Mesenchymal stem cells (MSCs): From isolation to scaling up

Mesenchymal stem cells (MSCs) are multipotent adult stem cells found in connective tissue. They are often also referred to as “mesenchymal stromal cells” by the scientific community.¹ MSCs are adherent, fibroblast-like cells with regenerative properties.^2,3 They also release cytokines and extracellular vesicles that communicate with surrounding cells to maintain tissue homeostasis.

Human MSCs show promise in regenerative medicine and stem cell therapy for treating autoimmune diseases, inflammatory conditions, and tissue damage.^2,3 The therapeutic value of MSCs stems from their ability to regulate immune responses and support tissue repair through paracrine signaling.

Explore our cell culture basics page on MSCs to learn more about MSC biology.

Where do mesenchymal stem cells come from?

Mesenchymal stem cells can be isolated from several tissue sources in the body. Common sources include the bone marrow, adipose tissue, and umbilical cord tissues like Wharton’s jelly.^4,5 Other sources include peripheral blood (although MSCs are found at a lower frequency), dental pulp, and placental tissues.^4,6

^{Isolation of MSCs is based on enrichment of stromal stem cell populations through various techniques:}

Density gradient separates mononuclear cells from whole tissue samples.^7,8
Plastic adherence then selects for the attachment properties of MSCs.⁷
Magnetic sorting and flow-based enrichment methods can further select for populations based on the presence of surface markers.^7,9

Large-scale isolation from human bone marrow requires significant time and resources, and its success depends on bone marrow sample quality and donor age.¹⁰

Figure 1: Sources of mesenchymal stem cells in the human body. Tissue sources of MSCs include umbilical cord and placenta, bone marrow, dental pulp, lungs, endometrium and menstrual blood, and adipose tissue.

How to culture mesenchymal stem cells?

Mesenchymal stem cells require a controlled culture environment, including the selection of an optimized MSC cell culture medium, to ensure that their biological properties are reproducible.¹¹ Serum can support cell growth, but it adds variability. Fetal bovine serum is complex and changes from lot to lot, which can influence the proliferation, morphology, and functional characteristics of cells.^11,12 Defined or reduced-serum media, such as xeno-free MSC culture systems, reduce variability and increase reproducibility.

Low-serum or serum-free media have the benefit of providing more standardized culture conditions over a long time span. Examples of low-serum or serum-free options include Mesenchymal Stem Cell Growth Medium XF, Mesenchymal Stem Cell Growth Medium 2, and the Excipient GMP-grade PromoExQ MSC Growth Medium XF (prf).

The use of supplements, the choice of extracellular matrix, and oxygen tension in cell culture can also influence mesenchymal stem cell characteristics. For example, extracellular matrix and oxygen tension can affect adhesion properties, proliferation kinetics, and differentiation potential of MSCs.^13,14

Passaging practices can also influence MSC quality and expansion. When using our Mesenchymal Stem Cell Growth Medium 2, seeding at 5,000 cells/cm² typically allows cultures to reach 80%–90% confluency within 4–6 days. High passage numbers can lead to reduced proliferation, altered morphology, and genetic changes that could compromise the therapeutic potential of MSCs.^15,16

From lab to manufacturing: Scaling up mesenchymal stem cells

Producing high-quality mesenchymal stem cells in sufficient quantities for therapeutic applications is challenging. Mesenchymal stem cells make up only 0.001%–0.01% of mononuclear cells in bone marrow from adult donors.¹⁷ Regenerative medicine applications require infusions containing several million cells per kilogram of body weight.¹⁸ MSCs are sensitive to their environment, and suboptimal conditions can lead to phenotypic drift, reduced proliferation, and morphological changes.¹⁷

The use of consistent, optimized protocols is key for successful scale-up of MSC cultures for clinical applications. Standardized procedures help maintain cell quality across production batches.^17,18 Process controls monitor parameters like cell density, medium composition, and environmental conditions. Quality assurance testing at multiple stages confirms that expanded populations retain their therapeutic properties and meet safety standards.^17,18

Long-term storage through cryopreservation allows MSC banking for future use. Nontoxic, xeno-free cryopreservation methods protect cell viability and function during freezing and thawing.^19,20 For researchers requiring reliable cryopreservation solutions, Freezing Medium Cryo-SFM Plus (for GMP environment: PromoExQ CellNAP) offers a serum-free option that maintains MSC viability and phenotype during storage.

Practical toolkit: Mesenchymal stem cell media and protocols

We at PromoCell offer several mesenchymal stem cell products that support high-quality MSCs and reproducible performance for both research and clinical applications. Our portfolio includes primary cells, culture media, and differentiation media.

Product	Size	Cat. No.
Mesenchymal Stem Cell Growth Medium 2 (Ready-to-use)	500 ml	C-28009
Mesenchymal Stem Cell Growth Medium 2 (Ready-to-use), phenol red-free	500 ml	C-28017
Mesenchymal Stem Cell Growth Medium XF * (Ready-to-use)	500 ml	C-28019
Mesenchymal Stem Cell Growth Medium XF * (Ready-to-use), phenol red-free	500 ml	C-28018
PromoExQ MSC Growth Medium XF * (Manufactured in compliance with the EXCiPACT™ GMP certification standard)	500 ml	EQ-C-28019

Table 1. MSC isolation and expansion media

* Fibronectin- or vitronectin-coated plates are necessary in conjunction with the xeno-free (XF) media

Product	Size	Cat. No.
MSC Adipogenic Differentiation Medium 2 (Ready-to-use)	100 ml	C-28016
Mesenchymal Stem Cell Chondrogenic Differentiation Medium (Ready-to-use) with or without inducers	100 ml each	C-28012/C-28014
Mesenchymal Stem Cell Osteogenic Differentiation Medium (Ready-to-use)	100 ml	C-28013
Mesenchymal Stem Cell Neurogenic Differentiation Medium (Ready-to-use)	100 ml	C-28015

Table 2. MSC differentiation media

For project-specific guidance on culture optimization or differentiation protocols, contact our scientific support team.

Related product categories

Mesenchymal Stem Cell Culture
Hematopoietic Progenitor Cell Culture

FAQs

1. Are mesenchymal stem cells (MSCs) adult stem cells?

Yes. MSCs are widely described as a multipotent type of “adult” (postnatal) stem/stromal cells found in multiple tissues (e.g., bone marrow, adipose, perinatal tissues) and are studied as an adult stem cell population in regenerative medicine.²¹

2. How are mesenchymal stem/stromal cells (MSCs) characterized?

MSCs are typically characterized using the ISCT minimal criteria: they must adhere to plastic, show tri-lineage differentiation (adipogenic, chondrogenic, osteogenic), and meet a defined surface marker profile—CD105/CD73/CD90 positive and CD45/CD34/CD14 or CD11b/CD79α or CD19/HLA DR negative (under standard conditions). In practice, characterization is commonly performed by flow cytometry (to confirm marker expression) and differentiation assays with lineage-specific staining to verify tri-lineage potential.²²

3. What are the various cell types MSCs can differentiate into?

According to the ISCT criteria, MSCs can differentiate into osteoblasts (bone), adipocytes (fat), and chondroblasts/chondrocytes (cartilage) in vitro (“tri-lineage differentiation”). Under specific conditions, some studies also report differentiation toward additional lineages (e.g., myogenic/neural-like), but tri-lineage differentiation capacity is the standard used for MSC characterization.²²

4. What quality attributes are typically assessed before MSCs are used for clinical application?

Typically, before MSCs are used for clinical applications, they undergo risk-based assessment that often includes: (a) Identity/phenotype (e.g., flow cytometry markers aligned with the ISCT criteria); (b) Viability (cell survival at release and/or post-thaw); (c) Microbiological safety, such as sterility, plus mycoplasma and endotoxin controls.²³

5. Is freezing an important consideration during MSC scale up for cell therapy development?

Yes. In cell therapy workflows, MSC batches often need to be banked and stored for extended periods, and freeze–thaw stress can affect their viability and phenotype, which can impact downstream consistency and comparability. Thus, the use of robust cryopreservation protocols is essential for long-term storage and clinical applications.²³

References

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Viswanathan S, Shi Y, Galipeau J, et al. (2019). Mesenchymal stem versus stromal cells: International Society for Cell & Gene Therapy (ISCT®) Mesenchymal Stromal Cell committee position statement on nomenclature. Cytotherapy, 21(10):1019-1024. doi:10.1016/j.jcyt.2019.08.002
Han X, Liao R, Li X, et al. (2025). Mesenchymal stem cells in treating human diseases: molecular mechanisms and clinical studies. Signal Transduct Target Ther., 10(1):262. doi:10.1038/s41392-025-02313-9
Dabrowska S, Andrzejewska A, Janowski M, Lukomska B. (2021). Immunomodulatory and regenerative effects of mesenchymal stem cells and extracellular vesicles: therapeutic outlook for inflammatory and degenerative diseases. Front Immunol. , 11:591065. doi:10.3389/fimmu.2020.591065
Costela-Ruiz VJ, Melguizo-Rodríguez L, Bellotti C, et al. (2022). Different sources of mesenchymal stem cells for tissue regeneration: a guide to identifying the most favorable one in orthopedics and dentistry applications. Int J Mol Sci., 23(11):6356. doi:10.3390/ijms23116356
Giai Via A, Frizziero A, Oliva F. (2012). Biological properties of mesenchymal stem cells from different sources. Muscles Ligaments Tendons J., 2(3):154-162.
Zhidu S, Ying T, Rui J, Chao Z. (2024). Translational potential of mesenchymal stem cells in regenerative therapies for human diseases: challenges and opportunities. Stem Cell Res Ther., 15(1):266. doi:10.1186/s13287-024-03885-z
Bieback K, Schallmoser K, Klüter H, Strunk D. (2008). Clinical protocols for the isolation and expansion of mesenchymal stromal cells.Transfus Med Hemotherapy., 35(4):4-4. doi:10.1159/000141567
Pierini M, Dozza B, Lucarelli E, et al. (2012). Efficient isolation and enrichment of mesenchymal stem cells from bone marrow. Cytotherapy, 14(6):686-693. doi:10.3109/14653249.2012.677821
Jia Z, Liang Y, Li X, et al. (2018). Magnetic-activated cell sorting strategies to isolate and purify synovial fluid-derived mesenchymal stem cells from a rabbit model. J Vis Exp., (138):57466. doi:10.3791/57466
Nallakumarasamy A, Shrivastava S, Ravi V, et al. (2025). Optimizing bone marrow harvesting sites for enhanced mesenchymal stem cell yield and efficacy in knee osteoarthritis treatment. World J Methodol., 15(2). doi:10.5662/wjm.v15.i2.101458
Najar M, Melki R, Khalife F, et al. (2022). Therapeutic mesenchymal stem/stromal cells: value, challenges and optimization. Front Cell Dev Biol., 9:716853. doi:10.3389/fcell.2021.716853
Renesme L, Pierro M, Cobey KD, et al. (2022). Definition and characteristics of mesenchymal stromal cells in preclinical and clinical studies: a scoping review. Stem Cells Transl Med., 11(1):44-54. doi:10.1093/stcltm/szab009
Estrada JC, Albo C, Benguría A, et al. (2012). Culture of human mesenchymal stem cells at low oxygen tension improves growth and genetic stability by activating glycolysis. Cell Death Differ., 19(5):743-755. doi:10.1038/cdd.2011.172
Salasznyk RM, Williams WA, Boskey A, Batorsky A, Plopper GE. (2004). Adhesion to vitronectin and collagen I promotes osteogenic differentiation of human mesenchymal stem cells. BioMed Res Int., 2004(1):24-34. doi:10.1155/S1110724304306017
Kim M, Rhee JK, Choi H, et al. (2017). Passage-dependent accumulation of somatic mutations in mesenchymal stromal cells during in vitro culture revealed by whole genome sequencing. Sci Rep., 7(1):14508. doi:10.1038/s41598-017-15155-5
Cai J, Miao X, Li Y, et al. (2014). Whole-genome sequencing identifies genetic variances in culture-expanded human mesenchymal stem cells. Stem Cell Rep., 3(2):227-233. doi:10.1016/j.stemcr.2014.05.019
Li J, Wu Z, Zhao L, et al. (2023). The heterogeneity of mesenchymal stem cells: an important issue to be addressed in cell therapy. Stem Cell Res Ther., 14(1):381. doi:10.1186/s13287-023-03587-y
Mizukami A, Swiech K. (2018). Mesenchymal stromal cells: from discovery to manufacturing and commercialization. Stem Cells International, 2018:1-13. doi:10.1155/2018/4083921
Rogulska O, Tykhvynska O, Revenko O, et al. (2019). Novel cryopreservation approach providing off-the-shelf availability of human multipotent mesenchymal stromal cells for clinical applications. Stem Cells International, 2019:1-11. doi:10.1155/2019/4150690
Lechanteur C, Briquet A, Giet O, Delloye O, Baudoux E, Beguin Y. (2016). Clinical-scale expansion of mesenchymal stromal cells: a large banking experience. Journal of Translational Medicine, 14(1):145. doi:10.1186/s12967-016-0892-y
Robert AW, Marcon BH, Dallagiovanna B, Shigunov P. (2020). Adipogenesis, osteogenesis, and chondrogenesis of human mesenchymal stem/stromal cells: A comparative transcriptome approach. Frontiers in Cell and Developmental Biology, 8:561. doi:10.3389/fcell.2020.00561
Dominici M, Le Blanc K, Mueller I, et al. (2006). Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy, 8(4):315-317. doi:10.1080/14653240600855905
Williams ME, Banche Niclot F, Rota S, et al. (2025). Optimizing mesenchymal stem cell therapy: from isolation to GMP-compliant expansion for clinical application. BMC Molecular and Cell Biology, 26(1):15. doi:10.1186/s12860-025-00539-7