Heterotypic interactions in the preservation of morphology and functionality of porcine hepatocytes by bone marrow mesenchymal stem cells in vitro

Abstract

Temporary replacement of specific liver functions with extracorporeal bioartificial liver has been hampered by rapid de-differentiation of porcine hepatocytes in vitro. Co-cultivation of hepatocytes with non-parenchymal cells may be beneficial for optimizing cell functions via mimicry of physiological microenvironment consisting of endogenous matrix proteins. However, the underlying mechanisms remain to be elucidated. A randomly distributed co-culture system composed of porcine hepatocytes and bone marrow mesenchymal stem cells was generated, and the morphological and functional changes of varying degrees of heterotypic interactions were characterized. Furthermore, contributions of extracellular matrix within this co-culture were evaluated. A rapid attachment and self-organization of three-dimensional hepatocyte spheroids were encouraged. Studies on hepatocyte viability showed a metabolically active, viable cell population in all co-culture configurations with occurrence of few dead cells. The maximal induction of albumin production, urea synthesis, and cytochrome P4503A1 activities was achieved at seeding ratio of 2:1. Immunocytochemical detection of various extracellular matrix confirmed that a high level of matrix proteins synthesis within distinct cells was involved in hepatocyte homeostasis. These results demonstrate for the first time that cell-matrix has synergic effects on the preservation of hepatic morphology and functionality in the co-culture of porcine hepatocytes with mesenchymal stem cells in vitro, which could represent a promising tool for tissue engineering, cell biology, and bioartificial liver devices.

Microbiological safety of a novel bio-artificial liver support system based on porcine hepatocytes: a experimental study

“…A co-culture system of porcine hepatocytes and MSCs was prepared as described previously [14]. In brief, bone marrow was aspirated from the iliac crest of the pigs, then the mononuclear cells were collected over a Ficoll histopaque layer by gradient centrifugation (20 minutes, 400 g, density 1.077 g/ml) and seeded at a density of 1 × 10 6 cells/cm 2 in growth medium containing low-glucose DMEM supplemented with 10% FBS, 100 IU/ml penicillin and 100 mg/ml streptomycin.…”

“…The bioreactor is composed of a stack of 65-layer round flat plates and a cylindrical container with an inlet on the top and an outlet on the bottom. A co-culture system of porcine hepatocytes and mesenchymal stem cells (MSCs), which had been established previously in our institute [14], was perfused into the bioreactor to act as functional cells, and a new extracorporeal BAL support system based on the novel multi-layer radial-flow bioreactor and the co-culture system of porcine hepatocytes and MSCs. was constructed.…”

Mesenchymal Stem Cells: Current Clinical Applications and Therapeutic Potential in Liver Diseases

“…Qihao et al showed the supportive effect of adult liver cells on hepatic differentiation of rat MSCs. They observed spheroid formation of the differentiated cells which could express liver specific markers such as albumin, α-fetoprotein and cytokeratin-18 at gene and protein levels [56]. Moreover, the heterotypic interaction of porcine hepatocytes/MSCs at ratio 2:1 was found as an optimal ratio for hepatic lineage induction.…”

Bioartificial liver devices: Perspectives on the state of the art

“…Our investigations indicated that co-cultivation could promote the activities and functions of hepatocytes by forming spherical aggregates, increasing direct contact between the liver cells and other cells; co-cultured non-parenchymal cells can promote the liver cells' function. The component of the extracellular matrix secreted by non-parenchymal cells could provide a favorable growth microenvironment for hepatocytes [15][16][17][18][19].…”