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Today, around 10 types of MSC products have gained approval for marketing globally, and this number is expected to grow. These products come in different dosages, including low, medium, and high doses, depending on the therapeutic requirements.

While MSCs from various tissue sources meet ISCT standards, research suggests that they exhibit differences, such as cell size, proliferation potential, secretory factor types and quantities, and immunosuppressive abilities. To address diverse medical indications, different tissue-source MSC products are used. However, the quality of these cells can vary significantly based on the separation technique employed.

The choice of an MSC manufacturing process primarily depends on two factors: the required cell dosage and whether the therapy involves autologous or allogeneic cells:

• Plate Manufacturing Process: For small and medium-scale clinical trials, plate manufacturing processes, including multi-layer plate culture systems or “cell factories,” are often employed. These systems can amplify up to 10 billion cells, offering a cost-effective solution. Automated plate manufacturing processes have also been developed, featuring online monitoring capabilities and closed cultures. However, they may require substantial space and automated handling devices for large-scale production (over 30 billion cells).
  
  

• Bioreactor Manufacturing Process: Bioreactor manufacturing processes are ideal for large-scale MSC production. They provide an environment that mimics in vivo growth conditions, ensuring high-efficiency proliferation, automation, mass production, and high cell viability. Two types of bioreactors—perfusion bioreactors and stirred suspension bioreactors—play significant roles in this process.
  
  

1. • Perfusion Bioreactors: These systems allow for continuous media exchange while minimizing cell shear forces. They offer a continuous surface for cell growth but typically yield lower cell numbers (up to 100 million cells) and are more suitable for autologous cell production.
     
     

1. • Stirred Suspension Bioreactors: These are the go-to choice for large-scale MSC production, with the potential to yield up to 500 billion cells per run. Microcarriers are used to support cell attachment and growth, making this process highly efficient and scalable.

Quality Control System for MSCs

Whether it’s the meticulous process in a petri dish or the intricate dance within a bioreactor, the production of MSC products demands unwavering consistency and stability. Researchers have crafted a toolkit of quality control measures to scrutinize every aspect of these cell products, including:

1. Sterility: Picture this: A journey from the initial tissue collection to the final cell product. At every juncture, the specter of microbial contamination looms. Traditional detection methods, though effective, are time-consuming. Enter modern marvels like BacT/ALERT and BACTEC, FDA-approved systems capable of swiftly spotting fungi, anaerobic, and aerobic bacteria. But that’s not all. Detecting mycoplasma using PCR or similar methods is vital throughout the process. And let’s not forget the quantitative measurement of endotoxin levels, ensuring the product’s purity.

    

1. Safety: While MSCs are generally regarded as safe, a shadow of doubt looms when it comes to tumorigenicity. Safety evaluations are imperative. Standard G-banded karyotype analysis is used to spot chromosomal abnormalities. To delve deeper, techniques like comparative genomic hybridization (CGH), fluorescent in situ hybridization (FISH), or PCR can unveil potential tumorigenicity. Senescence, while not linked to malignant transformation, is also monitored for safety and efficacy. In essence, ensuring the cells are as youthful as possible.

    

1. Cell Activity: Assessing cell viability is a pivotal step. The go-to method, trypan blue staining, relies on the integrity of the cell membrane to gauge viability. It’s cost-effective but time-consuming for large samples. For a more precise assessment, Annexin V/PI co-staining and flow cytometry reveal even early apoptotic cells, ensuring the utmost accuracy.

    

1. Cell Surface Markers: The ISCT set the stage by defining MSCs based on surface markers. The markers CD105, CD73, and CD90, along with the absence of CD45, CD34, CD14, CD11b, CD79α, CD19, and HLA-DR, provide a framework. However, these markers have limitations and can’t predict therapeutic properties. The hunt is on for new, MSC-specific markers that can better guide production and clinical transformation.

    

1. Product Purity: Purity isn’t just about safety; it impacts efficacy too. Residual contaminants must be quantified to ensure they’re within acceptable levels. Molecular pollutants, like residual proteins or cytokines, and cellular contaminants, such as non-MSCs, demand attention. A quantitative analysis of residual pollutants assures product safety and effectiveness.

    

1. Stability: MSCs are delicate beings, sensitive to their surroundings. Storage and transport conditions play a crucial role in maintaining cell quality. While fresh MSCs can endure for hours at 4°C in saline, longer storage diminishes their abilities. Once production is complete, swift transport to the clinical setting is vital to minimize cell loss.

    

The quest for MSC quality control is an integral part of the stem cell therapy journey. It intertwines with every step, from tissue collection to product release. Yet, achieving the pinnacle of preclinical efficacy goes beyond quality control. It ventures into the realm of biological efficacy, an intriguing concept we’ll explore in our next chapter.

Efficacy Evaluation

The potency of cell products, as defined by the FDA, encompasses the unique abilities or functions of a product based on clinical data and laboratory methods. It involves two critical facets: strength and effectiveness, referring to the achieved effect at a given concentration. However, assessing the potency of MSCs is complex due to their variability in tissue source, preparation processes, and characterization methods.

Initially, researchers believed that MSCs would differentiate in vivo to replace damaged cells. However, in vivo differentiation remains unproven, and current consensus suggests that MSCs primarily exert their effects through paracrine mechanisms. These mechanisms involve the secretion of various molecules, including cytokines, chemokines, growth factors, and extracellular vesicles.

To effectively evaluate MSC efficacy, it is crucial to consider specific indications. MSCs exhibit diverse therapeutic actions, primarily immune modulation and angiogenesis promotion. Hence, potency assays should align with these actions, ideally showing a positive correlation with treatment outcomes.

For instance, in immune-related diseases, MSCs’ ability to reduce inflammation and regulate the immune system is pivotal. The ISCT has issued guidelines recommending immune function testing as a release standard for MSC products. This includes the quantitative detection of mRNA, surface marker analysis, and assessing secretion-related proteins. Specific biomarkers like TSG-6 and PGE2 have been identified as indicators of immunomodulatory function.

Conversely, in indications related to angiogenesis, the evaluation should focus on promoting blood vessel growth. Quantifying the secretion of vascular endothelial growth factor (VEGF) and conducting in vitro assays to measure angiogenic activity are essential steps. Other factors like CXCL5 and IL-8 also play crucial roles in assessing angiogenesis capacity.

In some cases, understanding the precise mechanisms of MSC action remains a challenge. However, emerging research suggests that extracellular vesicles (EVs) and mitochondrial transfer may be involved. EVs, including exosomes and microvesicles, are carriers of signaling factors and RNA. Studies have shown that MSC-derived EVs can replicate the therapeutic effects of MSCs themselves. Mitochondrial transfer, wherein MSCs transfer mitochondria to target cells, is also being explored as a potential therapeutic mechanism.