Comparative Analysis of Mesenchymal Stem Cell Cultivation in Fetal Calf Serum, Human Serum, and Platelet Lysate in 2D and 3D Systems

Abstract

In vitro two-dimensional (2D) and three-dimensional (3D) cultivation of mammalian cells requires supplementation with serum. Mesenchymal stem cells (MSCs) are widely used in clinical trials for bioregenerative medicine and in most cases, in vitro expansion and differentiation of these cells are required before application. Optimized expansion and differentiation protocols play a key role in the treatment outcome. 3D cell cultivation systems are more comparable to in vivo conditions and can provide both, more physiological MSC expansion and a better understanding of intercellular and cell-matrix interactions. Xeno-free cultivation conditions minimize risks of immune response after implantation. Human platelet lysate (hPL) appears to be a valuable alternative to widely used fetal calf serum (FCS) since no ethical issues are associated with its harvest, it contains a high concentration of growth factors and cytokines and it can be produced from expired platelet concentrate. In this study, we analyzed and compared proliferation, as well as osteogenic and chondrogenic differentiation of human adipose tissue-derived MSCs (hAD-MSC) using three different supplements: FCS, human serum (HS), and hPL in 2D. Furthermore, online monitoring of osteogenic differentiation under the influence of different supplements was performed in 2D. hPL-cultivated MSCs exhibited a higher proliferation and differentiation rate compared to HS- or FCS-cultivated cells. We demonstrated a fast and successful chondrogenic differentiation in the 2D system with the addition of hPL. Additionally, FCS, HS, and hPL were used to formulate Gelatin-methacryloyl (GelMA) hydrogels in order to evaluate the influence of the different supplements on the cell spreading and proliferation of cells growing in 3D culture. In addition, the hydrogel constructs were cultivated in media supplemented with three different supplements. In comparison to FCS and HS, the addition of hPL to GelMA hydrogels during the encapsulation of hAD-MSCs resulted in enhanced cell spreading and proliferation. This effect was promoted even further by cultivating the hydrogel constructs in hPL-supplemented media.

Keywords: differentiation; fetal calf serum; gelatin methacryloyl (GelMA); human serum; hydrogel; medium supplements; mesenchymal stem cells; platelet lysate.

Figure 1

Schematic representation of 3D hydrogel-based cultivation: first GelMA hydrogels were formulated with 50% FCS, 50% HS, or 50% hPL followed by cultivation in medium supplemented with 10% FCS, 10% HS, or 2.5% hPL.

Figure 2

The influence of cell culture supplements on the long-term cultivation of hAD-MSCs. Cells from four donors [(A) Donor 1, (B) Donor 2, (C) Donor 3 and (D) Donor 4] were cultivated over five passages in 2.5% hPL (black line), 10% HS (light gray dotted line) or 10% FCS (gray dashed line) and cumulative cell numbers were calculated. *p < 0.05, **p < 0.01, ***p < 0.001 (*, **, *** indicates significant difference to FCS); ^¤p < 0.05, ^¤¤p < 0.01 (¤, ¤¤ indicates significant difference to HS).

Figure 3

(A) Alizarin red staining of the hAD-MSCs from four donors differentiated in the presence of 2.5% hPL, 10% HS, or 10% FCS. The influence of cell culture supplements on the calcium deposition evaluated by Alizarin red extraction, 10× objective, scale bar 100 μm. (B) and on the cell viability during the differentiation (C). Data represent the mean ± SD of a threefold determination for four donors.

Figure 4

(A) Alkaline phosphatase staining of differentiated hAD-MSCs cultivated for 7, 14, and 21 days and differentiated in medium supplemented with FCS, HS and hPL, 10× objective, scale bar 100 μm. (B) Measured ALP activity in the supernatant of hAD-MSCs cultivated with FCS, HS and hPL for 7, 14, and 21 days. Data represent the mean ± SD of a threefold determination for two donors.

Figure 5

(A) The influence of cell culture supplements on the calcium deposition evaluated by using a trained metric phase object confluence mask for microscopic pictures analyzed by the IncuCyte Live-Cell Imaging System and Software (Essen BioScience, Ann Arbor, MI). Data represent the mean ± SD for a fourfold determination. (B) Microscopic pictures taken by an IncuCyte Live-Cell Imaging System and blended with a trained metric phase object confluence mask, which indicates typical osteogenic changes of the cells, scale bar 400 μm.

Figure 6

(A) Alcian Blue staining of the hAD-MSCs from four donors differentiated in the presence in 2.5% hPL, 10% HS, or 10% FCS. 10× objective, scale bar 100 μm. (B) Determination of the GAG deposition in cells (in μg GAG/μg DNA) differentiated for 7, 14, and 21 days under influence of FCS, HS, and hPL. Data represent the mean ± SD of two independent experiments in threefold determination for two donors.

Figure 7

Morphological examination (A–C) and cell viability (D) of hAD-MSCs encapsulated with a UV dose of 1.2 J/cm² in 5% GelMA with 50% degree of functionalization (DoF) formulated with 50% PBS and 50% FCS, 50% HS, or 50% hPL. The hydrogels were cultivated in (A) FCS, (B) HS, or (C) hPL supplemented medium. After cultivating the cells for 1, 3, and 7 days, they were stained with calcein-AM; 4× objective, scale bar 500 μm. (D) The CellTiter-Blue (CTB) assay was performed on day 1, day 3, and day 7 of cultivation. Data represent the mean ± SD for a threefold determination. *p < 0.05, **p < 0.01, ***p < 0.001.