Effect of fructose and dithiothreitol on cell viability and pluripotency of cryopreserved bone marrow mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.2016.41.001

Abstract

Abstract:

BACKGROUND: Cell cryopreservation is required for clinical use of stem cells, and the current process of cryopreservation however may be harmful to cell viability, pluripotency and differentiation capacity.
OBJECTIVE: To explore the effect of fructose and dithiothreitol on pluripotency and osteogenesis of cryopreserved bone marrow mesenchymal stem cells.
METHODS: Bone marrow mesenchymal stem cells were isolated from the bone marrow of Sprague-Dawley rats and pretreated with fructose (200 μmol/L), dithiothreitol (500 μmol/L) or combined components before cryopreservation. Then the cells were cryopreseved for 6 months and the morphology of cells was observed by inverted microscopy. The cell viability was evaluated by MTT, and real-time PCR was used to detect the mRNA expression of Nanog, OCT4 and Sox2. Alkaline phophatase activity assay and alizarin red staining were utilized to detect the osteogenic capacity of bone marrow mesenchymal stem cells.
RESULTS AND CONCLUSION: Images captured by inverted microscopy showed no significant difference in cell morphology between groups. The MTT results indicated that fructose and combined pretreatment could promote the cell viability of bone marrow mesenchymal stem cells after cryopreservation, while the real-time PCR results demonstrated that dithiothreitol significantly facilitated the expression of Naogo and Sox2 in bone marrow mesenchymal stem cells. Moreover, ALP activity assay and alizarin red staining confirmed the positive effects of fructose, dithiothreitol and combined pretreatment on osteogenic capacity of bone marrow mesenchymal stem cells after cryopreservation, and the best effects were found after pretreatment with dithiothreitol and combined components. Overall, these findings indicate that fructose pretreatment is beneficial for cell viability of cryopreseved bone marrow mesenchymal stem cells, and dithiothreitol contributes to maintaining the pluripotency and osteogenesis capacity of cryopreseved bone marrow 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: Bone Marrow, Mesenchymal Stem Cells, Freezing, Fructose, Dithiothreitol, Tissue Engineering

CLC Number:

R394.2

Zheng Xin-tong, Liu Qin, Zhang Jing-xia, Luo Qing, Chen Zhe, Song Guan-bin. Effect of fructose and dithiothreitol on cell viability and pluripotency of cryopreserved bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(41): 6085-6091.

Figures/Tables 1

2.1 骨髓间充质干细胞复苏后形态学观察复苏细胞贴壁24 h后，倒置显微镜下观察可见细胞仍呈长梭态，符合骨髓间充质干细胞一般形态特征，见图1。进一步发现果糖、二硫苏糖醇或果糖+二硫苏糖醇联合处理组细胞整体形态与对照组无明显差别。 2.2 复苏骨髓间充质干细胞活性利用MTT实验检测骨髓间充质干细胞冻存后细胞活性，结果显示果糖或果糖+二硫苏糖醇预处理有助于冻存后细胞活性维持，与对照组比较差异有显著性意义，但保护程度有限。二硫苏糖醇单独预处理对冻存后细胞活性维持无明显促进作用，见图2。 2.3 复苏骨髓间充质干细胞多能性基因表达定量PCR结果显示，二硫苏糖醇预处理有助于骨髓间充质干细胞冻存后Nanog基因表达，而果糖预处理无明显作用，见图3A。果糖或二硫苏糖醇预处理对骨髓间充质干细胞冻存后Oct4基因的表达无显著影响，见图3B。二硫苏糖醇亦促进了冻存骨髓间充质干细胞复苏后Sox2基因的表达，见图3C。 2.4 复苏骨髓间充质干细胞成骨分化潜能经成骨诱导体系诱导7 d，检测各组细胞碱性磷酸酶活性。与对照组相比，果糖、二硫苏糖醇及果糖+二硫苏糖醇预处理均可一定程度上调复苏骨髓间充质干细胞碱性磷酸酶活性，其中果糖+二硫苏糖醇预处理组效果最为明显(P < 0.05)，见图4。成骨诱导14 d时，茜素红染色结果显示，果糖、二硫苏糖醇及果糖+二硫苏糖醇预处理可明显促进钙节点形成，体现为钙节点数量增多、面积(团块状)增大，其中二硫苏糖醇及果糖+二硫苏糖醇组尤为明显，见图5。因碱性磷酸酶活性及钙节点分别是骨髓间充质干细胞成骨分化的早期及晚期标志，故以上结果说明果糖+二硫苏糖醇预处理可有效维持冻存骨髓间充质干细胞成骨分化能力。"

References

[1] See EY, Toh SL, Goh JC. Multilineage potential of bone-marrow-derived mesenchymal stem cell cell sheets: implications for tissue engineering. Tissue Eng Part A. 2010;16(4):1421-1431.
[2] Zhang Q, Zhao YH.Therapeutic angiogenesis after ischemic stroke: Chinese medicines, bone marrow stromal cells (BMSCs) and their combinational treatment. Am J Chin Med. 2014;42(1):61-77.
[3] Hang HL, Xia Q. Role of BMSCs in liver regeneration and metastasis after hepatectomy. World J Gastroenterol. 2014;20(1):126-132.
[4] Liu P, Feng Y, Wang Y, et al. Therapeutic action of bone marrow-derived stem cells against acute kidney injury. Life Sci. 2014;115(1-2):1-7.
[5] Acker JP. Biopreservation of cells and engineered tissues. Adv Biochem Eng Biotechnol. 2007;103: 157-187.
[6] Rowley SD.Hematopoietic stem cell processing and cryopreservation.J Clin Apher. 1992;7(3):132-134.
[7] Chong YS, Chan ML, Tan HH, et al. Human embryo cryopreservation: one-step slow freezing does it all. J Assist Reprod Genet. 2014;31(7):921-925.
[8] Kajiura S, Mii S, Aki R, et al. Cryopreservation of the Hair Follicle Maintains Pluripotency of Nestin- Expressing Hair Follicle-Associated Pluripotent Stem Cells. Tissue Eng Part C Methods. 2015;21(8): 825-831.
[9] Kang YH, Lee HJ, Jang SJ, et al. Immunomodulatory properties and in vivo osteogenesis of human dental stem cells from fresh and cryopreserved dental follicles. Differentiation. 2015;90(1-3):48-58.
[10] Escobar CH, Chaparro O. Xeno-Free Extraction, Culture, and Cryopreservation of Human Adipose- Derived Mesenchymal Stem Cells. Stem Cells Transl Med. 2016;5(3):358-365.
[11] Pirnia A, Parivar K, Hemadi M, et al. Stemness of spermatogonial stem cells encapsulated in alginate hydrogel during cryopreservation. Andrologia. 2016 Jul 29.[Epub ahead of print]
[12] Rao W, Huang H, Wang H, et al. Nanoparticle- mediated intracellular delivery enables cryopreservation of human adipose-derived stem cells using trehalose as the sole cryoprotectant. ACS Appl Mater Interfaces. 2015;7(8):5017-5028.
[13] Gurruchaga H, Ciriza J, Saenz Del Burgo L, et al. Cryopreservation of microencapsulated murine mesenchymal stem cells genetically engineered to secrete erythropoietin. Int J Pharm. 2015;485(1-2): 15-24.
[14] Yong KW, Wan Safwani WK, Xu F, et al. Cryopreservation of Human Mesenchymal Stem Cells for Clinical Applications: Current Methods and Challenges. Biopreserv Biobank. 2015;13(4):231-239.
[15] de Lima Prata K, de Santis GC, Orellana MD, et al. Cryopreservation of umbilical cord mesenchymal cells in xenofree conditions. Cytotherapy. 2012;14(6): 694-700.
[16] Sparks AE.Human embryo cryopreservation-methods, timing, and other considerations for optimizing an embryo cryopreservation program.Semin Reprod Med. 2015;33(2):128-144.
[17] Windrum P, Morris TC, Drake MB, et al. Variation in dimethyl sulfoxide use in stem cell transplantation: a survey of EBMT centres. Bone Marrow Transplant. 2005;36(7):601-603.
[18] Yango P, Altman E, Smith JF, et al. Optimizing cryopreservation of human spermatogonial stem cells: comparing the effectiveness of testicular tissue and single cell suspension cryopreservation. Fertil Steril. 2014;102(5):1491-1498.
[19] Lindemann D, Werle SB, Steffens D, et al. Effects of cryopreservation on the characteristics of dental pulp stem cells of intact deciduous teeth. Arch Oral Biol. 2014;59(9):970-976.
[20] Gole MD, Poulsen D, Marzo JM, et al. Chondrocyte viability in press-fit cryopreserved osteochondral allografts. J Orthop Res. 2004;22(4):781-787.
[21] Kotobuki N, Hirose M, Machida H, et al. Viability and osteogenic potential of cryopreserved human bone marrow-derived mesenchymal cells. Tissue Eng. 2005;11(5-6):663-673.
[22] Fang JK, Zhou YP, Li ML. Research progress on effects of traditional Chinese medicines on proliferation, apoptosis and differentiation of bone marrow mesenchymal stem cells. Zhongguo Zhong Yao Za Zhi. 2014;39(15):2834-2837.
[23] Nemeth K, Mezey E. Bone marrow stromal cells as immunomodulators. A primer for dermatologists. J Dermatol Sci. 2015;77(1):11-20.
[24] García-García A, de Castillejo CL, Méndez-Ferrer S. et al. BMSCs and hematopoiesis. Immunol Lett. 2015; 168(2):129-135.
[25] Gasbarrini A, Borle AB, Farghali H, et al. Fructose protects rat hepatocytes from anoxic injury. Effect on intracellular ATP, Ca2+i, Mg2+i, Na+i, and pHi. J Biol Chem. 1992;267(11):7545-7552.
[26] Aghdai MH, Jamshidzadeh A, Nematizadeh M, et al. Evaluating the Effects of Dithiothreitol and Fructose on Cell Viability and Function of Cryopreserved Primary Rat Hepatocytes and HepG2 Cell Line. Hepat Mon. 2013;13(1):e7824.
[27] Su X, Ling Y, Liu C, et al. Isolation, Culture, Differentiation, and Nuclear Reprogramming of Mongolian Sheep Fetal Bone Marrow-Derived Mesenchymal Stem Cells. Cell Reprogram. 2015; 17(4):288-296.
[28] Fan YX, Gu CH, Zhang YL, et al. Oct4 and Sox2 overexpression improves the proliferation and differentiation of bone mesenchymal stem cells in Xiaomeishan porcine. Genet Mol Res. 2013;12(4): 6067-6079.
[29] Ren Y, Wu H, Wang X, et al. Analysis of the stem cell characteristics of adult stem cells from Arbas white Cashmere goat. Biochem Biophys Res Commun. 2014;448(2):121-128.
[30] Pashaiasl M, Khodadadi K, Kayvanjoo AH, et al. Unravelling evolution of Nanog, the key transcription factor involved in self-renewal of undifferentiated embryonic stem cells, by pattern recognition in nucleotide and tandem repeats characteristics. Gene. 2016;578(2):194-204.