Development of a 3D system to study endothelial cell-smooth muscle cell interactions

1,2 1 1Jonathan Sheard ,* Graeme Cottrell and Darius Widera

[1] Stem Cell Biology and Regenerative Medicine Group, School of Pharmacy, University of Reading, Whiteknights campus, Reading, RG6 6AP, United Kingdom[2] Sheard BioTech Ltd, 20-22, Wenlock Road London N17GU, United Kingdom* Correspondence: Jonathan Sheard - E-mail: jonathan@sheardbiotech.com

Introduction. Blood vessels are composed of two main cell types, endothelial cells (ECs) and smooth muscle

cells (SMCs), which make up a dual layered structure. An elastic membrane, (internal elastic lamina) separates a

single concentric layer of ECs from the secondary surrounding layer of SMCs. Aim. We hypothesise that ECs

not only communicate via extracellular vesicles (EVs) over intermediate and long distances but also signal to

neighbouring SMCs in the myoendothelial junction (MEJ) using the same paracrine mechanism. The overall goal

of this project is to use nanofibrillar cellulose hydrogels (NFC) to create a physiological model of the EC and SMC

layers, whilst studying EV-mediated communication between these ECs and SMCs.

Endothelial Cells and Smooth Muscle Cells are viable and grow within ® ®GrowDex and GrowDex T in 3D

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® ®ECs and SMCs show distinctly different morphologies when seeded within either GrowDex or GrowDex T whilst 4 4maintaining viability compared to 2D. ECs and SMCs were seeded at different seeding concentrations (5x10 or 10x10

® ®cells/well) within different densities of GrowDex (GDx) or GrowDex T (GDxT- 0.1%, 0.2% and 0.4%). Cell morphology (A) was

observed and cell viability (B) tested with XTT assays following 48hrs of culture and it was noted that ECs are more viable in ®GrowDex T. Bar: all images 1000mm.

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ECs and SMCs were cultured as semi 2D cultures ON TOP of either GrowDex or GrowDex T. ECs and SMCs were seeded ® ®

at different seeding concentrations (1x10 , 2.5x10 , 5x10 or 10x10 cells/well) on top of either GrowDex (GDx 1.5%) or 4 4 4 4 ®

GrowDex T (GDxT 1.0%). Cell morphology (A) was observed and cell viability (B) tested with XTT assays following 48hrs of ®

culture and it was noted that ECs and SMCs are more viable on GrowDex T than GrowDex . Bar: all images 1000mm.® ®

Endothelial Cells and Smooth Muscle Cells are viable and grow ON © ©TOP of GrowDex and GrowDex T in Semi 2D

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©Endothelial Cells bind to 3D GrowDex T tubules

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ECs adhere to GrowDex T tubules, forming a confluent monolayer of cells. ECs were seeded onto tubules formed from ®

GrowDex T (GDxT 1.0%). Confocal imaging shows detailed tubule morphology, cell morphology, cell-cell organisation, as well ®

as actin fillament organisation (phalloidin) in 2D maximum intensity projection images (A) and 3D reconstruction images (B). ECs

did not migrate into the GrowDex T tubule.®

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Viral Transduction of SMCs with and RFP protein allows easier identification during EC/SMC co-culture

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SMCs were transduced with an RFP-blastocidin resistance lenti virus, whilst EC and SMC viability was tested within

alternative culture conditions. Blastocidin resistant (B) SMCs express RFP (A) allowing easier identification within co-culture

conditions. XTT viability was measured from ECs and SMCs seeded at different seeding concentrations within either MCDB131

(ECs basal media) or DMEM (SMCs basal media).

Results. We used GFP expressing HMEC-1 cells as model ECs and A10 cells as model SMCs. We have been

able to determine the optimal 3D cultivation conditions and viability of ECs and SMCs within and on the surface of

GrowDex® and GrowDex®T. We have successfully transduced A10 cells (SMCs) with an RFP construct for

better identification when in co-culture. Finally, we have used GrowDex®T to form long tube like constructs,

coated the outer layer of the constructs with a thick layer of ECs and cultured them for up to 4 weeks. Summary. In

summary, we have been able to develop a novel co-culture model of GFP-ECs and RFP-SMCs to study the cell-

cell interactions on an NFC based scaffold.