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

Design of bioreactors suitable for plant cell and tissue cultures

Plant cell suspension cultures and hairy roots are potential sources of secondary metabolites and recombinant proteins. In contrast to traditionally grown "whole wild plants” or "whole transgenic plants”, their production in bioreactors guarantees defined controlled process conditions and therefore minimizes or even prevents variations in product yield and quality, which simplifies process validation and product registration. Moreover, bioreactors and their configuration significantly affect cultivation results by accomplishing and controlling the optimum environment for effective cell growth and production of bioactive substances. This review highlights the main design criteria of the most widely used bioreactor types, both for plant cell suspension cultures and for hairy roots, and outlines suitable low-cost disposable bioreactors which have found increasing acceptance over the last 10year

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

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

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 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

Disposable bioreactors: the current state-of-the-art and recommended applications in biotechnology

Disposable bioreactors have increasingly been incorporated into preclinical, clinical, and production-scale biotechnological facilities over the last few years. Driven by market needs, and, in particular, by the developers and manufacturers of drugs, vaccines, and further biologicals, there has been a trend toward the use of disposable seed bioreactors as well as production bioreactors. Numerous studies documenting their advantages in use have contributed to further new developments and have resulted in the availability of a multitude of disposable bioreactor types which differ in power input, design, instrumentation, and scale of the cultivation container. In this review, the term "disposable bioreactor” is defined, the benefits and constraints of disposable bioreactors are discussed, and critical phases and milestones in the development of disposable bioreactors are summarized. An overview of the disposable bioreactors that are currently commercially available is provided, and the domination of wave-mixed, orbitally shaken, and, in particular, stirred disposable bioreactors in animal cell-derived productions at cubic meter scale is reported. The growth of this type of reactor system is attributed to the recent availability of stirred disposable benchtop systems such as the Mobius CellReady 3L Bioreactor. Analysis of the data from computational fluid dynamic simulation studies and first cultivation runs confirms that this novel bioreactor system is a viable alternative to traditional cell culture bioreactors at benchtop scal

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