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Human mesenchymal stem cells (hMSCs) are a valuable source of cells for clinical applications (e.g., treatment of acute myocardial infarction or inflammatory diseases), especially in the field of regenerative medicine. However, for autologous (patient-specific) and allogeneic (off-the-shelf) hMSC-based therapies, in vitro expansion is necessary prior to the clinical application in order to achieve the required cell numbers. Safe, reproducible, and economic in vitro expansion of hMSCs for autologous and allogeneic therapies can be problematic because the cell material is restricted and the cells are sensitive to environmental changes. It is beneficial to collect detailed information on the hydrodynamic conditions and cell growth behavior in a bioreactor system, in order to develop a so called “Digital Twin” of the cultivation system and expansion process. Numerical methods, such as Computational Fluid Dynamics (CFD) which has become widely used in the biotech industry for studying local characteristics within bioreactors or kinetic growth modelling, provide possible solutions for such tasks. In this review, we will present the current state-of-the-art for the in vitro expansion of hMSCs. Different numerical tools, including numerical fluid flow simulations and cell growth modelling approaches for hMSCs, will be presented. In addition, a case study demonstrating the applicability of CFD and kinetic growth modelling for the development of an microcarrier-based hMSC process will be shown.

The optimization of operating conditions is vital for cell culture process development. In the present study, the pilot-scale single-use bioreactor XcellerexTM XDR-200, which is typically used in cell culture scale-up, is investigated. This unbaffled reactor is equipped with an off-centered, bottom mounted impeller, and has a maximum working volume of 200 L. The flow field for this sparsely investigated bioreactor configuration is studied with computational fluid dynamics. Moreover, numerical simulations of the mixing time and the volumetric oxygen mass transfer coefficient for seven different test conditions are validated against experimental data. Mixing times across all tested conditions are found to remain below 30 s, and the volumetric oxygen mass transfer coefficient is between 2.0 to 20.0 h−1, which is a typical range for animal cell culture processes. These values of the oxygen transfer coefficient are found for as low as 0.004 to 0.125 L L−1 min−1 sparging rates with the microporous sparger. The hydrodynamic stress remains below critical values for cell damage for all conditions investigated. Vortex formation is suppressed in the bioreactor even though it is unbaffled due to off-centered impeller. Across the test conditions, the highest investigated impeller speed of 200 rpm is the most favorable due to fast mixing and high oxygen transfer.

In this study, Euler-Euler coupled computational fluid dynamics (CFD), species transport and population balance model (PBM) simulations have been used to model multiphase hydrodynamics and mass (O2) transfer in a single use bioreactor (SUB) for mAb production. In simulations, drag was estimated by Schiller-Neuman equations. O2(g) local volume fraction was estimated via species transport model (STM). The PBM equations were solved using quadrature method of moments (QMOM) to get bubble size distribution (BSD) and thereby the Sauter mean diameter,d32. Also, the mean diameter over surface (d21) and De Brouckere mean (d43) were estimated for calculation of the BSD. The mass transfer coefficient (kLa) was predicted using species transport model (STM) and Higbie’s penetration theory. The experimental estimation of kLa was performed via simplified dynamic pressure method. Total stress (τ) and energy dissipation rate (e) were estimated as 34.17 Pa and 1.352 m2.s−3, respectively. Calculations of the d32, d21 and d43 indicate that the BSD becomes more dispersed with increase in agitation speed at all aeration rates. Further, the kLa values of STM were within 0.47% to 4.21% of experimental data. In summary, with error between 4 and 10%, the proposed approach offers better visualization of O2 transport mechanism across gas–liquid interface than conventional approaches.

Allogeneic cell therapy products, such as therapeutic cells derived from pluripotent stem cells (PSCs), have amazing potential to treat a wide variety of diseases and vast numbers of patients globally. However, there are various challenges related to the manufacturing of PSCs in large enough quantities to meet commercial needs. This manuscript addresses the challenges for the process development of PSCs production in a bioreactor, and also presents a scalable bioreactor technology that can be a possible solution to remove the bottleneck for the large-scale manufacturing of high-quality therapeutic cells derived from PSCs.

During the past 10 years, single-use bioreactors have been well accepted in modern biopharmaceutical production processes targeting high-value products. Up to now, such processes have mainly been small- or medium-scale mammalian cell culture-based seed inoculum, vaccine or antibody productions. However, recently first attempts have been made to modify existing single-use bioreactors for the cultivation of plant cells and tissue cultures, and microorganisms. This has even led to the development of new single-use bioreactor types. Moreover, due to safety issues it has become clear that single-use bioreactors are the “must have” for expanding human stem cells delivering cell therapeutics, the biopharmaceuticals of the next generation. So it comes as no surprise that numerous different dynamic single-use bioreactor types, which are suitable for a wide range of applications, already dominate the market today. Bioreactor working principles, main applications, and bioengineering data are presented in this review, based on a current overview of greater than milliliter-scale, commercially available, dynamic single-use bioreactors. The focus is on stirred versions, which are omnipresent in R&D and manufacturing, and in particular Sartorius Stedim’s BIOSTAT family. Finally, we examine development trends for single-use bioreactors, after discussing proven approaches for fast scaling-up processes.

Background: To improve cultivation conditions for human bone-marrow-derived mesenchymal stem cells, we redesigned the commercially available UniVessel® SU bioreactor using results obtained from computational fluid dynamics. The goal was to produce ≥1 × 109 cells and to achieve expansion factors ≥30. Screening studies suggested that microcarrier solid fractions of at least 0.3% are required to reach the appropriate cell densities. Results: The fluid flow pattern found in the most promising modification (#2) was altered by increasing the impeller blade angle and lowering the off-bottom clearance. As a result, the maximum required specific power input was reduced by a factor of 2.2–4.6, depending on the microcarrier concentration, and peak cell densities were 3.4-times higher than in the standard version. Conclusion: The peak cell number of nearly 1.1 × 109 cells (expansion factor = 35), which was achieved in our low-serum cultivations, indicates an improvement in the redesigned UniVessel® SU configuration f...

This study presents the concept of the travelling wave single-use bioreactor, based on an orbitally shaken, annular-shaped vessel. A numerical model was developed for early design studies in order to reduce the number of prototypes. The flow characteristics in two different vessel shapes were investigated. It was shown that the orbital motion combined with the toroidal shape of the bioreactor create the desired wave characteristics in the contents of the vessel.

Stirred single-use bioreactors can be used as substitutes for their conventional counterparts made of glass or stainless steel in the development and production of biopharmaceuticals wherever possible. Various studies have confirmed their comparability in cell growth as well as in product quantity and quality. However, information about their engineering characteristics is still rare. This study focuses on the stirred Mobius® CellReady 3L bioreactor. The main engineering parameters for typical operation conditions used in animal cell cultivations are presented for the first time. Numerical simulations with a commercial CFD package (Fluent 6.3) were accomplished to obtain data on the single- and multi-phase fluid flow, power input, mixing time and oxygen mass transfer. The results, which were compared with data from experiments and from the literature, reveal the suitability of the Mobius® CellReady 3L bioreactor for cell expansion and protein production with animal cell cultures. Furthermore, the data enable comparisons with other single-use and reusable cell culture bioreactors at bench-scale.

Driven by global competition and rising cost pressure in the pharmaceutical industry, over the last ten years single-use bioreactors have been increasingly used for animal cell cultivations in screening experiments, seed inoculum and seed train productions as well as in small and medium scale production processes of proteins (in particular, antibodies and vaccines). In contrast to re-usable bioreactors made of glass or stainless steel, single-use bioreactors consist of a flexible or rigid cultivation vessel which has been gamma-sterilized and made ready for the purchaser to use. After harvest, the cultivation vessel is discarded, which results in a lower risk of cross-contamination, and eliminates the need for steam sterilization and cleaning [Eibl et al. (2010)]. Nowadays a multitude of single-use bioreactors are commercially available, which can be categorized according to the type of power input into static and dynamic systems. The latter can be further subdivided into hydraulically, pneumatically and mechanically driven bioreactors and their combinations, the so-called hybrid systems [Eibl et al. (2011)]. Mechanically driven single-use bioreactors represent the largest group and are mixed by either orbital-shaken, wave-induced or stirrer motion. While wave-mixed bioreactors (e.g. from GE Healthcare, Sartorius Stedim Biotech, Applikon Biotechnology) were initially dominant, nowadays stirred bag systems (such as Hyclone® Single Use Bioreactor S.U.B., BIOSTAT® CultiBag STR, XDRTM-DSTB animal) are more frequently used, mainly due to the broad experience already obtained with conventional stirred cell culture bioreactors [Eibl et al. (2011)]. The flexible cultivation bags are fixed and shaped by a temperature-controlled stainless steel container. This design is also used in the BIOSTAT® CultiBag STR from Sartorius Stedim Biotech, which is available at different scales from 50 to 1,000 L. The bag geometry, the impellers and the aeration system are designed in a similar way to Sartorius Stedim’s re-usable stirred cell culture bioreactors to ensure comparability of single-use and classical processes, and thus facilitate integration of singleuse bioreactor technologies into modern cultivation processes [De Wilde et al. (2009); Noack et al. (2011)]. Until summer 2009, stirred bag bioreactors were available only at working volumes exceeding 50 L for manufacturing and cost reasons. The introduction of the first single-use stirred bioreactors with a rigid cultivation vessel (Mobius® CellReady 3L bioreactor and CelliGen® BLU SUB) bridged the gap for stirred systems between laboratory and pilot scale

CFD calculations were performed for a series of stirred, single use bioreactor vessels using both rotating reference frame and sliding mesh model approaches. Comparisons of quantities such as flow patterns, power numbers, and mixing times are presented. Calculations to predict mass transfer coefficients for a sparged 250L vessel were also performed using the rotating reference frame model. Results presented include those from a series of single, fixed bubble diameter calculations, and those which employed a population balance model consisting of 9 discrete bubble diameters.Copyright © 2010 by ASME