(B-47) Development of an Alginate-based Cell-Adhesive Microbeads

Introduction:: Topological features of the scaffold material influence the cellular behavior on the surface [1]. To elucidate basic rules of how such topological feature change the cellular response, curved surface has drawn considerable attention because curvature is a useful parameter defining the scaffold-surface geometry simply and systematically. Numerous studies have recently shown that curved surfaces at a several-hundred micron scale, which is an order of magnitude larger than a single cell, have a significant influence on cellular behaviors including tissue organization and differentiation [2]. But the molecular biological mechanisms remain unexplained. A new cell culture technique is desirable for clarifying the molecular mechanisms underlying such substrate-curvature-dependent cellular behaviors. This study aims to develop a new scaffold material that allows us to culture cells on a curved surface with a defined radius and harvest them for potential biochemical analysis.


Materials and Methods:: To culture cells on a curved surface with a defined radius, this study produces alginate-based hydrogel beads whose sol-gel transition can be simply controlled by Ca²⁺ concentration, applying a centrifuge-based microdroplet formation method [3]. Sodium alginate solution was filled inside a capillary stabilized to the rid of a centrifuge tube. CaCl₂ solution was then prepared inside a centrifuge tube. Centrifugation forces the sodium alginate solution to drop from the capillary into the CaCl₂ solution and it immediately solidified into calcium alginate beads. By varying the rotational speed and the distance from the capillary tip to the CaCl₂ solution surface (surface distance), beads with different diameters and circularity were produced. In addition, cRGD-modified alginate was added to the naïve alginate solution (weight ratio 1:3) to obtain cell-adhesive beads. Human aortic vascular smooth muscle cells (HASMC) were cultured on cRGD-mixed microbeads of 1000 µm diameter to check the cytotoxicity.

Results, Conclusions, and Discussions:: We first examined the controllability of the diameter of the microbeads. By sweeping the centrifuge speed from 200 to 1200 rpm, the diameter of the beads decreased monotonically from 1300 to 400 µm. When the liquid surface distance was 1.6 cm, beads enough to be called true spheres, i.e., 0.95 or more circularity of the peripheral shape, were produced at all the centrifuge speeds. We produced microbeads of 1000 µm diameter containing cRGD-modified alginate and then checked the cytocompatibility by culturing HASMCs on the beads. HASMCs seeded on the microbeads spread well on the bead surface and could be cultured for 72 hours (Figure 1); this observation confirms that the beads provide a suitable environment for cells to adhere and did not show cytotoxicity to the cells. This new microbead platform provides a new method to culture cells on curved surfaces of defined radius and thus contribute to the studies investigating the curvature-dependent cellular behaviors.
This study reported a new method to produce alginate-based cell-adhesive microbeads with defined radii using a centrifugation-driven droplet generation method. The cytocompatibility of the microbeads was confirmed. This method would contribute to the studies pursuing curvature-dependent unique cellular behaviors.

Acknowledgements (Optional): :

References (Optional): : [1] C. A. Dessalles et al., Commun. Biol., 2021, 4:764

[2] S. J. P. Callens et al., Biomaterials, 2020, 232:119739

[3] S. M. Kang et al, Sci. Rep., 2019, 9:12776