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Biocompatibility and cytotoxicity study

For this in vitro experiment, particle-conditioned media was introduced to the cells and incubated over time with images taken at different time points. Particle-conditioned media was prepared by dissolving the particles in the aforementioned Caco-2 culture media at 1mg/mL for 24 hours at 37ºC in an incubator. The mixture was filter-sterilized through a 0.22μm syringe filter, since our particles should all be smaller than 0.22μm in diameter.

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Cells were detached from the T75 flask using trypsin, counted using trypan blue, and then seeded into a 24-well plate at a density of about 10,000cells/well. The plate was incubated for one day to allow the cells to attach to the wells and then the normal media was replaced with the particle-conditioned media as previously described. At three different time points (8, 24, 48 hours), optical images were taken to assess cell growth and morphology in the particle-conditioned media.

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Figure 4 (a-c): Optical microscopy images of Caco-2 incubated in particle-conditioned media at time points 8 hours, 24 hours, and 48 hours.

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Figure 4 shows the results of the in vitro assay at the three predetermined time points. As we can see, the cells migrated and aggregated around the silk microparticles. In Figure 4a, some microparticles are visible with Caco-2 cells floating around them. In Figures 4b and 4c, however, we can see the cells clumping up around the microparticles and become less spread out. This is due to a couple of factors. First, there was not much volume of media in each well, so the cells migrated to the edges of the well. The second conclusion that can be taken from these images is that the microparticles have an affinity for the cells, and vice versa. Figure 4c was taken with lower magnification so that the cellular aggregation around the microparticles could be more clearly seen.

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These results showed that the particle-conditioned media had no harmful effects on the cells, as they continued to proliferate, and the microparticles and cells had an affinity to each other. This affinity is an important aspect of this project, as the basis of this delivery system relies on the microparticles adsorbing to the lung epithelium and releasing a drug compound.

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