Albumin Production Activity Research Articles

Evaluation of cell aggregation induced peptide for 3D culture

Event Abstract Back to Event Evaluation of cell aggregation induced peptide for 3D culture Yoshiaki Hirano1*, Yudai Futaki1* and Sachiro Kakinoki1* 1 Kansai University, Faculty of Chemistry, Materials and Bioengineering, Japan Introduction: Most cells within the human body interact with neighboring cells and extracellular matrix (ECM) components to establish a unique three-dimensional (3D) organization. These cell-cell and cell-ECM interactions form a complex communication network of biochemical and mechanical signals that are critical for normal cell physiology. The behavior of cells in a 3D environment is fundamentally different from that of cells in monolayer culture, however. For instance, both primary articular chondrocytes and hepatocytes rapidly lose their normal phenotype once taken out of the body and placed in two-dimensional (2D) cell culture, but this loss can be attenuated or even reversed using 3D culturing methodologies[1]-[3]. Aggregation can affect cell-cell interactions, resulting in enhanced biological functions. Conventional cell culture involves the use of 2D systems, which differ significantly from the local environment of cells in living tissues. Therefore, 3D cell culture technologies have been developed. However, when a cell aggregate becomes too large, the cells inside the aggregate tend to die due to a lack of oxygen and nutrients. Such conditions often make it difficult to culture cells in vitro for long periods of time, thereby impeding basic research into cell differentiation and organization. The periodic peptide induces the formation of fibroblast[4], hepatocyte, human mesenchymal stem cell (hMSC) aggregates. In the present study, we evaluated (Lys-Pro)12 [KP24] derivative periodic peptides, such as (Lys-Pro-Pro)8[KPP24] and (Lys-Lys-Pro)8[KKP24], with respect to the relationship between the structure of the peptide chain and the function of hepatocyte, and hMSC aggregates. Materials and Methods: Four different periodic peptides were synthesized using a solid phase peptide synthesis procedure. All peptides were characterized by HPLC, MADI-TOF-MS, and amino acid analysis. The effect of the periodic peptides on cell aggregation was evaluated using albumin production of hepatocyte, and differentiation activity of hMSC for osteogenesis. Results and Discussion: IIn the presence of peptide in cell culture medium, cell aggregation was observed beginning on the third day of culture, and the cell aggregations formed maintained their shape over the 7-day culture period. The KP24, and KPP24 peptides induced the formation of uniformly sized cell aggregates. Albumin is regarded as a key biomarker of mature hepatocytes. Albumin production was comparable in cell aggregation and 2D monolayer cultures on day 1, increased dramatically on day 3, and maintained in a relatively stable level from day 3 to 7 in both cultures. Four types periodic peptides induced cell aggregation exhibited a relatively higher albumin production with the 2D monolayer. Results of ALP activity about hMSC were presented on the day. Conclusions: Periodic peptides induced cell aggregation exhibited a relatively higher albumin production activity with the 2D monolayer on hepatocyte. This result was applied for drug metabolism, cell transplantation and micro array. This work was partly supported Grant-in-Aid for Scientific Research (C)「KAKENHI-25350556」from Japan Society for the Promotion of Science (JSPS)

Development of hepatocyte-embedded hydrogel-filled macroporous scaffold cultures using transglutaminase

We aimed to develop cell-embedded gel-filled macroporous scaffold (CGS) culture technology as a fundamental technology for the construction of large-scale tissue-like structures, which will be indispensable for practical liver tissue engineering. Tissue transglutaminase (tTGase) from guinea pig liver showed no cytotoxicity toward primary hepatocytes, and hepatocyte-embedded gelatin gel cultures could be realized. The albumin production activities in tTGase-mediated hepatocyte-embedded gelatin gel cultures were similar to those in collagen gel cultures, representing an established hepatocyte culture method for the expression of liver-specific functions. Therefore, it was expected that a hepatocyte-embedded gelatin gel culture system enabling high liver-specific function expression could be created. The CGS culture system was created by incubating a suspension of hepatocytes and tTGase-containing gelatin-filled hydrophilic-treated scaffolds at 37 °C. The albumin production activities in CGS with poly( l-lactic acid) macroporous scaffold (porosity = 68.4%) were inferior to those in tTGase–gelatin gel cultures because of a diffusion problem. However, the activities were similar between CGS with macroporous polyurethane foam (porosity = 98.8%) and tTGase–gelatin gel cultures, even under stationary conditions. On the other hand, further functional improvements of hepatocytes were achieved in HGF- or HGF/heparin-containing gel cultures. Based on these results, tTGase-mediated CGS cultures are expected to become a fundamental technology for the creation of engineered liver tissues.

Albumin production activity of primary rat hepatocytes is improved on type V collagen

Primary rat hepatocytes were cultured on type V collagen. Hepatocyte spreading on type V collagen was inferior compared with that on type I collagen. However, the albumin production rates of hepatocytes cultured on type V collagen were approximately twice as high as those of hepatocytes cultured on type I collagen.

Composition of culture medium is more important than co-culture with hepatic non-parenchymal cells in albumin production activity of primary rat hepatocytes, and the effect was enhanced by hepatocytes spheroid culture in collagen gel

Co-culture of primary rat hepatocytes with hepatic non-parenchymal cells or sinusoidal endothelial cells for albumin production activity as an index of liver-specific function was studied. The co-cultures were effective for the expression and maintenance of albumin production activity. However, the co-culture effect was not observed when we used a suitable culture medium, which had already been reported to be sufficient for albumin production activity. Albumin production of dispersed cells in collagen gel culture was higher than that of spheroid culture. In addition, albumin production of spheroids in collagen gel culture was higher than that of spheroid culture and dispersed cell collagen gel culture with a suitable culture medium. We found that culture medium composition was more important than co-culture for expression and maintenance of albumin production. Furthermore, we found that cell–cell interaction was effective for the expression of albumin production, but heterotypic cell–cell interaction was not necessary.

Development of a practical small-scale circulation bioreactor and application to a drug metabolism simulator

We developed an easy-to-use, small-scale circulation-type bioreactor system that enables the simultaneous evaluation of many specimens. Medium flow was generated by a magnetic stirrer in this system. Primary rat hepatocytes formed a monolayer, and there were no morphological differences between cells in circulation and stationary cultures. The mitochondrial activity of hepatocytes in the circulation culture was 23% lower than that in the stationary culture after 2 days of culture. On the other hand, albumin production activity in the circulation culture after 2 days of culture was 1.4 times higher than that in the stationary culture. Albumin production activity per cell in the circulation culture was 1.9 times higher than that in the stationary culture after 2 days of culture. In addition, lidocaine metabolism rate per cell in the circulation culture was 1.3 times higher than that in the stationary culture. The lidocaine clearance of the circulation culture in our circulation-type bioreactor was 1.3 times higher than that of the stationary culture. It was shown that this bioreactor is suitable for the expression of the liver-specific functions of primary rat hepatocytes. Therefore, we can expect that this circulation-type bioreactor system will be a practical drug metabolism simulator.

Monolayer culture of primary rat hepatocytes on an Arg-Gly-Asp (RGD)-immobilized polystyrene dish express liver-specific functions of albumin production and p-acetamidophenol metabolism the same as for spheroid culture

The aim of this study was to evaluate the expression of albumin production and drug metabolism activities of primary rat hepatocytes under a two-dimensional monolayer culture condition formed on a RGD-immobilized culture dish. Albumin production activity on the RGD-immobilized dish (2500 ng cm −2 Pronectin F-coated dish) was 1.6 ( p < 0.05) and 1.2 ( p < 0.05) times higher than that on the collagen-coated dish and that of the spheroid culture, respectively. p-Acetamidophenol (APAP) metabolism rate of the collagen-monolayer at 5 days of culture was decreased to 16% of the activity at 1 day of culture. On the other hand, APAP metabolism activities of the RGD-monolayer and spheroid cultures were well maintained during 7 days of culture. However, we could not detect the effectiveness of the RGD-monolayer on lidocaine metabolism of hepatocytes. This is the first report suggesting that a hepatocyte monolayer on a RGD-immobilized culture substratum expresses albumin production and APAP metabolism activities equal to those of a hepatocyte spheroid culture.

Expression of a liver-specific function by a hepatoblastoma cell line cocultured with three-dimensional endothelialized tubes in collagen gels

A human hepatoblastoma cell line, Hep G2, showed albumin production activity, a hepatic function, when cocultured with three-dimensional endothelialized tubes in collagen gels. The albumin production rate of the collagen-based liver-like constructs increased with increasing length of the endothelialized tube in the construct.

Spheroid formation and expression of liver specific functions of primary rat hepatocytes co-cultured with bone marrow cells

We have developed a new co-culture system consisting of adhesive bone marrow cells (A-BMCs), non-adhesive bone marrow cells (NA-BMCs) and hepatocytes with improved hepatocyte immobilization efficiency and better maintenance of liver specific functions. The composition of the inoculated cells affected the morphology of hepatocytes. Spheroids formed spontaneously when rat hepatocytes were co-cultured with bone marrow cells (BMCs). However, the addition of NA-BMCs to existing hepatocyte monolayers did not change their morphologies to spheroids. On the other hand, NA-BMCs dramatically increased hepatocyte immobilization efficiency. Hepatocytes co-cultured with either NA-BMCs or A-BMCs maintained their albumin production activities significantly better than hepatocyte culture alone. Interestingly, the two fractions of BMCs appear to have combination effects on hepatocytes to maintain albumin production activity. We conclude that co-culture of hepatocytes and BMCs is an effective strategy to enhance hepatocyte immobilization efficiency and functions in vitro.