Efficient and high-density immobilization of animal cells by a microfiber with both swelling and cell adhesion properties and its application to exosome production

Abstract

Purpose: For high-density cell culture, we studied the development of optimal microfibers (MFs) with a 0.1-10 μm diameter, which due to their large surface area can serve as an immobilization carrier for animal cells. To date, few studies have used MFs as scaffolding for high-density cell culturing.

Results: Using six types of nonsoluble synthetic polymers, MF sheets were fabricated by electrospinning. The cellulose acetate, polyketone, and polyvinyl acetate MFs exhibited swelling and water retention capacities. Next, the six types of MF fragments were examined for immobilizing TKD2 mouse vascular endothelial cells. Although most cells were taken into the three MFs characterized by swelling, most leaked from the MFs without adhesion. To solve this, the MF sheets comprising cellulose acetate and polyketones were coated with gelatin. Although the adhesive capacity was enhanced, the swelling capacity decreased and almost all the immobilized mouse cells remained on the sheets' surfaces. Based on these results, we produced a novel MF sheet comprising a gelatin, cellulose acetate, and polyketone mixture (CPG). Since the cells were taken into the MFs by swelling and attached by the gelatin, the CPG fragment immobilized almost all the supplied cells with little loss and reached a high density of 3.2 × 10⁹ MF-g^-1, Furthermore, the immobilized cells continuously produced exosomes with a high productivity of 6-7 × 10¹⁰ particles ml^-1 after either 8 h or 16 h of culturing.

Conclusion: CPG-based MFs are expected to have a wide range of future applications, including exosome production from animal cells.

Keywords: Electrospinning; Exosomes; High-density immobilization; Microfiber; Swelling.

Fig. 1

SEM images of MFs

Fig. 2

Hydrophilic properties of MF sheets. a Average diameter of MFs. PS* is the spherical portion of the PS MF (n = 50, mean ± SD). b Static contact angles (initial contact angle at 0.1 ms) of MFs after droplet drop. c Percentage of the volume of water retained in the MF sheet without uptake relative to the volume of the initial droplet, when a droplet was dripped to the MF sheet. d Water retention ratio relative to MF mass (n = 3, mean ± SD)

Fig. 3

Immobilization characteristics of TKD2 cells to dishes, films, and MFs. a Image of TKD2 cells cultured on a dish. b Average growth per dish and glucose consumption in the medium when TKD2 cells were cultured on dishes (n = 2). c Cell number attached films made of synthetic polymers when TKD2 cells were cultured for 4 days (n = 3, mean ± SD). d The number of cells that were not taken into the MF fragments and the amount of rest medium when cell suspensions (1 × 10⁵ cells) were added into microtubes containing MF fragments (4.0 mg) and incubated for 16 h. e Change over time in the number of immobilized cells when TKD2 cells were cultured with 4 mg of MF fragments for 22 days. The number of inoculated cells is 1 × 10⁵. (mean ± SD, n = 3), and the number of immobilized cells was determined with Cell Counting Kit 8. f SEM images of immobilized TKD2 cells in PLA MF made (15 th day culture)

Fig. 4

Immobilization of cells with GEL-coated MF fragments. a Change over time in the number of immobilized cells when TKD2 cells were cultured with 4 mg of GEL-coated MF fragments made of CA and PK for 22 days. The number of inoculated cells is 1 × 10⁵ and the number of immobilized cells was determined with Cell Counting Kit 8. (mean ± SD, n = 3). b Images of MFs in 48 well plate and SEM images of immobilized TKD2 cells in GEL-coated PK- and CA MFs. (15 day culture). c Numbers of exosome particles per medium when TKD2 cells were immobilized in GEL-coated PK and CA MFs (4 mg) were respectively cultured in 0.5 ml of EV-up and ExoFBS-DMEM media for 2 days. The operation was repeated three times. (mean ± SD, n = 3)

Fig. 5

Immobilization of cells with CPG MF fragment. a Infrared spectra of CPG, PK, CA, and GEL MFs. b SEM images of CPG MF (upper side photo) and immobilized cells in CPG MF (12 day culture). c Procedure for immobilizing cells in CPG MF fragments. (1) MF fragment. (2) Drop cell suspension in the MF fragment. (3) Cultured MF swollen with cell suspension for over 6 h. d Change over time in the number of immobilized cells when TKD2 cells were cultured with 4 mg (0.60 mm thickness) and 0.3 mg (0.25 mm thickness) of CPG MF fragments. The number of inoculated cells is 1 × 10⁵ and the number of immobilized cells was determined with Cell Counting Kit 8 (mean ± SD, n = 3). e Numbers of exosome particles per medium when TKD2 cells immobilized in MFs were cultured in Exo-FBS-DMEM media. Three different conditions were applied: (i) 0.5 ml of medium per fragment and incubated for 2 days, (ii) 0.25 ml of medium per fragment and incubated for 1 day, and (iii) 0.125 ml of medium per fragment and incubated for 16 h and 8 h. The operation was repeated eight times for each condition