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

The need for a consistent hydrogel system for scale-up of 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.

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.

CONSISTENCY AND DIVERSITY WITH THE VITROGEL STEM SYSTEM

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.

Advantages of the VitroGel STEM

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 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.

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.