Biological Considerations in Scaling Up Therapeutic Cell Manufacturing

Abstract

Cell therapeutics - using cells as living drugs - have made advances in many areas of medicine. One of the most clinically studied cell-based therapy products is mesenchymal stromal cells (MSCs), which have shown promising results in promoting tissue regeneration and modulating inflammation. However, MSC therapy requires large numbers of cells, the generation of which is not feasible via conventional planar tissue culture methods. Scale-up manufacturing methods (e.g., propagation on microcarriers in stirred-tank bioreactors), however, are not specifically tailored for MSC expansion. These processes may, in principle, alter the cell secretome, a vital component underlying the immunosuppressive properties and clinical effectiveness of MSCs. This review outlines our current understanding of MSC properties and immunomodulatory function, expansion in commercial manufacturing systems, and gaps in our knowledge that need to be addressed for effective up-scaling commercialization of MSC therapy.

Keywords: biomanufacturing; bioreactors; cell therapy; immunomodulatory; mesenchymal stromal cells; microcarriers; secretome.

Figure 1

Immunosuppressive effects of live and apoptotic MSCs. (A) In the presence of pro-inflammatory cytokines, resting MSCs become “licensed” to secrete key anti-inflammatory molecules, including PD-L1/PD-L2 (Davies et al., 2017), TGF-β (Niu et al., 2017) and IDO (Kim et al., 2018). Licensed MSCs also secrete homing molecules that promote MSC migration and Treg cell recruitment to tissue injury sites (Yu et al., 2011; Lunardi et al., 2014). Surface expression of various molecules on MSCs mediates interactions with T cells and guides MSC migration into inflammatory tissues (Ren et al., 2008; Chinnadurai et al., 2014). MSC cargo, in the form of extracellular vesicles (EVs) and subcellular components, such as mitochondria, may also play a role in MSC-mediated immunosuppression. (B) Following intravenous administration, MSCs can become apoptotic and are engulfed by circulating phagocytes, triggering the expression and release of immunomodulatory molecules (Galleu et al., 2017; de Witte et al., 2018; Cheung et al., 2019). Apoptotic cells can secrete immunosuppressive cargo packaged in extracellular vesicles (Caruso and Poon, 2018). Together, the host response elicited by live and engulfed MSCs leads to broader downstream effects on immune cell function.

Figure 2

A visual comparison of expansion strategies for human MSCs. Inputs to the process include cells, media, supplements, a culture surface (flask, cell stacker, microcarriers) and other additives including growth factors. Figure adapted from (Kropp et al., 2017). Traditional culturing methods encompass 2D, planar technologies such as expanding MSCs in a culture dish or flask by continual passaging. Scale out of this approach uses cell stackers or multilayered flasks which work in this manner through multiplication of the culturing flask. In comparison, scale-up manufacturing methods involve MSCs forming aggregates or being seeded onto microcarrier in suspension in bioreactor systems such as stirred tank, vertical wheel or wave bag bioreactors. Downstream processes such as cell harvesting cell washing, cell concentration, finish and fill and storage through cryopreservation are also critical parts of the manufacture of MSCs for clinical applications.

Figure 3

Mechanisms of MSC attachment to microcarriers. (A) Cell attachment is facilitated through non-specific protein adsorption on the surface of microcarriers that do not contain a coating of any description (e.g., Solohill Plastic). (B) Microcarriers that contain a coating of a biologically derived molecule (e.g., gelatin) which facilitates cell attachment through native cell attachment motifs. (C) Microcarriers which contain a synthetic coating with a chemically synthesized cell attachment motif, for example a short peptide sequence (e.g., Synthemax^®).

Figure 4

Visual representation of MSC expansion on microcarriers over time within a bioreactor. Figure adapted from (Caruso et al., 2014). MSCs initially attach at low coverage in a rounded morphology then flatten and spread over the induction period. The cells then enter a growth phase and expand to cover a large proportion of the microcarrier surface area.