Mesenchymal Stromal Cell (MSC) Culture

Cell culture is an essential tool in biomedical research, enabling the study of different cell types for therapeutic and scientific applications. In this context, MSC culture has gained great importance due to its regenerative and immunomodulatory capacity, the relative ease of obtaining them from various sources such as bone marrow, adipose tissue, and umbilical cord, as well as their potential to differentiate into osteoblasts, chondrocytes, adipocytes, and some other cell types.

To ensure the viability, proliferation, and functionality of MSCs, it is crucial to provide an appropriate environment by controlling aspects such as the culture medium, environmental conditions, and cell adhesion. Below, we will discuss the fundamental aspects necessary for successful MSC culture.

Culture Medium and Supplements

The culture medium is a nutrient-rich solution designed to provide cells in culture with the necessary nutrients, growth factors, and physicochemical conditions for their maintenance and proliferation. For MSCs, the most commonly used media are Dulbecco’s Modified Eagle Medium (DMEM), α-MEM, and Iscove’s Modified Dulbecco’s Medium (IMDM), enriched with fetal bovine serum (FBS). FBS plays a crucial role in cell culture, as it contains a broad range of growth factors, hormones, transport proteins, and other bioactive components that promote cell proliferation and viability. Additionally, it helps reduce cellular stress and maintain the structural integrity of cells in culture.

Currently, due to variability in its composition and the potential risk of pathogen transmission, its use in clinical applications is being regulated and, in some cases, replaced by serum-free defined media, which offer greater reproducibility and safety in clinical and advanced research settings.

To prevent contamination by microorganisms such as bacteria and fungi, antibiotics and antimycotic compounds—such as penicillin and streptomycin, in combination with amphotericin B—are incorporated. These agents help prevent pathogen growth, ensuring a sterile and stable environment that allows cells to develop under optimal conditions without external interference.

Environmental Conditions

MSCs must be maintained under strictly controlled conditions: a constant temperature of 37°C, an atmosphere with 5% CO₂, and 95% relative humidity.

• CO₂ is essential for maintaining a stable pH in the culture medium, ensuring an optimal environment for cell viability.
• Humidity prevents medium evaporation, preserving osmotic stability and avoiding solute concentration, which could negatively affect cell growth.
• Oxygen regulation is also critical; physiological levels (2-5%) minimize oxidative stress, contributing to the longevity of the culture.

Culture Surface: Substrate Characteristics and Cell Adhesion

Since MSCs are adherent cells, they require an adequate surface. Typically, plastic culture flasks with different surfaces (single or multilayer) are used, specifically designed for adherent cell cultures. These plastics are often treated with ionized gases, modifying their surface to increase hydrophilicity and improve cell adhesion.

It is important to note that all culture devices and containers allow for gas exchange, ensuring an appropriate environment for cell growth.

Passaging or Subculturing

When cell density reaches a critical point, it is necessary to divide and redistribute the cells into new flasks with fresh culture medium. This process, known as passaging or subculturing, is essential to:

• Prevent nutrient depletion and metabolic waste accumulation, which could compromise culture health.
• Facilitate cell expansion, increasing the number of cells available for research and therapeutic applications.

Isotonic Washes and Trypsinization

This process is essential in MSC culture and requires a combination of trypsin and EDTA:

• Trypsin is an enzyme that dissolves intercellular junctions and adhesion to the culture surface.
• EDTA chelates calcium ions, preventing cells from re-adhering or aggregating.

This process facilitates cell detachment without compromising their integrity. However, it is crucial to carefully control the exposure time to trypsin, as excessive digestion can affect cell viability and integrity. Once cells are detached, enzymatic activity must be stopped or neutralized.

Before this step, it is fundamental to remove the culture medium and perform isotonic washes to eliminate serum residues and other components that might interfere with trypsin activity.

Conclusions

To ensure a successful MSC culture, it is essential to:

• Optimize the culture medium.
• Maintain strict environmental control.
• Use appropriate surfaces for cell adhesion.
• Incorporate antibiotics and antimycotics to prevent contamination, ensuring a sterile environment.

Additionally, proper passaging and trypsinization contribute to maintaining MSC viability and functionality over time. Proper handling of these factors optimizes culture performance and enables its effective application in scientific research and advanced therapies.

References

• Freshney, R. I. (2021). Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications.
• Galipeau, J., & Sensebé, L. (2018). Mesenchymal stromal cells: Clinical challenges and therapeutic opportunities. Cell Stem Cell, 22(6), 824-833.
• Pittenger, M. F., Discher, D. E., Péault, B., Phinney, D. G., Hare, J. M., & Caplan, A. I. (2019). Mesenchymal stem cell perspective: Cell biology to clinical progress. NPJ Regenerative Medicine, 4, 22.
• World Health Organization (2021). Good Cell Culture Practice (GCCP) Guidelines.

 

Author: Bioeng. Francisco Javier Caro Moreno