Bioreactors for tissue engineering

Bioreactors are essential in tissue engineering, not only because they provide an in vitro environment mimicking in vivo conditions for the growth of tissue substitutes, but also because they enable systematic studies of the responses of living tissues to various mechanical and biochemical cues. The basic principles of bioreactor design are reviewed, the bioreactors commonly used for the tissue engineering of cartilage, bone and cardiovascular systems are assessed in terms of their performance and usefulness. Several novel bioreactor types are also reviewed.     

The role of bioreactors in cartilage tissue engineering

Cartilage tissue engineering is concerned with developing in vitro cartilage implants that closely match the properties of native cartilage, for eventual implantation to replace damaged cartilage. The three components to cartilage tissue engineering are cell source, such as in vitro expanded autologous chondrocytes or mesenchymal progenitor cells, a scaffold onto which the cells are seeded and a bioreactor which attempts to recreate the in vivo physicochemical conditions in which cartilage develops. Although much progress has been made towards the goal of developing clinically useful cartilage constructs, current constructs have inferior physicochemical properties than native cartilage. One of the reasons for this is the neglect of mechanical forces in cartilage culture. Bioreactors have been defined as devices in which biological or biochemical processes can be re-enacted under controlled conditions e.g. pH, temperature, nutrient supply, O2 tension and waste removal. The purpose of this review is to detail the role of bioreactors in the engineering of cartilage, including a discussion of bioreactor designs, current state of the art and future perspectives.     

Three-dimensional culture and bioreactors for cellular therapies

A bioreactor is defined as a specifically designed vessel to facilitate the growth of organisms and cells through application of physical and/or electrical stimulus. When cells with therapeutic potential were first discovered, they were initially cultured and expanded in two-dimensional (2-D) culture vessels such as plates or T-flasks. However, it was soon discovered that bioreactors could be used to expand and maintain cultures more easily and efficiently. Since then, bioreactors have come to be accepted as an indispensable tool to advance cell and tissue culture further. A wide array of bioreactors has been developed to date, and in recent years businesses have started supplying bioreactors commercially. Bioreactors in the research arena range from stirred tank bioreactors for suspension culture to those with various mechanical actuators that can apply different fluidic and mechanical stresses to tissues and three-dimensional (3-D) scaffolds. As regenerative medicine gains more traction in the clinic, bioreactors for use with cellular therapies are being developed and marketed. While many of the simpler bioreactors are fit for purpose, others fail to satisfy the complex requirements of tissues in culture. We have examined the use of different types of bioreactors in regenerative medicine and evaluated the application of bioreactors in the realization of emerging cellular therapies.     

High‐throughput miniaturized bioreactors for cell culture process development: Reproducibility,scalability, and control

Decreasing the timeframe for cell culture process development has been a key goal toward accelerating biopharmaceutical development. Advanced Microscale Bioreactors (ambr?) is an automated micro‐bioreactor system with miniature single‐use bioreactors with a 10–15 mL working volume controlled by an automated workstation. This system was compared to conventional bioreactor systems in terms of its performance for the production of a monoclonal antibody in a recombinant Chinese Hamster Ovary cell line. The miniaturized bioreactor system was found to produce cell culture profiles that matched across scales to 3 L, 15 L, and 200 L stirred tank bioreactors. The processes used in this article involve complex feed formulations, perturbations, and strict process control within the design space, which are in‐line with processes used for commercial scale manufacturing of biopharmaceuticals. Changes to important process parameters in ambr? resulted in predictable cell growth, viability and titer changes, which were in good agreement to data from the conventional larger scale bioreactors. ambr? was found to successfully reproduce variations in temperature, dissolved oxygen (DO), and pH conditions similar to the larger bioreactor systems. Additionally, the miniature bioreactors were found to react well to perturbations in pH and DO through adjustments to the Proportional and Integral control loop. The data presented here demonstrates the utility of the ambr? system as a high throughput system for cell culture process development. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:718–727, 2014     

Simple bioreactors for mass propagation of plants

Bioreactors provide a rapid and efficient plant propagation system for many agricultural and forestry species, utilizing liquid media to avoid intensive manual handling. Large-scale liquid cultures have been used for micropropagation through organogenesis or somatic embryogenesis pathways. Various types of bioreactors with gas-sparged mixing are suitable for the production of clusters of buds, meristems or protocorms. A simple glass bubble-column bioreactor for the proliferation of ornamental and vegetable crop species resulted in biomass increase of 3 to 6-fold in 3–4 weeks. An internal loop bioreactor was used for asparagus, celery and cucumber embryogenic cultures. However, as the biomass increased, the mixing and circulation were not optimal and growth was reduced. A disposable pre-sterilized plastic bioreactor (2–5-l volume) was used for the proliferation of meristematic clusters of several ornamental, vegetable and woody plant species. The plastic bioreactor induced minimal shearing and foaming, resulting in an increase in biomass as compared to the glass bubble-column bioreactor. A major issue related to the use of liquid media in bioreactors is hyperhydricity, that is, morphogenic malformation. Liquid cultures impose stress signals that are expressed in developmental aberrations. Submerged tissues exhibit oxidative stress, with elevated concentrations of reactive oxygen species associated with a change in antioxidant enzyme activity. These changes affect the anatomy and physiology of the plants and their survival. Malformation was controlled by adding growth retardants to decrease rapid proliferation. Growth retardants ancymidol or paclobutrazol reduced water uptake during cell proliferation, decreased vacuolation and intercellular spaces, shortened the stems and inhibited leaf expansion, inducing the formation of clusters. Using a two-stage bioreactor process, the medium was changed in the second stage to a medium lacking growth retardants to induce development of the meristematic clusters into buds or somatic embryos. Cluster biomass increased 10–15-fold during a period of 25–30 days depending on the species. Potato bud clusters cultured in 1.5 1 of medium in a 2-l capacity bioreactor, increased during 10–30 days. Poplar in vitro roots regenerated buds in the presence of thidiazuron (TDZ); the biomass increased 12-fold in 30 days. Bioreactor-regenerated clusters were separated with a manual cutter, producing small propagule units that formed shoots and initiated roots. Clusters of buds or meristematic nodules with reduced shoots, as well as arrested leaf growth, had less distortion and were optimal for automated cutting and dispensing. In tuber-, bulb- and corm-producing plants, growth retardants and elevated sucrose concentrations in the media were found to enhance storage organ formation, providing a better propagule for transplanting or storage. Bioreactor-cultures have several advantages compared with agar-based cultures, with a better control of the contact of the plant tissue with the culture medium, and optimal nutrient and growth regulator supply, as well as aeration and medium circulation, the filtration of the medium and the scaling-up of the cultures. Micropropagation in bioreactors for optimal plant production will depend on a better understanding of plant responses to signals from the microenvironment and on specific culture manipulation to control the morphogenesis of plants in liquid cultures.     

Towards Self‐Regulated Bioprocessing: A Compact Benchtop Bioreactor System for Monitored and Controlled 3D Cell and Tissue Culture

Bioreactors are crucial tools for the manufacturing of living cell‐based tissue engineered products. However, to reach the market successfully, higher degrees of automation, as well as a decreased footprint still need to be reached. In this study, the use of a benchtop bioreactor for in vitro perfusion culture of scaffold‐based tissue engineering constructs is assessed. A low‐footprint benchtop bioreactor system is designed, comprising a single‐use fluidic components and a bioreactor housing. The bioreactor is operated using an in‐house developed program and the culture environment is monitored by specifically designed sensor ports. A gas‐exchange module is incorporated allowing for heat and mass transfers. Titanium‐based scaffolds are seeded with human periosteum‐derived cells and cultured up to 3 weeks. The benchtop bioreactor constructs are compared to benchmark perfusion systems. Live/Dead stainings, DNA quantifications, glucose consumption, and lactate production assays confirm that the constructs cultured in the benchtop bioreactor grew similarly to the benchmark systems. Manual regulation of the system set points enabled efficient alteration of the culture environment in terms of temperature, pH, and dissolved oxygen. This study provides the necessary basis for the development of low‐footprint, automated, benchtop perfusion bioreactors and enables the implementation of active environment control.     

Disposable bioreactors for plant liquid cultures at Litre‐scale

Driven by the demands of the market and the manufacturing industry, disposable bioreactors have gained in importance in cell culture‐based processes during the last 10 years. Today they are widely accepted in R&D and also in manufacturing where process simplicity, safety and flexibility have top priority. Although disposable bioreactors are mainly used for cell expansions, glycoprotein secretions and virus generations realised with mammalian and insect cell lines, there are several reports delineating their suitability for the cultivation of plant cell and tissue cultures. This review describes the current disposable bioreactor types suitable for growing plant cell suspensions and organ cultures (hairy roots, meristematic clusters, somatic embryos) at Litre‐scale. Based on a definition of the term “disposable bioreactor”, a categorisation of the prevalent types for plant liquid cultures is presented. We describe the bioreactor regimes, working principles and bioengineering parameters of mechanically and pneumatically agitated bag bioreactors, which have advantages of process scalability and efficiency. Furthermore, results from the literature and data from our own research (obtained during production of undifferentiated bioactive cells, expressions of secondary metabolites and glycoproteins, and micropropagations of plant tissues) are discussed.     

Dynamic modeling and simulation of continuous airlift bioreactors

For dynamic behaviors of continuous airlift bioreactors, a mathematical model based on a tanks-in-series model with backflow has been developed. The equations describing the dynamics of airlift bioreactors are material balances for micro-organism, substrate, dissolved oxygen and oxygen in gas-phase and heat balances. Non-ideal mixing of liquid and gas phases is taken into account using a tanks-in-series model with backflow. The batch operation, startup operation and the consequence of plant failure were simulated and the effects of design and operating parameters for an airlift bioreactor on its dynamic behaviors were discussed. The concentration profiles of micro-organism, substrate, dissolved oxygen and oxygen in gas-phase and the temperature profile in an airlift bioreactors and their dynamics were obtained. The computational results indicate that the transients of a chemostat in the case of bubble column bioreactor are slower compared with those in the case of airlift bioreactor. The proposed simulator is more precise as compared with models published previously in the literature and therefore provides more reliable and rational examination of continuous airlift bioreactor performance.     

Improvement of plastic-based disposable bioreactors for plant science needs

The present article describes two new applications of plastic-based cell culture systems in the plant biotechnology domain. Different types of bioreactors are used at Nestlé R&D Center-Tours for large scale culture of plants cells to produce metabolites or recombinant proteins and for mass propagation of selected plant varieties by somatic embryogenesis. Particularly, recent studies are directed to cut down the production costs of these two processes by developing disposable cell culture systems. For large scale culture, two novel flexible plastic-based disposable bioreactors have been developed from 10 to 100 l working volumes, validated with several plant species (“Wave and Undertow” and “Slug Bubble” bioreactors). Vegetative propagation of elite plant varieties is achieved through somatic embryogenesis in liquid medium. A pilot scale process has been recently set up for the industrial propagation of Coffea canephora (Robusta coffee). The current production capacity is 2.5–3.0 million embryos per year. The pre-germination of the embryos was previously conducted by temporary immersion in liquid medium in 10-l glass bioreactors. An improved process has been developed using a 10-l disposable bioreactor consisting in a bag containing a rigid plastic box (“Box-in-Bag” bioreactor), insuring, amongst other advantages, a higher light transmittance to the biomass due to its horizontal design.     

Microbial population dynamics in laboratory-scale solid waste bioreactors in the presence or absence of biosolids

Aims:  Decomposition of solid waste is microbially mediated, yet little is known about the associated structure and temporal changes in prokaryotic communities. Bioreactors were used to simulate landfill conditions and archaeal and bacterial community development in leachate was examined over 8 months.
Methods and Results:  Municipal solid waste (MSW) was deposited in laboratory bioreactors with or without biosolids and combustion residues (ash). The near-neutral pH fell about half a log by day 25, but recovered to ∼7·0 by day 50. Cell concentrations in bioreactors containing only MSW were significantly higher than those from co-disposal bioreactors. Archaeal and bacterial community structure was analysed by denaturing gradient gel electrophoresis targeting 16S rRNA genes, showing temporal population shifts for both domains. mcrA sequences retrieved from a co-disposal bioreactor were predominantly affiliated with the orders Methanosarcinales and Methanomicrobiales .
Conclusion:  Regardless of waste composition, microbial communities in bioreactor leachates exhibited high diversity and distinct temporal trends. The solid waste filled bioreactors allowed simulation of solid waste decomposition in landfills while also reducing the variables.
Significance and Impact of the Study:  This study advances the basic understanding of changes in microbial community structure during solid waste decomposition, which may ultimately improve the efficiency of solid waste management.     

Tubular bioreactors: case study of bioreactor performance for industrial production and scientific research

Application of bioreactors is dominated by industrial production with the consequence that bioreactors also for scientific purposes are mainly used following an empiric pragmatic approach. For the sake of a breakthrough in biotechnology in general, and especially for advanced process development, a more systematic approach is emphasized here. This methodology in bioreactor performance studies is explained and the meaning clarified in a case study of a new type of tubular bioreactor. The central role of so-called "model bioreactors" in bench-scale applications is illustrated as a powerful contribution to the optimal design of bioreactors in technical scale. Pilot plant data in case of a tubular reactor for the production of ethanol with Zymomonas and biopesticides with Bacillus thuringiensis are presented.     

Two new disposable bioreactors for plant cell culture: The wave and undertow bioreactor and the slug bubble bioreactor

The present article describes two novel flexible plastic-based disposable bioreactors. The first one, the WU bioreactor, is based on the principle of a wave and undertow mechanism that provides agitation while offering convenient mixing and aeration to the plant cell culture contained within the bioreactor. The second one is a high aspect ratio bubble column bioreactor, where agitation and aeration are achieved through the intermittent generation of large diameter bubbles, "Taylor-like" or "slug bubbles" (SB bioreactor). It allows an easy volume increase from a few liters to larger volumes up to several hundred liters with the use of multiple units. The cultivation of tobacco and soya cells producing isoflavones is described up to 70 and 100 L working volume for the SB bioreactor and WU bioreactor, respectively. The bioreactors being disposable and pre-sterilized before use, cleaning, sterilization, and maintenance operations are strongly reduced or eliminated. Both bioreactors represent efficient and low cost cell culture systems, applicable to various cell cultures at small and medium scale, complementary to traditional stainless-steel bioreactors.     

新型生物反应器结构研究进展

生物反应器是生物工程的核心设备,其结构的合理性直接决定反应器生物加工的效率。生物反应器的研究一直是生物工程的核心问题之一。随着青霉素的工业化生产,机械搅拌式生物反应器应运而生,此后,随着动植物细胞培养,高等真菌培养,藻类培养等生物过程的发展,人们相应开发了大量的生物反应器,其中以机械搅拌式生物反应器和气升式样生物反应器尤为突出,本文总结了近年来文献报道的新型生物反应器,主要阐述了机械搅拌式和气升式两类生物反应器结构的研究进展,对目前国内外报道的11种新型反应器典型结构进行了总结与分析。     

Silicone-tubing aerated bioreactors for somatic embryo production

Bioreactors equipped with silicone tubings for bubble free oxygen supply are suitable for culture of embryogenic cell suspensions. The advantages of bubble free aeration systems over various devices for dispersion of air bubbles are the lack of foam formation and the possibilities of precise control of the desired oxygen set point. The specification of silicone tubing (length, diameter, wall thickness) has to be adapted according to the amount of embryogenic biomass to be produced in the bioreactor. Cell suspensions of Euphorbia pulcherrima were cultured --2 l bioreactor at 60% pO₂, supplied by a silicone tubing system of 155 cm length, 4.0 mm diameter and 0.4 mm wall thickness. The oxygen concentration decreased when the packed cell volume exceeded 14% (=3.7 g l^-1 cell dry weight), indicating the upper limit of oxygen supply by the silicone tubing. Mathematical considerations for membrane aerated bioreactors are presented with the intention of enabling a more precise definition for the configuration of silicone tube systems in different bioreactor types.     

Application of bioreactor systems for large scale production of horticultural and medicinal plants

Automation of micropropagation via organogenesis or somatic embryogenesis in a bioreactor has been advanced as a possible way of reducing costs. Micropropagation by conventional techniques is typically a labour-intensive means of clonal propagation. The paper describes lower cost and less labour-intensive clonal propagation through the use of modified air-lift, bubble column, bioreactors (a balloon-type bubble bioreactor), together with temporary immersion systems for the propagation of shoots, bud-clusters and somatic embryos. Propagation of Anoectochilus, apple, Chrysanthemum, garlic, ginseng, grape, Lilium, Phalaenopsis and potato is described. In this chapter, features of bioreactors and bioreactor process design specifically for automated mass propagation of several plant crops are described, and recent research aimed at maximizing automation of the bioreactor production process is highlighted.     

Extruded Bioreactor Perfusion Culture Supports the Chondrogenic Differentiation of Human Mesenchymal Stem/Stromal Cells in 3D Porous Poly(ɛ‐Caprolactone) Scaffolds

Novel bioengineering strategies for the ex vivo fabrication of native‐like tissue‐engineered cartilage are crucial for the translation of these approaches to clinically manage highly prevalent and debilitating joint diseases. Bioreactors that provide different biophysical stimuli have been used in tissue engineering approaches aimed at enhancing the quality of the cartilage tissue generated. However, such systems are often highly complex, expensive, and not very versatile. In the current study, a novel, cost‐effective, and customizable perfusion bioreactor totally fabricated by additive manufacturing (AM) is proposed for the study of the effect of fluid flow on the chondrogenic differentiation of human bone‐marrow mesenchymal stem/stromal cells (hBMSCs) in 3D porous poly(?‐caprolactone) (PCL) scaffolds. hBMSCs are first seeded and grown on PCL scaffolds and hBMSC–PCL constructs are then transferred to 3D‐extruded bioreactors for continuous perfusion culture under chondrogenic inductive conditions. Perfused constructs show similar cell metabolic activity and significantly higher sulfated glycosaminoglycan production (≈1.8‐fold) in comparison to their non‐perfused counterparts. Importantly, perfusion bioreactor culture significantly promoted the expression of chondrogenic marker genes while downregulating hypertrophy. This work highlights the potential of customizable AM platforms for the development of novel personalized repair strategies and more reliable in vitro models with a wide range of applications.     

Plant Bioreactors for Pharmaceuticals

Abstract

Plant bioreactors are attractive expression systems for economic production of pharmaceuticals. Various plant expression systems or platforms have been tested with certain degrees of success over the past years. However, further development and improvement are needed for more effective plant bioreactors. In this review we first summarize recent progress in various plant bioreactor expression systems and then focus on discussing protein compartmentation to unique organelles and various strategies for developing better plant bioreactors.     

Microbial community analysis of a full-scale DEMON bioreactor

Full-scale applications of autotrophic nitrogen removal technologies for the treatment of digested sludge liquor have proliferated during the last decade. Among these technologies, the aerobic/anoxic deammonification process (DEMON) is one of the major applied processes. This technology achieves nitrogen removal from wastewater through anammox metabolism inside a single bioreactor due to alternating cycles of aeration. To date, microbial community composition of full-scale DEMON bioreactors have never been reported. In this study, bacterial community structure of a full-scale DEMON bioreactor located at the Apeldoorn wastewater treatment plant was analyzed using pyrosequencing. This technique provided a higher-resolution study of the bacterial assemblage of the system compared to other techniques used in lab-scale DEMON bioreactors. Results showed that the DEMON bioreactor was a complex ecosystem where ammonium oxidizing bacteria, anammox bacteria and many other bacterial phylotypes coexist. The potential ecological role of all phylotypes found was discussed. Thus, metagenomic analysis through pyrosequencing offered new perspectives over the functioning of the DEMON bioreactor by exhaustive identification of microorganisms, which play a key role in the performance of bioreactors. In this way, pyrosequencing has been proven as a helpful tool for the in-depth investigation of the functioning of bioreactors at microbiological scale.     

Concentric cylinder bioreactor for production of tissue engineered cartilage: effect of seeding density and hydrodynamic loading on construct development

A concentric cylinder bioreactor has been developed to culture tissue engineered cartilage constructs under hydrodynamic loading. This bioreactor operates in a low shear stress environment, has a large growth area for construct production, allows for dynamic seeding of constructs, and provides for a uniform loading environment. Porous poly-lactic acid constructs, seeded dynamically in the bioreactor using isolated bovine chondrocytes, were cultured for 4 weeks at three seeding densities (60, 80, 100 x 10(6) cells per bioreactor) and three different shear stresses (imposed at 19, 38, and 76 rpm) to characterize the effect of chondrocyte density and hydrodynamic loading on construct growth. Construct seeding efficiency with chondrocytes is greater than 95% within 24 h. Extensive chondrocyte proliferation and matrix deposition are achieved so that after 28 days in culture, constructs from bioreactors seeded at the highest cell densities contain up to 15 x 10(6) cells, 2 mg GAG, and 3.5 mg collagen per construct and exhibit morphology similar to that of native cartilage. Bioreactors seeded with 60 million chondrocytes do not exhibit robust proliferation or matrix deposition and do not achieve morphology similar to that of native cartilage. In cultures under different steady hydrodynamic loading, the data demonstrate that higher shear stress suppresses matrix GAG deposition and encourages collagen incorporation. In contrast, under dynamic hydrodynamic loading conditions, cartilage constructs exhibit robust matrix collagen and GAG deposition. The data demonstrate that the concentric cylinder bioreactor provides a favorable hydrodynamic environment for cartilage construct growth and differentiation. Notably, construct matrix accumulation can be manipulated by hydrodynamic loading. This bioreactor is useful for fundamental studies of construct growth and to assess the significance of cell density, nutrients, and hydrodynamic loading on cartilage development. In addition, studies of cartilage tissue engineering in the well-characterized, uniform environment of the concentric cylinder bioreactor will develop important knowledge of bioprocessing parameters critical for large-scale production of engineered tissues.     

Hosting the plant cells in vitro: recent trends in bioreactors

Biotechnological production of high-value metabolites and therapeutic proteins by plant in vitro systems has been considered as an attractive alternative of classical technologies. Numerous proof-of-concept studies have illustrated the feasibility of scaling up plant in vitro system-based processes while keeping their biosynthetic potential. Moreover, several commercial processes have been established so far. Though the progress on the field is still limited, in the recent years several bioreactor configurations has been developed (e.g., so-called single-use bioreactors) and successfully adapted for growing plant cells in vitro. This review highlights recent progress and limitations in the bioreactors for plant cells and outlines future perspectives for wider industrialization of plant in vitro systems as “green cell factories” for sustainable production of value-added molecules.