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.
Micro and small bioreactors are well described for use in bioprocess development in pre-production manufacture, using ultra-scale down and microfluidic methodology. However, the use of bioreactors to understand normal and pathophysiology by definition must be very different, and the constraints of the physiological environment influence such bioreactor design. This review considers the key elements necessary to enable bioreactors to address three main areas associated with biological systems. All entail recreation of the in vivo cell niche as faithfully as possible, so that they may be used to study molecular and cellular changes in normal physiology, with a view to creating tissue-engineered grafts for clinical use; understanding the pathophysiology of disease at the molecular level; defining possible therapeutic targets; and enabling appropriate pharmaceutical testing on a truly representative organoid, thus enabling better drug design, and simultaneously creating the potential to reduce the numbers of animals in research. The premise explored is that not only cellular signalling cues, but also mechano-transduction from mechanical cues, play an important role.
Fat grafting is emerging as a promising alternative to silicon implants in breast reconstruction surgery. Unfortunately, this approach does not provide a proper mechanical support and is affected by drawbacks such as tissue resorption and donor site morbidity. Synthetic scaffolds can offer a valuable alternative to address these challenges, but poorly recapitulate the biochemical stimuli needed for tissue regeneration. Here, we aim at combining the positive features of a structural, synthetic polymer to an engineered, devitalized extracellular matrix (ECM) to generate a hybrid construct that can provide a mix of structural and biological stimuli needed for adipose tissue regeneration. A RGD-mimetic synthetic scaffold OPAAF, designed for soft tissue engineering, was decorated with ECM deposited by human adipose stromal cells (hASCs). The adipoinductive potential of the hybrid ECM-OPAAF construct was validated in vitro, by culture with hASC in a perfusion bioreactor system, and in vivo, by subcutaneous implantation in nude mouse. Our findings demonstrate that the hybrid ECM-OPAAF provides proper mechanical support and adipoinductive stimuli, with potential applicability as off-the-shelf material for adipose tissue reconstruction.
Knowledge of leveraging biomass characteristics is essential for achieving a microbial community with a desired structure to optimize anaerobic bioreactor performance. This study investigates the successive granule transformations in a high-rate anaerobic system with intermittent gas sparging and sequential increases in organic loading rates (OLRs), by establishing the correlations between the granule microstructures and reactor operating parameters. Over the course of a 196-day lab-scale trial, the granules were visualized in various stages using scanning electron microscopy, and digital image processing was applied for further quantifying their surface properties. Correlation analyses revealed that irregularities of the granule microstructures (surface properties, specific surface area and pore volume) emerged at stage 4 when the OLR was 13.31 kg COD/m3·day and in stage 5 in the absence of gas sparging. The loading ratio (substrate surface loading to upward velocity) was identified to be the main parameter controlling the granule transformations, and the surface structures were classified into three categories for further interpretation. Confocal laser scanning microscopy analyses showed that the granule core started to hollow out from stage 4. It is also found that a rough granule surface helped accelerate the growth of the granular diameter under gas sparging. Overall, this study not only establish quantitative correlations between the granules microstructures and reactor operating parameters, but also shed light on the use of intermittent gas sparging to control the surface properties of anaerobic granules in high-rate anaerobic bioreactors.
In tissue engineering, the generation and functional maintenance of dense voluminous tissues is mainly restricted due to insufficient nutrient supply. Larger three-dimensional constructs, which exceed the nutrient diffusion limit become necrotic and/or apoptotic in long-term culture if not provided with an appropriate vascularization. Here, we established protocols for the generation of a pre-vascularized biological scaffold with intact arterio-venous capillary loops from rat intestine, which is decellularized under preservation of the feeding and draining vascular tree. Vessel integrity was proven by marker expression, media/blood reflow and endothelial LDL uptake. In vitro maintenance persisted up to 7 weeks in a bioreactor system allowing a stepwise reconstruction of fully vascularized human tissues and successful in vivo implantation for up to 4 weeks, although with time-dependent decrease of cell viability. The vascularization of the construct lead to a 1.5× increase in cellular drug release compared to a conventional static culture in vitro. For the first time, we performed proof-of-concept studies demonstrating that 3D tissues can be maintained within a miniaturized vascularized scaffold in vitro and successfully implanted after re-anastomosis to the intrinsic blood circulation in vivo. We hypothesize that this technology could serve as a powerful platform technology in tissue engineering and regenerative medicine.