Cell Culture Bioreactor Research Articles

Design, experimental optimization, and flow analysis of a novel bioreactor for dynamic mammalian cell culture at laboratory scale using Box-Behnken design

Improving the quality of dynamic cell culture in laboratories is an important field in bioengineering. In this study, a novel lab-scale bioreactor using a vibrating agitator and a modified flask has been introduced to create a strong mixing at low shear stress. This bioreactor has been optimized using Box-Behnken design based on three dimensionless important structural factors including disc diameter, vibration amplitude, and the height of the disc placement. Three growth indicators including the specific growth rate, the natural logarithm of the maximum cell density, and productivity have been considered as biological responses. The results show that the disc diameter has the most important role in these indicators. If the disc diameter, vibration amplitude, and the height of disc placement are set to 0.24, 0.02, and 0.4 of the flask diameter, respectively, the values of the specific growth rate, the maximum cell density, and productivity at this optimum settings are 0.033 (h−1), 13.11, and 5133 (cells/(mL.h)), respectively. These values of the indicators are high and indicate the better performance of this bioreactor than other lab-scale bioreactors. In addition, investigating Reynolds number in the fluid flow indicates that in the range of 780 up to 1150, growth indices are high.

Real-time multivariate statistical monitoring of biopharmaceutical processes with no prior product-specific history

Biologics drug substance manufacturing normally comprises several processing steps including cell culture, purification, etc. Real-time Multivariate Statistical Process Monitoring (RT-MSPM) is an integral need in order to achieve early fault detection and prevention to guarantee robust and efficient process performance. As a part of RT-MSPM framework, Batch Evolution Models (BEMs), which take into account the transient phase of a batch prior to reaching full maturity (steady state), are the standard methods for real-time early fault detection in batch processes. Prior product-specific knowledge and training dataset are critical in order to model time (batch maturity) dependencies. Unlike traditional settings, modern biopharmaceutical manufacturing and clinical facilities use modular designs to enable simultaneous production of multiple medicines and quickly pivot from producing one medicine to another where no prior product-specific historical data might be available. In this article, first, a framework is proposed to detect the steady state operation of a manufacturing scale cell culture bioreactor. This is critical to achieve the ultimate goal to demonstrate feasibility of developing a continuous statistical process monitoring framework to monitor the steady state operation of a batch with no prior product specific history. Steady state time series data from previous step are processed following a systematic approach. The requirements for statistical properties of the processed time series in order to leverage data from different products to monitor a new product are outlined. The new framework establishes a guideline to develop models using datasets from other products with different recipes to monitor new products with no prior product-specific history. A novel software product is developed to test the framework. The framework is successfully applied for real-time monitoring of fast-rate sensors of a manufacturing cell culture bioreactor.

Study of hydrodynamic stress in cell culture bioreactors via lattice-Boltzmann CFD simulations supported by micro-probe shear stress method

Mammalian cell cultivation in pharmaceutical industry can last up to units of weeks and requires proper transport of nutrients and oxygen for cell growth and production. Given the long time period, cells experience flow fields from all bioreactor’s zones, where the energy dissipation rate (ε) varies substantially. Shear sensitive micro-probes with size comparable to cells and Kolmogorov eddies are used for the determination of the maximum hydrodynamic stress (τmax) in bioreactors. For the very first time, the micro-probe method is applied successfully not only to laboratory (3 L) and pilot scale (80 L and 200 L), but also to industrial production scale bioreactor (12,500 L) with Rushton turbine and pitched-blade (RT-PB) impeller configuration. Experimentally obtained data are used for the validation of comprehensive CFD scale-up study, using the Lattice-Boltzmann large eddy simulation (LB-LES) method. Besides τmax, this work also focuses on the study of mixing time and flow field attributes.

Validation of a CFD model for cell culture bioreactors at large scale and its application in scale-up

Among all the operating parameters that control the cell culture environment inside bioreactors, appropriate mixing and aeration are crucial to ensure sufficient oxygen supply, homogeneous mixing, and CO2 stripping. A model-based manufacturing facility fit approach was applied to define agitation and bottom air flow rates during the process scale-up from laboratory to manufacturing, of which computational fluid dynamics (CFD) was the core modeling tool. The realizable k-ε turbulent dispersed Eulerian gas-liquid flow model was established and validated using experimental values for the volumetric oxygen transfer coefficient (kLa). Model validation defined the process operating parameter ranges for application of the model, identified mixing issues (e.g., impeller flooding, dissolved oxygen gradients, etc.) and the impact of antifoam on kLa. Using the CFD simulation results as inputs to the models for oxygen demand, gas entrance velocity, and CO2 stripping aided in the design of the agitation and bottom air flow rates needed to meet cellular oxygen demand, control CO2 levels, mitigate risks for cell damage due to shear, foaming, as well as fire hazards due to high O2 levels in the bioreactor gas outlet. The recommended operating conditions led to the completion of five manufacturing runs with a 100% success rate. This model-based approach achieved a seamless scale-up and reduced the required number of at-scale development batches, resulting in cost and time savings of a cell culture commercialization process.

Enhancing real-time cell culture monitoring: Automated Raman model optimization with Taguchi method.

Raman spectroscopy has found widespread usage in monitoring cell culture processes both in research and practical applications. However, commonly, preprocessing methods, spectral regions, and modeling parameters have been chosen based on experience or trial-and-error strategies. These choices can significantly impact the performance of the models. There is an urgent need for a simple, effective, and automated approach to determine a suitable procedure for constructing accurate models. This paper introduces the adoption of a design of experiment (DoE) method to optimize partial least squares models for measuring the concentration of different components in cell culture bioreactors. The experimental implementation utilized the orthogonal test table L25(56). Within this framework, five factors were identified as control variables for the DoE method: the window width of Savitzky-Golay smoothing, the baseline correction method, the order of preprocessing steps, spectral regions, and the number of latent variables. The evaluation method for the model was considered as a factor subject to noise. The optimal combination of levels was determined through the signal-to-noise ratio response table employing Taguchi analysis. The effectiveness of this approach was validated through two cases, involving different cultivation scales, different Raman spectrometers, and different analytical components. The results consistently demonstrated that the proposed approach closely approximated the global optimum, regardless of data set size, predictive components, or the brand of Raman spectrometer. The performance of models recommended by the DoE strategy consistently surpassed those built using raw data, underscoring the reliability of models generated through this approach. When compared to exhaustive all-combination experiments, the DoE approach significantly reduces calculation times, making it highly practical for the implementation of Raman spectroscopy in bioprocess monitoring.

A assembled plate column bioreactor for adherent cell culture

The ideal bioreactor should be highly efficient, cost-effective and able to promote cells adhesion growth. In this study, we constructed a plate-tower bioreactor with S-loop in each layer based on multilayer plate bioreactor and plate column theory. Human hepatocellular carcinomas(HepG2) and human normal liver cell L02 (L02) cells were employed evaluation of bioreactor performance. The results show that cells were able to adhere to the plates and the density reached to 1～1.2 × 105/cm2. When circulating flow rate of the reactor is 8 ml/min and the oxygen supply concentration is 40 %, the sufficient material and gas exchange in bioreactor, the amount of albumin, urea and cytochrome P450(CYP450) content in the hyperoxia group were 3.31, 2.58 and 2.6 times higher than that of the control group, there was no obvious cell damage. This condition up the regulation of genes in more metabolic pathways and improved the metabolic capacity of hepatocytes. The single-layer area and the number of bioreactors can be flexibly adjusted, and the manufacturing cost of single-layer is close to that of ordinary plastic cell culture dishes. Either batch or continuous mode can be used in the bioreactor. In view of the above characteristics, the bioreactor is more conducive to practical application.

An artificial neural network-based machine learning approach to correct coarse-mesh-induced error in computational fluid dynamics modeling of cell culture bioreactor

Computational Fluid Dynamics (CFD) is a valuable tool for studying fluid environments within cell culture bioreactors and optimizing processing parameters, but it can be computationally expensive. This study developed an artificial neural network (ANN)-based machine learning model to predict and correct the coarse-mesh-induced errors in CFD modeling of a spinner flask bioreactor. A baseline ANN model was trained to predict the velocity error function between the coarse and optimized reference mesh results at one rotational speed (90 rpm), demonstrating that the ANN-based approach could correct the coarse-mesh velocity with RMSE values of nodal velocities improved by an average of ∼20% at different rotational speeds. The effect of ANN structure, input data normalization, and training dataset combinations on prediction performance was evaluated. More neurons and hidden layers generated better results but required more computational time for training. The model’s generalization capabilities were further evaluated in case studies of correcting velocity and Kolmogorov length at different fluid viscosity and bioreactor impeller geometry conditions. Results suggested that the ANN model had better generalization in correcting Kolmogorov length than velocity. This research provides insights into using a machine learning approach to enhance CFD modeling in bioreactor applications, contributing to advancing tissue engineering processes.

Development of a scalable single process for producing SARS-CoV-2 RBD monomer and dimer vaccine antigens.

We have developed a single process for producing two key COVID-19 vaccine antigens: SARS-CoV-2 receptor binding domain (RBD) monomer and dimer. These antigens are featured in various COVID-19 vaccine formats, including SOBERANA 01 and the licensed SOBERANA 02, and SOBERANA Plus. Our approach involves expressing RBD (319-541)-His6 in Chinese hamster ovary (CHO)-K1 cells, generating and characterizing oligoclones, and selecting the best RBD-producing clones. Critical parameters such as copper supplementation in the culture medium and cell viability influenced the yield of RBD dimer. The purification of RBD involved standard immobilized metal ion affinity chromatography (IMAC), ion exchange chromatography, and size exclusion chromatography. Our findings suggest that copper can improve IMAC performance. Efficient RBD production was achieved using small-scale bioreactor cell culture (2L). The two RBD forms - monomeric and dimeric RBD - were also produced on a large scale (500L). This study represents the first large-scale application of perfusion culture for the production of RBD antigens. We conducted a thorough analysis of the purified RBD antigens, which encompassed primary structure, protein integrity, N-glycosylation, size, purity, secondary and tertiary structures, isoform composition, hydrophobicity, and long-term stability. Additionally, we investigated RBD-ACE2 interactions, in vitro ACE2 recognition of RBD, and the immunogenicity of RBD antigens in mice. We have determined that both the monomeric and dimeric RBD antigens possess the necessary quality attributes for vaccine production. By enabling the customizable production of both RBD forms, this unified manufacturing process provides the required flexibility to adapt rapidly to the ever-changing demands of emerging SARS-CoV-2 variants and different COVID-19 vaccine platforms.

Construction of a novel kinetic model for the production process of a CVA6 VLP vaccine in CHO cells.

The online version contains supplementary material available at 10.1007/s10616-023-00598-8.

Engineering a sustainable protein revolution: Recent advances in cultured meat production

AbstractEmerging as a food of the future, cultured meat is created by growing animal cells outside the organism instead of relying on traditional animal rearing and slaughtering practices. This innovative approach shows great promise in addressing challenges associated with resource utilization, environmental pollution, and public health concerns often encountered in conventional livestock production. In the past decade, popularity of cultured meat has grown enough to be considered as a novel food in regions like Europe, the United Kingdom, and China. This paper discusses the recent advancements in technologies for sustainable cultured meat production. It examines the latest developments in cell sources, cell culture media optimization, bioreactor design, 3D printing, and tissue engineering approaches, which have greatly enhanced the efficiency and scalability of cultured meat production. This food can have a tremendous application as a novel functional food. This review explores the future possibility of applying cultured meat matrix for delivery of hydrophobic nutraceuticals, prebiotics, and probiotics (Bacillus sp).

Advancing our understanding of bioreactors for industrial-sized cell culture: health care and cellular agriculture implications.

Bioreactors are advanced biomanufacturing tools that have been widely used to develop various applications in the fields of health care and cellular agriculture. In recent years, there has been a growing interest in the use of bioreactors to enhance the efficiency and scalability of these technologies. In cell therapy, bioreactors have been used to expand and differentiate cells into specialized cell types that can be used for transplantation or tissue regeneration. In cultured meat production, bioreactors offer a controlled and efficient means of producing meat without the need for animal farming. Bioreactors can support the growth of muscle cells by providing the necessary conditions for cell proliferation, differentiation, and maturation, including the provision of oxygen and nutrients. This review article aims to provide an overview of the current state of bioreactor technology in both cell therapy and cultured meat production. It will examine the various bioreactor types and their applications in these fields, highlighting their advantages and limitations. In addition, it will explore the future prospects and challenges of bioreactor technology in these emerging fields. Overall, this review will provide valuable insights for researchers and practitioners interested in using bioreactor technology to develop innovative solutions in the biomanufacturing of therapeutic cells and cultured meat.

Production of High-Value Proteins under Stringent Cost Constraints—The Case of Hollow Fiber Technology for Cell Culture

For decades, the benefits of utilizing hollow fiber bioreactors for continuous cell culture to produce monoclonal antibodies have been widely recognized. However, the suitability of this technology for laboratories or centers with limited resources and expertise seeking to expand their production capacity is uncertain, mainly due to unknown cost-effectiveness. In this study, a hollow fiber bioreactor with a 4.7-mL culture volume was used to culture a hybridoma clone producing immunoglobulin G antibody specific to hemoglobin F (HbF). The antibody reached a maximum concentration of 1.22 g/L and totaled 21 mg over a 44-day culture period. This preliminary production data was used to estimate the cost of consumables required for using the hollow fiber bioreactor to produce 130 mg of monoclonal antibodies, which was found to be THB35.8k (£880). The cost was slightly more expensive than batch cultivation in typical culture dishes, which ranged from THB27.8 to 30.2k (£680 to 740). Despite the advantages in terms of reduced hands-on time, shorter production duration, and highly concentrated products, the primary challenges associated with using hollow fiber bioreactors were the cost and availability of the cartridges.

Ultrathin and Flexible Bioelectronic Arrays for Functional Measurement of iPSC-Cardiomyocytes under Cardiotropic Drug Administration and Controlled Microenvironments.

Emerging heart-on-a-chip technology is a promising tool to establish in vitro cardiac models for therapeutic testing and disease modeling. However, due to the technical complexity of integrating cell culture chambers, biosensors, and bioreactors into a single entity, a microphysiological system capable of reproducing controlled microenvironmental cues to regulate cell phenotypes, promote iPS-cardiomyocyte maturity, and simultaneously measure the dynamic changes of cardiomyocyte function in situ is not available. This paper reports an ultrathin and flexible bioelectronic array platform in 24-well format for higher-throughput contractility measurement under candidate drug administration or defined microenvironmental conditions. In the array, carbon black (CB)-PDMS flexible strain sensors were embedded for detecting iPSC-CM contractility signals. Carbon fiber electrodes and pneumatic air channels were integrated to provide electrical and mechanical stimulation to improve iPSC-CM maturation. Performed experiments validate that the bioelectronic array accurately reveals the effects of cardiotropic drugs and identifies mechanical/electrical stimulation strategies for promoting iPSC-CM maturation.

Improved control strategies for the environment within cell culture bioreactors

This paper describes the development of improved control strategies for the standard environmental conditions in a fed-batch bioreactor used for monoclonal antibody cell culture. The consequences of relying on fixed parameter PID based controllers are considered and poor performance is demonstrated as a consequence of non-linearity and loop interactions. The benefits from adopting a more sophisticated control strategy are considered. Model Predictive Control (MPC) relies on a process model that can be identified from small system perturbations. It considers the predicted longer-term response and consequently can deliver improved control and satisfy user defined constraints. Results from experimental trials demonstrate the capability of MPC and the merits are discussed with regards to industrial application.

Bubble column and airlift bioreactor systems for animal cell culture applications

AbstractMany biopharmaceutical industries have focused on the screening for novel cell lines and producers for manufacturing of advanced therapeutics. Commercial scale productions at reduced cost and improved productivity are only possible with the bioreactor systems. Animal cell culture bioreactors have all started to draw attention in view that 20th century and advanced significantly over the previous few a long time especially with the need for viral vaccine productions, monoclonal antibody productions and in vitro meat productions. To produce these high value‐added products in large scales, bioreactor configurations are advanced to reduce investment and operational costs, control the process parameters, enhance productivity, and make easier scale‐up. Although novel bioreactor systems have interested in recent years, conventional bubble column and airlift systems are still the most familiar and experienced bioreactors. Pneumatically agitated bioreactors have been studied for many years in animal cell culture. This current review intends to provide an overview of pneumatic bioreactor productions for animal cell culture applications in key items of cell culture and bioreactor technology, for example, design parameters, transport phenomena during cultivation, shear sensitivity of cells. Besides, the previous studies, investigated that the design parameters and process conditions for productivity and shear sensitivity, have been reviewed and summarized.

Real-time online monitoring of insect cell proliferation and baculovirus infection using digital differential holographic microscopy and machine learning.

Real-time, detailed online information on cell cultures is essential for understanding modern biopharmaceutical production processes. The determination of key parameters, such as cell density and viability, is usually based on the offline sampling of bioreactors. Gathering offline samples is invasive, has a low time resolution, and risks altering or contaminating the production process. In contrast, measuring process parameters online provides more safety for the process, has a high time resolution, and thus can aid in timely process control actions. We used online double differential digital holographic microscopy (D3HM) and machine learning to perform non-invasive online cell concentration and viability monitoring of insect cell cultures in bioreactors. The performance of D3HM and the machine learning model was tested for a selected variety of baculovirus constructs, products, and multiplicities of infection (MOI). The results show that with online holographic microscopy insect cell proliferation and baculovirus infection can be monitored effectively in real time with high resolution for a broad range of process parameters and baculovirus constructs. The high-resolution data generated by D3HM showed the exact moment of peak cell densities and temporary events caused by feeding. Furthermore, D3HM allowed us to obtain information on the state of the cell culture at the individual cell level. Combining this detailed, real-time information about cell cultures with methodical machine learning models can increase process understanding, aid in decision-making, and allow for timely process control actions during bioreactor production of recombinant proteins.

Recent Advances in the Development of Bioreactors for Manufacturing of Adoptive Cell Immunotherapies.

Harnessing the human immune system as a foundation for therapeutic technologies capable of recognizing and killing tumor cells has been the central objective of anti-cancer immunotherapy. In recent years, there has been an increasing interest in improving the effectiveness and accessibility of this technology to make it widely applicable for adoptive cell therapies (ACTs) such as chimeric antigen receptor T (CAR-T) cells, tumor infiltrating lymphocytes (TILs), dendritic cells (DCs), natural killer (NK) cells, and many other. Automated, scalable, cost-effective, and GMP-compliant bioreactors for production of ACTs are urgently needed. The primary efforts in the field of GMP bioreactors development are focused on closed and fully automated point-of-care (POC) systems. However, their clinical and industrial application has not yet reached full potential, as there are numerous obstacles associated with delicate balancing of the complex and often unpredictable cell biology with the need for precision and full process control. Here we provide a brief overview of the existing and most advanced systems for ACT manufacturing, including cell culture bags, G-Rex flasks, and bioreactors (rocking motion, stirred-flask, stirred-tank, hollow-fiber), as well as semi- and fully-automated closed bioreactor systems.

Critique – Simulation Apps

This critique discusses the simulation App of a bioreactor (Sartorius D-DCU 100 L) for cell culture, which was included in the article Laboratory-independent exploration of stirred bioreactors and their fluid dynamics by Stefan Seidel et al., Education for Chemical Engineers, xx, xxx.

Mini- and macro-scale direct perfusion bioreactors with optimized flow for engineering 3D tissues.

Bioreactors enabling direct perfusion of cell suspensions or culture media through the pores of 3D scaffolds have long been used in tissue engineering to improve cell seeding efficiency as well as uniformity of cell distribution and tissue development. A macro-scale U-shaped bioreactor for cell culture under perfusion (U-CUP) has been previously developed. In that system, the geometry of the perfusion chamber results in rather uniform flow through most of the scaffold volume, but not in the peripheral regions. Here, the design of the perfusion chamber has been optimized to provide a more homogenous perfusion flow through the scaffold. Then, the design of this macro-scale flow-optimized perfusion bioreactor (macro-Flopper) has been miniaturized to create a mini-scale device (mini-Flopper) compatible with medium-throughput assays. Computational fluid dynamic (CFD) modeling of the new chamber design, including a porous scaffold structure, revealed that Flopper bioreactors provide highly homogenous flow speed, pressure, and shear stress. Finally, a proof-of-principle of the functionality of the Flopper systems by engineering endothelialized stromal tissues using human adipose tissue-derived stromal vascular fraction (SVF) cells has been offered. Preliminary evidence showing that flow optimization improves cell maintenance in the engineered tissues will have to be confirmed in future studies. In summary, two bioreactor models with optimized perfusion flow and complementary sizes have been proposed that might be exploited to engineer homogenous tissues and, in the case of the mini-Flopper, for drug testing assays with a limited amount of biological material.

Micro and milli-chamber bioreactors for human induced pluripotent stem cell culture: Model and experimental validation

One of the main interests in developing microfluidic platforms is the lab-on-a-chip (LOC) applications, including the use of microdevices for the culture of prokaryotic and eukaryotic cells for different applications. In this context, the trapping and control of Micro/nano-scale bubbles have been considered as it becomes a field of growing concern addressing many challenges in LOC applications. In the present work, we present a novel microfluidic bioreactor based on the micropillar design that reduces stresses caused by bubbles on the cell growth in the microchip. The combination of one chamber with 3-pillars and 10-pillars design with the channel heights of 270 µm and 2 mm were evaluated. Computational fluid dynamics simulations (CFD) were conducted to investigate the flow dynamics in the new designs. Numerical values of velocity were compared with those obtained by experimental flow tests to evaluate the performance of the proposed microdevice. Finally, the functionality of the new milli-bioreactor design was validated by culturing human induced pluripotent stem cells (hiPSC) for 5 days. Cell growth, viability and sustain pluripotency were successfully proved using a new platform architecture. This multidisciplinary research provides new insights into the chemical engineering and operability of micro and milli-bioreactors with prospects for future industrial applications.