Adipose-derived stem cells (ADSCs) promote tissue regeneration and repair. Cryoprotective agents (CPAs) protect cells from cryodamage during cryopreservation. Safe and efficient cryopreservation of ADSCs is critical for cell-based therapy in clinical applications. However, most CPAs are used at toxic concentrations, limiting their clinical application. The aim of this study is to develop a non-toxic xeno-free novel CPA aiming at achieving high-efficiency and low-risk ADSC cryopreservation. We explored different concentrations of trehalose (0.3 M, 0.6 M, 1.0 M, and 1.25 M) and glycerol (10%, 20%, and 30% v/v) for optimization and evaluated and compared the outcomes of ADSCs cryopreservation between a combination of trehalose and glycerol and the commonly used CPA DMSO (10%) + FBS (90%). All samples were slowly frozen and stored in liquid nitrogen for 30 days. The effectiveness was evaluated by the viability, proliferation, migration, and multi-potential differentiation of the ADSCs after thawing. Compared with the groups treated with individual reagents, the 1.0 M trehalose (Tre) + 20% glycerol (Gly) group showed significantly higher efficiency in preserving ADSC activities after thawing, with better outcomes in both cell viability and proliferation capacity. Compared with the 10% DMSO + 90% FBS treatment, the ADSCs preserved in 1.0 M Tre + 20% Gly showed similar cell viability, surface markers, and multi-potential differentiation but a significantly higher migration capability. The results indicated that cell function preservation can be improved by 1.0 M Tre + 20% Gly. The 1.0 M Tre + 20% Gly treatment preserved ADSCs with a higher migration capability than 10% DMSO + 90% FBS and with viability higher than that with trehalose or glycerol alone but similar to that with 10% DMSO + 90% FBS and fresh cells. Moreover, the new CPA achieves stemness and multi-potential differentiation similar to those in fresh cells. Our results demonstrate that 1.0 M Tre + 20% Gly can more efficiently cryopreserve ADSCs and is a non-toxic CPA that may be suitable for clinical applications.

中文翻译：

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海藻糖和甘油的组合：冷冻保存人类脂肪干细胞的一种有效且无毒的配方

脂肪干细胞（ADSC）促进组织再生和修复。冷冻保护剂（CPA）可在冷冻保存过程中保护细胞免受冷冻损害。ADSCs的安全有效冷冻保存对于临床应用中基于细胞的治疗至关重要。但是，大多数CPA以有毒浓度使用，限制了其临床应用。这项研究的目的是开发一种无毒，无异种的新型CPA，旨在实现高效率和低风险的ADSC冷冻保存。我们探索了不同浓度的海藻糖（0.3 M，0.6 M，1.0 M和1.25 M）和甘油（10％，20％和30％v / v）进行优化，并评估和比较了两种组合之间ADSC冷冻保存的结果海藻糖和甘油，以及常用的CPA DMSO（10％）+ FBS（90％）。将所有样品缓慢冷冻并在液氮中保存30天。通过解冻后ADSC的生存力，增殖，迁移和多能分化来评估有效性。与使用单独试剂处理的组相比，1.0 M海藻糖（Tre）+ 20％甘油（Gly）组在融化后保留ADSC活性方面显示出更高的效率，并且在细胞活力和增殖能力上都有更好的结果。与10％DMSO + 90％FBS处理相比，保存在1.0 M Tre + 20％Gly中的ADSCs具有相似的细胞活力，表面标记和多潜能分化，但迁移能力明显更高。结果表明，通过1.0 M Tre + 20％Gly可以改善细胞功能的保存。1。0 M Tre + 20％Gly处理保存的ADSC具有比10％DMSO + 90％FBS更高的迁移能力，并且比单独使用海藻糖或甘油时具有更高的生存能力，但与10％DMSO + 90％FBS和新鲜细胞的生存能力相似。而且，新的CPA可以实现干细胞和多潜能分化，类似于新鲜细胞。我们的结果表明，1.0 M Tre + 20％Gly可以更有效地冷冻保存ADSC，并且是一种无毒的CPA，可能适用于临床应用。