Engineering characterisation of single-use bioreactor technology for mammalian cell culture applications

The thesis describes an experimental investigation of the fluid dynamics within novel single-use bioreactors (SUBs), including stirred, rocked and pneumatically driven mixing systems. Biological studies to ascertain the impact of hydrodynamic conditions within these systems, on the growth and protein productivity of a mammalian cell line, are also presented. Two-dimensional velocity measurements within different SU technology were acquired with the use of a whole flow field laser-based technique, Particle Image Velocimetry (PIV). Fluid dynamic characteristics including velocity, turbulence, turbulent kinetic energy and vorticity were determined from time-resolved and phase-resolved velocity measurements. Commercial bioreactor systems were modified, if needed, in order to perform experiments within bioreactors commonly used for cell culture experiments, in preference to using vessel mimics. The fluid flow characteristics in both the impeller region and bulk fluid of a single-impeller stirred bioreactor were investigated, facilitating an enhanced understanding of the spatial distribution of velocity and turbulence throughout the vessel. PIV was also used to study the flow in a dual-impeller stirred bioreactor, providing a rare examination of the interaction between the flow fields generated by two impellers. The whole flow field velocity and turbulence characteristics measured within a rocked bag and pneumatically driven vessel, allow a unique insight into the flow pattern and turbulence distribution within two novel cell culture systems. Cell viability, size, growth, protein productivity and metabolites concentration were monitored under different cell culture operating conditions. Cell culture experiments, combined with the hydrodynamic information acquired using PIV, offer an insight into the physiological response of the cells to highly disparate flow conditions. This information helped to understand how the hydrodynamics induced by novel commercially used mixing systems, can impact upon a mammalian cell line. Having implications for an augmented capacity for cross-compatibility, in addition to enhanced strategies for scale translation and optimal bioreactor design

Fluid Dynamic Characterization of a Laboratory Scale Rocked Bag Bioreactor

Single-use technology is being widely adopted for the manufacture of biotherapeutics and cell therapy products. Rocked single-use bioreactors in particular have been commonly used, however, the hydrodynamics have rarely been characterized and are poorly understood. In this work, phase-resolved Particle Image Velocimetry and high frequency visual fluid tracking were used to investigate the flow pattern and velocity characteristics for the first time. The studies were performed on an optically accessible mimic of a Sartorius 2L CultiBag at different conditions. Wave formation was observed and higher rocking speeds caused the fluid to move proportionately out of phase with respect to the platform. Dimensional comparisons of fluid velocities with conventional bioreactors suggest that similar fluid dynamics characteristics can be achieved between rocked and stirred configurations. These results provide a first insight into the fluid dynamics of a novel bioreactor type at relevant process conditions supporting the generation of scale translation laws

Modelling and optimisation of the one-pot, multi-enzymatic synthesis of chiral amino-alcohols based on microscale kinetic parameter determination

Advances in synthetic biology are facilitating the de novo design of complex, multi-step enzymatic conversions for industrial organic synthesis. This work describes the integration of multi-step enzymatic pathway construction with enzyme kinetics and bioreactor modelling, in order to optimise the synthesis of chiral amino-alcohols using engineered Escherichia coli transketolases (TK) and the Chromobacterium violaceum transaminase (TAm). The specific target products were (2S,3S)-2-aminopentane-1,3-diol (APD) and (2S,3R)-2-amino-1,3,4-butanetriol (ABT). Kinetic models and parameters for each of the enzymatic steps were first obtained using automated microwell experiments. These identified the TK-catalysed conversions as being up to 25 times faster than the subsequent TAm conversions and inhibition of TAm by the amino-donor used, (S)-(−)-α-methylbenzylamine (MBA), as limiting the overall conversion yields. In order to better ‘match’ the relative rates of the two enzymes an E. coli expression system, based on two compatible plasmids, was constructed to produce both enzymes in a single host. By control of induction time and temperature it was possible to produce six times more recombinant TAm than TK to help balance the reaction rates. To overcome MBA inhibition and an unfavourable reaction equilibrium, fed-batch addition of the amino-donor was introduced as well as the use of isopropylamine as an alternate amino-donor. Adopting these strategies, and using the kinetic models to optimise feeding strategies, the one pot syntheses of APD and ABT were successfully scaled-up to preparative scales. Excellent agreement was found between the kinetic profiles and yields predicted and those achieved experimentally at the larger scale. In this case the integration of these multi-disciplinary approaches enabled us to achieve up to a 6 fold greater yield using concentrations an order of magnitude higher than in previous preparative scale batch bioconversions carried out sequentially

Fluid dynamic characterization of a fluidized‐bed perfusion bioreactor with CFD–DEM simulation

BACKGROUND: In the recent development of regenerative medicine, the low yields of progenitor cells have limited the large-scale clinical applications. To overcome the limitation, a novel fluidized bed bioreactor has emerged. However, a detailed understanding of the fluid dynamics is still lacking. RESULTS: A three-dimensional modelling approach that couples computational fluid dynamics (CFD) and discrete element method (DEM) was used to simulate the liquid and solid flows in a bioreactor being designed for stem cells expansion. The model was validated by comparing the simulation results with literature experimental data (Chem. Eng. Sci. 60: 1889-1900 (2005)), which showed a good agreement. Using the validated model, the effects of the superficial liquid velocity, particle size and particle density on the solids volume fraction, shear stress on the particles and liquid-solid mass transfer coefficient of dissolved oxygen and glucose were analyzed. CONCLUSIONS: Simulation results show that particle size and density have an important impact on the shear stress distribution, and that the liquid velocity affects the shear stress distribution rather modestly when its value is beyond the minimum fluidization velocity. The liquid-particle mass transfer coefficient of dissolved oxygen and glucose can be improved by raising the liquid velocity, and the adoption of a high-density material allows the reactor to operate with higher liquid velocities before reaching shear stress heterogeneities. Furthermore, the two objectives, (i) maintaining lower and homogeneously distributed shear stress and (ii) improving mass transfer, pose conflicting requirements on certain design parameters which need to be carefully considered in the reactor design.</p

Fluid dynamic characterization of a fluidized‐bed perfusion bioreactor with CFD–DEM simulation

BACKGROUND: In the recent development of regenerative medicine, the low yields of progenitor cells have limited the large-scale clinical applications. To overcome the limitation, a novel fluidized bed bioreactor has emerged. However, a detailed understanding of the fluid dynamics is still lacking. RESULTS: A three-dimensional modelling approach that couples computational fluid dynamics (CFD) and discrete element method (DEM) was used to simulate the liquid and solid flows in a bioreactor being designed for stem cells expansion. The model was validated by comparing the simulation results with literature experimental data (Chem. Eng. Sci. 60: 1889-1900 (2005)), which showed a good agreement. Using the validated model, the effects of the superficial liquid velocity, particle size and particle density on the solids volume fraction, shear stress on the particles and liquid-solid mass transfer coefficient of dissolved oxygen and glucose were analyzed. CONCLUSIONS: Simulation results show that particle size and density have an important impact on the shear stress distribution, and that the liquid velocity affects the shear stress distribution rather modestly when its value is beyond the minimum fluidization velocity. The liquid-particle mass transfer coefficient of dissolved oxygen and glucose can be improved by raising the liquid velocity, and the adoption of a high-density material allows the reactor to operate with higher liquid velocities before reaching shear stress heterogeneities. Furthermore, the two objectives, (i) maintaining lower and homogeneously distributed shear stress and (ii) improving mass transfer, pose conflicting requirements on certain design parameters which need to be carefully considered in the reactor design.</p

An additive manufacturing approach to bioreactor design for mesenchymal stem cell culture

Bioreactor design is a challenging endeavour that aims to provide the most ideal environment in which cells can grow and biological reactions can occur. The emergence of regenerative medicine and stem cell therapies has led to the need for more diverse environmental requirements in the bioreactor design space. The study presented uses an additive manufacturing approach for the initial design phase of a packed/fluidized bed bioreactor for mesenchymal stem cell expansion. Combining 3D-printing with CFD for precision control over the bioreactor flow dynamics. Novel flow distributors were developed to engender swirling particle fluidization. The design was simulated and optimised using CFD, demonstrating an increase from 0.01 m/s to 0.02 m/s in the radial velocity of 3.0 mm macrocarriers (1080 kg/m3) at the minimum fluidization velocity. An autoclavable prototype was constructed to illustrate proof-of-concept in the use of swirling flow distribution to enhance cell attachment efficiency (compared to static culture system). Commercial Cytodex 1 carriers were tested: an improvement in attachment efficiency after 24 h from 50 % to 95 % was induced by the swirling flow distributor, with subsequent expansion of 2.4-fold after 6 days of culture. The computational design, modelling and 3D-printing of complex geometric architecture that control the flow dynamics within a bioreactor, provides a novel approach to bioprocess unit operation development for manufacturing novel ATMPs.</p

Development of thermo-responsive polycaprolactone macrocarriers conjugated with Poly(N-isopropyl acrylamide) for cell culture

Poly(N-isopropyl acrylamide) (PNIPAAm) is a well-known 'smart' material responding to external stimuli such as temperature. PNIPAAm was successfully conjugated to polycaprolactone (PCL) bead surfaces through amidation reaction. Functionalization steps were characterized and confirmed by Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, scanning electron microscopy and Energy Dispersion Spectroscopy. PNIPAAm-conjugated PCL allowed human dermal fibroblast cells (HDF) and mesenchymal stem cells (MSC) to adhere, spread, and grow successfully. By reducing the temperature to 30 °C, more than 70% of HDF were detached from PNIPAAm-conjugated PCL macrocarriers with 85% viability. The cell detachment ratio by trypsin treatment was slightly higher than that induced by reduced temperature, however, cell detachment from PNIPAAm-conjugated macrocarriers by lowering the temperature significantly reduced cell death and increased both cell viability and the recovery potential of the detached cells. HDF attachment and detachment were also observed by Live-Dead staining and phase contrast imaging. The expression of extracellular matrix proteins such as Laminin and Fibronectin was also affected by the trypsinization process but not by the reduced temperature process. Taken together, our results showed that thermo-responsive macrocarriers could be a promising alternative method for the non-invasive detachment of cells, in particular for tissue engineering, clinical applications and the use of bioreactors

On the use of 3D-printed flow distributors to control particle movement in a fluidized bed

3D-printing has emerged as a revolutionary tool for the rapid-prototyping of both conventional and novel products. Its use can foster innovative solutions to engineering challenges that previously would have been considered impractical. We propose the manipulation and control of multiphase systems (e.g. fluidized bed bioreactors) as one such use. The article presented investigates the particle flow and mixing within a fluidized bed induced by novel additive manufactured flow distributors. The fluidized bed is designed for adherent cell expansion on 3 mm diameter calcium alginate macrocarriers. Particle tracking was employed to assess the influence of flow channel angle and direction upon the radial flux of the carriers within the vessel. Uni-directional angled (45o) flow channels generated swirling fluidization of the macrocarriers; increasing particle radial velocities by up to 5.2 times (compared to their vertical flow channel counterparts) at a liquid superficial velocity of 0.0047 m/s. Swirling fluidization also generated particle bed heights up to 52% higher than vertical flow channels. Bi-directional flow channels improved the spatial uniformity of particle radial velocity. In addition, the angular flow channels generated axial velocity gradients that facilitate fluctuations in the height of fluidized particles, thus counteracting elutriation. Finally, lower liquid flow rates and interstitial velocities were required to mix the particles, thus leading to lower hydrodynamic stresses introduced into the system. The introduction of multi-directional flow channels provides novel options to the design and use of flow distributor technology. We foresee additional advancements in chemical engineering product design utilizing additive manufacturing to manipulate multiphase flows

On the use of 3D-printed flow distributors to control particle movement in a fluidized bed

3D-printing has emerged as a revolutionary tool for the rapid-prototyping of both conventional and novel products. Its use can foster innovative solutions to engineering challenges that previously would have been considered impractical. We propose the manipulation and control of multiphase systems (e.g. fluidized bed bioreactors) as one such use. The article presented investigates the particle flow and mixing within a fluidized bed induced by novel additive manufactured flow distributors. The fluidized bed is designed for adherent cell expansion on 3 mm diameter calcium alginate macrocarriers. Particle tracking was employed to assess the influence of flow channel angle and direction upon the radial flux of the carriers within the vessel. Uni-directional angled (45o) flow channels generated swirling fluidization of the macrocarriers; increasing particle radial velocities by up to 5.2 times (compared to their vertical flow channel counterparts) at a liquid superficial velocity of 0.0047 m/s. Swirling fluidization also generated particle bed heights up to 52% higher than vertical flow channels. Bi-directional flow channels improved the spatial uniformity of particle radial velocity. In addition, the angular flow channels generated axial velocity gradients that facilitate fluctuations in the height of fluidized particles, thus counteracting elutriation. Finally, lower liquid flow rates and interstitial velocities were required to mix the particles, thus leading to lower hydrodynamic stresses introduced into the system. The introduction of multi-directional flow channels provides novel options to the design and use of flow distributor technology. We foresee additional advancements in chemical engineering product design utilizing additive manufacturing to manipulate multiphase flows