Protein Expression via Transient Transfection of Mammalian Cells in a WAVE Bioreactor.

Large culture volumes are often required when expression constructs are particularly low-yielding or when end uses require significant amounts of material. In these cases, a single homogeneous culture is usually more convenient, in terms of both consistency of expression and labor/resource requirements, than multiple parallel cultures. Using a WAVE Bioreactor culture, volumes as high as 500L may be achieved in a single vessel. Here, we describe the transfection of Expi293F cells in a disposable 50L Cellbag on a WAVE Bioreactor platform to produce recombinant protein. The methods described herein may be adapted, with suitable optimizations, for other suspension-adapted mammalian cell lines.

Trends in Monoclonal Antibody Production Using Various Bioreactor Syst.

Monoclonal antibodies are widely used as diagnostic reagents and for therapeutic purposes, and their demand is increasing extensively. To produce these proteins in sufficient quantities for commercial use, it is necessary to raise the output by scaling up the production processes. This review describes recent trends in high-density cell culture systems established for monoclonal antibody production that are excellent methods to scale up from the lab-scale cell culture. Among the reactors, hollow fiber bioreactors contribute to a major part of high-density cell culture as they can provide a tremendous amount of surface area in a small volume for cell growth. As an alternative to hollow fiber reactors, a novel disposable bioreactor has been developed, which consists of a polymer-based supermacroporous material, cryogel, as a matrix for cell growth. Packed bed systems and disposable wave bioreactors have also been introduced for high cell density culture. These developments in high-density cell culture systems have led to the monoclonal antibody production in an economically favourable manner and made monoclonal antibodies one of the dominant therapeutic and diagnostic proteins in biopharmaceutical industry.

Protein Expression via Transient Transfection of Mammalian Cells in a WAVE Bioreactor.

Large culture volumes are often required when expression constructs are particularly low-yielding or when end-uses require significant amounts of material. In these cases, a single homogenous culture is usually more convenient, in terms of both consistency of expression and labour/resource requirements, than multiple parallel cultures. Using a WAVE Bioreactor culture volumes as high as 500 L may be achieved in a single vessel. Here we describe the transfection of 293-6E cells in a disposable 50 L Cellbag on a WAVE Bioreactor platform to produce recombinant protein. The methods described herein may be adapted, with suitable optimizations, for other suspension-adapted mammalian cell lines.

Rapid expansion in the WAVE bioreactor of clinical scale cells for tumor immunotherapy.

Cell-based immunotherapy using natural killer (NK) cells, cytokine-induced killer (CIK) cells and dendritic cells (DCs) is emerging as a potential novel approach in the auxiliary treatment of a tumor. However, non-standard operation procedure, small-scale cell number, or human error may limit the clinical development of cell-based immunotherapy. To simplify clinical scale NK cells, CIK cells and DCs expansions, we investigated the use of the WAVE bioreactor, a closed system bioreactor that utilizes active perfusion to generate high cell numbers in minimal volumes. We developed an optimized rapid expansion protocol for the WAVE bioreactor that produces clinically relevant number of cells for our adoptive cell transfer clinical protocols. The high proliferative rate, surface phenotypes, and cytotoxicity of these immune cells, as well as the safety of cultivation were analyzed to illuminate the effect of WAVE bioreactor. The results demonstrated that the benefit of utilizing modern WAVE bioreactors in cancer immunotherapy was simple, safe, and flexible production.

DROSOPHILA S2 cell culture in a WAVE Bioreactor: potential for scaling up the production of the recombinant rabies virus glycoprotein.

The transmembrane rabies virus glycoprotein (RVGP) is the main antigen of vaccine formulations used around the world to prevent rabies, the most lethal preventable infectious disease known. The objective of this work was to evaluate the potential of a bioreactor using wave-induced agitation in the initial steps of scaling up the rRVGP production process by a Drosophila melanogaster S2 cell line to produce rRVGP in sufficient quantities for immunization and characterization studies. Taking advantage of some remarkable features recognized in Drosophila S2 cells for scaling the culture process, a robust recombinant lineage (S2MtRVGPH-His) engineered by our group for the expression of rRVGP using a copper-inducible promoter was used in the bioreactor cultures. The WAVE Bioreactor was chosen because it represents an innovative approach to the cultivation of animal cells using single-use technology. For that purpose, we firstly established a procedure for culturing the S2MtRVGPH-His lineage in 100 mL Schott flasks. Using an inoculum of 5 × 105 cells/mL in culture medium (Sf900-III) induced with solution of CuSO4 (0.7 mM) and a convenient pH range (6.2-7.0), optimal parameter values such as time of induction (72 h) and temperature (28 °C) to increase rRVGP production could be defined. This procedure was reproduced in culture experiments conducted in a WAVE Bioreactor™ 2/10 using a 2 L Cellbag. The results in Schott flasks and in WAVE Bioreactor™ were very similar, yielding a maximum titer of rRVGP above of 1 mg.L-1. The immunization study showed that the rRVGP produced in the bioreactor was of high immunogenic quality.

Automated Closed-System Expansion of Pluripotent Stem Cell Aggregates in a Rocking-Motion Bioreactor.

Pluripotent stem cell suspension aggregates have proven to be an efficient and phenotypically stable means for expansion and directed differentiation. Bioreactor systems with automation of perfusion, fluidization, and gas exchange are essential for scaling up pluripotent stem cell cultures. Since stem cell pluripotency and differentiation are affected by both chemical and physical signals, we investigated a low-shear method for the expansion of cells in a rocking-motion bioreactor. The rocking motion drives continual mixing and aeration, and the single-use disposable bioreactors avoid issues around contamination during seeding, medium exchange, passage, and cell harvest. Serial passaging from a 150 mL to a 1 L scale was demonstrated, achieving cell densities of up to 4 million cells/mL. In an average of 13 experiments, pluripotent stem cell aggregates expanded 5.7-fold (with maximal 9.5-fold expansion) and maintained 97% viability over 4 days in a rocking bioreactor culture. In seven experiments with improved culture conditions, the average expansion was 6.8-fold. Maintenance of pluripotency was confirmed by differentiation to all three germ layers and surface marker expression, and the expanded aggregates maintained a stable normal karyotype. The automation associated with the rocking platform bioreactor required no user intervention during the 4-day culture, providing hands-off expansion of pluripotent stem cells.

Drosophila S2 cell culture in a wave bioreactor: potential for scaling up the production of the recombinant rabies virus glycoprotein

The transmembrane rabies virus glycoprotein (RVGP) is the main antigen of vaccine formulations used around the world to prevent rabies, the most lethal preventable infectious disease known. The objective of this work was to evaluate the potential of a bioreactor using wave-induced agitation in the initial steps of scaling up the rRVGP production process by a Drosophila melanogaster S2 cell line to produce rRVGP in sufficient quantities for immunization and characterization studies. Taking advantage of some remarkable features recognized in Drosophila S2 cells for scaling the culture process, a robust recombinant lineage (S2MtRVGPH-His) engineered by our group for the expression of rRVGP using a copper-inducible promoter was used in the bioreactor cultures. The WAVE Bioreactor was chosen because it represents an innovative approach to the cultivation of animal cells using single-use technology. For that purpose, we firstly established a procedure for culturing the S2MtRVGPH-His lineage in 100 mL Schott flasks. Using an inoculum of 5 x 10(5) cells/mL in culture medium (Sf900-III) induced with solution of CuSO4 (0.7 mM) and a convenient pH range (6.2-7.0), optimal parameter values such as time of induction (72 h) and temperature (28 degrees C) to increase rRVGP production could be defined. This procedure was reproduced in culture experiments conducted in a WAVE Bioreactor (TM) 2/10 using a 2 L Cellbag. The results in Schott flasks and inWAVE Bioreactor (TM) were very similar, yielding a maximum titer of rRVGP above of 1 mg.L-1. The immunization study showed that the rRVGP produced in the bioreactor was of high immunogenic quality.

The development of sexual stage malaria gametocytes in a Wave Bioreactor.

BACKGROUND: Blocking malaria gametocyte development in RBCs or their fertilization in the mosquito gut can prevent infection of the mosquito vector and passage of disease to the human host. A 'transmission blocking' strategy is a component of future malaria control. However, the lack of robust culture systems for producing large amounts of Plasmodium falciparum gametocytes has limited our understanding of sexual-stage malaria biology and made vaccine or chemotherapeutic discoveries more difficult. METHODS: The Wave BioreactorTM 20/50 EHT culture system was used to develop a convenient and low-maintenance protocol for inducing commitment of P. falciparum parasites to gametocytogenesis. Culture conditions were optimised to obtain mature stage V gametocytes within 2 weeks in a large-scale culture of up to a 1 l. RESULTS: We report a simple method for the induction of gametocytogenesis with N-acetylglucosamine (10 mM) within a Wave Bioreactor. By maintaining the culture for 14-16 days as many as 100 million gametocytes (stage V) were produced in a 1 l culture. Gametocytes isolated using magnetic activated cell sorting (MACS) columns were frozen in aliquots for storage. These were revitalised by thawing and shown to retain their ability to exflagellate and infect mosquitoes (Anopheles stephansi). CONCLUSIONS: The production of gametocytes in the Wave Bioreactor under GMP-compliant conditions will not only facilitate cellular, developmental and molecular studies of gametocytes, but also the high-throughput screening for new anti-malarial drugs and, possibly, the development of whole-cell gametocyte or sporozoite-based vaccines.

A re-usable wave bioreactor for protein production in insect cells.

Wave-mixed bioreactors have increasingly replaced stainless steel stirred tank reactors in seed inoculum productions and mammalian cell-based process developments. Pre-sterilized, single-use plastic bags are used for cultivation, eliminating the risk of cross-contamination and cleaning procedures. However, these advantages come with high consumable costs which is the main barrier to more uptakes of the technology by academic institutions. As an academic Core Facility that faces high demand in protein production from insect cells, we have therefore developed a cost-effective alternative to disposable wave bags. In our study we identified: •A re-usable wave shaken polycarbonate bioreactor for protein production in insect cells achieves protein yields comparable to disposable bags.•The advantages of this re-usable bioreactor are low costs, long life cycle, flexible configuration of accessories and convenient handling due to its rigid shape.

Design of fuzzy PID control algorithm and simulink simulation for temperature control system of wave bioreactors / 军事医学

Objective To design a temperature control strategy for wave bioreactors.Methods According to the requirements of temperature control precision and response speed of wave bioreactors,the traditional PID control method was combined with fuzzy control method which was used to adjust the parameters of the PID control in real time online in order to strengthen the ability of the temperature control strategy to regulate temperature.Results A fuzzy PID controller was completed and simulation results were compared with the traditional PID controller.Conclusion The fuzzy PID control method has a smaller overshoot and shorter stability than the traditional one, so it has a higher temperature control performance.

Baculovirus-mediated expression of GPCRs in insect cells.

G-protein-coupled receptors (GPCRs) are a large family of seven transmembrane proteins that influence a considerable number of cellular events. For this reason, they are one of the most studied receptor types for their pharmacological and structural properties. Solving the structure of several GPCR receptor types has been possible using almost all expression systems, including Escherichia coli, yeast, mammalian, and insect cells. So far, however, most of the GPCR structures solved have been done using the baculovirus insect cell expression system. The reason for this is mainly due to cost-effectiveness, posttranslational modification efficiency, and overall effortless maintenance. The system has evolved so much that variables starting from vector type, purification tags, cell line, and growth conditions can be varied and optimized countless ways to suit the needs of new constructs. Here, we present the array of techniques that enable the rapid and efficient optimization of expression steps for maximal protein quality and quantity, including our emendations.

Simplified protocol for clinical-grade tumor-infiltrating lymphocyte manufacturing with use of the Wave bioreactor.

BACKGROUND AIMS: The high level of complexity of current Good Manufacturing Practice-compliant methods of manufacturing hampers rapid and broad application of treatment with tumor-infiltrating lymphocytes (TILs). METHODS: To ensure higher applicability of TIL production to laboratory routine, a practical and simple protocol of TIL manufacturing with the use of a closed-system bioreactor was developed and implemented at our institution. RESULTS: This protocol enabled significant work load reduction during the most labor-intense step of TIL expansion, and allowed generation of high-quality TIL products, which mediated clinical regression in patients with metastatic melanoma. CONCLUSIONS: Implementation of simplified methods of TIL expansion will speed up dissemination of TIL methods worldwide and will increase patient access to this highly effective treatment.