Computer-controlled Bioreactor Research Articles

Computer controlled expansion of equine cord blood mesenchymal stromal cells on microcarriers in 3L vertical-wheel® bioreactors.

Mesenchymal stromal cells (MSCs) are an ideal cell source for allogenic cell therapy due to their immunomodulatory and differentiation properties. Equine MSCs (eMSCs) have been found to be a promising treatment for equine joint injuries including meniscal injuries, cartilage degradation, and osteoarthritis. Although the use of eMSCs has shown efficacy in preliminary studies, challenges associated with biomanufacturing remain. To achieve the required cell numbers for clinical application, bioreactor-based processes are required. Initial studies have shown that eMSCs can be cultivated in microcarrier-based, stirred suspension bioreactor culture at the laboratory 0.1L scale using a Vertical-Wheel® (VW) bioreactor. However, investigations regarding scale up of these processes to the required biomanufacturing scales are required. This study investigated the scale-up of a equine cord blood MSC (eCB-MSC) bioprocess in VW bioreactors at three scales. This included scale-up from the 0.1-0.5L bioreactor, scale-up from static culture to the 3L computer-controlled bioreactor, and scale-up into the 3L computer-controlled bioreactor using a mock clinical trial process. Results from the various scale-up experiments demonstrated similar cell expansion at the various tested scales. The 3L computer-controlled system resulted in a final cell densities of 1.5 × 105 cells/cm2 on average, achieving 1.5 × 109 harvested cells. Biological testing of the cells showed that cell phenotype and functionality were maintained after scale-up. These findings demonstrate the scalability of an eCB-MSC bioprocess using microcarriers in VW bioreactors to achieve clinically relevant cell numbers, a critical step to translate MSC treatments from research to clinical applications. This study also represents the first known published study expanding any cell type in the 3L VW bioreactor.

Large-Scale Expansion of Human Umbilical Cord-Derived Mesenchymal Stem Cells in a Stirred Suspension Bioreactor Enabled by Computational Fluid Dynamics Modeling.

Human umbilical cord-derived mesenchymal stem cells (hUCMSCs) hold great potential to generate novel and curative cell therapy products. However, the current large-scale cultivation of hUCMSCs is based on empirical geometry-dependent methods, limiting the generation of high-quantity and high-quality hUCMSCs for clinical therapy. Herein, we develop a novel scale-up strategy based on computational fluid dynamics (CFD) to effectively expand the hUCMSCs in a 3D tank bioreactor. Using a standardized hUCMSCs line on microcarriers, we successfully translated and expanded the hUCMSCs from a 200 mL spinner flask to a 1.5 L computer-controlled bioreactor by matching the shear environment and suspending the microcarrier. Experimental results revealed that the batch-cultured hUCMSCs in bioreactors with an agitation speed of 40 rpm shared a more favorable growth and physiological state, similar to that run at 45 rpm in a 200 mL spinner flask, showing comparability in both culture systems. Notably, the maximum cell density reached up to 27.3 × 105 cells/mL in fed-batch culture, 2.9 folds of that of batch culture and 20.2 times of seeding cells. As such, efficient process optimization and scale-up expansion of hUCMSCs were achieved in the microcarrier-based bioreactor system by the developed CFD simulation strategy, which provided an alternative toolbox to generate massive and standardized curative cell therapy products.

Multiomic analysis of stretched osteocytes reveals processes and signalling linked to bone regeneration and cancer

Exercise is a non-pharmacological intervention that can enhance bone regeneration and improve the management of bone conditions like osteoporosis or metastatic bone cancer. Therefore, it is gaining increasing importance in an emerging area of regenerative medicine—regenerative rehabilitation (RR). Osteocytes are mechanosensitive and secretory bone cells that orchestrate bone anabolism and hence postulated to be an attractive target of regenerative exercise interventions. However, the human osteocyte signalling pathways and processes evoked upon exercise remain to be fully identified. Making use of a computer-controlled bioreactor that mimics exercise and the latest omics approaches, RNA sequencing (RNA-seq) and tandem liquid chromatography-mass spectrometry (LC-MS), we mapped the transcriptome and secretome of mechanically stretched human osteocytic cells. We discovered that a single bout of cyclic stretch activated network processes and signalling pathways likely to modulate bone regeneration and cancer. Furthermore, a comparison between the transcriptome and secretome of stretched human and mouse osteocytic cells revealed dissimilar results, despite both species sharing evolutionarily conserved signalling pathways. These findings suggest that osteocytes can be targeted by exercise-driven RR protocols aiming to modulate bone regeneration or metastatic bone cancer.

Mechanical Stimulation Of Osteocyte-like Cells Changes Their Secretome - Implications For Regenerative Medicine

Osteocytes are secretory bone cells that regulate bone homeostasis and for this reason, are often coined as the “brain of the bone”. In vitro studies demonstrated that mechanically stimulated osteocytes release interleukins and growth factors that help coordinating bone formation and resorption, however, their secretome remains largely unknown. PURPOSE: To investigate WNT signalling and the secretome of mouse and human osteocyte-like cells. Insights from this study could help to devise informed therapeutic exercise regimen e.g. aiming to preserve bone mass across ageing or accelerate bone fracture healing. METHODS: The murine MLO-Y4 (Kerafast) cell line was cultured according to Kerafast instructions. Human adipose stem cells (ATCC® PCS -500-011™) were expanded and differentiated into osteocyte-like cells (hOC) according to ATCC instructions. Cells were cultured in a computer-controlled bioreactor (Flexcell Int) for mechanical loading (3.4%, 2Hz, 5h). Static cultures were used as control. Relative expression of 84 key genes of the WNT signalling pathway (Sabiosciences) was quantified by RT-qPCR. Relative protein expression was estimated by western blotting. The secretome was analysed by quantitative mass spectrometry (TripleTOF 6600, SCIEX) using SWATH and IDA and processed using OneOmics (SCIEX) software. RESULTS: The relative gene expression remained unchanged in mechanically MLO-Y4 and hOC. Regarding protein quantification, active β-catenin and Cyclin D1 showed an up-regulation trend in mechanically stimulated MLO-Y4 but this was not statistically significant. A total of 917 proteins were identified in the MLO-Y4 secretome, ~12% present exclusively under mechanical active conditions. The secretome obtained under loading contained 14 cyclin-dependent kinases (CDKs) including CDK6, a critical regulator of osteoblasts and osteoclasts differentiation. A total of 329 proteins were identified in the supernatant of hOC, ~9% present exclusively under mechanical stimulation. Unlike MLO-Y4, no CDKs were identified in this cell type. The small ubiquitin-like modifier (SUMO) 2 and 3 were present in the secretomes of mechanically loaded MLO-Y4 and hOC. CONCLUSION: Mechanically stimulated osteocyte-like cells secrete a specific set of proteins which could impact bone health and regeneration.

Computer-Controlled Biaxial Bioreactor for Investigating Cell-Mediated Homeostasis in Tissue Equivalents.

Soft biological tissues consist of cells and extracellular matrix (ECM), a network of diverse proteins, glycoproteins, and glycosaminoglycans that surround the cells. The cells actively sense the surrounding ECM and regulate its mechanical state. Cell-seeded collagen or fibrin gels, so-called tissue equivalents, are simple but powerful model systems to study this phenomenon. Nevertheless, few quantitative studies document the stresses that cells establish and maintain in such gels; moreover, most prior data were collected via uniaxial experiments whereas soft tissues are mainly subject to multiaxial loading in vivo. To begin to close this gap between existing experimental data and in vivo conditions, we describe here a computer-controlled bioreactor that enables accurate measurements of the evolution of mechanical tension and deformation of tissue equivalents under well-controlled biaxial loads. This device allows diverse studies, including how cells establish a homeostatic state of biaxial stress and if they maintain it in response to mechanical perturbations. It similarly allows, for example, studies of the impact of cell and matrix density, exogenous growth factors and cytokines, and different types of loading conditions (uniaxial, strip-biaxial, and biaxial) on these processes. As illustrative results, we show that NIH/3T3 fibroblasts establish a homeostatic mechanical state that depends on cell density and collagen concentration. Following perturbations from this homeostatic state, the cells were able to recover biaxial loading similar to homeostatic. Depending on the precise loads, however, they were not always able to fully maintain that state.

Sintezna biologija za proizvodnjo biobutanola

In 2015, the first-ever Slovenian high school team competed at the iGEM (international Genetically Engineered Machine) competition in synthetic biology. The team was carefully selected from a list of candidates proposed by their high school teachers of chemistry and biology. Composed of eight students from 7 high schools from across the country, the team split into two groups, working in two institutions but with regular common meetings. The group of four students who worked at the National Institute of Chemistry focused on biotechnological production and the second group was preparing genetic constructs at the University of Ljubljana Faculty of Chemistry and Chemical Technology. The central problem that students tangled was the conversion of C-4 acids that are side-products of anaerobic waste degradation, into the corresponding alcohol. In the biotechnological part of the project students tested butanoic acid production in an anaerobic fermentation broth using a 15-channel computer-controlled bioreactor system, configured to allow online analysis of gaseous products (by GC) and frequent analysis of dissolved compounds (by HPLC). Next, at optimal butanoic acid production conditions (c = 1 g/L butanoic acid) growth of E. coli was examined in terms of medium composition, temperature and pH in order to obtain optimal operational parameters for biotechnological transformation of butanoic acid to butanol. Overall, E. coli growth was analysed in 72 different growth media in microtiter plates. Students followed inhibition of E. coli growth by butanol, acetone and isobutanol in the presence of butanoic acid and glycerol. The synthetic biology group started with PCR amplification of three genes ( ctf A and ctf B encoding two polypeptide chains of Co-A transferase, and bdh B encoding butyraldehide dehydrogenase for a two.stage conversion of butyril CoA to butanol) from anaerobic microbial community. Amplification failed, probably due to contaminants in the broth and a low number of bacteria harbouring these genes. Consequently, synthetic genes were designed with appended sequences for easier cloning, for a hexahistidine tag for detection of recombinant proteins using specific antibodies. Synthetic genes were inserted into pSB1C3 vectors and for each of them a strong promoter- ribosome binding site region was inserted in front of the enzyme coding region. Such combined constructs were further transferred into vectors with double terminators of transcription. In total, 9 different DNA constructs were prepared and deposited in the Registry of biological parts. All final expression constructs efficiently directed production of recombinant enzymes in E. coli DH5α under aerobic conditions. The giant jamboree of competing teams took place in Boston, USA, from September 24 to 28 with more than 260 teams from around the globe. In the high school track the Slovenian team presented the project entitled “From waste to fuel: Reprogrammed E. coli for sustainable production of biobutanol from butanoic acid”. The project received high recognition, as it was awarded a gold medal for completed tasks and was nominated among five best teams in categories Best Integrated Human Practices, Best Wiki, Best Presentation and Best High School Project.

Construction of Computer Controlled Bioreactor

Bioprocess automation is developing fast for the reasons of quality control, production cost reduction et. al.. Bioreactor is the central equipment of bioprocess. Construction of computer controlled bioreactor is need for bioprocess automation. In this research, a two level hierarchical structure computer control bioreactor system is designed and constructed. The lower level plays the major role of set-point feedback control of process variables and the higher level plays the major role of graphical human interface and biological mathematical model solution. This control system has advantages of high reliability and flexibility than the single level digital control system, and has plenty of functions and high practicality compared with a normal commercial one.

Successful Development of Small Diameter Tissue-Engineering Vascular Vessels by Our Novel Integrally Designed Pulsatile Perfusion-Based Bioreactor

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 O2 and CO2 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.

A non-rotational, computer-controlled suspension bioreactor for expansion of umbilical cord blood mononuclear cells

The proliferation and differentiation characteristics of umbilical cord blood mononuclear cells were examined in a non-rotational suspension bioreactor with a fishtail mixer. The system consisted of a glass vessel, a mixer that moved vertically, a data acquisition and control system to continuously monitor pH, temperature and dissolved O(2). The bioreactor provided superior expansion of total HSCs and not total cell number, as well as expression of stemness-related genes which followed with increasing in number of colony-forming cells during 14days of culture compared to T -lask culture. Vertical agitation thus reduces the total cell number, which may be related to increased shear stress, but has no effect on HSC function.

Design and validation of a bi-axial loading bioreactor for mechanical stimulation of engineered cartilage

A mechanical-conditioning bioreactor has been developed to provide bi-axial loading to three-dimensional (3D) tissue constructs within a highly controlled environment. The computer-controlled bioreactor is capable of applying axial compressive and shear deformations, individually or simultaneously at various regimes of strain and frequency. The reliability and reproducibility of the system were verified through validation of the spatial and temporal accuracy of platen movement, which was maintained over the operating length of the system. In the presence of actual specimens, the system was verified to be able to deliver precise bi-axial load to the specimens, in which the deformation of every specimen was observed to be relatively homogeneous. The primary use of the bioreactor is in the culture of chondrocytes seeded within an agarose hydrogel while subjected to physiological compressive and shear deformation. The system has been designed specifically to permit the repeatable quantification and characterisation of the biosynthetic activity of cells in response to a wide range of short and long term multi-dimensional loading regimes.

A Novel Cylindrical Biaxial Computer-Controlled Bioreactor and Biomechanical Testing Device for Vascular Tissue Engineering

It is becoming evident that tissue-engineered constructs adapt to altered mechanical loading, and that specific combinations of multidirectional loads appear to have a synergistic effect on the remodeling. However, most studies of mechanical stimulation of engineered vascular tissue engineering employ only uniaxial stimulation. Here we present a novel computer-controlled bioreactor and biomechanical testing device designed to precisely and simultaneously control mean and cyclic values of transmural pressure (at rates up to 1 Hz and ranges of 40 mmHg), luminal flow rate, and axial length (or load) applied to gel-derived, scaffold-derived, and self-assembly-derived tissue-engineered blood vessels during culture, while monitoring vessel geometry with a resolution of 6.6 mum. Intermittent monitoring of the extracellular matrix and cells is accomplished on live tissues using multi-photon confocal microscopy under unloaded and loaded conditions at multiple time-points in culture (on the same vessel) to quantify changes in cell and extracellular matrix content and organization. This same device is capable of performing intermittent cylindrical biaxial biomechanical testing at multiple time-points in culture (on the same vessel) to quantify changes in the mechanical behavior during culture. Here we demonstrate the capabilities of this new device on self-assembly-derived and collagen-gel-derived tissue-engineered blood vessels.

Bioreactor Maintained Living Skin Matrix

Numerous reconstructive procedures result in wounds that require skin grafting. Often, the amount of tissue available from donor sites is limited. In vivo tissue expanders have been used clinically to generate larger sections of skin, and other methods exist to cover large wounds, but all have significant limitations. We investigated whether these difficulties could be overcome by increasing the surface area of skin in vitro while maintaining tissue viability. Human foreskin was incrementally expanded in a computer-controlled bioreactor system over 6 days to increase its surface dimensions under culture conditions. Morphological, ultrastructural, and mechanical properties of the foreskin were evaluated before and after expansion using histology, scanning electron microscopy, mercury porosimetry, and tensile testing. The surface area of the tissue was 110.7% +/- 12.2% greater, with maintenance of cell viability and proliferative potential. Histomorphological and ultrastructural analyses showed that dermal structural integrity was preserved. The pore diameter of the expanded skin was 64.49% +/- 32.8% greater. The mechanical properties were not adversely affected. These findings show that expansion of living skin matrices can be achieved using a computer-controlled bioreactor system. This technique provides an opportunity to generate large amounts of skin for reconstructive procedures.

Endothelialization of Heart Valve Matrix Using a Computer-Assisted Pulsatile Bioreactor

Although calcification remains as the main clinical concern associated with bioprosthetic heart valve replacement surgery, there is evidence that tissue deterioration leads to thromboembolism. In such instances, measures that prevent thrombosis may be beneficial. To minimize thrombosis, endothelialization of the valve surface before implantation has been proposed to facilitate coverage. In this study we aimed to define the optimal flow parameters for the endothelialization of decellularized heart valves using endothelial progenitor cell (EPC)-derived endothelial cells (ECs). We assessed the thrombogenic characteristics of the endothelialized heart valve surface using a bioreactor. EPC-derived ECs were seeded on decellularized porcine valve scaffolds. A computer-controlled bioreactor system was used to determine the optimal flow rates. Successful endothelialization was achieved by preconditioning the cell-seeded valves with stepwise increases in volume flow rate up to 2 L/min for 7 days. We show that decellularized valve scaffolds seeded with EPC-derived ECs improved the anti-thrombotic properties of the valve, whereas the scaffolds without ECs escalated the coagulation process. This study demonstrates that preconditioning of ECs seeded on valve matrices using a bioreactor system is necessary for achieving uniform endothelialization of valve scaffolds, which may reduce thrombotic activity after implantation in vivo.

Cyclic Mechanical Preconditioning Improves Engineered Muscle Contraction

The inability to engineer clinically relevant functional muscle tissue remains a major hurdle to successful skeletal muscle reconstructive procedures. This article describes an in vitro preconditioning protocol that improves the contractility of engineered skeletal muscle after implantation in vivo. Primary human muscle precursor cells (MPCs) were seeded onto collagen-based acellular tissue scaffolds and subjected to cyclic strain in a computer-controlled bioreactor system. Control constructs (static culture conditions) were run in parallel. Bioreactor preconditioning produced viable muscle tissue constructs with unidirectional orientation within 5 days, and in vitro-engineered constructs were capable of generating contractile responses after 3 weeks of bioreactor preconditioning. MPC-seeded constructs preconditioned in the bioreactor for 1 week were also implanted onto the latissimus dorsi muscle of athymic mice. Analysis of tissue constructs retrieved 1 to 4 weeks postimplantation showed that bioreactor-preconditioned constructs, but not statically cultured control tissues, generated tetanic and twitch contractile responses with a specific force of 1% and 10%, respectively, of that observed on native latissimus dorsi. To our knowledge, this is the largest force generated for tissue-engineered skeletal muscle on an acellular scaffold. This finding has important implications to the application of tissue engineering and regenerative medicine to skeletal muscle replacement and reconstruction.

Quantifying microbial lag phenomena due to a sudden rise in temperature: a systematic macroscopic study

The microbial lag phase is a complex and yet not completely understood phenomenon. Many studies on the microbial lag phase have been published but few report a systematic study; moreover, previous lag studies have involved the effect of multiple confounded factors. Here, the effect of sudden temperature rises on an exponentially growing Escherichia coli culture is systematically investigated. Experiments are performed in a computer-controlled bioreactor where E. coli K12 MG1655 is grown under aerobic conditions in Brain Heart Infusion (BHI) broth. This experimental set-up is used to characterise the effect of (i) the amplitude of the temperature shift, (ii) the pre-shift temperature level and (iii) the post-shift temperature level on the occurrence and length of a lag phase. Besides temperature, no other environmental changes take place at the moment of the temperature shift. To quantify the length of the induced lag phase, the experimental data are described with a common growth model. Depending on the three factors tested, a lag phase of more or less 1 h is induced or not. This lag/no lag behaviour can largely be explained by the existence of a normal physiological temperature range but also the amplitude of the temperature rise plays a role. It can be concluded that for the microorganism under study the lower boundary of the normal range lies approximately between 22.78 and 23.86 °C. It is shown that this boundary is no cut-off point, but rather a transition zone. Even more, repeated experiments at this boundary have yielded different results (lag or no lag). This observation points out that the mechanism triggering this lag phase is not absolute but may be subject to biological variability.

Iron deficiency leads to inhibition of oxygen transfer and enhanced formation of virulence factors in cultures of Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa PAO1 was recently found to exhibit two remarkable physiological responses to oxidative stress: (1) a strong reduction in the efficiency of oxygen transfer from the gas phase into the liquid phase, thus causing oxygen limitation in the culture and (2) formation of a clear polysaccharide capsule on the cell surface. In this work, it has been shown that the iron concentration in the culture plays a crucial role in evoking these phenomena. The physiological responses of two P. aeruginosa PAO1 isolates (NCCB 2452 and ATCC 15692) were examined in growth media with varied iron concentrations. In a computer-controlled bioreactor cultivation system for controlled dissolved oxygen tension (pO2), a strong correlation between the exhaustion of iron and the onset of oxygen limitation was observed. The oxygen transfer rate of the culture, characterized by the volumetric oxygen transfer coefficient, kLa, significantly decreased under iron-limited conditions. The formation of alginate and capsule was more strongly affected by iron concentration than by oxygen concentration. The reduction of the oxygen transfer rate and the subsequent oxygen limitation triggered by iron deficiency may represent a new and efficient way for P. aeruginosa PAO1 to adapt to growth conditions of iron limitation. Furthermore, the secretion of proteins into the culture medium was strongly enhanced by iron limitation. The formation of the virulence factor elastase and the iron chelators pyoverdine and pyochelin also significantly increased under iron-limited conditions. These results have implications for lung infection of cystic fibrosis patients by P. aeruginosa in view of the prevalence of iron limitation at the site of infection and the respiratory failure leading to death.

Optimal temperature input design for estimation of the Square Root model parameters: parameter accuracy and model validity restrictions

As part of the model building process, parameter estimation is of great importance in view of accurate prediction making. Confidence limits on the predicted model output are largely determined by the parameter estimation accuracy that is reflected by its parameter estimation covariance matrix. In view of the accurate estimation of the Square Root model parameters, Bernaerts et al. have successfully applied the techniques of optimal experiment design for parameter estimation [Int. J. Food Microbiol. 54 (1–2) (2000) 27]. Simulation-based results have proved that dynamic (i.e., time-varying) temperature conditions characterised by a large abrupt temperature increase yield highly informative cell density data enabling precise estimation of the Square Root model parameters. In this study, it is shown by bioreactor experiments with detailed and precise sampling that extreme temperature shifts disturb the exponential growth of Escherichia coli K12. A too large shift results in an intermediate lag phase. Because common growth models lack the ability to model this intermediate lag phase, temperature conditions should be designed such that exponential growth persist even though the temperature may be changing. The current publication presents (i) the design of an optimal temperature input guaranteeing model validity yet yielding accurate Square Root model parameters, and (ii) the experimental implementation of the optimal input in a computer-controlled bioreactor. Starting values for the experiment design are generated by a traditional two-step procedure based on static experiments. Opposed to the single step temperature profile, the novel temperature input comprises a sequence of smaller temperature increments. The structural development of the temperature input is extensively explained. High quality data of E. coli K12 under optimally varying temperature conditions realised in a computer-controlled bioreactor yield accurate estimates for the Square Root model parameters. The latter is illustrated by means of the individual confidence intervals and the joint confidence region.