[3S06e] 16. Cell and Tissue Engineering for Biomedical Process Innovation - Part 3

5:30 PM - 5:50 PM JST(8:30 AM - 8:50 AM UTC)

[3CTBP-12] Development of Porous Scaffolds Using Functional Nanofibers with Controlled Cell Aggregates Dimension for Tissue Engineering

○Yasuhiro Ikegami¹, Hiroyuki Ijima¹ (1. Kyushu Univ. (Japan))

Keywords:

Electrospun nanofiber,Growth factor,Porous scaffold

[Purpose]
Cell aggregates allows cells to survive and maintain their specific functions after transplantation. However, oxygen and nutrients can reach the interior of the aggregates only by diffusion. Therefore, controlling the distance from the surface to the center of each aggregate is essential for efficient substance supply. In this study, we developed a morphology-controlled porous construct composed of electrospun nanofibers (Designed Fiber Porous Construct, dFPC). Biodegradable polymer fibers were fragmented and molded into designed shapes, enabling controlling of both cell aggregate distribution and substance transport through the inter-fiber voids. Moreover, the electrospun fibers were functionalized with heparin which has binding affinity with various kinds of growth factors to enhance their local concentration, thereby facilitating the construction of highly functional tissues for transplantation.
[Method]
Electrospun fibers prepared with 7wt% PCL/gelatin solution were fragmented in followed by ultrasonication. The resulted short fibers, 3wt% gelatin aqueous solution, and ethanol were sequentially poured into a 3D-printed mold to entangle the fibers. The dFPCs were obtained by incubating the constructs at 55°C for 1h and subsequently freeze-dried. The resulting dFPCs were insolubilized using chemical crosslinkers (i. e. EDC and NHS) followed by functionalization using a mixture of heparin and chemical crosslinkers to grant a growth factor immobilizability. Primary rat hepatocytes and their cell aggregates (i.e. spheroid) were seeded in heparin-functionalized dFPC and cultured with growth factors. The cell distribution within the porous scaffold was observed through H&E staining, and their gene expression on hypoxia and apoptosis was tested to assess the cell survival. Liver-specific functions (e.g. albumin secretion and drug metabolism) of the hepatocytes were evaluated with ELISA and EROD assay after 7-days of culture.
[Results & Consideration]
The dFPC replicated the designed mold geometry while retaining its porous structure on the scaffold surface. Furthermore, permeability test demonstrated that the dFPC exhibited a higher mass diffusion coefficient than a gelatin sponge and showed no significant decrease compared with diffusion coefficient in water. Primary rat hepatocytes cultured on the dFPC formed aggregates that localized within the scaffold grooves. Lower level of hypoxia and apoptosis marker of hepatocytes in dFPC suggested that the porous scaffold enhanced the cell survival more than other scaffolds with the same shape, that implying that the fibrous porous structure promoted abundant exchange of oxygen, medium components, and cellular metabolites. Also, hepatocytes in heparin-functionalized dFPC showed higher liver-specific functions compared to that of heparin-free dFPC, which might be caused by enhanced local concentration of the growth factor immobilization.
[Conclusion]
Taken together, the heparin-functionalized dFPC achieved both controlled distribution of cell aggregates and efficient substance exchange to maintain survived cell and enhanced their liver-specific functions via growth factor immobilization for superior cell transplantation.