Dataset Information

Dynamic in vitro culture of cryopreserved-thawed human ovarian cortical tissue using a microfluidics platform does not improve early folliculogenesis.

ABSTRACT: Current strategies for fertility preservation include the cryopreservation of embryos, mature oocytes or ovarian cortical tissue for autologous transplantation. However, not all patients that could benefit from fertility preservation can use the currently available technology. In this regard, obtaining functional mature oocytes from ovarian cortical tissue in vitro would represent a major breakthrough in fertility preservation as well as in human medically assisted reproduction. In this study, we have used a microfluidics platform to culture cryopreserved-thawed human cortical tissue for a period of 8 days and evaluated the effect of two different flow rates in follicular activation and growth. The results showed that this dynamic system supported follicular development up to the secondary stage within 8 days, albeit with low efficiency. Surprisingly, the stromal cells in the ovarian cortical tissue were highly sensitive to flow and showed high levels of apoptosis when cultured under high flow rate. Moreover, after 8 days in culture, the stromal compartment showed increase levels of collagen deposition, in particular in static culture. Although microfluidics dynamic platforms have great potential to simulate tissue-level physiology, this system still needs optimization to meet the requirements for an efficient in vitro early follicular growth.

SUBMITTER: Del Valle JS

PROVIDER: S-EPMC9372461 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Dynamic <i>in vitro</i> culture of cryopreserved-thawed human ovarian cortical tissue using a microfluidics platform does not improve early folliculogenesis.

Del Valle Julieta S JS Mancini Vanessa V Laverde Garay Maitane M Asseler Joyce D JD Fan Xueying X Metzemaekers Jeroen J Louwe Leoni A LA Pilgram Gonneke S K GSK van der Westerlaken Lucette A J LAJ van Mello Norah M NM Chuva de Sousa Lopes Susana M SM

Frontiers in endocrinology 20220729

Current strategies for fertility preservation include the cryopreservation of embryos, mature oocytes or ovarian cortical tissue for autologous transplantation. However, not all patients that could benefit from fertility preservation can use the currently available technology. In this regard, obtaining functional mature oocytes from ovarian cortical tissue <i>in vitro</i> would represent a major breakthrough in fertility preservation as well as in human medically assisted reproduction. In this s ...[more]

PMID: 35966050

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Project description:The use of biomarkers to infer drug response in patients is being actively pursued, yet significant challenges with this approach, including the complicated interconnection of pathways, have limited its application. Direct empirical testing of tumor sensitivity would arguably provide a more reliable predictive value, although it has garnered little attention largely due to the technical difficulties associated with this approach. We hypothesize that the application of recently developed microtechnologies, coupled to more complex 3-dimensional cell cultures, could provide a model to address some of these issues. As a proof of concept, we developed a microfluidic device where spheroids of the serous epithelial ovarian cancer cell line TOV112D are entrapped and assayed for their chemoresponse to carboplatin and paclitaxel, two therapeutic agents routinely used for the treatment of ovarian cancer. In order to index the chemoresponse, we analyzed the spatiotemporal evolution of the mortality fraction, as judged by vital dyes and confocal microscopy, within spheroids subjected to different drug concentrations and treatment durations inside the microfluidic device. To reflect microenvironment effects, we tested the effect of exogenous extracellular matrix and serum supplementation during spheroid formation on their chemotherapeutic response. Spheroids displayed augmented chemoresistance in comparison to monolayer culturing. This resistance was further increased by the simultaneous presence of both extracellular matrix and high serum concentration during spheroid formation. Following exposure to chemotherapeutics, cell death profiles were not uniform throughout the spheroid. The highest cell death fraction was found at the center of the spheroid and the lowest at the periphery. Collectively, the results demonstrate the validity of the approach, and provide the basis for further investigation of chemotherapeutic responses in ovarian cancer using microfluidics technology. In the future, such microdevices could provide the framework to assay drug sensitivity in a timeframe suitable for clinical decision making.

Project description:BackgroundOvarian tissue cryopreservation and transplantation are novel therapeutic approaches for fertility preservation. However, follicle loss caused by ischemic and hypoxic damage is one of the issues after frozen-thawed ovarian tissue transplantation. Promoting angiogenesis in grafts is the key to restore cryopreserved ovarian function. Mesenchymal stem cells (MSCs) have been reported to facilitate angiogenesis in the cryopreserved ovarian tissue transplantation. However, the risk of embolization, immunogenic effect and tumorigenesis hinders the clinical application of MSCs to human organ transplantation. In this study, we established an in vitro ovarian culture system to restore frozen-thawed ovarian function before transplantation with the application of umbilical cord mesenchymal stem cells (UC-MSCs), and explored the effects of UC-MSCs on frozen-thawed ovaries in vitro ovarian culture system and the mechanisms of UC-MSCs on the angiogenesis of frozen-thawed ovaries.MethodsA simple in vitro three dimensional (3D) ovarian culture system using Matrigel was established to support to an ideal niche, and ovary was alone cultured in the 24-well plate as a control. We also evaluated the effects of UC-MSCs treatment on ovarian function with or without Matrigel support. All thawed ovaries were randomly divided into control group (Matrigel-/UC-MSCs-), Matrigel group (Matrigel+/UC-MSCs-), UC-MSCs group (Matrigel-/UC-MSCs+) and UC-MSCs + Matrigel group (Matrigel+/UC-MSCs+). HE staining was used to detect the histological structure of follicles and TUNEL staining was used to detect cell apoptosis. The number of microvessels was counted to evaluate neovascularization. The mRNA expression of VEGFA, IGF1 and ANGPT2 were detected by RT-PCR. Western blotting was used to measure the expression of GSK-3β, β-catenin and p-β-catenin.ResultsIn the absence of UC-MSCs, 3D culture system supported by Matrigel showed significantly improved follicular development and microvascular number. Additionally, UC-MSCs were also found to effectively improve follicular development and microvascular number regardless of the culture condition used. However, alleviated follicular apoptosis, increased mRNA expression of angiogenesis-related gene and activated Wnt/β-catenin pathway occurred only in the UC-MSCs + Matrigel group. Besides, with the application of IWP-2 in UC-MSCs + Matrigel group, Wnt//β-catenin pathway could be blocked by IWP-2 serving as one of Wnt/β-catenin pathway inhibitors.ConclusionsThis in vitro study showed the beneficial effects of UC-MSCs on thawed ovaries and explored a potential mechanism inducing angiogenesis. In particular, 3D ovarian culture system supported by Matrigel further improved UC-MSCs treatment. The in vitro culture system using Matrigel and UC-MSCs may provide a potential treatment strategy for improving the success rate of thawed ovaries transplantation.

Dataset Information

Dynamic in vitro culture of cryopreserved-thawed human ovarian cortical tissue using a microfluidics platform does not improve early folliculogenesis.

Publications

Dynamic <i>in vitro</i> culture of cryopreserved-thawed human ovarian cortical tissue using a microfluidics platform does not improve early folliculogenesis.

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