production-use-customizable-agarose-molds-for-scaffold-free-mouse

Production and Use of Customizable Agarose Molds for Scaffold-Free Mouse Ovarian Follicle Culture

The ovarian follicle is the functional unit of the ovary that produces hormones and gametes needed to sustain female reproductive function and health. The ...

Northwestern University

Since the beginning of clinical medicine, the human uterus has held the fascination of clinicians and researchers, given its critical role in the reproduction of our species. The endometrial lining provides residence for the embryo; however, this symbiotic interaction can be disrupted if the timing is not correct and the endometrium is not receptive. Diseases associated with the endometrium interfere with the reproductive process and cause a life-altering burden of pain and even death. With the advancement of technologies and new insights into the biology of the endometrium, much has been uncovered about the dynamic and essential changes that need to occur for normal endometrial function, as well as aberrations that lead to endometrial diseases. As expected, the more that is uncovered, the more the complexity of the endometrium is made evident. In this study, we bring together three areas of scientific advancement that remain in their infancy, but which together have the potential to mirror this complexity and enable understanding. Studies on induced pluripotent stem cells, three-dimensional tissue mimics, and microfluidic culture platforms will be reviewed with a focus on the endometrium. These unconventional approaches will provide new perspectives and appreciation for the elegance and complexity of the endometrium. Impact statement The ability of the human endometrium to regenerate on a monthly basis for ∼4 decades of reproductive years exemplifies its complexity as well as its susceptibility to disease. Restrictions on the types of research that can be done in the human endometrium motivate the development of new technologies and model systems. The three areas of technological advancement reviewed here-induced pluripotent stem cells, three-dimensional model systems, and microfluidic culture systems-will highlight some of the tools that can be applied to studying the human endometrium in ways that have not been done before.

Microphysiological Modeling of the Human Endometrium.

The oviducts (fallopian tubes in mammals) function as the site of fertilization and provide necessary support for early embryonic development, mainly via embryonic exposure to the tubal microenvironment. The main objective of this study was to create an oviduct-specific extracellular matrix (oviECM) hydrogel rich in bioactive components that mimics the native environment, thus optimizing the developmental trajectories of cultured embryos. Rabbit oviducts were decellularized through SDS treatment and enzymatic digestion, and the acellular tissue was converted into oviductal pre-gel extracellular matrix (ECM) solutions. Incubation of these solutions at 37 °C resulted in stable hydrogels with a fibrous structure based on scanning electron microscopy. Histological staining, DNA quantification and colorimetric assays confirmed that the decellularized tissue and hydrogels contained no cellular or nuclear components but retained important components of the ECM, e.g. hyaluronic acid, glycoproteins and collagens. To evaluate the ability of oviECM hydrogels to maintain early embryonic development, two-cell rabbit embryos were cultured on oviECM-coated surfaces and compared to those cultured with standard techniques. Embryo development was similar in both conditions, with 95.9% and 98% of the embryos reaching the late morula/early blastocyst stage by 48 h under standard culture and oviECM conditions, respectively. Metabolomic analysis of culture media in the presence or absence of embryos, however, revealed that the oviECM coating may include signalling molecules and release compounds beneficial to embryo metabolism.

Development of Decellularized Oviductal Hydrogels as a Support for Rabbit Embryo Culture.

Organoids are three-dimensional (3D) multicellular tissue models that mimic their corresponding in vivo tissue. Successful efforts have derived organoids from primary tissues such as intestine, liver, and pancreas. For human uterine endometrium, the recent generation of 3D structures from primary endometrial cells is inspiring new studies of this important tissue using precise preclinical models. To improve on these 3D models, we decellularized pig endometrium containing tissue-specific extracellular matrix and generated a hydrogel (EndoECM). Next, we derived three lines of human endometrial organoids and cultured them in optimal and suboptimal culture expansion media with or without EndoECM (0.01 mg/mL) as a soluble additive. We characterized the resultant organoids to verify their epithelial origin, long-term chromosomal stability, and stemness properties. Lastly, we determined their proliferation potential under different culture conditions using proliferation rates and immunohistochemical methods. Our results demonstrate the importance of a bioactive environment for the maintenance and proliferation of human endometrial organoids.

Improved Models of Human Endometrial Organoids Based on Hydrogels from Decellularized Endometrium.

Each month during a woman's reproductive years, the endometrium undergoes vast changes to prepare for a potential pregnancy. Diseases of the endometrium arise for numerous reasons, many of which remain unknown. These endometrial diseases, including endometriosis, adenomyosis, endometrial cancer and Asherman syndrome, affect many women, with an overall lack of efficient or permanent treatment solutions. The challenge lies in understanding the complexity of the endometrium and the extensive changes, orchestrated by ovarian hormones, that occur in multiple cell types over the period of the menstrual cycle. Appropriate model systems that closely mimic the architecture and function of the endometrium and its diseases are needed. The emergence of organoid technology using human cells is enabling a revolution in modelling the endometrium in vitro. The goal of this Review is to provide a focused reference for new models to study the diseases of the endometrium. We provide perspectives on the power of new and emerging models, from organoids to microfluidics, which have opened up a new frontier for studying endometrial diseases.

Strategies for modelling endometrial diseases.

Research aimed at preserving female fertility is increasingly using bioengineering techniques to develop new platforms capable of supporting ovarian cell function in vitro and in vivo. Natural hydrogels (alginate, collagen, and fibrin) have been the most exploited approaches; however they are biologically inert and/or biochemically simple. Thus, establishing a suitable biomimetic hydrogel from decellularized ovarian cortex (OC) extracellular matrix (OvaECM) could provide a complex native biomaterial for follicle development and oocyte maturation. The objectives of this work were (i) to establish an optimal protocol to decellularize and solubilize bovine OC, (ii) to characterize the histological, molecular, ultrastructural, and proteomic properties of the resulting tissue and hydrogel, and (iii) to assess its biocompatibility and adequacy for murine in vitro follicle growth (IVFG). Sodium dodecyl sulfate was identified as the best detergent to develop bovine OvaECM hydrogels. Hydrogels added into standard media or used as plate coatings were employed for IVFG and oocyte maturation. Follicle growth, survival, hormone production, and oocyte maturation and developmental competence were evaluated. OvaECM hydrogel-supplemented media best supported follicle survival, expansion, and hormone production, while the coatings provided more mature and competent oocytes. Overall, the findings support the xenogeneic use of OvaECM hydrogels for future human female reproductive bioengineering.

Advances of xenogeneic ovarian extracellular matrix hydrogels for in vitro follicle development and oocyte maturation.

To accurately phenocopy human biology , researchers have been reducing their dependence on standard, static two-dimensional (2D) cultures and instead are moving towards three-dimensional (3D) and/or multicellular culture techniques. While these culture innovations are becoming more commonplace, there is a growing body of research that illustrates the benefits and even necessity of recapitulating the dynamic flow of nutrients, gas, waste exchange and tissue interactions that occur . However, cost and engineering complexity are two main factors that hinder the adoption of these technologies and incorporation into standard laboratory workflows. We developed LATTICE, a plug-and-play microfluidic platform able to house up to eight large tissue or organ models that can be cultured individually or in an interconnected fashion. The functionality of the platform to model both healthy and diseased tissue states was demonstrated using 3D cultures of reproductive tissues including murine ovarian tissues and human fallopian tube explants (hFTE). When exogenously exposed to pathological doses of gonadotropins and androgens to mimic the endocrinology of polycystic ovarian syndrome (PCOS), subsequent ovarian follicle development, hormone production and ovulation copied key features of this endocrinopathy. Further, hFTE cilia beating decreased significantly only when experiencing continuous media exchanges. We were then able to endogenously recreate this phenotype on the platform by dynamically co-culturing the PCOS ovary and hFTE. LATTICE was designed to be customizable with flexibility in 3D culture formats and can serve as a powerful automated tool to enable the study of tissue and cellular dynamics in health and disease in all fields of research.

A new tissue-agnostic microfluidic device to model physiology and disease: the lattice platform.

Ex vivo follicle growth is an essential tool, enabling interrogation of folliculogenesis, ovulation, and luteinization. Though significant advancements have been made, existing follicle culture strategies can be technically challenging and laborious. In this study, we advanced the field through development of a custom agarose micromold, which enables scaffold-free follicle culture. We established an accessible and economical manufacturing method using 3D printing and silicone molding that generates biocompatible hydrogel molds without the risk of cytotoxicity from leachates. Each mold supports simultaneous culture of multiple multilayer secondary follicles in a single focal plane, allowing for constant timelapse monitoring and automated analysis. Mouse follicles cultured using this novel system exhibit significantly improved growth and ovulation outcomes with comparable survival, oocyte maturation, and hormone production profiles as established three-dimensional encapsulated in vitro follicle growth (eIVFG) systems. Additionally, follicles recapitulated aspects of in vivo ovulation physiology with respect to their architecture and spatial polarization, which has not been observed in eIVFG systems. This system offers simplicity, scalability, integration with morphokinetic analyses of follicle growth and ovulation, and compatibility with existing microphysiological platforms. This culture strategy has implications for fundamental follicle biology, fertility preservation strategies, reproductive toxicology, and contraceptive drug discovery.

Article	Year
Microphysiological Modeling of the Human Endometrium. Tissue engineering. Part A\| PubMed ID: 32348708	2020
Development of Decellularized Oviductal Hydrogels as a Support for Rabbit Embryo Culture. Reproductive sciences (Thousand Oaks, Calif.)\| PubMed ID: 33511539	2021
Improved Models of Human Endometrial Organoids Based on Hydrogels from Decellularized Endometrium. Journal of personalized medicine\| PubMed ID: 34205034	2021
Strategies for modelling endometrial diseases. Nature reviews. Endocrinology\| PubMed ID: 36050476	2022
Advances of xenogeneic ovarian extracellular matrix hydrogels for in vitro follicle development and oocyte maturation. Biomaterials advances\| PubMed ID: 37267748	2023
A new tissue-agnostic microfluidic device to model physiology and disease: the lattice platform. Lab on a chip\| PubMed ID: 37846545	2023
Three-Dimensionally Printed Agarose Micromold Supports Scaffold-Free Mouse Ex Vivo Follicle Growth, Ovulation, and Luteinization. Bioengineering (Basel, Switzerland)\| PubMed ID: 39061801	2024

Hannes Campo

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