Expansion of Mesenchymal Stem Cells in Blood-free, Chemically Defined Media

Last month’s ISCT conference held in Montreal, Canada featured many novel approaches for cell therapy research and manufacturing. One of the posters presented at the conference, “Inclusion of Recombinant Albumin and Transferrin Enables the Blood-Free Expansion of Mesenchymal Stem Cells in Chemically Defined Media,” highlighted the importance of blood-component free media for cell therapies and provided data on the use of a blood-free media in mesenchymal stem cell (MSC) culture.

Stem Cell Media

The inclusion of human plasma or plasma-derived proteins, such as albumin and transferrin, in stem cell culture media is common practice because the addition of these proteins have demonstrated benefits in multiple cell types. These proteins often serve as a substitute for fetal bovine serum (FBS) in the culture and permit a higher level of media definition than FBS. However these proteins, even when isolated from human serum, can still create problems associated with variability, stabilizing reagents, and the possibility of introducing contamination with adventitious agents.

Thus there has been much interest in creating cell culture media for therapeutic cell types that would be free of any animal or human blood sourced components. In this poster, authors’ present advances in recombinant protein expression that has permitted production of recombinant versions of serum albumin and transferrin. Non-mammalian expressed human serum albumin (Cellastim S) and recombinant human serum transferrin (Optiferrin) have been shown to be both biologically active as well as economically viable¹. Incorporating these proteins can produce media formulations that may simplify research and development with a higher degree of chemical definition as well as streamline the regulatory approval process for clinical products due the absence of blood-derived components. In the study, authors utilized Cellastim S and Optiferrin in the formulation of a blood-free media for mesenchymal stem cell (MSC) culture.

Poster Data Highlights

The blood-free media created using the recombinant albumin and transferrin was capable of efficiently expanding mesenchymal stem cells (MSC) isolated from multiple tissues when compared to xeno-free and serum- containing media. Additionally, MSCs cultured in this blood-free medium retained critical positive and negative MSC markers with minimal spontaneous differentiation after several subcultures.

Mesenchymal Stem Cell Culture Performance

Authors’ compared the use of serum-derived albumin and transferrin to the recombinant versions, Cellastim S and Optiferrin, using a quantifiable functional performance assay in bone marrow-derived MSCs. This permitted a better understanding of how these proteins perform. The albumins and transferrins were formulated into a SFM specifically optimized for MSCs at a predetermined inclusion level and MSCs were allowed to expand in these different SFMs for 96 hrs using alphaMEM + 10% FBS as a standard control. Cells were harvested and counted and data was normalized against the 0 protein control (Figure 1).

The functional screen determined that the recombinant versions of albumin and transferrin exhibited equivalent or superior performance to that of the native serum derived versions in expanding low passage MSC isolated from human bone marrow. Both native and recombinant versions, however, exhibited improved performance from the 0 protein and FBS controls.

Blood free mesenchymal cell-culture media performance
Serum free media formulations and albumin levels for MSC were optimized prior to the studies described here. Mesenchymal stem cells (MSC) from human bone marrow were obtained from a commercial source and banked. Cells were previously expanded in alpha MEM + 10% FBS. Cells were passaged from this media directly into the in-house SFM containing native or recombinant albumins and transferrins. For the transferrin assay media, recombinant albumin was incorporated in the assay media. Similarly, the assay media for albumin incorporated the recombinant transferrin. Cells were harvested and data was normalized to the SFM without albumin or transferrin. (A) The bone marrow-derived MSC demonstrated a 133 ± 17 percent control increase when serum-derived transferrin was incorporated versus 197 ± 63 percent control with Optiferrin. (B) Serum-derived albumin exhibited almost a 2 fold increase over the 0 albumin control while the cells expanded in Cellastim S expanded 283 ± 63 percent of control. **, * p ≤ 0.01, p ≤ 0.05 by One Way ANOVA, respectively.

To further confirm these initial findings, mesenchymal stem cells derived from human bone marrow and adipose tissue were expanded in serum-free media containing the recombinant proteins, Cellastim S and Optiferrin, and compared to commercially available xeno-free serum- free media in the isolation and expansion of MSC from both tissue sources.

Again, the blood-free
 media containing Cellastim S and Optiferrin exhibited equivalent expansion capability to media containing human serum albumin and transferrin (Figure 2).

Blood free mesenchymal cell-culture media performance
MSC were isolated from human bone marrow (n=2) or adipose tissue (n+2) and seeded on T-flasks coated with CellSTART (1/50) at P0 in either a commercially available xeno free serum free media or the blood-free media containing Cellastim S and Optiferrin. Cells were subsequently transferred to CellBIND-treated T-flasks at P1. After colonies appeared and were ready for passage, cells were passaged at least 4 times. At each passage, population doublings were determined and the cumulative population doublings were calculated. For bone marrow MSC, cells doubled a total of 9.26 ± 0.26 and of 10.52 ± 1.19 in the xeno free SFM versus 9.86 ± 0.94 and 9.87 ± 0.61 in the blood-free media containing Cellastim S and Optiferrin, respectively. Adipose-derived MSC exhibited similar trends in that cells cultured in the xeno free SFM doubled 19.01 ± 1.62 and 17.02 ± 1.28 times for each donor while the blood-free media doubled 20.25 ± 1.52 and 16.72 ± 1.05 times.

Mesenchymal Stem Cell Morphology and Phenotype

expanded in Cellastim S and Optiferrin also appeared 
to be morphologically normal during expansion. Cells also exhibited normal phenotype of bone marrow and adipose derived MSC. (See figure 3 & 4 on poster for supporting data)

Please see poster for complete data and methods – Inclusion of Recombinant Albumin and Transferrin Enables the Blood-Free Expansion of Mesenchymal Stem Cells in Chemically Defined Media

Inclusion of Recombinant Albumin and Transferrin Enables the Blood-Free Expansion of Mesenchymal Stem Cells in Chemically Defined Media
Also, don’t miss the InVitria webinar on this topic:

Using Blood-Free Insulin, Transferrin, and Albumin Supplements to Reduce and Eliminate FBS in Research & Manufacturing

Thursday, June 28th 12-1pm MT

See How Blood-Free Supplements Add Consistency & Reduce Variability

It is well known that serum and other animal-derived components can create inconsistency in cell culture, leading to variability in cell growth, phenotype, and functional performance. Therefore, it is critical to both manufacturing and research applications to reduce or completely eliminate the use of these ingredients.

InVitria offers blood-free cell culture supplements that effectively reduce the amount of serum needed in routine cell culture by replacing the insulin, transferrin, and albumin found in serum with recombinant versions. These supplements, known as ITSE AF, ITS AF, and ITSE + A AF, also serve as a suitable foundation for fully serum and blood-free cell expansion.


  1. Generating Recombinant Versions of Human Serum-Derived Proteins – Transferrin and Albumin, Cell Culture Dish
  2. Albumin in Cell Culture Media – An examination of quality and function, Cell Culture Dish
  3. Albumin Fatty Acid Profiles for cell culture media – Enabling Albumin Optimization for Cell Culture Media

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