Introduction:: Most cells within the human body interact with neighboring cells and extracellular matrix (ECM) components to establish a unique 3D organization. These cell–cell and cell–ECM interactions form a complex communication network of biochemical and mechanical signals critical for normal cell physiology. Cells in a 3D environment behave fundamentally differently from cells in monolayer (2D) culture [1-3]. Aggregation can manipulate influence cell-cell interactions, resulting in enhanced cell proliferation [1-4]. Conventional cell culture is performed in 2D systems, which is very different from the local environment of cells in living tissues. Therefore, 3D cell culture technologies have been developed [4,5]. We have previously reported that KP24 is a peptide that induces uniform-sized cell aggregates only in cell suspension [6,7]. By immobilizing this peptide on the substrate, we thought that it could be applied to a three-dimensional culture substrate that can induce cell aggregates with controlled size. Therefore, in this study, mouse fibroblasts (L929) and human Mesenchymal Stem Cells (hMSC) were seeded on a glass substrate on which KP24 was immobilized, and the cell behavior was observed. In this study, KP24 is chemically fixed on the surface of the substrate; so, it is possible to trace the mechanism of interaction between KP24 and the cells surface. These results can contribute to the development of regenerative medicine such as cell transplantation substrates and microarrays.
Materials and Methods:: KP24, a 12-cycle repeating sequence consisting of Lys and Pro, was synthesized manually by the Fmoc solid-phase synthesis method. All peptides were purified by RP-HPLC, subsequently, characterized by HPLC, MALDI-TOF-MS, NMR, and amino-acid analysis. By immobilizing the synthesized KP24 from the end, two types of KP24-immobilized base materials having different surface functional groups were prepared (Figure 1). Immobilization of KP24 was evaluated by analyzing the wettability of the substrate surface and surface elements. In the cell experiment, L929 was seeded on the KP24-immobilized substrate to observe whether the cell aggregate could be induced on the substrate. Furthermore, the viability of cells involved in cell assembly formation was evaluated by Live Dead staining. Three-dimensional analysis of cell aggregates induced on the P24-immobilized substrate was performed using Mil-Cell (Sumitomo Electric Industries, Ltd. Osaka, Japan) and 3D imaging analysis software. Based on the observation results of Mil-Cell, the three-dimensional shape and sphericity of each cell aggregate were compared.
Results, Conclusions, and Discussions:: On the first day after seeding, L929 was adherent and extended over all substrate’s surfaces, and no changes were observed between substrates. However, on day 4 after seeding, cell aggregates were induced on two KP24-immobilized substrates, KP24 (COOH) and KP24 (NH2) substrate. Observation on day 7 after seeding showed that induction of cell aggregates was complete. The sequence of KP24 contains 12 residues of lysine, a basic amino acid with a positive charge. This positive charge was thought to induce the cell through electrostatic interaction with the negative charge of the cell membrane. However, the amino groups on the main and side chains of KP24 interacted with the carboxyl groups on the CMTES (COOH) substrate surface, weakening the positive charge and inhibiting the induction of cell assembly. It is speculated that the reason for the formation of cell aggregates on the KP24 (COOH) substrate is that some amino groups of KP24 do not interact with the carboxyl groups on the substrate. Most of the cell aggregates induced on the KP24 (COOH) substrate were less than 100 µm in diameter. In contrast, many cell aggregates larger than 100 µm in diameter were induced on the KP24 (NH2) substrate. The average diameter of L929 cell aggregates induced on KP24 (COOH) substrate was 86.6 µm. On the KP24 (NH2) substrate, cell aggregates with an average diameter of 122.9µm were induced. Live/Dead staining results showed a small number of dead cells in all substrates as well as inside the cell aggregates. The outer cells of the cell assembly are in direct contact with the culture medium and are easily supplied with factors such as nutrients and oxygen. However, the inner cells have difficulty receiving these factors. Therefore, a small number of dead cells were observed inside the cell aggregates.
In experiments with L929 seeded on the prepared substrates, the formation of cell aggregates was observed on the KP24 immobilized substrates. When hMSC cells were seeded onto the KP24-immobilized substrate, cell aggregation was observed around day 4, as in the case of L929, and aggregation was observed to be complete by day 7.
Acknowledgements (Optional): : This work was financially supported by MEXT–Private University Research Branding Project (2016–2019), the Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Scientific Research and Kansai University Research Branding Project.
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