[摘要]目的：骨髓基质干细胞（BMSCs）是构建组织工程化骨主要的种子细胞，实现BMSCs的深低温保存对骨组织工程具有重要意义。目前，玻璃化冻存是最有发展前景的冻存方法，因此希望通过改进玻璃化液和预处理条件来提高BMSCs的玻璃化冻存效果。方法：采用不同组成及浓度的玻璃化液在不同的预处理条件下对第2（P2）代成骨诱导的犬骨髓基质干细胞（cBMSCs）进行玻璃化冻存实验，通过复苏后的细胞存活率来选择一种理想的玻璃化液及适合的预处理条件，并在此基础上再与常规冻存的实验结果进行比较，同时考虑冻存过程对细胞成骨活性的影响。结果：将VS226作为玻璃化液，在0℃/5min的预处理条件下玻璃化冻存P2代成骨诱导的cBMSCs，与常规冻存后的细胞存活率相比无统计学差异，且冻存过程对此细胞的成骨能力没有影响。结论：采用VS226来玻璃化冻存BMSCs，可实现为骨组织工程提供大量种子细胞的目的。
[关键词]深低温保存；玻璃化冻存；骨组织工程；骨髓基质干细胞
[中图分类号]Q813.1 [文献标识码]A [文章编号]1008-6455（2009）03-0318-05
Experimental study of vitrification preservation for BMSCs
YIN Hong-yu1, SUN Jian3, LIU Guang-peng3,CUI Lei2,3,CAO Yi-lin1,2,3
(1. Research Center Of Plastic Surgery Hospital, Chinese Academy of Medical Sciences& Peking Union Medical College, Beijing 100041, China; 2.Department of Plastic and Reconstructive Surgery, Shanghai Ninth People"s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200011, China; 3.Shanghai Tissue Engineering Research and Development Center, Shanghai 200234, China.)
Abstract: Objective Bone marrow stromal cells (BMSCs) have become the main cell source for tissue-engineered bone. There is of great significance for bone tissue engineering to cryopreserved BMSCs. At present, vitrification preservation is the most promising methods of cryopreservation for cells. The effect of vitrification method for BMSCs was wished to enhance by improving the vitrified solution and the pretreatment condition. Methods Using the different composition and concentration of vitrification fluid, the freezing experiments on osteogenically induced canine bone marrow stromal cells (cBMSCs) at passage 2 (P2) were performed under the different preconditioning. According to the cellular survival rate after rewarming, the ideal vitrification media and optimal pretreatments were chosen to cryopreserve the cells in order to compare with the experimental result of slow-freezing. Simultaneously, the influence of vitrification protocol on osteogenic activity of the cells needed to be considered. Results Taking the VS226 as a vitreous cryoprotectant, osteo-induced cBMSCs at P2 were banked under 0℃/5min of the pretreatment condition. There was no statistical significance of percent cell viability post-thaw between routine and vitrification method, and the cooling process did not affect osteogenic ability of the cells. Conclusions Based on these results, it can be concluded that BMSCs frozen by vitrification with VS226 can be used to provide the massive seed cell for bone tissue engineering.
Key words: cryopreservation; vitrification; bone tissue engineering; bone marrow stromal cells
组织工程学是一门以细胞生物学和材料学相结合, 进行体外和体内构建组织或器官的新兴学科[1]。构建的方法主要是在支架材料上接种高浓度的种子细胞进行复合培养。骨组织工程是组织工程的主要分支，其种子细胞以成骨诱导的BMSCs为主，因此组织工程化骨的构建迫切需要进行大量种子细胞的冷冻贮备。玻璃化冻存技术是近年来低温生物学领域发展最快的研究方向之一。本文通过采用一种新研制的玻璃化液对成骨诱导的cBMSCs进行玻璃化冻存的研究，以期能使组织工程化骨的种子细胞获得理想的玻璃化冻存效果。
1材料和方法
1.1材料
1.1.1 动物：2岁龄Beagle犬3条，雌雄不限，体重约20kg，上海农学院提供。
1.1.2 试剂：Percoll原液：77237 Percoll （sigma公司，美国）；BM Purple染色剂（Boehringer Mannheim公司，德国）；茜素红染液（sigma公司，美国）。
1.2方法
1.2.1 cBMSCs的分离和体外诱导培养：将实验犬以5 %的戊巴比妥钠（0.5ml/Kg）麻醉后，抽取骨髓2.5ml。根据以前报道的方法[2]进行Percoll液的配制和犬骨髓单个核细胞的分离提纯。将原代细胞接种培养48h后开始应用含有地塞米松（10mmol/L）、β-磷酸甘油钠（2.16g/L）和2-磷酸抗坏血酸（37.5mg/L）的成骨条件培养液，置37℃、5% CO2的培养箱内进行成骨诱导培养。细胞培养至第8天，细胞克隆形成单位的中心生长达密集时用0.25 %胰蛋白酶-0.02%EDTA进行第1次传代[3]。以1.6×104/cm2的细胞密度接种，培养至P2代。
1.2.2 冻存液的制备：常规冻存液由体积比为10%二甲基亚砜（DMSO）、50%胎牛血清（FBS）和40%细胞培养液（DMEM）组成；根据文献[4]制备玻璃化液VS55；其它玻璃化液参见表1（根据文献[4]制备EC液）。
1.2.3细胞的深低温保存方法：①常规冻存：将P2代成骨诱导的cBMSCs消化离心后倒除上清液，用0.5ml常规冻存液重悬并置入2ml冻存管中，先在4℃预冷30min，再置于-20℃ 2h，经过24h的-80℃保存后投入液氮（-196℃）；②玻璃化冻存：将同样方法获得的细胞用同体积的玻璃化液重悬于冻存管中，经过不同的预处理方案，包括将冻存管放入冰水混合物(0℃)中或置于室温（25℃）下分别保持5min或15min后再直接投入液氮，或立即投入液氮（0min）。
1.2.4 深低温保存细胞的复苏方法：从液氮中取出冷冻保存1天的冻存管，在37℃ 水浴中快速摇晃进行复温，将复温后的细胞溶液迅速转移到离心管中并加入成骨条件培养液进行稀释，离心（1 500 转/min，5 min）后去上清液，共洗涤2次。
1.2.5 深低温保存细胞复苏后的细胞存活率[5]：将3×105个P2代成骨诱导的cBMSCs连续培养6天，用血球计数板计算出培养后的细胞总数，根据公式：增殖率=细胞总数÷(3×105)，来获得未冻存细胞的增殖率。然后深低温保存3×105个此细胞，冻存1天后复苏，继续成骨诱导培养6天，同样获取培养后的细胞总数，利用公式：细胞存活率=培养后细胞总数÷(3×105×增殖率)，以得出冻存细胞的存活率。
1.2.6 生化检测：根据以前报道的方法[2]分别将P2代成骨诱导的cBMSCs进行冻存前后的碱性磷酸酶（ALP）染色和茜素红染色。。
1.2.7 统计学分析：数据采用EXCEL软件处理，行t检验。每个检测样本重复3次，数据以均数±标准差表示,P15min>0min（P 为将玻璃化冻存中的细胞毒性损伤减小到最低限度,当然还需要严格控制细胞与冷冻保护剂溶液接触的温度和时间，加快降温和复温的速率。实验结果指出预处理温度为0℃的玻璃化冻存效果好于25℃，这是因为低温环境可减少DMSO的细胞毒性[20]。而预处理时间以5min为最佳，这是由于将细胞与冷冻保护剂预先接触可降低冻存过程中的渗透压突变对细胞的打击，而过长的接触时间又会增加冻存液对细胞的毒性损伤。
自从Vajta[21]发明了开放型拉细冻存麦管(open pulled straw, OPS)，冷冻速率增至20000℃/min，大大加快了降温复温速率和提高了细胞存活率。但因吸入管内液体仅为2μl，极大的限制了冻存细胞的数量，然而组织工程所需的细胞数量又较大，可见OPS法是很难满足的。但常规的2ml冻存管却可实现此要求，因此实验选用冻存管成装细胞悬液。同时为增加热交换率，实验仅用0.5ml的冻存液来混合细胞，以希望获得更大的降温和复温的速率。但毕竟此方法无法达到OPS法的冷冻速率，这也说明了本实验的细胞存活率不是非常高的原因。
由于在液氮（-196℃）温度下，细胞、组织内各种酶的活力及代谢很低，几乎为零，生命处于所谓的“停滞”状态。在此温度下冻存的细胞，理论上可无限期保存[22]。鉴于深低温保存的时间并不是问题，因此本实验选用1天作为冻存的时间。
Kadiyala等[3]研究指出cBMSCs从P2代以后的细胞活性即开始降低，这也就是为什么选用P2的cBMSCs进行本实验的原因。目前，成骨诱导的BMSCs是骨组织工程重要的种子细胞，冻存细胞不但要保证高的细胞存活率，而且还要保持细胞的成骨活性。本实验经过生化检测验证了冻存过程并未影响成骨诱导cBMSCs的成骨能力。因此玻璃化冻存完全可以满足骨组织工程中对细胞冷冻保存的要求。
总之，用VS226作为玻璃化液，经0℃/5min的预处理后对P2代成骨诱导的cBMSCs进行玻璃化冻存，复苏后的细胞存活率达到了与常规冻存相同的效果。
[参考文献]
[1]曹谊林.组织工程学理论与实践[M].上海:上海科学技术出版社, 2004:3.
[2]尹宏宇, 刘广鹏, 崔磊，等. Percoll法分离犬骨髓单个核细胞与体外成骨诱导培养的实验研究[J]. 组织工程与重建外科杂志, 2007, 3(5):277-279.
[3]Kadiyala S, Young RG, Thiede MA, et al. Culture expanded canine mesenchymal stem cells possess osteochondrogenic potential in vivo and in vitro[J]. Cell Transplant, 1997, 6(2):125-134.
[4]Mehl PM. Nucleation and crystal growth in a vitrification solution tested for organ cryopreservation by vitrification[J]. Cryobiology, 1993, 30(5):509-518.
[5]Liu BL, McGrath J, McCabe L, et al. Response of murine osteoblasts and porous hydroxyapatite (HA) scaffolds to two-step, slow freezing and vitrification processes[J]. Cell Preservation Technol, 2002, 1(1):33-44.
[6]Kuleshovaa LL, Gouka SS, Hutmacher DW. Vitrification as a prospect for cryopreservation of tissue-engineered constructs[J]. Biomaterials, 2007, 28(9):1585-1596.
[7]Bianchi V, Coticchio G, Fava L, et al. Meiotic spindle imaging in human oocytes frozen with a slow freezing procedure involving high sucrose concentration[J]. HumReprod, 2005, 20(4):1078-1083.
[8]Fahy GM, Macfarlane DR, Angell CA, et al. Vitrification as an approach to cryopreservation[J]. Cryobiology, 1984, 21(4):407-426.
[9]Rall WF, Fahy GM. Ice-free cryopreservation of mouse embryos at -196℃ by vitrification[J]. Nature, 1985, 313(6003):573-575.
[10]Son WY, Lee SY, Chang MJ, et al. Pregnancy resulting from transfer of repeat vitrified blastocysts produced by in-vitro matured oocytes in patient with polycystic ovary syndrome[J]. Reprod Biomed Online, 2005,10(3):398-401.
[11]Kyono K, Fuchinoue K, Yagi A, et al. Successful pregnancy and delivery after transfer of a single blastocyst derived from a vitrified mature human oocyte[J]. Fertil Steril, 2005, 84(4):1017.
[12]Zhou CQ, Mai QY, Li T, et al. Cryopreservation of human embryonic stem cells by vitrification[J]. Chin Med J, 2004, 117(7):1050-1055.
[13]Cai X Y, Chen GA,Lian Y, et al. Cryoloop vitrification of rabbit oocytes[J]. Hum Reprod, 2005, 20(7):1969-1974.
[14]Selman H, Angelini A, Barnocchi N, et al. Ongoing pregnancies after vitrification of human oocytes using a combined solution of ethylene glycol and dimethyl sulfoxide[J]. Fert Steril,2006, 86(4):997-1000.
[15]Sheehan CB, Lane M, Gardner DK, et al. The CryoLoop facilitates revitrification of embryos at four successive stages of development without impairing embryo growth[J]. Hum Reprod,2006, 21(11):2978-2984.
[16]Gorlin J. Stem cell cryopreservation[J]. J Infus Chemother,1996,6(1):23-27.
[17]Hausen B, Beuke M, Schroeder F, et al. In vivo measurement of lung preservation solution efficacy: comparison of LPD, UW, EC and low K+-EC following short and extended ischemia[J]. Eur J Cardiothorac Surg,1997,12(5):771-780.
[18]Jassem W, Armeni T, Quiles JL, et al. Protection of mitochondria during cold storage of liver and following transplantation: comparison of the two solutions, University of Wisconsin and Eurocollins[J]. J Bioenerg Biomembr,2006,38(1):49-55.
[10]Shim H, GutierrezAdan A, Chen LR, et al. Isolation of pluripotent stem cells from cultured porcine primordial germ cells[J]. Biol Reprod, 1997, 57(5):1089-1095.
[20]Wusteman MC, Pegg DE. Differences in the requirements for cryopreservation of porcine aortic smooth muscle and endothelial cells[J]. Tissue Eng,2001,7(5):507-518.
[21]Vajta G,Holm P,Kuwayama M,et al. Open Pulled Straw (OPS) vitrification : a new way to reduce cryoinjuries of bovine ova and embryos[J]. Mol Reprod Dev, 1998, 51(1): 53-58.
[22]Karlsson JO, Toner M. Long-term storage of tissues by cryopreservation: critical issues[J]. Biomaterials,1996,17(3):243-256.
[收稿日期]2008-11-12[修回日期]2009-02-08
编辑/张惠娟