Functionally Enhanced Human Stem Cell Derived Hepatocytes in Galactosylated Cellulosic Sponges for Hepatotoxicity Testing.

Pluripotent stem cell derived hepatocyte-like cells (hPSC-HLCs) are an attractive alternative to primary human hepatocytes (PHHs) used in applications ranging from therapeutics to drug safety testing studies. It would be critical to improve and maintain mature hepatocyte functions of the hPSC-HLCs, especially for long-term studies. If 3D culture systems were to be used for such purposes, it would be important that the system can support formation and maintenance of optimal-sized spheroids for long periods of time, and can also be directly deployed in liver drug testing assays. We report the use of 3-dimensional (3D) cellulosic scaffold system for the culture of hPSC-HLCs. The scaffold has a macroporous network which helps to control the formation and maintenance of the spheroids for weeks. Our results show that culturing hPSC-HLCs in 3D cellulosic scaffolds increases functionality, as demonstrated by improved urea production and hepatic marker expression. In addition, hPSC-HLCs in the scaffolds exhibit a more mature phenotype, as shown by enhanced cytochrome P450 activity and induction. This enables the system to show a higher sensitivity to hepatotoxicants and a higher degree of similarity to PHHs when compared to conventional 2D systems. These results suggest that 3D cellulosic scaffolds are ideal for the long-term cultures needed to mature hPSC-HLCs. The mature hPSC-HLCs with improved cellular function can be continually maintained in the scaffolds and directly used for hepatotoxicity assays, making this system highly attractive for drug testing applications.

A novel design of bioartificial kidneys with improved cell performance and haemocompatibility.

Treatment with bioartificial kidneys had beneficial effects in animal experiments and improved survival of critically ill patients with acute kidney injury in a Phase II clinical trial. However, a Phase II b clinical trial failed. This and other results suggested various problems with the current design of bioartificial kidneys. We propose a novel design to improve various properties of device, including haemocompatibility and cell performance. An important feature of the novel design is confinement of the blood to the lumina of the hollow fibre membranes. This avoids exposure of the blood to the non-haemocompatible outer surfaces of hollow fibre membranes, which usually occurs in bioartificial kidneys. We use these outer surfaces as substrate for cell growth. Our results show that commercial hollow fibre membranes can be directly applied in the bioreactor when human primary renal proximal tubular cells are grown in this configuration, and no coatings are required for the formation of robust and functional renal epithelia. Furthermore, we demonstrate that the bioreactor unit produces significant amounts of interleukins. This result helps to understand the immunomodulatory effects of bioartificial kidneys, which have been observed previously. The novel bioartificial kidney design outlined here and the results obtained would be expected to improve the safety and performance of bioartificial kidneys and to contribute to a better understanding of their effects.

Tethered primary hepatocyte spheroids on polystyrene multi-well plates for high-throughput drug safety testing.

Hepatocyte spheroids are useful models for mimicking liver phenotypes in vitro because of their three-dimensionality. However, the lack of a biomaterial platform which allows the facile manipulation of spheroid cultures on a large scale severely limits their application in automated high-throughput drug safety testing. In addition, there is not yet a robust way of controlling spheroid size, homogeneity and integrity during extended culture. This work addresses these bottlenecks to the automation of hepatocyte spheroid culture by tethering 3D hepatocyte spheroids directly onto surface-modified polystyrene (PS) multi-well plates. However, polystyrene surfaces are inert toward functionalization, and this makes the uniform conjugation of bioactive ligands very challenging. Surface modification of polystyrene well plates is achieved herein using a three-step sequence, resulting in a homogeneous distribution of bioactive RGD and galactose ligands required for spheroid tethering and formation. Importantly, treatment of polystyrene tethered spheroids with vehicle and paradigm hepatotoxicant (chlorpromazine) treatment using an automated liquid handling platform shows low signal deviation, intact 3D spheroidal morphology and Z' values above 0.5, and hence confirming their amenability to high-throughput automation. Functional analyses performance (i.e. urea and albumin production, cytochrome P450 activity and induction studies) of the polystyrene tethered spheroids reveal significant improvements over hepatocytes cultured as collagen monolayers. This is the first demonstration of automated hepatotoxicant treatment on functional 3D hepatocyte spheroids tethered directly on polystyrene multi-well plates, and will serve as an important advancement in the application of 3D tethered spheroid models to high throughput drug screening.

Characterization of membrane materials and membrane coatings for bioreactor units of bioartificial kidneys.

The bioreactor unit of bioartificial kidneys contains porous membranes seeded with renal cells. For clinical applications, it is mandatory that human primary renal proximal tubule cells (HPTCs) form differentiated epithelia on the membranes. Here, we show that HPTCs do not grow and survive on a variety of polymeric membrane materials. This applies also to membranes consisting of polysulfone/polyvinylpyrrolidone (PSF/PVP), which have been used in the bioreactor unit of bioartificial kidneys after coating with an extracellular matrix (ECM). Our data reveal that coating with just an ECM does not sufficiently improve HPTC performance on non-HPTC-compatible membrane materials. On the other hand, we have characterized the effects of a variety of surface treatments and coatings, and found that double coating with 3,4-dihydroxy-l-phenylalanine and an ECM markedly improves HPTC performance and results in the formation of differentiated epithelia on PSF/PVP membranes. We have also synthesized alternative membrane materials, and characterized membranes consisting of polysulfone and Fullcure. We found that these membranes sustain proper HPTC performance without the need for surface treatments or coatings. Together, our data reveal that the materials that have been previously applied in bioartificial kidneys are not suitable for applications with HPTCs. This study elucidates the types of membrane materials and coatings that are favorable for the bioreactor unit of bioartificial kidneys.