Dynamic in vitro culture of bovine and human ovarian tissue enhances follicle progression and health

Abstract

In vitro ovarian cortical tissue culture, followed by culture of isolated secondary follicles, is a promising future option for production of mature oocytes. Although efforts have been made to improve the culture outcome by changing the medium composition, so far, most studies used static culture systems. Here we describe the outcome of 7 days cultures of bovine and human ovarian cortical tissue in a dynamic system using a novel perifusion bioreactor in comparison to static culture in conventional and/or gas permeable dishes. Findings show that dynamic culture significantly improves follicle quality and viability, percentage and health of secondary follicles, overall tissue health, and steroid secretion in both species. Model predictions suggest that such amelioration can be mediated by an enhanced oxygen availability and/or by fluid-mechanical shear stresses and solid compressive strains exerted on the tissue.

Enhanced solute transport and steady mechanical stimulation in a novel dynamic perifusion bioreactor increase the efficiency of the in vitro culture of ovarian cortical tissue strips

“…Some materials and methods used for this study are very similar to those used in other studies published by our group (Talevi et al, 2018;Barbato et al, 2023), and their description may occasionally reproduce verbatim that has been previously reported.…”

“…Such deformation depends on the strip thickness and the viscoelastic behaviour of the tested material. To minimize possible bias, a tissue cutter was developed that reliably delivers tissue strips of 600-μm thickness ( Barbato et al, 2023 ). Accounting for the viscoelastic behaviour of the tissue and nets would have required a thorough characterization of the ovarian tissue's and nets' mechanical behaviour and the development and validation of a suitable mechanical model of the bioreactor set-up, which is beyond the scope of this work.…”

“…Cortical tissue slices were reliably dissected from the bovine ovaries with a custom-made tissue slicer and were soaked in handling medium (i.e., Leibovitz's L-15 medium supplemented with 2 mM glutamine, 3 mg/mL BSA, 1% Pen-Strep, and 1 μg/mL amphotericin B) at T room , discarding zones containing antral follicles. Then, 1 mm × 1 mm large, 0.6 mm, i.e., 588 ± 56-μm-thick ( Barbato et al, 2023 ) cortical tissue strips were reproducibly prepared with a tissue chopper (Mcilwain, Mickle Laboratory Engineering Company, Ltd., Surrey, UK). Then, the strips were rinsed twice in fresh handling medium and were used for the culture experiments.…”

Thiol-yne click crosslink hyaluronic acid/chitosan hydrogel for three-dimensional in vitro follicle development

“…While it is possible to support the growth of primordial follicles using this approach, it inhibits the development of follicles beyond the pre-antral stage. Although most studies have been conducted growing follicles after the secondary follicle stage, only a limited number of studies have explored the culture of primordial follicles within ovarian cortex tissue [ [86] , [87] , [88] , [89] ].…”

Silk-Ovarioids: Establishment and characterization of human ovarian primary cells 3D-model system

“…Obtaining in vitro matured metaphase II oocytes from in vitro grown follicles remains a challenge for both basic research and clinical implementation in ART programs (Hu et al, 2023;Barbato et al, 2023;Hao et al, 2020). In fact, in vitro follicle growth methodology has been developed mainly using animal models, and its application is still limited due to many drawbacks.…”