Automated manufacture of functional human liver tissue

Introduction

At present, most advanced tissue models are manufactured one at a time by-hand in a laboratory setting. Commercial-level production of even simple tissues (e.g. cells on a scaffold), much less tissues with more complex architectures and multiple components, require more effective manufacturing solutions. Using an agile, robotic biomanufacturing platform developed by our group, we manufactured a thick, human liver tissue and compared the outcomes to liver tissues fabricated by hand. The platform involves a 6-axis robotic arm, capable of multiple automatic tool exchanges, with a modular tissue culture incubator and a confocal scanner uniquely integrated for the automated manufacturing of tissues and organs.

Methods

Fabrication of the vascularized liver tissue involves the 3D printing of a sacrificial mold to structure the tissue and provide surfaces for oxygen/nutrient exchange. The tissue itself is comprised of primary human hepatocytes, non-parenchymal cells, and adipose-derived microvessel fragments. The fabrication entails multiple tasks involving 3D printing, cell dispensing, media pipetting, incubation, and imaging. After loading the platform with the cells and media, the automated manufacturing recapitulates these tasks automatically, in a closed, clean environment. Liver tissue constructs were grown for up to two weeks with a media exchange every 24 hours. Culture supernatants were collected and analyzed for the presence of urea and LDH.  At the end of each experiment liver tissues were snap-frozen or fixed and analyzed for gene expression via real-time PCR or were sectioned and stained with αSMA, UEA-1, and CD45. Comparisons were made between tissues built with and without human microvessels and between manual and automated fabrication.

Results

We have leveraged a 3D printed, sacrificial molding technique to create vertical channels within a thick, dense hepatocyte-rich tissue. The presence of these channels increased surface area to tissue volume ratios for oxygen/nutrient exchange. Analysis of culture supernatants indicated a slight drop in urea levels from day 1 to day 14. LDH levels, an indicator of cell injury, were high on day 1 and reduced to near zero at day 7 and day 14. Fabricated liver tissues express albumin, MRP2, and Cyp3A4 genes. Finally, there was no difference in these measurements between liver tissues fabricated manually or automatically using the robotic platform.  

Conclusion

In this study, we demonstrated, the automated manufacture of thick, human, liver tissue constructs from primary cell sources. The liver tissues are cell-dense and fabricated using an approach amenable to changes in composition and scaling in construct number and size. Importantly, the automated fabrication of the vascularized liver tissue did not compromise tissue health or function. We expect the automation fabrication of the vascularized liver tissue, at the point of use and using off-the-shelf platforms, eases building the complex model and increases its utility.