3D Static Suspension Culture and Scale-up of Human Pluripotent Stem Cells

The need for a consistent hydrogel system for scaling up hPSC populations

The use of human pluripotent stem cells (hPSCs) in biomedical research is expanding exponentially. hPSCs provide an excellent system for studying basic human development and function, as well as providing a robust platform for testing stem cell-based therapies in laboratory and clinical settings. Recent advances in hPSCs procedures have opened the door for use in clinical trials. hPSCs have an immense potential for use in regenerative medicine, as they may hold the key to reversing tissue damage caused by diseases and injuries. With this great potential comes the need for scaling up, as high throughput experiments are becoming commonplace due to the ever-increasing demand for big data.

Current stem cell maintenance and expansion methods require plating cells on 2D matrix coated culture vessels with large surface areas, which can be time-consuming and expensive. Moreover, the matrix can be temperature sensitive. This can lead to an uneven coating of the culture vessel, thereby producing inconsistent stem cell colony adhesiveness and size. Therefore, 3D expansion systems offer a more consistent means of stem cell expansion. Besides expansion, stem cell researchers are discovering the many applications of 3D hPSC cultures. Perhaps, the most exciting of these is the development of sophisticated tissue organoids with laminar organization. However, current 2D culture methods present some challenges to producing consistent, high-quality organoids. Traditional methods have included “lifting” of stem cell colonies with harsh enzymes, such as trypsin, which can have deleterious effects on cell viability and produce stem cell spheroids with inconsistent sizes. Thus, current 2D stem cell methods often require long periods of troubleshooting to overcome these inconsistencies. Many established organoid protocols also require tissue-like precursors that have to be developed in a 2D system and after germ layer differentiation, the precursors must be lifted and embedded into a 3D matrix hydrogel. These multiple, complicated steps can ultimately result in inconsistent and malformed organoids.

For both small laboratories and large biotechnology companies, fast and dependable expansion of stem cell lines is becoming a critical need. However, current methods for scaling up stem cell populations require expensive and cumbersome equipment, such as shakers, spinning flasks, or bioreactors. These protocols also typically call for the use of microcarriers to aid in stem cell expansion. In many protocols, to ensure the formation of spheroidal stem cell aggregates, shakers or bioreactors are used at high speeds, which can elicit spheroidal shearing, resulting in impaired growth and cell viability.

Consistency and diversity with the VitroGel STEM system

VitroGel STEM is a Xeno-free hydrogel system developed to improve the performance of three-dimensional (3D) static suspension cultures and scale-up hPSCs populations to create a high-throughput system to model various tissue and disease states. This hydrogel system is ready-to-use with an optimized formulation that fully supports the rapid expansion of high-quality 3D stem cell spheroids with pluripotent properties. hPSCs directly thawed from liquid nitrogen or passaged from 2D matrix coated culture vessels can be immediately mixed with the hydrogel solution for static suspension cultures. Moreover, the optimization protocol is ideal for time-sensitive experiments, as it does not require excessive medium exchanges, which can ultimately save on time and materials. This hydrogel system is compatible with most hPSC culture media and tissue culture vessels.

Furthermore, in cases where hPSC expansion is needed, this system does not require any special, expensive suspension culture vessels. Due to the unique static suspension culture procedure, the requirement for microcarriers for large-scale bioreactors is eliminated, making cell harvesting simple and effective. The 3D stem cell spheroids that are developed using this system can be used for further sub-culturing, patterned differentiating, or re-establishing 2D culture morphologies.

Advantages of the VitroGel STEM

Undifferentiated stem cells can easily be mixed with VitroGel STEM to form cell-hydrogel mixtures, which can efficiently transfer to multiple different types of cell culture vessels, including 96-well plates, T-flasks, shaking flasks, and bioreactors. VitroGel STEM is compatible with various stem cell culture media. Moreover, after expansion, using the VitroGel STEM system, stem cell spheroids can easily be sub-cultured in 3D for expansion or differentiation, as well as a re-established 2D culture on a matrix coating plate.

VitroGel® STEM offers the ability to directly culture stem cells from liquid nitrogen in 3D suspension cultures for the expansion of stem cell pools. Multi-passaged stem cells cultured on 2D culture vessels, such as tissue culture plates or flasks, can also be easily transitioned to 3D using the VitroGel® STEM platform. Upon expansion, cells can be efficiently harvested or sub-cultured, without the requirement of additional reagents, for further differentiation.

Stem cell populations can be scaled with VitroGel® STEM in combination with bioreactors. At ultra-low agitation speeds, stem cell suspension cultures can be expanded with high cell viability and excellent cell growth rates. Using VitroGel® STEM, expanded stem cell pools maintain full pluripotent properties.

VitroGel® STEM is not similar to common stem cell culture systems that require expensive matrix coating procedures, which can be laborious and time-consuming, or microcarriers. With VitroGel® STEM, there is also no need for typical extraneous laboratory equipment, such as shakers or stirrers, to successfully scale up stem cell populations.

While 2D matrix coated culture vessels are still widely used in most stem cell expansion and differentiation protocols, the passaging of cells 2D requires extensive media exchanges and handling of cells, which can introduce possible human errors and contaminations. Using VitroGel® STEM, cells from liquid nitrogen or 2D matrix coated cultures can be directly mixed with the hydrogel solution to create consistent static suspension cultures. The optimized protocol reduces the need for medium exchanges, thereby saving on time and materials. The VitroGel® STEM system is compatible with most stem cell culture media and does not require any particular culture vessels for stem cell expansion. The unique static suspension culture procedure also eliminates the need for microcarriers for large-scale bioreactors and makes cell harvesting extremely simple and fast. The expansion process is quick and flexible, with either a 3/4-day culture cycle or a 7-day culture cycle, depending on the desired amount of expansion.

In short, newly thawed or passaged stem cell clumps, or aggregates, are mixed with the hydrogel solution and then suspended in stem cell media supplemented with ROCK inhibitor. After 3 to 4 days, stem cell aggregates expand substantially in size. In the 7-day culture cycle, stem cell media is again added on day 3/4. After the desired amount of expansion, stem cell spheroids are prepared for sub-culture or cell harvesting. With the aid of a cell strainer to remove single or dead cells, spheroids are collected and transferred to a conical tube. The spheroids can then be centrifuged and resuspended and mixed with VitroGel® STEM for subculture or further expansion.

A great 3D system for static suspension cultures and scaling up hPSC populations

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 the 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 effectively control stem cell spheroid size. 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.
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 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).
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.

VitroGel STEM promotes cell viability and maintenance of pluripotency

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) I
mages 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.

The VitroGel STEM system offers easy passaging hPSC static suspension cultures

Expansion of stem cell pools requires regular passaging, which can often be time-consuming, expensive, and exposes cultures to possible contaminations. However, with the VitroGel STEM system, stem cell static suspension cultures can easily be passaged without excessive materials (Figure 7). In short, after the initial expansion (Figure 7A), hPSC spheroids are transferred from the culture vessel to a reversible strainer to filter out single and dead cells. The large, healthy hPSC spheroids are then transferred forced through a 40 or 70 µm strainer a slow rate to mechanically dissociated the spheroids into smaller clumps. These smaller clumps can then be used for subsequent passages. After the smaller clumps are collected, they are again mixed with VitroGel STEM for subculturing. Figure 7B illustrates to an abundant expansion of hPSC cultures after just one passage. This technique can exponentially expand stem cell populations allowing for quick scaling up on a small laboratory or large industrial scale.

Figure 7. Passaging hPSC spheroids with 40 µm or 70 µm strainer.
Stem cell spheroids were dissociated into small clumps after passing through the cell strainer and grew into large spheroids after 4 days.

Applications of the VitroGel STEM system

The VitroGel STEM offers the quick and efficient expansion of stem cell lines to be used in a number of applications. Stem cell suspension cultures can be differentiated into tissue-specific spheroid and more sophisticated organoids. Spheroids are typically more homogenous and have been developed to investigate the cellular behaviors of a number of different cell types. Conversely, organoids have a greater number of different tissue-specific cell types and often possess some amount of 3D structure. The development of tissue organoids has been shown to be highly dependent upon the size, density, and pluripotency of the initial stem cell spheroids. Therefore, with the ability of VitroGel STEM to effectively control stem cell spheroid size, researchers can quickly determine the ideal spheroid starting size for their desired organoid. As such, using the VitroGel STEM system to produce consistent, large stem cell spheroids actually can actually accelerate the organoid formation process. Moreover, unwanted and unpredictable differentiation can often plague 2D stem cell cultures. Any amount of undefined differentiation can have amplified effects on the generation of organoids. Since VitroGel STEM precisely maintains pluripotency in hPSC static suspension cultures, organoids can be generated with confidence. With the VitroGel STEM, researchers have a platform to generate large numbers of stem cells that can be used to study human disease.