Bioreactor Parameters for Microcarrier-Based Human MSC Expansion under Xeno-Free Conditions in a Vertical-Wheel System

Abstract

Human mesenchymal stem/stromal cells (hMSCs) have been investigated and proven to be a well-tolerated, safe therapy for a variety of indications, as shown by over 900 registered hMSC-based clinical trials. To meet the commercial demand for clinical manufacturing of hMSCs, production requires a scale that can achieve a lot size of ~100B cells, which requires innovative manufacturing technologies such as 3D bioreactors. A robust suspension bioreactor process that can be scaled-up to the relevant scale is therefore crucial. In this study, we developed a fed-batch, microcarrier-based bioreactor process, which enhances media productivity and drives a cost-effective and less labor-intensive hMSC expansion process. We determined parameter settings for various stages of the culture: inoculation, bioreactor culture, and harvest. Addition of a bioreactor feed, using a fed-batch approach, was necessary to replenish the mitogenic factors that were depleted from the media within the first 3 days of culture. Our study resulted in an optimized hMSC culture protocol that consistently achieved hMSC densities between 2 × 10⁵-6 × 10⁵ cells/mL within 5 days with no media exchange, maintaining the final cell population doubling level (PDL) at 16-20. Using multiple hMSC donors, we showed that this process was robust and yielded hMSCs that maintained expansion, phenotypic characteristic, and functional properties. The developed process in a vertical-wheel suspension bioreactor can be scaled to the levels needed to meet commercial demand of hMSCs.

Keywords: bioprocess; bioreactor; hMSCs; microcarrier.

Figure 1

Fed-batch bioreactor culture paradigm. (A) Comparison study of a xeno-free (XF) bioreactor process utilizing batch and fed-batch processes shows a distinct advantage of the fed-batch process on final cell yield (n = 3). (B) Media productivity is enhanced using a fed-batch process. (C) Images indicating typical cell growth on microcarriers during a 5-day culture in a vertical-wheel bioreactor (* p ≤ 0.05). (D) Schematic of the bioreactor culture workflow using a fed-batch process.

Figure 2

Determination of cell and microcarrier inoculation number. (A) Growth kinetics in bioreactors for various cell numbers and microcarrier during seeding, as well as media exchanges. Cell counts were obtained from sampling. (B) Final harvest density obtained from in-vessel harvest. At the highest tested cell-to-media ratio, cell growth can only be supported through media exchanges. (C) Media productivity comparison shows the disadvantage of media exchanges despite the high cell yield. Tested groups are 1×: 2.1 × 10⁶ cells and 1.25 g; 2×: 4.2 × 10⁶ cells and 2.5 g; 3×: 6.3 × 10⁶ cells and 3.75 g; and 3×ΔΔ: 6.3 × 10⁶ cells and 3.75 g with media exchanges on Days 3 and 4.

Figure 3

Optimization of bioreactor seeding parameters at fixed microcarrier amount (A; 1.25 g), or fixed cell number (B; 2.1 × 10⁶ cells). (A) Effect of cell seeding numbers on bioreactor culture yield with 1.25 g microcarriers, and (B) effect of microcarrier density on bioreactor culture yield with 2.1 × 10⁶ cells seeded. For both optimizations, we presented hMSC growth kinetics in bioreactors based on cell density from daily sampling (i), final density obtained from an in-vessel harvest (ii), expansion of cells harvested from the bioreactors compared to the 2D control (iii), and analysis of nutrient and waste in medium during culture (iv). Both seeding parameter studies support bioreactor seeding with 1.25 g of microcarriers and 2.1 × 10⁶ cells (4667 cells/cm²) to optimize for the cell yield and post-bioreactor expansion, while minimizing costs associated with materials.

Figure 4

Optimization of bioreactor culture process. (A) Effect of bioreactor agitation speed on hMSC growth. Higher agitation speed (45 rpm) results in lower final cell yield. (B) Cell health following bioreactor culture. Cells harvested from the bioreactor on Day 6 showed 30% reduced expansion potential. (C,D) Effect of bioreactor feed timing on hMSC growth. Addition of feed on Day 3 results in the highest cell yield. (E) Effect of microcarrier addition during culture. Addition of 1.25 g of microcarriers (additional 450 cm² of surface area) to increase growth surface area during culture did not influence overall hMSC growth.

Figure 5

Effect of surfactants on hMSC growth in bioreactor. Addition of pluronic PF68 shows no effect on cell growth (A), however a high concentration of PF68 (2 g/L) markedly reduces post-bioreactor expansion (B).

Figure 6

Effect of various bioreactor harvest conditions on post-harvest cell health: (A) agitation duration and speed during harvest in TrypLE, (B) duration in quench solution, and (C) temperature of quench solution.

Figure 7

Validation of optimized fed-batch bioreactor process and hMSC characterization following bioreactor culture. (A) Parameters for fed-batch culture in a bioreactor. (B) Validation of optimized bioreactor culture process in five hMSC donors. (C–G) Verification of cell health, critical quality attributes, and functionality following bioreactor culture: XF hMSCs expanded in the bioreactor maintained their cell surface marker expression identity (C), tri-lineage differentiation potential to osteo-, adipo-, and chondrocytes (E), inducible indoleamine 2,3-dioxygenase (IDO) activity when stimulated with IFN_γ (F), and cytokine secretion profile (G). The health, critical quality attributes, and functionality of three hMSC donors are comparable for cells harvested from bioreactor and cells harvested from its respective 2D flask control (In panel E, Scale bar = 400 µm).