Bioreactor Engineering Fundamentals for Stem Cell Manufacturing

CHAPTER 3

Bioreactor Engineering Fundamentals for Stem Cell Manufacturing A.W. Nienow1, 2, 3, K. Coopman1, T.R.J. Heathman1, Q.A. Rafiq1, 3, C.J. Hewitt1, 3 1

Loughborough University, Leicestershire, United Kingdom; 2University of Birmingham, Birmingham, United Kingdom; Aston University, Birmingham, United Kingdom

3

3.1 INTRODUCTION Cell-based therapies have the potential to address currently unmet patient care and thus effective manufacture of these products is essential. There are, however, many challenges that must be overcome before this can become a reality and a better definition of the manufacturing requirements for cell-based products must be obtained. A review [1] has indicated a total of w1350 active cell-based therapy clinical trials based on cell type, target indication, and phase. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) are able to differentiate into any mature cell type of the body, and thus offer an array of cell-replacement therapy opportunities. However, though recently the successful culture of iPSCs as aggregates in spinner flasks using completely defined and xeno-free media has been reported [2], in general, their clinical usage has been undermined by the difficulty associated with expansion while maintaining pluripotency, their innate tumorigenicity (ie, ability to form teratomas upon implantation), lack of efficient culture systems to control their differentiation, and for hESCs, ethical constraints due to the destruction of the embryo [3,4]. As a result, the vast majority of these trials have been conducted using human mesenchymal stem cells (hMSCs) (Fig. 3.1), which are free from such constraints. Until recently, the approach to growing stem cells has been on a planer surface in Tflasks. For autologous therapies (ones where the donor and the recipient are the same individual), such an approach may be feasible and increasing cell numbers may be met by using multiple flasks (scale-out) with T-flasks in cell factories or stacks, preferably fully automated [5]. However, for allogeneic cell therapy (one where a single donor provides the cells for a multiplicity of patients), it has been estimated that 1016 cells would be needed for the treatment of 250,000 patients for cardiomyocyte replacement and that this number would require approximately 286 million T-175 tissue culture flasks [6]. Thus, it is clear that another approach is required [7]. Essentially, this approach is one of scale-up where the surface area per unit volume of bioreactor on which the cells grow is greatly enhanced by orders of magnitude by the addition of microcarriers (small particles of the order of 100e200 mm in suspension, of Stem Cell Manufacturing ISBN 978-0-444-63265-4, http://dx.doi.org/10.1016/B978-0-444-63265-4.00003-0

© 2016 Elsevier B.V. All rights reserved.

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44 Stem Cell Manufacturing

1. Ease of isolation and potential for ex vivo expansion 2. Immunomodulatory properties and universal transplantation 3. Secretion of trophic factors to initiate tissue and organ repair NEUROLOGY

MESENCHYMAL STEM CELLS

CARDIOLOGY Phase 1 Phase 2 Phase 3 Phase 4

MENTAL DISABILITY HUNTINGTON'S DISEASE ATAXIA PARKINSON'S DISEASE UNSPECIFIED MOTOR NEURON DISEASE AUTISM CEREBAL PALSY MULTIPLE SCLEROSIS STROKE

INFECTION RENAL HEMATOLOGY OPTHALMOLOGY DERMAL & WOUND PULMONARY OTHER RECONSTRUCTION

CONGENITAL DISEASE ANGINA PERIPHERAL VASCULAR DISEASE CONGESTIVE HEART FAILURE ACUTE CORONARY SYNDROME

0

3

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5 10 15 20 25 30 35

SPINAL INJURY ONCOLOGY

RHEUMATOLOGY

MUSKCULOSKELETAL DIABETIS HEPATIC VASCULAR RHEUMATOLOGY IMMUNOLOGY CARDIOLOGY NEUROLOGY 0

10

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30

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IMMUNOLOGY

UNSPECIFIED SJOGREN'S SYNDROME ANKYLOSING SPONDYLITIS ULCERATIVE COLITIS VASCULITIS SCLEROSIS RHEUMATOID ARTHRITIS LUPUS ERYTHEMATOSUS OSTEOARTHRITIS

IMMUNODEFICIENCY TRANSPLANTATION

CROHN'S DISEASE GRAFT VS. HOST DISEASE 0

3

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Figure 3.1 The advantages of using hMSCs for stem cell therapies leading to them be the most common in clinical trials. (Modified from T.R.J. Heathman, A.W. Nienow, M.J. Mccall, K. Coopman, B. Kara, C.J. Hewitt, The translation of cell-based therapies: clinical landscape and manufacturing challenges, Regen. Med. 10 (2015) 49e64.)

Bioreactor Engineering Fundamentals for Stem Cell Manufacturing

which there are a huge variety as discussed later) suspended in bioreactors similar to those in which animal cells are grown in free suspension [8]. Though many types of bioreactors have been proposed, for many years, the stirred bioreactor has been the most common for the largest commercial free-suspension culture (w25 m3) [9] and it is rapidly becoming so even at a small scale for clone selection using the 15 mL ambr15Ô (advanced microscale bioreactor) [10]. Economic analysis of manufacturing technology for adherent cell-based therapies has also shown that these suspension bioreactors are necessary in order to achieve cost effectiveness, a key consideration for any therapeutic [11]. Though there has been a major increase in the utilization of single-use bioreactors (SUBs) for such purposes, again the most common type involve the use of stirrers to provide the energy to meet the many different processing aspects required of a bioreactor [12]. Thus, this chapter will concentrate on the use of stirred bioreactors containing microcarriers for growing hMSCs. Indeed, such a configuration is also the one most reported in the literature [3]. The underlying issues requiring consideration for a successful cultivation of all cell types in free suspension are very similar, but for stem cells, there is one major difference. In growing cells for regenerative medicine (and for diagnostics and drug development purposes), the cells themselves form the basis of the product. Thus, when growing cells in free suspension to produce a therapeutic protein, cultivation can only be considered successful, no matter how high the viable cell density produced, if the protein has the correct quality attributes, most importantly, the glycosylation pattern [13,14]. With stem cells, it is the cells that must be shown to maintain their critical quality attributes at all the processing stages up to and including cryopreservation, thaw, and delivery to the patient [15]. The advantages of stirred bioreactors compared to T-flask culture are the potential ease of scale-up rather than scale-out, so that the unit cost of producing cells goes down with increasing scale; their inherent flexibility with respect to agitation intensity, stirring objectives, and aeration techniques (headspace or bubble sparging); and their process monitoring and control capability (pH, dissolved oxygen (dO2), temperature, and potentially, though less common, pCO2). They can also be used with various feeding strategies. All these aspects explain why bioreactor culture is so widespread in the biopharmaceutical industry. The ability to undertake microcarrier culture in stirred bioreactors is a further measure of their flexibility. Though less common, it is still used as a large-scale expansion technique for the culture of adherent cells in vaccine production and was first described in 1967, where positively charged DEAE-Sephadex beads were used to culture rabbit embryonic skin cells and human embryonic lung cells [16]. The use of microcarriers waned in the 1980s when free suspension culture was established for the production of monoclonal antibodies and therapeutic proteins. Embryoid bodies (three-dimensional aggregates of pluripotent embryonic stem cells, sometimes called spheroids) have also been considered for large-scale stem cell

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Stem Cell Manufacturing

production but in addition to the ethical considerations, the size of the bodies is difficult to control in bioreactors. There is also concern that these differences in size can lead to nutrient variation in the embryoid bodies, including oxygen deprivation in the center of the largest; and also to different differentiation paths [17]. Nevertheless, there are reports of success using that approach using human iPSCs with aggregates of about 500 cells [2,18,19]. It is also worth pointing out that when aggregate culture in a spinner flask with hMSCs was tried in our laboratory, growth was poor, although a recent review of other studies has indicated improved hMSC differentiation and function [20]. However, most of the latter cultures have been done at very small scale such as hanging drops [21] or a rotary orbital shaker [22]. On balance, it is clear that, at present, there are strong reasons for choosing stirred bioreactors containing microcarriers with hMSCs for commercial stem cell culture.

3.2 STIRRED BIOREACTOR BASICS The stirred bioreactor is the most common type of bioreactor in use and regardless of the scale or precise fabrication details, they all work on essentially the same principal, namely that the energy needed to undertake the many different processes required for successful culture is imparted to the medium by a rotating stirrer. Thus, spinner flasks are also essentially stirred bioreactors, and in our work cultivating hMSCs, in addition to spinner flasks (Fig. 3.2), we have used the 15 mL ambrÔ (Fig. 3.3), 250 mL DASGIP (Fig. 3.4), and 5 L Sartorius bioreactors (Fig. 3.5), the latter being the largest to date in the peer-reviewed literature [23]. At larger scales, SUBs (often a stainless steel container that has a plastic bag inserted into it within which the cultivation is undertaken) are proving popular and economically superior [24], and at conferences, success in scales up to 50 L have been reported by academia (Fig. 3.6) [25] and even 1000 L by Jannsen [26].

Figure 3.2 Unbaffled spinner flasks; (A) agitated by a circular bar; (B) agitated by a bar plus flat plate.

Bioreactor Engineering Fundamentals for Stem Cell Manufacturing

(A)

(B)

Figure 3.3 The 15 mL operating volume ambr bioreactor (vessel body, 63 mm high
31 mm wide
18 mm deep) (compared to the ambr for free suspension culture; for stem cell culture, the sparge tube has been removed since oxygen demand is extremely low and its removal eases microcarrier suspension (lowers NJS)): (A) front view; (B) side view.

Figure 3.4 The impeller and sample port in the 250 mL Dasgip.

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Figure 3.5 The 5 L Sartorius bioreactor showing impeller, baffles, gas sparger, and various probes.

Figure 3.6 The Sartorius 50 L BIOSTAT Cultibag bioreactor.

Table 3.1 lists the many different physical aspects of stirred bioreactors that need to be understood and considered for bioreactors containing low, constant viscosity, m (Newtonian), media in which cells are grown in free suspension regardless of the cell type or scale [27]; the special additional features associated with cultivation on microcarriers are highlighted in bold. Except when undertaking stem cell culture, in such fluids the flow in bioreactors is typically turbulent (inertial forces dominate viscous ones) and the Reynolds number, Re (¼rND2/m) (conceptually the ratio of these two forces) > 2
104, in all sizes from the bench scale to the industrial scale. The reason for such

Bioreactor Engineering Fundamentals for Stem Cell Manufacturing

Table 3.1 Important Generic Physical Aspects of Agitation for Microcarrier Culture (Some May be Studied Without an Actual Culture; Highlighted Topics Most Significant at Present State of the Art)

1. Mass transfer to and from cells in suspension and on microcarriersa 2. Bulk fluid mixing 3. Unaerated power draw, P ¼ PorLN3D5 (or mean specific energy dissipation rate, εT ) 4. Variation in local specific energy dissipation rates, εT W/kg 5. Microcarrier suspension and abrasion characteristicsa 6. Flow close to the agitator-single and air-liquid 7. Air dispersion capability  
8. Reduced power draw on aeration, Pg εT g ¼ Pg =M 9. Heat transfer (main issue; heat release f T3; cooling area f T2) a

Actual values are specific to the biological system.

Re values is because the oxygen demand of the cells in free suspension in aerobic fermentations whether Escherichia coli, yeast, or even animal cells for example, is sufficiently high that the agitator speed required to meet it leads to such Re values. In stem cell culture, the oxygen demand is extremely small as discussed later. Also, again as will be discussed, once cells are attached to microcarriers, their culture is potentially very sensitive to fluid dynamic stresses, which makes significant cell doubling difficult. The outcome of these two special features of stem cell culture [23,28e32] and indeed culture of other cells such as CHO cells on microcarriers [33e37] is that in all cases reported in the peerreviewed literature, Re < 104. Nevertheless, because there are not, at present, any means theoretically of dealing with such transitional flows, the flow in stirred bioreactors culturing stem cells is, when necessary, treated as though it was turbulent because it certainly is not laminar. An feature that typically distinguishes spinner flasks from other stirred bioreactors is that the former do not contain baffles. These devices protrude w10% of the diameter of the bioreactor in from the wall to prevent the simple rotational motion found with all impeller types if they are not present. Spinner flasks are also stirred by either a circular bar (Fig. 3.2A) or a bar plus flat paddle (Fig. 3.2B); both lead to a somewhat unique rotational flow pattern. In most other bioreactors, the swirling motion is damped out by the presence of probes (dO2, pH, temperature, etc.) as in the DASGIP bioreactor (Fig. 3.4) or in addition and even more effectively, by the use of baffles (Fig. 3.5). Though their presence has been considered to give rise to regions in the bioreactor of high fluid, dynamically generated stresses that might damage cells, a detailed analysis of the flow field in such cases gives no evidence to support this concept [27]. Compared to the unbaffled case, the use of baffles in combination with down-pumping angled (Fig. 3.5) or profiled (Fig. 3.4) blade impellers gives rise to an axial flow pattern. Such a flow pattern enables microcarriers to be suspended at a relatively lower specific energy dissipation rate, thus

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reducing the fluid dynamic stresses on the microcarriers and cells. In the case of the ambrÔ , the rectangular cross-section (Fig. 3.3) prevents the swirling motion and again an axial flow is generated [10], but because of the rectangular shape and the position of the impeller, microcarrier suspension is quite difficult. The other major difference between the spinner flasks and the other bioreactors illustrated in Figs. 3.3 to 3.6 is the level of measurement and control available. With all the others, temperature, dO2, and pH can all be monitored and controlled in situ, which enables much more precise culture protocols to be followed. Fresh medium can also generally be fed as appropriate. In spinner flasks, oxygen concentration in the incubator in which they sit can be controlled but the level in the spinner headspace and in the medium within it is entirely dependent on the resistance to mass transfer through the cap of the spinner and through the upper interface of the medium in the flask, respectively [38]. Similarly, the pH can be followed roughly by means of indicators but not accurately, and not controlled. Having discussed stirred bioreactors in general, the various points in Table 3.1 will now be discussed in detail, especially in relation to hMSC cultivation. For further information on stirred bioreactors in general, refer to more general literature sources [9,27,39,40].

3.3 SPECIAL FEATURES OF STIRRED BIOREACTORS FOR hMSC CULTURE ON MICROCARRIERS 3.3.1 Introduction All the aspects mentioned in Table 3.1 are important but to varying degrees. However, for the purposes of this chapter, those aspects in bold will be treated in more detail. The order in which they will be addressed will be the order in which they impact the operation of the stirred bioreactor, though that may well not be the most important or the most challenging.

3.3.2 Preparing the Bioreactor for Culture The scale at which the hMSCs have been cultivated on microcarriers is small, with 5 L being the largest reported at the time of writing (early 2016) in the peer-reviewed literature in a stirred bioreactor [23]. In our work in the glass bioreactors illustrated in Figs. 3.2, 3.4, and 3.5, it was necessary to coat their internal surfaces with Sigmacote (Sigma Aldrich, UK) to siliconize the surface, thereby preventing microcarrier/cell attachment to it. Sigmacote solution was first applied to the entire vessel and impeller surface area and after a short interval, aspirated; the vessels were then left overnight to dry in a fume hood and rinsed with distilled water after 24 h. Though made of plastic, such treatment also proved necessary for the 15 mL ambrÔ if microcarrier attachment to surfaces was to be prevented. In larger SUBs such as that shown in Fig. 3.6 where plastic bag

Bioreactor Engineering Fundamentals for Stem Cell Manufacturing

inserts are placed inside steel containers, such coating does not appear to be required. In our microcarrier screening studies conducted in multiwell plates, ultralow attachment systems were successfully used.

3.3.3 Medium and Medium Exchange The medium has to provide all the nutrients required by the cells for them to grow and proliferate in culture. Until recently, though many different types have been available, they have all contained some form of animal-derived supplement (serum). However, especially in free suspension culture for therapeutic products because of concerns for possible contamination and batch-to-batch variability, there has been a growing trend to move toward serum-free and chemically defined media. This trend is also happening in hMSC culture and is particularly important since the cells form the basis of the product and it is not possible to expose them to as stringent a purification chain as therapeutic proteins before using them in patients. In addition, studies have shown distinct improvement in culture performance using a serum-free medium with respect to maximum cell numbers and also culture consistency between different donors [15,41]. As cells grow, they utilizse the energy source (usually mainly glucose) and trace supplements in the medium and the products of metabolism (mainly lactate and ammonia) and large numbers of other molecules in small amounts [42] are discharged into it. In order to accommodate these changes replacing the nutrients and diluting the metabolites, it is usual to undertake medium exchange. In T-flasks, 100% exchange is typical but that is not possible with microcarriers as these, too, get removed with the medium. However, it has been shown by comparing T-flask performance with 100%, 50%, and 0% exchange that there is no significant difference in culture performance between 50% and 100% but with 0%, it is significantly worse [38]. Therefore, in general, in our work discussed in this chapter, 50% medium exchange has been employed during culture. Though of great importance, further discussion of medium development is beyond the scope of this chapter.

3.3.4 Microcarrier Selection Employing the most appropriate microcarrier for hMSC expansion is a critical component of a microcarrier-based expansion process. Yet, very few studies have been undertaken where the cultivation of hMSCs from different donors on large numbers of microcarriers has been compared. In Rafiq et al. [43], cells from three different donors were cultivated in monolayer and on 13 commercially available microcarriers under static conditions in ultralow attachment microwell plates and in spinner flasks. The systematic study showed that the three donor cell lines each performed differently with respect to cumulative population doublings over three passages in monolayer, but the performance of each cell line maintained the same relative order on the microcarriers, whether static or

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stirred. In addition, the performance on the microcarriers when stirred was consistently better than when they were static. Finally, there were major differences in performance between microcarriers. Overall, the SoloHill plastic microcarrier was selected as the optimal microcarrier for hMSC expansion based on the following criteria: (1) extent of cell proliferation on the microcarrier, (2) amenability for xeno-free processing, and (3) the ability to effectively harvest the cells from the microcarrier in the spinner flasks [23,32] without any detrimental effect on cellular immunophenotype and differentiation capacity. Overall, the protocol outlined [43] seems appropriate as a way of establishing the best donor cell/microcarrier match. This approach also suggests that once a microcarrier has been selected following such a rigorous screening protocol, a process can be built around that particular microcarrier and there may not be the need to develop an entirely new one should the original donor cell bank be depleted and a new donor cell bank introduced. In addition, while an agitated bioreactor comparison study would be definitive, a microwell screening study may be sufficient for a high-throughput comparison of multiple microcarriers.

3.3.5 Cell and Microcarrier Concentrations The literature indicates that seeding density has an effect on the proliferation of hMSCs grown as a monolayer, with lower seeding densities (100 cells/cm2) demonstrating increased proliferation compared to higher seeding densities (5000 cells/cm2) [44,45]. With respect to the cell-to-bead ratio, similar results have been found. Thus, Hewitt et al. [31] used 5 and 10 cells/bead with the lower figure giving the best results, the same ratio as that proposed by Yuan et al. [46]. A similar result was obtained by Forestell et al. [47] who found a minimum ratio of 3e4 cells/bead. Thus, a ratio of approximately 5 cells/bead for cell inoculation provides a balance between ensuring there are a sufficient number of cells to make the initial attachment and proliferate to achieve desired cell densities/cm2 without requiring a large cell number of cells for inoculation. Such values were used by Rafiq et al. [23] at the 5 L scale, which is equivalent to 6000 cells/cm2, similar to that used in standard T-flask culture where the seeding density used is typically 5000 cells/cm2. Hewitt et al. [31] also investigated the impact of microcarrier concentration using both 3000 and 7500 Cytodex 3 microcarriers mL1 in spinner flasks. The former concentration was chosen because the total surface area of the microcarriers in the spinner flasks approximately matched that available in a standard T175 flask; the latter to see if a higher concentration and hence surface area would give improved cell numbers. Overall, it was concluded that the lower concentration gave a better culture performance and a similar concentration was used in a 5 L bioreactor [23]. This issue is addressed again later in this chapter.

Bioreactor Engineering Fundamentals for Stem Cell Manufacturing

3.3.6 Attachment Protocol It is important to ensure that cells are attached to the microcarriers efficiently and a priori it is not obvious how this should be done. Clearly, if the microcarriers in the bioreactor are not suspended at all during attachment, the cells will tend to attach only to the upper surface of the microcarriers. Furthermore, if there is sufficient agitation to cause suspension of the microcarriers, it will increase cellemicrocarrier contacts due to the relative motion of both microcarriers and cells in the near-turbulent flow of the medium. However, attachment may not occur effectively if the time of contact is too short or the energy dissipated by the flow is too great. This balance is similar to that found in orthokinetic flocculation where typically with increasing speed from a very low level, particles adhere to each other more rapidly but above a certain optimum speed, the equilibrium size decreases as the flow pulls them apart [48]. Thus, Dos Santos et al. [49] opted to employ an intermittent agitation strategy whereby during the first 24 h, the culture was agitated for 15 min at 25 rpm after which followed a period of nonagitation for 2 h. After this, the culture was agitated constantly at 40 rpm for the duration of the culture. Schop et al. [50] instead employed an agitation strategy of constant agitation at 30 rpm for 18 h, after which the culture was constantly agitated at 40 rpm [50]. In spite of these considerations, Hewitt et al. [31] found that when compared to continuous agitation at NJS (the minimum speed to ensure all microcarriers were suspended [51]), an impeller delay of 1 day was introduced, and the effect on maximum cell number was generally markedly positive on maximum cell number per microcarrier. Rafiq et al. [23] adopted a similar approach with an initial delay of 18 h. More recently, we have revisited this question and found that by a judicious choice of periods of stirring followed by periods of rest during the first 3 h post inoculation in combination with a reduced starting medium volume (50% of intended final working volume), an improved attachment protocol could be achieved leading to a significant improvement in culture performance.

3.3.7 Use of Coatings to Enhance Attachment With serum-containing media with components such as fetal bovine serum, cells attach satisfactorily to microcarriers aided by the presence of the animal-derived elements such as fibronectin and vitronectin that are naturally present in the serum and promote cell attachment. However, with the recognition of the need by media development companies to go serum-free as discussed earlier, coating of microcarriers has been found to be important to ensure satisfactory attachment. With a coating on plastic microcarriers such as PRIME-XV human fibronectin (Irvine Scientific, USA) in conjunction with an appropriate serum-free medium from the same supplier, it has been shown that cell attachment is highly efficient and the cell proliferation obtained was a factor of 4 better in serum-free media compared to serum-containing [15,41]. Indeed we have had success

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with other recombinant protein surface coatings, for example, LN-521 (BioLamina, Sweden), and no doubt more coatings will be developed. They clearly add to the cost of goods but, if the proliferation is sufficiently improved, such costs are fully justified.

3.3.8 The Minimum Speed for Suspension, NJS and Associated Mean Specific Energy Dissipation Rate, εT 3.3.8.1 General Aspects If the bioreactor is to work effectively, it is essential that the microcarriers are at least fully suspended in such a way that though they may regularly touch and move on the bottom, they do not remain stationery for any significant period of time, typically 5 s. That condition requires a certain minimum speed to achieve it and was first defined by Zwietering [52] for stirred tank reactors in general. It is usually given the symbol, NJS (rev/s). Under these conditions, the cells on the microcarrier are always surrounded by medium and therefore able to take up nutrients (including O2) from it and discharge waste metabolites into it. The link between speed and mean specific energy dissipation rate, εT , which is numerically equal to the specific power, P/M, imparted to the medium at this point is given by:  εT ¼ P=M ¼ PorL N 3 D5 M [3.1] where Po is the impeller power number, D is the impeller diameter, and M is the mass of medium and microcarriers in the vessel. Po is dependent on the impeller type and also weakly on the precise size of the impeller and its position in the vessel. However, provided there are baffles present in the bioreactor or swirling flow is essentially damped out in some other way; at Re values above about 1000, Po is constant for a particular geometry. As can be seen from Eq. [3.1], small increases in impeller speed can cause a large increase in εT . Much work has been carried out to determine NJS and it can be calculated from [52]. NJS ¼ Sn0:1 dp0:2 ðgDr=rL Þ0:45 X 0:13 D0:85

[3.2]

where S is a dimensionless parameter, independent of scale but related to the system geometry; S values are available for a variety of impeller types [51]. Unfortunately, most of the studies have been undertaken with particles of much greater density than microcarriers, which are only just above neutrally buoyant compared to growth media. Only one study has concentrated on them [53] and because at the time, their use for vaccine production was the application in mind, the microcarrier concentration was of the order of 20 wt%. The study suggested that the behavior of such light particles is somewhat different than those generally studied, thus making the use of Eq. [3.2] to give precise values of NJS somewhat questionable. However, many different studies have shown that down-pumping impellers such as pitched

Bioreactor Engineering Fundamentals for Stem Cell Manufacturing

blade turbines, “elephant ears,” propellers, or axial hydrofoils, with D ¼ w0.4 T (where T is the bioreactor diameter) at a clearance off the base of about one-quarter the liquid height, require the lowest values of ( εT )JS [51]. Increases in speed above NJS produce little or no increase in the rates of transfer to or from the cells, so for these aspects of cultivation, there is no advantage in using a higher
speed [54]. However, the value of εT is important because if it is too high, it may lead JS

to the culture being hindered by fluid dynamic stresses generated on the microcarrier and/or on the cells while attached, which may adversely impact cell functionality/ viability or detach the cells from the microcarrier surface. On the other hand, if it is too low, the rate of transfer of oxygen from the gas phase to the medium and then to the cells to satisfy their specific oxygen demand might also be insufficient, particularly during latter stages of the culture when higher cell densities are achieved. It also impacts the mixing time of the medium. All these aspects are discussed in more detail later (see also Nienow [27], Nienow [9]). 3.3.8.2 NJS Considerations in hMSC Culture There is much emphasis in the literature on the sensitivity of cells grown on microcarriers to high fluid dynamic stresses generated by agitation such that the culture performance is poor whether dealing with animal cells for vaccine production or stem cells. This issue is discussed in more detail later. However, as pointed out earlier, there is little point agitating the bioreactor unless the microcarriers are satisfactorily suspended. Therefore, we decided to use the NJS condition as the starting point for cultivation studies. Unfortunately, because the literature available for predicting NJS has been carried out with such different geometries and particles, it is necessary to determine it in each configuration. At the scales we have studied to date, this speed can be determined visually, which is also the usual method used to study particle suspension in the mixing literature [51e53]. The visually determined NJS values for the spinner flasks with the different spinners shown in Fig. 3.2A,B were 50 rpm and 30 rpm, respectively [31], when using Cytodex 3 (GE Healthcare, Sweden), a solid microcarrier, mean diameter 175 mm with a collagen surface over a dextran matrix. This same agitation criterion was used for the bioreactors in Figs. 3.3e3.5, and the NJS values used are given in Table 3.2. For the 5-L bioreactor, NJS was determined for both the three-blade 45 degrees-pitch segment impeller (with a similar geometry to the optimum described earlier as determined by Ibrahim and Nienow [53]) and a Rushton turbine impeller with six vertical blades. The impellers had the same NJS of 75 rpm but the latter required a much higher specific energy dissipation rate due to its much higher power number [55], thus confirming the choice of the down-pumping impeller; the same type of impeller was used in the DASGIP. For the ambrÔ , a much higher speed was required to suspend the microcarriers at the end away from the impeller

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Table 3.2 Agitation Parameters During Culture and Detachment in a Range of Bioreactors ðεT ÞJS max (lK)JS NJS Culture Volume/ Power (sL1) (W/kg) (mm) Detachment No., a Expansion Parameters D (m)/T (m) Culture Platform Volume Po/F

15 mL TAP ambr 125 mL Spinner flask 250 mL DASGIP bioreactor 5 L Sartorius bioreactor a

15 mL/6 mL 100 mL/60 mL

2.1b/18 1.0c/10

100 mL/70 mL

d

2.5 L/NA

0.011/0.023 0.055/w0.08

6.67 0.5

0.142 6.3
10

1.5 /18

0.030/0.063

1.92

0.046

1.5e/25

0.070/0.16

1.25

0.049

3

52 112

ND (sL1)

ðεT ÞD max (W/kg)

(lK)D (mm)

Detachment Parameters

13.3/10.8 2.5

2.83/1.50 1.31

24/29 30

68

6.25

2.23

26

67

NA

NA

NA

Estimated from Zhou and Kresta [70]. From Nienow et al. [10]. From Hewitt et al. [31]. d Estimated from Nienow et al. [32] as agitators in the DASGIP and Sartorius bioreactors are similar three-blade, pitched turbines. e From Nienow et al. [32]. Modified from A. W. Nienow, C.J. Hewitt, T.R.J. Heathman, V.A.M. Glyn, G.N. Fonte, M.P. Hanga, K. Coopman, Q.A. Rafiq, Agitation conditions for the culture and detachment of hMSCs from microcarriers in multiple bioreactor platforms, Biochemical Engineering Journal, 108 (2016) 24e29. b c

Bioreactor Engineering Fundamentals for Stem Cell Manufacturing

due to the geometry of the vessel and to avoid clumping in the corners so that overall, NJS was very high. In all cases, the cells grew well, in general reaching cell densities up to >3
105 cells/mL in each type of bioreactor, with quality attributes meeting the ISCT criteria (as shown in the following example) [56]. When measured, the dO2 never fell below 50% of saturation with respect to air except when controlled to a lower level. Thus, at the agitation intensities associated with the NJS criterion and without sparging, the oxygen demand of the cells was satisfactorily met and there was not any sign of cell damage. The issue of oxygen demand will be discussed next followed by cell sensitivity to damage due to fluid dynamic stresses.

3.3.9 Oxygen Demand, Mass Transfer, and Optimum Dissolved Oxygen 3.3.9.1 General Considerations For satisfactory operation, the maximum oxygen demand of the cells (ODmax) must be met and it is related to the cell-specific oxygen uptake rate (SOD) and maximum cell concentration (Xmax) by ODmax ¼ SOD$Xmax

[3.3]

Thus, for stable operation, OD (or OUR) needs to be met by the oxygen transfer rate (OTR). Thus,   ODmax ¼ OURmax ¼ OTR ¼ kL ðA=V Þ Cg*  CL ¼ kL aDCL [3.4] where CL is the oxygen concentration in the medium, which should be held at a suitable level for satisfactory operation. Cg* depends on the partial pressure of oxygen in the gas phase, pg, which can be related to the total pressure, Pg, from Dalton’s law of partial pressures: pg ¼ Pg y

[3.5]

where y is the mole (volume) fraction of oxygen in the gas phase and Pg is the total pressure; that is, in stem cell culture to date in stirred bioreactors, atmospheric pressure. Finally,  Cg* ¼ pg H [3.6] where H is the Henry’s law constant, which relates the partial pressure in the gas phase to that in the liquid at equilibrium (ie, the solubility). kL can be considered as a parameter, called the mass transfer coefficient, that links the area available for transfer from the gas phase to the motion of the fluid around that surface. In stem cell culture in the type of bioreactors shown in Figs. 3.2e3.5 to date, the surface area available has generally meant the upper surface of the medium, A (surface aeration) and V is the volume of the medium. Thus, as the scale increases, the

57

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Stem Cell Manufacturing

area/volume ratio, A/V, of the medium decreases. kL increases with increases in velocity at that surface, roughly in proportion with the square root of the agitator speed, kL f N0.5 [57]. In free suspension animal cell culture, air or air enriched with oxygen is usually sparged into the bioreactor, leading to bubble formation. In that case, it is the specific area, a m2/m3 of the bubbles that most contributes to kLa while the contribution to oxygen transfer through the upper surface of the medium is negligible. This type of aeration has been shown to be lethal to cells as bubbles burst and requires the addition of a surfactant, Pluronic F68, to prevent it [9]. With the level of oxygen demand of hMSCs and the scales used to date, bubbling aeration at the current cell densities achievable is not required. However, as the ability to grow to higher cell densities, which will surely follow as with animal cells (from w105 cells/mL in the mid-1980s to >107 cells/mL today [58]), bubbling aeration will be required, if only to strip out CO2 that is generated as the oxygen is consumed by the cells [59]. Under those conditions, the use of Pluronic F68 may well be required, which may raise an issue when cells are to be used for therapeutic purposes. All these topics have been discussed in detail elsewhere for free suspension cell culture and the reader is referred to that article for more information [12]. 3.3.9.2 Application to hMSC Culture In Eq. [3.4], the concentrations should be expressed in mass/unit volume or mole/unit volume. However, the solubility of oxygen is dependent on the composition of the medium and temperature and its precise value is not known. However, the concentration in practice is generally measured in bioreactors by probes (oxygen electrodes (Fig. 3.6) or patches (Fig. 3.3)), which give the concentration as a percent of the saturation concentration (% dO2) [38]. Therefore, in this chapter, when oxygen concentration in the media is quantified, % dO2 will be used.1 In animal cell culture, CL should be > (CL)crit where (CL)crit is the critical oxygen concentration below which the rate of cell proliferation begins to deteriorate, whereas above it, the performance is zero order with respect to dO2 concentration (ie, independent of it) [9]. However, in stem cell culture, there are a number of reports that indicate 1

Unfortunately, there is, in general, a disparity between the nomenclature used to describe the oxygen concentration present in the cells’ growth medium in the bioprocess engineering and life sciences fields, which may lead to confusion. In bioreactors including those used for mammalian cell culture, the concentration of dissolved oxygen in the medium is usually measured and given as a percent with respect to saturation when in contact with atmospheric air (ie, 20e21% v/v oxygen in the gas phase gives 100% dO2). In the life sciences, when the concentration in the gas phase is 20e21 v/v% as in T-flasks or spinner flasks, the concentration in the medium is called normoxic. If the concentration in the gas phase is less than 20%, it is called hypoxic, which would lead to a dO2 < 100% (eg, 2e4% v/v ¼ 10e20% dO2). Since the use of dO2 is standard in stirred bioreactors, as indicated above, that convention will be used here.

Bioreactor Engineering Fundamentals for Stem Cell Manufacturing

Table 3.3 Growth Parameters for 100% and 10% dO2 for Both Collagen and Plastic 100% dO2 10% dO2

Specific growth rate (h1) Doubling times (h) Fold increase

Plastic

Collagen

Plastic

Collagen

0.013 55.2 6.1

0.013 54.5 6.3

0.011 62.8 4.9

0.010 70.7 4.1

Q.A. Rafiq, Developing a standardised manufacturing process for the clinical-scale production of human mesenchymal stem cells, PhD thesis, Loughborough University, UK, 2013.

culture is improved by operating at low dO2 [60e64]. Other studies, however, have demonstrated that 10e25% dO2 can have an impact on either cell quality by attenuating cell differentiation [65] or cell quantity by reducing cell proliferation [66] in comparison to 100% dO2. In our work, we have studied cells from a range of donors in T-flasks [38], spinner flasks, and other stirred bioreactors. Cell proliferation was inferior at 20% dO2 (and worse at 10% [38]) compared to 100% dO2 expressed as cumulative cell number, with the former consuming more glucose and producing more lactate and ammonium. The latter observation therefore suggests that different metabolic pathways employed as hMSCs adapt to lower dO2 conditions. Tests were also undertaken on 13 microcarriers in microwells in low attachment plates and in spinner flasks at NJS with air in the incubator (100% dO2) [43]. In all cases, viable cell numbers were higher in spinner flasks than under static conditions and overall plastic 102-L microcarriers (Pall/SoloHill) were selected as the best because of the cell densities obtained and because they are xeno-free. Subsequently, cells were also cultivated with 10% dO2 with plastic and collagen (Pall/SoloHill) microcarriers, which had also performed well in the microcarrier screening investigations but not selected as the optimal microcarrier for large-scale expansion due to the animal-derived (porcine) collagen coating. The results are summarized in Table 3.3. It can again be clearly seen that the performance was better at the higher dO2 concentration. Also, the lower dO2 again consumed more glucose and produced more lactate and ammonium. When culturing in the 5 L stirred bioreactor, it was operated throughout without bubbling or headspace flushing with air, though additional dissolved O2 entered the bioreactor in the medium during its exchange. During this time, the same cells proliferated at dO2 values (now based on an oxygen electrode in the medium) from 100% down to w50%. By monitoring the dO2 in this way and determining the number of cells as a function of time, the specific oxygen demand was determined and shown to be w8
1015 mol O2 cell1 h1, which is around one order of magnitude less than with animal cells in free suspension. Over this range of dO2, cells proliferated consistently well, though at the lower values, they again consumed more glucose and produced more

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Stem Cell Manufacturing

Table 3.4 Growth Parameters in 5 L Bioreactor and 100 mL Spinner Flasks Bioreactor 1 Bioreactor 2 Spinner Flask 1

Fold increase Doubling time (h) Max specific growth rate (h1)

7.02 76.8 0.014

6.02 83.4 0.014

3.66 128.0 0.006

Spinner Flask 2

5.00 103.4 0.013

Modified from Q.A. Rafiq, K.M. Brosnan, K. Coopman, A.W. Nienow, C.J. Hewitt, Culture of human mesenchymal stem cells on microcarriers in a 5 l stirred-tank bioreactor, Biotechnol. Lett. 35 (2013) 1233e1245.

lactate and ammonium. The performance in the bioreactor was also better than in spinner flasks as can be seen in Table 3.4. Returning to Eqs. [3.4] and [3.6], this very low specific oxygen demand for hMSCs has some implications for dO2 control in bioreactors. Generally, to meet the oxygen demand of cells, either kLa (by adjusting stirrer speeds or bubbling rate [12]), Cg* (via pg), or desired CL can be adjusted. However, the oxygen demand can be achieved at NJS with 100% dO2 by headspace aeration with air. Thus, if it is desired to operate a bioreactor controlling CL to a dO2 <100% (as some literature suggests), then the only way to do so at the present cell densities achievable is to blend the incoming air in the headspace with nitrogen to give the appropriate headspace composition for that dO2. Other studies from our group using the DASGIP bioreactor system has enabled closer control of the dO2 concentration than in the published studies [38,43] where in the latter cases, dO2 control was briefly lost during sampling and media exchange. With this closer control, lower dO2 concentrations (typically 10e25%) led to higher hMSC proliferation. This ability to control the bioreactor parameters, as highlighted previously, is important for ensuring a consistent manufacturing process. In addition to increased proliferation, the hMSCs harvested from these low dO2 conditions were smaller in size and demonstrated enhanced quality characteristics, such as increased outgrowth kinetics and colony forming potential. However, the cells in the most recent study were also from a different donor, so the difference in performance may also be donor cell specific. Work is continuing to clarify this position. Overall, given the variations in the literature and in our own work on the impact of differences in dO2, it would seem to be a parameter that should be explored in depth if a particular donor cell line is chosen for scale-up. After all, such experiments are not difficult.

3.3.10 Fluid Dynamically Generated Stresses and Cell Proliferation 3.3.10.1 General Considerations There are a number of mechanisms that can give rise to mechanical stress on microcarriers and the cells on them; fluid motion relative to the microcarrier [9,35], impacts between microcarriers and the impeller or between microcarriers [32,34], and from bursting bubbles associated with the cells’ oxygen demand [67,68]. When growing cells, there

Bioreactor Engineering Fundamentals for Stem Cell Manufacturing

are two ways in which culture may be compromised; one is growth and proliferation and the other, of critical importance, is cell quality. These issues in turn may arise for two reasons: the stress will detach the cells from the microcarriers and subsequently performance will be compromised or the stress will damage the cell while still on the microcarrier [32]. Some underlying theory related to these different damage mechanisms will now be discussed. As previously pointed out, cell culture on microcarriers has generally been undertaken at Re < 2
104, so the flow is not turbulent. Nevertheless, Kolmogorov’s theory of isotropic homogeneous turbulence has been assumed applicable when considering the issue of stress due to fluid flow. This theory suggests that provided the size of the biological entity, dE, that is suspended in the flow is less than the Kolmogorov scale, lK, then the entity should not be damaged where 
1=4 lK ¼ n3 εT max fN 3=4 [3.7] and εT max is the maximum local specific energy dissipation rate close to an impeller and n is the kinematic viscosity. In addition, εT max ¼ FεT

[3.8]

where F depends rather weakly on the impeller type (it is very similar with both so-called high-shear Rushton turbines and low-shear hydrofoil impellers and more on impeller diameter/vessel diameter ratio, D/T) [69,70]. A value of F used for studies of a range of biological entities has been about 30 [71]. Alternatively, if F is unknown, εT max can be estimated by assuming that all the energy dissipation occurs in the impeller zone, a volume equal to that swept out by the impeller [72]. This approach also implies that F increases with smaller D/T ratios. Thus, provided the impeller power number is known, it is possible to estimate the Kolmogorov scale. This theory has been successfully applied to bacteria, yeast, and animal cells [71]. However, early work for cells on microcarriers, perhaps because the structure of the entity (cells on microcarriers) is different or perhaps because of the Re number indicating flow in the transitional regime, it was shown that cell growth is not compromised provided lK  w0.6 dmicro [36]. Little has been reported on the cell damage from microcarriers by impact with other microcarriers or with the rotating impeller. On the other hand, much has been done in relation to damage to crystals (the removal of tiny nuclei, a process known as secondary nucleation, from the surface of relatively large crystals, typically the size of microcarriers) from such mechanisms. Theories for the two cases of particleeparticle impacts [73] and particleeimpeller impacts [74] for secondary nucleation are similar to those developed later for those two mechanisms for microcarrier culture by [34]. For microcarriereimpeller impacts based on a model related to the frequency of circulation of microcarriers through the impeller zone, the kinetic energy of impact should impact occur, and the probability of

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impact, the potential for damage, DM-I is related to the agitation parameters by the functionality, DM-I fεT NfN 4

[3.9]

For microcarrieremicrocarrier impaction on the other hand, the model is based on an energy cascade, and the potential for damage, DM-M, is related to agitation and other parameters by 6 DM-M fdmicrocarrier εT1:5 X 2 fN 4:5

[3.10]

where dmicrocarrrier is the diameter of the microcarrier. The equations developed by Cherry and Papoutsakis [34] are very similar. The details of these impact models are beyond the scope of this chapter but in essence they suggest that damage (whether destroying cells or just removing them from microcarriers) is more sensitive to mean specific energy dissipation rate and agitator speed than turbulent eddies. The other mechanism, which may cause damage, arises from bursting bubbles. Though it is the mechanism that is most damaging in free suspension cell culture, it can readily be prevented by the use of the surfactant Pluronic F68. This surfactant prevents cells attaching to bubbles and thereby being in the region close to the bubble where enormously high energy dissipation rates occur [9,67,68]. 3.3.10.2 Application to hMSC Culture At present, because the oxygen demand is so low, the culture of hMSCs does not require bubbling aeration (sparging) to enhance kLa to meet it. Nevertheless, it has been used in 1.3 L stirred bioreactors at a flow rate of w0.04 vvm with hMSCs [75] and of w0.08 vvm with mouse ESCs [30]. These flow rates are quite high when compared to those used in free suspension culture and in neither article was the use of Pluronic mentioned. Both cultures were reported to be successful in terms of viable cell densities and quality attributes. These results are encouraging since, inevitably, as significant increases in cell density are achieved, bubbling aeration will almost certainly become necessary. Clearly, however, if bubbling aeration does appear desirable in order to meet the oxygen demand, the sensitivity to damage due to this mechanism will need to be investigated. In our work in spinner flasks [31], the spinners in Fig. 3.2A,B had Kolmogorov scales of 131 and 183 mm, respectively, at the appropriate NJS value with 175 mm Cytodex 3 microcarriers. Growth was achieved with both spinner types and in both cases, lK  w0.6 dmicro. However, the growth performance was a little better with the spinners in Fig. 3.2B. It was also shown that the agitation of naked beads without cells for 7 days did not produce any plastic fragments that could prove problematic for therapeutic use. Our later work in spinner flasks of the type shown in Fig. 3.2B with plastic microcarriers gave almost identical results (Table 3.2) [23,32].

Bioreactor Engineering Fundamentals for Stem Cell Manufacturing


In the 5 L bioreactor (2.5 L working volume) at NJS (75 rpm), εT ¼ JS w1:6
103 W=kg assuming power number of 1.5 for the pitched blade impeller [23]. Thus, assuming F ¼ 30, ðεT Þmax ¼ w0:05W=kg and lK ¼ 118 mm, again w0.6 times the size of the 200 mm plastic P102-L microcarriers, in good accord with our spinner flask study [31] and other earlier work [35]. For the DASGIP, on
the other hand, εT was notably higher and (lK)JS significantly smaller at around JS

30% dmicro. Finally, the poor geometry for suspension in the ambr led to a much higher
εT and as a result, (lK)JS ¼ w0.25 dmicro. Even so, in both cases, the cells maintained JS

their desired quality attributes [76]. Clearly, these results are very encouraging because having to work at N < NJS would certainly remove many of the advantages of using stirred bioreactors. Overall, it seems that ðεT ÞJS max can be significantly larger and (lK)JS smaller than was originally proposed but there is still need for further work in this area to establish the upper limits of agitation intensity that stem cells on microcarriers can tolerate. It will almost certainly be donor specific (due to the relative changes in attachment rates between donors) and the protocol set out here working at NJS seems to offer a suitable one; if the cell/microcarrier/ bioreactor combination does not allow satisfactory proliferation at NJS, it is probably not worth further work on it.

3.3.11 Fluid Dynamically Generated Stresses and Their Application to Cell Harvesting Most studies have concentrated on the culture of hMSCs covering a range of commercially available microcarriers; in addition to the ones mentioned here, they include Cytodex-1, Cultispher-S, and Cultispher-G [32]. However, in the majority of cultivation studies, there has been little focus on the harvesting procedure, especially with increasing scale. Yet, detachment or dissociation of cells from the microcarrier surface and subsequent retention of cell quality is equally as important as cell attachment and proliferation given that the product of interest for cell therapies is the cell itself. It is also important to recognize that, independent of whether the cells are to be reused for further expansion following a passage or stored prior to use for therapeutic or other purposes, harvesting involves two steps. First, the cells are detached from microcarriers to produce a cellemicrocarrier suspension; second, a further separation step leaves the cells in suspension without the microcarriers present. It is the first of these steps that at present appears to be capable of effective implementation within the bioreactor [32]. The typical approach for detachment for a vaccine-producing process is to do it enzymatically. However, while the enzyme is in contact with the cells, cell damage occurs, so

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Stem Cell Manufacturing

that it is critical to minimize this time, though it is typically tens of minutes [77]. However, because the cell is not the product, the potential damage is tolerable [78]. With stem cells as the basis of the product, keeping this time low is extremely critical. Nevertheless, the enzymatic approach has been generally recommended for stem cells but until recently, the scale at which detachment has been studied has been very small, typically 5 mL [79]. They used a range of microcarriers and enzymes and ascertained that some combinations did much better than others; plastic P102-L with trypsin or trypsin-accutase is particularly good. For the larger quantities of microcarriers and medium associated with spinner flasks, one recommended harvesting procedure for plastic microcarriers has been suggested by their manufacturers (Pall/SoloHill). In summary, it involves stopping agitation, allowing the microcarriers to settle, and removing the medium. Trypsin and EDTA is then added to the spinner flask, allowed to stand without agitation for 15 min, and then diluted with growth medium to minimize the impact of trypsin on the quality of the cells. Finally, the cell suspension is gently pipetted up and down (aspirated) to expose the cells to elongational shear stresses and thus dislodge the cells. However, when attempted in our work, the detachment efficiency was <5%. In order to improve on this approach, use was made of the detachment potential implied by Eqs. [3.7], [3.9], and [3.10]. The precise form of these equations is not important. What is important is that the impaction mechanisms do not apply to free suspension cells because they are too small to cause either type of impact to occur. Also, the mechanism associated with the Kolmogorov scale (Eq. [3.7]) is only weakly dependent on specific power or on agitator speed whereas both impact mechanisms (Eqs. [3.9] and [3.10]) are strongly dependent. Thus, it should be possible to dramatically increase parameters that lead to cell detachment by increasing agitation intensity (speed) while keeping the Kolmogorov scale greater than the cell size once it has been removed. Thus, detachment has been carried out at the 100 mL scale in a spinner flasks by significantly increasing the speed for a short time to enhance detachment but keeping it less than that at which bubbles would be dragged into the suspension through the upper surface, thereby avoiding bubble disengagement damage without Pluronic F68 [9]. Thus, as indicated in Table 3.2, a speed of 150 rpm (compared to NJS of 30 rpm) and a time of 7 min were selected. Under these conditions, the fivefold increase in impeller speed increased the detachment mechanisms, DMI and DMM by factors of w625 and w1400, respectively. However, once detachment was complete, while the value of ðεT ÞDmax increased significantly to w0.13 W/kg, the Kolmogorov scale, ((lK)D), is w30 mm, well above the size of the cell so that theoretically, damage should not occur. This approach led to >95% detachment efficiency with cells able to grow when reseeded back into monolayer and microcarrier platforms. They also met the usual quality attribute tests and demonstrated typical fibroblastic morphology as expected of hMSCs when grown as a monolayer [32].

Bioreactor Engineering Fundamentals for Stem Cell Manufacturing

Since then a similar detachment protocol has been used for the ambrÔ and DASGIP bioreactors [76] using the speeds shown in Table 3.2. The higher of the two speeds chosen for the ambr was close to the maximum available and the lower gave ðεT ÞDmax similar to that used in the spinner flask. For the DASGIP, the speed chosen was one, which ensured bubble entrainment did not occur to avoid potential damage from the bubble burst mechanism. Detachment was in every case effective and (lK)D > dcell; and the cells maintained their desired quality attributes. Clearly, being based on sound theoretical mixing principles, it should be scalable to in situ detachment at much bigger bioreactor scales.

3.4 FUTURE ISSUES 3.4.1 Increasing Cell Density At present the cell density achieved is limited by the microcarrier concentration being utilized. This concentration has been selected because at higher and lower concentrations, cell proliferation was found to be less good. One of the aims of our current work is try to increase the microcarrier concentration by the addition of microcarriers during culture. There is some dispute in the literature as to whether bead-to-bead transfer occurs but in our earliest work with hMSCs it clearly did so [31] as also found by Cherry and Papoutsakis [34] with bovine embryonic kidney cells. Our later studies also suggest that such transfer is taking place, though it is unclear whether it arises from cells detaching and then reattaching to new microcarriers; or from cellemicrocarrier aggregates forming entrapping naked microcarriers, which thereby gain cells on aggregate fragmentation. Assuming that bead-to-bead transfer occurs by whatever mechanism, therefore, the next requirement is optimizing the mode of microcarrier addition to ensure the cells remain in the exponential phase for as long as possible, investigating the time, frequency, and quantity during culture. This aspect is currently under study.

3.4.2 Oxygen Demand and Mass Transfer at Higher Cell DensitySparging and Higher Agitator Speeds At present, the cell densities and the specific oxygen demand of hMSCs is so low that the OTR can be easily met by surface aeration from the headspace of the bioreactor certainly up to the 5 L scale. If by use of increased microcarrier concentrations to give a greater surface for cell attachment before confluence, higher oxygen demands arise, it initially can be met by increasing the agitator speed, which enhances kL, the mass transfer coefficient for the top surface, area A, such that kL fN 0:5

[3.11]

provided the cells are not detached or damaged. Little work has been done to date to establish how much N can be increased above NJS before culture deteriorates; or to

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ascertain the mechanism causing it. Increasing airflow through the headspace also enhances kL. Of course, if it is desired to work at <100 dO2, then it may be necessary to operate with nitrogen-enriched air with a suitable composition to satisfy Eq. [3.4] while ensuring that CL is held at the desired level for the particular cell, assuming that different hMSCs will perform best at different values of dO2. Further increases in oxygen demand can be met by a combination of increased stirrer speed and the introduction of oxygen-enriched air to the headspace and/or sparging. In that case, Eq. [3.4] can be written as   OTR ¼ kL ðA=V Þ Cg*  CL ¼ kL aDCL [3.12] where the use of oxygen-enriched air will increase DCL; and, if sparged, a is the specific area of bubbles in the bioreactor (m1). kLa is the specific mass transfer coefficient, dimensions s1 if SI units are used but usually expressed for free suspension animal cell culture in h1 (typically
where εT

g

 g

is the mean specific energy dissipation rate, W/kg (though it is also often

expressed as kW/m3, the two being numerically equal in media of density ¼ 1000 kg/m3), from agitation and sparging, and vS is the superficial gas velocity, m/s (numerically equal to the volumetric sparged flow rate, QG (m3/s), divided by the cross-sectional area of the bioreactor, A m2). Critically, since this is a dimensional equation, the numerical value of A is dependent on the units used to express the parameters in Eq. [3.13]. Such equations have been shown to apply to free suspension culture and other aerobic bioprocesses and are expected to apply to hMSC culture. If the use of N > NJS is required, as with free suspension, sensitivity of hMSCs on microcarriers to agitation intensity and bubbling rate (with or without Pluronic F68) will need to be investigated [9,12].

3.4.3 Carbon Dioxide, Osmolality, and pH With cell densities in free suspension culture getting up to 107 cells/mL and higher, the oxygen demand and hence transfer rates is increased. This increase may be met by raising agitation intensities and sparge rates as indicated by Eq. [3.13] as just discussed; but there are concerns that such a change may lead to cell or product damage. As a result, the increase in OTR has often been met by increasing the driving force by using

Bioreactor Engineering Fundamentals for Stem Cell Manufacturing

oxygen-enriched air and/or reducing bubble size using a fritted sparger to increase the specific surface area, a. However, both the latter approaches reduce the gas flow rate, leading to higher carbon dioxide (pCO2) levels in the medium, which if high enough may cause a significant fall in cell and product yield. It is essential to maintain a sufficient sparge rate if the pCO2 level is to be controlled. This issue is currently a major one at the large commercial scale for free suspension culture and is discussed at length elsewhere [12,59]. Since the concern for damage to cells is even more acute in stem cell culture, when stem cell density or the production scale reaches the level that headspace aeration is not sufficient to meet the cells’ oxygen demands, there will again be a reluctance to meet that demand by increased agitation and aeration rates. However, it will be important to investigate that way of meeting the oxygen demand as well as enhancing it by using a greater driving force and/or specific area.

3.4.4 Human-Induced and Embryonic Pluripotent Stem Cells It is clear that it is hMSCs that are currently by far the most common stem cell type with respect to representation in clinical development and other large-scale applications, probably because of the problems discussed earlier with respect to human iPSCs (hiPSCs) and human embryonic pluripotent stem cells (hEPSCs). Nevertheless, some success has been achieved in developing the proliferation of hiPSCs and hEPSCs in stirred bioreactors on microcarriers. Indeed, hEPSCs have been successfully cultivated on Cultispher-S microcarriers in a 250 mL stirred bioreactor, albeit with a poorly characterized impeller [80]. They were passaged onto fresh microcarriers and then expanded without any feeder layer while maintaining pluripotency. The method required, as usual, careful optimization of initial seeding parameters by which microcarrier aggregation was minimized. Overall, a sevenfold expansion was achieved with doubling times similar to monolayer culture with a feeder layer. Following the discovery of ways of inducing pluripotency [81], hiPSCs have also been successfully cultivated on xeno-free microcarriers in 15 mL spinner flasks, again retaining their pluripotency markers [82]. Thus, for the hiPSCs in the spinner flask, the scale was very small and for the hEPSCs, the configuration of the bioreactor was so unusual that it would be very difficult to scale up with confidence. It is suggested that if the scale-up of these pluripotent cell types is shown to be desirable for clinical use or the testing of the efficacy of other pharmaceutical products, the most likely configuration that will be used is the stirred bioreactor. In addition, some progress is being made with hiPSCs in bioreactors as aggregates [2] without the use of microcarriers, as well as hMSCs (spheroids) [20]. If successful, the removal of the need to use microcarriers would offer a distinct advantage as the detachment of cells in order to harvest them would no longer be required and the possibility of getting microcarrrier fragments in the product would be eliminated.

67

Table 3.5 Combination of Bioreactor, Microcarrier (Coated and Uncoated), hMSC Cells and Media Used Along With Detachment Enzyme (in All Cases, >95% Cells Were Recovered With a Viability >95% After Harvest and the ISCT Quality Criteria Were met) Number Vessel Type Microcarrier Type Surface Coating Cell Line Dissociation Reagent Culture Medium

1 2 3 4 5 6 7 8 9

100 mL Spinner flask 100 mL Spinner flask 100 mL Spinner flask 100 mL Spinner flask 100 mL Spinner flask 100 mL Spinner flask 100 mL Spinner flask 100 mL Spinner flask 100 mL Spinner flask

Solohill Solohill Solohill Solohill Solohill Solohill Solohill Solohill Solohill

10

100 mL Spinner flask

Solohill plastic

11

100 mL Spinner flask

Solohill plastic

12

100 mL Spinner flask

Solohill plastic

13 14 15 16 17

100 mL Spinner flask 100 mL Spinner flask 100 mL Spinner flask 100 mL Spinner flask 100 mL DASGIP bioreactor 15 mL ambr bioreactor 15 mL ambr bioreactor 15 mL ambr bioreactor 15 mL ambr bioreactor 5 L Bioreactor (harvest in 100 mL spinner flask)

Solohill Solohill Solohill Solohill Solohill

18 19 20 21 22

plastic plastic plastic plastic plastic plastic plastic plastic plastic

None None None None None None None None None

BM-hMSC BM-hMSC BM-hMSC BM-hMSC BM-hMSC BM-hMSC BM-hMSC BM-hMSC BM-hMSC

Fibronectin (Irvine Scientific) Fibronectin (Irvine Scientific) Fibronectin (Irvine Scientific) LN-521 (BioLamina) LN-521 (BioLamina) None None None

DMEM DMEM DMEM DMEM DMEM DMEM DMEM DMEM DMEM

BM-hMSC 2

Trypsin/EDTA Trypsin/EDTA Trypsin/EDTA Trypsin/EDTA TrypLE express TrypLE express TrypLE express Accutase Trypsin/EDTA þ Accutase Trypsin/EDTA

BM-hMSC 2

TrypLE express

SFM (Irvine Scientific)

BM-hMSC 1

TrypLE express

SFM (Irvine Scientific)

BM-hMSC1 BM-hMSC 1 BM-hMSC 1 BM-hMSC 2 BM-hMSC 2

Trypsin TrypLE express Trypsin/EDTA Trypsin/EDTA Trypsin/EDTA

DMEM (10% FBS) SFM (Irvine Scientific) DMEM (10% FBS) DMEM (10% FBS) DMEM (10% FBS)

Solohill plastic

None

BM-hMSC 1

Trypsin/EDTA

DMEM (10% FBS)

Solohill plastic

None

BM-hMSC 1

TrypLE express

SFM (Irvine Scientific)

Solohill plastic

Fibronectin (Irvine Scientific) Fibronectin (Irvine Scientific) None

BM-hMSC 1

Trypsin/EDTA

DMEM (10% FBS)

BM-hMSC 1

TrypLE express

SFM (Irvine Scientific)

BM-hMSC 1

Trypsin/EDTA

DMEM (10% FBS)

plastic plastic collagen collagen plastic

Solohill plastic Solohill plastic

1 2 3 4 2 3 1 1 1

(10% (10% (10% (10% (10% (10% (10% (10% (10%

FBS) FBS) FBS) FBS) HPL) HPL) FBS) FBS) FBS)

DMEM (10% FBS)

BM-hMSC 1, 20 years old, black male; BN-hMSC 2, 19, black female; BM-hMSC 3, 24, Caucasian male. BM-hMSC 4, 25, Hispanic female. DMEM, Dulbecco’s Modified Eagle’s Medium; FBS, fetal bovine serum. Modified from A. W. Nienow, C.J. Hewitt, T.R.J. Heathman, V.A.M. Glyn, G.N. Fonte, M.P. Hanga, K. Coopman, Q.A. Rafiq, Agitation conditions for the culture and detachment of hMSCs from microcarriers in multiple bioreactor platforms, Biochemical Engineering Journal, 108 (2016) 24e29.

Bioreactor Engineering Fundamentals for Stem Cell Manufacturing

If any or all of these developments occur, the concepts discussed in this chapter should also be highly relevant to their design and operation (including consideration of aggregate size control [48]) with regard to cell culture, and if microcarriers are involved, cell detachment.

3.5 CONCLUSIONS For stem cell bioprocessing to provide the number of cells required for allogeneic therapies, there is a need to maximize the target cell output and minimize production costs while maintaining close control of the production environment and complying with a stringent regulatory landscape. Since the cells form the basis of the product and are to be introduced into patients, it is essential that they can be manufactured with a minimum risk of forming teratomas upon implantation. As a result, the use of hMSCs has become the favored cell type because though not pluripotent, they are multipotent, not tumorigenic, and do not initiate a patient immune response. However, such cells at present need a surface on which to grow and in order to provide a large surface area per volume of highly expensive growth medium in the bioreactor, microcarriers of about 200 mm diameter are placed within it. The cells then need to be attached to the microcarriers and in order for all the surface area to be available for transfer of nutrients to and metabolites from the attached cells, the microcarriers need to be suspended. Taking these basic concepts into account has led to the choice of stirred bioreactors containing microcarriers on which hMSCs are cultured as the system of choice. This chapter, therefore, concentrates on basic generic stirred bioreactor fluid dynamic concepts plus certain special features associated with unique aspects of stem cell culture. It is shown that because of the extremely low oxygen demand of the cells, headspace aeration is sufficient to meet that need and microcarrier suspension is the most critical of the many tasks that the stirrer in a stirred tank bioreactor has to achieve, at least at the scales being routinely utilized at present (up to 50 L). In order to passage cells during culture and at the end of culture before purification and cryopreservation, the hMSCs need to be detached from the microcarriers. It has been found that detachment can be achieved by a short period of intense agitation in the same bioreactor. Again the basic concepts behind this technique are discussed. Finally, Table 3.5 lists the different cases that have been reported where the two protocols outlined here have been used successfully in stirred bioreactors for proliferation and detachment. The agitation conditions under which proliferation and detachment were undertaken are shown in Table 3.2 and involved the 15 mL ambrÔ , 125 mL spinner flasks, 250 mL DASGIP and 5 L Sartorius Stedim bioreactors. Altogether (Table 3.5), some 21 combinations in the three smallest bioreactors have been successfully cultured and detached in situ, with cells from four donors plus different media (with and without serum), microcarriers (uncoated and with two types of coating), and two detachment

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enzymes. In every case, >95% cells with >95% viability meeting the full ISCT quality attributes criteria were recovered after harvesting and resuspension [76]. The successful use of the ambrÔ is very interesting because its unusual geometry makes ðεT ÞJS max at NJS very high, leading to (lK)JS ¼ w25% dmicro, much smaller than earlier work that suggested (lK)JS must be >w60% dmicro if cell damage is to be avoided. Yet successful culture was undertaken. However, sensitivity to fluid dynamic stresses is likely to be microcarrier/donor cell specific. Thus, the use of devices like the ambr offers a way of quickly establishing whether a particular combination is appropriate. If efficient cell proliferation on a specific microcarrier/donor pairing is satisfactory in the ambr, it should be in other bioreactor configurations, which in general are able to suspend microcarriers at much lower ðεT ÞJS max .

NOMENCLATURE a A CL D D DM-I DM-M g H kL kLa M N OD OTR OUR pg P Pg Po QG Re S SOD t T vS V y X

Specific area of bubbles, m1 Surface area for mass transfer (m2); or dimensional constant in Eq. [3.13] Concentration in the liquid phase, mol/m3 Agitator diameter, m Size of particle, m Microcarrier-impeller impact parameter; see Eq. [3.9] Microcarrieremicrocarrier impact parameter; see Eq. [3.10] Gravitational constant, 9.81 m/s2 Bioreactor fill level, m; or Henry’s law constant, atm m3/mol Liquid film mass transfer coefficient, m/s Specific mass transfer coefficient, s1 or h1 Mass of media, kg Agitator speed, s1 or rpm Oxygen demand, mol/m3 s Oxygen transfer rate, mol/m3 s Oxygen uptake rate, mol/m3 s Partial pressure, atm Power, W Total pressure, atm Power number, dimensionless Airflow rate, m3/s Reynolds number, dimensionless Suspension parameter, dimensionless Specific oxygen demand, mol/s cell Temperature,  C; or time, s Bioreactor diameter, m Superficial gas velocity, m/s Volume of media, m3 Mol fraction, dimensionless Cell density, cells/m3; or mass of microcarriers/mass of media
100, dimensionless

Bioreactor Engineering Fundamentals for Stem Cell Manufacturing

Greek letters a,b Exponents DC Concentration driving force, mol/m3 εT Local specific energy dissipation rate, W/kg εT Mean specific energy dissipation rate, W/kg F εT max εT , dimensionless lK Kolmogoroff turbulence scale, m m Viscosity, Pa s n Kinematic viscosity, m2/s Liquid density, kg/m3 rL qm Mixing time, s Subscripts cell Related to the cell crit Critical oxygen concentration i At the interface JS Just fully suspended in Entering at the sparger g When air is sparged; or in the gas phase max Maximum micro Microcarrier Superscript * At equilibrium

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