Cell culture media: ask the experts

Written by RegMedNet

In this ‘Ask the Experts’ feature, a panel of international experts share their perspective on current obstacles and future developments in cell culture media.

Cell culture is a vital step in the development, manufacture and commercialization of any cell-based therapy. Inadequate consideration and planning can lead to inconsistencies in your cell therapy and difficulties in moving it through the regulatory pipeline. Selecting the most suitable cell culture media is crucial to this process.

In this ‘Ask the Experts’ feature, we’ve collected insights from experts around the world to discuss the development of cell culture media and what still needs to be done.

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Please introduce yourself and your institution/company?

Ulla Schultz (US): I am Head of R&D Cell Culture Systems at CellGenix (Freiburg, Germany). I joined CellGenix in 2006 and as head of R&D Cell Culture Systems am primarily responsible for the development of new serum-free media and other cell culture reagents.

CellGenix is a leading global supplier of high quality raw materials for the expanding cell and gene therapy and regenerative medicine space. We develop, manufacture and market human cytokines and growth factors in preclinical and GMP quality, along with GMP serum-free media for further manufacturing of ATMPs.

With 25 years of experience we are experts in the GMP manufacturing of raw materials for the cell and gene therapy space. As a former ATMP developer and manufacturer we gained in-depth cell culture knowledge and superior regulatory expertise. With this unique background we understand the high requirements our customers face during product development and the regulatory approval process. By offering expert technical and regulatory support we can help simplify raw material qualification and validation efforts.

Robert Newman (RN): I’m Chief Scientific Officer for FUJIFILM Irvine Scientific

(CA, USA) –a global leader with decades of industry insight to deliver advanced cell culture media solutions for use in medical and translational research, biopharmaceuticals, regenerative medicine, clinical diagnostics, and reproductive medicine.

Jon Rowley (JRR): I am Founder and Chief Product Officer of RoosterBio (MD, USA). I did my PhD training in Tissue Engineering, and I have spent my 18 years in Industry mostly as a regenerative medicine product development and bioprocess engineer. I have been a lifelong student of entrepreneurship and technology commercialization.
RoosterBio was founded in 2013 with the goal of radically simplifying regenerative medicine product development. My company provides standardized, scalable human mesenchymal stem/stromal cell bioprocess solutions to regenerative medicine product developers that remove years of time and millions of dollars from their product development timelines. We are tremendously impacting the field by industrializing the regenerative medicine supply chain, and we aim to have the same influence on this field that Intel had on the computer industry.

Sara Pijuan-Galito (SPG): I am is a Sir Henry Wellcome Postdoctoral Fellow working between the School of Pharmacy and the School of Medicine at the University of Nottingham (UK). I got my PhD in Uppsala University (Sweden); my thesis focused on stem cell signaling pathways and culture optimization. I was awarded the Swedish Research Council International Postdoctoral grant in 2015, and moved to the University of Nottingham to continue my work on in vitro cell expansion methods and the study of the stem cell “niche” and its repercussions in cell phenotype and expansion capacity. In 2016, I was awarded the Sir Henry Wellcome Postdoctoral grant by the Wellcome Trust, with a project focused on the study of the cell-biomaterial interface, and the cell adhesion events in different extracellular matrix compositions and surface chemistries.

Jeff Ross (JRM): I am CEO of Miromatrix (USA), a Minnesota-based biotechnology company on a mission to save and drastically improve patients’ lives by eliminating the organ transplant waiting list. Through our proprietary perfusion decellularization and recellularization technology, we are developing fully implantable human organs including livers, kidneys, lungs and hearts.

There are, however, some evolutions, such as cell culture under different oxygen concentrations, which has literally exploded in recent years. In the atmosphere, therefore in standard cell culture conditions, oxygen is present for 20%. It has been discovered that culturing cells in lower oxygen concentrations, such as 5% and even 2%, changed dramatically their behavior and properties. The reason is that genes encoding proteins named hypoxia inducible factors (HIFs) are activated under lower oxygen concentration (hypoxia). Changing cell properties under hypoxia is not a mere culture artefact though, but reflects the fact that, depending on the extent of vascularization, some tissues in the body are naturally hypoxic. A whole area of pathobiologic research tries to understand how oxygen concentration regulates cell function, which is of high relevance in organ development and regeneration, as well as cancer.

In another perspective, cell culture is increasingly combined with biomaterial science to determine optimal cell survival and growth conditions, in two and three dimensions. To this aim, cells are cultivated in the presence of adhesion molecules, synthetic matrices and scaffolds. Beyond basic biology, this research aims to determine optimal “packaging” conditions for therapeutic cells to be transplanted into patients.

What are some common mistakes made when choosing or using cell culture media?

US: The initial focus when choosing a cell culture media is the functionality of the media: cell expansion, phenotype, viability and functionality. What’s often forgotten is that at a later stage in the development process, such as the clinical stages and commercial manufacturing, the cell culture media needs to meet international regulatory guidelines and local requirements. We therefore recommend choosing a cell culture media that is compliant with international regulatory guidelines already during early stage preclinical research. Since individual countries can have special local requirements, it is also beneficial to choose a supplier who can give customized regulatory support.

In order to meet global regulatory requirements, it is advantageous to establish a cell culture protocol that is serum-free. This is also a critical consideration for the commercial manufacturing stage because of the high cost, supply limitations, safety issues and high lot-to-lot variation of human serum. Human serum is however still frequently used to enhance cell expansion and promote cell adherence. As a cell culture media developer, we focus on designing serum-free media that offer these functionalities without the need for serum addition.

RN: Media is more than an ancillary agent–it is an important part of the entire manufacturing process and there are several issues that can hinder scale-up. One example is choosing media that works at a small scale without considering its scale-up potential. If a thorough assessment of how media performs isn’t conducted at an early time point, it can lead to issues with manufacturing later on–and a poor product.

Additionally, a lack of understanding regulatory requirements with regard to media composition and its impact on variability in performance can also yield costly mistakes. If a manufacturer selects a serum-containing formula at the outset, its variability between lots can negatively affect the need for consistency at scale-up, in addition to the cost involved with sourcing serum-containing cell culture media as there are only a select few who manufacture it. This is why chemically-defined media, which may be more expensive to use at the outset, becomes cost-effective with scale-up because it delivers consistent, reliable results.

JRR: We believe there are three common mistakes when choosing and using cell culture media, and these are:

  1. not placing a high importance on the quality of the media and media additives, such as growth factors;
  2. grossly underestimating the impact of media cost on the overall cost of your product and;
  3. not monitoring media productivity as a key metric in decision making.

First, anyone hoping to move into clinical development needs to make sure that their media system has a direct path to cGMP sourcing, or the FDA may not accept your investigational new drug (IND) application. “GMP” quality media cannot simply be “turned on” once you are ready for manufacturing, and there can be several months, if not multiple quarters, of experiments and supplier qualification to perform — so choosing high quality suppliers for both medium and ancillary materials, such as growth factors, cytokines and other media additives that are translation-friendly is key. Ideally, a media supplier will have both research use only (RUO), which is usually lower cost, and a cGMP-manufactured version of their media that can simplify this decision-making process.

Second is around cost, which is a four-letter word in manufacturing. Media is always the largest cost driver of both research programs and manufacturing processes. Media itself is expensive, but there are many additional hidden costs associated with media that do not come to light until a process has transitioned into cGMP manufacturing, including labor, waste disposal and storage. I gave a talk at ISCT 2019 on this and the take home message is to minimize total media volume used when growing cells. This concept of minimizing media use is a lesson learnt from the monoclonal antibody industry and can be established in media system design requirements. This should be strongly considered when working with your cell culture media supplier.

Lastly, due to the significant cost of media, many scientists will focus on the per liter cost of media. However, the focus should be on cost of media per cells produced — or more simply, focus on media productivity. The antibody industry focuses on grams of product produced per liter of media, or g/L of therapeutic protein. The analogous metric for cell therapy is millions of cells produced per liter, or M cells/L. It is actually okay if a medium is more costly per liter, if the number of cells generated from that medium is more than the incremental cost increase. That being said, any bioprocess media supplier should be able to talk confidently about the productivity of their media and the impact that this has on your overall final product costs.

SPG: Using media that relies on complex or undefined components, such as using animal-derived additives, without investigating their impact in your results. While many efforts have been directed to the discovery of defined systems for cell culture, a great number of laboratories are still using undefined components routinely, either for economical or historical reasons. One such component is Matrigel, an extracellular matrix solution derived from a mouse sarcoma.

Many studies on human cell differentiation and maturation are currently being performed on Matrigel-coated substrates. However, how could those ever be translatable to clinical applications? We should be more careful over the choice of each component in our protocols.

The extracellular matrix is emerging as a key modulator of cell behavior; its composition, spatial organization and physical properties all have a major impact in cellular spreading, proliferation, migration potential, and even differentiation and organ formation. As such, the choice of extracellular matrix or adhesive substrate is just as important as the media composition and will have a major impact on the resulting research.

JRM: To fully answer this question, we must consider that cultured cells are removed from their native environments, purified and often placed into a 2-dimensional culture system, such as a tissue culture flask. In these environments, cell culture media have been developed for specific cell types often at the detriment of other cells that were viewed as “containing” cell populations.

Under this pretense, it is important to ask “What are you trying to achieve when choosing a cell culture media?” If it is the culturing of a well-characterized cell line, often the media that has been used for many years is adequate. If you are performing primary cell isolation, a common mistake is to use the most readily available cell culture media, which can often decrease cellular functionality, or the use of high serum concentrations to overcome deficiencies of the base cell culture media.  One way to overcome this challenge is to screen multiple base media formulations early on to identify the best media with a defined output such as cell doubling time, metabolism rates, protein secretion, etc.

Another common mistake is in the development and culturing of advanced tissues that involve multiple cell types. Cell culture media has traditionally been developed for a single cell type, which makes culturing multiple cell types difficult since what one cell type favors could inhibit the growth of another cell type. To overcome this, screen many cell culture media and add the appropriate supplements to overcome these challenges.

Cell culture media decisions are often made early on. How can and should the later stages of development be considered when developing a cell culture workflow?

US: Global regulatory guidelines should be considered. We recommend identifying the appropriate serum-free GMP cell culture media prior to clinical development of your cell therapy. This will prevent the need for expensive and time-consuming clinical comparability studies to prove process adaptations or cell culture media changes do not alter the final ATMP. A study performed by the Tufts Center for the Study of Drug Development (MA, USA) estimated that the costs of an amendment for a Phase III trial costs more than three times as much as an amendment for a Phase II trial.

In addition, it’s important to think about how the cell culture workflow will be scaled up or out. Is the cell culture media suitable for production in a different or larger cell culture device? Is the cell culture media available in all global regions where you want to manufacture your cell & gene therapy? Is there a secured supply chain available?

RN: Have your regulatory plan clearly outlined and start gathering regulatory documents in the early stages of development. This way, when you are required to begin submitting regulatory documents in the later stages, you’ll be prepared without having to scramble. If you choose a medium without a drug master file (DMF), you may find yourself having to procure a new media supplier later down the road. To address this, select cell culture media from a manufacturer that maintains strict requirements for raw material control and sourcing, as well as adheres to current good manufacturing practices (cGMP). These efforts assure cell therapy manufacturers of high-quality products that can be scaled-up and transferred to clinical applications and commercial manufacturing. Plus, there is no need to start all over to meet regulatory requirements.

It’s also wise to select an easier-to-use media format and have it dialed-in before scale-up. Less components are better and help to reduce the possibility of adding too many components during the manufacturing process. Plus, there is less chance for errors or mistakes.

JRR: It’s no exaggeration to say that the early decision can ultimately determine the success or failure of a project. We have actually written a series of blog posts (links here and here) that outline how to create a ballpark estimation of what an eventual lot size would be for a successful product, then how to work backwards towards determining a production technology, such as flasks, multi-layer vessels, or bioreactors, needed to achieve those lot sizes, and from there the type of media system is dictated. By performing product development using a medium designed for scalability and demonstrating comparability between flask and bioreactors, it is possible for a program to save years of time and millions of dollars in downstream product development. Therefore, we think it is critically important, and totally possible with today’s products, to have a seamless transition from early stage development all the way through late-stage clinical development.

Learn more about building effective multi-year process development programs on the RoosterBio blog, including estimating hMSC lot size ranges for clinical manufacturing and the evolution of technology platform decisions based on lot size>>

SPG: Even in the early stages of protocol design and optimization, it is important to keep in mind the end-point goal of the research study. For example, if your research is trying to improve maturation of cardiomyocytes for clinical applications and drug screening, the matrix and the media need to be as close to the in vivo conditions as possible and, as such, complexity would be acceptable but animal-derived components would be a major issue for your endpoint goal. Alternatively, if you are looking to gain insight into specific signaling events evoked by a particular molecule or component, highly defined conditions are preferable, so that you can study what each individual component does to the cell and isolate each of them for accurate results.

JRM: A critical early step in development is the successful definition of design criteria. While we often specify design criteria on the function of the cells, it is necessary to add design criteria such as the ability to ensure the media formulation is amenable to GMP and scalable. In this, you will set a priority on not only screening cell culture media that provides you with the appropriate cellular response but also a formulation that will allow it to be scaled and be a part of the final validation.

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What are the challenges relating to cell culture media when scaling up or out a process?

US: Transferring conditions believed to be defined to a new facility is always challenging. It is therefore of great importance to use highly consistent GMP reagents.

Large scale manufacturing requires the use of automated and closed processes. When a process is transferred from a small-scale static culture to a rocking system with greater process controls, process optimization is often required. In some cases, it is even necessary to choose a different cell culture media. The same applies for scaling up to a larger cell culture volume. Increased cell numbers lead to increased chance of inhomogeneity of culture which can change cellular performance.

RN: Having appropriate packaging is key. With automated processes, customers are typically using bag formats, while at smaller scale they are using bottles. When scaling up, stability is also a greater concern as you generally have to buy a lot of media up front, schedule your manufacturing process, and consider the amount of space available to keep product on-hand and ready for the next run. It is also important that your media supplier offer reliable timelines for supplying product. This is vital especially for CAR-T, where fresh cells require enough media for expansion and valuable time necessary to manufacture a patient’s therapy becomes lost waiting for a delivery.

JRR: Scaling up and scaling out cell manufacturing processes are two very different challenges. Scaling up is going from a 3 liter bioreactor to a 50 liter and eventually to a volume of several hundred liters. You can write a book on the challenges of scaling up while maintaining cell health and functional critical quality attributes, so I will avoid these topics. As the challenges related to media systems, they are:

  1. Media exchanges are very challenging, if not impossible, when scaling to the several hundred-liter scale. It would take hours to perform a partial or complete media exchange in a single vessel, leaving a very large number of cells that were expensive to produce in precariously low media volumes that could threaten the entire production run. Some process developers move towards media perfusion; however, this can be a terribly expensive solution as it can take several more total media volumes to generate the same cell densities — thus cratering your cell productivity metrics, measure in M cells/L. We highly recommend moving toward the best practices of CHO production and implementing a fed-batch media system. These systems have demonstrated good effectiveness with hMSCs and are really taking over the market.
  2. Another challenge is moving from open to closed, bagged media systems takes a significant transition for many organizations. New infrastructure is required to weld bags to vessels, to manage an inventory of bagged media and reagents, as well as even moving 50 liters of media from cold storage, warming it up and starting a successful cell culture! These challenges are best addressed over time and can be expensive. Having a supplier that is anticipating your transition into closed systems is very important, as designing the disposable connections between each unit operation can take many months to get right. This is a typical unanticipated bottleneck in many programs.
  3. The biggest challenge of scaling out production processes is also associated with a transition towards closed systems but is compounded by the sheer volume of inventory units. If one scales out a 10-layer process to 80-120 vessels, managing the inventory and movement of 80-120 bags of basal media, plus supplementing each bag with frozen boosters — not to mention all of the other bagged reagents such as PBS, harvest reagents and quench — can become horribly cumbersome. Thus, scaling up into bioreactors is really worth the investment for late-stage clinical development. We still recommend early phase production in 10 layers when lot sizes are less than 10 billion, but when you require 20 billion+ cells then bioreactor scale up is the technology path of choice.

SPG: One of the biggest challenges when scaling up is the cost. In small, laboratory scale research we can use relatively costly media without it having a major impact, but that would not be economically viable when expanding cells in bioreactors for clinical applications, as the spend in media would increase exponentially.

Another major issue is the cell-phenotype control and the optimization of their growth without inducing other changes, such as genomic mutations that derive into cancerous cells. While bioreactor-based expansion is the most cost- and volume-efficient method for large cell expansion, the conditions are known to induce stress on the cells, be that due to shear-stress generated through the mixing strategies used to infuse oxygen or nutrients, or due to higher cell/media ratio, which increases oxidative molecules over the cell population. Any nutritional or physical stress applied onto the cells will have an unavoidable response, typically an adaptation event that will favor cells with a higher adaptable and proliferative phenotype.

A third issue is the difficulty to generate appropriate matrix-adherent conditions for cells grown in large-scale. As much as we have gained insight into the major role that the adhesive and extracellular matrix environment plays in cell behavior, the translation of this knowledge to large-scale culture is still a roadblock, it is much more difficult to control in large-scale processes, as they still require a simplified cell-adhesive method or an adhesive-free culture to be able to work efficiently.

JRM: One of the biggest challenges is to have access to the actual recipe or the ability to ensure the base media can meet GMP specifications. Often, early development is achieved on various commercial-grade premade media without consideration to a much larger scale where hundreds or thousands of liters of media are required weekly depending on the application. A common pitfall is to proceed with these media without the knowledge of the base formulation or access to the powder form to control the manufacturing and input in a larger scaled process. By achieving these considerations, the manufacturing can complete internal validations and reduce handling risks, saving valuable time and money.

What’s the best/most effective technology innovation you’ve seen in the last 5 years?

US: The development of different closed cell culture systems and workflows, such as Cocoon from Lonza (Basel, Switzerland) or the G-Rex closed system (Wilson Wolf Manufacturing, MN, USA). This greatly simplifies the cell culture process and enables large scale manufacturing. We are currently evaluating other product formats that can provide a direct sterile connection of our GMP raw materials to such a closed cell culture system.

RN: There have been some very innovative breakthroughs in the past 5 years including CAR-T therapies, CRISPR gene editing and 3D bioprinting. CAR-T therapies have shown safety and efficacy in the clinic for treating hematological malignancies. CRISPR gene editing technologies provide a simpler approach for editing the human genome which can be used to develop therapies as well as advanced in vitro models for drug discovery. In addition, 3D bioprinting is emerging as an innovative technology for generating human organs and there have been initial successes using sheets of 3D bioprinted skin and cardiac tissue with the potential for developing even more complex organs such as the retina, liver and lungs.

Regarding cell culture media-specific innovations, organoid technology–where mini organs are created in a dish–is a fascinating breakthrough. This technology came out of Hans Clever’s lab in the Netherlands, with his initial success being the creation of gastrointestinal organoids. A number of groups have since become involved with this technology, even creating mini-brains in a dish.

JRR: I think the most effective technology innovation in the last 5 years has been the introduction of plug and play cell and media systems for cGMP manufacturing of cells for therapy. Have cGMP “off the shelf” bioprocess media and working cell banks for purchase with FDA Master Files supporting them removes hundreds of pages from an IND, millions of dollars in development and production costs, and years of time from standard product development timelines. The time of cellular components being as readily available as microchips is going to catapult the regenerative medicine industry forward and accelerate the entire industry!

SPG: The coating-free culture of human stem cells. When Chen et al showed that stem cell media could be reduced to the 8 components that make the Essential 8 formulation in 2011, the media requirements for human stem cells were reduced to a minimal formulation and the scientific efforts were then shifted towards the extracellular matrix. For years, Matrigel had been the preferred replacement for the feeder-cells used in the derivation of the very first human embryonic stem cells lines.

However, scientists started to recognize that this formulation derived from an animal sarcoma was unfit for clinical translation and, therefore, efforts were put towards the development of more defined, easier and simpler ways to accommodate human stem cell growth. The elimination of feeder cells and time-consuming coating procedures has now opened up the possibility to use stem cells in a larger scale and in combination with complex scaffolds, as well as simplifying the protocol for their use by a wider, nonspecialized community of researchers.

JRM: Related to cell culture and media, it has been the focus placed on 3-dimensional culturing surfaces and the use of larger bioreactors to further scale up cell production. By mimicking native microenvironment cells, these innovations have demonstrated greater functionality. In addition, as therapies continue to advance, the need for larger cell sources is required. Consider the bioengineering of organs and the billions of cells that each construct will require. The advancement of these technologies enables the scaling of cell production and just a decade ago was considered cost-prohibitive.

What is the biggest obstacle remaining in the field?

US: To yield a highly reproducible effective product for every patient no matter how variable donor cells may be. This should be achieved with highly defined raw materials in an automated process at an affordable price.

RN: There are actually several obstacles. One is that there is no standard, all-purpose manufacturing format for bioprocessing, which makes the development of “one-size-fits-all” manufacturing reagents a challenge. Another is the cost of cell therapies–to have lower costs, reagents would need to be less expensive. Costs involved with creating these therapies is also high because the scale of manufacturing is small for most clinical trials, and especially for autologous treatments, so the e