**3. Additional processes that require MSCs**

MSCs are typically the sole product of any MSC cultivation process, but in some applications, the MSCs are used as helper cells to deliver a different product or they are used as a vehicle to produce a specific cellular component. In each case, the CQAs differ significantly from the standard MSC manufacturing process and other CPPs must therefore be considered. We discuss two examples below.

#### **3.1 Production of MSC-derived extracellular vesicles**

MSCs are potent therapeutics, but researchers are seeking new ways to achieve the same therapeutic effect without the drawbacks associated with MSC manufacturing, such as the limited availability of potent cells, the complex transfusion process, and the entrapment of MSCs in nontarget organs [89]. As discussed earlier, the therapeutic effect of MSCs reflects the secretion of cytokines, growth factors, and other paracrine signaling molecules, particularly via the release of extracellular vesicles that interact directly with target cells and deliver their contents into the cytosol. The advantage of these vesicles over whole MSCs is their much greater stability, which means they can be manufactured, stored, and shipped without losing therapeutic efficacy [90, 91].

The large-scale manufacturing of extracellular vesicles requires the cultivation of MSCs, which secrete these vesicles directly in the culture medium. Scalable production methods are not yet available, and vesicles are currently produced in T-flasks or cell factories without process monitoring. Bioreactors could be used to scale up production, and given there is no need to harvest the MSCs, it would be possible to consider a wider range of bioreactor systems than the relatively narrow selection favored for MSC manufacturing. A fixed bed bioreactor has been used for the continuous production and harvesting of extracellular vesicles, which increased the yield 10-fold compared to T-flasks [92]. Stirred tank reactors with microcarriers might also be suitable, but they have not yet been used for vesicle production [57]. The cells would be exposed to shear forces caused by the impellers and air bubble cavitation, and this may influence vesicle production and potency [93].

The effect of different process parameters on the production of MSC-derived extracellular vesicles has been investigated at the laboratory scale. For example, hMSCs and their vesicles are primed by hypoxic conditions or changes in medium composition, such as the removal of FCS or the addition of priming factors like IFNγ and TNFα [34, 35]. The yield of extracellular vesicles can also be increased by preparing spheroids that mimic *in vivo* conditions, for example by laying down an extracellular matrix and supplying appropriate signaling molecules [89]. Cell density, passage number, and cell origin also affect the vesicle yield. The immortalized cell line hMSC-TERT is more stable than primary MSCs, but the immortalization process has an impact on vesicle production, which must be investigated individually for each cell line because it is not related to the immortalization method [93].

**217**

**4. Conclusions**

*Bioprocess Development for Human Mesenchymal Stem Cell Therapy Products*

There is currently no standardized large-scale production platform for primed

purification process would be required. The laboratory-scale purification of vesicles captured from the culture medium is currently based on a combination of ultracentrifugation, dead-end filtration, precipitation, and size exclusion chromatography, which are difficult to scale up [94, 95]. However, tangential-flow filtration can also be used for large-scale purification, washing, and buffer exchange, and this method should be investigated in more detail for vesicle purification [94, 96]. Extracellular vesicles are even more sensitive to process changes than MSCs, so the influence of multiple cell-dependent, culture, and process parameters on the potency of these

The ability of MSCs to restore the activity of dysfunctional cells *in vivo* is the basis of their therapeutic efficacy, but the same interactions can also be exploited *in vitro*. One key example is the interaction between MSCs and pancreatic beta cells, which are widely used for drug screening and cell therapy in the context of diabetes. In both applications, large numbers of functional beta cells are required, but beta cells rapidly lose their functionality when expanded *in vitro*. The loss of beta cell functionality *in vitro* can be prevented by cocultivation with MSCs, which not only stimulate beta cell proliferation but also enhance their glucose-dependent

The major challenge of cocultivation is to balance the demands of two completely different cell types. In large scales, the distribution of cells becomes heterogeneous, which can lead to instability within the bioreactor and lower cell viability. A well-balanced and tightly controlled culture environment is needed to stabilize large-scale cocultures. Because secreted factors are important for the cocultivation of MSCs and beta cells, the hydrodynamic forces in bioreactors, which influence the distribution of secreted molecules, must be considered at an early stage [100]. Furthermore, the optimal cocultivation ratio of the cells must be determined. Established processes can be modified to achieve a new process setup for cocultivation, but it is often beneficial to separate cell expansion from cocultivation (i.e., first expand the pure cultures to generate the cells needed for the coculture and then combine them to improve the function of beta cells in a second process step). For the expansion step, it can be sufficient to improve the growth of beta cells using conditioned medium from the cultivation of MSCs. Alternatively, the expansion and functionalization of beta cells can be combined in one process step [101]. The CPPs for such a complex process can be difficult to identify, but the CQAs of the beta cells are most relevant if the aim of the process is to produce functionalized beta cells for drug screening or cell therapy. Even so, the potency of the MSCs must not be neglected because they are required to stimulate the beta cells. Accordingly, the MSCs must be expanded under controlled and standardized conditions that maximize their beneficial impact on beta cells. In the future, cocultivation bioreactor concepts for MSCs and beta cells must be tested to allow the completely aseptic

MSCs are potent therapeutic agents, but their complexity and environmental sensitivity make the GMP-compliant manufacturing of MSC products extremely challenging. Given the range of tissue sources, isolation procedures, and expansion

hMSC-derived vesicles, but even if such a platform existed, a corresponding

*DOI: http://dx.doi.org/10.5772/intechopen.90029*

vesicles must be determined.

secretion of insulin [97–99].

**3.2 Cocultivation of MSCs with other cells**

expansion and cocultivation of both cell types.

#### *Bioprocess Development for Human Mesenchymal Stem Cell Therapy Products DOI: http://dx.doi.org/10.5772/intechopen.90029*

There is currently no standardized large-scale production platform for primed hMSC-derived vesicles, but even if such a platform existed, a corresponding purification process would be required. The laboratory-scale purification of vesicles captured from the culture medium is currently based on a combination of ultracentrifugation, dead-end filtration, precipitation, and size exclusion chromatography, which are difficult to scale up [94, 95]. However, tangential-flow filtration can also be used for large-scale purification, washing, and buffer exchange, and this method should be investigated in more detail for vesicle purification [94, 96]. Extracellular vesicles are even more sensitive to process changes than MSCs, so the influence of multiple cell-dependent, culture, and process parameters on the potency of these vesicles must be determined.

### **3.2 Cocultivation of MSCs with other cells**

*New Advances on Fermentation Processes*

**3. Additional processes that require MSCs**

potency of MSCs [88].

The production of MSCs with standardized properties would be facilitated by the development of standardized validated potency assays so that results obtained in different laboratories are truly comparable. The ISCT has taken steps in this direction by publishing standards for the harmonization of potency assays. In a matrix assay approach, they propose to use quantitative RNA analysis for selected gene products, flow cytometry to detect functionally-relevant surface markers, and protein-based assays to map the secretome and determine the immunomodulatory

MSCs are typically the sole product of any MSC cultivation process, but in some applications, the MSCs are used as helper cells to deliver a different product or they are used as a vehicle to produce a specific cellular component. In each case, the CQAs differ significantly from the standard MSC manufacturing process and other

MSCs are potent therapeutics, but researchers are seeking new ways to achieve the same therapeutic effect without the drawbacks associated with MSC manufacturing, such as the limited availability of potent cells, the complex transfusion process, and the entrapment of MSCs in nontarget organs [89]. As discussed earlier, the therapeutic effect of MSCs reflects the secretion of cytokines, growth factors, and other paracrine signaling molecules, particularly via the release of extracellular vesicles that interact directly with target cells and deliver their contents into the cytosol. The advantage of these vesicles over whole MSCs is their much greater stability, which means they can be manufactured, stored, and shipped without

The large-scale manufacturing of extracellular vesicles requires the cultivation of MSCs, which secrete these vesicles directly in the culture medium. Scalable production methods are not yet available, and vesicles are currently produced in T-flasks or cell factories without process monitoring. Bioreactors could be used to scale up production, and given there is no need to harvest the MSCs, it would be possible to consider a wider range of bioreactor systems than the relatively narrow selection favored for MSC manufacturing. A fixed bed bioreactor has been used for the continuous production and harvesting of extracellular vesicles, which increased the yield 10-fold compared to T-flasks [92]. Stirred tank reactors with microcarriers might also be suitable, but they have not yet been used for vesicle production [57]. The cells would be exposed to shear forces caused by the impellers and air bubble

cavitation, and this may influence vesicle production and potency [93].

The effect of different process parameters on the production of MSC-derived extracellular vesicles has been investigated at the laboratory scale. For example, hMSCs and their vesicles are primed by hypoxic conditions or changes in medium composition, such as the removal of FCS or the addition of priming factors like IFNγ and TNFα [34, 35]. The yield of extracellular vesicles can also be increased by preparing spheroids that mimic *in vivo* conditions, for example by laying down an extracellular matrix and supplying appropriate signaling molecules [89]. Cell density, passage number, and cell origin also affect the vesicle yield. The immortalized cell line hMSC-TERT is more stable than primary MSCs, but the immortalization process has an impact on vesicle production, which must be investigated individually for each cell line because it is not related to the immortalization method [93].

CPPs must therefore be considered. We discuss two examples below.

**3.1 Production of MSC-derived extracellular vesicles**

losing therapeutic efficacy [90, 91].

**216**

The ability of MSCs to restore the activity of dysfunctional cells *in vivo* is the basis of their therapeutic efficacy, but the same interactions can also be exploited *in vitro*. One key example is the interaction between MSCs and pancreatic beta cells, which are widely used for drug screening and cell therapy in the context of diabetes. In both applications, large numbers of functional beta cells are required, but beta cells rapidly lose their functionality when expanded *in vitro*. The loss of beta cell functionality *in vitro* can be prevented by cocultivation with MSCs, which not only stimulate beta cell proliferation but also enhance their glucose-dependent secretion of insulin [97–99].

The major challenge of cocultivation is to balance the demands of two completely different cell types. In large scales, the distribution of cells becomes heterogeneous, which can lead to instability within the bioreactor and lower cell viability. A well-balanced and tightly controlled culture environment is needed to stabilize large-scale cocultures. Because secreted factors are important for the cocultivation of MSCs and beta cells, the hydrodynamic forces in bioreactors, which influence the distribution of secreted molecules, must be considered at an early stage [100]. Furthermore, the optimal cocultivation ratio of the cells must be determined. Established processes can be modified to achieve a new process setup for cocultivation, but it is often beneficial to separate cell expansion from cocultivation (i.e., first expand the pure cultures to generate the cells needed for the coculture and then combine them to improve the function of beta cells in a second process step). For the expansion step, it can be sufficient to improve the growth of beta cells using conditioned medium from the cultivation of MSCs. Alternatively, the expansion and functionalization of beta cells can be combined in one process step [101]. The CPPs for such a complex process can be difficult to identify, but the CQAs of the beta cells are most relevant if the aim of the process is to produce functionalized beta cells for drug screening or cell therapy. Even so, the potency of the MSCs must not be neglected because they are required to stimulate the beta cells. Accordingly, the MSCs must be expanded under controlled and standardized conditions that maximize their beneficial impact on beta cells. In the future, cocultivation bioreactor concepts for MSCs and beta cells must be tested to allow the completely aseptic expansion and cocultivation of both cell types.
