Growth behavior of human adipose tissue-derived stromal/stem cells at small scale : numerical and experimental investigations

Human adipose tissue-derived stromal/stem cells (hASCs) are a valuable source of cells for clinical applications, especially in the field of regenerative medicine. Therefore, it comes as no surprise that the interest in hASCs has greatly increased over the last decade. However, in order to use hASCs in clinically relevant numbers, in vitro expansion is required. Single-use stirred bioreactors in combination with microcarriers (MCs) have shown themselves to be suitable systems for this task. However, hASCs tend to be less robust, and thus, more shear sensitive than conventional production cell lines for therapeutic antibodies and vaccines (e.g., Chinese Hamster Ovary cells CHO, Baby Hamster Kidney cells BHK), for which these bioreactors were originally designed. Hence, the goal of this study was to investigate the influence of different shear stress levels on the growth of humane telomerase reversed transcriptase immortalized hASCs (hTERT-ASC) and aggregate formation in stirred single-use systems at the mL scale: the 125 mL (= SP100) and the 500 mL (= SP300) disposable Corning® spinner flask. Computational fluid dynamics (CFD) simulations based on an Euler⁻Euler and Euler⁻Lagrange approach were performed to predict the hydrodynamic stresses (0.06⁻0.87 Pa), the residence times (0.4⁻7.3 s), and the circulation times (1.6⁻16.6 s) of the MCs in different shear zones for different impeller speeds and the suspension criteria (Ns1u, Ns1). The numerical findings were linked to experimental data from cultivations studies to develop, for the first time, an unstructured, segregated mathematical growth model for hTERT-ASCs. While the 125 mL spinner flask with 100 mL working volume (SP100) provided up to 1.68.10⁵ hTERT-ASC/cm² (= 0.63 × 10⁶ living hTERT-ASCs/mL, EF 56) within eight days, the peak living cell density of the 500 mL spinner flask with 300 mL working volume (SP300) was 2.46 × 10⁵ hTERT-ASC/cm² (= 0.88 × 10⁶ hTERT-ASCs/mL, EF 81) and was achieved on day eight. Optimal cultivation conditions were found for Ns1u < N < Ns1, which corresponded to specific power inputs of 0.3⁻1.1 W/m³. The established growth model delivered reliable predictions for cell growth on the MCs with an accuracy of 76⁻96% for both investigated spinner flask types

Growth Behavior of Human Adipose Tissue-Derived Stromal/Stem Cells at Small Scale: Numerical and Experimental Investigations

Human adipose tissue-derived stromal/stem cells (hASCs) are a valuable source of cells for clinical applications, especially in the field of regenerative medicine. Therefore, it comes as no surprise that the interest in hASCs has greatly increased over the last decade. However, in order to use hASCs in clinically relevant numbers, in vitro expansion is required. Single-use stirred bioreactors in combination with microcarriers (MCs) have shown themselves to be suitable systems for this task. However, hASCs tend to be less robust, and thus, more shear sensitive than conventional production cell lines for therapeutic antibodies and vaccines (e.g., Chinese Hamster Ovary cells CHO, Baby Hamster Kidney cells BHK), for which these bioreactors were originally designed. Hence, the goal of this study was to investigate the influence of different shear stress levels on the growth of humane telomerase reversed transcriptase immortalized hASCs (hTERT-ASC) and aggregate formation in stirred single-use systems at the mL scale: the 125 mL (=SP100) and the 500 mL (=SP300) disposable Corning® spinner flask. Computational fluid dynamics (CFD) simulations based on an Euler–Euler and Euler–Lagrange approach were performed to predict the hydrodynamic stresses (0.06–0.87 Pa), the residence times (0.4–7.3 s), and the circulation times (1.6–16.6 s) of the MCs in different shear zones for different impeller speeds and the suspension criteria (Ns1u, Ns1). The numerical findings were linked to experimental data from cultivations studies to develop, for the first time, an unstructured, segregated mathematical growth model for hTERT-ASCs. While the 125 mL spinner flask with 100 mL working volume (SP100) provided up to 1.68 × 105 hTERT-ASC/cm2 (=0.63 × 106 living hTERT-ASCs/mL, EF 56) within eight days, the peak living cell density of the 500 mL spinner flask with 300 mL working volume (SP300) was 2.46 × 105 hTERT-ASC/cm2 (=0.88 × 106 hTERT-ASCs/mL, EF 81) and was achieved on day eight. Optimal cultivation conditions were found for Ns1u < N < Ns1, which corresponded to specific power inputs of 0.3–1.1 W/m3. The established growth model delivered reliable predictions for cell growth on the MCs with an accuracy of 76–96% for both investigated spinner flask types

Nutzung von Sensor Spots zur Bestimmung der Gelöstsauerstoffkonzentration in Single-use Systemen im Labormassstab

Einleitung: Der wirtschaftlich gewinnbringende Einsatz biotechnologischer Verfahren setzt eine explizite Kenntnis des verwendeten Systems voraus. Für aerobe Kultivierungen bedeutet dies insbesondere die Überwachung und Optimierung der Gelöstsauerstoffkonzentration im Bioreaktor. Dies gilt sowohl für die zunehmend an Bedeutung gewinnenden Single-Use Systeme als auch für klassische Bioreaktoren, wobei sich für erstere durch ihre besonderen Eigenschaften Chancen und Herausforderungen ergeben. Einsatzgebiete: Der Einsatz klassischer Messsonden in Single-Use Systemen widerspricht dem zugrundeliegenden out of the box Konzept und erhöht gleichzeitig den Aufwand in Vorbereitung und das Risiko einer Kontamination. Für Systeme wie Schüttelkolben oder Bagreaktoren ist der Einsatz dieser Messsonden aus geometrischen und strömungstechnischen Gründen zudem wenig sinnvoll. In diesem Anwendungsfeld (Laborbioreaktoren im Milliliter- und Litermaßstab) bieten Sensor Spots eine einfache und vielversprechende Alternative zur Sauerstoffüberwachung. In der vorliegenden Posterpräsentation wird der Einsatz von Sensor Spots zur Bestimmung des volumenbe-zogenen Sauerstoffübergangskoeffizienten (kLa-Wert) in verschiedenen Systemen (beispielsweise in Thomson Optimum Growth™ Schüttelkolben oder TubeSpin® Bioreaktoren) illustriert. Im Fokus steht dabei die Optimierung der Prozessparameter für pflanzliche und tierische Zellkulturen. Anwendungsbeispiele: Den Abschluss dieser Posterpräsentation bildet die Vorstellung verschiedener Auswertungsmöglichkeiten sowie die Demonstration der Einsatzmöglichkeiten unter verschiedenen Gesichtspunkten. Die Anwendung wird am Beispiel einer Kultivierungsstudie für CHO (Chinese Hamster Ovary) Zellen demonstriert

On-line-pH- und -DO-Messungen in Mikrocarrier-basierter hMSC Kultur

Erworben im Rahmen der Schweizer Nationallizenzen (http://www.nationallizenzen.ch)Spinner flasks are often used for microcarrie-based cultivations of human mesenchymal stem cells (hMSCs). Normally, they are not equipped with pH and dissolved oxygen (DO) probes. This application note describes the cultivation of hMSCs in single-use spinner flasks equipped with optical pH (SP-HP8) and DO (SP-PSt3) sensors for the first time. While reaching peak cell numbers between 4.1 × 107 cells and 5.9 × 107 cells in two cultivation runs, reliable DO and pH data were delivered

How to use computational fluid dynamics in the development of cell therapeutics ?

Computational Fluid Dynamics (CFD) is an established method in fluid mechanics that allows fluidic problems to be solved through numerical methods. In recent years, CFD has established itself as a useful tool in biochemical engineering, where it is mainly used to characterise and optimize devices (e.g. bioreactors, pumps, etc.). By using CFD, fundamental bioengineering parameters (e.g. turbulent dissipation rates, shear gradients) can be predicted independently of time and location. This allows process related parameters to be defined in silico, which reduces the number of experiments and costs. This is particularly important for the development of cell therapeutics, where the starting cell material is restricted and the batch costs are high. Recent economic reports have predicted a significant increase in cell therapeutics over the next few years, especially for human mesenchymal stem cells (hMSCs). This situation can also be seen in the high number of clinical trials (269 trails, August 2016; clinicaltrails.gov) that are currently focusing on using hMSCs for the treatment of illnesses such as myocardial infarction, Crohn’s disease and graft versus host disease. However, large amounts of hMSCs are required for one single therapeutic dose (35-350 million cells per dose), which explains the demand for efficient and scalable in vitro expansion procedures. Following a brief introduction to CFD, we aim to highlight the capabilities of CFD for the development of bioprocesses and scale-up procedures. For this purpose, we will show how CFD data can be used to support the scale-up of a microcarrier-based hMSC expansion process in stirred and wave-mixed single-use bioreactors. Our presented investigations involve Computational Fluid Dynamics (CFD) simulations and data verification using Particle Image Velocimetry (PIV) measurements, suspension studies in a serum-reduced culture medium with a suitable polystyrene microcarrier, and expansion studies with human adipose tissue-derived stromal/stem cells (hASCs). This combination of biochemical engineering and biological expertise enabled the establishment of a MC-based hMSC expansion process that resulted in up to 1.25 x 106 hMSCs/mL in stirred single-use bioreactors. Initial proof-of-concept expansions of hASCs in wave-mixed single-use bioreactors at a rocking angle/ -rate combination of 4° and 31 rpm resulted in the harvest of 2.85 x 108 hASCs after 9 days of cultivation without changes to the stem cell characteristics. All the investigations performed showed that the suspension criteria NS1U for stirred and NS1UW for wave-mixed bioreactors are beneficial for the cultivation of hMSCs. References: • Jossen, V., Pörtner, R., Kaiser, S.C., Kraume, M., Eibl, D., Eibl, R. (2014) Mass Production of Mesenchymal Stem Cells – Impact of Bioreactor Design and Flow Conditions on Proliferation and Differentiation. In: Eberli D. (ed.), Regenerative Medicine and Tissue Engineering, InTech. ISBN 978-953-51-4114-3 • Jossen, V., Schirmer, C., Mostafa, D.S., Eibl, R., Kraume, M., Pörtner, R., Eibl, D. (2016) Theoretical and Practical Issues That Are Relevant When Scaling Up hMSC Microcarrier Production Processes. Stem cells International, Article ID 47641

Successfully employing single-use bioreactors for different expression systems

The single-use bioreactor market was estimated to have grown at a CAGR of 20% in 2014. Wave-mixed bioreactors and stirred bioreactors represent the largest segment of today`s single-use bioreactor market, and are preferred by process developers and manufacturers of preclinical and clinical samples when high-value products up to medium scale and products requiring high safety demands are in focus. However, wave-mixed and stirred single-use bioreactors are also becoming increasingly important for commercial biopharmaceutical productions. This is ascribed to the availability of high-productivity cell lines, which require smaller bioreactors. In addition, the availability of bioengineering data and cultivation bags with improved films enables optimized operations with wave-mixed and stirred single-use bioreactors, whereby the risks of leachable migration and adsorption of hydrophobic components are reduced. Based on a brief description of the current single-use bioreactor market, we aim to highlight the predominance of wave-mixed and stirred versions, and their main applications. In addition, their advantages and limitations are summarized. An insight into our long-term work with wave-mixed and stirred single-use bioreactors is given in the main part of our presentation, and results from cultivations with different expression systems covering plant cells, insect cells and mesenchymal stem cells (1-4) are provided. In this context, the advantageous combination of bioengineering and cell biological expertise is demonstrated for both process development and scale-up

Growth behavior of human adipose tissue-derived stromal/stem cells in single-use spinner flasks: Numerical and experimental investigations

Human adipose tissue-derived stromal/stem cells (hASC) represent a valuable source of cells for clinical applications, especially in the field of regenerative medicine. Therefore, it comes as no surprise that interest in hASCs has increased greatly over the last decade. However, in order to use hASCs successfully in clinical applications, in vitro expansion is required. Single-use bioreactors in combination with microcarriers (MC) have been shown to be suitable systems for this task (1-3). However, hASCs are prone to higher shear sensitivity than conventional cell lines (e.g. CHO, BHK) that are normally expanded in these systems. Hence, the goal of this study was to investigate the influence of different shear stress levels on the growth of hASCs in small scale single-use spinner flasks. For this purpose, Computational Fluid Dynamics simulations based on a Euler-Euler and Euler-Lagrange approach were performed to predict the hydrodynamic stresses (0.06 – 0.87 Pa), the residence times (0.4 – 7.3 s) and the circulation times (1.6 - 16.6 s) of the MCs in various high shear zones. The numerical findings were combined with experimental data from cultivation studies (0.29 – 1.1∙106 hASC/mL) in order to develop a segregated mathematical growth model for the prediction of MC-associated hASC growth in small scale single-use spinner flasks. V., Jossen, R., Pörtner, S.C., Kaiser, M., Kraume, D., Eibl, R., Eibl. Mass Production of Mesenchymal Stem Cells – Impact of Bioreactor Design and Flow Conditions on Proliferation and Differentiation. In: Cells and Biomaterials in Regenerative Medicine, D. Eberli (ed.), 119-174, InTech 2014. C., Schirmaier, V., Jossen, S.C., Kaiser, F., Jüngerkes, S., Brill, A., Safavi-Nab, A., Siehoff, C., van den Bos, D. Eibl, R., Eibl. Scale-up of adipose tissue-derived mesenchymal stem cell production in stirred single-use bioreactors under low-serum conditions. Eng. Life Sci. 2014, 14: 292-303 T., Lawson, D.E., Kehoe, A.C., Schnitzler, P.J., Rapiejko, K.A., Der, K., Philbrick, S., Punreddy, S., Rigby, R., Smith, Q., Feng. Biochem Eng J. 2017, 120: 49-6

In-line monitoring and control of glucose concentration with single-use sensors in CHO and stem cell applications

Nearly 20 years ago, the Food and Drug Administration (FDA) published a guidance document to describe a regulatory framework for process analytical technologies and to promote innovation in pharmaceutical development, manufacturing, and quality assurance [1], in which process analysis and control tools play an important role. Since then, the capabilities for on-line, at-line, and in-line measurement of various parameters have continuously evolved. One of the most critical parameters in mammalian and stem cell cultivation is glucose, the most important carbon source, alongside pH and dissolved oxygen, which are already measured and controlled in-line in many bioreactors. While high glucose levels can be inhibitory, limitation rapidly leads to apoptosis in most cell types. In this work, CITSens Bio sensors (C-CIT Sensors AG) were used for in-line measurement of glucose in T25-flasks during hASC expansion (ASC52telo, ATCC) in a serum- and xeno-free stem cell culture medium (UrSuppe [2-3]). The hASC expansion process ran for 6 days in batch mode. The glucose concentration measured in-line was in good agreement with the measurements determined off-line (Cedex Bio, Roche). In further experimental runs, the sensor was used for the automated control of the glucose concentration in a fed-batch IgG production process with CHO suspension cells (ExpiCHO-S, Gibco). For this purpose, a glucose solution was pumped into the stirred bioreactor (2 L working volume) in addition to a continuous supply of feed solution, in order to constantly regulate the concentration to a minimum of 1 g/L. The glucose concentration was successfully measured throughout the 21-day process and regulated to values between 1.12 g/L and 1.38 g/L from day 8 to day 21 of the cultivation. The IgG titer achieved in this automated fed-batch process was 3.7 g/L, comparable to the conventional processes using a daily bolus feeding. References [1] U.S. Department of Health and Human Services, Food and Drug Administration: Guidance for industry: PAT - a framework for innovative pharmaceutical development, manufacturing, and quality assurance. 2004, https://www.fda.gov/media/71012/download [2] Jossen et al. 2020, An approach towards a GMP compliant in vitro expansion of human adipose stem cells for autologous therapies, doi: 10.3390/bioengineering7030077 [3] Panella et al. 2021, Chemically defined xeno- and serum-free cell culture medium to grow human adipose stem cells, doi: 10.3390/cells1002046

Manufacturing human mesenchymal stem cells at clinical scale: Process and regulatory challenges

There is an obvious increasing interest in human mesenchymal stem cell (hMSC)-based therapies for regenerative medicine (e.g. neurology, cardiology, immunology, orthopaedics). At the beginning of May 2018, there were 253 registered clinical trials using hMSCs (www.clinicaltrials.gov). Despite the large number of current clinical studies, only 13 hMSC-based products have received regulatory approval. In order to efficiently manufacture hMSC-based products, not only must the targeted cell quantity and quality be taken into account, but the production costs must also be considered. In general, autologous and allogeneic stem cell products are characterized by similar upstream processing (USP), downstream processing (DSP), formulation, and fill & finish operations. Typical USP operations are manufacturing of the Master Cell Bank (MCB) and the Working Cell Bank (WCB), seed cell production, and subsequent cell expansion. The DSP steps include cell harvesting, cell detachment, cell separation, washing and concentration procedures, and medium exchange. However, before hMSCs can be administered as an Advanced Therapeutic Medicinal Product (ATMP), additional formulation, and fill and finish steps have to be carried out. The main differences between allogeneic and autologous manufacturing approaches are the number of therapeutic doses generated in each batch and the number of patients treated. Therefore, it is unsurprising that allogeneic therapies are the more cost-effective method in terms of hMSC production. Furthermore, various economic studies have demonstrated that USP and in particular, hMSC expansion, represent the main cost drivers when examining the entire manufacturing process. In order to achieve the high cell numbers of up to 1013 cells per batch needed in allogeneic hMSC manufacturing processes, manufacturers have to move away from traditional planar cultivation systems. Many reports over the last years have shown that instrumented, single-use bioreactors in combination with microcarriers are promising systems for this task. Even though different procedures and equipment for USP and DSP are already available and established for allogeneic production of hMSCs, various challenges still exist. Therefore, the authors intend to highlight the current state of the art of allogeneic hMSC manufacturing and show the current main process and regulatory challenges for USP and DSP operations

Standardized expansion of human adipose tissue-derived stromal/stem cells (hASCs) in wave-mixed single-use bioreactors with one- dimensional motion

The large number of realized and ongoing clinical trials with human mesenchymal stem cells (200 by end of June 2016; www.clinicaltrial.gov) demonstrate their great potential in the field of regenerative medicine. However, new and standardized production technology is necessary to generate the number of cells required at the cell quality desired. The previously used parallelized plastic shells that consist of multiple layers (e.g. stacked plate systems) need to be replaced by instrumented, dynamically-mixed and scalable single-use bioreactors. In these systems the growth surface for the adherent growing cells is provided by microcarriers. While the first users have already worked with stirred single-use bioreactors (e.g. Lonza in Walkersville), there have only been two approaches described in the specialist literature for one-dimensional wave-mixed bioreactors, both of which were not completely successful. This is surprising, since these single-use bioreactors have dominated seed inoculum production for some time due to their uncomplicated operation. Furthermore, wave-mixed single-use bioreactors have proven to be successful in microcarrier-based vaccine production. The investigations we present consist of Computational Fluid Dynamics (CFD) simulations and data verification by Particle Image Velocimetry (PIV) measurements, suspension studies in a serum-reduced culture medium with a suitable polystyrene microcarrier, and expansion studies with human adipose tissue-derived stromal/stem cells (hASCs) based on biochemical engineering investigations. They provide an important contribution to the development and production of stem cell-based cell therapeutics in single-use bioreactors with one-dimensional motion, and for the first time demonstrate the suitability of this type of reactor for the expansion of hASCs when working with culture specific suspension criteria (NS1UW and NS1W). The first proof-of-concept expansions of hASCs at a rocking angle/rocking rate combination of 4° and 31 rpm allowed 2.85 x 108 hASCs to be harvested after 9 days of cultivation without a change in the stem cell characteristics. Moreover, the results generated contribute to a better understanding of the parameter-dependent, hydrodynamic cell stress caused by mechanical stress in hASC expansions with microcarriers in wave-mixed bioreactors with one-dimensional motion, and allow for direct comparison with stirred single-use bioreactors, which was previously not possible. Finally, the results obtained are also beneficial for other microcarrier-based production processes, such as those for vaccines (e.g. shortening of process development time)