Creating a semi-opened micro-cavity ovary through sacrificial microspheres as an in vitro model for discovering the potential effect of ovarian toxic agents

The bio-engineered ovary is an essential technology for treating female infertility.Especially the development of relevant in vitro models could be a critical step in a drug study.Herein,we develop a semi-opened culturing system(SOCS)strategy that maintains a 3D structure of follicles during the culture.Based on the SOCS,we further developed micro-cavity ovary(MCO)with mouse follicles by the microsphere-templated technique,where sacrificial gelatin microspheres were mixed with photo-crosslinkable gelatin methacryloyl(GelMA)to engineer a micro-cavity niche for follicle growth.The semi-opened MCO could support the follicle growing to the antral stage,secreting hormones,and ovulating cumulus-oocyte complex out of the MCO without extra manipulation.The MCO-ovulated oocyte exhibits a highly similar transcriptome to the in vivo counterpart(correlation of 0.97)and can be fertilized.Moreover,we found that a high ROS level could affect the cumulus expansion,which may result in anovulation disorder.The damage could be rescued by melatonin,but the end of cumulus expansion was 3h earlier than anticipation,validating that MCO has the potential for investigating ovarian toxic agents in vitro.We provide a novel approach for building an in vitro ovarian model to recapitulate ovarian functions and test chemical toxicity,suggesting it has the potential for clinical research in the future.

Coaxial Embedded Printing of Gelatin Methacryloyl-alginate Double Network Hydrogel for Multilayer Vascular Tubes

The reconstruction of vascular-like tissues exhibiting a typical three-layer structure in vitro is vital to bio-fabrication research.It enables the realization of more complicated micro-environments,such as myocardium,liver,and tumor,which enables us to investigate their specific physiological phenomena or pathological mecha-nisms.Herein,we propose a coaxial embedded printing method,where the gelatin methacrylate(GelMA)-alginate composite hydrogel and sacrificial materials are extruded from a coaxial nozzle into a cylinder mold.By applying this method,we achieve the rapid fabrication of multilayer tube structures with inner diameters ranging from 400 to 1000μm.In addition,myoblasts are encapsulated in the hydrogel,and the cells show high viability.Moreover,we encapsulate smooth muscle cells(SMCs)and the human umbilical vein endothelial cells-T1(HUVEC-T1)cell line in the hydrogel to form vascular-like tissues,and the cells exhibit good morphology and protein expression.These results suggest that a vascular tube fabricated using the proposed method can serve as a vascular model for in vitro studies.