Biology subjects

Murphy, A. R.

Publications and source records attributed to Murphy, A. R..

9 recordsLinked to original sources

bioRxiv2026

Marrow-mimicry MEW scaffolds recapitulate erythroblastic islands for high-density cRBC biomanufacturing

Biomanufactured cell therapies are delivering a step-change in modern medicine, but their high costs have prevented widespread adoption. Cultured red blood cells (cRBCs) are one type of cell therapy in clinical trials for off-the-shelf blood transfusion but remain limited by the exorbitant costs of manufacturing trillions of RBCs required for a blood transfusion. These costs could be mitigated if culture platforms could grow cells at higher density, like those found in the bone marrow, requiring less media and lower concentrations of expensive growth factors per cell. We engineered melt electrowritten (MEW) polycaprolactone (PCL) scaffolds in geometries which imitate marrow trabeculae structures and were then plasma-treated to enable peripheral blood mononuclear cells (PB MNC) attachment. PB MNC monocytes first adhered to the scaffold and rapidly differentiated into CD68+ macrophage-like cells. These macrophage-loaded scaffolds adhered CD71+ erythroblasts, which then grew from 10 to 25% between days 4 and 8 and produced CD235a+ erythroid cells by day 20, under erythrogenic culture conditions. MEW PCL scaffolds in trabecular-like geometries had greater surface area, adhered more macrophages, and generated more erythroblasts than traditional MEW crosshatch geometries. The MEW scaffolds continuously released less-adherent cells to the supernatant, providing a 74% increase in supernatant cell numbers versus liquid suspension controls with identical cell seeding numbers and media volumes. High-cell-density scaffolds exhibited increased metabolism and endogenous production of self-supportive cytokines, which may offer cost-saving opportunities through future media optimisation. This study reports marrow trabeculae-mimetic MEW PCL designs capable of selectively adhering PB MN cells to develop macrophage-coated scaffolds, which provided physical and paracrine support for cRBC production. This high-cell-density culture platform may be useful for future cell therapy biomanufacturing protocols.

bioengineering↗

bioRxiv2026

Spatially distributed growth factor environments for evaluating hydrogel-based CD34+ cell culture conditions

Suspension culture is the gold standard for ex vivo hematopoietic stem cell (HSC) expansion; however, high media requirement results in expensive cell therapy products. Hydrogel-based HSC culture represents a more realistic in vitro representation of human in vivo HSC microenvironments and may support a more efficient use of media during ex vivo HSC expansion. However, media conditions used for hydrogel-supported cell culture often simply mimic that which are currently used in suspension culture approaches, potentially resulting in an oversupply of cell growth proteins. In this work, we present a microfluidic culture system capable of supporting hydrogel-based cell culture and generating spatial distributions of independent growth factor combinations. Using this device, we image, in real-time, the behaviour of human umbilical cord blood-derived CD34+ cells, within fibrin-based hydrogels, in response to spatial gradients of the human recombinant growth factors stem cell factor, thrombopoietin, angiopoietin-2 and insulin-like growth factor-II. Using live microscopy and image-based single-cell segmentation and quantification, local changes in cell density over time in relation to spatial concentration of factors were identified. This novel microfluidic device has the potential to screen combinations of growth factors required for hydrogel-supported CD34+ cell expansion and inform culture media formulations of future hydrogel-based cell manufacturing processes.

bioengineering↗

bioRxiv2026

Identifying cytokine-release signatures of flow-driven endothelial remodelling in an intracranial aneurysm cell culture model

Intracranial aneurysm (IA) rupture is catastrophic, yet current models of rupture-risk inadequately capture underlying IA remodelling mechanisms. Endothelial-haemodynamic interactions are central to these processes, but in vitro flow platforms often lack vessel-relevant geometry or long-term perfusion. Here, temporal and spatial endothelial responses to haemodynamic stress were investigated across idealised and patient-specific vascular models. Polydimethylsiloxane models were endothelialised with human aortic endothelial cells then perfused at up to 1.6 Pa wall shear stress for five days. IA models were exposed to steady or cardiovascular flow waveforms, with endothelial phenotype assessed by immunofluorescence and cytokine profiling. Flow initiation induced a transient inflammatory response, with elevated MCP-1 and TNF- at day two, followed by a resolution of cytokine levels by day five, including a [~]7.5-fold reduction in MCP-1, despite increased haemodynamic loading. Endothelial cells retained a cobblestone-like morphology with eNOS undetected, resembling a partially activated phenotype. Compared with steady flow, cardiovascular flow reduced TGF-{beta}1 and IL-8 secretion and decreased FGF-b consumption ([~]2.5 fold), suggesting enhanced phenotypic stability. This study presents the first in vitro IA model incorporating a cardiovascular flow waveform and identifies cytokine signatures with potential utility as biomarkers of IA remodelling, highlighting the importance of long-term perfusion for modelling chronic vascular disease. Table of Contents FigureAn in vitro model of an intracranial aneurysm was developed to investigate how fluid flow dynamics impact endothelial remodelling and inflammation. Pulsatile cardiac flow promoted stabilisation of inflammatory signalling, which was sustained under a steady flow regime. Cytokine signatures emerged with potential utility as biomarkers of IA remodelling, highlighting the importance of long-term perfusion for modelling chronic vascular disease. The schematic of the cytokine release dynamics used in the graphical abstract below was generated with the assistance of AI-based tools including ChatGPT (v5.5) and M365 Copilot to align with key results from this manuscript. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=182 SRC="FIGDIR/small/733289v1_ufig1.gif" ALT="Figure 1000"> View larger version (80K): org.highwire.dtl.DTLVardef@709204org.highwire.dtl.DTLVardef@825f81org.highwire.dtl.DTLVardef@14c345eorg.highwire.dtl.DTLVardef@222a8_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

Microglial Fructose Metabolism Is Essential for Glioblastoma Growth

Glioblastoma (GBM) is most common and aggressive primary brain tumor in adults, for which standard of care hasnt changed in twenty years. GBM tumor associated macrophages (TAMCs), consisting of infiltrating myeloid cells from the periphery and resident microglia cells, are pro-tumorigenic, promoting tumor growth. Fructose is one of the most abundant metabolites in the GBM tumor microenvironment (TME), as well as in the healthy central nervous system (CNS). In the CNS and GBM, microglia are the predominant expressors of the fructose transporter GLUT5. Mice lacking the GLUT5 transporter (GLUT5-KO) survive significantly longer after orthotopic implantation of two glioma cell lines than wildtype mice, which is not due to dietary fructose or peripherally derived TAMCs. Investigation of the TME showed that GLUT5-KO mice have more highly activated and inflammatory innate and adaptive immune compartments. Microglia cultured in fructose have a decreased phagocytic ability and exhibit decreased inflammatory capacity due to the polyol pathway promoting redox homeostasis.

cancer biology↗

bioRxiv2025

Additive manufacturing of patient-specific intracranial aneurysm cell culture models

Intracranial aneurysms (IAs) are present in 2-6% of the global population. While rare, rupture results in mortality rates of 30-50% and lifelong disabilities in survivors. While treatment of unruptured IAs carries its own risk of mortality, there are no rigid guidelines indicating which IA presentation is at greater risk of rupture. We develop and evaluate the suitability of various additive manufacturing processes to fabricate patient-specific IA culture models for understanding IA pathophysiology and thereby support future development of a rupture risk prediction tool. Material compatibility of several 3D printed resins, polydimethylsiloxane (PDMS) and collagen gel with immortalised human brain endothelial cells (HBECs) were investigated. Patient angiograms were segmented to produce in vitro models via two fabrication approaches: stereolithography (SLA) 3D printing versus chocolate injection moulding of a sacrificial core embedded in PDMS. These 3D arterial models were then cellularised with HBECs, and geometric accuracy and distension properties evaluated. PDMS, collagen gel and the elastic50A resin materials supported cellularisation on material surfaces with high cell viability and proliferation. Both the 3D printed resin and injection moulding techniques successfully fabricated patient-specific basilar artery IA models with a Dice-Sorensen coefficient of over 90%. However, only PDMS models offered complete cell coverage in 3D geometries. This exceeds current benchmarks on IA fabrication accuracy. Through controlling IA model wall thickness, we demonstrate the ability to create localised distension under pressure in the context of thin- and thick-walled aneurysms. In vitro IA models present a promising platform for investigating IA pathophysiology and rupture risk. The novel chocolate sacrificial core technique offers a biocompatible, support-free fabrication method suitable for 3D cultures. Given the independent effects of fluid dynamics and mechanical strain on cell behaviour, it is essential to characterise distension under pressure and ensure accurate fabrication for reliable analysis into cell behaviour.

bioengineering↗

bioRxiv2025

The Batch-Resourcing Angiogenesis Tool (BRAT) to enable high-content microscopy screening of microvascular networks.

Vessel forming assays are a valuable in vitro technology to evaluate the vasculogenic and angiogenic potential of different cell types, matrix proteins, and soluble factors. Recent advances in high-content microscopy allow for vascular morphogenesis assays to be captured in real-time and in high-throughput screening purposes. Unfortunately, existing microvascular network quantification algorithms are either inaccurate, not user-friendly, or manually analyse one image at a time, unfavourable to high-content screening applications. This manuscript introduces BRAT, the Batch-Resourcing Angiogenesis Tool, a computer algorithm with an open-source graphic user interface to efficiently segment, skeletonize, and analyse large batches of vascular network images with high accuracy. Benchmarked across diverse clinical and cultured microvascular network images, BRAT is the most sensitive vascular network image analysis tool (94.5%) and exhibits leading accuracy (93.3%). BRATs multi-threaded processing automatically analysed 886 microscopy images at a speed of 0.17 seconds/image (2:29 minutes) on a performance computer or 2.31 seconds/image (34:04) on a typical laptop. This is 10-to-100 fold more time-efficient than existing tools, which require 12 to 16 seconds of direct user input per image. BRAT is broadly useful to analyse vessel networks of different endothelial cell types cultured on 2D substrates and within 3D biomaterials. BRAT represents a powerful approach for the accurate and high-content screening of vessel forming assays for disease models, regenerative medicines, and therapeutic testing.

bioengineering↗

bioRxiv2025

Fabricating microfluidic co-cultures of immortalised cell lines uncovers robust design principles for the simultaneous formation of patterned, vascularised, and stem cell-derived adipose tissue.

In vitro culture processes supporting the simultaneous formation of vessel networks alongside differentiation towards mature parenchymal tissue have numerous clinical and agricultural applications but remain unrealised due to contrasting culture requirements. Of specific interest is lab-grown vascularised adipose tissue to study metabolic syndrome, diabetes, obesity, cardiovascular diseases, and to advance cultivated meat technologies. We report a microfluidic 3D hydrogel culture device capable of supporting live-imaging of fluorescent reporter cell lines and generating counter-current gradients of vasculogenic and adipogenic growth factors. for the first time, we report experimental conditions capable of reproducibly forming diverse microvascular networks from telomerase immortalised endothelial and mesenchymal stem cells in both 2D and 3D hydrogel-embedded cultures. Using our novel microfluidic culture design, we demonstrate the generation of growth factor environments which support the 3D co-formation of integrated robust microvascular networks and lipid-producing adipocytes after 31 days gradient culture. We demonstrate microvascular networks substantially support parenchymal stromal cell differentiation to mature adipose tissue (67.4% lipid coverage), unachieved in avascular cultures (1.86% lipid coverage). We attempt to validate our co-culture model by applying inhibitors of vessel-mediated lipogenesis (spermidine and VO-OHpic), which are demonstrated to be ineffective in our novel human preclinical model. HighlightsO_LIAn optimised co-culture protocol for vasculogenesis of immortalised cell lines. C_LIO_LIImmortalised co-cultures form reproducible microvessel networks up to 31 days. C_LIO_LIGradient co-culture enables simultaneous MSC adipogenesis and EC vasculogenesis. C_LIO_LIMSC differentiation to dense, lipid-laden adipose tissue relies on EC co-culture. C_LIO_LIHuman vascularised adipose tissue formation is unaffected by a murine regulator. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/634386v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@4b3567org.highwire.dtl.DTLVardef@19f0278org.highwire.dtl.DTLVardef@5b3d1eorg.highwire.dtl.DTLVardef@9a5c9_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2022

Vitamin D receptor cistrome-transcriptome analyses establishes quantitatively distinct receptor genomic interactions in African American prostate cancer regulated by BAZ1A

BackgroundAfrican American (AA) prostate cancer (PCa) appears uniquely sensitive to 1,25(OH)2D3 signaling, compared to European American (EA) PCa, but the extent and impact of vitamin D receptor genomic functions remain poorly defined. ResultsA panel of EA and AA prostate epithelial cells (EA: HPr1-AR, LNCaP, AA: RC43N, RC43T, RC77N, RC77T) were analyzed with RIME to reveal the cell-specific composition of the VDR- complex. 1,25(OH)2D3-dependent ATAC-Seq revealed the greatest impact on nucleosome positioning in RC43N and RC43T, with gain of nucleosome-free at enhancer regions. VDR ChIP-Seq identified stronger and more frequent VDR binding in RC43N and RC43T that was enriched for a larger and distinct motif repertoire, than EA cells. VDR binding significantly overlapped with core circadian rhythm transcription factors in AA cell line models. RNA-Seq also revealed significantly stronger 1,25(OH)2D3 dependent VDR transcriptional responses enriched for circadian rhythm and inflammation networks in AA cells. Whilst RC43N was most responsive, RC43T displayed distorted responses. Significantly reduced BAZ1A/SMARCA5 in AA PCa samples was identified, and restored BAZ1A expression uniquely and significantly increased 1,25(OH)2D3-regulated VDR targets in AA cells. These VDR- dependent cistrome-annotated genes were also uniquely and most significantly identified in three cohorts of AA PCa patients. ConclusionThese data suggest VDR transcriptional control in the prostate is more potent and dynamic in AA men, and primed to govern inflammatory and circadian pathways. Reduced BAZ1A/SMARCA5 expression and/or reduced environmentally-regulated serum vitamin D3 levels suppress these actions. Therefore, the VDR axis lies at the cross-roads of biopsychosocial processes including stress responses, access to quality early detection and treatment, social determinants and that collectively contribute to PCa health disparities.

cancer biology↗

bioRxiv2019

In utero estrogenic endocrine disruption alters the stroma to increase extracellular matrix density and mammary gland stiffness.

In utero endocrine disruption is linked to increased risk of breast cancer later in life. Despite numerous studies establishing this linkage, the long-term molecular changes that predispose mammary cells to carcinogenic transformation are unknown. Several lines of evidence indicate the stroma mediates endocrine disruption following early-life (or in utero) exposure. Herein, we utilized BPA as a model of estrogenic endocrine disruption to analyze the long-term consequences in the stroma. Using RNA-seq transcriptional profiling of adult primary fibroblasts isolated from female mice exposed to BPA in utero, we identified deregulated genes associated with the extracellular matrix. Specifically, multiple collagen genes had increased expression in exposed mice. In line with the transcriptional data, collagen deposition is increased in adult BPA-exposed mice. We further demonstrate in vitro that fibroblasts exposed to BPA in utero remodel a collagen matrix, thereby decreasing permeability of the collagen matrix. These alterations to the mammary gland resulted in increased gland stiffness in the adult mice. Our data connects early life endocrine disruption to breast density. Interestingly, increased collagen deposition and gland stiffness were not observed in the developing glands of younger mice, suggesting risk factors for breast cancer continue to develop throughout life following these exposures. Finally, we assessed whether in utero exposure to two other endocrine disruptors, BPS and DES, also increase breast stiffness in adult mice. While DES increased breast stiffness, BPS did not, suggesting this BPA alternative may in fact pose less breast cancer risk than its predecessor. As breast stiffness, extracellular matrix density, and collagen deposition have been directly linked to breast cancer risk, these data mechanistically link endocrine disruptor exposures and molecular alterations to increased disease susceptibility in the gland.

cancer biology↗