Liver-specific functions

To be useful to both, clinicians and the pharmaceutical industry, a bioartificial liver will need to maintain a large number of hepatocytes at high cell densities and in a fully differentiated state for prolonged periods of time. Development of such a system has been impeded by three principal problems a) a requirement for large numbers of cells (>25 10 ) b) loss of liver-specific functions in cultured cells (primary and immortalized) c) nutrient and waste product gradients in high density cultures leading to lowered cell viability and impaired function. 

Cell lines established from human liver cancer cells can be derived from primary hepatocellular carcinoma or hepatoblastoma cells [10]. For example, the well known HepG2 fine was derived from hepatoblastoma cells. Such cells can be employed effectively if the function of normal liver cells has been highly preserved. In practice, however, such cells contain a high proportion of abnormal genetic component, which inhibits their ability to express normal protein synthesis and enzyme activity. Potential problems, such as the loss of liver specific functions and the possibifity of metastasis, which could arise from the use of hepatoma cells have not yet been satisfactorily discussed [13]. 

The answer to the shortage of hver tissue is to evolve from a dependence on whole and partial organs to the use of hepatic cehs. Ceh therapies range from the injection of ceh colonies with the hope that they wih take up residence and become chnicahy active to the development of implantable or extracorporeal devices. Such approaches must consider both the sources of hepatocytes and stabilization of liver-specific functions. 

The performance of modified and unmodified membranes evaluated by analyzing the expression of liver-specific functions in terms of albumin production is shown in Figure 19.3. Hepatocytes cultured on unmodified PES membranes produced... 

While primary hepatocytes maintained under conventional culture conditions tend to undergo rapid loss of liver-specific functions, great progress has been made in the last decade to slow this process. In our opinion, the three most important factors in retaining CYP responsiveness in primary hepatocyte are media formulation, matrix composition, and cell-cell contacts (10-13). 

Hepatocytes are attachment-dependent cells [3, 25]. Without adequate extracellular matrix they lose their liver-specific functions. Therefore, it has been studied intensively which types of matrix, hydrogels, and scaffolds support differentiated hepatocyte functions and which are the key properties. One of the first commercially available hydrogels was Matrigel, an extract from mouse sarcoma cells [26]. Later Extragel, another collagen-based hydrogel [27] Alimatrix, a porous 3D scaffold [28] and PuraMatrix... 

B.J. Kane, M.J. Ziimer, M.L. Yarmush and M. Toner, Liver-specific functional studies in a microfluidic array of primary mammalian hepatocytes, Analytical Chemistry, 78(13), 4291-1298 (2006). 

FSS, whereby a FSS higher than 5 dyns/cm can impede their liver-specific functions [23]. FSS also plays a role in the extravasations of tumour cells as tumour-endothelial cell interactions are dependent on shear [24]. 

Stably immobilized 3D flat aggregates and exhibited superior cell bioactivity with higher levels of liver-specific function maintenance in terms of albumin secretion, urea synthesis and cytochrome P-450 enzyme than 3D spheroid aggregates formed on GC films. These results suggested that the GC-based nanofibrous scaffolds could be useful for various applications such as bioartificial liver-assist devices and tissue engineering for liver regeneration as primary hepatocytes culture substrates. 

It is known that when hepatocytes are freshly isolated and cultured under conventional conditions on plastic or collagen coated dishes, gene transcription is drastically depressed and liver specific mRNA declines while the mRNA of structure related genes increases many fold. By contrast, hepatocyte culture on extracellular matrix (ECM), rich in laminin, type IV collagen, and heparin sulfate proteoglycan exhibits increased longevity and maintenance of several liver-specific functions. 

Coculture is necessary to establish the necessary heterotypic interactions for performance of liver-specific functions. Hepatocytes and fibroblasts were patterned using microcontact printing [7]... 

De Bartolo, L., Morelli, S., Lopez, L.C. et al. 2005. Biotransformation and liver-specific functions of human hepatocytes in culture on RGD-immobiUzed plasma-processed membranes. 26 4432-4441. 

Ranucci, C. S., and Moghe, P. V. (1999), Polymer substrate topography actively regulates the multicellular organization and liver-specific functions of cultured hepatocytes. Tissue Eng. 5(5) 407-420. 

De Bartolo et al. reported the viability of human hepatocytes in a multibore hollow-fiber bioreactor and their liver specific functions, when human hepatocytes were cultured in the intraluminal compartment of the bioreactor. Human hepatocytes maintained their round shape on the membranes and showed focal adhesions as sites of interaction with the membrane surface they expressed fiver functions, including synthetic and detoxification activity. The results demonstrated that human hepatocytes cultured in the multibore fiber bioreactor are able to sustain the same in vivo fiver functions in vttro. ... 

Gotoh, Y., Ishizuka, Y., Matsuura, T., Niimi, S., 2011. Spheroid formation and expression of liver-specific functions of human hepatocellular carcinoma-derived FLC-4 cells cultured in lactose-silk fibroin conjugate sponges. Biomacromolecules 12, 1532—1539. 

Figure 6.6 Comparison of liver-specific functions such as albumin secretion (a), ammonia elimination (b), and EROD activities (c) in AL and AL/GC sponges for monoculture and co-culture of hepatocytes with NIH3T3. (a) Albumin secretion rates for both systems were measured over 28 days in culture. ELISA specific for mouse albumin were used to quantify protein in 24h conditioned medium, (b) Ammonia elimination was compared between both systems on day 5 and 16 of culture, (c) EROD deethylase activity by cytochrome P4501A1 was compared between both systems on day 6 and 16 of culture. EROD activity was measured using the highly fluorescent resomfin in both systems. All data are represented as mean SDj jj (n = 6). Statistics (two-tailed paired Smdent s f-test) compared monoculture with co-culture P<0.05 and "P <0.01.

In addition, liver-specific functions of hepatocytes in the alginate/XG scaffolds were slowly decreased with culture time, whereas those were rapidly decreased in the alginate ones. 

Mice hepatic-layered cell sheets, which express human al-antitrypsin, are transplanted into mice subcutaneous locations with neovascular networks (Ohashi et al., 2007). After transplantation, liver-specific functions such as albumin production and drug metabolism are improved and kept for longer than 200 days. In addition, the transplanted cell sheet has an ability to proliferate and grow in response to a regenerative stimulus, which is induced by the resection of two-thirds of the host liver (Ohashi et al., 2007). 

Kim, K., Ohashi, K., Utoh, R., Kano, K., Okano, T. (2012). Preserved liver-specific functions of hepatocytes in 3D co-culture with endothelial cell sheets. Biomaterials, 33, 1406-1413. 

Liu, Y., li, H., Yan, S., et al. Hepatocyte cocultures with endothelial cells and fibroblasts on micropattemed fibrous mats to promote liver-specific functions and capillary formation capabilities. Biomacromolecirles 15,1044—1054 (2014). doi 10.1021/bm401926k... 

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