The Effects of Static and Dynamic Culture Systems on Cell Proliferation and Conditioned Media of Umbilical Cord-derived Mesenchymal Stem Cells

Preclinical and clinical studies have demonstrated the therapeutic effects of umbilical cord-derived mesenchymal stem cells (UC-MSCs) and secretome to cure various degenerative diseases. Thus, the mass-scale production of MSCs is necessary to ensure their availability and cost-effectiveness. In the current study, we evaluated the effect of dynamic 3D and static 2D culture systems on cell proliferation and conditioned media of UC-MSCs.  The lysate of concentrated thrombocyte was used to substitute animal-derived serum in the culture media. From two experimental sets with different UC and lysates of concentrated thrombocyte donors, it was found that the shortest PDTs for experimental set 1 were 12.3 h (2D culture) and 14.8 h (3D culture), whereas in experimental set 2, they were 17.7 h (2D culture) and 16.9 h (3D culture). Microscopic observation confirmed the formation of cell aggregates in the 3D system, particularly during the exponential phase. SDS-PAGE analysis revealed similar protein profiles of conditioned media from both culture systems. An anti-inflammatory cytokine, namely tumor necrosis factor beta (TGF-?), was analyzed using ELISA to evaluate the effect of culturing methods on TGF-? release. Interestingly, the relative TGF-? contents in the 2D culture were stagnant throughout the incubation times, whereas a higher accumulation of TGF-? was detected in the 3D culture, which was most likely caused by shear stress. Our study confirmed that a dynamic culture system with a microcarrier-supported bioreactor is a promising approach to scaling up MSC and secretome productions.

Bioreactor; Dynamic culture; Mesenchymal stem cells; Proliferation; Secretome

Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into various types of adult cells, such as osteocytes, chondrocytes, adipocytes, and others (Jiang et al., 2002; Rizal et al., 2020). Preclinical and clinical studies have uncovered therapeutic effects of MSC therapy in curing various degenerative diseases (Parekkadan and Milwid, 2010). In addition to MSCs, secretome is a promising resource for regenerative medicine due to its rich supply of growth factors and cytokines (Pawitan et al., 2017). Secretome can be collected from the conditioned media as a product of MSC culture. MSC secretome contains various growth factors and cytokines, including Epidermal Growth Factor (EGF), Vascular Endothelial Growth Factor (VEFG), Nerve Growth Factor (NGF), and Placental Growth Factor (PIGF) (Pawitan et al., 2017). Other studies have suggested that MSCs from the umbilical cord also secrete transforming growth factor (TGF)-? (Farias et al., 2018) that plays a direct role in cell growth, proliferation, differentiation, anti-inflammatory effects, immunomodulation, and wound healing (Phelps et al., 2018).

The immense potential of MSCs and secretome in therapeutic applications requires mass-scale production to ensure its availability and cost effectiveness. MSCs, similar to other stem cells, are limited in their native tissues; therefore, several methods have been developed for facilitating in vitro cell cultures. The conventional MSC culture uses 2-dimensional (2D) surface-treated containers, such as a flask or dish, to facilitate cell attachment. Static 2D culture is feasible for small-scale MSC expansion; however, this method is costly and quite challenging to adopt for large-scale cell production (Mizukami et al., 2018). The 3D culture system is a promising method for scaling up cell production using various types of bioreactors (Tsai et al., 2019; Sibuea et al., 2020; Nadhif et al., 2020). Dissolved oxygen in conventional culture can be maintained by limiting media depth. Meanwhile, in a 3D culture system, the oxygen level can be improved via agitation or oxygen infusion, which may increase hydrodynamic stress for cells.

Our current study was designed to evaluate the effects of static 2D and dynamic 3D culture methods on the MSCs’ proliferation and secretome profile. Lysed human thrombocyte/platelet concentrate was used to substitute for animal-derived serum. A stirred bioreactor was employed in this study due to its adequate capacity, ease of adjustment, homogeneous conditions, ease of scale-up, and ease of control (Mizukami et al, 2018). The TGF-? level in conditioned media was measured to compare the effects of culturing methods to the specific cytokine release.

    UC-MSCs could grow effectively in thrombocyte-supplemented culture with dynamic 3D and static 2D systems. Based on two experimental sets with different UC and lysates of concentrated thrombocyte donors, it was found that the shortest PDTs for experimental set 1 were 12.3 h (2D culture) and 14.8 h (3D culture), whereas in experimental set 2, they were 17.7 h (2D culture) and 16.9 h (3D culture). Microscopic observation suggested that cells formed aggregates in the 3D system, particularly after the cells reached the exponential phase. Decreased cell proliferation after day 5 was caused by cell-microcarrier aggregates rather than nutrient depletion or waste accumulation. The relative TGF-? contents in the 2D culture were stagnant throughout the incubation process, whereas higher TGF-? accumulation was detected in the 3D culture. Our study demonstrated that dynamic culture systems could improve specific cytokine production from UC-MSCs. Further research is necessary to elucidate the effects of hydrodynamic stress on cytokine production.

    This work was supported by Q2 Scheme Research Grant (Hibah Publikasi Terindeks Internasional (PUTI Q2 2020)) No. NKB-1545/UN2.RST/HKP.05.00/2020 and IMERI research cluster grant 2018 (Hibah Klaster Riset IMERI 2018) from Universitas Indonesia.

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