COMPARATIVE STUDIES ON DIFFERENT CRYOPRESERVATION PROTOCOLS OF HUMAN AMNIOTIC FLUIDDERIVEDMESENCHYMAL STEM CELLS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

WANG Yiru ¹ , BAI Jing ² , CHEN Jie ¹ , LIU Lifeng ¹ , WANG Yu ¹

1 Institute of Geriatric Cardiology, General Hospital of Chinese PLA, Beijing, 100853, P.R.China;;
2 Department of Cardiology, the First Affiliated Hospital ofGeneral Hospital of Chinese PLA.;

Corresponding author：

Keywords：

Human amniotic fluid-derived mesenchymal stem cells , Cryopreservation and resuscitation, Biocharacteristic

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【Abstract】 Objective To isolate and culture human amniotic fluid-derived mesenchymal stem cells (HAFMSCs),
to investigate a better cryopreservation protocol of HAFMSCs and to observe the biocharacteristics and the multi-potential of HAFMSCs after cryopreservation for the further fundamental researches and cl inical appl ications. Methods HAFMSCs
were isolated from the amniotic fluid of pregnant women during the second trimester by the improved two-step method.
HAFMSCs were cryopreserved with different cryopreservation protocols (containing different contents of FBS and DMSO at
cryoprotectant) in l iquid nitrogen for 12 weeks. The biocharacteristics of the HAFMSCs after cryopreservation were analyzed. The growth characteristics were observed by MTT method and the growth curves were drawn. The surface antigens of HAFMSCs were detected using flow cytometry, including CD29, CD34, CD44, CD45, CD73, and CD90. The adi pogenic and osteogenic differentiation abil ities of HAFMSCs were observed. The mRNA levels of Oct-4 and Nanog of the HAFMSCs were compared between before and after cryopreservations. Results At 12 weeks after cryopreservation, different protocols had different effects on the cell viabil ity; the better formula of cryoprotectant was 50% DMEM, 40% FBS, and 10% DMSO. After cryopreservation, the cells proliferated rapidly and the growth curves showed “S” shape, which was the same as the cells before cryopreservation. Phenotype showed that HAFMSCs were positive for the surface markers CD29, CD44, CD73, and CD90, and negative for CD34 and CD45. After 21 days of adi pogenic differentiation, the l ipid droplets were observed by oil red O staining. After 21 days of osteogenic differentiation, the calcium mineralizations were verified by von Kossa staining. There was no significant difference (P gt; 0.05) in the mRNA levels of Oct-4 and Nanog between before and after cryopreservations. Conclusion HAFMSCs have rapid proliferation and multi-potential in vitro. The cells have high viabil ities and no changes of the biocharacteristics and differentiation potential ities after cryopreservation for 12 weeks. Cryoprotectant containing 50% DMEM, 40% FBS, and 10% DMSO is a better cryopreservation protocol.

Citation： WANG Yiru,BAI Jing,CHEN Jie,LIU Lifeng,WANG Yu. COMPARATIVE STUDIES ON DIFFERENT CRYOPRESERVATION PROTOCOLS OF HUMAN AMNIOTIC FLUIDDERIVEDMESENCHYMAL STEM CELLS. Chinese Journal of Reparative and Reconstructive Surgery, 2012, 26(2): 141-145. doi: Copy

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12.	Decembrini S, Cananzi M, Gualdoni S, et al. Comparative analysis of the retinal potential of embryonic stem cells and amniotic fluid-derived stem cells. Stem Cells Dev, 2011, 20(5): 851-863.
13.	Hilfiker A, Kasper C, Hass R, et al. Mesenchymal stem cells and progenitor cells in connective tissue engineering and regenerative medicine: is there a future for transplantation. Langenbeck Arch Surg, 2011, 396(4): 489-497.
14.	Hauser PV, De Fazio R, Bruno S, et al. Stem cells derived from human amniotic fluid contribute to acute kidney injury recovery. Am J Pathol, 2010, 177(4): 2011-2021.
15.	Teodelinda M, Michele C, Sebastiano C, et al. Amniotic liquid derived stem cells as reservoir of secreted angiogenic factors capable of stimulating neo-arteriogenesis in an ischemic model. Biomaterials, 2011, 32(15): 3689-3699.
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1. 1 Prusa AR, Marton E, Rosner M, et al. OCT-4 expressing cells in human amniotic fluid: a new source for stem cell research? Hum Reprod, 2003, 18(7): 1489-1493. 2 Tsai MS, Lee JL, Chang YJ, et al. Isolation of human multipotent mesenchymal stem cells from second-trimester amniotic fluid using a novel two-stage culture protocol. Hum Reprod, 2004, 19(6): 1450-1456.
2. Roubelakis MG, Pappa KI, Bitsika V, et al. Molecular and proteomic characterization of human mesenchymal stem cells derived from amniotic fluid: comparison to bone marrow mesenchymal stem cells. Stem Cells Dev, 2007, 16(6): 931-952.
3. Sessarego N, Parodi A, Podesta M, et al. Multipotent mesenchymal stromal cells from amniotic fluid: solid perspectives for clinical application. Haematologica, 2008, 93(3): 339-346.
4. Ghionzoli M, Cananzi M, Zani A, et al. Amniotic fluid stem cell migration after intraperitoneal injection in pup rats: implication for therapy. Pediatr Surg Int, 2010, 26(1): 79-84.
5. Baghaban Eslaminejad M, Jahangir S, Aghdami N. Mesenchymal stem cells from murine amniotic fluid as a model for preclinical investigation. Arch Iran Med, 2011, 14(2): 96-103.
6. Shaw SW, David AL, De Coppi P, et al. Clinical applications of prenatal and postnatal therapy using stem cells retrieved from amniotic fluid. Curr Opin Obstet Gynecol, 2011, 23(2): 109-116.
7. Gekas J, Walther G, Skuk D, et al. In vitro and in vivo study of human amniotic fluid-derived stem cell differentiation into myogenic lineage. Clin Exp Med, 2010, 10(1): 1-6.
8. Tsai MS, Hwang SM, Tsai YL, et al. Clonal amniotic fluid-derived stem cells express characteristics of both mesenchymal and neural stem cells. Biol Reprod, 2006, 74(3): 545-551.
9. In’t Anker PS, Scherjon SA, Kleijburg-van der Keur C, et al. Amniotic fluid as a novel source of mesenchymal stem cells for therapeutic transplantation. Blood, 2003, 102(4): 1548-1549.
10. De Coppi P, Bartsch G Jr, Siddiqui MM, et al. Isolation of amniotic stem cells with potential for therapy. Nat Biotechnol, 2007, 25(1): 100-106.
11. Cananzi M, Atala A, De Coppi P. Stem cells derived from amniotic fluid: new potentials in regenerative medicine. Reprod Biomed Online, 2009, 18 Suppl 1: 17-27.
12. Decembrini S, Cananzi M, Gualdoni S, et al. Comparative analysis of the retinal potential of embryonic stem cells and amniotic fluid-derived stem cells. Stem Cells Dev, 2011, 20(5): 851-863.
13. Hilfiker A, Kasper C, Hass R, et al. Mesenchymal stem cells and progenitor cells in connective tissue engineering and regenerative medicine: is there a future for transplantation. Langenbeck Arch Surg, 2011, 396(4): 489-497.
14. Hauser PV, De Fazio R, Bruno S, et al. Stem cells derived from human amniotic fluid contribute to acute kidney injury recovery. Am J Pathol, 2010, 177(4): 2011-2021.
15. Teodelinda M, Michele C, Sebastiano C, et al. Amniotic liquid derived stem cells as reservoir of secreted angiogenic factors capable of stimulating neo-arteriogenesis in an ischemic model. Biomaterials, 2011, 32(15): 3689-3699.
16. 赵春华. 干细胞原理、技术与临床. 北京: 化学工业出版社, 2006: 309-310.
17. 王有为, 韩之波, 严淑琳, 等. 人脐带间充质干细胞冻存复苏后的生物学特征. 中国组织工程研究与临床康复, 2010, 14(10): 1729-1733.
18. Karlmark KR, Freilinger A, Marton E, et al. Activation of ectopic Oct- 4 and Rex-1 promoters in human amniotic fluid cells. Int J Mol Med, 2005, 16(6): 987-992.

Chinese Journal of Reparative and Reconstructive Surgery

COMPARATIVE STUDIES ON DIFFERENT CRYOPRESERVATION PROTOCOLS OF HUMAN AMNIOTIC FLUIDDERIVEDMESENCHYMAL STEM CELLS

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