Decellularization and recellularization of the ovary for bioengineering applications; studies in the mouse

Abstract

Background Fertility preservation is particularly challenging in young women diagnosed with hematopoietic cancers, as transplantation of cryopreserved ovarian cortex in these women carries the risk for re-introducing cancer cells. Therefore, the construction of a bioengineered ovary that can accommodate isolated small follicles was proposed as an alternative to minimize the risk of malignancy transmission. Various options for viable bioengineered scaffolds have been reported in the literature. Previously, we reported three protocols for producing mouse ovarian scaffolds with the decellularization technique. The present study examined these scaffolds further, specifically with regards to their extracellular composition, biocompatibility and ability to support recellularization with mesenchymal stem cells. Material and methods Three decellularization protocols based on 0.5% sodium dodecyl sulfate (Protocol 1; P1), or 2% sodium deoxycholate (P2), or a combination of the two detergents (P3) were applied to produce three types of scaffolds. The levels of collagen, elastin and sulfated glycosaminoglycans (sGAGs) were quantified in the remaining extracellular matrix. Detailed immunofluorescence and scanning electron microscopy imaging were conducted to assess the morphology and recellularization efficiency of the constructs after 14 days in vitro utilizing red fluorescent protein-labelled mesenchymal stem cells. Results All protocols efficiently removed the DNA while the elastin content was not significantly reduced during the procedures. The SDS-protocol (P1) reduced the sGAG and the collagen content more than the SDC-protocol (P2). All scaffolds were biocompatible and recellularization was successful, particularly in several P2-derived scaffolds. The cells were extensively distributed throughout the constructs, with a denser distribution observed towards the ovarian cortex. The cell density was not significantly different (400 to 550 cells/mm 2 ) between scaffold types. However, there was a tendency towards a higher cell density in the SDC-derived constructs. Scanning electron microscope images showed fibrous scaffolds with a dense repopulated surface structure. Conclusions While there were differences in the key structural macromolecules between protocols, all scaffolds were biocompatible and showed effective recellularization. The results indicate that our SDC-protocol might be better than our SDS-protocol. However, additional studies are necessary to determine their suitability for attachment of small follicles and folliculogenesis.

Induced Pluripotent Stem Cells from Animal Models: Applications on Translational Research

“…Organ engineering based on recellularization with patient-derived iPSCs offers the unique potential to promote autologous treatment, and are also promising as tools for animal production, once piPSCs differentiated into germinative cells could be used to re-colonize depleted ovaries or testicles in order to spread the desired genetics in other animals [112]. Also, the use of muscle differentiation from iPSCs in scaffolds would benefit not only the cellular therapy for injured muscles in general, but opens new possibilities regarding in vitro meat production.…”

A Review on Biomaterials for Ovarian Tissue Engineering

“…Recently, Alshaikh et al [170] investigated the decellularization of mouse ovarian tissue 431 using two different types of detergents: 0.5% sodium dodecyl sulfate and 2% sodium 432 deoxycholate. While both protocols showed high biocompatibility, the latter produced DECM 433 that appeared to be slightly more advantageous due to a higher recellularization efficiency.…”

Novel Decellularization Method for Tissue Slices

“…In fact, to assess the quality of the resulting scaffold it is required to analyze the effects of the decellularizing agent on the ECM components. At times, only components of the interstitial ECM are analyzed, like collagen type I and elastin ( O’Neill et al, 2013a ; Balestrini et al, 2015 ; Alshaikh et al, 2020 ). However, the evaluation of the basement membrane is of paramount importance especially for tissue engineering and cell culture applications since these components heavily influence cell adhesion and differentiation ( Rasmussen and Karsdal 2016 ).…”

“…Additionally, most of the available methods fail in the preservation of some ECM components. For instance, significant decreases in elastin ( Maghsoudlou et al, 2013 ; Alshaikh et al, 2020 ), collagen ( Hill et al, 2015 ; Alshaikh et al, 2020 ), glycosaminoglycans ( Mendoza-Novelo et al, 2011 ; Lü et al, 2014 ), laminins ( Hill et al, 2015 ; Wüthrich et al, 2020 ) and proteoglycans ( Hill et al, 2015 ; Calle et al, 2016 ) have been described. In addition to these limitations, the current protocols are still not particularly flexible or accessible.…”