Creating a lung epithelial cell barrier on flexible, porous poly(trimethylene carbonate) (PTMC) membranes
Overig (New in vitro models)INTRODUCTION
Organs-on-chips can more accurately mimic in vivo situations than traditional in vitro models since they introduce mechanical forces and microfluidics to cells. The membranes on which the cells grow, however, often lack the necessary cytocompatibility and mechanical properties for a given tissue due to the choice of material (1). Therefore, flexible, porous membranes made of more cytocompatible materials could stimulate in vitro studies in lung research.
In this study, poly(trimethylene carbonate) (PTMC), a flexible, cytocompatible polymer was used to fabricate porous membranes via evaporation-induced phase separation (EIPS) (2) to determine if Calu-3 lung epithelial cell layers could be cultured on them. Moreover, another goal was to determine if the membranes could be tailored to positively influence the behaviour of the cells by changing the porosity, permeability and coating of the membranes.
Polymer solutions were made, including among others PTMC and hexanol. The porosity and permeability of the membranes were altered by changing the hexanol amount. The solutions were cast on silicon wafers, after which EIPS was performed. UV-light was then used to crosslink the membranes, after which they were washed and dried. Membranes were placed in Transwell® inserts after which membranes were left uncoated or provided with a cell-adhesive coating based on a catecholamine and extracellular matrix proteins. Calu-3 cells were then seeded on the membranes, left submerged until confluence and then grown at the air-liquid interface. Cell morphology, viability and barrier function were then determined via immunofluorescent staining, live/dead staining and transepithelial electrical resistance (TEER) measurements, respectively. Commercial inserts with poly(ethylene terephthalate) (PET) membranes were used as controls.
Porous PTMC membranes were made by EIPS. Changing the amount of hexanol in the polymer solution resulted in membranes with different porosities and permeabilities. Calu-3 cells only formed confluent cell layers on membranes containing the cell-adhesive coating. Proper cell morphology was only seen on membranes with higher porosity and permeability. Live/dead staining showed no difference between cells on these highly porous/permeable PTMC membranes and cells in inserts with PET membranes. Moreover, the barrier function of cells on these membranes was also similar, with TEER values for Calu-3 cells on coated PET membranes and coated, highly porous/permeable PTMC membranes of 179,8 ± 16,4 Ω*cm2 and 186,2 ± 87,4 Ω*cm2, respectively.
Providing the membranes with the cell-adhesive coating was required for proper cell attachment. Although PTMC is cytocompatible, the fabrication process probably resulted in a high hydrophilicity of the membranes which hampers cell attachment. Our data suggest that the higher porosity and permeability of the membranes increased access of the cells to nutrients and allowed for better waste disposal, which is favourable for cell culture. These characteristics of the cell culture substrate are thus very relevant for the outcome of in vitro studies.
Calu-3 cell layers can be cultured and maintained on PTMC membranes with an appropriate coating, porosity and permeability. These membranes can be used as novel, adaptable substrates for creating lung cell barriers in vitro, e.g. in a lung-on-a-chip.
1. Pasman T. et al., Flat and microstructured polymeric membranes in organs-on-chips. J R Soc Interface. 2018.
2. Zhao J. et al., Preparation of microporous silicone rubber membrane with tunable pore size via solvent evaporation-induced phase separation. ACS Appl Mater Interfaces. 2013.
The authors would like to thank the Lung Foundation Netherlands (Grant no: 6.1.14.010) for providing financial support to this project.