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Components, types and selection of mesenchymal stem cells [culture medium]

Components, types and selection of mesenchymal stem cells [culture medium]

  • Categories:Company News
  • Author:CytoNiche
  • Origin:CytoNiche
  • Time of issue:2022-11-29
  • Views:633

(Summary description)Cell culture is one of the most common and most complex techniques used in life sciences, and culture medium is a key factor for maintaining the health and proliferation of mesenchymal stem cells (MSC

Components, types and selection of mesenchymal stem cells [culture medium]

(Summary description)Cell culture is one of the most common and most complex techniques used in life sciences, and culture medium is a key factor for maintaining the health and proliferation of mesenchymal stem cells (MSC

  • Categories:Company News
  • Author:CytoNiche
  • Origin:CytoNiche
  • Time of issue:2022-11-29
  • Views:633

Cell culture is one of the most common and most complex techniques used in life sciences, and culture medium is a key factor for maintaining the health and proliferation of mesenchymal stem cells (MSCs). When completely broken down, all MSCs culture media essentially contain the same basic components: basic culture media, buffer system, glutamine, specific growth factors and additional supplements, serum (or serum substitutes).

This article gives a brief overview of some of the main components of the culture media, the types of media and their selection.


[Main components of MSCs culture media]

Part1. Basic culture media

Classic basic media (DMEM, MEM, RPMI, etc.) are chemically defined, each of which can be used to support specific cell lines or culture conditions. The main differences between different classical basic media are the properties and amounts of buffers, salts and cell growth additives.

Many basic media were originally developed with mouse fibroblasts, HeLa or CHO cell lines. Over time, basic media suitable for various cell types have been established. For example, DMEM/F12, a common stem cell basal medium, is a 1:1 mixture of Ham's F12 and DMEM (containing higher concentrations of amino acids and vitamins) in a composite medium.

Part2. Buffer system

The MSCs medium should not only meet the basic nutritional requirements for culturing cells, but also be suitable for the culture conditions used. Most mammalian cells grow well at a pH of 7.4, and maintaining this pH is critical for stem cell culture.

When gas-permeable culture flasks or dishes are used, atmospheric carbon dioxide is dissolved into the cell culture medium and equilibrium with HCO₃⁻ is established. Because carbon dioxide is acidic, the pH of the medium decreases. While salts and amino acids in the media can provide some buffering capacity, additional buffering compounds still need to be added.

①Sodium bicarbonate

To maintain a stable pH of the medium in CO2 incubators, sodium bicarbonate (NaHCO3), a non-toxic natural buffer, is added to the stem cell culture medium.
Studies find that 1.5 g/L NaHCO3 is required to maintain the pH of the medium at 7.4 when cells are cultured in a 5% CO2 incubator.
H2O + CO2 ↔ H2CO3 ↔ H+ + HCO3-NaHCO3 ↔ Na+ + HCO3-


HEPES is a zwitterionic organic buffer that is also used to maintain the physiological pH of cell culture media.
When the cell culture system is very sensitive, the use of HEPES is recommended to increase the buffering capacity and stabilize the pH in the range of 7.2 to 7.6, regardless of the CO2 level.

③ Phenol red

Besides CO2 level, various factors such as cell metabolites and medium additives can also affect the pH value of MSCs culture media. Therefore, phenol red is commonly added into stem cell culture media as a visual pH indicator.

Medium containing phenol red appears red at pH 7.4 and varies from bright yellow to dark purple as the pH fluctuates. Medium that appears yellow or pink/purple is generally unsuitable for stem cells. At this time, it is necessary to immediately exchange the medium, subculture or adjust gases in the incubator.

Figure 1. pH diagram of phenol red  The medium containing phenol red appears red at pH 7.4 and then changes from yellow to purple along with pH changes.

Part3. Glutamine

The nitrogen is needed for cells to build nucleotides, amino acids and vitamins. L-Glutamine, an essential amino acid, serves as an essential additive to most cell culture media, facilitating the storage of nitrogen in culture media and nitrogen transfer into cells. Glutamine is also one of the most readily available amino acids when glucose levels are low and energy demands are high, especially for rapidly proliferating cells or cells cultured under hypoxic conditions. However, free L-glutamine is quite unstable when dissolved in a liquid medium. L-glutamine in cell culture media can degrade to toxic ammonium and pyroglutamate by-products at temperatures over 4°C. Because of its short half-life, L-glutamine stock solution must be frozen at-20°C and added to the freshly prepared culture medium before use.

Part4. Growth factors and other additives

① Growth factors

Growth factors, cytokines, and chemokines, serving as chemical messengers for cells, are small protein molecules naturally secreted by cells and induce physiological effects (proliferation, growth, or differentiation) on cells that receive signals. Different stem cells require media supplemented with specific growth factors to maintain the health, proliferation and differentiation of the cultures. For example, basic fibroblast growth factor (bFGF) is a key growth factor for maintaining the pluripotency of human embryonic stem (ES) and the versatility of induced pluripotent stem (iPS) cells, while leukemia inhibitory factor (LIF) is a major regulator of ES and iPS cells in mice.

The quality of the growth factors used in the stem cell culture medium has a major impact on the culture effect. Growth factors used in cell culture media are mostly recombinant proteins that can be synthesized in a variety of cell types and species, including E. coli, CHO, and human lines. Optimal results can be obtained by using growth factors with high activity, high purity, appropriate protein folding and post-translation modification. Growth factors and cytokines are stable when stored as lyophilized powders, but have a short shelf life once added to culture media. To avoid degradation of these components, small aliquots of their concentrated stock solution are prepared and added to the medium prior to use for best results.

② Amino acids

In addition to the essential amino acids provided in the basic medium and supplemented glutamine and growth factors, non-essential amino acids (NEAA) are often added to the medium to stimulate cell proliferation and prolong the viability of cultured cells. Although cells can naturally synthesize NEAA, supplementing the culture medium with NEAA can reduce potential side effects that can affect cell growth when amino acid levels are low.

③ Antibiotics

Antibiotics are often supplemented in laboratories to control the growth of possible bacterial pollutants in cell culture environment. Except for primary cell isolation, antibiotics are not required or encouraged in stem cell culture. Routine use of antibiotics can mask low-level bacterial contamination, generate drug-resistant strains, mask the effects of mycoplasma contamination, and even interfere with cellular metabolism.

Part5.Serum and serum substitutes

Conventional MSCs medium is typically supplemented with fetal bovine serum (FBS), which contains growth factors, hormones, adhesion proteins and other factors essential for cell proliferation and maintenance.

However, the use of animal-derived fetal bovine serum is only suitable for in vitro studies and not for human clinical applications, mainly because:

① Fetal bovine serum with xenogeneic components can cause an immune response when injected into humans.

② Fetal bovine serum might carry animal-derived pathogens, such as bacteria or viruses, which can spread to mesenchymal stem cells during cell culture.

③ Unidentified components in fetal bovine serum and batch-to-batch differences can affect the accuracy of research results and treatment results [1].

Therefore, it is crucial to replace the traditional FBS-containing MSCs medium with a safer, serum-free medium that can effectively maintain the characteristics of MSCs for clinical applications.


[Types of serum-free medium for MSCs].

There are two main directions of research on serum-free media.

① The culture medium does not contain any added components of animal origin.

② The culture medium does not contain any unspecified additive components.

According to the research directions, they can be grouped into four categories (Figure 2):

①Serum-free medium (SF) in the general sense: The chemical components of the additives are not clear, which might contain a large amount of animal-derived protein.

② Xeno-free culture medium (XF): It does not contain animal-derived components, but might contain human-derived components.

③Animal component-free (ACF) medium: It does not contain any components from animals or humans.

④ Chemically defined (CD) medium: All chemical components are clear and their concentrations are determined.

Figure 2: Development direction of culture medium

With the increasing demand for MSCs for clinical applications, novel techniques for the preparation of MSCs culture media have emerged. Currently, there are two strategies for developing serum-free media for MSCs.

Strategy 1: Replace FBS with "human-derived" supplements (such as human platelet lysate, HPL) from human blood or hydrolyzed vegetable protein (HVP, such as wheat protein hydrolysates).


HPL is a xeno-free, animal serum-free cell culture medium supplement derived from human platelets for the development of serum-free/xeno-free media. HPLs can be collected from blood banks where expired platelets not suitable for patient transfusion are available.

HPLs contain large amounts of substances such as cytokines, growth factors, and proteins (Table 1), which can promote the growth of MSCs while maintaining their differentiation potential and immunomodulatory properties.


HVP is a product obtained by hydrolyzing wheat, soybean, rice and other plant raw materials under the catalysis of acid or enzyme preparations, and then processing the resulting intermediate mixed colloid of amino acids and amino acid polypeptides.

For example, wheat protein hydrolyzate as a serum substitute is rich in dipeptides and polypeptides, which are preferred nutrients for MSCs compared to free amino acids. Moreover, wheat protein hydrolyzate is rich in stable glutamate containing polypeptides that is considered to be an important nucleic acid raw material in cell culture, and is also an excellent source of energy. Plant protein hydrolyzates can be used to develop animal component-free media.

Strategy 2: Only synthetic, recombinant or human-derived purified substances are used to prepare the culture medium, without serum or serum substitutes as supplements.

This kind of culture medium is chemically defined medium. Its main advantages are clear chemical components and concentrations, accurate quantification, and guaranteed stability for each test, without batch-to-batch variation. Also, it avoids the possibility of introducing animal-derived pathogens. Due to the lack of adhesion proteins in such media, most of them require adding suitable encapsulating solution to promote the adhesion and migration of MSCs.

Currently, several serum-free/xeno-free (SF/XF) and chemically defined (CD) media have been marketed for the culture and amplification of MSCs (Table 2), but the effectiveness of these media is debatable [2]. For example, studies have shown that primary MSCs are poorly cultured in StemPro MSC SFM or mTeSR medium, and additional human AB serum is required for culture. Moreover, the subculture of mesenchymal stem cells requires a high cell inoculation density, because lower densities can slow down cell proliferation. MesenCult-XF reportedly also does not provide sound support for the isolation, amplification, phenotype and differentiation potential of mesenchymal stem cells.  


[Selection of culture medium for MSCs].

An ideal culture medium for MSCs shall meet the following criteria.

① Serum-free.

② Xeno-free components.

③ Chemically defined.

④ Supports the primary culture of MSCs.

⑤ Supports the subculture of MSCs at low inoculation density levels.

⑥ Able to promote the adherent growth of MSCs without pre-encapsulating.

⑦ The medium performance is greater than or similar to the performance of FBS-containing medium.

MSCs are widely used in various disease scenarios such as trauma tissue repair, immune diseases, diabetes, and cancer, making the demand for MSCs rise sharply. For cell therapy, safety, efficacy and reproducibility are primary considerations. Clinical-grade media preparations can significantly improve the safety and quality of transplanted stem cells, and serum-free/xeno-free media with consistent batches and defined components appear to be optimal for clinical applications. This will be a challenging process due to the complexity of the culture media.



[ 1 ]H. T. H. Bui, Nguyen, L. T., Than, U. T. T. Influences of Xeno-Free Media on Mesenchymal Stem Cell Expansion for Clinical Application[J]. Tissue Eng Regen Med,2021,18(1):15-23.

[ 2 ]X. Wu, Kang, H., Liu, X., Gao, J., Zhao, K., Ma, Z. Serum and xeno-free, chemically defined, no-plate-coating-based culture system for mesenchymal stromal cells from the umbilical cord[J]. Cell Prolif,2016,49(5):579-588.


Beijing CytoNiche Biotechnology Co., Ltd. was established by the research team of Professor Du Yanan from Tsinghua University School of Medicine, and was jointly established by Tsinghua University through equity participation. The core technologies were derived from the transformation of scientific and technological achievements of Tsinghua University. CytoNiche focuses on building an original 3D cell "smart manufacturing" platform, as well as providing overall solutions for the 3D microcarrier-based customized cell amplification process.

CytoNiche's core product, 3D TableTrix® Microcarrier Tablet (Microcarrier), is an independent innovation and the first pharmaceutical excipient grade microcarrier that can be used for cell drug development. It has obtained the certificate of analysis from relevant authoritative institutions such as National Institutes for Food and Drug Control, and obtained 2 qualifications for pharmaceutical excipients from the National Medical Products Administration (CDE approval registration number: F20210000003, F20200000496). Moreover, the product has obtained the DMF qualification for pharmaceutical excipients from U.S. FDA (DMF: 35481). 

Products and services of CytoNiche can be widely used in the upstream process development of gene and cell therapy, extracellular vesicles, vaccines, and protein products. At the same time, it also has broad prospects for applications in the fields of regenerative medicine, organoids, and food technology (cell-cultured meat, etc.).

Our company has a R&D and transformation platform of 5,000 square meters, including a CDMO platform of more than 1,000 square meters, a GMP production platform of 4,000 square meters, and a new 1200 L microcarrier production line. The relevant technologies have obtained more than 100 patents and more than 30 articles about the technologies in international journals have been published. The core technology projects have obtained a number of national-level project support and applications.


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Copyright: Beijing CytoNiche Biotechnology Co., Ltd.
Copyright: Beijing CytoNiche Biotechnology Co., Ltd.