Speaker
Description
While both, regenerative medicine and tissue engineering shift from 2D based models towards 3D models, problems related to imaging of those difficult constructs became more relevant than ever. One of the most attractive technics used by the aforementioned fields is 3D bioprinting based on hydrogel scaffolds. Unfortunately, due to the complexity of the growth kinetic of cells in the printed hydrogels imaging choices and approaches can vary, especially due to multiple morphological architypes of cell growth.
We have tested multiple Gelatin/Alginate based hydrogels as a growth scaffold for two cell lines with different growth kinetics to determine the tendencies of scaffold population and morphological preferences of tested cells. We have also determined best imaging practices for such hydrogels, scaffolds maintaining sterile conditions.
Mouse Fibroblasts (3T3 cell line) and Human Keratinocytes (KerCT cell line) were grown in hydrogels for a maximum of 4 weeks and observations of key growth events were performed both with live imaging and fixated sample imaging using fluorescence technics. Gel biocompatibility was tested using custom Calcein AM staining. Observations of the residual cell layer post- hydrogel transfer was also performed during the experiment.
Hydrogels containing 6% Gelatin and 2% Alginate used in this study were based on the, recommended growth medium: DMEM/F12K for 3T3 and KBM Gold for KerCT cell line. After adding DAS (Dialdehyde starch) / SQ (Squaric acid) 0,5 milion cells / ml of gel were added and mixed. The final step was crosslinking the hydrogels with calcium chloride and washing. Hydrogels were incubated in a cell growth CO2 controlled incubator with the dedicated growth medium and the media was replaced every 72hours. When excessive cell growth was observed on the bottom of the culture vessel, hydrogel scaffolds were migrated to a fresh vessel.
Both hydrogel types were found biocompatible with the tested cell lines. We have observed a crucial relationship between both, growth kinetics and the morphological architypes of cells depending on the hydrogel thickness and degradation level. The 3T3 cell line favored rapid growth in damaged areas of hydrogels where the observed morphology was most comoplex. KerCT cells did also exhibit faster growth in the same areas, but due to the naturally slower growth kinetics it was less visible. Cell growth observed within the thicker areas of gels (above 200 µm) was considerably slower, and occurred after partial gel degradation nearing the third week of cell culture. For both cell lines spheroid formation was occurring near the peripheral areas, but was more common for hydrogels crosslinked using SQ.
The research was funded by NCBiR (MATURO 3D project) and from internal CM UMK idub equipment grant.
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