Human Adipose Stem Cells (hASCs) Grown on Biodegradable Microcarriers in Serum- and Xeno-Free Medium Preserve Their Undifferentiated Status

Abstract

Human adipose stem cells (hASCs) are promising candidates for cell-based therapies, but they need to be efficiently expanded in vitro as they cannot be harvested in sufficient quantities. Recently, dynamic bioreactor systems operated with microcarriers achieved considerable high cell densities. Thus, they are a viable alternative to static planar cultivation systems to obtain high numbers of clinical-grade hASCs. Nevertheless, the production of considerable biomass in a short time must not be achieved to the detriment of the cells' quality. To facilitate the scalable expansion of hASC, we have developed a new serum- and xeno-free medium (UrSuppe) and a biodegradable microcarrier (BR44). In this study, we investigated whether the culture of hASCs in defined serum-free conditions on microcarriers (3D) or on planar (2D) cell culture vessels may influence the expression of some marker genes linked with the immature degree or the differentiated status of the cells. Furthermore, we investigated whether the biomaterials, which form our biodegradable MCs, may affect cell behavior and differentiation. The results confirmed that the quality and the undifferentiated status of the hASCs are very well preserved when they grow on BR44 MCs in defined serum-free conditions. Indeed, the ASCs showed a gene expression profile more compatible with an undifferentiated status than the same cells grown under standard planar conditions.

Keywords: UrSuppe; biodegradable microcarrier (MC); biomaterial; human adipose stem cells (hASCs); serum- and xeno-free cell culture medium.

Figure 1

SEM microphotographs to show the morphology of the BR44 and PNF MCs. (A) BR44: (1) 200×, scale bar 500 µm (2) 600×, scale bar 100 µm (3) 500×, scale bar 200 µm (broken MCs). (B) PNF: (1) 200×, scale bar 500 µm (2) 600×, scale bar 100 µm (3) 400×, scale bar 200 µm (broken MCs).

Figure 2

SEM microphotographs to show the stability of BR44 MC at 4 °C in water. (A1) BR44 newly produced: (A2) BR44 after 2 months in water at 4 °C. (250×, scale bar 300 µm).

Figure 3

SEM microphotographs to show the erodibility of BR44 MC at 37 °C in cell culture medium UrSuppe and under stirred conditions. (A1) BR44 on day 0: (A2) BR44 on day 1, (A3) BR44 on day 4, (A4) BR44 on day 9. (500×, scale bar 200 µm).

Figure 4

DAPI stained samples of hASCs cultured on MCs in a static condition after 1, 2, and 4 days of culture. (A) BR44 MCs, 100×, scale bar 300 µm (B) PNF MCs, magnification: 200×, scale bar 150 µm.

Figure 5

SEM microphotographs to confirm the presence of hASCs on MCs. BR44 MCs (A) and PNF MCs (B) after 1, 2, and 4 days of static culture. Fold magnification: 250×, scale bar 300 µm.

Figure 6

Analysis of 7-AAD stained nuclei released from MCs-based cultures. The nuclei can be divided into three categories: nuclei with a DNA content of 2N (G₀ and G₁ phases), those with a DNA quantity corresponding to 4N (G₂ phase), and finally, those with an intermediate DNA content (S phase). The three phases of interest, G₁, G₂, and S, are distinguishable by flow cytometry. (A) Volumetric flow cytometry was employed to establish the number of nuclei released from the carrier-cells aggregates. Graphical representation of the nuclei counts of three patients hASCs’ grown on BR44 MC (green bar) and PNF MC (orange bar). The data represent cell densities [cells/cm²] and are shown in function of four measurements (days 1, 2, 4, and 7), (n = 3, error bars represent S.E.M.). No significant differences were observed between cells cultured on BR44 or PNF microcarriers. (B) Graphical representation of the active proliferation percentage (APP): the percentages of nuclei found in G₁, G₂, or in the S phase were determined for hASCs grown on BR44 MC and PNF MC. A slightly significant difference in APP was founded only on day 2 (p = 0.042). The measurements were done after days 1, 2, 4, 7, and APP percentage was calculated as follows: APP = [(G₂ + S)/G₁] × 100. (n = 3, error bars represent S.E.M.).

Figure 7

Percentages of primary hASCs positive for the eight cell surface markers picked out for this analysis. Bar chart recapitulates flow cytometry average of data obtained from three different donors of hASCs. Cell culture condition: blue bar, standard 2D cell culture vessels; green bar, Figure 3. D condition with BR44 MCs; orange bar, defined static 3D with PNF MC. The cell culture medium used: UrSuppe. (n = 3, error bars represent S.E.M.). The single-parameter histograms are shown in the Supplementary Materials, Figures S3–S6. * Reduction of expression compared to 2D culture are statistically significant: p-value < 0.0001.

Figure 8

Time-dependent profiles of cell density (A), substrate/metabolite concentrations (B), and DAPI stained microscopic pictures during the proof-of-concept cultivation in the spinner flask (C). A partial medium exchange of 30% was performed on day 5. The symbols represent the experimentally measured values collected from offline measurements. The lines represent the simulated time courses, which were calculated according to [23]. Scale bar for fluorescence microscopic pictures = 650 μm.

Figure 9

Relative expression levels of some genes regulating the adipocyte differentiation process measured by RT-qPCR. Primary hASCs from three different donors were grown in standard 2D cell culture vessels, in static 3D cell culture system on BR44 MC (A), in static 3D cell culture system on PNF MC (B), and in dynamic 3D cell culture system on BR44 (C). The calculated expression levels of the different markers for the 3D tests are related to the values obtained in the 2D conditions. (n = 3 error bars represent S.E.M.). * Statistically significant: p-value < 0.0001.

Figure 10

Representative quantitative secretome analysis of hASCs cultured in defined xeno- and serum-free conditions (UrSuppe medium). Human ASCs’ supernatants of cells at passage 2 were analyzed using a commercially available “Proteome Profiler Human Adipokine Array.” The three cell culture conditions tested were standard 2D cell culture system, blue bars; static 3D conditions on BR44 MC, green bars; dynamic 3D conditions on BR44 MC, red bars. Data are represented as relative Mean Pixel Intensity (MPI) of fluorescence. Background fluorescence was subtracted from each measured value and then normalized by the positive control intensity to obtain the relative MPI. This experiment was performed with hASCs obtained from the biopsy of one donor. More information about these adipokines could be found in Supplementary Materials Tables S9 and S10.

Figure 11

Amino acid profile overview. The values were calculated relative to the initial amino acid concentrations in the UrSuppe medium. Red: consumed amino acids; Green: produced amino acids. Time-dependent profiles of each amino acid are shown in Supplementary Materials Figure S9.