Agitation strategies for the culture and detachment of human mesenchymal stem cells (hMSCs) from microcarriers in multiple bioreactor platforms

Unlike cell culture for biopharmaceuticals, where the product of interest is usually a recombinant protein, for regenerative medicine, the cells form the basis of the therapeutic. For the expansion of cells for allogeneic purposes, work, mainly in spinner flasks, has led to culture on microcarriers. For processes involving mass transfer and reaction with particles in stirred reactors (e.g., crystallization, catalytic reaction), it is essential that particles are at least just fully suspended, agitator speed NJS, for effective mass transfer to and from the particles. Though gentle agitation has generally been recommended, it has not previously been defined in such a precise way as the minimum effective agitation intensity for cell culture on microcarriers where transfer of nutrients to and metabolites from them is essential. This criterion has been applied here for four sizes of stirred bioreactor (15 mL ambrTM (Sartorius Stedim), 125 mL spinner flask, 250 mL DASGIP (Eppendorf) and 5 L Sartorius Stedim). If the agitation intensity at NJS for the particular bioreactor adversely affects the quality and quantity of the cells, then that configuration is inappropriate for cell culture. In addition, it is critical that the stem cells are successfully detached and separated from the microcarriers in a manner that again does not adversely affect cell quality or the quantity. Indeed, effective cell recovery will reduce overall cost of goods by increasing process efficiency and enabling process intensification. However, surprisingly, few published studies have harvested greater than millilitre samples of the microcarrier culture, typically by enzymatic digestion aided by extensional flow using a pipette. At larger scales, such an approach becomes impractical and in addition, the enzymes can also damage the cells if exposure is prolonged. Thus, a new method is required. Given the sensitivity of particles of the size of microcarriers to abrasion (or if crystals, to secondary nucleation), it was decided to try a short period of intense agitation at agitator speeds significantly greater than NJS to enhances the removal of the cells by the action of the enzyme. This presentation outlines our work using NJS for cell culture in the four different bioreactors and the new technique for detaching cells in-situ in the three smallest. In total, cells from four donors were used with two microcarrier with and without surface coatings (two types), four enzymes and three growth media (with and without serum), a total of 22 different combinations. Stresses on cells on microcarriers may come from turbulence and from microcarrier impacts with themselves and with impellers. For turbulence, it has generally been considered that if the Kolmogorov scale of turbulence, lK is greater than ~ 60% of the size of the microcarrier (dmicro = ~200 mm), damage to attached cells should not occur. For the stresses from impacts, they increase very dramatically with increases in agitator speed, N (µ N~4). The latter concept led to the use of an enhanced agitator speed (~ 2 to 5NJS) being used during enzymic detachment for 7 minutes. Once detached, the cells were smaller than lK and thus cells should not be damaged. To suspend cells in the rectangular ambrTM required a high NJS which led to lK = ~ 0.25dmicro, much smaller than has generally been accepted can be used without impacting process performance. Yet the cells grew well and maintained the desired quality attributes. With the spinner flask, lK = ~ 0.6dmicro but the growth was similar and again the quality attributes were maintained. The results were essentially the same in both the DASGIP and Sartorius bioreactors though lK = ~ 0.3dmicro. After detachment, cells were separated from the microcarriers by filtration and in each case, \u3e 95% cells were recovered regardless of the bioreactor, the detachment enzyme, the microcarrier or the donor. In addition, the cells always met the desired quality attributes and were able to proliferate. These criteria for culture and detachment, well grounded in agitation theory seem a promising approach to scale up; and for comparing the effectiveness of different bioreactors. The relatively high agitation intensities at NJS leading to lK values much smaller than generally accepted as appropriate for cell culture is rather notable. That finding along with the new detachment technique may also interest manufacturers using microcarrier culture with other animal cells such as CHO for vaccines

The scale-up of microbial batch and fed-batch fermentation processes

Micro-organisms are important for both human health and to industry so the fed-batch cultivation of microbial strains, often over expressing recombinant or natural proteins, to high cell density has become an increasingly important technique throughout the field of biotechnology, from basic research programmes to large-scale pharmaceutical production processes (Hewitt et al., 1999). The scale-up of such a process is usually the final step in any research and development programme leading to the large-scale industrial manufacture of such products by fermentation (Einsele, 1978). It is important to understand that the process of scaling-up a fermentation system is frequently governed by a number of important engineering considerations and not simply a matter of increasing culture and vessel volume. Therefore, it is perhaps surprising when the large-scale does not perform as well as the small-scale laboratory process. It is often observed that the biomass yield and any growth associated products are often decreased on the scale-up of an aerobic process (Enfors et al., 2001). For Saccharomyces cerevisiae, the biomass yield on molasses increased by 7% when the process was scaled-down from 120 m3 to 10 l even when a seemingly identical strain, medium and process were employed (George et al., 1993). In an E. coli fed-batch recombinant protein process, the maximum cell density reached was found to be 20% lower when scaling-up from 3l to 9 m3 and the pattern of acetic acid formation had changed. (Bylund et al., 1998). During another study (Enfors et al., 2001), the performance of a recombinant strain of E. coli during fed-batch culture was found to vary on scale-up from the lab-scale to 10-30 m3 industrial bioreactors. This included lower biomass yields, recombinant protein accumulation and surprisingly perhaps a higher cell viability. These findings are typical of those found when scaling up most fermentation processes yet only a few mechanisms have been presented that can satisfactorily explain these phenomena. In this Chapter, we will briefly discuss the main engineering considerations involved in fermentation scale-up and then critically review those mechanisms thought to be responsible for any detrimental change in bioprocessing at the largerscale. Though it addresses mainly E. coli fed-batch fermentations, much of the discussion also applies to batch and other single celled aerobic microbial fermentations too

Scale-down studies for the scale-up of a recombinant Corynebacterium glutamicum fed-batch fermentation:loss of homogeneity leads to lower levels of cadaverine production

BACKGROUND: The loss of efficiency and performance of bioprocesses on scale-up is well known, but not fully understood. This work addresses this problem, by studying the effect of some fermentation gradients (pH, glucose and oxygen) that occur at the larger scale in a bench-scale two-compartment reactor [plug flow reactor (PFR) + stirred tank reactor (STR)] using the cadaverine-producing recombinant Corynebacterium glutamicum DM1945 Δact3 Ptuf-ldcC_OPT. The new scale-down strategy developed here studied the effect of increasing the magnitude of fermentation gradients by considering not only the average cell residence time in the PFR (τPFR), but also the mean frequency at which the bacterial cells entered the PFR (fm) section of the two-compartment reactor. RESULTS: On implementing this strategy the cadaverine production decreased on average by 26%, 49% and 59% when the τPFR was increased from 1 to 2 min and then 5 min respectively compared to the control fermentation. The carbon dioxide productivity was highest (3.1-fold that of the control) at a τPFR of 5 min, but no losses were observed in biomass production. However, the population of viable but non-culturable cells increased as the magnitude of fermentation gradients was increased. The new scale-down approach was also shown to have a bigger impact on fermentation performance than the traditional one. CONCLUSION: This study demonstrated that C. glutamicum DM1945 Δact3 Ptuf-ldcC_OPT physiological response was a function of the magnitude of fermentation gradients simulated. The adaptations of a bacterial cell within a heterogeneous environment ultimately result in losses in fermentation productivity as observed here

Application of the migratory nature of human mesenchymal stem cells to optimise microcarrier-based expansion processes

As the number of mesenchymal stem cell based therapies proceeding through clinical trials increases so does the demand for well characterized, scalable expansion technologies that can yield the estimated number of cells required. Microcarriers used in conjunction with stirred tank bioreactors provide a suitable platform for this large scale expansion. Research has proven that mesenchymal stem cells migrate between microcarriers during culture in agitated systems. A series of experiments have been conducted using Pall SoloHill microcarriers to determine whether this bead-to-bead transfer mechanism can be exploited to streamline various unit operations of the expansion process such as the initial bioreactor inoculation. Please click Additional Files below to see the full abstract

Process development of human mesenchymal stem cell microcarrier culture using an automated high-throughput microbioreactor

Improvements to process development technology will have a significant impact in reducing the overall costs associated with the manufacture and scale-up of human cell-based therapies. Small-scale models, including microbioreactors, play a critical role in this regard as they reduce reagent requirements and can facilitate high-throughput screening of process parameters and culture conditions. Here we have demonstrated, for the first time, the amenability of the automated ambr15 cell culture microbioreactor system (originally designed for free suspension culture) for adherent hMSC microcarrier culture. We also demonstrated that the ambr15 could be used for bioprocess development of a microcarrier process which was subsequently validated with larger-scale spinner flask studies. The results were achieved by a combination of strategies including adapting the free suspension design of the vessel to improve the suspension and mixing of the microcarriers. A more effective cell attachment method was also developed by using only 50% of the final working volume of medium for the first 24 h combined with an intermittent agitation strategy. These improvements led to a reduction in the initial lag phase which in turn resulted in \u3e 150 % increase in viable cell density after 24 h compared to the original process (no agitation for 24 h and 100 % working volume). Using the same methodology as in the ambr 15, similar improvements were obtained in larger scale spinner flask studies. Finally, this improved bioprocess methodology, which was developed for a serum-based medium process, was applied to a serum-free process in the ambr15; this resulted in \u3e 250% increase in yield compared to the ambr15 serum-based process. The use of the ambr15, with its improved control compared to the spinner flask, reduced the coefficient of variation on viable cell density in the serum containing medium from 7.65% to 4.08%, and the switch to the serum free medium further reduced these to 1.06% and 0.54% respectively. The combination of both serum-free and automated processing improved the consistency more than 10-fold compared to the initial manual, serum-based spinner flask work. The findings of this study demonstrate that the ambr15 microbioreactor is an effective tool for bioprocess development of hMSC microcarrier cultures and that a combination of serum-free medium and automation improves both process yield and consistency. Please click Additional Files below to see the full abstract

Studies supporting the use of mechanical mixing in large scale beer fermentations

Brewing fermentations have traditionally been undertaken without the use of mechanical agitation, with mixing being provided only by the fluid motion induced by the CO2 evolved during the batch process. This approach has largely been maintained because of the belief in industry that rotating agitators would damage the yeast. Recent studies have questioned this view. At the bench scale, brewer’s yeast is very robust and withstands intense mechanical agitation under aerobic conditions without observable damage as measured by flow cytometry and other parameters. Much less intense mechanical agitation also decreases batch fermentation time for anaerobic beer production by about 25% compared to mixing by CO2 evolution alone with a small change in the concentration of the different flavour compounds. These changes probably arise for two reasons. Firstly, the agitation increases the relative velocity and the area of contact between the cells and the wort, thereby enhancing the rate of mass transfer to and from the cells. Secondly, the agitation eliminates spatial variations in both yeast concentration and temperature, thus ensuring that the cells are maintained close to the optimum temperature profile during the whole of the fermentation time. These bench scale studies have recently been supported by results at the commercial scale from mixing by an impeller or by a rotary jet head, giving more consistent production without changes in final flavour. It is suggested that this reluctance of the brewing industry to use (adequate) mechanical agitation is another example where the myth of shear damage has had a detrimental effect on the optimal operation of commercial bioprocessing

A potentially scalable method for the harvesting of hMSCs from microcarriers

The use of hMSCs for allogeneic therapies requiring lot sizes of billions of cells will necessitate large-scale culture techniques such as the expansion of cells on microcarriers in bioreactors. Whilst much research investigating hMSC culture on microcarriers has focused on growth, much less involves their harvesting for passaging or as a step towards cryopreservation and storage. A successful new harvesting method has recently been outlined for cells grown on SoloHill microcarriers in a 5L bioreactor [1]. Here, this new method is set out in detail, harvesting being defined as a two-step process involving cell 'detachment' from the microcarriers' surface followed by the 'separation' of the two entities. The new detachment method is based on theoretical concepts originally developed for secondary nucleation due to agitation. Based on this theory, it is suggested that a short period (here 7min) of intense agitation in the presence of a suitable enzyme should detach the cells from the relatively large microcarriers. In addition, once detached, the cells should not be damaged because they are smaller than the Kolmogorov microscale. Detachment was then successfully achieved for hMSCs from two different donors using microcarrier/cell suspensions up to 100mL in a spinner flask. In both cases, harvesting was completed by separating cells from microcarriers using a Steriflip® vacuum filter. The overall harvesting efficiency was >95% and after harvesting, the cells maintained all the attributes expected of hMSC cells. The underlying theoretical concepts suggest that the method is scalable and this aspect is discussed too

Agitation conditions for the culture and detachment of hMSCs from microcarriers in multiple bioreactor platforms

In our recent work in different bioreactors up to 2.5L in scale, we have successfully cultured hMSCs using the minimum agitator speed required for complete microcarrier suspension, N JS. In addition, we also reported a scaleable protocol for the detachment from microcarriers in spinner flasks of hMSCs from two donors. The essence of the protocol is the use of a short period of intense agitation in the presence of enzymes such that the cells are detached; but once detachment is achieved, the cells are smaller than the Kolmogorov scale of turbulence and hence not damaged. Here, the same approach has been effective for culture at N JS and detachment in-situ in 15mL ambr™ bioreactors, 100mL spinner flasks and 250mL Dasgip bioreactors. In these experiments, cells from four different donors were used along with two types of microcarrier with and without surface coatings (two types), four different enzymes and three different growth media (with and without serum), a total of 22 different combinations. In all cases after detachment, the cells were shown to retain their desired quality attributes and were able to proliferate. This agitation strategy with respect to culture and harvest therefore offers a sound basis for a wide range of scales of operation

Scale‐down studies for the scale‐up of a recombinant Corynebacterium glutamicum fed‐batch fermentation; loss of homogeneity leads to lower levels of cadaverine production

BACKGROUNDThe loss of efficiency and performance of bioprocesses on scale‐up is well known, but not fully understood. This work addresses this problem, by studying the effect of some fermentation gradients (pH, glucose and oxygen) that occur at the larger scale in a bench‐scale two‐compartment reactor (Plug flow reactor (PFR) + Stirred tank reactor (STR)) using the cadaverine‐producing recombinant Corynebacterium glutamicum DM1945 Δact3 Ptuf‐ldcC_OPT. The new scale‐down strategy developed here studied the effect of increasing the magnitude of fermentation gradients by considering not only the average cell residence time in the PFR (τPFR), but also the mean frequency at which the bacterial cells entered the PFR (fm) section of the two‐compartment reactor.RESULTSOn implementing this strategy the cadaverine production decreased on average by 26 %, 49 % and 59 % when the τPFR was increased from 1 min to 2 min and then 5 min respectively compared to the control fermentation. The CO2 productivity was highest (3.1‐fold that of the control) at a τPFR of 5 min, but no losses were observed in biomass production. However, the population of viable but non‐culturable cells increased as the magnitude of fermentation gradients was increased.CONCLUSIONThis study demonstrated that C. glutamicum DM1945 Δact3 Ptuf‐ldcC_OPT physiological response was a function of the magnitude of fermentation gradients simulated. The adaptations of a bacterial cell within a heterogeneous environment ultimately result in losses in fermentation productivity as observed here.</div