Advancements in mesenchymal stem cell (MSC) research are shedding light on how these stem cells may someday be used in various clinical applications, including immunomodulatory therapies and cell replacement therapies for mesenchymal tissues such as bone and cartilage [1,2]. For more than a decade, we have provided key resources to address challenges in MSC research. Designed to work together, our portfolio of GIBCO® stem cell products and services supports and accelerates the path to discovery.

Mesenchymal Stem Cells: A Small Population With Big Potential

MSCs, also known as adult stem cells, were recognized by Dr. Alexander Friedenstein and colleagues in the 1970s as a population of fibroblast precursors located within the bone marrow [3]. By the 1980s, these cells were found to be capable of differentiating into adipocytes, osteoblasts,  and chondrocytes [4]. Today, this mesodermal lineage is found to include tenocytes, myoblasts, and even neural cell types, all of which share the potential to impact research in basic biology, cancer biology, genomics, drug discovery, and cell therapy.

It is estimated that human MSCs constitute just 0.0001% to 0.01% of total bone marrow nucleated cells, and as a result, they require robust in vitro cell culture expansion to obtain sufficient numbers for basic research and clinical applications. The GIBCO® brand provides the broadest selection of cGMP-compliant complete culture systems for MSCs, many of which are free of animal-derived components. Our diverse portfolio of MSC culture systems includes serum-containing, reduced-serum, serum-free, and serum-free/xeno-free formulations.

Growing MSCs in Reduced-FBS or Serum-Free Conditions

Classical medium for MSCs contains DMEM and 10% FBS. MesenPRO RS™ Medium is a reduced-serum formulation (2%) that allows increased consistency of MSC growth. Compared with MSCs grown in classical medium, MSCs grown in MesenPRO RS™ Medium exhibit similar gene expression patterns (Figure 1).

Growing MSCs in serum-free conditions enhances the synergistic effect on human bone marrow (BM)-MSC proliferation by permitting the use of an optimized mix of recombinant growth factors. StemPro® MSC SFM [5–7] supports a more spindle-shaped morphology with high-density cell growth. This optimized formulation allows continual propagation and superior efficiency of human MSC expansion (Figure 2) at high cell densities, requiring less medium, surface area, and time when compared with classical medium (DMEM + 10% FBS). Expanded BM-MSCs grown with StemPro® MSC SFM exhibit trilineage differentiation (Figure 3).

MesenPRO RS™ medium Figure 1. MesenPRO RS™ medium maintains global mRNA expression of MSCs. MSCs were grown for three subpassages (at 3 x 103 cells/cm2 per subpassage) in MesenPRO RS™ Medium or DMEM + 10% FBS, total RNA was isolated, and then mRNA expression was assessed using Illumina BeadArray™ technology. Data were analyzed using Illumina BeadStudio software; a pairwise comparison of the two samples is represented as a scatter plot. Genes with greater than 0.99 detection units are in blue; points lying outside the red lines represent genes with greater than 2-fold differences. The R2 correlation of 0.94 indicates similar gene expression patterns between MSCs grown in the two media types.
CELLstart™ CTS™ substrate 
Figure 2. hMSCs grown on CELLstart™ CTS™ substrate–coated dishes in StemPro® MSC SFM exhibit a 166% improvement in expansion over 10 passages compared to classical medium. Average net total cell number per T25 flask was calculated for human MSCs growing in StemPro® MSC SFM and classical medium (n = 3). Cultures were grown with a seed density of 1 x 104 cells/cm2, a split frequency of 3 days, and a medium change every 2 days.

serum-free expanded human MSCs
Figure 3. Differentiation capacity of serum-free expanded human MSCs. (Top panel) Day 14 differentiation cultures from cells expanded for eight additional passages in SFM revealed trilineage mesoderm differentiation, as shown by positive (a) oil red O staining for adipocytes, (b) alcian blue staining for chondrocytes, and (c) alkaline phosphatase staining for osteoblasts. (a, c) 10x, (b) 20x objective. (Bottom panel) Toluidine blue staining of day 21 chondrogenic micromass-differentiation cultures after three passages in (a) serum-free medium or (b) serum-containing medium. Image reproduced from Stem Cell Res Ther (2010) 1:8.

Xeno-Free MSC Culture

StemPro® MSC SFM contains xenogenic components not amenable to some clinical applications; therefore, the need for a second-generation product exists. StemPro® MSC SFM XenoFree offers a completely animal origin–free system when used in conjunction with CELLstart™ CTS™ substrate. Expansion of human MSCs and adipose-derived stem cells (ADSCs) [8] in StemPro® MSC SFM XenoFree is comparable to classical medium (DMEM +10% MSC-Qualified FBS) in terms of morphology and growth characteristics (Figure 4). In addition, primary isolation of BM-MSCs may be achieved using StemPro® MSC SFM XenoFree supplemented with 2.5% pooled human AB-Human Serum. This culture system can also be used to generate induced pluripotent stem cells [9].

StemPro® MSC SFM XenoFree
Figure 4. Multipassage expansion of human bone marrow–derived MSCs in StemPro® MSC SFM XenoFree. Passage 5 human bone marrow MSCs (4-donor pool) were used as input cells.

A Lipid Supplement for Enhanced MSC Growth

Lipoproteins play a critical role in the regulation and maintenance of cellular growth and metabolism. StemPro® LipoMAX™ is a human-derived lipoprotein-based cell culture supplement for MSCs and adipose-derived stem cells (ADSCs). Addition of StemPro® LipoMAX™ supplement under serum-free conditions can improve expansion of ADSCs by ~50% after 3 passages (Figure 5).

ADSCs in StemPro® LipoMAX™ supplement 
Figure 5. Enhanced growth of adipose-derived stem cells (ADSCs) in StemPro® LipoMAX™ supplement. (Top panel) ADSCs expanded in StemPro® MSC SFM with 1:100 StemPro® LipoMAX™ exhibit an improvement in expansion of ~50% over 3 passages compared to StemPro® MSC SFM alone. (Bottom panel) Morphology of ADSCs expanded for 3 passages in StemPro® MSC SFM with StemPro® LipoMAX™ supplement.

Complete Solutions for Mesenchymal Stem Cell Research

Learn more about the complete range of products for mesenchymal stem cell research, and the new GIBCO® CTS™ brand for use in cell therapy applications.

References

  1. Chamberlain G, Fox J, Ashton B et al. (2007) Stem Cells 25:2739–2749.
  2. Aggarwal S, Pittenger M (2005) Blood 105:1815–1822.
  3. Friedenstein A, Chailakhyan R, Latsinik N et al. (1974) Transplantation 17:331–340.
  4. Bab I, Ashton B, Gazit D et al. (1986) J Cell Sci 84:139–151.
  5. Chase L, Lakshmipathy U, Solchaga L et al. (2010) Stem Cell Res Ther 1:8.
  6. Agata H, Watanabe N, Ishii Y et al. (2009) Biochem Biophys Res Commun 382:353–358.
  7. Ng F, Boucher S, Koh S et al. (2008) Blood 112:295–307.
  8. Lindroos B, Boucher S, Chase L et al. (2009) Cytotherapy 11:958–972.
  9. Sugii S, Kida Y, Kawamura T et al. (2010) Proc Natl Acad Sci U S A 107:3558–3563.
For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

For Research Use Only. Not for use in diagnostic procedures.