Michael M. Gepp1,2, Petra Geßner3, Isabelle Sébastien1,2, Karina Ivaskevica1,2, Julia C. Neubauer1,2 and Heiko Zimmermann1,4,5

1. Fraunhofer Institute for Biomedical Engineering IBMT, Sulzbach, Germany
2. Fraunhofer Project Center for Stem Cell Process Engineering, Würzburg, Germany
3. Alginatec GmbH, Riedenheim, Germany
4. Molecular and Cellular Biotechnology/Nanotechnology, Saarland University, Saarbrücken, Germany
5. Facultad de Ciencias del Mar, Universidad Católica del Norte, Coquimbo, Chile

Three-dimensional (3D) cell culture technology is becoming increasingly important in research and the biotechnology industry. In particular, the expansion and differentiation of pluripotent stem cells on an industrial scale is the focus of many academic and industrial research groups. Although optimised protocols and culture media for lineage-specific 3D cultivation already exist, further developments in biomaterials and cell scaffolds are required. It is known that microcarriers can significantly increase the productivity of the bioprocess due to the higher surface to volume ratio (Figure 1a). For example, less cell culture medium is required, and more cells can be produced in a single bioreactor system. However, there are few commercially available soft microcarrier systems for stem cells. It is known that the mechanical properties of the biomaterial, such as elasticity, have a significant impact on cell culture behaviour. Therefore, alginate microcarriers with tuneable mechanical properties are advantageous for novel cell models in tissue engineering as they better mimic the physiological properties of the in vivo situation (Figure 1b). In this application note, we describe the properties of soft alginate microcarriers and their integration into human induced pluripotent stem cell (hiPSC) expansion processes using the CERO suspension bioreactor system.

General properties and handling of alginate microcarriers

The growth area of the alginate microcarriers can be adjusted by pipetting and centrifuging the corresponding volume. Each vial indicates the growth area per millilitre, enabling a quick and dependable inoculation process. The microcarriers are ready-to-use and stored in isotonic NaCl solution at 4°C until use, eliminating the need for rehydration. The microcarriers are compatible with poloxamers at low concentrations. This minimises adsorption of the microcarriers to plastic surfaces. HiPSC on transparent, non-auto-fluorescent alginate microcarriers can be imaged for documentation purposes and analysed using various microscopic techniques, such as phase contrast or confocal laser scanning microscopy. Fluorescent dyes and antibodies can be used for staining, and imaging can be done without modifying the manufacturer's staining protocol. Alginate microcarriers, unlike other commercial microcarriers, are soft and can imitate the mechanical properties of the in vivo environment. The diameter of the microcarriers is adjustable, and microcarriers ranging from 100 µm to 500 µm can be produced. Standard laboratory equipment can be used for cell harvest when working with these microcarriers. Separating the medium and microcarriers overgrown with cells can be achieved through centrifugation and sieving. Alginate microcarriers have a density of about 1014 kg/m³, which causes them to sediment slowly. However, this also means that less energy is required for stirring in stirred bioreactors. Cell detachment can be achieved using trypsin, TrypLE™, accutase, or EDTA. Additionally, commercially available alginate lyases can be used to degrade hydrogels, allowing for the recultivation of expanded cells on standard 2D cell culture surfaces. The viscosity and elastic modulus of alginate change over time, depending on storage conditions and production mould. Varying the geometry and modulus (2.0-8.0 kPa) of the cell scaffold does not affect the efficiency or quality of hiPSC-3D cultivation.

3D expansion of hiPSC in CERO bioreactors

The alginate microcarriers were used to expand hiPSC in CERO suspension bioreactors. Microcarriers with different mechanical properties (increasing E-Modul) have been studied and after 4 days viability and total cell number has been measured. Overall, the data (see Figure 2 a-c) show an excellent compatibility with hiPSC independent from the alginate’s formulation. The viability after expansion was greater than 85% and a total cell number of at least 6.0x106 ± 1,8x106 cells was achieved. The results lead to the conclusion, that there are no adverse effects of different moduli of elasticity on the proliferation of UKBi005-A cells. Neither the total cell number nor the viability were affected by the mechanical properties of the cell scaffold. The expanded cells were also analysed by flow cytometry (FACS). No deviations in the expression pattern of the pluripotency markers could be detected as a function of the Young's modulus of the cell scaffolds. In addition to the qualitative evaluation of the proliferation and pluripotency of the UKBi005-A cells, a microscopic evaluation was also carried out during the entire cultivation process. The representative images from last day of cultivation are shown in Figures 3.

In this study, alginate microcarriers coated with Matrigel™ were characterised as an important biomaterial for stem cell expansion in suspension bioreactors on soft microcarriers. New insights were gained into the mechanical properties of alginate under the influence of environmental conditions, the correlation between viscosity and elastic modulus, and the interaction between alginate and iPSC lines. It was found that despite the different viscosities of the mixture components, the alginate microcarriers had a modulus of elasticity of 2.5 to 4.5 kPa, which did not affect the efficiency of hiPSC expansion. The hiPSCs cultured on these cell scaffolds showed high viability (85-95%) and excellent adhesion. In additional studies could be demonstrated that the alginate microcarriers can be part of complex stem cell processes (see [7]) and are compatible with various human cell systems. Thus, the alginate microcarriers can be integrated into cultivation protocols of mesenchymal stem cells [1] and expansion and differentiation protocols of hiPSC for renal proximal tubular epithelial cells [5], neural cells [3], and 3,56 kPa 2,83 kPa 2,49 kPa 4,44 kPa 2,97 kPa 4,13 kPa

the cultivation of cardiomyocytes. Furthermore, the alginate microcarrier has emerged as stable in vitrification studies of adherent hiPSC [6]. The key benefits of UHV-alginate microcarriers in stem cell processes are (not an exhaustive list):