3D Stem Cell Culture

With the increasing need for high throughput experiments in laboratory settings, VitroGel® STEM was created to allow for the expansion of stem cell lines while reducing time and resources. The system offers an innovative approach to scaling up stem cell lines by mixing stem cell aggregates directly with a bioactive hydrogel to form stem cell spheroids, in the absence of differentiation factors.

Stem cell-derived static suspension cultures from 2D matrix cultures

To initiate 3D static suspension culture from 2D matrix coating culture, stem cell clumps, or aggregates, that have been lifted from 2D cultures are easily mixed with VitroGel® STEM to form nascent cell-hydrogel spheroids. As shown in Figure 1, after 24 hours, small hPSC spheroids form. From day 1 to 6, suspension cultures quickly grow larger, leading to the generation of healthy and high-quality stem cell spheroids. As shown in Figure 1, after day 3, cell number grows exponentially (Figure 1B) and spheroid size steadily increases (Figure 1C). The hPSC spheroids display characteristics of shallow craters or pockmarks, indicating expression of hPSC markers and successful expansion of healthy and high-quality stem cell spheroids. The resulting spheroids provide researchers with large numbers of healthy hPSCs for further experiments.

Figure 1.  3D static suspension culture of hPSC at 7-day culture circle.
A) Start the suspension culture by using healthy and high-quality cells from 2D matrix coating culture. The cell spheroids formed and kept growing from day 1 to day 6. The cell spheroid shows characters of shallow craters or pockmarks, which indicate the high expression of hPSC markers and good expansion of cells at healthy and high-quality states.  B) The chart shows the growth of the cell number from day 1 to day 7 after being seeded for 3D static suspension culture (The cell proliferation assay was tested by CCK8 assay). C) The increasing of cell sizes of hPSC spheroid from around 60 µm at day 1 to over 250 µm at day 7.

Importantly, the VitroGel® STEM system can be used to control stem cell spheroid size effectively. This feature adds flexibility for researchers who may require faster-developing, larger stem cell spheroids. Figure 2 shows that spheroids can be formed from an array of initial cell densities for efficient expansion of the stem cell population. All densities tested show increased spheroid diameter at each time point (Figure 2B). Therefore, the VitroGel® STEM system can be used independently of the initial stem cell population size.

Figure 2. Different sizes of hPSC spheroids from different cell seeding densities.
A) The morphology of the hPSC growth from day 1 to day 3 at initial densities of cell clump suspension at 3.6×10⁵ cells/mL, 9×10⁵ cells/mL, 1.8×10⁶ cells/mL, and 3.6×10⁶ cells/mL, respectively.  The initial sizes of the cell spheroids are different according to different cell seeding density. B) The increasing of cell sizes of hPSC spheroid with different initial cell seeding densities.

Stem cell-derived static suspension cultures directly from liquid nitrogen

VitroGel® STEM is also extremely effective in expanding stem cell populations directly from liquid nitrogen stocks. After removal of any cell dissociation reagent or cell freezing solution, stem cells that were stored in liquid nitrogen are resuspended in stem cell media and subsequently gently mixed with VitroGel® STEM to form nascent spheroids. As shown in Figure 3A, hPSC-hydrogel aggregates successfully form healthy spheroids after 1 day in culture. The hPSC spheroids continue to expand from day 1 to 6 (Figure 3B). The resulting hPSC spheroids also show hallmark features of healthy and high-quality stem cell spheroids, i.e., shallow craters or pockmarks. Figure 3B shows that hPSC static suspension cultures from liquid nitrogen are positive for Alkaline Phosphatase, indicating the successful expansion of healthy stem cell populations.

Figure 3. 3D static suspension culture of hPSC directly from Liquid Nitrogen (LN2).
A) start the suspension culture by using healthy and high-quality cells directly from LN2. The cell spheroids formed and kept growing from day 1 to day 6. The cell spheroid shows characters of shallow craters or pockmarks, which indicate the high expression of hPSC markers and good expansion of cells at the healthy and high-quality states.  B) AP staining image of hPSC spheroids show healthy stem cells with a high level of AP expression.

The simplicity of VitroGel® STEM ensures that the resulting static suspension cultures are fully supported, and, as seen in Figure 4, very little cell death is observed. VitroGel® STEM is compatible with multiple different hPSC media ensuring high expansion rates and cell viability in any laboratory platform.

Figure 4.  Live/Dead assay of hPSC spheroids.
The health and live cell spheroids show in the green color. The dead cells show as small clumps or individual cells surrounding the big cell spheroids.

VitroGel® STEM also guards against unwanted differentiation, which can affect the purity of differentiated target cell types. Loss of pluripotency, due to uncontrolled differentiation, can negatively affect multiple applications of stem cell research, including gene editing, tissue organoid development, cell reprogramming, biochemical assays, and DNA/RNA sequencing. Therefore, VitroGel® STEM ensures the undifferentiated state of stem cell lines during scaling up. As shown in Figure 5, hPSC aggregates mixed with VitroGel® STEM hydrogel retain pluripotency after 7 days, evidenced by the expression of key pluripotent stem cell markers, SSEA4, OCT4, SOX2, and TRA-1-60.

Figure 5.  Immunofluorescence images of hPSC spheroids with key pluripotent stem cell markers.
A) Images of hPSC spheroids with the SSEA4 and OCT4 expression, B) Images of hPSC spheroids with the SOX2 and TRA-1-60 expression.

Within 7 days, hPSC number expands exponentially, thereby creating a pool of stem cells ready to be harvested for 3D sub culturing. If desired, stem cell spheroids can also be used to re-establish 2D culture by adding on 2D matrix coated tissue vessels (Figure 6). The resulting expanded hPSC populations have tri-lineage potential and can be further differentiated into any cell type.

Figure 6.  Re-establish 2D colonies from 3D hPSC spheroids.
The hPSC spheroids cultured in 3D static suspension (A) can be harvested and seeded to 2D matrix coating surface. The 2D cell colonies (B) formed within 24 hours after attaching back to the matrix coating surface.