保存解决方案，可以保持大藻酸盐胶囊的完整性并产生高活力和效力。重要的是，该配方无血清、无毒，可以支持临床可转化的封装细胞疗法的开发。