P–452 The challenge of ovarian tissue culture: 2D versus 3D
Authors: Almeid. Santos A T , Pais A S , Reis S , Laranjo M , Caramelo F , Silva F , Botelho F 
Published: August 2021

Abstract

Study question
Does an alginate matrix scaffold improve ovarian tissue culture?

Summary answer
Ovarian tissue culture within an alginate scaffold has no advantage over conventional culture, being more time consuming and less reproductible

What is known already
Cryopreservation of ovarian tissue is a powerful technique for preserving female fertility, as it can restore fertility and endocrine function. Several studies have been carried out aiming to increase the longevity of the transplant and decrease the risk of reimplantation of neoplastic cells. For in vitro follicle culture, recent research has shifted from two dimensional (2D) toward the use of three-dimensional (3D) structures. The use of a matrix maintains the architecture and mimics in vivo conditions, with a variable access to oxygen and nutrients. This bridges the gap between conventional cell culture and animal models.

Study design, size, duration
Ovarian tissue fragments were divided into 2 groups: conventional culture (2D culture) and culture using an alginate matrix scaffold (3D culture). Tissue was evaluated at four time-points: immediately after thawing and after 24, 48 and 72 hours of culture.

Participants/materials, setting, methods
Rat ovarian tissue was cryopreserved and thawed with validated protocols. Follicular analysis was conducted after haematoxylin and eosin staining, regarding density, classification and degeneration. Tissue viability was assessed using lactate dehydrogenase (LDH) levels in supernatants and histological score. Three parameters were considered, namely, interstitial oedema, follicular cell degeneration and percentage of tissue in necrosis. Apoptosis was assessed by caspase 3 immunostaining. Proliferating cells were identified using Ki67 immunohistochemical labelling.

Main results and the role of chance
Follicular density, cell proliferation and apoptosis both in follicles and stroma was similar in both culture conditions. Stromal cells proliferation was stable in conventional culture but decreased in 3D culture (p = 0.001), which can be explained by the rigidity of alginate matrix. At 24 hours of culture, cytotoxicity was lower in the 3D model (p = 0.006), due to low levels of LDH in the supernatant, that may be related to retention within the matrix. As culture time increased cytotoxicity seemed to be similar. Degradation of the tissue was suggested by the histological score analysis of tissue during 72 hours of culture. Tissue injury was greater (p = 0.01) in 3D culture due to higher interstitial oedema (p = 0.017) and tissue necrosis (p = 0.035). In the interior of the alginate scaffold, the bioavailability of oxygen and nutrients may be limited, affecting cell survival over time and conditioning higher level of necrosis and release of intracellular content.

Limitations, reasons for caution
There are two major limitations that should be addressed in future research, namely the study of the tissue-matrix interactions and culture medium supplementation to decrease follicular atresia.
Wider implications of the findings: There is no advantage in the use of an alginate matrix scaffold for ovarian tissue culture, as it is more time consuming, difficult to perform and less reproductible.

Trial registration number
Not applicable