Small-diameter (<4 mm) vascular constructs are urgently needed for patients requiring replacement of their peripheral vessels. However, successful development of constructs remains a significant challenge. In this study, we successfully developed small-diameter vascular constructs with high patency using our integrally designed computer-controlled bioreactor system. This computer-controlled bioreactor system can confer physiological mechanical stimuli and fluid flow similar to physiological stimuli to the cultured grafts. The medium circulating system optimizes the culture conditions by maintaining fixed concentration of O(2) and CO(2) in the medium flow and constant delivery of nutrients and waste metabolites, as well as eliminates the complicated replacement of culture medium in traditional vascular tissue engineering. Biochemical and mechanical assay of newly developed grafts confirm the feasibility of the bioreactor system for small-diameter vascular engineering. Furthermore, the computer-controlled bioreactor is superior for cultured cell proliferation compared with the traditional non-computer-controlled bioreactor. Specifically, our novel bioreactor system may be a potential alternative for tissue engineering of large-scale small-diameter vascular vessels for clinical use.
In recent years, several automated scale-down bioreactor systems have been developed to increase efficiency in cell culture process development. ambr™ is an automated workstation that provides individual monitoring and control of culture dissolved oxygen and pH in single-use, stirred-tank bioreactors at a working volume of 10-15 mL. To evaluate the ambr™ system, we compared the performance of four recombinant Chinese hamster ovary cell lines in a fed-batch process in parallel ambr™, 2-L bench-top bioreactors, and shake flasks. Cultures in ambr™ matched 2-L bioreactors in controlling the environment (temperature, dissolved oxygen, and pH) and in culture performance (growth, viability, glucose, lactate, Na(+), osmolality, titer, and product quality). However, cultures in shake flasks did not show comparable performance to the ambr™ and 2-L bioreactors.
Tendon and ligament injury is a worldwide health problem, but the treatment options remain limited. Tendon and ligament engineering might provide an alternative tissue source for the surgical replacement of injured tendon. A bioreactor provides a controllable environment enabling the systematic study of specific biological, biochemical, and biomechanical requirements to design and manufacture engineered tendon/ligament tissue. Furthermore, the tendon/ligament bioreactor system can provide a suitable culture environment, which mimics the dynamics of the in vivo environment for tendon/ligament maturation. For clinical settings, bioreactors also have the advantages of less-contamination risk, high reproducibility of cell propagation by minimizing manual operation, and a consistent end product. In this review, we identify the key components, design preferences, and criteria that are required for the development of an ideal bioreactor for engineering tendons and ligaments.
: The cultivation of hairy roots for the production of secondary metabolites offers numerous advantages; hairy roots have a fast growth rate, are genetically stable, and are relatively simple to maintain in phytohormone free media. Hairy roots provide a continuous source of secondary metabolites, and are useful for the production of chemicals for pharmaceuticals, cosmetics, and food additives. In order for hairy roots to be utilized on a commercial scale, it is necessary to scale-up their production. Over the last several decades, significant research has been conducted on the cultivation of hairy roots in various types of bioreactor systems. In this review, we discuss the advantages and disadvantages of various bioreactor systems, the major factors related to large-scale bioreactor cultures, process intensification technologies and overview the mathematical models and computer-aided methods that have been utilized for bioreactor design and development.
Bone Tissue Engineering (TE) aims to develop reproducible and predictive three-dimensional (3D) TE constructs, defined as cell-seeded scaffolds produced by a controlled in vitro process, to heal or replace damaged and non-functional bone. To control and assure the quality of the bone TE constructs, a prerequisite for regulatory authorization, there is a need to develop non-invasive analysis techniques to evaluate TE constructs and to monitor their behavior in real time during in vitro culturing. Most analysis techniques, however, are limited to destructive end-point analyses. This study investigates the use of the non-toxic alamarBlue® (AB) reagent, which is an indicator for metabolic cell activity, for monitoring the cellularity of 3D TE constructs in vitro as part of a bioreactor culturing processes. Within the field of TE, bioreactors have a huge potential in the translation of TE concepts to the clinic. Hence, the use of the AB reagent was not only evaluated in static cultures, but also in dynamic cultures in a perfusion bioreactor set-up. Hereto, the AB assay was successfully integrated in the bioreactor-driven TE construct culture process in a non-invasive way. The obtained results indicate a linear correlation between the overall metabolic activity and the total DNA content of a scaffold upon seeding as well as during the initial stages of cell proliferation. This makes the AB reagent a powerful tool to follow-up bone TE constructs in real-time during static as well as dynamic 3D cultures. Hence the AB reagent can be successfully used to monitor and predict cell confluence in a growing 3D TE construct.
Large numbers of Mesenchymal stem/stromal cells (MSCs) are required for clinical relevant doses to treat a number of diseases. To economically manufacture these MSCs, an automated bioreactor system will be required. Herein we describe the development of a scalable closed-system, packed bed bioreactor suitable for large-scale MSCs expansion. The packed bed was formed from fused polystyrene pellets that were air plasma treated to endow them with a surface chemistry similar to traditional tissue culture plastic. The packed bed was encased within a gas permeable shell to decouple the medium nutrient supply and gas exchange. This enabled a significant reduction in medium flow rates, thus reducing shear and even facilitating single pass medium exchange. The system was optimised in a small-scale bioreactor format (160 cm2) with murine-derived green fluorescent protein-expressing MSCs, and then scaled-up to a 2800 cm2 format. We demonstrated that placental derived MSCs could be isolated directly within the bioreactor and subsequently expanded. Our results demonstrate that the closed system large-scale packed bed bioreactor is an effective and scalable tool for large-scale isolation and expansion of MSCs.
Simultaneous multi-metal leaching from industrial pyrite ash is reported for the first time using a novel bioreactor system that allows natural diffusion of atmospheric O2 and CO2 along with the required temperature maintenance. The waste containing economically important metals (Cu, Co, Zn & As) was leached using an adapted consortium of meso-acidophilic Fe2+ and S oxidising bacteria. The unique property of the sample supported adequate growth and activity of the acidophiles, thereby, driving the (bio) chemical reactions. Oxido-reductive potentials were seen to improve with time and the system’s pH lowered as a result of active S oxidation. Increase in sulphur dosage (>1g/L) and agitation speed (>150rpm) did not bear any significant effect on metal dissolution. The consortium was able to leach 94.01% Cu (11.75% dissolution/d), 98.54% Co (12.3% dissolution/d), 75.95% Zn (9.49% dissolution/d) and 60.80% As (7.6% dissolution/d) at 150rpm, 1g/L sulphur, 30°C in 8days.
The ex vivo engineering of autologous cartilage tissues has the potential to revolutionize the clinical management of joint disorders. Yet, high manufacturing costs and variable outcomes associated with tissue-engineered implants are still limiting their application. To improve clinical outcomes and facilitate a wider use of engineered tissues, automated bioreactor systems capable of enhancing and monitoring neotissues are required. Here, we developed an innovative system capable of applying precise uni- or biaxial mechanical stimulation to developing cartilage neotissues in a tightly controlled and automated fashion. The bioreactor allows for simple control over the loading parameters with a user-friendly graphical interface and is equipped with a load cell for monitoring tissue maturation. Applying our bioreactor, we demonstrate that human articular chondrocytes encapsulated in hydrogels composed of gelatin methacryloyl (GelMA) and hyaluronic acid methacrylate (HAMA) respond to uni- and biaxial mechanical stimulation by upregulation of hyaline cartilage-specific marker genes. We further demonstrate that intermittent biaxial mechanostimulation enhances accumulation of hyaline cartilage-specific extracellular matrix. Our study underlines the stimulatory effects of mechanical loading on the biosynthetic activity of human chondrocytes in engineered constructs and the need for easy-to-use, automated bioreactor systems in cartilage tissue engineering.
Over the last decades, anaerobic bioreactor technology proved to be a competitive technology for purifying wastewater while producing biogas. Methanogens perform the crucial final step in methane production, and monitoring their activity is of paramount importance for system understanding and management. Cofactor F430 is an essential prosthetic group of the methyl-coenzyme M reductase (MCR) enzyme catalysing this final step. This research investigates whether the quantification of cofactor F430 in bioreactor systems is a viable intermediate-complexity monitoring tool in comparison to the conventional biogas and volatile fatty acid (VFA) concentration follow-up and molecular genetic techniques targeting the mcrA gene encoding the MCR protein or its transcripts. Cofactor F430 was quantified in a lab-scale anaerobic membrane bioreactor (AnMBR) using liquid chromatography. The system was subjected to two organic loading rate shocks, and the F430 content of the sludge was followed up alongside mcrA gene copy and transcript numbers and classical performance monitoring tools. The research showed for the first time the combined mcrA gene transcript and F430 content dynamics in an anaerobic bioreactor system and reveals their significant positive correlation with in situ methane production rate. The main difference between the two monitoring methods relates to the cofactor’s slower degradation kinetics. The work introduces the use of cofactor F430 as a biomarker for methanogenic activity and, hence, as a monitoring tool that can be quantified within half a working day, yielding information directly related to in situ methanogenic activity in methanogenic reactors.
Monoclonal antibody production in commercial scale cell culture bioprocessing requires a thorough understanding of the engineering process and components used throughout manufacturing. It is important to identify high impact components early on during the lifecycle of a biotechnology derived product. While cell culture media selection is of obvious importance to the health and productivity of mammalian bioreactor operations, other components such as antifoam selection can also play an important role in bioreactor cell culture. Silicone polymer based antifoams were known to have negative impacts on cell health, production, and downstream filtration and purification operations. High throughput screening in micro-scale bioreactors provides an efficient strategy to identify initial operating parameters. Here, we utilized a micro-scale parallel bioreactor system to study an IgG1 producing CHO cell line, to screen Dynamis, ProCHO5, PowerCHO2, EX-Cell Advanced and OptiCHO media, and 204, C, EX-Cell, SE-15 and Y-30 antifoams and their impacts on IgG1 production, cell growth, aggregation, and process control. This study found ProCHO5, EX-Cell Advanced and PowerCHO2 media supported strong cellular growth profiles, with an IVCD of 25-35 x 10(6) cells-d/mL, while maintaining specific antibody production (Qp>2 pg/cell-d) for our model cell line and a monomer percentage above 94%. Antifoams C, EX-Cell and SE-15 were capable of providing adequate control of foaming while antifoam 204 and Y-30 noticeably stunted cellular growth. This work highlights the utility of high throughput micro bioreactors and the importance of identifying both positive and negative impacts of media and antifoam selection on a model IgG1 producing CHO cell line. This article is protected by copyright. All rights reserved.