Cellular and Molecular Bioengineering

Anti-angiogenic cancer therapies attempt to withhold necessary nutrients and oxygen from growing tumors by targeting the major promoters of endothelial cell (EC) angiogenesis: vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR-2). Unfortunately, these treatments are often insufficient, even when coupled with chemotherapies, and fail to significantly increase survival rates. The tumor microenvironment (TME) is mechanically distinct compared to normal tissue, including increased matrix deformations or strains caused by cancer-associated fibroblasts (CAFs). In this report, we detail the specific and independent roles of two tyrosine residues, Y1054 and Y1214, on mechanical activation of VEGFR-2. Furthermore, we characterize CAF biochemical and mechanical signaling and demonstrate how ECs exhibit decreased vessel growth when co-cultured with CAFs and treated with a contractility inhibitor. Using non-phosphorylatable VEGFR-2 mutants, we reveal Y1054 and Y1214 are each necessary for EC angiogenesis, particularly in response to strain. Overall, this research highlights the need to study how mechanics in the TME promote vessel growth and thus tumor progression, which is important to consider when developing future anti-angiogenic therapies.

Oncofetal fibronectin (oFN) is a known biomarker of cancer progression and is involved in events such as epithelial mesenchymal transition and multidrug resistance. It is also understood that fibronectin (FN) and its splice variants are differentially arranged by cancer associated fibroblasts (CAFs) to support cancer cell proliferation and migration. Contrastingly, little is understood about the mechanobiology of oFN, a post-translationally modified FN isoform defined by O-glycosylation of a specific threonine residue. To investigate the architectural features of the oFN matrix, CAFs were simulated by culturing human mammary fibroblasts (HMFs) on stiff polyacrylamide gels (20 kPa) in media collected from HCC-1937 (primary site breast adenocarcinoma) and MDA-MB-231 (metastatic breast adenocarcinoma) cells. As a control, HMFs were also cultured on softer gels representing healthy adipose tissue (2 kPa) in media not exposed to cancer cells. After immunocytochemical analysis, tumor conditioned HMFs displayed an expected CAF-like phenotype that was most prominent with MDA-MB-231 conditioned media. Matrices produced by tumor-conditioned HMFs contained more oFN with structural features mostly following that of the overall dense, aligned FN matrix. Cancer cells reseeded on their corresponding decellularized tumor-associated matrices had greater F-actin/oFN colocalization than control matrix. The lack of distinct oFN topographical cues may indicate that oFN O-glycosylation is more directly involved in biochemical signaling than mechanosignaling through matrix architecture. Together, this study established a platform for investigating stromal oFN which revealed that tumor-conditioned media sourced from cancer cells with different metastatic potential influences HMF activation, leading to distinct total FN and thus oFN matrix microarchitecture features.

In breast cancer, collagen fiber alignment and the second harmonic generation forward-to-backward ratio (F/B), an optical measurement sensitive to fibril-scale organization within collagen fibers, have each been associated with metastatic outcome. Although both are measured at the tumor-stromal interface, in vitro studies have largely examined them separately, because experimental systems have not allowed the two to be varied independently. To address this technical gap, we used a microfluidic biofabrication approach to generate paired aligned and unaligned regions within 3D collagen hydrogels via extensional strain and varied measured F/B across gels by changing the pre-gel pH. The resulting library covered alignment values reported in breast tumors and F/B values associated with poorer metastatic outcome in invasive ductal carcinoma at matched matrix stiffness, pore diameter, and fiber fraction. In MDA-MB-231 cells, alignment biased migration along the fiber axis without changing migration speed, as expected, but the magnitude of the contact guidance response varied with measured F/B. In contrast, MCF-7 cells showed little overall contact guidance, yet migrated ~30% faster in high F/B than low F/B gels in both aligned and unaligned regions. Together, these results show that cells migrated differently in matrices with similar fiber alignment. Thus, including measured F/B may provide additional information to help anticipate how cells migrate in collagen environments that appear similar based on alignment alone.

Synthetic blood contacting devices frequently fail due to the lack of requisite biochemical and biomechanical cues needed to support transanastamotic endothelialization. During transanastomotic endothelialization, endothelial cells experience dynamic changes in extracellular mechanical cues as they migrate from compliant native vessels onto stiffer blood contacting device surfaces. However, how substrate stiffness and mechanical memory from prior mechanical environments influence temporal integrin remodeling and downstream endothelialization processes necessary to establish a stable endothelial layer remains poorly understood. In this study, human coronary artery endothelial cells (HCECs) were cultured on substrates spanning physiologically relevant stiffnesses to determine how substrate mechanics regulate collagen binding integrins and endothelialization. Increasing substrate stiffness promoted time dependent upregulation of 2{beta}1 integrin expression, whereas 1{beta}1 expression remained unchanged. Enhanced 2{beta}1 expression on stiff substrates was accompanied by increased vinculin associated focal adhesion maturation and accelerated endothelialization, characterized by increased proliferation, migration, and progression to confluence prior to reaching quiescence after 1 week. To better model transanastomotic migration and investigate mechanical history effects, cells initially expanded on compliant hydrogels were transferred to stiff substrates. Although these cells exhibited transient reductions in 2{beta}1 expression at early timepoints compared with tissue culture polystyrene expanded controls, no persistent differences in focal adhesion maturation, proliferation, migration, confluence, or quiescence were observed. Collectively, these findings demonstrate that substrate stiffness is a primary regulator of the early endothelialization processes required to establish a stable endothelial monolayer, whereas the influence of mechanical history is transient and ultimately superseded by the current mechanical environment. These findings also identify 2{beta}1 mediated mechanotransduction as a potential design target for blood contacting biomaterials that promote rapid endothelialization while supporting long-term endothelial cell quiescence.

Nanoparticle albumin-bound paclitaxel (Abraxane) was among the first clinically approved nanomedicines and is a first-line chemotherapeutic. It enhances therapeutic efficacy and reduces side effects of paclitaxel. Despite its extensive clinical use, mechanisms of Abraxane's therapeutic effects are the subject of ongoing study. This work posits a new mechanism for Abraxane's effects in lung cancers and metastasis. Tracing the albumin component of Abraxane with radioisotopes or fluorophores, we find that Abraxane homes to the lungs. Abraxane's lung tropism is elevated by inflammation and is eliminated in complement protein C3 knockout mice, showing that complement pathway drives the uptake. Flow cytometry and histology show that neutrophils are the dominant cell type taking up Abraxane in the lungs. We demonstrate Abraxane's tropism to pulmonary neutrophils in mice and in human donor lungs. We note previously undocumented side effects associated with Abraxane's lung tropism, including pro-thrombotic and pro-inflammatory responses in acute lung inflammation. We show that Abraxane's efficacy against lung metastases is affected when lung uptake is eliminated by C3 knockout. Our results provide insight into why Abraxane is effective in lung cancer and metastases. These findings may be used to improve outcomes in patients receiving Abraxane and help design next-generation cancer nanomedicines.

During tumor growth and progression, cancer cells are exposed to sustained physical confinement and volumetric compression that can alter cell volume, cytoskeletal organization, mechanotransduction, and invasive behavior. However, whether breast cancer cells retain a compression-induced mechanical memory after release from sustained volumetric compression, and how this memory influences subsequent migration and invasion, remains poorly understood. Here, by controlling cell volume using PEG - mediated volumetric compression, we investigated the compression and post-compression recovery responses of MCF-7 breast cancer cells. Cells were compressed for four days, followed by four days of recovery after PEG removal, and analyzed using daily morphological tracking, single-cell time-lapse imaging, F-actin and YAP staining, wound healing assays, and 3D spheroid invasion assays. We show that sustained volumetric compression shifts MCF-7 cells into a compact, jammed-like, low-motility state characterized by reduced morphodynamic remodeling, suppressed collective migration, and limited spheroid invasion. In contrast, post-compression recovery induces a distinct mechanobiological state marked by increased cell area and perimeter, altered single-cell trajectories, heterogeneous F-actin remodeling, enhanced YAP nuclear localization in enlarged recovered cells, accelerated wound closure, and increased spheroid invasion and cell dissemination. These findings suggest that prior volumetric compression can prime breast cancer cells for enhanced migration and invasion after stress release, supporting post-compression recovery as a form of mechanical memory that may contribute to tumor dissemination.

Pannexin 1 (PANX1) is a channel-forming glycoprotein that has been implicated in regulating melanoma cell properties such as growth, migration, and invasion. Here we report that the CRISPR-Cas9 mediated ablation of PANX1 or channel inhibition with probenecid shifted A375-P cell cycle distribution towards G2/M, while channel inhibition with spironolactone stalled cell cycle progression at the G1/S checkpoint regardless of PANX1 expression. Bulk RNA sequencing revealed cell cycle related gene expression was downregulated with PANX1 targeting strategies and this was exacerbated when PANX1 deficient cells were treated with spironolactone. When grown as spheroids, we found the deletion and inhibition of PANX1 disrupted spheroid compaction, with spheroids displaying poorly defined borders and reduced circularity. The deletion of PANX1 increased spheroid fragmentation, and this phenotype was intensified when spheroids devoid of PANX1 were treated with spironolactone, highlighting a potential synthetic lethal effect. Using liquid chromatography mass spectrometry, we sought to determine whether PANX1 had binding partners that may act to stabilize spheroid integrity and found that PANX1 co-immunoprecipitated various cell adhesion molecules such as {beta}-actin and F-actin-capping protein subunit -1, suggesting PANX1 and its interacting partners may stabilize cell-cell contacts. When control and PANX1 ablated cells were grown in chick chorioallantoic membrane xenografts to assess their tumour growth characteristics, we discovered that the deletion of PANX1 reduced tumour surface area and viability. Our findings indicate that PANX1 regulates cell cycle progression and the structural architecture of A375-P melanoma tumours, highlighting the therapeutic utility of targeting PANX1 in melanoma.

Metastatic dissemination is initiated by tumor cells interpreting spatially organized biochemical and biophysical cues that remain difficult to reproduce using conventional migration assays. Here, we developed a computationally guided metastasis-on-a-chip (MET-on-a-chip) platform based on the concept of the Minimally Functional Unit (MFU), in which only the biological components required to answer a defined experimental question are incorporated. The platform consists of two independent culture chambers connected through an array of confined microchannels that permits diffusion of soluble factors while constraining tumor cell migration. Rather than relying on empirical optimization, finite-element COMSOL simulations were first used to predict molecular transport, define growth factor loading conditions, identify biologically relevant exposure regions, and guide the rational design of the microfluidic assay. Computational predictions were experimentally validated using 70-kDa FITC-dextran diffusion and VEGF release studies, confirming the formation of stable spatial concentration gradients across the microfluidic platform. The simulations further demonstrated that both growth factor loading and cell positioning relative to the predicted gradients critically influenced assay performance, leading to the optimization of the platform through spatial reconfiguration of the tumor compartment. Using the optimized configuration, we compared the migratory responses of neuroblastoma, Ewing sarcoma, and osteosarcoma cells to vascular (VEGF-A165) and lymphatic (VEGF-C) chemotactic cues. VEGF-C significantly increased migration through the microchannel array in Ewing sarcoma and osteosarcoma cells, whereas VEGF-A165 produced no significant effect. In contrast, neuroblastoma cells exhibited minimal migration under either condition, revealing tumor-specific differences in responsiveness to VEGF signaling. Together, these findings establish a computationally guided workflow for the rational design of metastasis-on-a-chip assays, in which predictive modeling informs experimental design before biological validation. By substantially reducing empirical trial-and-error while enabling quantitative control over growth factor exposure, this strategy provides a robust framework for developing minimally functional microphysiological systems capable of dissecting individual steps of the metastatic cascade under experimentally defined conditions. Translational Impact StatementMetastatic dissemination remains one of the greatest clinical challenges in pediatric oncology, yet experimental models capable of quantitatively evaluating early migratory events remain limited. The computationally guided MET-on-a-Chip workflow presented here provides a human-relevant platform in which soluble microenvironmental cues can be systematically investigated under controlled and predictive conditions. Although demonstrated here using VEGF-A165 and VEGF-C, the platform can be readily adapted to study virtually any chemotactic factor, cytokine, extracellular vesicle population, or therapeutic candidate involved in metastatic dissemination. The modular MFU design allows biological complexity to be incorporated progressively as dictated by the scientific question, providing a flexible framework for future applications. In the longer term, this workflow could be combined with patient-derived tumor cells, organoids, or biopsy material to investigate patient-specific metastatic behavior and evaluate anti-metastatic therapeutic strategies in a personalized setting. Beyond identifying pro-migratory signaling pathways, the platform may serve as a preclinical tool to prioritize compounds capable of preventing tumor cell dissemination before evaluation in more complex animal models or clinical studies.

Lymphatic endothelial cell (LEC) function is strongly tied to transitions between button- and zipper-like junctions that control fluid flow into vessels. Published methods quantifying junction morphology are limited to single cell analysis or use few quantitative metrics to describe junctions across LEC monolayers. Therefore, a novel 3-step image analysis pipeline was developed to identify new metrics that quantify differences in button- and zipper-like junctions in LECs. The pipeline used Cellpose, CellProfiler, and Fiji to analyze VE-cadherin expression across lymphatic vessels within a 3D microfluidic chip and quantify VE-cadherin radial intensity, number of objects, object size, branchpoints, end-points, and junction thickness. Tumor necrosis factor (TNF)- exposure produced but-ton-like junctions, and unexposed cells were zippered. Radial intensity was heightened at the cell membrane in zipper-like junctions while perinuclear intensity increased for but-ton-like junctions. Increased branchpoints, endpoints, objects per cell, and junction thick-ness were associated with junction buttoning, while increased object size was associated with junction zippering. Principle component analysis results reinforced these trends and identified metric combinations that represented each junctional state. Overall, this semi-automated pipeline quantified differences in VE-cadherin junction morphology across LEC monolayers and generated new quantitative metrics to expand the tools available for studying junction morphology in lymphatic vasculature.

ObjectiveStructural adaptations of capillary networks, through angiogenesis, arterialization, and regression, are implicated in many diseases, and gaining a better understanding of the cell-cell interactions that underpin these adaptations may lead to novel therapeutic discoveries for disease management. Endothelial cells and pericytes are the two cell types that comprise capillary networks. Experimental model systems have been developed to study the dynamic interactions between endothelial cells and pericytes, providing valuable insights into capillary development, cell-to-cell communication, and responses to growth factors and therapeutic agents. MethodsIn this study, we present a novel and simple co-culture system that uses commercially available primary human endothelial cells and pericytes, does not require microfluidic perfusion, and allows simultaneous observation of cell morphologies and interactions over time in 60 samples, enabling high-throughput analysis of multiple culture conditions with replicates. ResultsImage analysis pipelines were created to quantify microvascular adaptations, including one to measure colocalization between endothelial cells and pericytes, capturing dynamic coupling and uncoupling associated with capillary stability, angiogenesis, and regression. We validated the ability of our co-culture system to reproducibly represent the effects of fibrotic and angiogenic activation signals, including an FDA-approved drug, on endothelial cells, pericytes, and their coupling. ConclusionThis novel, high-throughput microvascular screening assay enables quantification of microvascular dynamics in response to disease-relevant stimuli and therapeutics in a repeatable, real-time manner.

Radiotherapy (RT) significantly improves outcomes and reduces the risk of recurrence in breast cancer. However, in patients that experience recurrence despite treatment, the irradiated breast tissue may create a pre-metastatic niche that promotes tumor cell infiltration. Because current models of metastasis lack key physiological and microenvironmental cues, we developed a mammary vasculature-on-chip (MVoC) to model how the vasculature responds to RT and impacts immune and tumor cell behavior in mammary tissue. Murine and human MVoCs incorporated endothelial cells (ECs) cultured under physiologic shear stress and mammary-specific fibroblasts to mimic the vascular-fibrous stroma interface. MVoCs exhibited a transient, acute response to RT that led to a persistent phenotypic shift. Neutrophils, which play key roles in pre-metastatic niche formation, adhered more to irradiated ECs and induced persistent damage to the endothelial barrier, preventing recovery. Neutrophils also significantly increased tumor cell adhesion to the endothelium in MVoCs. These findings demonstrate that MVoCs can be used to study RT-induced pre-metastatic niche formation and suggest a positive feedback loop between damaged ECs and immune cell activation that may facilitate tumor cell colonization. MVoCs provide a biologically relevant approach to probe molecular and cellular interactions, improving translation toward developing novel therapies that improve patient outcomes.

The tumor microenvironment plays a pivotal role in tumor development, harboring elements such as endothelial cells, immune cells, fibroblasts, and soluble factors such as transforming growth factor-{beta} (TGF-{beta}) family. TGF-{beta} family regulates cell development and promotes tumor invasion, metastasis, angiogenesis, and endothelial-to-mesenchymal transition (EndoMT). Here, we investigate the effects of TGF-{beta} signaling on vascular remodeling using a three-dimensional (3D) vascular network in a microfluidic device. Using both a co-culture (3D-Co) and simplified endothelial monoculture (3D-CM), we demonstrate that TGF-{beta} signaling reduces the quality and functionality of the vasculature by regressing them. In addition, we observed the upregulation of EndoMT-related markers in mRNA and protein expressions, suggesting the induction of EndoMT in 3D vascular networks. The increased vascular permeability stimulated by TGF-{beta}2 also supports the loss of endothelial identity in the 3D-Co. Transcriptomic analysis revealed the coordinated activation of pathways associated with cell migration and EndoMT, along with the suppression of cell cycle progression. A comparative analysis of two-dimensional (2D) and 3D cultures revealed a fundamentally distinct endothelial response to TGF-{beta}2 in the 3D context, including metabolic reprogramming. These findings demonstrate that the 3D microenvironment critically modulates endothelial responses to TGF-{beta} and enables the emergence of vascular phenotypes not captured in 2D systems. This study provides a more physiologically relevant platform to investigate endothelial dysfunction and vascular remodeling.

Patient-derived tumor organoids provide a physiologically relevant 3D disease model for preclinical drug discovery, surpassing the limitations of conventional 2D cell lines. To better capture the dynamic nature of organoid drug responses, we developed a new systematic evaluation method called SCOPE (Systematic Classification of Organoids for Phenotypic Evaluation), harnessing phenotypic assessments from multi-timepoint 3D imaging data. By integrating artificial intelligence (AI)-based image analysis of organoid viability with tracking and mathematical modeling of organoid growth over time, we captured temporal-and dose-dependent dynamics of phenotypic changes, culminating in two novel metrics: a combined growth and viability (GV) score as well as a cytostatic-cytotoxic transition range (CCTR) that separates drug effects on organoid growth and viability. Our approach supports classification of specific drug responses into four distinct phenotypic groups: (1) cytotoxic, (2) cytostatic plus cytotoxic, (3) late cytotoxic, and (4) cytostatic. This novel drug evaluation system can identify previously unknown drug effects or new therapeutic use cases for existing drugs, facilitating the design of alternative therapeutic options to overcome efficacy or drug resistance challenges and improving the clinical applicability of organoid-based drug discovery results.

{gamma}{delta} T-cells, which are predominantly enriched in epithelial tissues, have been used in cancer therapy because of their capacity for rapid cytotoxicity in an MHC-independent manner. Current paradigms are largely agnostic to the role of tissue mechanical cues in regulating {gamma}{delta} T-cell function. Here, we investigated the role of matrix viscoelasticity in modulating {gamma}{delta} T-cell migration, differentiation state, phenotype, and function. Using a tunable collagen-based gel system, we found that encapsulation in highly-elastic (slow-relaxing) matrices preserved a less differentiated phenotype, as evidenced by CD27 and CD45RA expression. Slow-relaxing matrices also increased expression of Fas and PD-1, while decreasing expression of CD11a. Despite increased PD-1 expression, these cells remained functional, as demonstrated by high levels of TNF- and IFN-{gamma} relative to PD-1-negative cells. Proteomic analysis revealed that {gamma}{delta} T-cells respond to changes in viscoelasticity through actin remodeling and shifts in metabolic machinery. Overall, when compared to non-encapsulated cells (2D culture), encapsulated {gamma}{delta} T-cells showed increased expression of cytotoxic programs. Functionally, cells encapsulated in slow-relaxing gels showed improved control of tumor growth in an aggressive HCT116 tumor model. Together, these findings establish matrix viscoelasticity as an important regulator of post-thymic {gamma}{delta} T-cell differentiation and function.

Introduction: Placental tissue allografts are widely utilized in the management of chronic wounds, including diabetic foot ulcers. In addition to serving as wound coverings, these products may influence wound repair through extracellular matrix architecture and biologically active signaling molecules native to human amniotic tissue. Manufacturing and processing methodologies vary considerably across commercially available products and may substantially impact biologic activity, tissue remodeling, and wound-healing trajectories. In this study, a rodent wound-healing model was utilized to compare a proprietary processed placental tissue allograft with a conventionally processed placental tissue comparator. Methods: Two arms of a five-arm pilot rodent wound-healing study were evaluated to compare a proprietary processed human amniotic tissue allograft against a conventionally processed comparator; the remaining arms evaluated test articles under development and will be reported separately. Male rats (n=8 per arm), matched for weight, age, and diet, each received two full-thickness dorsal excisional wounds approximately 10 mm in diameter and 2 mm deep. Test articles were reapplied upon resorption over a 14-day period. At Day 14, sections were scored semi-quantitatively (0-4 Severity Injury Score) across histologic and immunohistochemical parameters spanning epidermal repair, inflammation, angiogenesis, proliferation, and remodeling. Cranial and caudal wounds were averaged within each animal, retaining the animal as the experimental unit. A Day 14 Histology Resolution Score was derived as a signed composite of pro- and anti-regenerative markers and evaluated alongside percent wound closure. Inferential statistics (Welch ANOVA with Games-Howell post hoc) were derived from the full five-arm cohort (n=40). Results: Animals treated with the proprietary processed allograft showed more advanced progression toward wound resolution than those receiving the conventionally processed comparator, across both surface and tissue-level endpoints. Mean animal-level wound closure at Day 14 was 95.2% with the proprietary processed allograft versus 89.9% with the conventional comparator. The integrated Day 14 Histology Resolution Score, a composite of epidermal repair, inflammation, angiogenesis, proliferation, and remodeling endpoints was 8.94 versus 5.13, corresponding to a shift from the poor-resolution range into the partial-resolution range on the pre-defined interpretive scale. Welch ANOVA across the full five-arm cohort (n=40) detected a significant overall effect of treatment on this composite (p = 0.010). The pairwise contrast between these two arms did not reach significance under Games-Howell correction across ten comparisons (p = 0.337); at n=8 per arm the study was not powered to resolve individual pairwise differences, and the observed standardized difference was large (Hedges' g = 0.93). Injury endpoints were consistently lower in the proprietary processed allograft: 69% lower acute inflammation, 60% lower dermal necrosis, approximately 57% lower epithelial erosion, 46% lower ulceration, and 42% less hemorrhage. Importantly, these reductions in residual tissue injury occurred while maintaining robust granulation tissue formation, angiogenesis, collagen remodeling, cellular proliferation, and macrophage recruitment. Discussion: These findings suggest that processing methodologies can materially influence the biologic performance of placental tissue allografts. The proprietary processed allograft was associated with lower residual tissue injury and greater regenerative and reparative activity relative to a conventionally processed comparator. These observations support the concept that placental tissue allografts should not necessarily be viewed as biologically equivalent and may have important implications for clinical outcomes and product selection in wound care.

Seroma is a common complication following abdominoplasty and other procedures involving extensive soft tissue dissection. Although animal seroma models have been reported, the stromal pathology underlying post-abdominoplasty seroma, particularly the role of oxidative stress, remains poorly characterized. Here, we developed a rat abdominal seroma model by surgically separating the subcutaneous tissue from the underlying muscle fascia and investigated the temporal evolution of the associated stromal response. Seroma developed in 9 of 20 operated rats, corresponding to a formation rate of 45.0%. Longitudinal ultrasound demonstrated that fluid accumulation peaked around postoperative day (POD) 7 and largely resolved by POD30. Despite macroscopic fluid resolution, substantial stromal remodeling persisted. Compared with normal abdominal tissue, seroma-associated tissues at POD14 and POD35 exhibited increased collagen deposition, macrophage accumulation, oxidative injury, and vascular remodeling. Additionally, at POD35, macrophage accumulation and 4-hydroxynonenal staining were greater than at POD14, whereas collagen deposition and vascular density remained elevated with no significant differences between postoperative time points. To our knowledge, this study presents the first rat model specifically designed to reproduce post-abdominoplasty seroma and the first temporal characterization of oxidative stress within seroma-associated tissue. These findings reveal that seroma fluid resolution does not coincide with stromal recovery and identify persistent oxidative stress, inflammation, fibrosis, and vascular remodeling as important features of seroma pathology.

The outer blood-retinal barrier (OBRB) is the primary interface through which systemically circulating drugs reach the retina. A tool that measures delivery across this barrier would support the development of targeted therapies as alternatives to repeated intravitreal injection, and the screening of drugs that reach the retina as an off-target toxicity. Such a tool should deliver drugs fluidically through a vascular compartment, measure transport across the retinal pigment epithelium (RPE), and display disease phenotypes relevant to efficacy. Here we adapt the SiM platform, which places epithelium and endothelium in direct juxtaposition across a permeable, optically transparent silicon nitride nanomembrane. ARPE-19 and human umbilical vein endothelial cells (HUVECs) were used as development cell sources. ARPE-19 monocultures reached a transepithelial electrical resistance of 68 {+/-} 26 {Omega} cm2 by 28 days, and ARPE-19 + HUVEC co-cultures reached a small-molecule permeability of 6.34 {+/-} 1.3 x 10-4 cm min-1 within 14 days, a state reported elsewhere only after longer culture. The barriers developed an intervening basement membrane. Drugs perfused through the basal vascular channel crossed into an open apical well, where sampling and mass spectrometry showed transport correlating with lipophilicity, as reported in vivo. The device also displayed two clinically relevant phenotypes. Digoxin at a clinically toxic concentration reduced viability in the co-barrier by about half and doubled permeability. In a vascularized configuration, VEGF drove endothelial invasion of the RPE layer, as seen in neovascular AMD. The SiM-OBRB therefore satisfies basic design criteria for measurement of drug bioavailability, toxicity, and efficacy.

The ability to generate vascularized tissue constructs supplied by surgically accessible vessels remains a major limitation in reconstructive tissue engineering. Previous work demonstrated that positioning porous biomaterials around a vein can induce extensive vascular sprouting and generate a vascularized tissue volume without creation of an arteriovenous loop. However, this phenomenon has principally been demonstrated using rigid calcium-phosphate scaffolds, limiting its direct relevance to soft-tissue reconstruction. Here, we investigated whether a flexible decellularized dermal matrix could similarly be vascularized around a single unmodified vein and whether autologous dermal fibroblasts could further enhance this response. Decellularized rat dermis, with or without autologous dermal fibroblast seeding, was implanted either subcutaneously in the dorsal region or around the femoral vein for 8 weeks. Following Microfil perfusion, three-dimensional micro-CT demonstrated an extensive branching vascular network within perivenous constructs. Vessel volume relative to tissue volume was 0.2 +/- 0.1% in subcutaneous constructs without fibroblasts (n = 11) and 0.6 +/- 0.2% with fibroblasts (n = 9), compared with 6.+/- 1.4 % in perivenous constructs without fibroblasts (n = 10) and 7.3+/- 1.2% with fibroblasts (n = 10). Histology and immunohistochemistry similarly showed greater cellular repopulation and substantially higher CD31- and -SMA-positive vessel densities following perivenous implantation. Integrated two-way ANOVA of the same 8-week data confirmed a dominant effect of implantation site across the vascular outcomes, while fibroblast seeding significantly affected vascular volume, dermal remodeling, and nuclear density. The effect of fibroblast seeding on vascular volume was site dependent, with a significant implantation site fibroblast interaction. At 8 weeks, the vascularized dermal construct was completely dissected free from the surrounding tissues while maintaining its connection to the femoral vein. A small superficial incision into the isolated dermis produced visible bleeding, supporting an acute functional connection of the intradermal vascular network to the host circulation while the femoral venous connection was preserved. After flap elevation, perivenous constructs retained substantially greater vascular volume and showed more complete surface healing and organized skin architecture than subcutaneous constructs. Fibroblast seeding accelerated wound closure, while its effect on post-elevation vascular volume depended on implantation site: VV/TV decreased in subcutaneous constructs but increased in perivenous constructs. These findings show that single-vein vascularization can extend from rigid calcium-phosphate biomaterials to a compliant biological extracellular-matrix scaffold and may provide a simplified axial vascularization strategy without arterialization or incorporation of an arterial vessel.

Three-dimensional (3D) cell culture models, particularly multicellular tumor spheroids, have become essential tools for studying cancer biology, drug screening, and preclinical testing due to their ability to mimic physiological tumor microenvironments. However, traditional invasion assays, such as Boyden-chamber- or Transwell-based systems, often suffer from variability introduced by spheroid handling and transfer, compromising data reproducibility. Here, we present a novel, 3D-printed migration and invasion platform --the MQm-sert-- designed to standardize and streamline spheroid-based invasion assays while maintaining spheroid integrity. Fabricated via fused filament fabrication using biobased polylactic acid, the MQm-sert integrates a hanging-drop spheroid culture system (MQm-sert) with a membrane-based invasion chamber (M-sert), eliminating the need for disruptive spheroid transfer steps. Using synthetic tumor environment mimics (STEMs) composed of breast cancer cells (MDA-MB-231 and MCF7), mesenchymal stromal cells (MSCs), and human pulmonary microvascular endothelial cells (HPMECs), we quantified invasion dynamics and cellular interactions. This innovation significantly reduces experimental variability, as demonstrated by lower variance in invasive cell mass dimensions and cell density compared to conventional workflows. Beyond biological insights, the platform aligns with sustainability goals by leveraging cost-effective, open-source 3D printing, reducing reliance on commercial labware, and addressing key challenges in 3D cell culture standardization.

The cortical neuron is a fundamental building block of the mammalian brain, and the morphology of its axonal projections is central to how functional circuits assemble. The trajectory along which an axon grows is a key determinant of connectivity, yet the kinematics of cortical axon outgrowth remain poorly quantified. Characterizing these dynamics is most tractable in vitro, where axonal growth can be measured directly and under controlled, reproducible conditions. Even in culture, however, this remains challenging because cortical neurons require dense plating for viability, and their soma is motile, so growth behavior is highly sensitive to local density and population context, complicating reproducible measurement of intrinsic dynamics. To overcome these limitations, we used size-controlled cortical neurospheres, which provide a fixed spatial origin and a reproducible environment, together with a custom semi-automated tracking pipeline to quantify single-axon kinematics across two functionalized substrates and two developmental phases. This approach revealed a substrate-dependent divergence in outgrowth: during the later developmental phase, axons on poly-D-lysine with laminin (PDL-LA) substrate grew faster than those on PDL, with a mean step size of 0.436 versus 0.339 {micro}m /min. Decomposing trajectories into Katz dynamic states, we built a generative biased random walk model that reproduces axonal behavior at both microscopic (single-axon) and macroscopic (network topology) scales. This open, reproducible framework links single-axon kinematics to network architecture, enabling the structural connectivity of neurospherebased circuits in vitro to be predicted from measurable growth dynamics, a necessary foundation for future studies linking circuit structure to emergent function.