Culture of human ovarian tissue in xeno-free conditions using laminin components of the human ovarian extracellular matr
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Culture of human ovarian tissue in xeno-free conditions using laminin components of the human ovarian extracellular matrix J. Hao 1,2,3 & A. R. Tuck 2,4 & C. R. Prakash 2 & A. Damdimopoulos 5 & M. O. D. Sjödin 4,6 & J. Lindberg 4,6 & B. Niklasson 2,7 & K. Pettersson 2 & O. Hovatta 2 & P. Damdimopoulou 2,4 Received: 27 April 2020 / Accepted: 8 July 2020 # Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract Purpose Our purpose was to identify human ovarian extracellular matrix (ECM) components that would support in vitro culture of human ovarian tissue and be compatible with possible future clinical applications. We characterized ovarian expression of laminins and selected three laminin tripeptides for culture experiments to be compared with Matrigel, an undefined and animalbased mixture of ECM components. Methods Expression of the 12 laminin genes was determined on transcript and protein levels using cortical tissue samples (n = 6), commercial ovary RNA (n = 1), follicular fluid granulosa cells (n = 20), and single-cell RNA-sequencing data. Laminin 221 (LN221), LN521, LN511, and their mixture were chosen for a 7-day culture experiment along with Matrigel using tissue from 17 patients. At the end of the culture, follicles were evaluated by scoring and counting from serial tissue sections, apoptosis measured using in situ TUNEL assay, proliferation by Ki67 staining, and endocrine function by quantifying steroids in culture media using UPLC-MS/MS. Results Approximately half of the cells in ovarian cortex expressed at least one laminin gene. The overall most expressed laminin α-chains were LAMA2 and LAMA5, β-chains LAMB1 and LAMB2, and γ-chain LAMC1. In culture experiments, LN221 enhanced follicular survival compared with Matrigel (p < 0.001), whereas tissue cultured on LN521 had higher proportion of secondary follicles (p < 0.001). LN511 and mixture of laminins did not support the cultures leading to lower follicle densities and higher apoptosis. All cultures produced steroids and contained proliferating cells. Conclusions LN221 and LN521 show promise in providing xeno-free growth substrates for human ovarian tissue cultures, which may help in further development of folliculogenesis in vitro for clinical practices. The system could also be used for identification of adverse effects of chemicals in ovaries. Keywords Human ovary . Tissue culture . Laminins . Xeno-free . Matrigel
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10815-020-01886-4) contains supplementary material, which is available to authorized users. * J. Hao [email protected] 1
Department of Reproductive Medicine, Xiangya Hospital, Central South University, Changsha 410008, Hunan, People’s Republic of China
2
Division of Obstetrics and Gynecology, Department of Clinical Science, Intervention and Technology, Karolinska Institutet and Karolinska University Hospital, 14186 Stockholm, Sweden
3
Present address: Clinical Research Center for Women’s Reproductive
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