C. Koehler1, W. Zhang1, J. R. Walsh1, R. A. Naqvi1, A. M. Chen1, E. Gramelspacher1, L. J. Smith2,3, P. Li1, B. Ekser1 1Indiana University School Of Medicine,Transplant Surgery/Department Of Surgery,Indianapolis, IN, USA 2Indiana University School Of Medicine,Department Of Radiology And Imaging Sciences,Indianapolis, IN, USA 3Indiana University School Of Medicine,3D-Bioprinting Core,Indianapolis, IN, USA
Introduction: Hepatic stellate cells (HSC), which compromise ~2-8% of liver cells, are vital to hepatocellular function. Scaffold-free 3D-bioprinting (SF3DBP) offers an avenue for the creation of realistic organ models without the use of biomaterials. Therefore, we hypothesized that co-culturing primary hepatocytes with HSC in SF3DBP liver model would uphold hepatocyte function over time, providing us a better 3D-liver model for research.
Methods: We used freshly thawed primary pig hepatocytes and immortalized pig HSC to generate spheroids with (i) hepatocytes alone, (ii) HSC alone, or (iii) a combination of hepatocytes and HSC. Spheroids were formed using low adhesion plates, then characterized for distance from well center, diameter, roundness, and smoothness. A column of spheroids was printed using a Regenova 3D-bioprinter. Remaining loose spheroids were incubated over two weeks for functionality assays (albumin secretion, mRNA transcription, urea clearance). Optimized spheroids of hepatocytes and HSC were cultured and printed to create a SF3DBP liver model.
Results: Co-cultures of hepatocytes and HSC (2.5:1 ratio) formed spheroids within 48 hours, as did HSC alone spheroids. Spheroids composed of hepatocytes alone failed to form round spheroids (Figure 1). The combination spheroids increased in roundness and decreased in diameter between characterizations over 6 days. Functional assays showed maintenance of albumin secretion and increased urea clearance over 14 days in spheroids formed by combined cells. Real-Time PCR demonstrated increased albumin (hepatocyte marker) and CRBP-1 (HSC marker) mRNA expressions in combination spheroids. Optimized spheroids (hepatocyte/HSC) enabled the creation of a SF3DBP construct on day 4. The 3D construct fused by day 3 and was able to be removed from temporary microneedle support by day 6 (Figure 1).
Conclusion: Spheroids formed by HSC alone proved too large to print at 48 hours. SF3DBP of spheroids (formed by hepatocyte:HSC in 2.5:1 ratio) would be viable by day 6. Optimization in centrifuging and incubation time allowed combination spheroids (2:1 ratio) to print earlier. Being able to print spheroids on day 4 as compared to day 6 increases the utility of future constructs for pharmacological, immunological, and hepatotoxicity testing. Maintenance of functionality of gene expression and albumin secretion emphasizes the utility of the 3D-bioprinted model over a period of 14 days. Further optimization of spheroids using different cell ratios including HSC, hepatocytes, liver sinusoidal endothelial cells, cholangiocytes, and fibroblasts will allow for production and printing of more physiologically accurate liver models.
