Effect of cold storage time on cellular viability of umbilical cord mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.0446

Abstract

Abstract:

BACKGROUND: With the in-depth basic research and clinical research on mesenchymal stem cells, mesenchymal stem cells have wide clinical prospects. However, little is reported on the temporary storage conditions and refusion time of mesenchymal stem cells.
OBJECTIVE: To explore the effect of cold storage time on the cell mass and viability of umbilical cord mesenchymal stem cells.
METHODS: Frozen-thawed and unfrozen umbilical cord mesenchymal stem cells were prepared and stored at 0-4 ℃. The cell viable cell rate, cell doubling time and colony forming efficiency were detected after 0, 6, 12, and 24 hours.
RESULTS AND CONCLUSION: Unfrozen cells could maintain the cell biological activity at 0-4 ℃ until dead cells appeared with the presence of decreased cell viability at 12 hours after storage. Frozen-thawed cells were unable to be stored at 0-4 ℃ for a long time, and cells began to die and the cell viability was weakened at 6 hours after storage. These findings indicate that umbilical cord mesenchymal stem cells should be injected into patients within 6 or 12 hours after preparation, to ensure the best therapeutic effects. If there is a longer transport distance, it is preferred to use unfrozen mesenchymal stem cells.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Stem Cells, Mesenchymal Stem Cells, Cryopreservation, Tissue Engineering

CLC Number:

R349.2

Yan Shan-shan, Li Yi-jia, Chen Xi, Liu Li-jun. Effect of cold storage time on cellular viability of umbilical cord mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(5): 748-753.

Figures/Tables 2

2.1 细胞形态未经过冻存的脐带间充质干细胞于 0-4 ℃保存12 h之内，细胞贴壁效率无明显差别，大部分细胞都能够正常贴壁，未贴壁的悬浮细胞很少，保存24 h后的贴壁效率明显下降，可以观察到较多未贴壁的悬浮细胞，但贴壁后的增殖能力均较好(图1)。冻存复苏的脐带间充质干细胞于0-4℃保存6 h之内，细胞贴壁效率无明显差别，大部分细胞都能较好的贴壁，悬浮细胞不多，保存12 h后细胞贴壁效率明显下降，能够看到大量未贴壁的死细胞；保存24 h后细胞不仅贴壁效果差，只能看到很少的细胞贴壁，增殖能力也明显降低(图2)。 2.2 活细胞率未冻存的脐带间充质干细胞在0-4 ℃储存0，6，12，24，36，48 h后的活细胞率分别为96%，94%，91%，77%，73%，59%，储存12 h后活细胞率明显降低；冻存复苏的脐带间充质干细胞在0-4 ℃储存0，6，12，24 h后的活细胞率分别为94%，92%，69%，64%，储存6 h后活细胞率明显降低(图3)。 2.3 倍增时间两种细胞生长曲线见图4，5。未冻存的脐带间充质干细胞在0-4 ℃储存0，6，12，24 h后细胞倍增时间分别为20.2，25.92，30.35，31.4 h；冻存复苏的脐带间充质干细胞在0-4 ℃储存0，6，12，24 h后细胞倍增时间分别为21.31，32.02，43.94，46.87 h，随着储存时间的延长，间充质干细胞倍增时间不断增加，冻存复苏的间充质干细胞与未冻存的间充质干细胞相比，倍增时间均较长。 2.4 细胞克隆率 Giemsa染色后能够清晰看到形成的细胞克隆团，随着存储时间的延长，克隆团数量随之减少。未冻存的脐带间充质干细胞0-4 ℃储存12 h内克隆形成率在89%以上，储存12 h后细胞克隆团数量明显减少，储存24 h后只能看到少量着色较深的克隆团(图6)。冻存复苏的脐带间充质干细胞0-4 ℃储存6 h内克隆形成率在80%以上，与未冻存的间充质干细胞形成的克隆相比，克隆团也较小，储存6 h后细胞克隆团数量明显减少，24 h后仍具有克隆形成能力的细胞更少(图7)。"

References

[1] Semenov OV, Koestenbauer S, Riegel M, et al. Multipotent mesenchymal stem cells from human placenta: critical parameters for isolation and maintenance of stemness after isolation. Am J Obstet Gynecol. 2010;202(2):193.e1-193.e13.

[2] Winkler S, Borkham-Kamphorst E, Stock P, et al. Human mesenchymal stem cells towards non-alcoholic steatohepatitis in an immunodeficient mouse model. Exp Cell Res.2014;326(2):230-239.

[3] Karahuseyinoglu S, Cinar O, Kilic E, et al. Biology of stem cells in human umbilical cord stroma: in situ and in vitro surveys. Stem Cells. 2007;25(2):319-331.

[4] Teixeira FG, Carvalho MM, Sousa N, et al. Mesenchymal stem cells secretome: a new paradigm for central nervous system regeneration. Cell Mol Life Sci. 2013;70(20): 3871-3882.

[5] 朱婷,李爱斌,曹景云,等.胎盘间充质干细胞的应用研究[J].中国组织工程研究,2015,19(32):5236-5242.

[6] Moodley Y, Atienza D, Manuelpillai U, et al. Human umbilical cord mesenchymal stem cells reduce fibrosis of bleomycin-induced lung injury. Am J Pathol. 2009;175(1): 303-313.

[7] 李清,唐笛,时玉舫,等.间充质干细胞与免疫的相互作用：从基础研究到临床应用[J].生命科学,2016,28(8):933-940.

[8] Teixeira FG, Carvalho MM, Neves-Carvalho A, et al. Secretome of mesenchymal progenitors from the umbilical cord acts as modulator of neural/glial proliferation and differentiation. Stem Cell Rev. 2015;11(2):288-297.

[9] Liu WH, Song FQ, Ren LN, et al. The multiple functional roles of mesenchymal stem cells in participating in treating liver diseases. J Cell Mol Med. 2015;19(3):511-520.

[10] Ribeiro A, Laranjeira P, Mendes S, et al. Mesenchymal stem cells from umbilical cord matrix, adipose tissue and bone marrow exhibit different capability to suppress peripheral blood B, natural killer and T cells. Stem Cell Res Ther. 2013; 4(5):125.

[11] 袁宝珠,张可华,纳涛. 建立间充质干细胞制剂质量控制评价体系[C].2013年中国药学大会,2013.

[12] 中华人民共和国国家卫生和计划生育委员会.《干细胞制剂质量控制及临床前研究指导原则( 试行) 》( 征求意见稿) 解读[J].转化医学杂志,2013,2(6):321-322.

[13] 关于征求干细胞临床研究管理办法(试行)意见的函[J].中国组织工程研究,2015,19(10):1640.

[14] 中华人民共和国国家卫生和计划生育委员会.《干细胞临床试验研究管理办法( 试行) 》(征求意见稿) 解读[J].转化医学杂志, 2013,2(6):322-323.

[15] 周琪. 中国及中国科学院干细胞与再生医学研究概述[J].生命科学,2016,28(8):833-838.

[16] 姜天娇,孙金海. 国外干细胞产品监管现状及对我国的启示[J].中国社会医学杂志,2016,33(2):117-120.

[17] Daley GQ, Hyun I, Apperley JF, et al. Setting Global Standards for Stem Cell Research and Clinical Translation: The 2016 ISSCR Guidelines. Stem Cell Reports. 2016;6(6): 787-797.

[18] Ramdasi S, Sarang S, Viswanathan C. Potential of Mesenchymal Stem Cell based application in Cancer. Int J Hematol Oncol Stem Cell Res. 2015;9(2):95-103.

[19] Bárcia RN, Santos JM, Teixeira M, et al. Umbilical cord tissue-derived mesenchymal stromal cells maintain immunomodulatory and angiogenic potencies after cryopreservation and subsequent thawing. Cytotherapy. 2017;19(3):360-370.

[20] Li Q, Yao D, Ma J, et al. Transplantation of MSCs in combination with netrin-1 improves neoangiogenesis in a rat model of hind limb ischemia. J Surg Res. 2011;166(1): 162-169.

[21] Nejad-Moghaddam A, Ajdary S, Tahmasbpour E, et al. Immunomodulatory Properties of Mesenchymal Stem Cells Can Mitigate Oxidative Stress and Inflammation Process in Human Mustard Lung. Biochem Genet. 2016;54(6):769-783.

[22] Tyndall A. Mesenchymal stem cell treatments in rheumatology: a glass half full. Nat Rev Rheumatol. 2014;10(2):117-124.

[23] Zhou HX, Liu ZG, Liu XJ,et al.Umbilical cord-derived mesenchymal stem cell transplantation combined with hyperbaric oxygen treatment for repair of traumatic brain injury.Neural Regen Res. 2016;11(1):107-113.

[24] Wang D, Huang S, Yuan X, et al. The regulation of the Treg/Th17 balance by mesenchymal stem cells in human systemic lupus erythematosus. Cell Mol Immunol. 2017; 14(5):423-431.

[25] Hajivalili M, Pourgholi F, Kafil HS, et al. Mesenchymal Stem Cells in the Treatment of Amyotrophic Lateral Sclerosis. Curr Stem Cell Res Ther. 2016;11(1):41-50.

[26] Paulini J, Higuti E, Bastos RM, et al. Mesenchymal Stem Cells as Therapeutic Candidates for Halting the Progression of Diabetic Nephropathy. Stem Cells Int. 2016;2016:9521629.

[27] 陈俊宇,王建忠.成骨细胞、破骨细胞与酒精性股骨头坏死的关联性：预防与靶向治疗的新思路[J].中国组织工程研究,2016, 20(46):6963-6969.

[28] 张香梅,田中良哉,单保恩. 间充质干细胞应用于类风湿关节炎组织修复的研究进展[J].中国全科医学,2016,19(32): 4014-4018.

[29] 顾问,郭雪君,徐卫国.间充质干细胞治疗呼吸系统疾病的研究进展[J].中华肺部疾病杂志,2016,9(1):82-86.

[30] Chang Z, Mao G, Sun L, et al. Cell therapy for cerebral hemorrhage: Five year follow-up report. Exp Ther Med. 2016;12(6):3535-3540.

[31] Heidemann R, Mered M, Wang DQ, et al. A new seed-train expansion method for recombinant mammalian cell lines. Cytotechnology. 2002;38(1-3):99-108.