Department of Clinical Immunology, Rigshospitalet, University of Copenhagen, 2100, Copenhagen, Denmark.
Department of Clinical Immunology, Rigshospitalet, University of Copenhagen, 2100, Copenhagen, Denmark.
Cryobiology. 2020 Oct;96:207-213. doi: 10.1016/j.cryobiol.2020.05.014. Epub 2020 Jun 22.
Mesenchymal stromal/stem cells (MSCs) derived from bone marrow, umbilical cord and especially adipose tissue are increasingly being explored for their therapeutic potential to treat a wide variety of diseases. A prerequisite for most allogeneic off-the-shelf and some autologous MSC therapies is the ability to safely and efficiently cryopreserve cells during production or for storage prior to treatment. Dimethyl sulfoxide (MeSO) is still the commonly used gold standard cryoprotectant (CPA). However, undesirable cellular impacts and side effects of MeSO have led to an increasing demand for the development of safe and effective alternatives. This study investigated the effect of pentaisomaltose as a CPA for cryopreservation of adipose-derived stromal/stem cells (ASCs). We compared pentaisomaltose-based freezing media containing 1% MeSO (PIM1) or 2% MeSO (PIM2) to our in-house freezing media formulation containing 10% MeSO (STD10) and to CryoStor freezing media containing 2% or 10% MeSO (CS2 and CS10). We assessed the recovery of viable ASCs, their phenotype, differentiation potential, proliferation potential, and migratory potential. Further, their immunomodulatory potential was assessed by measuring their ability to suppress T cell proliferation and express immunomodulatory markers. The results showed that the post-thaw viability of ASCs cryopreserved with STD10, CS10 and PIM2 was improved compared to that of CS2. The recovery of ASCs with PIM1 and PIM2 was also improved compared to that of CS2. Proliferation and migration were comparable among the tested freezing media. The results showed no difference in the induction of PDL1, PDL2 or IDO1 expression. Nevertheless, the potential of cryopreserved ASCs to suppress T cell proliferation was reduced when the MeSO concentration was reduced (CS10>STD10>CS2 and PIM2>PIM1). Altogether, the migratory and immunomodulatory potential combined with improved recovery indicate that the addition of pentaisomaltose in the freezing media may allow for the reduction of the MeSO concentration to 2% while retaining a more potent cell product that what is recovered using comparable freezing media. With the desire to reduce the amount of MeSO, these results suggest that 2% and potentially even 1% MeSO in combination with 10% pentaisomaltose could be an effective and less toxic alternative to comparable freezing media.
骨髓、脐带和特别是脂肪组织来源的间充质基质/干细胞（MSCs）因其治疗多种疾病的治疗潜力而越来越受到关注。大多数同种异体现成和一些自体 MSC 治疗的前提是在生产过程中或在治疗前安全有效地冷冻保存细胞的能力。二甲基亚砜（MeSO）仍然是常用的金标准冷冻保护剂（CPA）。然而，MeSO 的不良细胞影响和副作用导致对开发安全有效的替代品的需求不断增加。本研究调查了五异麦芽糖作为冷冻保护剂用于脂肪来源的基质/干细胞（ASCs）的冷冻保存的效果。我们将基于五异麦芽糖的冷冻培养基与含有 1% MeSO（PIM1）或 2% MeSO（PIM2）的培养基进行比较，与我们含有 10% MeSO 的内部冷冻培养基配方（STD10）和含有 2%或 10% MeSO 的 CryoStor 冷冻培养基（CS2 和 CS10）进行比较。我们评估了冷冻保存后活 ASC 的恢复、其表型、分化潜力、增殖潜力和迁移潜力。此外，通过测量其抑制 T 细胞增殖和表达免疫调节标志物的能力来评估其免疫调节潜力。结果表明，与 CS2 相比，用 STD10、CS10 和 PIM2 冷冻保存的 ASC 的解冻后活力得到了提高。与 CS2 相比，PIM1 和 PIM2 中 ASC 的恢复也得到了提高。在测试的冷冻培养基中，增殖和迁移没有差异。结果表明，PDL1、PDL2 或 IDO1 表达的诱导没有差异。然而，当 MeSO 浓度降低时（CS10>STD10>CS2 和 PIM2>PIM1），冷冻保存的 ASC 抑制 T 细胞增殖的潜力降低。总的来说，迁移和免疫调节潜力以及恢复能力的提高表明，在冷冻培养基中添加五异麦芽糖可以将 MeSO 浓度降低到 2%，同时保持更有效的细胞产物，与使用可比冷冻培养基恢复的产物相比。为了减少 MeSO 的用量，这些结果表明，2%甚至 1% MeSO 与 10%五异麦芽糖的组合可能是一种有效的、毒性更小的替代方案。
