Modified isolation and culture methods of human ovarian granulosa cells

doi:10.3969/j.issn.2095-4344.2015.46.015

Abstract

Abstract:

BACKGROUND: To build up an effective method of isolating and culturing granule cells is a pivotal step to enhance fertilization-embryo transfer rate. Current studies mainly focus on the isolation methods of human ovarian granulosa cells rather than cell counting, purity and subsequent growth.
OBJECTIVE: To establish the effective methods of isolating, purifying and culturing human ovarian granulosa cells in vitro.
METHODS: Follicular fluid was harvested from women undergoing fertilization-embryo transfer procedures. Human ovarian granulosa cells were obtained from the follicular fluid by lysis treatment, precipitation method or density gradient centrifugation. Granulosa cell mucus masses were digested with type I collagen enzyme or hyaluronidase and then cultured in the culture medium with or without autologous follicular fluid.
RESULTS AND CONCLUSION: Lysis treatment yielded the largest amount of granulosa cells compared to the precipitation method and density gradient centrifugation (P > 0.05, P < 0.05, respectively). Cells prepared by the three methods showed the same cell viability. After 24 hours of culture, the precipitation method obtained the largest amount of adherent granulosa cells (P < 0.05); and the density gradient centrifugation obtained the least
amount of cells (P < 0.05). Compared with type I collagen enzyme, hyaluronidase took less time to digest the cells thoroughly. Autologous follicular fluid could promote the growth and survival of granulosa cells. These findings indicate that the precipitation method, though time-consuming, can obtain the highest cell viability and harvested the largest amount of granulosa cells after culture; hyaluronidase is more suitable for digesting granulosa cell mucus mass than type I collagen enzyme; autologous follicular fluid added into the culture medium is more conducive to granulosa cell growth.
中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Ovary, Cells, Cultured, Fertilization in Vitro

Chen Dong-si, Qi Xiu-juan, Liu Jian-xin, Ding Yu, Ma Wen-cong . Modified isolation and culture methods of human ovarian granulosa cells[J]. Chinese Journal of Tissue Engineering Research, 2015, 19(46): 7456-7460.

Figures/Tables 3

References

[1] Jancar N, Kopitar AN, Ihan A, et al. Effect of apoptosis and reactive oxygen species production in human granulosa cells on oocyte fertilization and blast development. J Assist Reprod Genet.2007;24( 2-3) : 91-97.

[2] Shi Ft, Cheung AP, Huang HF, et al. Growth differentiation factor 9 (GDF9) suppresses follistatin and follistatin-like 3 production in human granulose-lutein cells. PloS One.2011; 6(8):e22866.

[3] Yata A, Nakabayashi K, Wakah ashi S, et al. Suppression of progesterone production by stresscop in/ urocortin 3 in cultured human granulose-lutein cells. Hum Reprod, 2009; 24(7):1748-1753.

[4] Ma M, Wang J, Xu L,et al.Effects of two human chorionic gonadotropin doses administered to the ovarian states during the <i>in vitro</i> fertilization and embryo transfer program. Biomed Rep. 2015;3(2):215-219.

[5] Liang Y, Du HL, Chang XF,et al.[Effect of bushen tiaojing recipe on the quality of the oocytes and reproductive hormones in the follicular fluid in IVF-ET patients].Zhongguo Zhong Xi Yi Jie He Za Zhi. 2014;34(8):911-916.

[6] Simopoulou M, Nikolopoulou E, Dimakakos A,et al. Cell adhesion molecules and in vitro fertilization.In Vivo. 2014; 28(5):683-690.

[7] Song CM, Yang HM, Lu J,et al. [Effect of Bushen Tiaojing Recipe containing serum on FSH/cAMP-PKA pathway in in vitro cultured human ovarian granular cells].Zhongguo Zhong Xi Yi Jie He Za Zhi. 2014;34(3):317-323.

[8] Gao X, Chang X, Du H,et al.Effect of soothing liver therapy on oocyte quality and growth differentiation factor-9 in patients undergoing in vitro fertilization and embryo transfer.J Tradit Chin Med. 2013;33(5):597-602.

[9] Mehta BN, Chimote MN, Chimote NN, et al. Follicular-fluid anti-Mullerian hormone (FF AMH) is a plausible biochemical indicator of functional viability of oocyte in conventional in vitro fertilization (IVF) cycles.J Hum Reprod Sci.2013;6(2): 99-105.

[10] Jin HX, Xin ZM, Song WY,et al.Effects of human cumulus cells on in vitro fertilization outcomes and its significance in short-term insemination.J Reprod Med. 2013;58(1-2):51-54.

[11] Karuputhula NB, Chattopadhyay R, Chakravarty B,et al. Oxidative status in granulosa cells of infertile women undergoing IVF.Syst Biol Reprod Med. 2013;59(2):91-98.

[12] Havelock JC, Rainey WE, Carr BR. Ovarian granulose cell lines. Mol Cell Endocrinol.2004;228(1): 67-78.

[13] 叶婧,丁婷,杜小芳,等.不同分离方法对人卵巢颗粒细胞体外培养的影响 [J].中国现代医学杂志,2013,23(17):1-5.

[14] Sutton ML, Gilchrist RB, Thompson JG. Effects of in-vivo and in-vitro environments on the metabolism of the cumulus-oocyte complex and its influence on oocyte developmental capacity. Hum Reprod Update. 2003;9(1): 35-48.

[15] Liu JY, Qian Y, Mao YD, et al. In vitro maturation, fertilization and embryo transfer of human immature oocyte. Zhonghua Fu Chan Ke Za Zhi.2003 ;38(4):230-232.

[16] Somigliana E, Viganò P, La Sala GB,et al. Follicular fluid as a favourable environment for endometrial and endometriotic cell growth in vitro. Hum Reprod.2001; 16(6):1076-1080.

[17] Malamitsi-Puchner A, Sarandakou A, Baka SG, et al. Concentrations of angiogenic factors in follicular fluid and oocyte-cumulus complex culture medium from women undergoing in vitro fertilization: association with oocyte maturity and fertilization. Fertil Steril.2001;76(1):98-101.