Automated Bioreactor System Research Articles

Integration of pH Control into Chi.Bio Reactors and Demonstration with Small-Scale Enzymatic Poly(ethylene terephthalate) Hydrolysis.

Small-scale bioreactors that are affordable and accessible would be of major benefit to the research community. In previous work, an open-source, automated bioreactor system was designed to operate up to the 30 mL scale with online optical monitoring, stirring, and temperature control, and this system, dubbed Chi.Bio, is now commercially available at a cost that is typically 1-2 orders of magnitude less than commercial bioreactors. In this work, we further expand the capabilities of the Chi.Bio system by enabling continuous pH monitoring and control through hardware and software modifications. For hardware modifications, we sourced low-cost, commercial pH circuits and made straightforward modifications to the Chi.Bio head plate to enable continuous pH monitoring. For software integration, we introduced closed-loop feedback control of the pH measured inside the Chi.Bio reactors and integrated a pH-control module into the existing Chi.Bio user interface. We demonstrated the utility of pH control through the small-scale depolymerization of the synthetic polyester, poly(ethylene terephthalate) (PET), using a benchmark cutinase enzyme, and compared this to 250 mL bioreactor hydrolysis reactions. The results in terms of PET conversion and rate, measured both by base addition and product release profiles, are statistically equivalent, with the Chi.Bio system allowing for a 20-fold reduction of purified enzyme required relative to the 250 mL bioreactor setup. Through inexpensive modifications, the ability to conduct pH control in Chi.Bio reactors widens the potential slate of biochemical reactions and biological cultivations for study in this system, and may also be adapted for use in other bioreactor platforms.

MyoBio: An Automated Bioreactor System Technology for Standardized Perfusion-Decellularization of Whole Skeletal Muscle.

Decellularizing solid organs is a promising top-down process to produce acellular bio-scaffolds for 'de novo' regrowth or application as tissue 'patches' that compensate, e.g., large volumetric muscle loss in reconstructive surgery. Therefore, generating standardized acellular muscle scaffolds marks a pressing area of need. Although animal muscle decellularization protocols were established, those are mostly manually performed and lack defined bioreactor environments and metrologies to assess decellularization quality in real-time. To close this gap, we engineered an automated bioreactor system to provide chemical decellularization solutions to immersed whole rat gastrocnemius medialis muscle through perfusion of the main feeding arteries. Perfusion control is adjustable according to decellularization quality feedback. This was assessed both from (i) ex situ assessment of sarcomeres/nuclei through multiphoton fluorescence and label-free Second Harmonic Generation microscopy and DNA quantification, along with (ii) in situ within the bioreactor environment assessment of the sample's passive mechanical elasticity. We find DNA and sarcomere-free constructs after 72 h of 0.1% SDS perfusion-decellularization. Furthermore, passive elasticity can be implemented as additional online decellularization quality measure, noting a threefold elasticity decrease in acellular constructs. Our MyoBio represents a novel and useful automated bioreactor environment for standardized and controlled generation of acellular whole muscle scaffolds as a valuable source for regenerative medicine.

Comprehensive assessment of host cell protein expression after extended culture and bioreactor production of CHO cell lines.

The biomanufacturing industry is advancing toward continuous processes that will involve longer culture durations and older cell ages. These upstream trends may bring unforeseen challenges for downstream purification due to fluctuations in host cell protein (HCP) levels. To understand the extent of HCP expression instability exhibited by Chinese hamster ovary (CHO) cells over these time scales, an industry-wide consortium collaborated to develop a study to characterize age-dependent changes in HCP levels across 30, 60, and 90 cell doublings, representing a period of approximately 60 days. A monoclonal antibody (mAb)-producing cell line with bulk productivity up to 3 g/L in a bioreactor was aged in parallel with its parental CHO-K1 host. Subsequently, both cell types at each age were cultivated in an automated bioreactor system to generate harvested cell culture fluid (HCCF) for HCP analysis. More than 1500 HCPs were quantified using complementary proteomic techniques, two-dimensional electrophoresis (2DE) and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). While up to 13% of proteins showed variable expression with age, more changes were observed when comparing between the two cell lines with up to 47% of HCPs differentially expressed. A small subset (50 HCPs) with age-dependent expression were previously reported to be problematic as high-risk and/or difficult-to-remove impurities; however, the vast majority of these were downregulated with age. Our findings suggest that HCP expression changes over this time scale may not be as dramatic and pose as great of a challenge to downstream processing as originally expected but that monitoring of variably expressed problematic HCPs remains critical.

Getting closer to the in vivo environment with human AB serum

Watch the video or read the poster to learn: The ideal donor characteristics and pool sizes for high quality serum lotsThe different production methods that address specific research needsThe impact of processing methods on reducing lot-to-lot variabilityThe current and future role of human AB serum in cell and gene therapyTrevor Smith is the Product Manager for the Immune Cell Portfolio at BioIVT. He has worked extensively in cell and gene therapy and has published methods of T cell expansion on automated bioreactor systems. He has a deep understanding of how to scale-up therapies through process development, GMP lab design and personnel training. In his current role, he uses this knowledge and his immunology background to help customers commercialize new therapies that will provide better patient outcomes.

Automated multi-scale cascade of parallel stirred-tank bioreactors for fast protein expression studies

Automation, parallelization and autonomous operation of standard lab equipment, usually applied for manual bioprocess development, is considered as the key for reduction of bioprocess development time and costs. An automated bioreactor system with 4 stirred-tank bioreactors on a L-scale was combined with a custom-made biomass transfer system to distribute the cell suspensions produced on the L-scale into 48 parallel stirred-tank bioreactors on a mL-scale. Afterwards parallel protein expression studies automated by a liquid handling system with integrated fluorescence reader were performed. Isopropyl β-D-1-thiogalactopyranoside-induced (IPTG) expression of the red fluorescence protein mCherry was studied as an example of using fed-batch processes with recombinant Escherichia coli. In a first automated study, IPTG concentrations were varied in 48 parallel fed-batch processes with E. coli cells produced at a growth rate of 0.1 h−1 on an L-scale and transferred automatically to the mL-scale. The mCherry expression rate increased with increasing inducer concentration until the highest protein expression rate was observed at > 9 μM IPTG. In a second automated study, the growth rate of E. coli was varied between 0.1−0.2 h-1 in parallelly-operated stirred-tank bioreactors on a L-scale. The cells were automatically transferred and distributed into the stirred-tank bioreactors on a mL-scale and the concentration of the inducer IPTG was varied as before in parallel fed-batch processes. An increased growth rate during the production of the recombinant E. coli cells and/or higher cell densities during protein expression resulted in the increased IPTG concentrations necessary to achieve identical expression rates compared to a growth rate of 0.1 h−1 with the exception of very low inducer concentrations and inducer concentrations in excess. The new automated multi-scale cascade of parallel stirred-tank bioreactors should easily be applicable for performing fast optimisation studies with other microbial production systems and will have the potential to reduce bioprocess development time and staff assignment considerably.

Automated Bioreactor System for the Cultivation of Autologous Tissue-Engineered Vascular Grafts.

In an aging society, diseases associated with irreversible damage of organs are frequent. An increasing percentage of patients requires bioartificial tissue or organ substitutes. Tissue engineering products depend on a well-defined process to ensure successful cultivation while meeting high regulatory demands. The goal of the presented work is the development of a bioreactor system for the cultivation of tissue-engineered vascular grafts (TEVGs) for autologous implantation and transition from a lab scale setup to standardized production. Key characteristics include (i) the automated reliable monitoring and control of a wide-range of parameters regarding implant conditioning, (ii) easy and sterile setup and operation, (iii) reasonable costs of disposables, and (iv) parallelization of automated cultivation processes. The presented prototype bioreactor system provides comprehensive physiologically conditioning, sensing, and imaging functionality to meet all requirements for the successful cultivation of vascular grafts on a productional scale.

Coating strategies for expansion of mesenchymal stromal cells in a hollow-fibre bioreactor

Background & Aim Background The increasing request for culture expanded mesenchymal stromal cells (MSC) in the last years discloses the need to reduce costs and man power in the manufacturing process. This can be achieved by the use of closed, (semi-)automated bioreactor systems. We have decided to test a functionally closed hollow-fibre bioreactor system (Quantum®). Surveillance of metabolic parameters and adaptation of feeding in dependence of these is possible. However, the nature of the hollow-fibre surface does not support attachment of MSC without coating. Several biological substances for coating are available, either as isolated proteins (e.g. fibronectin, vitronectin), or as complex protein mixtures derived from human blood (e.g. cryoprecipitate, platelet lysate). The use of such closed systems additionally reduces the risk of contaminations. Methods, Results & Conclusion Methods MSC were cultured as conventional cell culture or in a platelet lysate (PL), cryoprecipitate (CP), vitronectin (VN), or fibronectin (FN) coated Quantum® bioreactor (TerumoBCT) for 5 - 15 days in alphaMEM (Lonza), supplemented with 5% - 10% platelet lysate (PL from IKT Ulm) and 1 i.u. heparin (B.Braun). The surface of the Quantum® bioreactor is 21,000 cm2. For Quantum®, the feed rate was adjusted based on daily measured lactate or glucose levels. For conventional cell culture, mainly 2 chamber CellSTACK® (Corning) with a surface of 1272 cm2 were used and complete medium exchange was performed twice a week. TrypZEAN® (Lonza) was used for detachment of cells. Results For results of the cell expansion experiments (N ≥ 3) using the different coating reagents refer to Table 1. Conclusion We showed that the Quantum® bioreactor is suitable for expansion of MSC using PL, CP, FN or VN as coating reagent. Despite higher doubling times for cells grown in the Quantum®, this system may help to save costs since less man power is needed to manage the system and several steps do not necessarily have to be performed in a class A cabinet. In addition, clinically relevant cell numbers could be achieved by the use of a single bioreactor (data not shown). Perspective Protocols for cell expansion in the Quantum® still may have to be optimized in order to increase its potential in saving man power and consumables.

A fully automated bioreactor system for precise control of stem cell proliferation and differentiation

The high proliferation ability and pluripotency of stem cells are ideal for application in stem cell therapy and tissue engineering. However, the clinical use of human mesenchymal stem cells (hMSCs) is limited due to the technical difficulties associated with mass production, high manufacturing costs, and contamination. Bioreactor systems can modulate the environmental and biochemical cues and also induce biomechanical cues, such as shear stress and hypoxia, during mass production of stem cells. In this study, we developed a fully automated bioreactor system (fABS) for precisely controlling the stem cell fate. We used this system for the proliferation and differentiation of human bone derived MSCs (hBMSCs). The fABS mainly consists of five systems: a monitoring system, the primary control system, a medium feeding system, a mass flow controller, and a cell culture system. We evaluated the precision with which the fABS regulated the stem cell environment. We observed that the shear stress induced by the fABS enhanced the hBMSC proliferation and osteogenic differentiation. Moreover, hypoxia induced by the fABS enhanced the chondrogenic differentiation of hBMSCs. As this system can have various applications, such as mass production, osteogenic differentiation, and chondrogenic differentiation, the fABS can aid in advancing the stem cell technologies.

Method for Modeling the Small Intestine and Resident Microbiome Through in Vitro Fermentation (P20-001-19)

ObjectivesThe human small intestine is a complex and dynamic organ tasked with enzymatic digestion and absorption of nutrients. Design of a small intestine model can provide detailed systematic knowledge of these processes; model design challenges include differential pH and oxygen availability along the length of the small intestine, food-dependent host secretion of digestive compounds, complex nutrient absorption processes, and microbiome interactions with both food and host. Numerous in vitro models have been developed to simulate the small intestine, but physiological relevance is limited. Here, we present an in vitro fermentation model of the small intestine to include microbiota and enhance physiological relevance. MethodsA stepwise biofidelic model design approach was implemented with initial stages consisting of simulating ileum conditions, including pH and residence time, utilizing an automated bioreactor system for real-time monitoring and control of fermentation parameters, with incorporation of digestive enzymes and bile acids for breakdown of food inputs. Nutrient absorption, simulated using hollow-fiber columns to emulate passive diffusion, was initially optimized using small molecules to mimic dietary digestion byproducts; validation with food components, such as starch or whey powder, is planned. A mock microbial community, with organisms selected to represent major phyla and functions of the small intestine microbiota, was designed, implemented, and characterized in fermentations representing “fed-state” ileum conditions. ResultsDesign and validation of the model with mock food components will be presented, along with steps taken to integrate in situ nutrient absorption and mock microbial community. Initial characterization of the microbial community indicates synergistic growth dynamics and nutrient utilization under “fed-state” conditions. ConclusionsThese efforts will be the foundation for our long-term goal of simulating the small intestine to complement our large intestine fermentation model, jA2COB, creating a complete in vitro fermentation model of the lower GI tract. Insight gleaned from this model, alone or in concert with in vivo studies, can inform nutritional strategies to restore and maintain host gut homeostasis. Funding SourcesFunded by U.S. Army NSRDEC core applied research funds.

Full Thickness Skin Expansion ex vivo in a Newly Developed Reactor and Evaluation of Auto-Grafting Efficiency of the Expanded Skin Using Yucatan Pig Model.

Skin grafts are required in numerous clinical procedures, such as reconstruction after skin removal and correction of contracture or scarring after severe skin loss caused by burns, accidents, and trauma. The current standard for skin defect replacement procedures is the use of autologous skin grafts. However, donor-site tissue availability remains a major obstacle for the successful replacement of skin defects and often limits this option. The aim of this study is to effectively expand full thickness skin to clinically useful size using an automated skin reactor and evaluate auto grafting efficiency of the expanded skin using Yucatan female pigs. We developed an automated bioreactor system with the functions of real-time monitoring and remote-control, optimization of grip, and induction of skin porosity for effective tissue expansion. We evaluated the morphological, ultra-structural, and mechanical properties of the expanded skin before and after expansion using histology, immunohistochemistry, and tensile testing. We further carried out in vivo grafting study using Yucatan pigs to investigate the feasibility of this method in clinical application. The results showed an average expansion rate of 180%. The histological findings indicated that external expansion stimulated cellular activity in the isolated skin and resulted in successful grafting to the transplanted site. Specifically, hyperplasia did not appear at the auto-grafted site, and grafted skin appeared similar to normal skin. Furthermore, mechanical stimuli resulted in an increase in COL1A2 expression in a suitable environment. These findings provided insight on the potential of this expansion system in promoting dermal extracellular matrix synthesis in vitro. Conclusively, this newly developed smart skin bioreactor enabled effective skin expansion ex vivo and successful grafting in vivo in a pig model.

A novel bioreactor system for biaxial mechanical loading enhances the properties of tissue-engineered human cartilage

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.

Automated bioreactor system for cartilage tissue engineering of human primary nasal septal chondrocytes.

An automated bioreactor system for three-dimensional (3D) cultivation of facial cartilage replacement matrices (e.g. whole human auricles) with automatised medium exchange, gas flow and temperature control was developed. The measurement of O2 saturation and pH value in the medium was performed with a non-invasive optical method. The whole system can be observed via remote monitoring worldwide. First results demonstrated that the complete system remained sterile throughout a period of 42 days. Human chondrocytes migrated into the employed cartilage replacement matrix consisting of decellularised porcine nasoseptal cartilage (pNSC). Furthermore, an improved migration and new synthesis of aggrecan was detected. A first evaluation of the system was conducted by comparison of the results from laboratory analysis with computational fluid dynamics (CFD).

New bioreactor vessel for tissue engineering of human nasal septal chondrocytes

AbstractCultivation of human nasal septal chondrocytes in a self-established automated bioreactor system with a new designed reactor glass vessel and the results of a computational fluid dynamics model are presented. The first results show the effect of a homogeneous fluidic condition of the continuous medium flow and the resulting stresses on the scaffolds’ surface and their influence on the migration of the cells into the scaffold matrix under these conditions. For this purpose computational models, generated with the computational fluid dynamics software STAR-CCM+, and the results of alcian blue staining for newly synthesized sulphated glycosaminoglycans have been compared during cultivation in the new and a first version of the glass reactor vessel with inhomogeneous fluidic conditions, with the same automated bioreactor system and under similar cultivation conditions.

Packed Bed Bioreactor for the Isolation and Expansion of Placental-Derived Mesenchymal Stromal Cells.

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.

An automated perfusion bioreactor for the streamlined production of engineered osteogenic grafts.

A computer-controlled perfusion bioreactor was developed for the streamlined production of engineered osteogenic grafts. This system automated the required bioprocesses, from the initial filling of the system through the phases of cell seeding and prolonged cell/tissue culture. Flow through chemo-optic micro-sensors allowed to non-invasively monitor the levels of oxygen and pH in the perfused culture medium throughout the culture period. To validate its performance, freshly isolated ovine bone marrow stromal cells were directly seeded on porous scaffold granules (hydroxyapatite/β-tricalcium-phosphate/poly-lactic acid), bypassing the phase of monolayer cell expansion in flasks. Either 10 or 20 days after culture, engineered cell-granule grafts were implanted in an ectopic mouse model to quantify new bone formation. After four weeks of implantation, histomorphometry showed more bone in bioreactor-generated grafts than cell-free granule controls, while bone formation did not show significant differences between 10 days and 20 days of incubation. The implanted granules without cells had no bone formation. This novel perfusion bioreactor has revealed the capability of activation larger viable bone graft material, even after shorter incubation time of graft material. This study has demonstrated the feasibility of engineering osteogenic grafts in an automated bioreactor system, laying the foundation for a safe, regulatory-compliant, and cost-effective manufacturing process.

Growth Factors for Clinical-Scale Expansion of Human Articular Chondrocytes: Relevance for Automated Bioreactor Systems

The expansion of chondrocytes in automated bioreactors for clinical use requires that a relevant number of cells be generated, starting from variable initial seeding densities in one passage and using autologous serum. We investigated whether the growth factor combination transforming growth factor beta 1/fibroblast growth factor 2/platelet-derived growth factor BB (TFP), recently shown to enhance the proliferation capacity of human articular chondrocytes (HACs), allows the efficiency of chondrocyte use to be increased at different seeding densities and percentages of human serum (HS). HACs were seeded at 1,000, 5,000, and 10,000 cells/cm2 in medium containing 10% fetal bovine serum or 10,000 cells/cm2 with 1%, 5%, or 10%HS. The chondrogenic capacity of post-expanded HACs was then assessed in pellet cultures. Expansion with TFP allowed a sufficient number of HACs to be obtained in one passage even at the lowest seeding density and HS percentage and variability in cartilage-forming capacity of HACs expanded under the different conditions to be reduced. Instead, larger variations and insufficient yields were found in the absence of TFP. By allowing large numbers of cells to be obtained, starting from a wide range of initial seeding densities and HS percentages, the use of TFP may represent a viable solution for the efficient expansion of HACs and addresses constraints of automated clinical bioreactor systems.

Design of an automated bioreactor system to screen immobilised whole cell catalysts

An automated CSTR system has been developed to study the stability of immobilised whole cell biocatalysts for ammonium acrylate production. Two potentially useful amidase-active rhodococci have been identified: One possesses an amidase of much higher activity than the other but of much lower stability. The activity and stability of each biocatalyst over a range of temperatures has been determined and used to compare the productivity of the biocatalysts in a bioconversion.