Cell Culture Media in Cell Therapy: Towards Serum Free and Beyond

Cell media in Petri dish

Cell Therapies [CT] are a new class of medicine administered to patients as living cells and represent a paradigm shift in our ability to treat chronic, disabling, and degenerative diseases. These therapies have shown great promise in the clinic, but the industry now faces the challenge of commercialisation. Success hinges on the ability to produce a consistent product at a commercially viable cost. To achieve this, manufacturing technologies must improve, and COGs (Cost of Goods) need to be significantly reduced.

Cell culture media is one of the most critical factors in CT manufacturing, influencing production both biologically and economically. It is estimated that media accounts for between 15-20% of total development and manufacturing costs of CT products1,2. While Biologics media predominantly serves to feed production cells, formulations used in the expansion of CTs, like Mesenchymal Stem Cell (MSC) based therapies, play a far greater role. Along with providing nutrition, stem cell media must stimulate cell expansion while maintaining quality as it related to therapeutic Mode of Action (MOA). The expansion process is a sensitive balancing act governed primarily by the media.

An Industry Reliant on Serum
Stem cell media is under-developed in that it still relies heavily on animal- or human-derived supplements that are ill-defined, inconsistent and can pose a serious risk to patient safety.

Conventional cell production processes, established in academic centres to enable clinical trials, have made use of animal derived supplements to support cell proliferation in vitro. Basal media formulations (DMEM or alpha-MEM) are usually supplemented with 10% Foetal Bovine Serum (FBS), providing essential components like hormones and growth factors that are required for cell attachment, proliferation, and maintenance 3.

Today, it is estimated that over 2 million bovine foetuses are harvested annually to produce 800,000 litres of FBS, with the serum market size projected to exceed $1 Billion by 2030 (Allied Market Research).

However, the use of FBS in scaled CT manufacturing is problematic for the following reasons:

Safety: Use of animal derived materials in manufacturing presents the risk of pathogen transmission, xenogenic infections or immunological reactions to bovine proteins.
Heterogeneity: FBS is without a specific or clear composition and lacks production consistency. The resulting batch-to-batch variability influences the reproducibility of the manufacturing process and consequently of the final cell product.
Supply Chain: The FBS worldwide supply chain is a limiting factor for cell manufacturing processes.
Ethical: There is considerable opposition to the harvesting process by animal welfare and ethical associations worldwide.

In 2014, the FDA reported that 80% of all regulatory submissions (INDs) described the use of FBS in their MSC manufacturing processes4, despite being considered a high-risk material (USP < 1043 > ancillary material risk tier 4). Regulatory agencies currently recommend the use of non-animal materials where possible, and the continued use of FBS in pre-clinical and early clinical studies is concerning. Changing a component as integral to the process as cell culture media at phase II, III or beyond is complex, costly, and extremely risky. Beginning with the end goal in mind, it is important to select the right high-quality, low-risk starting material.

In search of Xeno-Free (XF) alternatives, the industry has moved towards the use of human platelet lysate (hPL) or albumin and transferrin purified from human serum. These supplements are comparable if not superior to FBS in many culture systems, for example MSCs expanded in hPL generally display higher replicative potential than those cultured in FBS5,6

Typically, hPL is obtained by pooling expired human plasma (within 7 days of collection) and activated through various methods (e.g., freeze/thaw cycles, sonication etc.) to ensure the release of growth factors and proteins from platelet concentrate (PC). Individual proteins like albumin and transferrin, that are used to sustain basic cellular processes, can also be purified directly from human plasma.

As with FBS, challenges remain with the use of hPL for commercial scale CT manufacturing:

Batch Consistency: There is a lack of standardisation in the methods used to produce hPL, with variation in time allowed for PC preparation following plasma harvest/expiration, numbers of pooled samples, and activation methods.
• Safety: Like any other blood derived product, hPL presents a transmission risk for human blood-borne pathogens such as bacteria, viruses, fungi, and prions. Regulatory agencies recommend limiting donor pool sizes to reduce this transmission risk or inclusion of Pathogen Reduction Treatments (PRTs) in the hPL production process.
Quality Control: Performance testing methods need to be established to account for the current variabilities in hPL production systems.

Achieving Reproducibility Through Media Control
As outlined above, media formulations containing serum or undefined components can add variability to the cell production system on top of the already present biological complexity. Therefore, development of Serum-Free (SF), Xeno Free (XF), Animal Component Free (ACF), and Chemically Defined (CD) (Table 1. 6) media for the clinical production of CTs is a high priority within the industry.

Table 1: Media Classification

Classification Definition
Serum-containing mediaContains animal or human serum (i.e., FBS or hPL)
Serum-Free media (SF)Does not contain animal or human serum or plasma as direct/primary ingredients. Media may still contain proteins purified from the blood (i.e., BSA and HSA)
Xeno-Free media (XF)Contains human-derived blood components as direct ingredients (i.e., hPL, human serum) and may contain human proteins purified from human blood (i.e., HSA) and human recombinant growth factors. 
Animal Component Free media (ACF)Does not contain any human or animal components. Does not contain human recombinant proteins and growth factors. Could contain biological proteins expressed in plant and rice (i.e., soy hydrolysate) 
Chemically Defined Media (CD)Media formulation with known chemical components and structures. Does not contain any proteins or complex raw materials. 
Taken from Jayaraman et al., 2021 6

Multiple vendors have commercialised SF, XF or ACF media formulations for CT applications, for example Stem Cell Technologies (Mesencult-ACF), Lonza (Thera Peak) and ThermoFischer (STEMPRO MSC SFM) all offer alternative MSC media for both translational and basic research application. However, barriers to the widespread use of these formulations persist as they frequently fail to generate comparable numbers of high quality cells, and their compositions are often confidential. This is a major drawback for clinical grade MSC production, as all media components needs to be traceable and require strict quality control. 

Recombinant proteins can also serve as an alternative to human plasma derived proteins. For example, in the absence of serum, cell-adhesion factors are often included to promote in vitro attachment and expansion. These can be produced in both mammalian and non-mammalian cell production systems, each with associated quality requirements (ICHQ5A/D, USP < 1043 > ANCILLARY MATERIALS FOR CELL, GENE, AND TISSUE-ENGINEERED PRODUCTS) 7. However, the high concentrations required along with the complex structure and dimension of these proteins make their production and isolation an expensive and complicated process, driving up the COGs of a serum free cell manufacturing process.

Finally, the use of CD media formulations is becoming an increasingly attractive prospect in the CT manufacturing industry. A CD media is a synthetic media formulation, containing active chemical components for which the quantity and the composition is known. This has the potential to minimises the introduction of unknown or undesirable components into the cell expansion system, in turn minimising the impact of ancillary material variability on the final therapeutic product. 

Small molecules as media supplements are an attractive tool as they are generally easily available, chemically defined and tuneable. Chemical approaches are currently used in various stem cell modulation applications, including the reprogramming of somatic cell towards a stemness phenotype, differentiation of MSCs towards lineage-specific phenotypes, or for drug screening in the context of cancer stem cells. A step forward in developing fully CD media is represented by emerging Animal Component Free (ACF) formulations, which do not have any human or animal derived components (e.g. serum, plasma, cytokines or growth factors).

Media Development & Qualification 

Developing the next generation of CT media is a gruelling challenge, with current strategies constrained by very low-throughput cell assays that can take months to test just a few candidate media formulations. Commonly used screening approaches like Design of Experiment (DOE) or One-factor-at-a-time (OFAT) offer limited throughput, and are only as good as the analytical tools employed to assess cell quality in each condition. It is clear that to develop better CT media, we need faster and richer cell assays. 

Once defined, a second challenge lies in ensuring media consistency throughout manufacturing. Persistent use of supplements like FBS and hPL means that therapy manufacturers need to establish stringent qualification programmes for these ancillary materials, to ensure comparability between manufacturing runs. Understanding how media variation impacts cell product quality requires simple, rapid and biologically informative assays of cell function. 

At ValitaCell, we are developing faster and more efficient cell characterisation tools to support media development and qualification programmes in Cell Therapy manufacturing. Connect with us to find out more about how our ChemStress and CellAi technologies could improve your media performance and consistency. 


1. ten Ham, R. M. T. et al. Estimation of manufacturing development costs of cell-based therapies: a feasibility study. Cytotherapy 23, 730–739 (2021).

2. Lipsitz, Y. Y. et al. A roadmap for cost-of-goods planning to guide economic production of cell therapy products. Cytotherapy 19, 1383–1391 (2017).

3. Subbiahanadar Chelladurai, K. et al. Alternative to FBS in animal cell culture – An overview and future perspective.Heliyon 7, e07686 (2021).

4. Mendicino, M., Bailey, A. M., Wonnacott, K., Puri, R. K. & Bauer, S. R. MSC-Based Product Characterization for Clinical Trials: An FDA Perspective. Cell Stem Cell 14, 141–145 (2014).

5. Bieback, K. et al. Human Alternatives to Fetal Bovine Serum for the Expansion of Mesenchymal Stromal Cells from Bone Marrow. Stem Cells 27, 2331–2341 (2009).

6. Jayaraman, P., Lim, R., Ng, J. & Vemuri, M. C. Acceleration of Translational Mesenchymal Stromal Cell Therapy Through Consistent Quality GMP Manufacturing. Frontiers in Cell and Developmental Biology 9, 636 (2021).

7. Solomon, J. et al. Current perspectives on the use of ancillary materials for the manufacture of cellular therapies. Cytotherapy 18, 1–12 (2016).

Dr. Stephanie Davies
Stephanie Davies, PhD
Head of Cell Therapy

Stephanie Davies leads the application of all Valitacell technology into The Cell Therapy Space, with particular focus on their integration into advanced MSC, & Exosome manufacturing platforms. Stephanie has a primary degree in Genetics and PhD in Molecular Cell Biology from University College Cork with advanced training in cell biology, biochemistry and genetics with extensive experience in the design and optimisation of cell-based systems.

Marilina Piemontese
Marilina Piemontese. PhD
Senior Scientist, Cell Therapy

Marilina Piemontese is Senior Scientist at ValitaCell and she manages and executes the R&D programme within the cell therapy division, with a focus on media development/optimization and novel cell characterization assays. Marilina holds a Master in Biotechnology and a PhD in Molecular Pathology with experience in cell-based assay, screening and imaging and a long standing interest in field of regenerative medicine

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