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Tissue Engineering

Tissue Engineering - Poster Session A

Poster K17 - Bioreactor Optimization for Supporting Mesenchymal Stromal Cell Expansion with Microcarriers

Thursday, October 24, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Mesenchymal stromal cells (MSCs) are a type of adult/somatic stem cell with the ability to differentiate into multiple cell types. MSCs have a wide range of therapeutic applications, including tissue regeneration and repair, immunomodulation, disease modeling, drug testing, gene therapy, and drug delivery. High numbers of MSCs are required for clinical use (>1x10^6 cells/kg body weight), although there are typically low quantities found in the tissue of origin (i.e., < 0.1% of bone marrow). Therefore, strategies for quick cell expansion and reproducible manufacturing are needed for achieving high cell yields. Various bioreactor systems exist and have been applied for the expansion of MSCs, including perfusion bioreactors. In perfusion bioreactors, the shear stress experienced by the MSCs from the constant media exchange has been shown to prime MSCs for quicker osteogenic differentiation.

A novel modular perfusion-based bioreactor system was previously developed by our group for the expansion of MSCs. This bioreactor has exchangeable inserts to support either monolayer or microcarrier culture (Figure 1A). Feasibility of microcarrier culture was previously demonstrated using the mesh-based insert, however, limitations with the microcarrier insert affected culture efficacy thus necessitating further insert design optimization. The goals for this work were: 1) to design a new microcarrier insert that will maintain consistent shape, keep the microcarriers from being swept into the pump, and avoid impeding the flow of the media; and 2) to experimentally confirm that the bioreactor system with the new microcarrier insert supports robust MSC microcarrier culture.

Materials and

Methods:  Prototype designs were developed using SolidWorks® and 3D-printed to test their fit within the bioreactor vessel, with multiple iterations performed. Prototype design was informed by computational fluid dynamics (CFD) modeling used to determine the optimal distance between the bioreactor media inlet and insert and the optimal flow rate.

Testing of the final design was conducted in two stages. First, static trials were done to assess proliferation of murine bone marrow-derived MSCs (D1, ATCC) on Synthemax® II Low Concentration microcarriers (Corning®) in the bioreactor, without the variable of a flow rate. Once successful static culture was confirmed, trials with a flow rate were performed to assess how the perfusion bioreactor compared to a spinner flask gold-standard control for MSC expansion.

During both static and dynamic trials, the cells were cultured with the microcarriers for an 18-hour static attachment period. The microcarriers were then transferred to the bioreactor or the spinner flask and cultured for another 48 hours (without a flow rate for the static trials and with a constant flow rate for the dynamic trials). Samples of microcarriers were then transferred to a well plate for LIVE/DEAD™ staining to confirm cell survival. In dynamic trials only, cells were harvested from the microcarriers post-expansion for a cell count and then seeded in a well plates to assess their ability to undergo osteogenic differentiate. After a 12-hour attachment period the media in the well plates was changed to osteogenic differentiation media. After 6 days Alizarin Red staining was done to assess differentiation.

Results, Conclusions, and Discussions: The final design of the microcarrier insert consisted of a circular piece of steel mesh sandwiched between two rings that were magnetically held together (Figure 1B). The height of the top ring was 3 mm, the determined optimal distance for the inlet to sit away from the cells based on CFD modeling. This height allowed for the inlet to sit directly on the insert, enclosing the microcarriers to prevent their escape into the vessel and attached pump tubing.

LIVE/DEAD™ viability staining for each trial showed that cells successfully adhered to and survived on the microcarriers when cultured in the bioreactor and the spinner flask (Figure 1D). The cell count showed that the perfusion bioreactor had a consistently higher cell yield than the spinner flask cultures. Qualitative analysis of the Alizarin Red staining showed that the MSCs from both the bioreactor and the spinner flask were visibly further along in the process of osteogenic differentiation than is typical at a 6-day timepoint, when compared to cells from static controls. Destaining of the wells and measurement of the destained solution absorbance showed a significantly higher amount of Alizarin Red in the cells harvested from the bioreactor. These results indicate that microcarrier culture in the perfusion bioreactor system better primes MSCs for osteogenic differentiation than microcarrier culture in a spinner flask (Figure 1E).

Acknowledgements (Optional):  This material is based upon work supported by the National Science Foundation under Grant No. EEC-1648035 for NSF Engineering Research Center for Cell Manufacturing Technologies (CMaT).