Frontiers in endocrinology · 2025
Assessment of molecular and morphological dynamics during long-time in vitro cultivation of cryopreserved human ovarian tissue: risk of genetic alterations.
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Wang Wanxue, Todorov Plamen, Isachenko Evgenia, Rahimi Gohar, Merzenich Markus, Mallmann-Gottschalk Nina, Zhou Yang, Yao Jilong, Li Xuemei, Isachenko Volodimir
Аннотация
BACKGROUND: Cryopreservation of human ovarian tissue is a technology for patients undergoing aggressive anticancer treatments. This technology includes the following stages: saturation by permeable cryoprotectants, freezing, thawing, removal of cryoprotectants, as well as tissues in vitro or in situ culture.
OBJECTIVE: Evaluation of quality of tissue after cryopreservation and in vitro culture with the aim of detection of genetic and molecular changes in cells.
METHODS: Ovarian tissue was frozen in 6% ethylene glycol and 6% dimethyl sulfoxide with speed of cooling 0.3°C/min and thawed at 100°C. After removal of cryoprotectants tissue fragments were in vitro cultured with the soluble extract of basement membrane protein (Matrigel) 3-D culture system for 7 days. Morphological and functional assessments were conducted using microscopic observation and RNA-Sequencing. Comparative analysis of tissue morphology before and after culture was performed with bioinformatics for gene expression and variant analysis, including functional annotation and study of protein-protein interaction.
RESULTS: DNA and RNA analyses after cultivation indicated a rise in gene fusion and alternative splicing events, potentially affecting gene expression and cellular functions.
CONCLUSION: Long-time in vitro culture of human ovarian tissue results in substantial changes in its morphology and genetic alteration.
OBJECTIVE: Evaluation of quality of tissue after cryopreservation and in vitro culture with the aim of detection of genetic and molecular changes in cells.
METHODS: Ovarian tissue was frozen in 6% ethylene glycol and 6% dimethyl sulfoxide with speed of cooling 0.3°C/min and thawed at 100°C. After removal of cryoprotectants tissue fragments were in vitro cultured with the soluble extract of basement membrane protein (Matrigel) 3-D culture system for 7 days. Morphological and functional assessments were conducted using microscopic observation and RNA-Sequencing. Comparative analysis of tissue morphology before and after culture was performed with bioinformatics for gene expression and variant analysis, including functional annotation and study of protein-protein interaction.
RESULTS: DNA and RNA analyses after cultivation indicated a rise in gene fusion and alternative splicing events, potentially affecting gene expression and cellular functions.
CONCLUSION: Long-time in vitro culture of human ovarian tissue results in substantial changes in its morphology and genetic alteration.