Flow characterization of a wavy-walled bioreactor for cartilage tissue engineering

Cartilage tissue engineering requires the use of bioreactors in order to enhance nutrient transport and to provide sufficient mechanical stimuli to promote extracellular matrix (ECM) synthesis by chondrocytes. The amount and quality of ECM components is a large determinant of the biochemical and mechanical properties of engineered cartilage constructs. Mechanical forces created by the hydrodynamic environment within the bioreactors are known to influence ECM synthesis. The present study characterizes the hydrodynamic environment within a novel wavy-walled bioreactor (WWB) used for the development of tissue-engineered cartilage. The geometry of this bioreactor provides a unique hydrodynamic environment for mammalian cell and tissue culture, and investigation of hydrodynamic effects on tissue growth and function. The flow field within the WWB was characterized using two-dimensional particle-image velocimetry (PIV). The flow in the WWB differed significantly from that in the traditional spinner flask both qualitatively and quantitatively, and was influenced by the positioning of constructs within the bioreactor. Measurements of velocity fields were used to estimate the mean-shear stress, Reynolds stress, and turbulent kinetic energy components in the vicinity of the constructs within the WWB. The mean-shear stress experienced by the tissue-engineered constructs in the WWB calculated using PIV measurements was in the range of 0-0.6 dynes/cm2. Quantification of the shear stress experienced by cartilage constructs, in this case through PIV, is essential for the development of tissue-growth models relating hydrodynamic parameters to tissue properties.     

Location of scaffolds in bioreactors modulates the hydrodynamic environment experienced by engineered tissues

Physical forces experienced by engineered-tissues during in vitro cultivation influence tissue growth and function. The hydrodynamic environment within bioreactors plays a decisive role in providing the necessary physical stimuli and nutrient transport to support tissue development. Our overall goal is to investigate interrelationships between the local hydrodynamic environment in the bioreactor and the structural and functional tissue properties in order to optimize the production of clinically relevant engineered-tissues. To this end, we used computational fluid dynamics (CFD) modeling to characterize the complex hydrodynamic environment in a wavy-walled bioreactor used for cultivation of tissue-engineered cartilage constructs and examined the changes in the flow field due to the presence of constructs. The flow-induced shear stress range experienced by engineered constructs cultivated in the wavy-walled bioreactor (0-0.67 dyn/cm(2)) was found to be significantly lower than that in the spinner flask (0-1.2 dyn/cm(2)), and to be modulated by the radial or axial position of the constructs. These CFD results are validated by experimental particle-image velocimetry (PIV) measurements previously reported by our group. Results from the present study indicate that the location of constructs in the bioreactor not only affected the magnitude and distribution of the shear stresses on the constructs, but also other hydrodynamic parameters, such as the directional distribution of the fluid velocity and the degree of fluid recirculation, all of which may differentially influence the development of tissue-engineered constructs.     

Enhancing cell seeding of scaffolds in tissue engineering through manipulation of hydrodynamic parameters

The seeding of cells onto biocompatible scaffolds is a determinant step in the attainment of functional properties of engineered tissues. Efficient, fast and spatially uniform cell seeding can improve the clinical potential of engineered tissue templates. One way to approach these cell seeding requirements is through bioreactor design. In the present study, bovine chondrocytes were seeded (2.5, 5.0 or 10.0 million cells per scaffold) onto polyglycolic acid scaffolds within the hydrodynamic environments of wavy-walled and spinner flask bioreactors. Previous characterizations of the hydrodynamic environment in the vicinity of constructs cultivated in these bioreactors suggested decreased flow-induced shear stress as well as increased recirculation and magnitude of the axial fluid velocities in the wavy-walled bioreactor. Here we report more efficient and spatially uniform cell seeding in the wavy-walled bioreactor, and at intermediate initial cell densities (5 million cells per scaffold). This study constitutes an important step towards the achievement of functional tissue-engineered implants by (i) increasing our understanding of the influence of hydrodynamic parameters on the efficiency and spatial distribution of cell attachment to scaffolds and the production of extracellular matrix and (ii) introducing a comprehensive approach to the investigation of the effects of bioprocessing conditions on tissue morphology and composition.     

Characterization of mixing in a novel wavy-walled bioreactor for tissue engineering

The wavy-walled bioreactor (WWB) possesses a novel geometry comprised of walls with sinusoidal waves that mimic baffles in an effort to promote mixing. This geometry provides a unique hydrodynamic environment suitable for the cultivation of mammalian cells and tissues and the investigation of fluid mechanical effects on cell and tissue growth and development. In the present study, mixing in WWB was characterized and compared to that in a conventional spinner flask (SF). The key parameters included in this characterization were mixing time, residence time distribution (RTD), and dissolved oxygen concentration during engineered cartilage tissue cultivation. Factors that influenced mixing in WWB included wave amplitude, agitation rate, and the ratio of the impeller diameter to the tank diameter (D/T). Data obtained from RTD and acid base neutralization studies confirmed the presence of different mixing zones in WWB. A theoretical comparison of WWB to a baffled spinner flask (BSF) using computational fluid dynamics (CFD) modeling predicted that while enhanced mixing was achieved in wavy-walled and BSF bioreactors, the shear stresses applied on tissue constructs were 15% lower in WWB. Improved mixing was achieved in WWB compared to the SF at similar D/T ratios, verified by improved oxygen transport and increased dispersion. However, for lower D/T ratios mixing in WWB was not necessarily improved. This study demonstrated the importance of characterization of mixing by showing the impact of even minor changes in bioreactor geometry and operating conditions.     

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.     

Comparisons of microcarrier cell culture processes in one hundred mini-liter spinner flask and fifteen-liter bioreactor cultures

Microcarrier cell culture process can be used to culture anchorange-dependent cells in large bioreactor vessels. The process performance in large bioreactors is usually less prominent than that in spinner flask vessels and bench scale reactors. In this study we investigated the microcarrier cell culture processes in 100?ml spinner flask and 15-liter bioreactor cultures, including the kinetics for cell attachment, cell growth and the production of Japanese encephaltilis vaccine strain (Beijing-1) virus. Under a fixed concentration of microcarrier and cell density used in inoculations, the attachment kinetics of Vero cells on Cytodex 1 microcarrier in a 15-liter bioreactor vessel was 2 folds slower than with 100?ml spinner flask culture. Virus replication in 15-liter bioreactor culture also revealed an approximately one day lag-time compared to 100?ml spinner flask culture. Findings presented herein provide valuable information for designing and operating microcarrier cell culture processes in large bioreactor vessels.     

造血细胞体外悬浮培养和生物反应器开发

为解决造血细胞的静态培养中由浓度梯度引起的培养不稳定、环境不均一、难放大等问题,首先采用转瓶对脐血单个核细胞进行了悬浮培养研究,结果表明,悬浮培养中总细胞、集落和CD34^＋细胞的扩增都高于静态的方瓶培养。在测试了所用材料生物相容性的基础上,开发了可以控制溶氧和pH的生物反应器,并将其应用到造血细胞的批培养中,结果表明反应器的培养环境均一,可实现较高密度的培养,而且总细胞、集落和CD34^＋细胞的扩增都优于静态培养。大规模的反应器培养有利于解决临床应用中细胞数量不足的问题。     

Fluid mechanics of a spinner-flask bioreactor

Spinner-flask bioreactors have been used for the production of articular cartilage in vitro. The dynamic environment within bioreactors is known to significantly affect the growth and development of the tissue. The present research focuses on the experimental and numerical characterization of the flow field within a spinner flask operating under conditions used to produce cartilage. Laboratory experiments carried out in a scaled-up model bioreactor employ particle-image velocimetry (PIV) to determine velocity and shear-rate fields in the vicinity of the construct closest to the stir bar, in addition to turbulence properties. Numerical computations calculated using FLUENT, a commercial software package, simulate the flow field in the same model bioreactor under similar operating conditions. In the computations, scaffolds were modeled as both solid and porous media with different permeabilities and flow rates through various faces of the construct nearest the stir bar were examined.     

On-line monitoring of oxygen in Tubespin, a novel, small-scale disposable bioreactor

A novel, optical sensor was fixed in a new type of disposable bioreactor, Tubespin, for the on-line (real-time) monitoring of dissolved oxygen concentrations during cell culture. The cell density, viability and volumetric mass transfer coefficient were also determined to further characterize the bioreactors. The k_La value of the Tubespin at standard conditions was 24.3 h⁻¹, while that of a spinner flask was only 2.7 h⁻¹. The maximum cell density in the Tubespin bioreactor reached 6 × 10⁶ cells mL⁻¹, which was two times higher than the cell density in a spinner flask. Furthermore, the dynamic dissolved oxygen level was maintained above 90% air-saturation in the Tubespin, while the value was only 1.9% in a spinner flask. These results demonstrate the competitive advantage of using the Tubespin system over spinner flasks for process optimization and scale-down studies of oxygen transfer and cell growth.     

Scale-up of mouse embryonic stem cell expansion in stirred bioreactors

The aim of this study was to develop a robust, quality controlled and reproducible large-scale culture system using serum-free (SF) medium to obtain vast numbers of embryonic stem (ES) cells as a starting source for potential applications in tissue regeneration, as well as for drug screening studies. Mouse ES (mES) cells were firstly cultured on microcarriers in spinner flasks to investigate the effect of different parameters such as the agitation rate and the feeding regimen. Cells were successfully expanded at agitation rates up to 60 rpm using the SF medium and no significant differences in terms of growth kinetics or metabolic profiles were found between the two feeding regimens evaluated: 50% medium renewal every 24 h or 25% every 12 h. Overall, cells reached maximum concentrations of (4.2 ± 0.4) and (5.6 ± 0.8) ×10(6) cells/mL at Day 8 for cells fed once or twice per day; which corresponds to an increase in total cell number of 85 ± 7 and 108 ± 16, respectively. To have a more precise control over culture conditions and to yield a higher number of cells, the scale-up of the spinner flask culture system was successfully accomplished by using a fully controlled stirred tank bioreactor. In this case, the concentration of mES cells cultured on microcarriers increased 85 ± 15-fold over 11 days. Importantly, mES cells expanded under stirred conditions, in both spinner flask and fully controlled stirred tank bioreactor, using SF medium, retained the expression of pluripotency markers such as Oct-4, Nanog, and SSEA-1 and their differentiation potential into cells of the three embryonic germ layers.     

Effect of a mechanical stimulation bioreactor on tissue engineered, scaffold-free cartilage

Achieving sufficient functional properties prior to implantation remains a significant challenge for the development of tissue engineered cartilage. Many studies have shown chondrocytes respond well to various mechanical stimuli, resulting in the development of bioreactors capable of transmitting forces to articular cartilage in vitro. In this study, we describe the production of sizeable, tissue engineered cartilage using a novel scaffold-free approach, and determine the effect of perfusion and mechanical stimulation from a C9-x Cartigen bioreactor on the properties of the tissue engineered cartilage. We created sizable tissue engineered cartilage from porcine chondrocytes using a scaffold-free approach by centrifuging a high-density chondrocyte cell-suspension onto an agarose layer in a 50 mL tube. The gross and histological appearances, biochemical content, and mechanical properties of constructs cultured in the bioreactor for 4 weeks were compared to constructs cultured statically. Mechanical properties were determined from unconfined uniaxial compression tests. Constructs cultured in the bioreactor exhibited an increase in total GAG content, equilibrium compressive modulus, and dynamic modulus versus static constructs. Our study demonstrates the C9-x CartiGen bioreactor is able to enhance the biomechanical and biochemical properties of scaffold-free tissue engineered cartilage; however, no additional enhancement was seen between loaded and perfused groups.     

Limiting cell aggregation during mesenchymal stem cell expansion on microcarriers

Mesenchymal stem cells (MSC) are known to be a valuable cell source for tissue engineering and regenerative medicine. However, one of the main limiting steps in their clinical use is the amplification step. MSC expansion on microcarriers has emerged during the last few years, fulfilling the lack of classical T‐flasks expansion. Even if the therapeutic potential of MSC as aggregates has been recently highlighted, cell aggregation during expansion has to be avoided. Thus, MSC culture on microcarriers has still to be improved, notably concerning cell aggregation prevention. The aim of this study was to limit cell aggregation during MSC expansion on Cytodex‐1^®, by evaluating the impact of several culture parameters. First, MSC cultures were performed at different agitation rates (0, 25, and 75 rpm) and different initial cell densities (25 and 50 × 10⁶ cell g^?1 Cytodex‐1^®). Then, the MSC aggregates were put into contact with additional available surfaces (T‐flask, fresh and used Cytodex‐1^®) at different times (before and after cell aggregation). The results showed that cell aggregation was partly induced by agitation and prevented in static cultures. Moreover, cell aggregation was dependent on cell density and correlated with a decrease in the total cell number. It was however shown that the aggregated organization could be dissociated when in contact with additional surfaces such as T‐flasks or fresh Cytodex‐1^® carriers. Finally, cell aggregation could be successfully limited in spinner flask by adding fresh Cytodex‐1^® carriers before its onset. Those results indicated that MSC expansion on agitated Cytodex‐1^® microcarriers could be performed without cell aggregation, avoiding a decrease in total cell number. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Computational fluid dynamics modeling of steady-state momentum and mass transport in a bioreactor for cartilage tissue engineering

Computational fluid dynamics (CFD) models to quantify momentum and mass transport under conditions of tissue growth will aid bioreactor design for development of tissue-engineered cartilage constructs. Fluent CFD models are used to calculate flow fields, shear stresses, and oxygen profiles around nonporous constructs simulating cartilage development in our concentric cylinder bioreactor. The shear stress distribution ranges from 1.5 to 12 dyn/cm(2) across the construct surfaces exposed to fluid flow and varies little with the relative number or placement of constructs in the bioreactor. Approximately 80% of the construct surface exposed to flow experiences shear stresses between 1.5 and 4 dyn/cm(2), validating the assumption that the concentric cylinder bioreactor provides a relatively homogeneous hydrodynamic environment for construct growth. Species mass transport modeling for oxygen demonstrates that fluid-phase oxygen transport to constructs is uniform. Some O(2) depletion near the down stream edge of constructs is noted with minimum pO(2) values near the constructs of 35 mmHg (23% O(2) saturation). These values are above oxygen concentrations in cartilage in vivo, suggesting that bioreactor oxygen concentrations likely do not affect chondrocyte growth. Scale-up studies demonstrate the utility and flexibility of CFD models to design and characterize bioreactors for growth of tissue-engineered cartilage.     

Anthocyanin production from Vaccinium pahalae: limitations of the physical microenvironment.

Physical microenvironmental parameters conducive to production of flavonoids in vitro from continuous Vaccinium pahalae suspension cultures were examined first in shake flask culture experiments, and results were used to guide adaptations of a bioreactor production environment. Anthocyanin pigments were primarily concentrated in smaller aggregates up to 519 microm diameter. Agitation at 150 rpm and routine use of a mechanical scraper to periodically retrieve cells thrown out of solution was sufficient to keep productive plant cell aggregates in bioreactor suspension. A set up for enhanced irradiance with mercury lamps providing an average of 240 micromol m(-2) s(-1) PPF at the inner surface of the bioreactor vessel was required to sustain anthocyanin productivity through a 10-day production cycle.     

Continuous cultivation of human hamstring tenocytes on microcarriers in a spinner flask bioreactor system

Tendon healing is a time consuming process leading to the formation of a functionally altered reparative tissue. Tissue engineering‐based tendon reconstruction is attracting more and more interest. The aim of this study was to establish tenocyte expansion on microcarriers in continuous bioreactor cultures and to study tenocyte behavior during this new approach. Human hamstring tendon‐derived tenocytes were expanded in monolayer culture before being seeded at two different seeding densities (2.00 and 4.00 × 10⁶ cells/1000 cm² surface) on Cytodex? type 3 microcarriers. Tenocytes' vitality, growth kinetics and glucose/lactic acid metabolism were determined dependent on the seeding densities and stirring velocities (20 or 40 rpm) in a spinner flask bioreactor over a period of 2 weeks. Gene expression profiles of tendon extracellular matrix (ECM) markers (type I/III collagen, decorin, cartilage oligomeric protein [COMP], aggrecan) and the tendon marker scleraxis were analyzed using real time detection polymerase chain reaction (RTD‐PCR). Type I collagen and decorin deposition was demonstrated applying immunolabeling. Tenocytes adhered on the carriers, remained vital, proliferated and revealed an increasing glucose consumption and lactic acid formation under all culture conditions. “Bead‐to‐bead” transfer of cells from one microcarrier to another, a prerequisite for continuous tenocyte expansion, was demonstrated by scanning electron microscopy. Type I and type III collagen gene expression was mainly unaffected, whereas aggrecan and partly also decorin and COMP expression was significantly downregulated compared to monolayer cultures. Scleraxis gene expression revealed no significant regulation on the carriers. In conclusion, tenocytes could be successfully expanded on microcarriers. Therefore, bioreactors are promising tools for continuous tenocyte expansion. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:142–151, 2014     

Minimization of aggregation of secreted bivalent anti-human T cell immunotoxin in Pichia pastoris bioreactor culture by optimizing culture conditions for protein secretion

In a bioreactor culture of genetically engineered Pichia pastoris secreting a bivalent immunotoxin, 64% of the secreted immunotoxin was present in aggregate forms and this resulted in a loss of bioactivity. Biochemical analyses of the secreted immunotoxin and an in vitro aggregation study using purified monomeric immunotoxin suggested that aggregation was primarily an extracellular event. By employing limited methanol feeding at 0.75 mlmin(-1) per 10l initial medium, oxygen consumption was reduced, permitting a lowering of the bioreactor agitation speed from 800 to 400 rpm. By increasing the anti-foam reagent to 0.6 mll(-1), the thickness of the air/liquid interfacial foam layer was reduced by 80%. These steps reduced the immunotoxin aggregates from 64% to 5%. Consequently immunotoxin purification yield was increased from 53.0% to 73.8%. Simultaneously this methodology enhanced immunotoxin secretion to 120 mgl(-1) at 163 h of methanol induction in a toxin resistant production strain. We conclude that minimizing shearing force and reducing the air/liquid interfacial foam area are crucial factors in reducing hydrophobic protein aggregation upon secretory expression in yeast bioreactor cultures.     

Computational fluid dynamics modeling of momentum transport in rotating wall perfused bioreactor for cartilage tissue engineering

In this study, computational fluid dynamics (CFD) analysis of a rotating-wall perfused-vessel (RWPV) bioreactor is performed to characterize the complex hydrodynamic environment for the simulation of cartilage development in RWPV bioreactor in the presence of tissue-engineered cartilage constructs, i.e., cell-chitosan scaffolds. Shear stress exerted on chitosan scaffolds in bioreactor was calculated for different rotational velocities in the range of 33-38 rpm. According to the calculations, the lateral and lower surfaces were exposed to 0.07926-0.11069 dyne/cm(2) and 0.05974-0.08345 dyne/cm(2), respectively, while upper surfaces of constructs were exposed to 0.09196-0.12847 dyne/cm(2). Results validate adequate hydrodynamic environment for scaffolds in RWPV bioreactor for cartilage tissue development which concludes the suitability of operational conditions of RWPV bioreactor.     

Lysozyme inactivation and aggregation in stirred-reactor

Mechanisms of enzyme inactivation and aggregation are still poorly understood. In this work, we are considering the characterisation of both inactivation and aggregation in stirred tank reactor, with lysozyme as the model enzyme.

The inactivation kinetics are first order. For stirring speeds in the range of 0–700 rpm, the kinetic constant is found to be proportional to the power brought by the impeller. It suggests that inactivation depends on collisions between enzyme molecules. Efficient collisions between native and inactive molecules induce native molecules to turn into inactive molecules and lead to lysozyme aggregation.

During inactivation, enzymes are found to aggregate as shown by light scattering measurements. The structure of aggregates was studied on samples treated for chemical denaturation and reduction. The aggregates are supramolecular edifices, mainly made up of inactivated enzymes linked by weak forces. But aggregates are also made up of dimers and trimers of lysozyme, linked by disulfide bridges. Dimers and trimers are 18% and 5%, respectively, of the total amount of lysozyme aggregates.

Whatever the stage of aggregate formation and the initial enzyme concentration are, these aggregates are irreversibly inactivated. Enzyme activity is definitely lost even if stirring is stopped and/or temperature decreased.

This study points out the importance of hydrodynamics in bioreactors and highlights the nature of the aggregates resulting from the interactions between native and inactive enzymes.     

Mechanobiology of engineered cartilage cultured under a quantified fluid-dynamic environment

Natural cartilage remodels both in vivo and in vitro in response to mechanical forces and hence mechanical stimulation is believed to have a potential as a tool to modulate extra-cellular matrix synthesis in tissue-engineered cartilage. Fluid-induced shear is known to enhance chondrogenesis on animal cells. A well-defined hydrodynamic environment is required to study the biochemical response to shear of three-dimensional engineered cell systems. We have developed a perfused-column bioreactor in which the culture medium flows through chondrocyte-seeded porous scaffolds, together with a computational fluid-dynamic model of the flow through the constructs' microstructure. A preliminary experiment of human chondrocyte growth under static versus dynamic conditions is described. The median shear stress imposed on the cells in the bioreactor culture, as predicted by the CFD model, is 3 × 10⁻³ Pa (0.03 dyn/cm²) at a flow rate of 0.5 ml/min corresponding to an inlet fluid velocity of 44.2 μm/s. Providing a fluid-dynamic environment to the cells yielded significant differences in cell morphology and in construct structure. Received: 22 December 2001 / Accepted: 18 February 2002     

Approach toward an efficient inoculum preparation stage for suspension BHK-21 cell culture

Mammalian cells are the most frequently used hosts for biopharmaceutical proteins manufacturing. Inoculum quality is a key element for establishing an efficient bioconversion process. The main objective in inoculation expansion process is to generate large volume of viable cells in the shortest time. The aim of this paper was to optimize the inoculum preparation stage of baby hamster kidney (BHK)-21 cells for suspension cultures in benchtop bioreactors, by means of a combination of static and agitated culture systems. Critical parameters for static (liquid column height: 5, 10, 15 mm) and agitated (working volume: 35, 50, 65 mL, inoculum volume percentage: 10, 30 % and agitation speed: 25, 60 rpm) cultures were study in T-flask and spinner flask, respectively. The optimal liquid column height was 5 mm for static culture. The maximum viable cell concentration in spinner flask cultures was reached with 50 mL working volume and the inoculum volume percentage was not significant in the range under study (10–30 %) at 25 rpm agitation. Agitation speed at 60 rpm did not change the main kinetic parameters with respect to those observed for 25 rpm. These results allowed for a schedule to produce more than 4 × 10⁹ BHK-21 cells from 4 × 10⁶ cells in 13 day with 1,051 mL culture medium.