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

Abstract

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 × 10⁵ 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 = 0.63 W/m³, τ_nt = 4.96 × 10^-3 Pa) acting at N_s1u (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 × 10⁵ 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.

Keywords: UrSuppe stem cell culture medium; autologous therapy; human adipose stem cells (hASCs); kinetic growth modeling; segregated and unstructured growth model; serum- and xeno-free conditions.

Figure 1

Principle of the growth model and influencing factors.

Figure 2

Light microscopic pictures of patient-derived Human Adipose Tissue Stem Cells (hASCs; a = donor 080, b = donor 085) during cell growth in T₂₅-flasks. Scale bar = 275 µm.

Figure 3

Time-dependent profiles of cell densities (a,c) and substrate/metabolite concentrations (b,d) in T₂₅-flasks. Donor 080 (upper row) and 085 (lower row). Partial medium exchanges of 40% and 60% were performed on days 4 and 8, respectively. The symbols represent the experimentally measured values collected from offline measurements. The lines represent the simulated time courses.

Figure 4

Time-dependent profiles of cell densities (a,c) and substrate/metabolite concentrations (b,d) in the Corning spinner flasks. Donor 080 (upper row) and 085 (lower row). A partial medium exchange of 50% was performed on day 5. The symbols represent the experimentally measured values collected by offline measurements. The lines represent the simulated time courses.

Figure 4

Time-dependent profiles of cell densities (a,c) and substrate/metabolite concentrations (b,d) in the Corning spinner flasks. Donor 080 (upper row) and 085 (lower row). A partial medium exchange of 50% was performed on day 5. The symbols represent the experimentally measured values collected by offline measurements. The lines represent the simulated time courses.

Figure 5

Fluorescence microscopic images during cell growth in Corning spinner flasks. Donors 080 (a) and 085 (b). DAPI-stained cells on Microcarriers (MCs) on day 1 (left), day 4 (middle) and day 9 (right). Scale bars: 275 µm (left and middle) and 650 µm (right).

Figure 6

MC-cell-aggregate diameter distributions (a) and photographic images (b,c) of MC-cell aggregates at the end of the cultivations (day 9). Donor 080 (b) and 085 (c).

Figure 7

Flow cytometry expression profile of selected markers. hASCs from donor 080 (a) and donor 085 (b) cultivated in 2D (T₂₅-flasks) or 3D (MC). hASCs were analyzed after harvesting on day 10. Mean fluorescence was calculated based on specific isotype controls (=relative marker expression).

Figure 8

Results of RT-qPCR measurements of donors 080 (I) and 085 (II). The investigated genes were subdivided into 3 groups: (a) stemness maintenance genes, (b) differentiation regulators/markers and (c) lineage hierarchy markers. Data are represented as 2^(-ΔΔCt) and related to the beginning of the culture (day 1). A single value for each experimental condition was calculated with this method. This figure is also depicted as “heat maps” and is shown in the “Supplementary Materials” (Figure S3).