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bioRxiv · 10.1101/2025.01.22.634386

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.

Murphy, A. R.·Franco, R. A.·Allenby, M. C.
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Abstract

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

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Murphy, A. R., Franco, R. A., Allenby, M. C.. 2025-01-24. 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.. https://doi.org/10.1101/2025.01.22.634386

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