**2. Expansion of human MSCs** *in vitro*

*New Advances on Fermentation Processes*

entity. Potency can also be measured using *in vitro* functional assays that determine MSC activity directly or via an indirect metric that correlates to MSC activity *in vivo*. An assay that measures differentiation potential is only appropriate to describe MSC potency if the therapeutic aim involves engraftment of the cells or tissue formation (notwithstanding the controversy over the assumption that MSCs differentiate *in vivo*, as discussed above). The FDA mandates that potency is measured using quantitative biological assays [27], so the standard approach is to differentiate MSCs *in vitro* by cultivating them in differentiation medium and then testing them after 21 days [17]. Staining for differentiation markers is nonquantitative, so alternative methods such as postdifferentiation RNA or protein analysis [28, 29], or the online monitoring

If the therapeutic effect of MSCs is conferred by the secretome, then the differentiation potential may not be the primary determinant of potency. The profile of secreted factors would be a more appropriate measure, and this could be determined by multiplex enzyme-linked immunosorbent assays (ELISAs) or mass spectroscopy [31]. However, a clear link between the secretome profile and *in vivo* efficacy must be established, so that animal models or cell-based assays can be used to determine the limits of the relevant factors. This is a typical way to move from a complex and highly variable *in vivo* assay to a multiassay approach combining the quantification of viability, target-specific cytotoxicity or cytokine release, surrogate biomarkers (morphological phenotype or released factors that correlate with function), bioactivity (e.g., presentation of surface markers), cell-based assays, and

Impurities are unwanted components from within the process, whereas contaminants come from outside the process. Impurities during MSC manufacturing include unwanted cell types, particles (e.g., residual microcarriers, or plastics and fibers from manufacturing equipment and materials), or components of culture medium. Contaminants include bacteria, fungi, viruses, endotoxins, and mycoplasma. The heterogeneity of MSCs makes it difficult to detect unwanted cell types. MSC preparations should ideally be pure, but fibroblasts are often present as impurities. Cell-specific sorting based on the marker CD166 (which is expressed at higher levels on MSCs) and CD9 (which is expressed at higher levels on fibroblasts) may help to achieve sufficient purity [33]. In other cases, it may be sufficient if most of the cells in the final product (>98%) fulfill the ISCT minimal criteria based on MSC surface markers. All other impurities and contaminants must be measured and the maximum residual levels must be defined to ensure safety and efficacy. A final sterilization step is not possible when the product is living cells, so the entire

MSC production process must be carried out under aseptic conditions.

From the QTPP list, CQAs must be identified, which directly influence the safety and efficacy of the MSC product. This means that a risk assessment is carried out to reduce the QTPP list to the most influential attributes based on impact and certainty. According to ICHQ8, a CQA is "*A physical, chemical, biological, or microbiological property or characteristic that should be within an appropriate limit, range, or distribution to ensure the desired product quality.*" Therefore, every process parameter "*whose variability has an impact on a CQA"* is a critical process parameter (CPP) that "*should be monitored or controlled to ensure that the process produces the desired quality."* There is no precise delimitation of the degree of impact required to define a CPP, so the broad definition of a CPP is generally divided into parameters that have a substantial impact on the CQAs and those with minimal or zero impact. Each process step has multiple CPPs. For example, during the *in vitro* expansion

of differentiation by Raman spectroscopy [30], are more suitable.

genomic, transcriptomic, and proteomic profiles [32].

*1.3.3 Sterility and purity*

**208**

Therapeutic applications of MSCs require at least 1 × 108 cells per dose, which is many more than can be isolated by tissue aspiration. All MSC production processes must therefore include an *in vitro* expansion. Having generated or isolated the starting cell population, *in vitro* expansion is followed by harvest, concentration, purification, formulation, fill and finish, storage, and shipping. The manufacturing steps of MSCs are therefore similar to the production of recombinant proteins, but MSCs are more challenging due to the variability of the starting material, the complexity of living cells as a product, an incomplete understanding of their mechanism of action, and the inherent difficulties encountered during product characterization.

#### **2.1 CPPs that affect MSC manufacturing**

The properties of MSCs are strongly influenced by the environment because MSCs in nature interact with surrounding cells and tissues, with the extracellular matrix and with various bioactive molecules. Even in an artificial environment like a bioreactor or T-flask, MSCs are very sensitive to their environment, and the most influential factors give rise to CPPs. By identifying CPPs that affect MSC quality, the process can be designed to favor the recovery of MSCs with specific phenotypes of interest, in this case those with the greatest therapeutic efficacy [34, 35]. The CPPs affecting MSC quality are discussed in more detail below.

### *2.1.1 Cell density and age*

During MSC isolation, the seeding density is important because all sources contain different quantities of MSCs. For example, only 1 in 100,000 bone marrow cells is an MSC, whereas in adipose tissue, the ratio is nearer to 1 in 100 [36]. If plastic adherence is selected as a strategy for MSC isolation, the number of adherent cells therefore differs according to the source if a similar number of tissue cells are seeded. Standardization during this step can be achieved by isolating MSCs using a strategy of surface marker sorting, allowing a defined number of cells to be seeded into the culture vessel. The seeding density selected for the *in vitro* expansion step is a CPP. MSCs can be seeded at a very low density (50–100 cells per cm<sup>2</sup> ) and will proliferate until they achieve confluence. This corresponds to a high expansion factor, but the process takes a long time and requires more rounds of cell division for each seeded cell, so the cells experience significant aging [37]. The aging of MSCs during expansion is a problem, because older cells lose competence to behave as stem cells and have a tendency to enter senescence or even to undergo transformation. The manufacturing of Prochymal provided a clear example of this issue: 10,000 or more doses were manufactured from one donor, and the corresponding expansion stress led to replicative senescence, in which the cells retained a typical

MSC surface marker profile but lost functionality [4]. Aging MSCs are more likely to activate a senescence-associated secretory phenotype and produce proinflammatory cytokines such as IL-1, IL-6, and IL-8, which inhibit the regenerative process. The duration of *in vitro* expansion must be considered not only because of senescence, but also due to the phenomenon of clonal impoverishment. MSCs are polyclonal, but prolonged expansion favors the growth of specific cell types or clones. Depending on the expansion time and expansion factor, the cell mixtures may completely differ in phenotype and also in potency. Therefore, although a high expansion factor in a short process time is desirable to achieve high product yields, *in vitro* expansion should never change the properties of MSCs to the extent that it compromises their functionality and potency.

#### *2.1.2 Culture medium*

Several basal media have been shown to influence MSC expansion and potency, including Dulbecco's modified Eagle's medium (DMEM), Iscove's modified Dulbecco's medium (IMDM), and MEM alpha (αMEM) [37]. One of the key components of these media is glucose, which is the main carbon source for MSCs. Glucose may be provided at physiological concentrations (1 g/L) or higher (up to 4.5 g/L), the latter variously described as having a negative effect on MSC proliferation and growth factor secretion [38] or no effect at all [39]. Glutamine as a second carbon source is present at concentrations of 2–4 mM and appears essential for MSC growth [40], but its impact on MSC properties is complex, with contradictory results [41–43]. Glutamine is unstable at 37°C and spontaneously degrades to form ammonia. GlutaMAX (dipeptide Ala-Gln) is recommended instead of glutamine to promote MSC expansion [44]. Lactate and ammonia are the most abundant waste products formed by MSCs, and both therefore have the potential to inhibit growth. It therefore follows that glucose, glutamine, lactate, and ammonia levels should be considered as CPPs for the production of MSCs. Several other amino acids may also be relevant, given that the amino acid metabolism of MSCs differs from that of commercial cell lines such as Chinese hamster ovary (CHO) cells [42].

Basal media formulations must be supplemented to achieve MSC expansion. The most important supplement is fetal calf serum (FCS), which is added to a final concentration of 5–20%. FCS strongly influences MSC growth and phenotype, but the specific effectors are unknown because the composition of FCS is variable and lot-dependent [45]. The use of FCS for the manufacture of clinical MSC products is discouraged nowadays, in line with the drive to eliminate all raw materials of animal origin. The complex, uncertain, and variable composition of FCS also makes it difficult to validate for GMP-compliant processes. Finally, the manufacturing process must accommodate steps to eliminate FCS from the final product to avoid potential immunogenicity and allergenicity [46]. FCS can be replaced with human serum and its derivatives, such as human platelet lysate, which promotes MSC growth [47]. However, the same lot-dependent quality issues described above for FCS also apply to human serum [48]. The most acceptable alternative is serum-free or preferably chemically-defined medium, the latter not only serum-free but also lacking any hydrolysates or supplements of unknown composition. MSCs grow well in several commercial serum-free media, including BD Mosaic MSC Serum-free (BD Biosciences), RoosterNourish (Rooster Bio), Mesencult-XF (Stemcell Technologies), StemPro MSC SFM Xeno-Free (Invitrogen), TheraPEAK MSCGM-CD (Lonza), and PPRF-msc6, STK1 and STK2 (Abion) [49]. Growth in chemically-defined medium has also been demonstrated [50]. However, although MSCs showed excellent growth in these serum-free media, they reached senescence earlier, and there were changes in morphology, surface marker profiles, and potency [51]. This does not mean that

**211**

*Bioprocess Development for Human Mesenchymal Stem Cell Therapy Products*

affect MSC identity and potency and must be carefully controlled.

serum-free and chemically-defined media should be avoided-it is still better to use these media for MSC expansion in order to meet GMP requirements-but further investigations are required to optimize the media composition. The development of serum-free media is mainly driven by companies, which tend not to disclose the precise composition, making it difficult for other researchers to build on the results. In serum-free and defined media, supplemental growth factors such as FGF2 and PDGF are needed to stimulate MSC proliferation, but they also influence MSC potency [18]. Accordingly, chemically-defined media would be preferable for the *in vitro* expansion of MSCs, but growth factor concentrations are important CPPs that

MSCs are aerobic cells and any culture vessel must therefore ensure an adequate supply of oxygen. However, the oxygen saturation in standard T-flasks (21% O2) is far removed from nature (5–7% O2) [34]. MSCs therefore tend to be oversaturated with oxygen, which can increase the concentration of damaging reactive oxygen species (ROS). Several studies have confirmed that hypoxia enhances MSC proliferation, stabilizes their cell fate, and prevents apoptosis by reducing the levels of caspase-3 [52]. However, rather than imposing hypoxia by preconditioning the cells, it may be better to impose hypoxia during the entire expansion phase, because

In addition to oxygen saturation, temperature and pH are CPPs in every process and can be monitored and controlled very easily. Typically, *in vitro* expansion is carried out at 37°C and neutral pH (7.2–7.4). Expansion at lower temperatures can be advantageous under certain circumstances because this reduces stress (ROS production and frequency of apoptosis) and may yield more potent MSCs. Although the expansion of MSCs has been achieved in the pH range 7.5–8.3 [54], it is unclear how significant variations in pH influence MSC metabolism and whether this affects the secretome. The optimal temperature and pH must be evaluated for each MSC product. Other CPPs include the parameters grouped under the term hydrodynamics, referring to the potential impact of aeration and agitation. Aeration is required to supply oxygen to the MSCs, but as well as affecting the oxygen saturation, it also generates forces that cause physical stress. In T-flasks, aeration is achieved by the diffusion of oxygen through the surface of the medium, whereas bioreactors must be actively aerated by, e.g., bubbling gas into the liquid. The bursting gas bubbles (cavitation) generate strong forces that can damage cells, although the stress can be reduced by controlling the bubble size [55]. Agitation in bioreactors is generally achieved with impellers, which help to disperse gas (and therefore contribute to aeration) but also maintain a homogenous suspension of cells and nutrients. The creation of a homogenous environment is advantageous because it avoids gradients of pH, nutrients, or waste products, whose effect on MSCs is unpredictable. Homogenization can also be achieved using pumps or is facilitated by air bubbles. Agitation always generates shear forces, so it is necessary to balance the homogeneity of the cultivation system and the impact of the hydrodynamic forces on the MSCs. Although excessive shear stress is detrimental, hydrodynamic forces can also stimulate MSC growth and increase potency [43]. For these reasons, the mode and rate of aeration and the method and intensity of agitation are CPPs that must be carefully optimized for each process.

MSCs are anchorage-dependent cells, so the properties of the growth surface also have a significant impact on the process and must be investigated and selected

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

*2.1.3 Conditions in the culture vessel*

this mimics their natural niche [53].

*2.1.4 Growth surface, cell harvest, and storage*

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

serum-free and chemically-defined media should be avoided-it is still better to use these media for MSC expansion in order to meet GMP requirements-but further investigations are required to optimize the media composition. The development of serum-free media is mainly driven by companies, which tend not to disclose the precise composition, making it difficult for other researchers to build on the results. In serum-free and defined media, supplemental growth factors such as FGF2 and PDGF are needed to stimulate MSC proliferation, but they also influence MSC potency [18]. Accordingly, chemically-defined media would be preferable for the *in vitro* expansion of MSCs, but growth factor concentrations are important CPPs that affect MSC identity and potency and must be carefully controlled.

### *2.1.3 Conditions in the culture vessel*

*New Advances on Fermentation Processes*

compromises their functionality and potency.

*2.1.2 Culture medium*

MSC surface marker profile but lost functionality [4]. Aging MSCs are more likely to activate a senescence-associated secretory phenotype and produce proinflammatory cytokines such as IL-1, IL-6, and IL-8, which inhibit the regenerative process. The duration of *in vitro* expansion must be considered not only because of senescence, but also due to the phenomenon of clonal impoverishment. MSCs are polyclonal, but prolonged expansion favors the growth of specific cell types or clones. Depending on the expansion time and expansion factor, the cell mixtures may completely differ in phenotype and also in potency. Therefore, although a high expansion factor in a short process time is desirable to achieve high product yields, *in vitro* expansion should never change the properties of MSCs to the extent that it

Several basal media have been shown to influence MSC expansion and potency, including Dulbecco's modified Eagle's medium (DMEM), Iscove's modified Dulbecco's medium (IMDM), and MEM alpha (αMEM) [37]. One of the key components of these media is glucose, which is the main carbon source for MSCs. Glucose may be provided at physiological concentrations (1 g/L) or higher (up to 4.5 g/L), the latter variously described as having a negative effect on MSC proliferation and growth factor secretion [38] or no effect at all [39]. Glutamine as a second carbon source is present at concentrations of 2–4 mM and appears essential for MSC growth [40], but its impact on MSC properties is complex, with contradictory results [41–43]. Glutamine is unstable at 37°C and spontaneously degrades to form ammonia. GlutaMAX (dipeptide Ala-Gln) is recommended instead of glutamine to promote MSC expansion [44]. Lactate and ammonia are the most abundant waste products formed by MSCs, and both therefore have the potential to inhibit growth. It therefore follows that glucose, glutamine, lactate, and ammonia levels should be considered as CPPs for the production of MSCs. Several other amino acids may also be relevant, given that the amino acid metabolism of MSCs differs from that of

commercial cell lines such as Chinese hamster ovary (CHO) cells [42].

Basal media formulations must be supplemented to achieve MSC expansion. The most important supplement is fetal calf serum (FCS), which is added to a final concentration of 5–20%. FCS strongly influences MSC growth and phenotype, but the specific effectors are unknown because the composition of FCS is variable and lot-dependent [45]. The use of FCS for the manufacture of clinical MSC products is discouraged nowadays, in line with the drive to eliminate all raw materials of animal origin. The complex, uncertain, and variable composition of FCS also makes it difficult to validate for GMP-compliant processes. Finally, the manufacturing process must accommodate steps to eliminate FCS from the final product to avoid potential immunogenicity and allergenicity [46]. FCS can be replaced with human serum and its derivatives, such as human platelet lysate, which promotes MSC growth [47]. However, the same lot-dependent quality issues described above for FCS also apply to human serum [48]. The most acceptable alternative is serum-free or preferably chemically-defined medium, the latter not only serum-free but also lacking any hydrolysates or supplements of unknown composition. MSCs grow well in several commercial serum-free media, including BD Mosaic MSC Serum-free (BD Biosciences), RoosterNourish (Rooster Bio), Mesencult-XF (Stemcell Technologies), StemPro MSC SFM Xeno-Free (Invitrogen), TheraPEAK MSCGM-CD (Lonza), and PPRF-msc6, STK1 and STK2 (Abion) [49]. Growth in chemically-defined medium has also been demonstrated [50]. However, although MSCs showed excellent growth in these serum-free media, they reached senescence earlier, and there were changes in morphology, surface marker profiles, and potency [51]. This does not mean that

**210**

MSCs are aerobic cells and any culture vessel must therefore ensure an adequate supply of oxygen. However, the oxygen saturation in standard T-flasks (21% O2) is far removed from nature (5–7% O2) [34]. MSCs therefore tend to be oversaturated with oxygen, which can increase the concentration of damaging reactive oxygen species (ROS). Several studies have confirmed that hypoxia enhances MSC proliferation, stabilizes their cell fate, and prevents apoptosis by reducing the levels of caspase-3 [52]. However, rather than imposing hypoxia by preconditioning the cells, it may be better to impose hypoxia during the entire expansion phase, because this mimics their natural niche [53].

In addition to oxygen saturation, temperature and pH are CPPs in every process and can be monitored and controlled very easily. Typically, *in vitro* expansion is carried out at 37°C and neutral pH (7.2–7.4). Expansion at lower temperatures can be advantageous under certain circumstances because this reduces stress (ROS production and frequency of apoptosis) and may yield more potent MSCs. Although the expansion of MSCs has been achieved in the pH range 7.5–8.3 [54], it is unclear how significant variations in pH influence MSC metabolism and whether this affects the secretome. The optimal temperature and pH must be evaluated for each MSC product.

Other CPPs include the parameters grouped under the term hydrodynamics, referring to the potential impact of aeration and agitation. Aeration is required to supply oxygen to the MSCs, but as well as affecting the oxygen saturation, it also generates forces that cause physical stress. In T-flasks, aeration is achieved by the diffusion of oxygen through the surface of the medium, whereas bioreactors must be actively aerated by, e.g., bubbling gas into the liquid. The bursting gas bubbles (cavitation) generate strong forces that can damage cells, although the stress can be reduced by controlling the bubble size [55]. Agitation in bioreactors is generally achieved with impellers, which help to disperse gas (and therefore contribute to aeration) but also maintain a homogenous suspension of cells and nutrients. The creation of a homogenous environment is advantageous because it avoids gradients of pH, nutrients, or waste products, whose effect on MSCs is unpredictable. Homogenization can also be achieved using pumps or is facilitated by air bubbles. Agitation always generates shear forces, so it is necessary to balance the homogeneity of the cultivation system and the impact of the hydrodynamic forces on the MSCs. Although excessive shear stress is detrimental, hydrodynamic forces can also stimulate MSC growth and increase potency [43]. For these reasons, the mode and rate of aeration and the method and intensity of agitation are CPPs that must be carefully optimized for each process.

### *2.1.4 Growth surface, cell harvest, and storage*

MSCs are anchorage-dependent cells, so the properties of the growth surface also have a significant impact on the process and must be investigated and selected carefully. However, unlike the parameters discussed above, the growth surface does not have to be monitored or controlled during MSC production, so it falls outside the technical definition of a CPP. The expression of certain surface markers by MSCs reflects the stiffness of the growth surface, so it is clear that the surface affects the phenotype [56]. As stated above, the ability to adhere to plastic surfaces is one of the minimal criteria that define MSCs, and tissue-culture plastic is therefore the most commonly-used growth surface. Although all commercial tissue-culture plasticware has a polypropylene base, the surface is often treated differently, and this changes the behavior and properties of the adherent MSCs [37]. MSCs further grow on other surface materials, e.g., glass [57] or dextran [58]. When MSCs are cultivated in serum-free medium, cell growth often requires that the surface is coated with further adhesion-promoting factors, such as fibronectin, vitronectin, or the peptide RGD.

Given that MSCs are anchorage-dependent cells, the harvesting of cells at the end of the *in vitro* expansion step requires an efficient cell detachment method that ideally does not affect functionality or potency. In the laboratory, MSCs can be detached from T-flasks by adding trypsin or other proteases, but this nonspecific proteolysis can affect cell viability and eliminate some MSC surface markers [59]. Proteolytic cleavage is incompatible with the larger-scale processes in bioreactors because longer incubation times are required for the enzymes to work, and even then, the efficiency of cell recovery is low [60]. More importantly, any negative effects of the enzymatic treatment on cell viability and potency are amplified by the longer exposure time, which can inhibit MSC differentiation [61]. These issues can be addressed by adjusting the hydrodynamic conditions to favor cell detachment after limited enzymatic treatment [62]. Alternatively, enzymatic treatment can be circumvented completely by promoting cell detachment using dissolvable growth surfaces [63] or thermosensitive surfaces that release cells following a temperature shift [64, 65]. However, unlike enzymatic treatments, these novel surfaces do not break direct cell-cell bonds and may be unsuitable if single cell is required. The formation of aggregates can be minimized by carefully monitoring the cell density and selecting a harvest point that favors the recovery of single cell, but this must be balanced against the efficiency of expansion given the need to harvest at lower cell densities. The so-called harvest problem, balancing the efficient release of cells against the recovery of cells with desirable properties, has yet to be solved. This highlights the importance of well-defined CPPs at the harvesting stage.

All the approved allogenic MSC products described earlier are cryopreserved, allowing them to be offered as off-the-shelf products that can be stored until quality control and batch release are completed. The use of cryopreserved allogeneic MSCs is the only feasible therapeutic strategy for acute tissue injury syndromes such as stroke, sepsis, or myocardial infarction, because the patient is likely to die before sufficient quantities of autologous MSCs could be prepared. However, cryopreservation and thawing have a massive impact on the potency of MSCs [66]. Indeed, even without optimization, fresh MSCs are much more potent than frozen ones [35]. A rule of thumb is to freeze the cells slowly (e.g., 1°C/min) but to thaw them quickly (e.g., direct transfer from storage to a 37°C water bath). The impact of multiple freeze-thaw cycles must be evaluated carefully [67]. The composition of the freezing medium is also important because it often contains dimethyl sulfoxide (DMSO) and FCS as cryoprotectants, the first being cytotoxic and the second undesirable for the reasons already discussed above. Nontoxic alternatives lacking DMSO and FCS have been tested and may be more compatible with MSCs intended for clinical applications [68–70].

In summary, the expansion of MSCs in bioreactors involves multiple CPPs including (i) the source of the initial MSCs before expansion, (ii) the impact of cell

**213**

*Bioprocess Development for Human Mesenchymal Stem Cell Therapy Products*

density and age, (iii) the effects of the culture medium, (iv) the properties of the bioreactor and aeration/agitation systems, and (iv) the method used for cell harvest and storage. The impact of these CPPs on the quality of MSCs can only be determined by designing robust assays for (i) *in vitro* senescence and genetic stability and (ii) relevant disease-specific mechanism of action and potency. It is clear that there is no one-size-fits-all MSC expansion process and that unique processes must be developed to match different therapeutic objectives. These processes may feature distinct CQAs, meaning that the CPPs may also differ on a case-by-case basis.

For the 989 interventional clinical trials involving MSCs reported thus far (www.clinicaltrials.gov, search term: mesenchymal stem cell OR mesenchymal stromal cell, 2019/09/27), the MSCs were expanded *in vitro* and in most cases were

The manufacture of protein therapeutics is almost always carried out in bioreactors because they are scalable, controllable via integrated process analytical technology, and most process steps can be automated. This is not the case for MSC products, and a survey of GMP manufacturing at US academic centers has revealed major differences in the various process steps (cell isolation, expansion, and characterization). In the context of cell expansion, 80% of the centers surveyed above used T-flasks or cell factories, whereas only 20% mainly used bioreactors. A broad range of seeding densities was used for cultivation (50–2500 cells/cm2

and the cultivation time ranged from 1 to 28+ days. The cultivation medium was supplemented with FCS (lot-selected or not) or donor-pooled human platelet lysate (in-house product or commercial product) [71]. All of the centers expanded MSCs under GMP conditions, but with huge variations in the protocol. The production of MSCs in T-flasks is adequate for a small number of patients (30 T-flasks each with

[72]). But for larger clinical trials with >100 patients, the resources required for cell culture would become unsupportable (assuming the conditions stated above, a trial with 140 patients would require 4200 T-flasks filling 32 standard 160-L incubators and 9 full-time personnel to handle the cells). Expansion in T-flasks might also be sufficient for autologous cell therapy, given that only a single patient is involved and it would not be necessary to produce more than 10 doses. However, even for small-scale manufacturing, an automated bioreactor system would offer several advantages over manual cultivation. Given that the entire manufacturing process must be aseptic, closed bioreactors provide much better insurance against contamination than an open culture system based on T-flaks. For allogenic MSC products, where up to 1 million doses are produced per batch, bioreactors are the only feasible

When an MSC product advances from research to commercial manufacturing, the *in vitro* expansion process must also change. Research is driven by the freedom to test different conditions, but the tests are typically conducted on a small scale. In contrast, commercial products must be manufactured using a standardized process to ensure robustness, and the scale is generally larger. Bioreactors play a key role in large-scale manufacturing because they offer greater traceability due to the control and monitoring of CPPs. The expansion of MSCs in bioreactors allows the

is dosed with 416 million cells and the harvesting efficiency is 8 × 104

would be required per patient, assuming each patient

cells per kilogram, never

)

cells/cm2

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

**2.2 MSC manufacturing for clinical trials**

exceeding 12 × 106

a growth surface of 175 cm2

manufacturing option (**Figure 2**).

**2.3 The expansion of MSCs in bioreactors**

transfused intravenously at typical doses of 1–2 × 106

cells/kg [3].

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

density and age, (iii) the effects of the culture medium, (iv) the properties of the bioreactor and aeration/agitation systems, and (iv) the method used for cell harvest and storage. The impact of these CPPs on the quality of MSCs can only be determined by designing robust assays for (i) *in vitro* senescence and genetic stability and (ii) relevant disease-specific mechanism of action and potency. It is clear that there is no one-size-fits-all MSC expansion process and that unique processes must be developed to match different therapeutic objectives. These processes may feature distinct CQAs, meaning that the CPPs may also differ on a case-by-case basis.

#### **2.2 MSC manufacturing for clinical trials**

*New Advances on Fermentation Processes*

or the peptide RGD.

carefully. However, unlike the parameters discussed above, the growth surface does not have to be monitored or controlled during MSC production, so it falls outside the technical definition of a CPP. The expression of certain surface markers by MSCs reflects the stiffness of the growth surface, so it is clear that the surface affects the phenotype [56]. As stated above, the ability to adhere to plastic surfaces is one of the minimal criteria that define MSCs, and tissue-culture plastic is therefore the most commonly-used growth surface. Although all commercial tissue-culture plasticware has a polypropylene base, the surface is often treated differently, and this changes the behavior and properties of the adherent MSCs [37]. MSCs further grow on other surface materials, e.g., glass [57] or dextran [58]. When MSCs are cultivated in serum-free medium, cell growth often requires that the surface is coated with further adhesion-promoting factors, such as fibronectin, vitronectin,

Given that MSCs are anchorage-dependent cells, the harvesting of cells at the end of the *in vitro* expansion step requires an efficient cell detachment method that ideally does not affect functionality or potency. In the laboratory, MSCs can be detached from T-flasks by adding trypsin or other proteases, but this nonspecific proteolysis can affect cell viability and eliminate some MSC surface markers [59]. Proteolytic cleavage is incompatible with the larger-scale processes in bioreactors because longer incubation times are required for the enzymes to work, and even then, the efficiency of cell recovery is low [60]. More importantly, any negative effects of the enzymatic treatment on cell viability and potency are amplified by the longer exposure time, which can inhibit MSC differentiation [61]. These issues can be addressed by adjusting the hydrodynamic conditions to favor cell detachment after limited enzymatic treatment [62]. Alternatively, enzymatic treatment can be circumvented completely by promoting cell detachment using dissolvable growth surfaces [63] or thermosensitive surfaces that release cells following a temperature shift [64, 65]. However, unlike enzymatic treatments, these novel surfaces do not break direct cell-cell bonds and may be unsuitable if single cell is required. The formation of aggregates can be minimized by carefully monitoring the cell density and selecting a harvest point that favors the recovery of single cell, but this must be balanced against the efficiency of expansion given the need to harvest at lower cell densities. The so-called harvest problem, balancing the efficient release of cells against the recovery of cells with desirable properties, has yet to be solved. This

highlights the importance of well-defined CPPs at the harvesting stage.

All the approved allogenic MSC products described earlier are cryopreserved, allowing them to be offered as off-the-shelf products that can be stored until quality control and batch release are completed. The use of cryopreserved allogeneic MSCs is the only feasible therapeutic strategy for acute tissue injury syndromes such as stroke, sepsis, or myocardial infarction, because the patient is likely to die before sufficient quantities of autologous MSCs could be prepared. However, cryopreservation and thawing have a massive impact on the potency of MSCs [66]. Indeed, even without optimization, fresh MSCs are much more potent than frozen ones [35]. A rule of thumb is to freeze the cells slowly (e.g., 1°C/min) but to thaw them quickly (e.g., direct transfer from storage to a 37°C water bath). The impact of multiple freeze-thaw cycles must be evaluated carefully [67]. The composition of the freezing medium is also important because it often contains dimethyl sulfoxide (DMSO) and FCS as cryoprotectants, the first being cytotoxic and the second undesirable for the reasons already discussed above. Nontoxic alternatives lacking DMSO and FCS have been tested and may be more compatible with MSCs intended

In summary, the expansion of MSCs in bioreactors involves multiple CPPs including (i) the source of the initial MSCs before expansion, (ii) the impact of cell

**212**

for clinical applications [68–70].

For the 989 interventional clinical trials involving MSCs reported thus far (www.clinicaltrials.gov, search term: mesenchymal stem cell OR mesenchymal stromal cell, 2019/09/27), the MSCs were expanded *in vitro* and in most cases were transfused intravenously at typical doses of 1–2 × 106 cells per kilogram, never exceeding 12 × 106 cells/kg [3].

The manufacture of protein therapeutics is almost always carried out in bioreactors because they are scalable, controllable via integrated process analytical technology, and most process steps can be automated. This is not the case for MSC products, and a survey of GMP manufacturing at US academic centers has revealed major differences in the various process steps (cell isolation, expansion, and characterization). In the context of cell expansion, 80% of the centers surveyed above used T-flasks or cell factories, whereas only 20% mainly used bioreactors. A broad range of seeding densities was used for cultivation (50–2500 cells/cm2 ) and the cultivation time ranged from 1 to 28+ days. The cultivation medium was supplemented with FCS (lot-selected or not) or donor-pooled human platelet lysate (in-house product or commercial product) [71]. All of the centers expanded MSCs under GMP conditions, but with huge variations in the protocol. The production of MSCs in T-flasks is adequate for a small number of patients (30 T-flasks each with a growth surface of 175 cm2 would be required per patient, assuming each patient is dosed with 416 million cells and the harvesting efficiency is 8 × 104 cells/cm2 [72]). But for larger clinical trials with >100 patients, the resources required for cell culture would become unsupportable (assuming the conditions stated above, a trial with 140 patients would require 4200 T-flasks filling 32 standard 160-L incubators and 9 full-time personnel to handle the cells). Expansion in T-flasks might also be sufficient for autologous cell therapy, given that only a single patient is involved and it would not be necessary to produce more than 10 doses. However, even for small-scale manufacturing, an automated bioreactor system would offer several advantages over manual cultivation. Given that the entire manufacturing process must be aseptic, closed bioreactors provide much better insurance against contamination than an open culture system based on T-flaks. For allogenic MSC products, where up to 1 million doses are produced per batch, bioreactors are the only feasible manufacturing option (**Figure 2**).

#### **2.3 The expansion of MSCs in bioreactors**

When an MSC product advances from research to commercial manufacturing, the *in vitro* expansion process must also change. Research is driven by the freedom to test different conditions, but the tests are typically conducted on a small scale. In contrast, commercial products must be manufactured using a standardized process to ensure robustness, and the scale is generally larger. Bioreactors play a key role in large-scale manufacturing because they offer greater traceability due to the control and monitoring of CPPs. The expansion of MSCs in bioreactors allows the

#### **Figure 2.**

*Manufacturing of autologous and allogenic MSC products. Autologous MSC products are isolated from the patient's own tissue, whereas for allogenic MSC products a healthy donor from the same specie donates cells. In the isolation and expansion, there are few differences between the two types of MSC products. Most common sources are bone marrow, adipose tissue, and umbilical cord (blood), all three giving different amount of MSCs. The expansion for both MSC product types differs in scale. Storage is only needed for allogenic MSC products. If we expect that 0.4 × 106 MSC are isolated per donation and one dose to treat a single patient is about 120 × 106 MSCs, an expansion factor of at least 300-fold is needed. If more doses should be produced from one isolate, e.g., because the patient needs several treatments or in case of allogenic MSC products, the expansion factor dramatically increases.*

precise control of the microenvironment, which has a profound influence on cell potency and therapeutic efficacy [73, 74]. For example, the dynamic cultivation of human MSCs in a bioreactor has been shown to induce the secretion of several beneficial growth factors, including BDNF, NGF, VEGF, and IGF-1 [75]. The use of bioreactors also means that the *in vitro* MSC expansion and harvesting steps can be automated, which improves the efficiency of both steps and reduces the amount of hands-on work. The elimination of operator-related errors and contamination risks makes the process more stable, avoiding batch-to-batch variability. Nutrient gradients and abrupt fluctuations in pH caused by manual medium exchange are also avoided. This enables the production of MSCs with consistent identity and potency (CQAs). Many different bioreactor types have been used for the *in vitro* expansion of MSCs, including fixed bed, fluidized bed, and stirred tank reactors, as well as newer innovations such as wave reactors, wall-rotating systems, and vertical wheel reactors [76]. However, most studies have involved only two types of reactor: stirred tank or fixed bed, and these are discussed in more detail below.

Stirred tank reactors are the most widely-used devices for large-scale MSC expansion. They are often used with microcarriers, which are small beads that increase the surface area available for cell attachment, although MSCs can also be grown in bioreactors as aggregates or spheroids. The expansion of MSCs growing on microcarriers is typically a batch-mode manufacturing process because the cells are harvested at a predetermined density. However, fed-batch processes involve a smaller inoculum (100 cells/cm2 , equivalent to five cells per microcarrier) and can thus achieve better economy and a higher expansion factor [77, 78]. There should be minimal (if any) agitation at the beginning of the expansion phase to allow for cell attachment to the microcarriers (if used) or otherwise for the formation of aggregates. However, agitation is required following attachment in order to homogenize the suspension and avoid the formation of large clumps. As discussed above, agitation is an important CPP and the parameters must be optimized based on the unique combination of

**215**

homogeneous.

*Bioprocess Development for Human Mesenchymal Stem Cell Therapy Products*

parameters must be optimized at different manufacturing scales [79].

N/cm2

aspect of bioreactor design that remains to be addressed [60, 86].

technology as a prerequisite for GMP manufacturing.

harvest can be monitored and controlled effectively.

lose functionality and therefore therapeutic potency.

system properties (e.g., impeller type/speed and microcarrier size/amount) to keep microcarriers or aggregates in suspension without causing shear damage, and these

Fixed bed reactors are also widely used for MSC expansion, and in this case, the cells are grown either on macrocarriers or as capsules (500 μm diameter), both of which form a stable bed at the reactor base. The production of homogeneous conditions in the bed can be frustrated by the development of channels and gradients in the bed, particularly in large-scale systems [80]. The shear forces in fixed bed

peaks near the impeller; the shear forces also remain constant at all scales [81]. The *in vitro* expansion of MSCs has been reported in several types of fixed bed reactors [82–85]. One of the major drawbacks of fixed bed and other reactor types compared to stirred tank reactors is the challenge of efficient harvesting. For example, in the reports above, the recovery of viable cells is rarely better than 70%, so this is a key

The earlier sections highlighted several challenges that must be overcome to develop robust processes for the expansion of MSCs in bioreactors, which are summarized briefly below. Furthermore, our current understanding of the CPPs affecting MSC production is rudimentary at best, and more work is required to determine the impact of hydrodynamic factors on the CQAs. Precise online monitoring tools are needed to control CPPs effectively and to measure their influence on cell viability, potency, and secretory profiles. An increase in process understanding will facilitate process modeling, to fulfill the requirements of process analytic

The major challenge for MSC therapy is the development of an *in vitro* expansion process that mimics the natural MSC niche, but nevertheless allows scaled up production for clinical trials without compromising CQAs such as cell functionality and potency. The development of a standardized process is frustrated by the heterogeneity of MSCs, which are isolated from different donors and different tissues, resulting in variable phenotypes and functions. The heterogeneity of primary MSCs can be avoided by working instead with induced pluripotent stem cells (iPSCs), which can differentiate into MSC-like cells with potent therapeutic properties [87]. However, well-controlled *in vitro* expansion processes in bioreactors can also help to reduce the batch-to-batch variation often encountered with MSCs, because parameters such as the seeding density, shear stress during cultivation, and cell density at

Polyclonal MSCs often show the most potent therapeutic effects, but clonal

Ultimately, even bioreactor-based processes for MSC expansion are constrained by the inbuilt replication limit of MSCs, which leads to senescence after a certain number of generations. Stem cells by definition have an unlimited capacity for selfrenewal, but this property is lost *in vitro*. The expansion stress that leads to replicative senescence generates MSCs that maintain their marker profiles but nevertheless

impoverishment occurs during lengthy expansion phases and this must be avoided if potency is compromised. However, even monoclonal MSCs become heterogeneous over time, generating subpopulations with different morphologies and surface marker profiles. The therapeutic outcome can only be predicted if the MSC pool does not change during expansion, and the well-controlled conditions in bioreactors can therefore help to ensure that the cell products remain

) and consistent throughout the reactor with no

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

reactors are low (~0.5 × 10<sup>−</sup><sup>5</sup>

**2.4 Remaining challenges**

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

system properties (e.g., impeller type/speed and microcarrier size/amount) to keep microcarriers or aggregates in suspension without causing shear damage, and these parameters must be optimized at different manufacturing scales [79].

Fixed bed reactors are also widely used for MSC expansion, and in this case, the cells are grown either on macrocarriers or as capsules (500 μm diameter), both of which form a stable bed at the reactor base. The production of homogeneous conditions in the bed can be frustrated by the development of channels and gradients in the bed, particularly in large-scale systems [80]. The shear forces in fixed bed reactors are low (~0.5 × 10<sup>−</sup><sup>5</sup> N/cm2 ) and consistent throughout the reactor with no peaks near the impeller; the shear forces also remain constant at all scales [81]. The *in vitro* expansion of MSCs has been reported in several types of fixed bed reactors [82–85]. One of the major drawbacks of fixed bed and other reactor types compared to stirred tank reactors is the challenge of efficient harvesting. For example, in the reports above, the recovery of viable cells is rarely better than 70%, so this is a key aspect of bioreactor design that remains to be addressed [60, 86].

#### **2.4 Remaining challenges**

*New Advances on Fermentation Processes*

precise control of the microenvironment, which has a profound influence on cell potency and therapeutic efficacy [73, 74]. For example, the dynamic cultivation of human MSCs in a bioreactor has been shown to induce the secretion of several beneficial growth factors, including BDNF, NGF, VEGF, and IGF-1 [75]. The use of bioreactors also means that the *in vitro* MSC expansion and harvesting steps can be automated, which improves the efficiency of both steps and reduces the amount of hands-on work. The elimination of operator-related errors and contamination risks makes the process more stable, avoiding batch-to-batch variability. Nutrient gradients and abrupt fluctuations in pH caused by manual medium exchange are also avoided. This enables the production of MSCs with consistent identity and potency (CQAs). Many different bioreactor types have been used for the *in vitro* expansion of MSCs, including fixed bed, fluidized bed, and stirred tank reactors, as well as newer innovations such as wave reactors, wall-rotating systems, and vertical wheel reactors [76]. However, most studies have involved only two types of reactor:

*Manufacturing of autologous and allogenic MSC products. Autologous MSC products are isolated from the patient's own tissue, whereas for allogenic MSC products a healthy donor from the same specie donates cells. In the isolation and expansion, there are few differences between the two types of MSC products. Most common sources are bone marrow, adipose tissue, and umbilical cord (blood), all three giving different amount of MSCs. The expansion for both MSC product types differs in scale. Storage is only needed for allogenic MSC products.* 

 *MSC are isolated per donation and one dose to treat a single patient is about* 

 *MSCs, an expansion factor of at least 300-fold is needed. If more doses should be produced from one isolate, e.g., because the patient needs several treatments or in case of allogenic MSC products, the expansion* 

stirred tank or fixed bed, and these are discussed in more detail below.

Stirred tank reactors are the most widely-used devices for large-scale MSC expansion. They are often used with microcarriers, which are small beads that increase the surface area available for cell attachment, although MSCs can also be grown in bioreactors as aggregates or spheroids. The expansion of MSCs growing on microcarriers is typically a batch-mode manufacturing process because the cells are harvested at a predetermined density. However, fed-batch processes involve a smaller inoculum

economy and a higher expansion factor [77, 78]. There should be minimal (if any) agitation at the beginning of the expansion phase to allow for cell attachment to the microcarriers (if used) or otherwise for the formation of aggregates. However, agitation is required following attachment in order to homogenize the suspension and avoid the formation of large clumps. As discussed above, agitation is an important CPP and the parameters must be optimized based on the unique combination of

, equivalent to five cells per microcarrier) and can thus achieve better

**214**

(100 cells/cm2

**Figure 2.**

*120 × 106*

*If we expect that 0.4 × 106*

*factor dramatically increases.*

The earlier sections highlighted several challenges that must be overcome to develop robust processes for the expansion of MSCs in bioreactors, which are summarized briefly below. Furthermore, our current understanding of the CPPs affecting MSC production is rudimentary at best, and more work is required to determine the impact of hydrodynamic factors on the CQAs. Precise online monitoring tools are needed to control CPPs effectively and to measure their influence on cell viability, potency, and secretory profiles. An increase in process understanding will facilitate process modeling, to fulfill the requirements of process analytic technology as a prerequisite for GMP manufacturing.

The major challenge for MSC therapy is the development of an *in vitro* expansion process that mimics the natural MSC niche, but nevertheless allows scaled up production for clinical trials without compromising CQAs such as cell functionality and potency. The development of a standardized process is frustrated by the heterogeneity of MSCs, which are isolated from different donors and different tissues, resulting in variable phenotypes and functions. The heterogeneity of primary MSCs can be avoided by working instead with induced pluripotent stem cells (iPSCs), which can differentiate into MSC-like cells with potent therapeutic properties [87]. However, well-controlled *in vitro* expansion processes in bioreactors can also help to reduce the batch-to-batch variation often encountered with MSCs, because parameters such as the seeding density, shear stress during cultivation, and cell density at harvest can be monitored and controlled effectively.

Polyclonal MSCs often show the most potent therapeutic effects, but clonal impoverishment occurs during lengthy expansion phases and this must be avoided if potency is compromised. However, even monoclonal MSCs become heterogeneous over time, generating subpopulations with different morphologies and surface marker profiles. The therapeutic outcome can only be predicted if the MSC pool does not change during expansion, and the well-controlled conditions in bioreactors can therefore help to ensure that the cell products remain homogeneous.

Ultimately, even bioreactor-based processes for MSC expansion are constrained by the inbuilt replication limit of MSCs, which leads to senescence after a certain number of generations. Stem cells by definition have an unlimited capacity for selfrenewal, but this property is lost *in vitro*. The expansion stress that leads to replicative senescence generates MSCs that maintain their marker profiles but nevertheless lose functionality and therefore therapeutic potency.

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 potency of MSCs [88].
