Human mesenchymal stem cell (hMSC)-based therapies are of increasing interest in the field of regenerative medicine. As economic considerations have shown, allogeneic therapy seems to be the most cost-effective method. Standardized procedures based on instrumented single-use bioreactors have been shown to provide billion of cells with consistent product quality and to be superior to traditional expansions in planar cultivation systems. Furthermore, under consideration of the complex nature and requirements of allogeneic hMSC-therapeutics, a new equipment for downstream processing (DSP) was successfully evaluated. This mini-review summarizes both the current state of the hMSC production process and the challenges which have to be taken into account when efficiently producing hMSCs for the clinical scale. Special emphasis is placed on the upstream processing (USP) and DSP operations which cover expansion, harvesting, detachment, separation, washing and concentration steps, and the regulatory demands.

The potential of human mesenchymal stem cells (hMSCs) for allogeneic cell therapies has created a large amount of interest. However, this presupposes the availability of efficient scale‐up procedures. Promising results have been reported for stirred bioreactors that operate with microcarriers. Recent publications focusing on microcarrier‐based stirred bioreactors have demonstrated the successful use of Computational Fluid Dynamics (CFD) and suspension criteria (N_S1u, N_S1) for rapidly scaling up hMSC expansions from mL‐ to pilot scale. Nevertheless, one obstacle may be the formation of large microcarrier‐cell‐aggregates, which may result in mass transfer limitations and inhomogeneous distributions of stem cells in the culture broth. The dependence of microcarrier‐cell‐aggregate formation on impeller speed and shear stress levels was investigated for human adipose derived stromal/stem cells (hASCs) at the spinner scale by recording the Sauter mean diameter (d₃₂) versus time. Cultivation at the suspension criteria provided d₃₂ values between 0.2 and 0.7 mm, the highest cell densities (1.25 × 10⁶ cells mL⁻¹ hASCs), and the highest expansion factors (117.0 ± 4.7 on day 7), while maintaining the expression of specific surface markers. Furthermore, suitability of the suspension criterion N_S1u was investigated for scaling up microcarrier‐based processes in wave‐mixed bioreactors for the first time.

Mass Production of Mesenchymal Stem Cells — Impact of Bioreactor Design and Flow Conditions on Proliferation and Differentiation | InTechOpen, Published on: 2014-11-26. Authors: Valentin Jossen, Ralf Pörtner, Stephan C. Kaiser, et

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.

Human Adipose Tissue Stem Cells (hASCs) are a valuable source of cells for clinical applications (e.g., treatment of acute myocardial infarction and inflammatory diseases), especially in the field of regenerative medicine. However, for autologous (patient-specific) and allogeneic (off-the-shelf) hASC-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 hASCs for autologous therapies is more problematic because the cell material changes for each treatment. Moreover, cell material is normally isolated from non-healthy or older patients, which further complicates successful in-vitro expansion. Hence, the goal of this study was to perform cell expansion studies with hASCs isolated from two different patients/donors (i.e., different ages and health statuses) under xeno- and serum-free conditions in static, planar (2D) and dynamically mixed (3D) cultivation systems. Our Primary aim was I) to compare donor variability under in-vitro conditions and II) to develop and establish an unstructured, segregated growth model as a proof-of-concept study. Maximum cell densities of between 0.49 and 0.65 × 10^5 hASCs/cm2 were achieved for both donors in 2D and 3D cultivation systems. Cell growth under static and dynamically mixed conditions was comparable, which demonstrated that hydrodynamic stresses (P/V = 0.63W/m3, τnt = 4.96 × 10^-3 Pa) acting at Ns1u (49 rpm for 10 g/L) did not negatively affect cell growth, even under serum-free conditions. However, donor-dependent differences in the cell size were found, which resulted in significantly different maximum cell densities for each of the two donors. In both cases, stemness was well maintained under static 2D and dynamic 3D conditions, as long as the cells were not hyperconfluent. The optimal point for cell harvesting was identified as between cell densities of 0.41 and 0.56 × 10^5 hASCs/cm2 (end of exponential growth phase). The growth model delivered reliable predictions for cell growth, substrate consumption and metabolite production in both types of cultivation systems. Therefore, the model can be used as a basis for future investigations in order to develop a robust MC-based hASC production process for autologous therapies.

Adipose tissue is an abundant source of stem cells. However, liposuction cannot yield cell quantities sufficient for direct applications in regenerative medicine. Therefore, the development of GMP-compliant ex vivo expansion protocols is required to ensure the production of a “cell drug” that is safe, reproducible, and cost-effective. Thus, we developed our own basal defined xeno- and serum-free cell culture medium (UrSuppe), specifically formulated to grow human adipose stem cells (hASCs). With this medium, we can directly culture the stromal vascular fraction (SVF) cells in defined cell culture conditions to obtain hASCs. Cells proliferate while remaining undifferentiated, as shown by Flow Cytometry (FACS), Quantitative Reverse Transcription PCR (RT-qPCR) assays, and their secretion products. Using the UrSuppe cell culture medium, maximum cell densities between 0.51 and 0.80 × 10⁵ cells/cm² (=2.55–4.00 × 10⁵ cells/mL) were obtained. As the expansion of hASCs represents only the first step in a cell therapeutic protocol or further basic research studies, we formulated two chemically defined media to differentiate the expanded hASCs in white or beige/brown adipocytes. These new media could help translate research projects into the clinical application of hASCs and study ex vivo the biology in healthy and dysfunctional states of adipocytes and their precursors. Following the cell culture system developers’ practice and obvious reasons related to the formulas’ patentability, the defined media’s composition will not be disclosed in this study.