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)
References:
[1] Lin, R.-Z., and Chang, H.-Y. (2008) Biotech. J., 7, 1172-1184.
[2] Griffith, L. G., Swartz, M. A. (2006) Nat. Rev. Mol. Cell Biol., 7, 211-224.
[3] Pampaloni, F., Reynaud, E. G., Stelzer, E. H. (2007) Nat. Rev. Mol. Cell Biol., 8, 839-845.
[4] Futaki, Y., and Hirano, Y. (2014) Peptide Science, 2013, 373-374.