Laying the foundations for a robust stem cell workflow

Understanding how to build robust stem cell workflows is becoming central to advancing safe, scalable regenerative therapies. However, the journey from research-stage workflows to translation-ready processes is fraught with challenges, requiring meticulous planning, robust standardization, and a deep understanding of cellular mechanisms.

In this interview, Hagen Wieland shares his expertise on navigating these complexities. From addressing variability in stem cell culture systems to ensuring smooth GMP transitions, Hagen provides invaluable insights into the critical decisions researchers must make early in development. He also highlights the importance of adopting defined systems, mitigating comparability risks during scale-up, and leveraging cryopreservation to maintain stem cell functionality.

This discussion sheds light on how forward-thinking strategies and a thorough understanding of technical and cellular processes can transform exploratory workflows into robust, scalable and clinically viable solutions.

This interview is part of the RegMedNet In Focus on stem cell therapy. Discover expert opinions on this topic by visiting our feature homepage.

What distinguishes a research-stage stem cell workflow from one that is truly translation-ready?

Research-stage stem cell workflows are often exploratory and not yet designed with translation in mind. As a result, they can introduce technological and regulatory risks that are sometimes underestimated.

At this stage, the main focus is often on achieving proof of concept. If parts of a protocol are not working as expected, different approaches are tested to resolve these issues, while other aspects such as standardization or scalability may be considered secondary.

In contrast, translation-ready workflows must meet a very different set of requirements. These include robustness, scalability, traceability, risk management, regulatory compliance and cost-effectiveness. All of these are essential for successful clinical development.

To enable efficient translation and avoid the need for major process redesign later, it is important to consider the final application of the workflow from the beginning. Early improvements in workflow design (e.g., choosing culture conditions with the highest possible grade of definition) can have a significant impact, helping to save time, reduce costs, and minimize risks in later stages of development.

Where do you see the greatest sources of variability in stem cell culture systems, and how can these be mitigated?

Variability is one of the main hurdles to overcome in order to obtain translation-ready workflows using stem cells. Two main risk factors for process variability are the use of non-standardized ingredients for the culture and differentiation of cells, as well as scaling effects.

Non-standardized ingredients, such as human platelet lysate or plasma-derived albumins, often show significant lot-to-lot variability. In many cases, their exact mode of action is not fully understood, and it remains unclear whether the component itself or trace contaminations of other molecules provide the active agent. This is clearly also a significant downside from a regulatory point of view.

The magnitude of effects evoked by a “simple” switch from small to large scale can be surprising. Steps in the workflow working perfectly in the research-stage process, e.g., the solubilization of difficult-to-dissolve media compounds or the expansion of a sufficient number of stem cells, have the potential to develop into true nightmares during scale-up. Completely different solubilization techniques or cell expansion in a bioreactor instead of flasks might be required for the large-scale process in order to achieve reproducible results.

How important is the early adoption of defined or animal component-free systems in supporting a smooth transition to GMP manufacturing?

Using only defined and animal component-free components in stem cell workflows is often not feasible for several reasons, such as non-availability of the respective ingredient in ACF-specification, unknown function of an animal-derived component (with a potential lack-of-function of its animal component-free equivalent) or simply cost considerations.

However, any opportunity to incorporate highly standardized single compounds with known function at an early stage may be of great benefit in the translation phase later on. Synthetic, chemically defined materials provide the highest level of standardization. Indeed, when it comes to proteins, recombinant variants of non-animal origin are usually a good choice.

Furthermore, relying on highly standardized and defined compounds as early as possible helps to exclude non-specific biological activities from the workflow. Such unwanted effects, which may even be functionally critical, are often difficult to control based on experience.

What are some critical early-stage decisions that researchers should make to ensure a successful GMP transition later in the development process?

Critical early-stage decisions made before or at the start of a GMP transition strongly influence cost, speed, regulatory risk and long-term robustness. Many of these decisions are difficult and/or expensive to reverse later.

Therefore, it is important to consider the intended GMP use and regulatory pathway early, and to select raw materials and define control strategies accordingly. These decisions can have a lasting impact on the success of the workflow. At this stage, the focus should not be on achieving a perfect process, but on identifying what must be controlled, justified and scalable, while preserving flexibility for further development.

How can teams reduce comparability risks when scaling their processes?

A deep understanding of your process and workflow is essential to control comparability risks during scale-up. Only well-characterized parameters can be controlled, modeled and supported by robust analytical data.

This includes the process environment, technical procedures and equipment, as well as the use of defined materials that minimize unintended side effects. In addition, a structured change-control system is critical when adapting processes during scale-up.

In summary, comparability risks can be reduced by combining deep process understanding, predictive models, disciplined change control and robust analytical evidence well before commercial scale is reached.

What role does cryopreservation play in enabling robust clinical workflows, and how can teams go beyond simple viability metrics to ensure functionality?

The impact of cryopreservation of stem cells or stem-cell-based cell products on their post-thaw functionality and performance is often underestimated. In addition to the physical freezing process, the composition of the cryopreservation medium plays a critical role.

It is a widespread misconception that post-thaw viability is the most suitable parameter to assess the quality and performance of cryopreservation media. Indeed, maintaining a high percentage of viable cells is crucial; however, the post-thaw viability rate cannot necessarily be equated with the overall performance of a cell freezing medium in terms of preserving stem cell functionality. A viability measurement cannot detect all kinds of late-effect cell damage that may occur during cryopreservation, e.g., oxidative injury by free radicals. Instead, delayed cell attachment and proliferation of cryopreserved cells after thawing are key indicators of suboptimal cell freezing procedures. Likewise, impairment or loss of biologically relevant cellular functions reflects inadequate cryopreservation quality.

In addition, the regulatory status of the cryopreservation medium is important, as it comes into direct contact with cells and must meet the same standards as other components in the workflow. As a result, protein-free, defined, and animal component-free cell freezing media without phenol red are increasingly considered best practice in stem cell workflows. Taken together, a suitable stem cell cryopreservation medium should be a defined formulation (without phenol red) that effectively protects and maintains stem cell functions, for example, through antioxidant protection strategies such as those implemented in PromoCells’ “CryoSFM Plus (prf)”.

When should researchers begin thinking about documentation and regulatory expectations and how can early planning support long-term success?

Researchers should consider documentation and regulatory expectations as early as possible, ideally from the beginning. What is (and is not) recorded during early discovery work determines whether the process can later be understood and reproduced. Even if a workflow is still exploratory, establishing good documentation habits early avoids costly rework when a program moves toward a regulated environment.

Early planning supports long-term success by aligning the workflow with its intended use (research tool vs. clinical/therapeutic manufacturing), enabling a risk-based control strategy, and building comparability into the process before scale-up. Defining critical process parameters documenting changes in a structured way also facilitates later GMP transition and regulatory interactions.

In practice, teams that plan early can evolve their process stepwise instead of having to rebuild it under time pressure when translation becomes imminent.

Which process parameters in stem cell culture are most critical to define early in development?

The most critical process parameters to define early are those that strongly influence cell identity, potency and genetic stability, and that are difficult to change later.

First, the starting material should be clearly defined, including the selection of an appropriate cell source and acceptance criteria. In addition, the choice of media and raw materials must align with the intended workflow. The culture format (2D vs. suspension/3D) and the potential need for extracellular matrix components, such as coatings, are also key considerations.

Early decisions in these areas have a significant impact on scalability, robustness, product quality and regulatory risk, and therefore strongly influence the success of later development stages.

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The opinions expressed in this interview are those of the interviewee and do not necessarily reflect the views of RegMedNet or Taylor & Francis Group.

In association with PromoCell.