Department of Obstetrics, University Hospital Zurich, Zurich, Switzerland.
Cell Transplant. 2011;20(8):1241-57. doi: 10.3727/096368910X547426. Epub 2010 Dec 22.
While therapeutic cell transplantations using progenitor cells are increasingly evolving towards phase I and II clinical trials and chemically defined cell culture is established, standardization in biobanking is still in the stage of infancy. In this study, the EU FP6-funded CRYSTAL (CRYo-banking of Stem cells for human Therapeutic AppLication) consortium aimed to validate novel Standard Operating Procedures (SOPs) to perform and validate xeno-free and chemically defined cryopreservation of human progenitor cells and to reduce the amount of the potentially toxic cryoprotectant additive (CPA) dimethyl sulfoxide (DMSO). To achieve this goal, three human adult progenitor and stem cell populations-umbilical cord blood (UCB)-derived erythroid cells (UCB-ECs), UCB-derived endothelial colony forming cells (UCB-ECFCs), and adipose tissue (AT)-derived mesenchymal stromal cells (AT-MSCs)-were cryopreserved in chemically defined medium supplemented with 10% or 5% DMSO. Cell recovery, cell repopulation, and functionality were evaluated postthaw in comparison to cryopreservation in standard fetal bovine serum (FBS)-containing freezing medium. Even with a reduction of the DMSO CPA to 5%, postthaw cell count and viability assays indicated no overall significant difference versus standard cryomedium. Additionally, to compare cellular morphology/membrane integrity and ice crystal formation during cryopreservation, multiphoton laser-scanning cryomicroscopy (cryo-MPLSM) and scanning electron microscopy (SEM) were used. Neither cryo-MPLSM nor SEM indicated differences in membrane integrity for the tested cell populations under various conditions. Moreover, no influence was observed on functional properties of the cells following cryopreservation in chemically defined freezing medium, except for UCB-ECs, which showed a significantly reduced differentiation capacity after cryopreservation in chemically defined medium supplemented with 5% DMSO. In summary, these results demonstrate the feasibility and robustness of standardized xeno-free cryopreservation of different human progenitor cells and encourage their use even more in the field of tissue-engineering and regenerative medicine.
虽然使用祖细胞的治疗性细胞移植越来越多地发展到 I 期和 II 期临床试验，并且已经建立了化学定义的细胞培养，但生物库的标准化仍处于起步阶段。在这项研究中，欧盟 FP6 资助的 CRYSTAL（用于人类治疗应用的干细胞低温保存）联盟旨在验证新的标准操作程序（SOP），以进行和验证无动物成分和化学定义的人类祖细胞冷冻保存，并减少潜在毒性的冷冻保护剂添加剂（CPA）二甲基亚砜（DMSO）的用量。为了实现这一目标，对三种人类成体祖细胞和干细胞群体-脐带血（UCB）衍生的红系细胞（UCB-ECs）、UCB 衍生的内皮集落形成细胞（UCB-ECFCs）和脂肪组织（AT）衍生的间充质基质细胞（AT-MSCs）-在补充有 10%或 5%DMSO 的化学定义培养基中进行冷冻保存。与标准的含有胎牛血清（FBS）的冷冻培养基相比，对解冻后的细胞回收、细胞再群体化和功能进行了评估。即使将 DMSO 的 CPA 减少到 5%，解冻后的细胞计数和活力测定表明与标准冷冻培养基没有总体显著差异。此外，为了比较冷冻保存过程中的细胞形态/细胞膜完整性和冰晶形成，使用了多光子激光扫描冷冻显微镜（cryo-MPLSM）和扫描电子显微镜（SEM）。在各种条件下，无论是 cryo-MPLSM 还是 SEM 都没有表明测试细胞群体的细胞膜完整性存在差异。此外，除 UCB-ECs 外，在化学定义的冷冻培养基中冷冻保存后，细胞的功能特性没有受到影响，UCB-ECs 在化学定义的培养基中添加 5%DMSO 后，其分化能力显著降低。总之，这些结果证明了不同人类祖细胞的无动物成分冷冻保存的可行性和稳健性，并鼓励在组织工程和再生医学领域更多地使用它们。
