The initiation of endometriotic lesions is not well understood or characterized because endometriosis is typically diagnosed at an advanced stage. Endometriotic lesions are most often found on pelvic tissues and organs, especially the ovaries. To investigate the role of tissue tropism on ovarian endometrioma initiation, a well-characterized polyacrylamide microarray system is adapted to investigate the role of tissue-specific extracellular matrix and adhesion motifs on endometriotic cell attachment, morphology, and size. The influence of cell origin (endometriotic vs non-endometriotic) is reported, substrate stiffness mimicking aging and fibrosis, and the role of multicellular (epithelial-stromal) cohorts on cell attachment patterns. Multiple ovarian-specific attachment motifs are identified that significantly increase endometriotic (vs non-endometriotic) cell cohort attachment that can be implicated in early disease etiology.

The differentiation of human liver tissue from induced pluripotent stem cells presents a powerful platform for drug screening and disease modeling. While 3D organoid systems effectively recapitulate tissue development, systematic investigation of microenvironmental parameters remains challenging. Here, complementary approaches are developed utilizing 2D cellular microarrays and 3D polyethylene glycol(PEG)-based microwells to examine human stem cell-derived hepatoblast differentiation. High-throughput microarray platform enables systematic evaluation of 60 distinct microenvironmental conditions, combining 15 ECM compositions with 4 growth factor treatments. Position-dependent expression of hepatocytic and cholangiocytic markers is observed including HNF4a, SOX9, and CK19, with differentiation patterns varying substantially across ECM and growth factor conditions. Based on these findings, specific ECM combinations are selected - Collagen 1, Fibronectin, and their combination - for integration into a modular 3D PEG hydrogel system. This 3D platform provides independent control over microtissue geometry and matrix composition, enabling investigation of spatial organization in hepatic differentiation. Through this integrated approach combining high-throughput screening and defined 3D culture, a framework is established for dissecting the microenvironmental regulation of human liver development.

Due to the extended period for clinical diagnosis, the etiology of endometriotic lesion initiation is not well understood or characterized. Endometriotic lesions are most often found on pelvic tissues and organs, especially the ovaries. To investigate the role of tissue tropism on ovarian endometrioma initiation, we adapted a well-characterized polyacrylamide microarray system to investigate the role of tissue-specific extracellular matrix and adhesion motifs on endometriotic cell attachment, morphology, and size. We report the influence of cell origin (endometriotic vs. non-endometriotic), substrate stiffness mimicking aging and fibrosis, and the role of multicellular (epithelial-stromal) cohorts on cell attachment patterns. We identify multiple ovarian-specific attachment motifs that significantly increase endometriotic (vs. non-endometriotic) cell cohort attachment that could be implicated in early disease etiology.

The trabecular meshwork (TM) tissue plays a crucial role in maintaining intraocular pressure (IOP) homeostasis. Increased TM contractility and stiffness are directly correlated with elevated IOP. Although cholesterol is known to be a determinant of glaucoma occurrence and elevated IOP, the underlying mechanisms remain elusive. In this study, we used human TM (HTM) cells to unravel the effects of cholesterol on TM stiffness. We achieved this by performing acute cholesterol depletion with Methyl-β-cyclodextrin (MβCD) and cholesterol enrichment/replenishment with MβCD cholesterol complex (CHOL). Interestingly, cholesterol depletion triggered notable actin depolymerization and decreased focal adhesion formation, while enrichment/replenishment promoted actin polymerization, requiring the presence of actin monomers. Using a specific reporter of phosphatidylinositol 4,5-bisphosphate (PIP2), we demonstrated that cholesterol depletion decreases PIP2 levels on the cell membrane, whereas enrichment increases them. Given the critical role of PIP2 in actin remodeling and focal adhesion formation, we postulate that cholesterol regulates actin dynamics by modulating PIP2 levels on the membrane. Furthermore, we showed that cholesterol levels regulate integrin α5β1 and αVβ3 distribution and activation, subsequently altering cell-extracellular matrix (ECM) interactions. Notably, the depletion of cholesterol, as a major lipid constituent of the cell membrane, led to a decrease in HTM cell membrane tension, which was reversed upon cholesterol replenishment. Overall, our systematic exploration of cholesterol modulation on TM stiffness highlights the critical importance of maintaining appropriate membrane and cellular cholesterol levels for achieving IOP homeostasis.

Chirality is an intrinsic cellular property that describes cell polarization biases along the left-right axis, apicobasal axis, or front-rear axes. Cell chirality plays a significant role in the arrangement of organs in the body as well as the orientation of organelles, cytoskeletons, and cells. Vascular networks within the endometrium, the mucosal inner lining of the uterus, commonly display spiral architectures that rapidly form across the menstrual cycle. Herein, we systematically examine the role of endometrial-relevant extracellular matrix stiffness, composition, and soluble signals on endometrial endothelial cell chirality using a high-throughput microarray. Endometrial endothelial cells display marked patterns of chirality as individual cells and as cohorts in response to substrate stiffness and environmental cues. Vascular networks formed from endometrial endothelial cells also display shifts in chirality as a function of exogenous hormones. Changes in cellular-scale chirality correlate with changes in vascular network parameters, suggesting a critical role for cellular chirality in directing endometrial vessel network organization.

The endometrium undergoes rapid cycles of vascular growth, remodeling, and breakdown during the menstrual cycle and pregnancy. Decidualization is an endometrial differentiation process driven by steroidal sex hormones that is critical for blastocyst-uterine interfacing and blastocyst implantation. Certain pregnancy disorders may be linked to decidualization processes. However, much remains unknown regarding the role of decidualization and reciprocal trophoblast-endometrial interactions on endometrial angiogenesis and trophoblast invasion. Here, we report an engineered endometrial microvascular network embedded in gelatin hydrogels that displays morphological and functional patterns of decidualization. Vessel complexity and biomolecule secretion are sensitive to decidualization and affect trophoblast motility, but that signaling between endometrial and trophoblast cells was not bi-directional. Although endometrial microvascular network decidualization status influences trophoblast cells, trophoblast cells did not induce structural changes in the endometrial microvascular networks. These findings add to a growing literature that the endometrium has biological agency at the uterine-trophoblast interface during implantation. Finally, we form a stratified endometrial tri-culture model, combining engineered microvascular networks with epithelial cells. These endometrial microvascular networks provide a well-characterized platform to investigate dynamic changes in angiogenesis in response to pathological and physiological endometrial states.

Nonalcoholic fatty liver disease affects 30% of the United States population and its progression can lead to nonalcoholic steatohepatitis (NASH), and increased risks for cirrhosis and hepatocellular carcinoma. NASH is characterized by a highly heterogeneous liver microenvironment created by the fibrotic activity of hepatic stellate cells (HSCs). While HSCs have been widely studied in 2D, further advancements in physiologically relevant 3D culture platforms for the in vitro modeling of these heterogeneous environments are needed. In this study, the use of stiffness-variable, extracellular matrix (ECM) protein-conjugated polyethylene glycol microgels as 3D cell culture scaffolds to modulate HSC activation is demonstrated. These microgels as a high throughput ECM screening system to identify HSC matrix remodeling and metabolic activities in distinct heterogeneous microenvironmental conditions are further employed. The 6 kPa fibronectin microgels are shown to significantly increase HSC matrix remodeling and metabolic activities in single or multiple-component microenvironments. Overall, heterogeneous microenvironments consisting of multiple distinct ECM microgels promoted a decrease in HSC matrix remodeling and metabolic activities compared to homogeneous microenvironments. The study envisions this ECM screening platform being adapted to a broad number of cell types to aid the identification of ECM microenvironments that best recapitulate the desired phenotype, differentiation, or drug efficacy.

Hepatic stellate cells (HSCs) are one of the primary drivers of liver fibrosis in non-alcoholic fatty liver disease. Although HSC activation in liver disease is associated with changes in extracellular matrix (ECM) deposition and remodeling, it remains unclear how ECM regulates the phenotypic state transitions of HSCs. Using high-throughput cellular microarrays, coupled with genome-wide ATAC and RNA sequenc-ing within engineered ECM microenvironments, we investigated the effect of ECM and substrate stiffness on chromatin accessibility and resulting gene expression in activated primary human HSCs. Cell microar-rays demonstrated the cooperative effects of stiffness and ECM composition on H3K4 and H3K9 methy-lation/acetylation. ATAC sequencing revealed higher chromatin accessibility in HSCs on 1kPa compared to 25kPa substrates for all ECM conditions. Gene set enrichment analysis using RNA sequencing data of HSCs in defined ECM microenvironments demonstrated higher enrichment of NAFLD and fibrosis-related genes in pre-activated HSCs on 1kPa relative to 25kPa. Overall, these findings are indicative of a microen-vironmental adaptation response in HSCs, and the acquisition of a persistent activation state. Combined ATAC/RNA sequencing analyses enabled identification of candidate regulatory factors, including HSD11B1 and CEBPb. siRNA-mediated knockdown of HSD11b1 and CEBPb demonstrated microenvironmental con-trolled reduction in fibrogenic markers in HSCs.Hepatic stellate cells (HSCs) are one of the primary drivers of liver fibrosis in non-alcoholic fatty liver disease. Although HSC activation in liver disease is associated with changes in extracellular matrix (ECM) deposition and remodeling, it remains unclear how ECM regulates the phenotypic state transitions of HSCs. Using high-throughput cellular microarrays, coupled with genome-wide ATAC and RNA sequencing within engineered ECM microenvironments, we investigated the effect of ECM and substrate stiffness on chromatin accessibility and resulting gene expression in activated primary human HSCs. Overall, these findings were indicative of a microenvironmental adaptation response in HSCs, and the acquisition of a persistent activation state. Combined ATAC/RNA sequencing analyses enabled identification of candidate regulatory factors, including HSD11B1 and CEBPb. siRNA-mediated knockdown of HSD11b1 and CEBPb demonstrated microenvironmental controlled reduction in fibrogenic markers in HSCs.& COPY; 2023 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Intercellular communication through secreted proteins is necessary in essential processes such as embryo and limb development, disease progression, and immune responses. There exist many techniques to study bulk solution protein concentrations, but there is a limited set of tools to study the concentrations of cell-secreted proteins in situ within diverse cell platforms while retaining spatial information. In this study, we have developed a microgel system that is able to quantitatively measure the cell-secreted protein concentration within defined three-dimensional culture configurations with single-cell spatial resolution, called μGeLISA (microgel-linked immunosorbent assay). This system, which is based on the surface modification of polyethylene glycol microgels, was able to detect interleukin 6 (IL-6) concentrations of 2.21-21.86 ng/mL. Microgels were also able to detect cell spheroid-secreted IL-6 and distinguish between low- and high-secreting single cells. The system was also adapted to measure the concentration of cell-secreted matrix metalloproteinase-2 (MMP-2). μGeLISA represents a highly versatile system with a straightforward fabrication process that can be adapted toward the detection of secreted proteins within a diverse range of cell culture configurations.

Cellular phenotypes and functional responses are modulated by the signals present in their microenvironment, including extracellular matrix (ECM) proteins, tissue mechanical properties, soluble signals and nutrients, and cell-cell interactions. To better recapitulate and analyze these complex signals within the framework of more physiologically relevant culture models, high throughput culture platforms can be transformative. High throughput methodologies enable scientists to extract increasingly robust and broad datasets from individual experiments, screen large numbers of conditions for potential hits, better qualify and predict responses for preclinical applications, and reduce reliance on animal studies. High throughput cell culture systems require uniformity, assay miniaturization, specific target identification, and process simplification. In this review, we detail the various techniques that researchers have used to face these challenges and explore cellular responses in a high throughput manner. We highlight several common approaches including two-dimensional multiwell microplates, microarrays, and microfluidic cell culture systems as well as unencapsulated and encapsulated three-dimensional high throughput cell culture systems, featuring multiwell microplates, micromolds, microwells, microarrays, granular hydrogels, and cell-encapsulated microgels. We also discuss current applications of these high throughput technologies, namely stem cell sourcing, drug discovery and predictive toxicology, and personalized medicine, along with emerging opportunities and future impact areas.

Abstract Disclosure: S.E. Hobbs: None. H. Kimmel: None. G. Underhill: None. S. Anakk: None. Hepatocellular Carcinoma (HCC), the primary form of liver cancer, is the second leading cause of cancer associated-associated deaths worldwide. Complex signaling mechanisms and several cellular processes are coordinated using scaffolding proteins. IQ motif-containing GTPase Activating Protein 1 (IQGAP1) is a scaffolding protein that is overexpressed in multiple epithelial cancers, including ovarian, breast, and liver. Previous work has shown that overexpression of IQGAP1 activates Yes-associated Protein (YAP) to promote HCC in mice. Extracellular matrix (ECM) stiffness, due to liver fibrosis in HCC, can activate YAP to promote tumor progression. Remarkably, our preliminary data show that IQGAP1 has increased localization at the plasma membrane when HepG2 cells (an HCC cell line) are seeded onto a stiff ECM (25 kPa). YAP is modulated by actin dynamics, which can be altered by changes in the plasma membrane. Therefore, IQGAP1’s movement to the plasma membrane can potentially activate YAP by modulating actin dynamics. We also found that the anti-HCC drug sorafenib, can induce the expression IQGAP1 and activate YAP in HepG2 cells grown on plastic. These sorafenib-mediated changes are counterproductive and can contribute to drug resistance of HCC. Finally, we found that inhibition of YAP in HepG2 cells significantly decreases IQGAP1 expression, indicating that there is bidirectional crosstalk between the two proteins. Future directions will elucidate if loss of IQGAP1 decreases ECM stiffness-induced YAP activation, proliferation, and migration in HCC cells. In addition, we will determine if ECM stiffness alters sorafenib-mediated IQGAP1 upregulation, as ECM stiffness has been linked to drug resistance in HCC. Overall, this work will shed light on the potential role of the IQGAP1-YAP axis in ECM stiffness-mediated HCC progression. Presentation: Thursday, June 15, 2023

49 The endometrium undergoes rapid cycles of vascular growth, remodeling, and breakdown 50 during the menstrual cycle and pregnancy. Decidualization is an endometrial 51 differentiation process driven by steroidal sex hormones that is critical for blastocyst- 52 uterine interfacing and blastocyst implantation. Certain pregnancy disorders may be linked 53 to decidualization processes. However, much remains unknown regarding the role of 54 decidualization and reciprocal trophoblast-endometrial interactions on endometrial 55 angiogenesis and trophoblast invasion. Here, we report an artificial endometrial 56 perivascular niche embedded in gelatin methacrylol hydrogels that displays morphological 57 and functional patterns of decidualization. We show vessel complexity and soluble factor 58 secretion are sensitive to decidualization and affect trophoblast motility. Importantly, we 59 demonstrate the engineered perivascular niche can be combined with epithelial cultures to 60 form a stratified endometrial model. This artificial perivascular niche provides a well- 61 characterized platform to investigate dynamic changes in angiogenesis in response to 62 pathological and physiological endometrial states. 63 an endometrial vessel model to understand endometrial vasculature cycle p p and p =0.011, respectively. These findings suggest that soluble factors from perivascular cultures increase trophoblast outgrowth for non-decidualized and decidualized conditions with P4 compared to the control condition. However, trophoblast outgrowth was We cell The of 23-25 stromal cells, endothelial cells form endothelial last only and fall apart over time, consistent with our results from our tube formation assay. We identified 2:1 ratio of endothelial to stromal cells allowed for 20+ days of culture. Our studies suggest that reduced initial stromal cell density may be more conducive to perivascular stability, likely due to the contractile nature of stromal cells which can cause the hydrogel cultures to contract and disintegrate over time, a result consistent with other reports in the literature 27 our in vitro

Abstract The cell-cell and cell-extracellular matrix (ECM) communication is central to maintain cellular functions, including proliferation. Scaffold proteins can mediate these communications by binding specific protein effectors of signaling pathways and tethering them into a stable complex. IQ-domain containing GTPase Activating Protein 1 (IQGAP1) is a multi-domain scaffold protein that is involved in cytokinesis, proliferation and apoptosis. Each of the five domains of IQGAP1 bind distinct effector proteins to transduce cellular signals. In the liver, supraphysiological levels of bile acids, a cholesterol metabolite, is linked to the increase in IQGAP1 levels and is positively associated with transcriptional co-activator YAP. However, how bile acids induce expression of IQGAP1 remains unknown. We hypothesize that supraphysiological levels of bile acids act on the cell microenvironment to activate IQGAP1. As bile acids are induced during liver regeneration, we examined and found that Iqgap1 levels are increased. Remarkably this increase in Iqgap1 transcript coincides with that of bile acids after 2/3 partial hepatectomy in mice. In addition, IQGAP1 demonstrated increased localization into puncta at the cell surface in HepG2 cells, seeded on ECM microarrays with higher stiffness indicating mechano-sensing signals can alter IQGAP1 protein levels. We will examine IQGAP1 localization during liver regeneration and investigate if there is a cross talk between ECM stiffness, bile acid and IQGAP1 expression. Using domain deletion constructs, we will also identify the IQGAP1 domain responsible for coordinating the bile acid and cell-ECM communication in the liver. Collectively, these results will help elucidate the IQGAP1-specific cell signaling pathways used in facilitating liver cell proliferation, and test if IQGAP1 scaffold can integrate metabolite cues (bile acids) to communication between liver cells and the cellular microenvironment. Presentation: Saturday, June 11, 2022 1:06 p.m. - 1:11 p.m., Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

In vitro human liver models are essential for drug screening, disease modeling, and cell-based therapies. Induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (iHeps) mitigate sourcing limitations of primary human hepatocytes (PHHs) and enable precision medicine; however, current protocols yield iHeps with very low differentiated functions. The composition and stiffness of liver's extracellular matrix (ECM) cooperatively regulate hepatic phenotype in vivo, but such effects on iHeps remain unelucidated. Here, we utilized ECM microarrays and high content imaging to assess human iHep attachment and functions on ten major liver ECM proteins in single and two-way combinations robotically spotted onto polyacrylamide gels of liver-like stiffnesses; microarray findings were validated using hydrogel-conjugated multiwell plates. Collagen-IV supported higher iHep attachment than collagen-I over 2 weeks on 1 kPa, while laminin and its combinations with collagen-III, fibronectin, tenascin C, or hyaluronic acid led to both high iHep attachment and differentiated functions; laminin and its combination with tenascin or fibronectin led to similar albumin expression in iHeps and PHHs. Additionally, several collagen-IV-, laminin-, fibronectin-, and collagen-V-containing combinations on 1 kPa led to similar or higher CYP3A4 staining in iHeps than PHHs. Lastly, collagen-I or -III mixed with laminin, collagen-IV mixed with lumican, and collagen-V mixed with fibronectin led to high and stable functional output (albumin/urea secretions; CYP1A2/2C9/3A4 activities) in iHep cultures versus declining PHH numbers/functions for 3 weeks within multiwell plates containing 1 kPa hydrogels. Ultimately, these platforms can help elucidate ECM's role in liver diseases and serve as building blocks of engineered tissues for applications. STATEMENT OF SIGNIFICANCE: We utilized high-throughput extracellular matrix (ECM) microarrays and high content imaging to assess the attachment and differentiated functions of iPSC-derived human hepatocyte-like cells (iHep) on major liver ECM protein combinations spotted onto polyacrylamide gels of liver-like stiffnesses. We observed that iHep responses are regulated in unexpected ways via the cooperation between ECM stiffness and protein composition. Using this approach, we induced mature functions in iHeps on substrates of physiological stiffness and select ECM coatings at higher levels over 3+ weeks than analogous primary human hepatocyte cultures, which is useful for building platforms for drug screening, disease modeling, and regenerative medicine.

Controlled in vitro multicellular culture systems with defined biophysical microenvironment have been used to elucidate the role of Notch signaling in the spatiotemporal regulation of stem and progenitor cell differentiation. In addition, computational models incorporating features of Notch ligand-receptor interactions have provided important insights into Notch pathway signaling dynamics. However, the mechanistic relationship between Notch-mediated intercellular signaling and cooperative microenvironmental cues is less clear. Here, liver progenitor cell differentiation patterning was used as a model to systematically evaluate the complex interplay of cellular mechanics and Notch signaling along with identifying combinatorial mechanisms guiding progenitor fate. We present an integrated approach that pairs a computational intercellular signaling model with defined microscale culture configurations provided within a cell microarray platform. Specifically, the cell microarray-based experiments were used to validate and optimize parameters of the intercellular Notch signaling model. This model incorporated the experimentally established multicellular dimensions of the cellular microarray domains, mechanical stress-related activation parameters, and distinct Notch receptor-ligand interactions based on the roles of the Notch ligands Jagged-1 and Delta-like-1. Overall, these studies demonstrate the spatial control of mechanotransduction-associated components, key growth factor and Notch signaling interactions, and point towards a possible role of E-Cadherin in translating intercellular mechanical gradients to downstream Notch signaling.

The endometrium undergoes rapid cycles of vascular growth, remodeling, and breakdown during the menstrual cycle and pregnancy. Decidualization is an endometrial differentiation process driven by steroidal sex hormones that is critical for blastocyst-uterine interfacing and blastocyst implantation. Certain pregnancy disorders may be linked to decidualization processes. However, much remains unknown regarding the role of decidualization and reciprocal trophoblast-endometrial interactions on endometrial angiogenesis and trophoblast invasion. Here, we report an artificial endometrial perivascular niche embedded in gelatin methacrylol hydrogels that displays morphological and functional patterns of decidualization. We show vessel complexity and soluble factor secretion are sensitive to decidualization and affect trophoblast motility. Importantly, we demonstrate the engineered perivascular niche can be combined with epithelial cultures to form a stratified endometrial model. This artificial perivascular niche provides a well-characterized platform to investigate dynamic changes in angiogenesis in response to pathological and physiological endometrial states. Teaser We describe an endometrial vessel model to understand endometrial vasculature in the menstrual cycle and pregnancy.

The endometrium undergoes profound changes in tissue architecture and composition, both during the menstrual cycle as well as in the context of pregnancy. Dynamic remodeling processes of the endometrial extracellular matrix (ECM) are a major element of endometrial homeostasis, including changes across the menstrual cycle. A critical element of this tissue microenvironment is the endometrial basement membrane, a specialized layer of proteins that separates the endometrial epithelium from the underlying endometrial ECM. Bioengineering models of the endometrial microenvironment that present an appropriate endometrial ECM and basement membrane may provide an improved environment to study endometrial epithelial cell (EEC) function. Here, we exploit a tiered approach using two-dimensional high-throughput microarrays and three-dimensional gelatin hydrogels to define patterns of EEC attachment and cytokeratin 18 (CK18) expression in response to combinations of endometrial basement membrane proteins. We identify combinations (collagen IV + tenascin C; collagen I + collagen III; hyaluronic acid + tenascin C; collagen V; collagen V + hyaluronic acid; collagen III; and collagen I) that facilitate increased EEC attachment, increased CK18 intensity, or both. We also identify significant EEC mediated remodeling of the methacrylamide-functionalized gelatin matrix environment via analysis of nascent protein deposition. Together, we report efforts to tailor the localization of basement membrane-associated proteins and proteoglycans in order to investigate tissue-engineered models of the endometrial microenvironment.

While cells are known to behave differently based on the size of micropatterned islands, and this behavior is thought to be related to cell size and cell-cell contacts, the exact threshold for this difference between small and large islands is unknown. Furthermore, while cell size and cell-cell contacts can be easily manipulated on small islands, they are harder to measure and continually monitor on larger islands. To investigate this size threshold, and to explore cell size, cell-cell contacts, and differentiation, we use a previously established simulation to plan experiments and explain results that we could not explain from experiments alone. We use five seeding densities covering three orders of magnitude over 25-500 µm diameter islands to examine markers of proliferation and differentiation in bone marrow-derived mesenchymal cells (cell line). We show that osteogenic markers are most accurately described as a function of confluence for larger islands, but a function of time for smaller islands. We further show, using results of the simulation, that cell size and cell-cell contacts are also related to confluence on larger islands, but only cell-cell contacts are related to confluence on small islands. This study uses simulations to explain experimental results that could not be explained from experiments alone. Together, the simulations and experiments in this study show different differentiation patterns on large and small islands, and this simulation may be useful in planning future studies related to this study.