Saadiya Nazli, M.D.
Cellular Therapy Fellow
Transfusion Medicine, Human Cellular Therapy Laboratory
Mayo Clinic, Rochester, MN, USA
Ashley Krull, Ph.D.
Associate Director, Cell Therapy Manufacturing and Engineering
Assistant Professor, Division of Hematology
Ohio State University Wexner Medical Center
Columbus, Ohio USA
Margaret (Maggie) DiGuardo, M.D.
Pathologist, Transfusion Medicine, Human Cellular Therapy Laboratory, Laboratory of Genetics and Genomics
Mayo Clinic, Rochester, MN USA
Disclaimer/Conflict of interest statement: none.
Microcarriers for Adherent Cell Culture
What?
Microcarriers are small, solid, or porous particles that are used in various fields, including cell therapy and biotechnology. Essentially, microcarriers are tiny beads ranging in size from 100 to 300 microns. They are typically made of biocompatible materials such as polystyrene, dextran, gelatin, or solubilizing material such as cross-linked polyglycolic acid (PGA).
When?
The purpose of the microcarrier is to provide a larger surface area for cell attachment, allowing expansion capacity to be amplified several fold compared to traditional flat cultures. Instead of being cultivated on a flat surface, cells are cultured on the surface of spherical microcarriers that remain in suspension during stirring, with each microcarrier carrying hundreds of cells.
Who?
Today, there are many researchers working with three dimensional microcarrier systems for MSC culture in a variety of fields, including regenerative medicine. The earliest successful MSC microcarrier expansion was reported only in 2007 by Frauenschuh and collaborators (1). Rafiq and collaborators performed a systematic review on 13 commercially available microcarriers to define an “ideal microcarrier”, and illustrated that the performance of the microcarriers when stirred was consistently better than when they were static (2). The SoloHill plastic microcarrier was chosen as the optimal microcarrier for human MSC expansion due to its xeno-free formulation, processing capability, and the ability to effectively harvest cells from the microcarrier in spinner flasks without compromising cellular immunophenotype and differentiation capacity. Pinto and collaborators described the use of plastic microcarriers in a vertical wheel bioreactor system to scale up adipose and umbilical cord matrix-derived MSCs (3). More recently, Miguel and collaborators described a serum-/xeno-free microcarrier-based culture system in a Vertical-WheelTM bioreactor (VWBR) that enabled the scalable production of MSC-derived extracellular vesicles (MSC-EVs) (4). Another useful resource for microcarrier research was written by Koh and collaborators who systematically reviewed three dimensional (3D) microcarrier systems in MSC culture, highlighting 14 articles that used microcarriers for MSC expansion (5).
Where? How?
There is a pressing need for scalable culture systems for MSCs due to the large number of cells required in clinical applications. MSCs are increasingly utilized for a myriad of clinical applications ranging from intrathecal treatment of spinal cord injury to incorporation into an implantable biosynthetic matrix to treat anal fistulas. MSCs can be isolated from various sources such as bone marrow, adipose tissue, and umbilical cord blood, and can be expanded in vitro for clinical applications. Bioreactors are widely used to culture MSCs on a large scale, and microcarriers enable the culture of anchorage‐dependent cells in stirred‐tank bioreactors (6). Microcarriers are also a convenient solution for producing vaccines and other biologics, including exosomes.
Here, we will discuss the different types of microcarriers used for MSC production with bioreactors, common commercially available microcarriers, and preferred xeno-free and GMP-grade microcarriers. Our lab had experience with Synthemax II (Corning) dissolvable microcarriers easily dissociated by EDTA & pectinase and, later, Star Plus microcarriers (SoloHill by Sartorius) that are xeno-free, GMP-grade, and come in a sterile, ready-to-use formulation that requires final filtration steps.
Non-Porous Microcarriers
Non-porous microcarriers are made of materials such as polystyrene or glass, and have a smooth surface that facilitates cell attachment. These microcarriers are typically used for adherent cells such as MSCs, and provide a stable surface for cell attachment, allowing for cell growth and proliferation. Non-porous microcarriers are easy to manufacture, have a high yield, and can be sterilized easily, making them ideal for large-scale production of MSCs.
Porous Microcarriers
Porous microcarriers are composed of a non-toxic, biocompatible material, such as collagen or gelatin. These microcarriers have a highly porous structure, which provides a larger surface area for cell attachment and nutrient exchange. The high porosity allows cells to attach into the inner part of the microcarrier and facilitates efficient mass transfer of oxygen and nutrients, promoting cell growth and proliferation.
Because of the high surface area-to-volume ratio of microcarrier systems, cell yield per unit culture medium volume is high. Furthermore, by adjusting the microcarrier concentration and adding fresh microcarriers, this technology allows for easy adjustment of the surface area available for cell development and bead-to-bead transfer of expanding cells.
Table. 1 Commercially available microcarriers suitable for use in the 3D milieu of Bioreactors.
Microcarrier
|
Manufacturer
|
Diameter
|
Matrix
|
Surface Coating
|
Surface Charge
|
Microcarrier porosity
|
Surface Area
|
Cytodex 1
|
Cytiva (Pall Life Science)
|
190 µm
|
Dextran beads
|
Hydrophilic DEAE exchanger
|
Positive charge
|
microporous
|
0.44m2/g
|
Cytopore
|
Cytiva (Pall Life Science)
|
230 µm
|
Cellulose
|
Hydrophilic DEAE exchanger
|
Yes
|
macroporous
|
1.1m2/g
|
Hillex® II
|
Sartorius
|
150 – 210 µm
|
Modified Polystyrene
|
No
|
Yes, Cationic charged
|
microporous
|
515 cm2
|
Plastic
|
Sartorius
|
125-212 µm
|
Cross-linked Polystyrene
|
No
|
No
|
Nonporous/solid
|
360-480 cm2
|
Plastic Plus
|
Sartorius
|
125 - 212 µm
|
Cross-linked Polystyrene
|
No
|
Yes, Cationic charged
|
Nonporous/solid
|
360 cm2
|
Star-Plus
|
Sartorius
|
125 - 212 µm
|
Cross-linked modified Polystyrene
|
No
|
Yes, Cationic charged
|
Rigid/spherical
|
360 cm2
|
Synthemax II
|
Corning
|
125 - 212 µm
|
Polystyrene
|
Low concentration synthemax II (Animal component free) or untreated
|
No
|
Nonporous
|
360 cm2/g
|
A look to the Future: With steps towards optimization and scale-up, the adoption of 3D culture systems will be on the rise, as will the demand for microcarriers. Employing the most appropriate microcarrier for cell expansion is a critical component of a microcarrier-based expansion process. Once the microcarrier has been selected following a rigorous screening protocol, a process can be built around that particular microcarrier for consistency and reproducibility.
In conclusion, microcarriers play a crucial role in the expansion of MSCs using bioreactor systems. Various types of microcarriers, including porous, non-porous, and dissolvable, are available for MSC production, each with unique advantages and disadvantages. Despite the benefits of using microcarriers for stem cell culture, there are some drawbacks to consider, such as the potentially harmful effects of shear stress and microcarrier clumping, as well as the increased operating costs associated with an additional downstream step for cell-microcarrier separation and microcarrier removal. In addition, supply chain difficulties produce variations that can also hinder progress. As more and more laboratories transition to bioreactor systems, the supply chain of GMP grade microcarriers would have to be strengthened to support the needs of the ever-growing field of cell therapy.
References
- Frauenschuh, S., Reichmann, E., Ibold, Y., Goetz, P.M., Sittinger, M. and Ringe, J. (2007), A Microcarrier-Based Cultivation System for Expansion of Primary Mesenchymal Stem Cells. Biotechnol Progress, 23: 187-193. https://doi.org/10.1021/bp060155w
- Rafiq QA, Coopman K, Nienow AW, Hewitt CJ. Systematic microcarrier screening and agitated culture conditions improves human mesenchymal stem cell yield in bioreactors. Biotechnol J. 2016 Mar;11(4):473-86. doi: https://doi.org/10.1002/biot.201400862. Epub 2016 Feb 29. PMID: 26632496; PMCID: PMC4991290.
- de Sousa Pinto, D., Bandeiras, C., de Almeida Fuzeta, M., Rodrigues, C.A.V., Jung, S., Hashimura, Y., Tseng, R.-J., Milligan, W., Lee, B., Ferreira, F.C., Lobato da Silva, C. and Cabral, J.M.S. (2019), Scalable Manufacturing of Human Mesenchymal Stromal Cells in the Vertical-Wheel Bioreactor System: An Experimental and Economic Approach. Biotechnol. J., 14: 1800716. https://doi.org/10.1002/biot.201800716
- Miguel de Almeida Fuzeta M, Bernardes N, Oliveira FD, Costa AC, Fernandes-Platzgummer A, Farinha JP, Rodrigues CAV, Jung S, Tseng RJ, Milligan W, Lee B, Castanho MARB, Gaspar D, Cabral JMS, da Silva CL. Scalable Production of Human Mesenchymal Stromal Cell-Derived Extracellular Vesicles Under Serum-/Xeno-Free Conditions in a Microcarrier-Based Bioreactor Culture System. Front Cell Dev Biol. 2020 Nov 3;8:553444. doi: https://doi.org/10.3389/fcell.2020.553444 PMID: 33224943; PMCID: PMC7669752.
- Koh, B., Sulaiman, N., Fauzi, M.B. et al. Three dimensional microcarrier system in mesenchymal stem cell culture: a systematic review. Cell Biosci 10, 75 (2020). https://doi.org/10.1186/s13578-020-00438-8
- Coral García-Fernández, Alba López-Fernández, Salvador Borrós, Martí Lecina, Joaquim Vives, Strategies for large-scale expansion of clinical-grade human multipotent mesenchymal stromal cells, Biochemical Engineering Journal, Volume 159, 2020, 107601, ISSN 1369-703X https://doi.org/10.1016/j.bej.2020.107601.
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