Speaker
Description
Introduction
Tubular tissues are ubiquitous in the body, from blood vessels and lymph ducts to bile and breast ducts. Evaluating drug efficacy for the treatment of pathophysiological conditions using in vitro models is crucial before initiating animal and clinical trials. A major problem with current in vitro models is the discrepancy between in vitro and in vivo results. This discrepancy exists mostly because the adopted in vitro models do not faithfully mimic the in vivo cellular and extracellular composition and dimensionality characteristics. Engineering a three-dimensional (3D) tubular model to mimic tubular tissues would be a significant step in reducing such discrepancies. We have manufactured a chip made of polymeric scaffolds to mimic the 3D architecture of tubular tissues. Currently, the model is being used to study breast and blood brain barrier tissues.
Methodology
Cell attachment: The chips were manufactured in the workshop and were covered with either collagen-I or poly-L-lysine. One milliliter of media with a cellular concentration of 300,000 cells per milliliter was placed on the chip. At different timepoints (0.5, 2 and 8 hours), the chips were washed with PBS and stained with DAPI/Calcein-AM to view and count the cells that attached.
Cell seeding: Cells were seeded in the channel at various densities (5, 10 and 20 million cells per milliliter) and monitored, with images taken on days 1, 4 and 7. On day 7, cells were stained with DAPI/Calcein-AM. Cell coverage of the channels was determined during the various days.
TEER measurements: Impedance spectroscopy was measured using a two-electrode setup with 2 Ag/AgCl electrodes. The impedance spectra were measured between 3 distinct points in the chip; the inlet, well, and the outlet.
Normal and cancerous breast cells co-culture: HMT-3522 S-1 and GFP labelled T-4 cells were co-cultured in the channel in a S-1:T-4 ratio of 10:1 to evaluate the interaction of these two cell lines. Cells were stained with DAPI and imaged after 7 days to monitor the growth of S-1 and T-4 cells.
Results
Cell attachment: Experiments showed that Collagen-I was a better attachment substrate than PLL.
Cell seeding: Seeding 20 million cells per milliliter proved most promising whereby cell coverage of the channel was adequate, and cells proliferated indicating metabolic activity. Lower seeding densities left vast areas of the channel lacking cellular coverage, even after 7 days in culture.
TEER measurements: TEER measurements showed that the impedance from either inlet/outlet to the center well were approximately half of the impedance from the inlet to the outlet of an empty chip. This measurement will allow us to investigate possible interferences of the channel structure on the TEER measurements to be carried out when complete cell coverage is achieved.
Normal and cancerous breast cells co-culture: HMT 3522 T-4 cells outgrew the S-1 cells significantly, with the vast majority of cells exhibiting GFP labelling.
Conclusion
The proposed tubular model shows promise to achieve biomimetic architecture of tubular tissues. This design would enable researchers to better evaluate the efficacy of drugs on tissues due to the improved biomimetic architecture.
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