University of Applied Sciences Giessen - Friedberg, Institute of Bioprocess Engineering and Pharmaceutical Technology, Wiesenstrasse 14, 35390 Giessen, Germany.
Cryobiology. 2011 Oct;63(2):67-75. doi: 10.1016/j.cryobiol.2011.05.002. Epub 2011 May 17.
Human mesenchymal stem cells (hMSCs) have great potential for clinical therapy and regenerative medicine. One major challenge concerning their application is the development of an efficient cryopreservation protocol since current methods result in a poor viability and high differentiation rates. A high survival rate of cryopreserved cells requires an optimal cooling rate and the presence of cryoprotective agents (CPA) in sufficient concentrations. The most widely used CPA, dimethylsulfoxide (Me(2)SO), is toxic at high concentrations at temperatures >4°C and has harmful effects on the biological functionality of stem cell as well as on treated patients. Thus, this study investigates different combinations of non-cytotoxic biocompatible substances, such as ectoin and proline, as potential CPAs in a systematic parametric optimization study in comparison to Me(2)SO as control and a commercial freezing medium (Biofreeze®, Biochrom). Using a freezing medium containing a low proline (1%, w/v) and higher ectoin (10%, w/v) amount revealed promising results although the highest survival rate was achieved with the Biofreeze® medium. Cryomicroscopic experiments of hMSCs revealed nucleation temperatures ranging from -16 to -25°C. The CPAs, beside Me(2)SO, did not affect the nucleation temperature. In most cases, cryomicroscopy revealed intracellular ice formation (IIF) during the cryopreservation cycle for all cryoprotocols. The occurence of IIF during thawing increased with the cooling rate. In case of hMSC there was no correlation between the rate of IIF and the post-thaw cell survival. After thawing adipogenic differentiation of the stem cells demonstrated cell functionality.
人骨髓间充质干细胞（hMSCs）在临床治疗和再生医学方面具有巨大的潜力。目前，应用 hMSCs 面临的一个主要挑战是开发高效的冻存方案，因为现有的方法会导致细胞存活率低和高分化率。冷冻保存细胞的高存活率需要最佳的冷却速率和足够浓度的冷冻保护剂（CPA）。目前最广泛使用的 CPA 二甲基亚砜（Me(2)SO）在>4°C 的高温下，浓度较高时具有毒性，对干细胞的生物功能以及接受治疗的患者都有有害影响。因此，本研究通过系统的参数优化研究，研究了非细胞毒性的生物相容性物质，如海藻糖和脯氨酸等不同组合，作为潜在的 CPA，与 Me(2)SO 作为对照和商业冷冻介质（Biofreeze®，BIOCHROM）进行比较。结果显示，使用含有低脯氨酸（1%，w/v）和高海藻糖（10%，w/v）量的冷冻介质的结果很有前景，尽管最高的存活率是用 Biofreeze®介质实现的。hMSCs 的低温显微镜实验显示成核温度范围为-16 至-25°C。除 Me(2)SO 外，这些 CPA 不会影响成核温度。在大多数情况下，低温显微镜显示在所有冷冻方案的冷冻保存循环中都会发生细胞内冰形成（IIF）。解冻过程中 IIF 的发生与冷却速率有关。在 hMSC 的情况下，IIF 的发生率与解冻后的细胞存活率之间没有相关性。解冻后，干细胞的成脂分化证明了细胞的功能。
