Cell and gene therapy (CGT) represents a paradigm shift in medicine, offering potential cures for diseases that traditional approaches struggle to address, particularly monogenic diseases. Unlike conventional treatments, CGT aims to restore or modify cells and genes, offering curative possibilities. These therapies, often described as “living drugs,” have the ability to heal and replace diseased organs. While still in early stages, CGT research and development are rapidly progressing towards preventing, treating, and curing both genetic and acquired diseases. Fundamental to CGT is the cultivation of cells and tissues, forming the cornerstone of these innovative biomedical applications.

Eppendorf recently released an eBook, “Optimizing Cell and Gene Therapy Workflows”, which describes in detail the important work being done to bring cell and gene therapies to patients that need them. Each articles examines aspects of the cell and gene therapy workflow to highlight optimal process development methods.

Cellular Therapy (CT) and Regenerative Medicine – Stem Cells

The first article describes how cellular therapy (CT) and regenerative medicine utilize human cells to replace or repair damaged tissue and cells in treating various diseases. These cells can be sourced from the patient (autologous) or a healthy donor (allogeneic) and are cultured and modified before infusion. Stem cells, particularly human pluripotent stem cells (hPSCs) like embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), are valuable for regenerative medicine due to their ability to differentiate into various cell types. Hematopoietic stem cells (HSCs) have been used in clinical therapies for decades, especially for treating blood cancers and hematologic conditions. CT with stem cells has diverse therapeutic applications, including autoimmune diseases, skeletal injuries, and neurological disorders. Large-scale production of lineage-specific cells (ranging in the billions) is necessary for research and development, requiring advanced culture systems for scalability. This type of production exceeds the capability of traditional methods like bags or T-flasks, which lack scalability, real-time control and often suffer from lower yields and poor adaptability. Thus, there’s a need for culture systems facilitating large-scale cell production with enhanced control and scalability.

Stem Cell Bioprocessing – Stirred Tank Bioreactors

The next article looks at stirred tank bioreactors and their role in stem cell bioprocessing in offering scalability and versatility to meet researchers’ diverse production needs. Stirred-tank bioreactors are ideal for suspension cells and microcarrier cultures, while packed-bed bioreactors are particularly well suited for adherent cells.

Single-use plastic bioreactors, like Eppendorf BioBLU® c Bioreactors, are gaining popularity for their convenience, eliminating the need for cleaning and sterilization processes, thus enabling faster turnaround times. Eppendorf bioreactors, both single-use and reusable, are compatible with their bioreactor controllers, facilitating efficient cell expansion beyond the limitations of conventional 2D-cell culture systems.

Widely used in the industry, bioreactor systems offer advantages like online monitoring and control of critical parameters such as oxygenation, pH, and temperature, essential for optimizing upstream bioprocesses in cellular therapy (CT) development. For example, the DASbox® Mini Bioreactor System enables parallel bioprocessing at a small scale.

The Eppendorf Bioprocess Unit, with its expertise in upstream bioprocessing, has contributed significantly to advancing stem cell cultivation in stirred-tank bioreactors. With real-time monitoring and scalable designs, the Eppendorf platform enables the transfer of small-scale bioprocess results to larger volumes, accelerating development timelines and time to market. Additionally, their bioprocess control software automates parameter control and routine tasks, freeing up time for valuable research.

Scalable Expansion of Human Pluripotent Stem Cells

The third article provides highlights of a case study that demonstrates how advanced bioreactor systems play a crucial role in scaling up cell cultures, particularly for the cultivation and differentiation of human pluripotent stem cells (hPSCs) in stirred-tank bioreactors.

In a 7-day expansion process, using equipment including the DASbox Mini Bioreactor System and BioBLU 0.3c Single-Use Bioreactors, a 4-fold increase in viable cell count of hPSCs was achieved using a fed-batch process. Flow cytometry confirmed that the majority of the cell population retained pluripotency-associated cell surface markers. This successful combination of bioreactor systems provides an excellent platform for process optimization and adaptation to lineage-specific hPSC differentiation processes. Building on this success, Dr. Robert Zweigerdt and his team from Hannover Medical School, Germany, further increased hiPSC yield in bioreactors, achieving a total culture yield of 35 million hiPSCs per mL.

This article leads into an interview with Dr. Zweigerdt where he explains how his team greatly increased the hiPSC yield in bioreactors.

The interview explains how Dr. Zweigerdt’s team was able to enhance human induced pluripotent stem cell (hiPSC) cultivation in bioreactors and achieved the milestone of 35 million cells per mL. Dr. Zweigerdt explained that the first hurdle which they attacked ten years ago was to transition cells from traditional 2D monolayer cultures to 3D suspension cultures. The second big step which they accomplished in collaboration with Eppendorf was the design of a modified stirring impeller design to support a more homogenous hiPSC aggregation and implementing a retention-filter system designed to keep hPSC cells in stirred suspension culture in the bioreactor upon automated perfusion feeding. Lastly, they were able to identify growth-limiting parameters like pH, glucose consumption, and lactate accumulation, and implement optimized perfusion feeding strategies.

Next Dr. Zweigerdt explained the translation of differentiation protocols from monolayer cultures to cell aggregates in bioreactors. He states that “the most significant challenges regarding directed differentiation in suspension culture include the impact of cell aggregates size, its heterogeneity, overall cell density, and defining mechanical and hydrodynamic parameters”. He also noted that the standard culture media com­ponents and differentiation-directing molecules that they are applying, for example the WNT pathway modulators used for mesoderm-induction and cardiac differentiation, have equivalent effects in 2D and in 3D cultures.

Then, he discussed feeding strategies. Perfusion feeding was favored over repeated batch feeding due to its ability to control growth-limiting parameters efficiently.

Following this, he reviewed the process optimization steps his team took to achieve such high yield. These steps included control of cell aggregation, pH, and nutrient supply, which led to a more than tenfold increase in hiPSC culture density.

Dr. Zweigerdt closed by sharing that despite their achievements, further scale-up is needed to meet the demand for cell therapy applications, particularly for heart-related treatments. Future efforts will focus on improving differentiation protocols and upscaling production to meet clinical needs.

Scalable Expansion of Human Bone Marrow-Derived Mesenchymal Stem Cell

The next article provides highlights of a case study that looks at the scalable expansion of human bone marrow-derived mesenchymal stem cells (hMSCs). Various equipment, including the DASbox Mini Bioreactor System and BioBLU 0.3c Single-Use Bioreactors, along with specific culture media and assays, were employed for this purpose. Rigid-wall, stirred-tank bioreactors offer precise control over critical process parameters like pH and dissolved oxygen, temperature, gas sparging, and agitation, thereby facilitating the homogeneous distribution of nutrients and gases along with the high process control necessary for stem cell cultivation in suspension.

The study demonstrated successful hMSC expansion using the Eppendorf DASbox Mini Bioreactor System equipped with BioBLU 0.3c Single-Use Bioreactors with Cytodex type 1 and type 3 microcarriers as growth surfaces. Results demonstrated a 17.5-fold expansion with a maximum cell density of 1 x 10⁸ cells/batch was achieved using Cytodex type 1, while a 11.5-fold expansion with a maximum of 7 x 10⁷ cells/bioreactor was obtained with Cytodex type 3. Importantly, the expanded hMSCs retained their multipotency and ability to differentiate into osteocytes and chondrocytes, indicating their suitability for therapeutic applications in regenerative medicine.

Optimization of CD4⁺ T Cells for Long Term Expansion

Another case study provided in the eBook looked at the optimization of CD4⁺ T cells for long-term expansion using the DASbox Mini Bioreactor System equipped with BioBLU 0.3c Single-Use Bioreactors. Adoptive cell therapy (ACT), focusing on the transplantation of immune cells like T cells for treating chronic viral infections and cancer, relies on the quality and scalability of T cell expansion protocols. The study utilized equipment such as incubators, flasks, and specific cell activators and expansion media. By controlling oxygen tension levels during culture, lower oxygen tension at 20% dissolved oxygen was found to positively impact CD4⁺ T cell proliferation rates by trend without affecting cell functionality. The production of interleukin 4 (IL-4), an important cytokine, was used as a quantitative measure of cell functionality. Results were reproducible across different donor T cells, confirming the efficacy of the optimized bioprocessing approach. These findings underscore the advantages of bioreactor-based culturing systems over static cultures in bags or flasks, highlighting the potential of the DASbox Mini Bioreactor System in combination with BioBLU 0.3c Single-Use Bioreactors for optimizing T cell culture conditions at a scalable level.

Exosomes Produced in Bioreactors – Bioreactors Provide the Ideal Environment for the Expansion and Large-Scale Production of Exosomes

The final feature in the eBook is an interview with Dr. Jorge Escobar, a senior research scientist in the applications lab at Eppendorf. The interview highlights the importance of ensuring the safety and efficacy of cell therapies, addressing concerns such as immune rejection and scalability in production. Exosomes, small extracellular vesicles containing bioactive molecules, are explored as potential substitutes for cell therapy, offering advantages in intercellular communication and therapeutic delivery. Bioreactors are identified as critical tools for large-scale exosome production, providing a controlled environment for optimal cell expansion and exosome yield. The role of Eppendorf in supporting researchers with bioreactor solutions and application notes is highlighted, with a commitment to advancing solutions in the field of extracellular vesicle research and cell therapy. The future focus of Eppendorf lies in addressing the evolving needs of the cell and gene therapy industry, aiming to provide innovative solutions to overcome development challenges and ultimately contribute to life-saving treatments.

The eBook emphasizes the pivotal role of cell and gene therapies in revolutionizing medical treatments by restoring or reconditioning cells or genes. Large-scale cultivation and production of stem cells are essential for the development of these therapies, given their ability to differentiate into various cell types. The Eppendorf portfolio of stirred-tank bioreactor systems, including BioBLU Single-Use Bioreactors and DASbox Mini Bioreactor Systems, offers efficient solutions for cell expansion beyond the limitations of conventional 2D-cell culture systems. These bioreactors, combined with bioprocess control software, demonstrate versatility in expanding various stem cell types such as CD4⁺ T cells, human pluripotent stem cells, and mesenchymal stem cells. Looking ahead, the Eppendorf platforms provide a reliable foundation for accelerating cell culture development, ultimately optimizing research efficiency.

To download a copy of the ebook, please see Optimizing Cell and Gene Therapy Workflows