Controlled release of basic fibroblast growth factor from a water-floatable polyethylene nonwoven fabric sheet for maintenance culture of iPSCs

Abstract

Basic fibroblast growth factor (bFGF) is an essential supplement for culture media to support the proliferation of human pluripotent stem cells, while preserving their pluripotency. However, it is extremely unstable under cell culture conditions at 37 °C. Therefore, a culture medium supplemented with bFGF needs to be changed every day to maintain an effective concentration of bFGF. This study aimed to create a bFGF-releasing material via simple bFGF adsorption following oxygen plasma treatment by using a water-floatable polyethylene (PE) nonwoven fabric sheet as a bFGF-adsorbent material. Preliminary oxygen plasma treatment enhanced bFGF adsorption onto the sheet by increasing its surface water wettability. Based on the bFGF concentration in the adsorption solution, the resulting bFGF-adsorbed sheet showed different bFGF-release profiles in the culture medium. The bFGF-adsorbed sheet prepared under optimum conditions released bFGF in a sustained manner, maintaining the bFGF concentration in the culture medium of human induced pluripotent stem cells (iPSCs) at ≥10 ng mL^-1 even without medium change for as long as 3 d. The bFGF released from the sheet retained its biological activity to support colony formation of iPSCs while preserving their pluripotency. This type of bFGF-releasing sheet can be used as a new form of bFGF supplement for the culture media of stem cells and would make a significant contribution to stem cell-based research and development.

Fig. 1. (a) Lower (upper) and higher (middle, lower) magnification SEM images and (b) contact angles (average + SD; n = 3; *p < 0.05) of an ultrapure water droplet on the surfaces of the P00, P05, P10, and P20 sheets. (c) Digital camera (upper) and side-view schematic (lower) images of the P00 (left) and P10 (right) sheets that were placed over the colored aqueous solution.

Fig. 2. (a) C_1s XPS spectra of the surfaces of the P00 and P10 sheets and (b) FT-IR spectra of their surfaces after 24 h adsorption using the 12 μg mL⁻¹ bFGF solution under shaking conditions. Inset in (a) shows C_1s XPS spectra enlarged in the y-axis direction to represent the formation of functional groups.

Fig. 3. Release profiles of bFGF in the acellular culture medium from the P00, P05, P10, and P20 sheets after 24 h adsorption using the 4 μg mL⁻¹ bFGF solution under static conditions (average ± SD; n = 3) compared with the concentration change of free bFGF (initial concentration = 10 ng mL⁻¹) in the culture medium.

Fig. 4. Variation with bFGF adsorption period of the bFGF residual rate in the 4 μg mL⁻¹ bFGF solution with and without the P10 sheet under (a) static and (b) shaking conditions (average ± SD; n = 3). Inset in (b) shows a comparison between the static (triangles) and shaking (circles) conditions with the P10 sheet.

Fig. 5. (a) Amounts of bFGF adsorbed on the surfaces of the P10F0, P10F0.5, P10F1, P10F2, P10F4, P10F8, and P10F12 sheets and (b) those plotted as a function of the equilibrated bFGF concentration in the bFGF solution after adsorption for 24 h (average ± SD; n = 3).

Fig. 6. Release profiles of bFGF in the acellular culture medium from the P10F0, P10F2, P10F4, P10F8, and P10F12 sheets (average ± SD; n = 3).

Fig. 7. (a) Release profiles of bFGF from the P10F12 sheet in the culture medium with and without iPSCs and (b) bFGF concentrations in the culture medium just before daily medium change at Days 1, 2, and 3 in the conventional culture of iPSCs with free bFGF (10 ng mL⁻¹) (positive control) in Culture 1 (average ± SD; n = 3; *p < 0.05).

Fig. 8. Phase contrast microscopic images of iPSCs cultured with the P10F12 sheet without medium change (upper) or with (positive control; middle) and without (negative control; lower) free bFGF (10 ng mL⁻¹) with daily medium change at Day 3 in Culture 1 (left) and Culture 2 (right). Insets show higher magnification images.

Fig. 9. (a) Number of viable iPSCs per well and (b) viability of iPSCs after Culture 1 with the P10F12 sheet without medium change or with free bFGF (10 ng mL⁻¹) with daily medium change (positive control) (average + SD; n = 3; *p < 0.05).

Fig. 10. Fluorescence microscopic images of iPSCs cultured with the P10F12 sheet without medium change (upper 2 rows) or with free bFGF (10 ng mL⁻¹) with daily medium change (positive control; lower 2 rows). The iPSCs were stained with FITC (green)-conjugated rBC2LCN (left), anti-Nanog (middle), and anti-Oct-3/4 (right) and their nucleus were counterstained with DAPI (blue) after Culture 2. Insets show higher magnification images.