Semipermeable cell culture membranes are commonly used in multilayered microfluidic devices to mimic the basement membrane in vivo and to create compartmentalized microenvironments for physiological cell growth and differentiation. However, existing membranes are predominantly made up of synthetic polymers, providing limited capacity to replicate cellular interactions with native extracellular matrices that play a crucial role in the induction of physiological phenotypes. Here we describe a new type of cell culture membranes engineered from native extracellular matrix (ECM) materials that are thin, semipermeable, optically transparent, and amenable to integration into microfluidic cell culture devices. Facile and cost-effective fabrication of these membranes was achieved by controlled sequential steps of vitrification that transformed three-dimensional (3D) ECM hydrogels into structurally stable thin films. By modulating the composition of the ECM, our technique provided a means to tune key membrane properties such as optical transparency, stiffness, and porosity. For microfluidic cell culture, we constructed a multilayered microdevice consisting of two parallel chambers separated by a thin membrane insert derived from different types of ECM. This study showed that our ECM membranes supported attachment and growth of various types of cells (epithelial, endothelial, and mesenchymal cells) under perfusion culture conditions. Our data also revealed the promotive effects of the membranes on adhesion-associated intracellular signaling that mediates cell–ECM interactions. Moreover, we demonstrated the use of these membranes for constructing compartmentalized microfluidic cell culture systems to induce physiological tissue differentiation or to replicate interfaces between different tissue types. Our approach provides a robust platform to produce and engineer biologically active cell culture substrates that serve as promising alternatives to conventional synthetic membrane inserts. This strategy may contribute to the development of physiologically relevant in vitro cell culture models for a wide range of applications.

A developmentally inspired tissue engineering method enables the construction of microphysiological musculoskeletal tissue models.

Xenohormesis proposes that phytochemicals produced to combat stressors in the host plant exert biochemical effects in animal cells lacking cognate receptors. Xenohormetic phytochemicals such as flavonoids and phytoalexins modulate a range of human cell signaling mechanisms but functional correlations with human pathophysiology are lacking. Here, potent inhibitory effects of grapefruit‐derived Naringenin (Nar) and soybean‐derived Glyceollins (Gly) in human microphysiological models of bulk tissue vasculogenesis and tumor angiogenesis are reported. Despite this interference of vascular morphogenesis, Nar and Gly are not cytotoxic to endothelial cells and do not prevent cell cycle entry. The anti‐vasculogenic effects of Glyceollin are significantly more potent in sex‐matched female (XX) models. Nar and Gly do not decrease viability or expression of proangiogenic genes in triple negative breast cancer (TNBC) cell spheroids, suggesting that inhibition of sprouting angiogenesis by Nar and Gly in a MPS model of the (TNBC) microenvironment are mediated via direct effects in endothelial cells. The study supports further research of Naringenin and Glyceollin as health‐promoting agents with special attention to mechanisms of action in vascular endothelial cells and the role of biological sex, which can improve the understanding of dietary nutrition and the pharmacology of phytochemical preparations.

Microphysiological systems (MPS) containing perfusable vascular beds unlock the ability to model tissue‐scale elements of vascular physiology and disease in vitro. Access to inexpensive stereolithography (SLA) 3D printers now enables benchtop fabrication of polydimethylsiloxane (PDMS) organ chips, eliminating the need for cleanroom access and microfabrication expertise, and can facilitate broader adoption of MPS approaches in preclinical research. Rapid prototyping of organ chip mold designs accelerates the processes of design, testing, and iteration, but geometric distortion and surface roughness of SLA resin prints can impede the development of standardizable manufacturing workflows. This study reports postprocessing procedures for manufacturing SLA‐printed molds that produce fully cured, flat, patently bonded, and optically clear polydimethyl siloxane (PDMS) organ chips. Injection loading tests were conducted to identify milliscale membrane‐free organ chip (MFOC) designs that allowed reproducible device loading by target end‐users, a key requirement for broad nonexpert adoption in preclinical research. The optimized milliscale MFOC design was used to develop tissue engineering protocols for (i) driving bulk tissue vasculogenesis in MFOC, and (ii) seeding the bulk tissue interfaces with a confluent endothelium to stimulate self‐assembly of perfusable anastomoses with the internal vasculature. Comparison of rocker‐ and pump‐based protocols for flow‐conditioning of anastomosed vascular beds revealed that continuous pump‐driven flow is required for reproducible barrier maturation throughout the 3D tissue bulk. Demonstrated applications include nanoparticle perfusion and engineering perfusable tumor vasculature. These easily adaptable methods for designing and fabricating vascularized microphysiological systems can accelerate their adoption in a diverse range of preclinical laboratory settings.

Purpose: Sex differences in cellular biology significantly influence cell responses in culture. Yet, the sex-specific effects of culture reagents such as fetal bovine serum (FBS) remain understudied. Increased adoption of cell-based models such as microphysiological systems (MPS) as replacements for animal models demands a greater understanding sex-specific responses to common media formulations. This study examined the effects of FBS and hormone-free charcoal-stripped serum (CSS) on male (XY) and female (XX) cells in 2D and 3D MPS culture models to demonstrate profound sex-specificity in bioassays and inform the development of future sex-specific cell culture protocols and methods. Methods: Primary human endothelial cells and fibroblasts from multiple organ sources were cultured in 2D and in 3D MPS models. Cells were cultured with either FBS or CSS. Endothelial specific gene expression, cytoskeletal spreading, and cell cycle status were analyzed in 2D culture. Vascular network formation, macromolecular leakage, and directional angiogenic sprouting were assessed in 3D MPS models. Results: FBS promoted significant upregulation of genes associated with endothelial function in XX endothelial cells, but the same gene clusters were downregulated in XY cells. FBS increased cytoskeletal spreading and cell cycle participation of XX endothelial cells and fibroblasts relative to culture with CSS. Conversely, culture with CSS increased these 2D metrics in XY cells. Measurement of 40 kDa FITC-dextran leakage in a single vessel MPS model revealed that culture with FBS significantly decreased XX endothelial barrier permeability relative to culture with CSS. In line with 2D assays, CSS conversely enhanced XY endothelial barrier permeability relative to culture with FBS. Culture with FBS increased metrics of vasculogenesis in XX tissues relative to CSS cultures, whereas prolonged cultured in CSS supported vasculogenesis in XY models. MPS angiogenesis assays revealed increased sprouting in XX tissues cultured with FBS, while only minimal sprouting was observed in all other conditions. Conclusions: FBS imparted significant sex-specific effects on the gene expression patterns, morphology, and cell cycle status of human endothelial cells and fibroblasts in 2D culture. Sex-specific effects measured in 2D culture assays carried over to 3D MPS assays of endothelial barrier function, vasculogenesis, and angiogenesis. Notably, FBS significantly enhanced XX cell functions relative to XY cells in all 2D and 3D MPS assays. Thus, accounting for the sex-specific effects of culture media components will be imperative to improve reproducibility and translational relevance of MPS in preclinical research.

Sex-specific cell culture methods and microphysiological systems can enhance our understanding of how biological sex influences health and disease. Here, we investigated the effects of estradiol and dihydrotestosterone on primary human lung and ocular fibroblasts as well as in human umbilical vein and retinal microvascular endothelial cells from both female and male donors. Treatment of female cells with estradiol and male cells with dihydrotestosterone in 2D culture significantly enhanced proliferation, mitochondrial membrane potential, and upregulated genes associated with bioenergetics and stress responses. Conversely, treatment of female cells with dihydrotestosterone and of male cells with estradiol decreased bioenergetic potential and inhibited cell proliferation. A microphysiological model of bulk tissue vasculogenesis revealed that estradiol enhances vasculogenesis in female tissues and inhibits vasculogenesis in male tissues. Collectively, these findings demonstrate that the sex hormone composition of culture medium significantly influences bioassay readouts in a sex-specific manner.