The rapid growth of translational applications for mesenchymal stem cells (MSCs) has heightened the need for efficient, scalable production systems. Central to this effort is the optimisation of media formulations, which directly affect MSC expansion, functionality, and overall therapeutic viability. By fine-tuning media composition, businesses can address critical challenges in scaling up production, ensuring that MSCs meet the stringent demands of clinical-grade applications without compromising quality or consistency.

The Importance of Media Optimisation for Scaling Up

The following sections explore how optimised media impacts these key areas, driving the success of MSC-based translational applications.

• Impact on Cell Yield and Quality

Optimised media formulations directly impact MSC growth rates, proliferation, and viability. High cell yields are essential to meet the requirements of MSC-based therapies, which often demand significant cell quantities. For instance, the recently approved MSC therapy RYONCIL (remestemcel-L) provides a clear example of these demands. 

In clinical applications, patients received intravenous infusions of RYONCIL at a dosage of 2 x 10⁶ MSCs/kg twice a week for four consecutive weeks, totalling eight infusions. An average adult weighing 70 kg translates to 1.12 billion MSCs per infusion or approximately 8.96 billion MSCs for the entire treatment course. 

Such substantial cell quantities highlight the critical need for media formulations that enable efficient and scalable MSC expansion while maintaining the functional integrity of the cells. Without optimised media, achieving these cell yields within the required timelines could become a significant bottleneck, limiting the feasibility of large-scale therapeutic applications.

Moreover, the quality of MSCs remains paramount. Media must support the preservation of functional properties, including differentiation potential, immunomodulatory effects, and the expression of key markers like CD73, CD90, and CD105. These attributes are vital for ensuring the therapeutic efficacy and safety of MSC-based treatments.

• Addressing Cost-Efficiency 

Scaling up MSC production involves considerable costs, particularly when transitioning from research-grade to clinical-grade processes. Media optimisation plays a pivotal role in controlling these expenses, ensuring that large-scale MSC expansion remains both feasible and accessible. 

Stem cell therapies, including MSC-based treatments, can be prohibitively expensive, with costs ranging anywhere from $5,000 to $50,000 per treatment course, depending on the complexity of the procedure, regulatory requirements, and production scale. These costs can limit the availability of life-changing therapies for many patients, creating a barrier to widespread adoption and impact. 

Optimised media formulations are key to reducing these costs. Businesses can significantly lower production costs by maximising cell yields and minimising reliance on expensive or redundant components. For example, transitioning from serum-based media to defined media can improve consistency while reducing risks of contamination and variability, which in turn decreases downstream costs associated with quality control and compliance. 

By prioritising cost-efficiency through media optimisation, businesses can achieve a dual objective: maintaining profitability while ensuring that more patients can benefit from the transformative potential of MSC-based therapies. 

• Regulatory Compliance

Regulatory requirements for MSC-based products are stringent, reflecting the critical need to ensure safety, efficacy, and consistency in therapeutic applications. Media composition is a focal point in this process, as it directly impacts the quality of the final MSC product.

Optimised media formulations play a crucial role in meeting these regulatory standards. By carefully selecting and standardising media components, manufacturers can minimise variability, ensuring that MSCs consistently meet quality benchmarks. This is particularly important for demonstrating reproducibility across batches, a key requirement for regulatory submissions.

The use of Good Manufacturing Practices (GMP)-grade media further enhances compliance by adhering to strict manufacturing and quality control standards. Such media are produced under controlled conditions, with robust documentation and traceability, ensuring they meet regulatory expectations for clinical-grade applications.

Aligning media development with regulatory expectations from the outset is a critical step for ensuring success in translational applications. Addressing compliance during the formulation stage helps minimise the risk of delays or rejections during the approval process, facilitating a smoother transition from research to clinical use.

Key Components in MSC Media Formulation

Optimising MSC media begins with understanding its key components and their functions. Here are the essential elements that support MSC growth and proliferation:

1. Basal Media
   • Basal media provide essential nutrients, including glucose, amino acids, vitamins, and minerals, that are critical for cell survival and growth. Different formulations of basal media, such as DMEM, α-MEM, and IMDM, are commonly used across laboratories, each offering distinct advantages depending on the specific requirements of the culture. While α-MEM-based media are often considered optimal for the isolation and expansion of MSCs, the ideal choice of basal media can vary depending on the specific type of MSCs being cultured. 
   • When selecting basal media, researchers should consider not only its impact on the proliferative capacity of MSCs but also its ability to preserve their intrinsic characteristics, such as differentiation potential and surface marker expression. Both aspects should be thoroughly evaluated to ensure that the chosen basal media supports both efficient expansion and the maintenance of MSC functionality. Therefore, it is important for researchers to test and assess these factors in relation to their specific MSC culture needs.
2. Serum or Serum Substitutes
   • Fetal bovine serum (FBS) has traditionally been a key component in MSC culture media due to its rich content of growth factors, hormones, and other nutrients that promote cell growth and expansion. However, the use of FBS is associated with several challenges, including ethical concerns related to its animal-derived origins, variability between different lots, and regulatory hurdles in clinical applications. These issues have led to a growing interest in alternative media formulations, particularly serum-free or defined media, which are seen as more consistent and suitable for large-scale, clinical-grade production. 
   • Human serum and human platelet lysates (HPL) have emerged as prominent alternatives to FBS. Human serum, derived from healthy donors, offers a more humanised environment for MSC culture and helps reduce the risk of immunogenic reactions that may arise from animal-derived components. HPL, in particular, has gained significant attention as a highly effective substitute for FBS. It is rich in growth factors, cytokines, and other components essential for MSC proliferation and maintenance. HPL usage has become an important step toward the development of xeno-free, humanised culture models, which are crucial for ensuring safety and regulatory compliance in clinical applications.
3. Growth Factors and Supplements
   • Growth factors such as fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF) are essential for MSC proliferation and differentiation. These factors exhibit pleiotropic effects, influencing various biological processes, including cell proliferation, morphology, immunophenotype, survival, and differentiation capacity. The optimal combination and concentration of growth factors to promote the physiological in vitro culture of MSCs are still areas of active research. 
   • In addition to growth factors, other supplements like insulin, transferrin, and selenium are commonly included in MSC media. These components support MSC metabolism, reduce oxidative stress, and improve cell viability, thereby enhancing overall culture conditions. The precise formulation of growth factors and supplements remains crucial for maintaining MSC functionality and ensuring successful expansion for downstream applications.

Optimising Media Formulations

Optimising medium formulation involves fine-tuning key components to support robust cell growth, maintain functionality, and ensure scalability. Advances in experimental design and innovative technologies such as machine learning are transforming traditional approaches, enabling precise and data-driven improvements in media performance. The following sections delve into experimental strategies, critical parameters, and challenges in optimising MSC media for both research and clinical use.

Experimental Design for Media Optimisation

Modern strategies for optimising MSC culture media focus on understanding the complex interplay between components such as carbon sources, amino acids, vitamins, and trace elements. These efforts balance traditional approaches with cutting-edge innovations to enhance efficiency and scalability.

1. Traditional Methods 

• OFAT (One-Factor-at-a-Time): Adjusts individual components to determine their effects but is time-intensive and overlooks interactions between variables. 

• Design of Experiments (DOE): Efficiently optimizes up to 10 components and identifies significant factors but struggles with highly complex systems. 

• Response Surface Methodology (RSM): Models relationships between variables to refine formulations but can oversimplify intricate interactions.

2. Machine Learning (ML) Innovations 

ML has transformed media optimisation by managing complex datasets and providing predictive insights that traditional methods cannot achieve. 

• Active Learning: Focuses on the most informative data points, reducing the experimental burden while improving prediction accuracy. 

• Gradient Boosting Decision Trees (GBDT): An interpretable algorithm that identifies the impact of individual medium components on cell growth and viability, offering actionable insights.

Leveraging ML tools like active learning and GBDT enables precise and efficient media optimisation, setting the stage for advancements in MSC culture and mammalian cell systems.  

Key Parameters to Monitor

Optimising media formulations for MSC culture involves comprehensive evaluation to ensure the medium supports robust growth, maintains cell health, and preserves the unique properties of MSCs. Below are the critical parameters to assess:

• Cell Growth Rates, Viability, and Population Dynamics 

A key indicator of media performance is its ability to sustain high cell growth rates while maintaining cell health. Metrics such as population doubling time (PDT) and population doubling level (PDL) are essential for evaluating the proliferative capacity of MSCs over multiple passages. 

• PDT reflects how quickly MSCs double in number and is a useful measure of the medium’s capacity to support rapid yet healthy proliferation. A prolonged PDT might indicate suboptimal media conditions or the onset of senescence. 

• PDL tracks the cumulative number of cell divisions and is vital for monitoring long-term culture stability. Maintaining a high PDL without compromising cell quality is crucial for large-scale expansion. 

Routine checks for early senescence are essential, as senescent cells lose their regenerative potential and differentiation capacity. Indicators of senescence include morphological changes, such as increased cell size, and expression of senescence-associated markers like β-galactosidase. Ensuring media supports healthy cell cycles helps minimise senescence and ensures consistent results in downstream applications.

• Phenotype and Adherence to Plasticware 

MSCs are characterised by their fibroblast-like morphology and plastic adherence, as per the ISCT criteria. The culture medium must enable MSCs to retain their typical spindle-shaped morphology and adhere uniformly to standard tissue culture plasticware. Loss of adherence or alterations in morphology, such as becoming rounded or irregular, could signal suboptimal media conditions or cellular stress. Regular microscopic examination of cell morphology and adherence properties is critical for confirming media suitability.

• Differentiation Potential 

One of the hallmark traits of MSCs is their ability to differentiate into osteogenic, adipogenic, and chondrogenic lineages. The media must preserve this multipotency throughout expansion. 

This can be tested by inducing differentiation into these lineages and evaluating lineage-specific markers using staining techniques (e.g., Alizarin Red for osteogenesis, Oil Red O for adipogenesis, and Alcian Blue for chondrogenesis) or molecular assays such as qPCR or immunohistochemistry. 

Media that compromises differentiation potential might lead to suboptimal outcomes in research or therapeutic applications, highlighting the importance of maintaining a physiological culture environment.

• Expression of MSC Markers 

Adherence to ISCT guidelines for MSC characterisation is crucial. These require MSCs to express markers such as CD73, CD90, and CD105 while lacking markers like CD14, CD19, CD34, CD45, and HLA-DR. Media formulations should support the stable expression of these surface markers over multiple passages.

Flow cytometry is the gold standard for assessing marker expression and verifying that cells meet the ISCT definition of MSCs. Consistent marker expression across passages indicates the preservation of MSC identity and quality.

Key Challenges in MSC Culture Media Optimisation and Solution 

There are several challenges that must be addressed to fine-tune formulations that support high-quality, viable MSCs. These challenges stem from the inherent complexity of MSC biology, donor variability, and the demands of large-scale production. Below are the key challenges researchers face when optimising MSC culture media:

• Component Balance 
  One of the most significant challenges in MSC culture is maintaining the proper balance of nutrients, including glucose, amino acids, and vitamins. Imbalances can lead to issues such as excessive lactate production, impaired cellular respiration, and compromised cell health, which can negatively impact downstream applications. To address this, researchers can conduct metabolic profiling to monitor nutrient consumption and waste accumulation, enabling precise adjustments to media composition. Dynamic media formulations combined with real-time monitoring systems can help maintain optimal nutrient levels during culture. Additionally, the use of buffering agents can mitigate lactate accumulation and associated cellular stress, ensuring a more stable environment for MSCs. 

• Specific Needs for MSCs from Different Sources 
  MSCs derived from bone marrow, adipose tissue, or umbilical cord exhibit distinct metabolic requirements and growth factor dependencies. A universal media formulation often fails to meet these diverse needs. To overcome this, media formulations must be tailored to the specific requirements of each MSC type. This can be achieved through detailed metabolic profiling and pre-optimisation experiments to identify the unique nutritional and environmental needs of each cell type. Incorporating targeted supplements, such as cytokines or specific amino acids, can further enhance growth and functionality. The development of flexible and customisable media solutions ensures that the specific characteristics of MSCs from diverse sources are adequately supported. 

• Donor Variability 
  Donor-to-donor variability poses a considerable challenge, as MSCs from different individuals, even within the same source, exhibit unique growth rates and metabolic profiles. This variability complicates the development of standardised media formulations. 

Addressing this challenge involves implementing screening and stratification methods to group donors with similar metabolic characteristics. Versatile additives, such as human platelet lysates or defined growth factors, can support a wide range of donor variations. Additionally, maintaining stringent quality control protocols ensures that media performance remains consistent across batches, mitigating the effects of donor heterogeneity.

• Process Conditions and Scalability 
  Scaling up MSC cultures from laboratory settings to large-scale production introduces complexities related to oxygen tension, pH, and nutrient availability. These changes can compromise cell viability and functionality. To tackle this, bioreactors equipped with real-time sensors for monitoring critical parameters provide precise control over culture conditions. Implementing fed-batch or perfusion culture systems can ensure a continuous supply of nutrients while effectively removing waste products. Furthermore, media formulations must be designed to remain stable and effective across various scales, supporting long-term culture durations required for therapeutic applications. 

• Cost Constraints 
  The high cost of MSC culture media, particularly due to expensive components like growth factors, presents a significant barrier to scalability. Reducing production costs without compromising cell performance is crucial. One solution is to substitute expensive components with cost-effective alternatives such as human platelet lysates. Purchasing media components in bulk and working with suppliers to develop custom formulations can also reduce costs. By achieving a balance between affordability and performance, cost-effective scalability becomes feasible for therapeutic applications.

• Optimisation Complexity

The iterative nature of media optimisation, which often involves high-throughput screening and computational modelling, can be time-consuming and resource-intensive. Researchers can streamline this process by employing high-throughput screening platforms that allow simultaneous testing of multiple formulations. DOE methodologies systematically evaluate component interactions, enabling efficient identification of optimal conditions. Machine learning algorithms provide an additional advantage by analysing historical data and predicting the best formulations, significantly reducing the need for repeated experimentation.

• Scale-Up Challenges 

Scaling up MSC cultures brings additional hurdles, such as ensuring consistent media performance, homogeneous cell growth, and stable nutrient levels in larger volumes. Media stability tests conducted under scale-up conditions are essential to verify consistent performance over extended culture periods. Advanced bioreactors with optimised mixing systems can ensure even distribution of nutrients and oxygen while preventing metabolic stress. Intermediate-scale validation trials help identify potential issues before full-scale production, enabling early troubleshooting. Moreover, optimising media concentrations to balance cost and quality ensures scalability remains economically viable.

Regulatory Guidelines for MSC Culture Media Manufacturing

The production of culture media MSC applications, particularly in translational and clinical settings, requires strict adherence to regulatory standards. These guidelines ensure the safety, consistency, and efficacy of the end products, which is critical for the success of MSC-based therapies. Below are the key regulatory considerations for MSC culture media manufacturing, along with insights into compliance practices and emerging trends. 

1. Compliance with GMP Standards: 

• Media used MSC culture in translational or clinical applications must adhere to GMP. This ensures consistent quality, safety, and efficacy of the end-product, especially when the MSCs are intended for therapeutic use. 

• GMP regulations cover all aspects of media production, from raw material sourcing to the final sterilisation and packaging processes. 

2. Raw Material Sourcing and Traceability: 

• Ingredients used in MSC media, such as basal media, growth factors, and supplements (e.g., human platelet lysate or serum substitutes), must meet regulatory-grade standards. 

• Traceability of raw materials is crucial to ensure compliance and address potential contamination risks, such as endotoxins, adventitious agents, or mycoplasma. 

3. Documentation and Validation: 

• Detailed documentation of the formulation and manufacturing process of MSC media is required to meet regulatory scrutiny. This includes batch records, material certificates of analysis (CoA), and process validation reports. 

• Validation involves demonstrating the sterility, stability, and consistency of media performance in supporting MSC expansion and maintaining their therapeutic properties (e.g., multipotency and immunomodulation). 

4. Compliance with ISO Standards: 

• Media for MSC culture must adhere to relevant ISO standards, such as ISO 13485, which outlines quality management systems for the production of medical devices, ensuring consistency, safety, and reliability in manufacturing processes. 

• Additionally, MSC culture media should conform to guidelines like USP <1043>, which provides recommendations for ancillary materials, emphasizing their quality, safety, and suitability for use in cellular and tissue-based therapies. 

• Together, these standards and guidelines ensure the media manufacturing process supports regulatory compliance, enhances product quality, and minimises risks related to contamination or variability. 

5. Alignment with Regional Regulatory Bodies: 

• MSC culture media manufacturing must align with the requirements of regional regulatory agencies, such as the Food and Drug Administration (FDA) in the United States, European Medicines Agency (EMA) in Europe, Pharmaceuticals and Medical Device Agency (PMDA) in Japan, or Health Sciences Authority (HSA) in Singapore. 

• Agencies may have specific requirements for MSC-based therapies, such as the need for detailed characterisation of the media’s role in maintaining the cells’ therapeutic attributes. 

6. Sterility Testing and End-Product Release Criteria: 

• Each batch of MSC media must undergo rigorous sterility testing, including assessments for bacterial, fungal, and mycoplasma contamination. 

• Release criteria should also evaluate the media’s ability to support MSC proliferation, viability, and functional properties. 

7. Emerging Trends and Guidance: 

• Regulatory guidelines are evolving to address the complexities of advanced therapy medicinal products (ATMPs), which include MSC-based therapies. 

• Manufacturers should stay informed about new guidance, such as the FDA’s recommendations on raw material risk assessments and the EMA’s standards for media used in cell and gene therapies. 

Key Trends Shaping the MSC Culture Media Development

The field of MSC culture media is undergoing rapid evolution to meet the growing demands of regenerative medicine, cell therapy, and advanced bioprocessing. These trends reflect a shift toward more precise, scalable, and ethically compliant solutions that enhance MSC growth, maintain functionality, and ensure regulatory compatibility. Below, we explore the major advancements driving innovation in MSC culture media development.

• Development of Chemically Defined Media:
  • The shift from serum-based to chemically defined media (CDM) is a pivotal advancement in MSC culture. By eliminating animal-derived components, CDM reduces variability, minimises contamination risks, and improves batch-to-batch consistency—essential for therapeutic applications. 
  • Regulatory guidelines favour CDM for traceability and reproducibility, as serum-based components carry risks of pathogen contamination. CDM also aligns with scalability requirements, supporting consistent MSC growth from small-scale research to clinical manufacturing. Advances in CDM formulations are driving adoption, with tailored media optimised for MSC expansion and differentiation. These formulations incorporate precise concentrations of growth factors and cytokines, ensuring robust cell growth and specific lineage differentiation. The use of recombinant or synthetic components further enhances safety, consistency, and compliance with ethical biomanufacturing standards.
• Increasing Focus on Personalised Medicine
  • The growing emphasis on personalised medicine is reshaping the requirements for MSC culture media. There is a rising demand for customised formulations tailored to expand MSCs derived from diverse tissue sources, including bone marrow, adipose tissue, and umbilical cord. These media formulations are designed to maintain the unique biological properties of MSCs, which are crucial for their therapeutic efficacy. 
  • Personalised MSC therapies, particularly autologous treatments, require media that not only support robust cell expansion but also ensure the retention of critical functional attributes, such as immunomodulatory or regenerative capabilities. Media customisation is increasingly directed toward specific therapeutic applications, such as enhancing tissue repair or modulating immune responses, enabling precision in MSC-based treatments. 
  • This trend is further driven by the need to address patient-specific variability, ensuring that media formulations align with the unique requirements of different donor profiles and disease contexts. As the field evolves, personalised culture media are set to play a pivotal role in advancing the safety, efficacy, and scalability of MSC therapies.
• Advancements in Cell Culture Technology 
  • Innovative bioprocessing techniques are revolutionising the production of MSCs, driving the need for specialised media formulations. Bioreactors, now widely used for large-scale MSC production, demand media that can sustain high-density cultures without compromising cell viability, phenotype, or therapeutic functionality. These formulations must also account for the unique metabolic requirements of MSCs in dynamic culture environments. 
  • Emerging technologies such as microcarrier-based culture systems and 3D culture platforms are further influencing media design. Microcarriers enable scalable adherent MSC expansion, while 3D culture systems aim to replicate in vivo conditions more closely. Both approaches require media that support efficient cell attachment, growth, and differentiation under mechanically active or spatially complex conditions. 
  • Additionally, the integration of automation and real-time monitoring tools into MSC manufacturing workflows is shaping the demand for adaptable media. These technologies require consistent, robust formulations that can perform reliably across various culture systems while minimising batch-to-batch variability. As these advancements continue to evolve, they highlight the importance of tailoring MSC culture media to the specific requirements of cutting-edge cell culture technologies, ultimately driving efficiency, scalability, and therapeutic potential in MSC applications.
• Emergence of Serum-Free and Xeno-Free Media
  • The shift toward serum-free and xeno-free media reflects a growing emphasis on producing clinical-grade MSCs suitable for therapeutic applications. Serum-free formulations avoid the variability and ethical concerns tied to FBS, while xeno-free media exclude non-human animal components, relying instead on human-derived supplements like human platelet lysate. This transition enhances reproducibility, mitigates the risk of zoonotic contamination, and ensures regulatory compliance, particularly for therapies requiring stringent traceability and safety standards.  
  • One notable advancement in this area is STEMGOLD, a xeno- and serum-free human MSC medium designed for the high-performance expansion of hMSCs across multiple lineages. This innovative medium maintains the trilineage differentiation potential of hMSCs while adhering to ISCT criteria. Its GMP-compliant manufacturing ensures minimal lot-to-lot variation and enhanced experimental reproducibility, making it an ideal choice for both research and therapeutic applications. STEMGOLD also eliminates the need for pre-coating, streamlining the MSC expansion process and improving efficiency. 
  • By leveraging technologies like STEMGOLD, researchers can achieve greater reproducibility, mitigate contamination risks, and meet the stringent regulatory requirements for MSC-based therapies. These advancements are critical for supporting the transition of MSC research from the laboratory to clinical settings. 

Optimising media formulations is a cornerstone of successful MSC expansion for translational applications. By understanding the core components, leveraging innovative optimisation strategies, and addressing scalability and regulatory challenges, bio-entrepreneurs can position themselves at the forefront of MSC-based therapies. Products like STEMGOLD offer valuable solutions, paving the way for consistent, scalable, and high-quality MSC production. 

References

https://www.ema.europa.eu/en/human-regulatory-overview/advanced-therapy-medicinal-products-overview/guidelines-relevant-advanced-therapy-medicinal-products

https://www.fda.gov/drugs/pharmaceutical-quality-resources/facts-about-current-good-manufacturing-practice-cgmp

https://www.hsa.gov.sg/ctgtp/regulatory-overview

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