Biology subjects

Franco, R. A.

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

4 recordsLinked to original sources

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

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↗