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

Abstract

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.

An Approach towards a GMP Compliant In-Vitro Expansion of Human Adipose Stem Cells for Autologous Therapies

“…An initial cell density of 15,000 cells/cm 2 (=54,000 cells/mL) and a Microcarrier (MC) concentration of 10 g/L (=1 g, 360 cm 2 ) were used to inoculate the spinner flasks. The MC concentration of 10 g/L was defined based on previous investigations by Schirmaier et al [29] and Jossen et al [1,30]. The cell inoculum was prepared in T 75 -flasks coated with r-fibronectin (5 µg/cm 2 ) and with cells from P1 (=080-PDL cum.…”

“…After the static attachment phase, the culture was continuously stirred at 49 rpm. The selected impeller speed, which corresponded to the N s1u criterion for 10 g/L MCs in the 125 mL disposable Corning spinner flask, was defined based on experimental and numerical fluid flow investigations by Kaiser et al [31] and Jossen et al [1,30]. The N s1u suspension criterion defines the lower limit of N s1 (=N js ), meaning that some MC beads are still in contact with the reactor bottom, but none of them were at rest [32].…”

“…Based on the findings from the static, planar growth experiments, an unstructured, segregated, simplistic growth model was developed and used to describe the hASC growth kinetics in the T 25 -flask cultures. A comparable model approach has already been successfully used by Jossen et al [1] to simulate the anchorage-dependent growth of hASCs on MCs during serum-reduced (5% FBS) expansion in single-use spinner flasks. The same model approach as that employed by Jossen et al [1] was also used with only minor modifications to simulate MC-based hASCs growth kinetics in this study.…”

“…A comparable model approach has already been successfully used by Jossen et al [1] to simulate the anchorage-dependent growth of hASCs on MCs during serum-reduced (5% FBS) expansion in single-use spinner flasks. The same model approach as that employed by Jossen et al [1] was also used with only minor modifications to simulate MC-based hASCs growth kinetics in this study. Detailed information about the MC-based growth model can be found in Jossen et al [1] and in "Supplementary Materials".…”

“…The successful development and application of cell-based therapies has the potential to treat a number of currently incurable diseases and to improve patient care. It is therefore not surprising that many research activities [1,2] are taking place all over the world in the field of regenerative medicine. However, despite the progress in this field, there are a number of challenges that remain before cell-based therapies can be performed more routinely in clinical practice.…”