Rogulska Olena, Petrenko Yuri, Petrenko Alexander
Department of Biochemistry, Institute for Problems of Cryobiology and Cryomedicine of National Academic of Sciences of Ukraine, Pereyaslavskaya 23, Kharkiv, 61015, Ukraine.
Institute of Experimental Medicine AS CR, v. v. i., Vídeňská 1083, 142 20, Prague 4-Krč, Czech Republic.
Cytotechnology. 2017 Apr;69(2):265-276. doi: 10.1007/s10616-016-0055-2. Epub 2016 Dec 24.
Off-the-shelf availability of human adipose-derived mesenchymal stromal cells (ASCs) for regenerative medicine application requires the development of nontoxic, safe, and efficient protocols for cryopreservation. Favorably, such cell processing protocols should not contain xenogeneic or toxic components, such as fetal bovine serum (FS) and dimethyl sulfoxide (DMSO). The objective of the study was to assess the sensitivity of ASCs to DMSO-free cryopreservation protocol depending on their expansion conditions: conventional, based on the application of FS or xeno-free, using PL as a medium supplement. ASCs expansion was carried out in α-MEM supplemented either with FS or PL. For DMSO- and xeno-free cryopreservation ASCs were pretreated with different concentrations of sucrose during 24 h of culture. Pretreated ASCs were cryopreserved in α-MEM containing 100-300 mM of sucrose with the cooling rate of 1 degree/min. ASCs were tested for survival (Trypan Blue test), viability (MTT test), recovery (Alamar Blue test), proliferation and ability to multilineage differentiation. The optimal concentrations of sucrose for ASCs pretreatment and as an additive in cryoprotective solution, which provided highest cell survival, comprised 100 and 200 mM, correspondingly. Survival and recovery rates of platelet lysate (PL)-expanded ASCs after DMSO-free cryopreservation comprised 59 and 51%, and were higher than in FS-cultured cells. After DMSO-free cryopreservation PL-processed ASCs had a shorter population doubling time and higher capacity for osteogenic differentiation than FS-processed cultures. The described DMSO- and xeno-free processing may form the basis for the development of safe and efficient protocols for manufacturing and banking of ASCs, providing their off-the-shelf availability for regenerative medicine applications.
用于再生医学的人脂肪间充质基质细胞（ASC）现货供应需要开发无毒、安全且高效的冷冻保存方案。有利的是，此类细胞处理方案不应包含异种或有毒成分，如胎牛血清（FS）和二甲基亚砜（DMSO）。本研究的目的是根据ASC的扩增条件评估其对无DMSO冷冻保存方案的敏感性：传统的基于FS应用或无动物源的，使用血小板裂解液（PL）作为培养基补充剂。ASC在补充有FS或PL的α-MEM中进行扩增。对于无DMSO和无动物源的冷冻保存，在培养24小时期间用不同浓度的蔗糖对ASC进行预处理。预处理后的ASC在含有100 - 300 mM蔗糖的α-MEM中以1℃/分钟的冷却速率进行冷冻保存。对ASC进行存活（台盼蓝试验）、活力（MTT试验）、复苏（alamar蓝试验）、增殖和多向分化能力的测试。用于ASC预处理以及作为冷冻保护溶液添加剂的蔗糖最佳浓度分别为100 mM和200 mM，这能提供最高的细胞存活率。无DMSO冷冻保存后，血小板裂解液（PL）扩增的ASC的存活和复苏率分别为59%和51%，高于FS培养的细胞。无DMSO冷冻保存后，PL处理的ASC的群体倍增时间比FS处理的培养物短，成骨分化能力更高。所描述的无DMSO和无动物源处理可为开发安全高效的ASC制造和储存方案奠定基础，为再生医学应用提供其现货供应。
