Cell retention in scalable, perfusion-based mesenchymal stem cell expansion processes: a proof of concept

Abstract

The production of clinically relevant quantities of human mesenchymal stromal cells (hMSCs) requires scalable and intensified manufacturing processes. For this reason, the applicability of alternating tangential flow filtration (ATF) and tangential flow depth filtration (TFDF) based cell retention systems for hMSC expansion on microcarriers (MCs) in perfusion mode was assessed. The processes were conducted in stirred tank bioreactors at a scale of 1.8 L and compared with repeated-batch cultivations. In the perfusion and repeated-batch control cultivations, competitive viable cell concentrations of ≈2.9 · 10⁶ cells mL^-1 were reached within a cultivation period of 5-7 days, resulting in an expansion factor of 41-57. The main difference between the operation modi was the aggregation behavior of the MCs. While the median MC aggregate diameter in the repeated-batch cultivation reached 470 μm, the ATF cell retention device constrained aggregate size to a median diameter of 250 µm. In the TFDF cultivation, the shear forces in the recirculation loop stripped most of the hMSCs from the MCs, resulting in the formation of spheroids that continued to proliferate, albeit at a decreased rate. While perfusion operation did not lead to increased productivity in this proof-of-concept study, manual handling and therefore contamination risk were reduced by replacing the repeated-batch process's daily 80% medium exchanges with automated perfusion operation. Additionally, the ATF system was shown to be useful for medium removal and washing of the MCs prior to adding the harvesting solution, which is highly valuable for cultivations conducted at larger scales. While the feasibility of ATF based cell retention for MC expansion processes could be demonstrated, increased growth area to medium ratios, i.e., higher MC concentrations, still need to be investigated to leverage the full potential of the perfusion process mode.

Keywords: alternating tangential flow filtration (ATF); hMSC; microcarrier; perfusion; single-use technology (SUT); stirred tank bioreactor; tangential flow filtration (TFF).

FIGURE 1

Overview of bioreactor and MC retention device setup for the different process modes.

FIGURE 2

Diaphragm pump of ATF-2 device installed upright (A) and upside down (B). Photographs were taken at the end of the pressurizing phase, showing MCs trapped between the pump housing and membrane.

FIGURE 3

(A) VCC and viability and (B) cell specific growth rate of the cultivations.

FIGURE 4

(A) Glucose, (B) glutamine, (C) lactate, (D) ammonium concentrations over cultivation time. Glutamine concentration represents the sum of free glutamine and alanyl-glutamine. (E) Lactate yield from glucose (C-metabolism) and ammonium yield from glutamine (N-metabolism). (F) Cell specific glucose consumption rate. (G) Cell specific glutamine consumption rate. (H) Cell specific lactate production rate. (I) Cell specific ammonium production rate. Because the concentration changes during the first 24 h were small compared to inherent inaccuracies of the analytical methods, the yields and cell specific production/consumption are not displayed for this period.

FIGURE 5

Applied perfusion rates during ATF and TFDF cultivation. On day 2, the respective perfusion devices were primed, and perfusion operation was started.

FIGURE 6

(A) Relative prevalence of cells on single MCs, aggregated MCs, and spheroids. The inhabitation ratio corresponds to the percentage of MCs showing at least 1 cell. (B) Size distribution of MC aggregates and aggregation ratio over time. For the TFDF cultivation, the boxplots show the spheroid diameters (green) instead of the MC aggregate diameters (white). A threshold of 240 µm was chosen to differentiate single MCs from MC aggregates based on diameter. Microscope images of samples taken from the TFDF cultivation on day 3 (C) and day 7 (D). The darker spheres are MCs while the spheroids are of lighter color. (E) Recirculation pump speed and flow during the TFDF cultivation. On day 3 and 4, the circulation loop was re-primed. (F) Picture of RB2 cultivation on harvest day. Visible are large MC aggregates stuck between reactor internals and wall.

FIGURE 7

Harvested hMSCs differentiated into adipocytes and stained with Oil Red O. (A) RB1, (B) RB2, (C) TFDF, (D) ATF, (E) negative control.

FIGURE 8

Examples of hMSCs differentiated into chondrocytes and stained with Alcian blue. (A) RB2, (B) negative control.

FIGURE 9

Harvested hMSCs differentiated into osteoblasts and stained with Alizarin Red S. (A) RB1, (B) RB2, (C) TFDF, (D) ATF, (E) negative control.