Stem cells have promising use in cell therapy applications because they have the potential to generate an entirely new organ. Mesenchymal stem cells (MSCs), despite various challenges due to their heterogeneity, have become increasingly popular for therapeutic use.
Mesenchymal stem cells can be defined as stem cells, stromal cells, or medicinal signaling cells. They are classified as advanced therapeutic medicinal products in the United States and Europe. In the U.S. this means that they are cell therapy products, but Europe further differentiates between somatic and tissue engineered products. These differences in classification dictate the regulatory processes in which cell manufacturing must be compliant.
The following article will summarize the therapeutic properties, quality attributes, and expansion methods of MSCs, as well as some major challenges these cells create when used in cell therapy applications.
Therapeutic properties of MSCs
MSCs can be isolated from bone marrow, adipose tissue, or umbilical cord blood. They can also be found in other adult, fetal, and perinatal tissues. These cells are heterogeneous and polyclonal with 3 subpopulations:
- Type I : spindle shaped, fibroblast-like
- Type II : large, flat, epithelial-like, senescent
- Type III : small, round, self-renewing
The minimal criteria for MSCs in cell therapy manufacturing are as follows:
- Show plastic adherence
- Can differentiatie into cartilage, bone, and fat tissue in vitro
- Express CD73, CD90, and CD105, but not CD11b, CD14, CD19, CD34, CD45, or HLA-DR
- Migrate to injury sites, secrete chemoattractants (cytokines, chemokines, growth factors, extracellular vesicles) that recruit stem cells to repair the damage
Quality Attributes of MSCs
The heterogeneity of these cells can pose challenges when implementing manufacturing guidelines. Thus, the establishment of Quality by Design (QbD) principles and Critical Process Parameters (CPPs) is essential.
Critical Quality Attributes of MSCs include the following:
Potency
Measured via:
- Percent viability
- In vitro functional assays such as MSC differentiation capacity (RNA/protein analysis and secretome analysis (ELISA, mass spectrometry)
Sterility, purity
- Purity guaranteed using cell-specific sorting
- Sterility tests
Expansion of MSCs in vitro (CPPs critical process parameter)
Seeding density — important to consider because of the variability of starting material and source yield (i.e. bone marrow aspirate vs. adipose tissue, etc.). Therapeutic applications require at least 1 x 108 cells per dose.
Culture age — expansion process should not allow the phenotypic changes of the MSCs to alter their original purpose for therapy (i.e. transformation, cytokine secretion, etc.)
Culture medium — Component considerations include, but are not limited to glucose, glutamine, fetal calf serum, human serum, and human platelet lysate. Glucose is the main carbon source for MSCs and can be provided at concentrations of 1 g/L- 4.5 g/L. Glutamine is a second carbon source (provide at 2-4 mΜ). It is recommended to use a more stable form of glutamine such as GlutaMAX to prevent unwanted byproducts such as ammonia. Purified serum proteins and lysates can be used in place of whole serum to reduce the amount of undefined components provided in the media. Serum-free media is the most optimal and GMP-compliant. However, the main issue is that cells cultured in serum-free media senesce more quickly.
Culture vessel conditions — Oxygen supply (21%) in tissue culture flasks is much higher than under physiological conditions (5-7%). This can increase Reactive Oxygen Species (ROS) and thus, should be adjusted accordingly. Temperature and pH must be optimized for each MSC subtype. Vessel material for culture is typically polypropylene, but the cells can also grow on glass or dextran. Cells cultured in serum-free conditions typically require additional coating with adhesion-promoting factors such as fibronectin or vitronectin.
Cell detachment — Standard use of non-specific, proteolytic methods such as trypsin can be damaging to cell markers and reduce viability. Enzymatic methods are more specific to the cell type. The use of dissolvable growth surfaces or thermosensitive surfaces are also an option, but can cause cell aggregation because they act on the cell surface rather than the cells themselves.
Freezing methods — freeze slowly, thaw quickly for allogeneic MSCs
MSC manufacturing for clinical trials
Surprisingly only about 20% of cell therapy centers reported the use of bioreactors, about 80% use T-flasks or cell factories
Advantages of bioreactor use include:
- Automation of expansion and harvest, reduces operation error and contamination risk, fluctuation in culture conditions, more batch to batch consistency
- Traceability – control and monitoring of CPPs
- Examples of bioreactors include fixed (macrocarriers) and fluidized bed, stirred tank (microcarriers), wave, wall-rotating, vertical wheel
- Use of microcarriers allow for higher expansion factor
Major challenges in MSC production for CT
The biggest challenge in using MSCs for cell therapy is being able to develop a process that mimics the natural MSC niche while allowing scale up that doesn’t compromise MSC properties. Due to their heterogeneic properties, it is difficult to standardize these cells. The International Society for Cell and Gene Therapy (ISCT) has published standards for harmonization of potency assays in attempt to overcome this issue.
Written by Angela
Scientific Support Specialist, Lonza Pharma-Bioscience Solutions at Lonza
