Effect of cultivation systems on the maintenance of human umbilical cord mesenchymal stem cell characteristics and their proliferation rate in vitro

doi:10.3969/j.issn.2095-4344.2017.13.010

Abstract

Abstract:

BACKGROUND: Preliminary data showed that the application of human platelet lysate to human umbilical cord mesenchymal stem cell culture can better maintain the characteristics of stem cells than the application of serum-free medium. However, the serum-free medium can better improve the proliferation of mesenchymal stem cells in vitro than the human platelet lysate.
OBJECTIVE: To screen out a better mesenchymal stem cell cultivation system that can greatly maintain the characteristics and proliferation rate of human umbilical cord mesenchymal stem cells.
METHODS: Six human umbilical cord specimens were inoculated in six culture systems, and the primary culture of umbilical cord mesenchymal stem cell was performed. These six culture systems were respectively MesenCult serum-free medium with 2% human platelet lysate (group A), StemPro serum-free medium with 2% human platelet lysate (group B), MesenCult serum-free medium (group C), StemPro serum-free medium (group D), low glucose-DMEM with 10% fetal bovine serum (group E), low glucose-DMEM with 10% human platelet lysate (group F). The cells were subcultured at 14 days after inoculation to compare the effects of different culture systems on the morphology, surface markers, differentiation and proliferation of human umbilical cord mesenchymal stem cells.
RESULTS AND CONCLUSION: (1) The morphology of passage 3 cells in group D was elongated and uneven in size. The morphology of passage 3 cells was flattened in groups E and F, but the cells in the other groups were spindle-shaped and uniform. There were no significant changes in morphology and size between passage 3 and 5 cells in A and B. In group C, the morphology of passage 5 cells was more flattened and uneven in size compared with passage 3 cells. In group D, the morphology of passage 5 cells was more elongated than that of passage 3 cell. In group E, the morphology of passage 5 cells was more flattened than that of passage 3 cells. There was no significant difference in morphology between passage 3 and 5 cells in group F. (2) The expression rate of cell surface markers had no significant difference at different passages in each group. (3) The adipoinduction and osteoinduction rates were relatively higher in groups A and B compared with groups E and F, and lowest in groups C and D. (4) The cell proliferation rate for each passages in group A was significantly higher than that in group C. The cell proliferation rate for each passage in group B was significantly higher than that in group D. The cell proliferation rate for each passage in groups E and F was significantly lower than that in groups A and B. To conclude, these results suggest that the combination of serum-free medium with human platelet lysate could better maintain the characteristics and the proliferation efficiency of 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, Umbilical Cord, Mesenchymal Stem Cells, Tissue Engineering

CLC Number:

R394.2

Sun Ya-ru, Zhang Bing-qiang, Wang Fu-bin, Xu Ping, Wang Er-pu, Li Cui-cui. Effect of cultivation systems on the maintenance of human umbilical cord mesenchymal stem cell characteristics and their proliferation rate in vitro[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(13): 2023-2028.

Figures/Tables 2

References

[1] Parekkadan B,Milwid JM.Mesenchymal stem cells as therapeutics.Annu Rev Biomed Eng.2010;12:87-117.

[2] Servais S,Beguin Y,Delens L,et al.Novel approaches for preventing acute graft-versus-host disease after allogeneic hematopoieticstem cell transplantation.Expert Opin Investig Drugs.2016;25(8):957-972.

[3] Wystrychowski W,Patlolla B,Zhuge Y,et al.Multipotency and cardiomyogenic potential of human adipose-derived stem cells from epicardium, pericardium, and omentum.Stem Cell Res Ther.2016;7(1):84-95.

[4] Jiang R,Han Z,Zhuo G,et al.Transplantation of placenta-derived mesenchymal stem cells in type 2 diabetes: a pilot study.Front Med.2011;5(1):94-100.

[5] Deng P,Torrest A,Pollock K,et al.Clinical trial perspective for adult and juvenile Huntington's disease using genetically-engineered mesenchymal stem cells.Neural Regen Res.2016;11(5):702-705.

[6] Labrador S,Alonso ML,Alvarez S,et al.Mesenchymal stem cell therapy in retinal and optic nerve diseases: An update of clinical trials.World J Stem Cells.2016;8(11):376-383.

[7] Moussa L,Pattappa G,Doix B, et al.A biomaterial-assisted mesenchymal stromal cell therapy alleviates colonic radiation-induced damage.Biomaterials.2017;115:40-52.

[8] Mao X,Liu Y,Chen C,et al.Mesenchymal Stem Cells and Their Role in Dental Medicine. Dent Clin North Am.2017;61(1): 161-172.

[9] Cui GH,Wang YY,Li CJ,et al.Efficacy of mesenchymal stem cells in treating patients with osteoarthritis of the knee: A meta-analysis.Exp Ther Med.2016;12(5):3390-3400.

[10] Oner A,Gonen ZB,Sinim N,et al.Subretinal adipose tissue-derived mesenchymal stem cell implantation in advanced stage retinitis pigmentosa: a phase I clinical safety study.Stem Cell Res Ther.2016;7(1):178.

[11] Dothel G,Raschi E,Rimondini R,et al.Mesenchymal stromal cell-based therapy: Regulatory and translational aspects in gastroenterology.World J Gastroenterol.2016; 22(41):9057- 9068.

[12] Can A,Balci D.Isolation,culture,and characterization of human umbilical cord stroma-derived mesenchymal stem cells. Methods Mol Biol.2011;698:51-62.

[13] Gong W, Han Z, Zhao H,et al.Banking human umbilical cord-derived mesenchymal stromal cells for clinical use.Cell Transplant.2012;21(1):207-216.

[14] 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.

[15] Guo ZY,Sun X,Xu XL,et al.Human umbilical cord mesenchymal stem cells promote peripheral nerve repair via paracrine mechanisms. Neural Regen Res. 2015;10(4): 651-658.

[16] Zhao Q,Wang XY,Yu XX,et al.Expression of human telomerase reverse transcriptase mediates the senescence of mesenchymal stem cells through the PI3K/AKT signaling pathway.Int J Mol Med.2015;36(3):857-864.

[17] Mizuno M,Katano H,Otabe K,et al.Platelet-derived growth factor (PDGF)-AA/AB in human serum are potential indicators of the proliferative capacity of human synovial mesenchymal stem cells.Stem Cell Res Ther.2015;6:243-253.

[18] Esmaeli A,Moshrefi M,Shamsara A,et al.Xeno-free culture condition for human bone marrow and umbilical cord matrix-derived mesenchymalstem/stromal cells using human umbilical cord blood serum.Int J Reprod Biomed (Yazd). 2016; 14(9):567-576.

[19] Fazzina R,Iudicone P,Fioravanti D,et al.Potency testing of mesenchymal stromal cell growth expanded in human platelet lysate from different human tissues. Stem Cell Res Ther.2016; 7(1):122.

[20] Smith JR,Pfeifer K,Petry F,et al.Standardizing Umbilical Cord Mesenchymal Stromal Cells for Translation to Clinical Use: Selection of GMP-Compliant Medium and a Simplified Isolation Method.Stem Cells Int.2016;2016:6810980.

[21] Kocaoemer A,Kern S,Klüter H,et al.Human AB serum and thrombin-activated platelet-rich plasma are suitable alternatives to fetal calf serum for the expansion of mesenchymal stem cells from adipose tissue.Stem Cells. 2007;25(5):1270-1278.

[22] Tateishi K,Ando W,Higuchi C,et al.Comparison of human serum with fetal bovine serum for expansion and differentiation of human synovial MSC: Potential feasibility for clinical applications.Cell Transplant.2008;17(5):549-557.

[23] Yilmaz M,Ovali E,Akdogan E,et al.Autologous serum is more effective than fetal bovine serum on proliferation of bone marrow derived human mesenchymal stem cells.Saudi Med J.2008;29(2):306-309.

[24] Juhl M,Tratwal J,Follin B,et al.Comparison of clinical grade human platelet lysates for cultivation of mesenchymal stromal cells from bone marrow and adipose tissue.Scand J Clin Lab Invest.2016;76(2):93-104.

[25] Stute N,Holtz K,Bubenheim M,et al.Autologous serum for isolation and expansion of human mesenchymal stem cells for clinical use.Exp Hematol.2004;32(12):1212-1225.

[26] Lange C,Cakiroglu F,Spiess AN,et al.Accelerated and safe expansion of human mesenchymal stromal cells in animal serum-free medium for transplantation and regenerative medicine.J Cell Physiol.2007;213(1):18-26.

[27] Chase LG,Lakshmipathy U,Solchaga LA,et al.A novel serum-free medium for the expansion of human mesenchymal stem cells.Stem Cell Res Ther.2010;1(1):8.

[28] Haack-Sorensen M,Friis T,Bindslev L,et al.Comparison of different culture conditions for human mesenchymal stromal cells for clinical stem cell therapy.Scand J Clin Lab Invest. 2008;68(3):192-203.

[29] Merlo B,Pirondi S,Iacono E,et al. Viability, in vitro Differentiation and Molecular characterization of equine adipose tissue-derived mesenchymal stem cells cryopreserved in serum and serum-free medium. Cryo Letters. 02016;37(4):243-252.

[30] Zanicotti DG,Coates DE.Growing Adipose-Derived Stem Cells Under Serum-Free Conditions.Methods Mol Biol. 2017; 1537: 439-446.

[31] 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.

[32] Wu X,Kang H,Liu X,et al.Serum and xeno-free, chemically defined, no-plate-coating-based culture system for mesenchymal stromal cells from the umbilical cord.Cell Prolif. 2016;49(5):579-88.

[33] Wu JY,Lu Y,Chen JS,et al.Pooled Umbilical Cord Blood Plasma for Culturing UCMSC and Ex Vivo Expanding Umbilical Cord Blood CD34? Cells.Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2015;23(4):1112-1119.

[34] Chase LG,Yang S,Zachar V,et al.Development and characterization of a clinically compliant xeno-free culture medium in good manufacturing practice for human multipotent mesenchymal stem cells.Stem Cells Transl Med. 2012;1(10):750-758.

[35] Simões IN,Boura JS,dos Santos F,et al.Human mesenchymal stem cells from the umbilical cord matrix: successful isolation and ex vivo expansion using serum-/xeno-free culture media. Biotechnol J.2013;8(4):448-458.

[36] Swamynathan P,Venugopal P,Kannan S,et al.Are serum-free and xeno-free culture conditions ideal for large scale clinical grade expansion of Wharton's jelly derived mesenchymal stem cells? A comparative study. Stem Cell Res Ther.2014; 5(4):88.

[37] Rakian R,Block TJ,Johnson SM,et al.Native extracellular matrix preserves mesenchymal stem cell "stemness" and differentiation potential under serum-free culture conditions. Stem Cell Res Ther.2015;6:235.

[38] Gottipamula S,Ashwin KM,Muttigi MS,et al.Isolation, expansion and characterization of bone marrow-derived mesenchymal stromal cells in serum-free conditions.Cell Tissue Res. 2014;356(1):123-135.

[39] Mizukami A,Fernandes-Platzgummer A,Carmelo JG,et al.Stirred tank bioreactor culture combined with serum-/xenogeneic-free culture medium enables an efficient expansion of umbilical cord-derived mesenchymal stem/stromal cells.Biotechnol J. 2016;11(8):1048-1059.

[40] Gerby S,Attebi E,Vlaski M,et al.A new clinical-scale serum-free xeno-free medium efficient in ex vivo amplification of mesenchymal stromal cells does not support mesenchymal stem cells.Transfusion.2016.doi: 10.1111/trf.13902. [Epub ahead of print]