Parallel-plate Bioreactor Research Articles

Fluid Flow Stimulation Modulates Expression of S100 Genes in Normal Breast Epithelium and Breast Cancer

IntroductionS100 proteins are intracellular calcium ion sensors that participate in cellular processes, some of which are involved in normal breast functioning and breast cancer development. Despite several S100 genes being overexpressed in breast cancer, their roles during disease development remain elusive. Human mammary epithelial cells (HMECs) can be exposed to fluid shear stresses and implications of such interactions have not been previously studied. The goal of this study was to analyze expression profiles of S100 genes upon exposing HMECs to fluid flow.MethodsHMECs and breast cancer cell lines were exposed to fluid flow in a parallel-plate bioreactor system. Changes in gene expression were quantified using microarrays and qPCR, gene-gene interactions were elucidated using network analysis, and key modified genes were examined in three independent clinical datasets.ResultsS100 genes were among the most upregulated genes upon flow stimulation. Network analysis revealed interactions between upregulated transcripts, including interactions between S100P, S100PBP, S100A4, S100A7, S100A8 and S100A9. Overexpression of S100s was also observed in patients with early stage breast cancer compared to normal breast tissue, and in most breast cancer patients. Finally, survival analysis revealed reduced survival times for patients with elevated expression of S100A7 and S100P.ConclusionThis study shows that exposing HMECs to fluid flow upregulates genes identified clinically to be overexpressed during breast cancer development, including S100A7 and S100P. These findings are the first to show that S100 genes are flow-responsive and might be participating in a fundamental adaptation pathway in normal tissue that is also active in breast cancer.

Fluid flow exposure promotes epithelial-to-mesenchymal transition and adhesion of breast cancer cells to endothelial cells

BackgroundMechanical interactions between tumor cells and microenvironments are frequent phenomena during breast cancer progression, however, it is not well understood how these interactions affect Epithelial-to-Mesenchymal Transition (EMT). EMT is associated with the progression of most carcinomas through induction of new transcriptional programs within affected epithelial cells, resulting in cells becoming more motile and adhesive to endothelial cells.MethodsMDA-MB-231, SK-BR-3, BT-474, and MCF-7 cells and normal Human Mammary Epithelial Cells (HMECs) were exposed to fluid flow in a parallel-plate bioreactor system. Changes in expression were quantified using microarrays, qPCR, immunocytochemistry, and western blots. Gene–gene interactions were elucidated using network analysis, and key modified genes were examined in clinical datasets. Potential involvement of Smads was investigated using siRNA knockdown studies. Finally, the ability of flow-stimulated and unstimulated cancer cells to adhere to an endothelial monolayer, migrate and invade membrane pores was evaluated in flow and static adhesion experiments.ResultsFluid flow stimulation resulted in upregulation of EMT inducers and downregulation of repressors. Specifically, Vimentin and Snail were upregulated both at the gene and protein expression levels in flow stimulated HMECs and MDA-MB-231 cells, suggesting progression towards an EMT phenotype. Flow-stimulated SNAI2 was abrogated with Smad3 siRNA. Flow-induced overexpression of a panel of cell adhesion genes was also observed. Network analysis revealed genes involved in cell flow responses including FN1, PLAU, and ALCAM. When evaluated in clinical datasets, overexpression of FN1, PLAU, and ALCAM was observed in patients with different subtypes of breast cancer. We also observed increased adhesion, migration and invasion of flow-stimulated breast cancer cells compared to unstimulated controls.ConclusionsThis study shows that fluid forces on the order of 1 Pa promote EMT and adhesion of breast cancer cells to an endothelial monolayer and identified biomarkers were distinctly expressed in patient populations. A better understanding of how biophysical forces such as shear stress affect cellular processes involved in metastatic progression of breast cancer is important for identifying new molecular markers for disease progression, and for predicting metastatic risk.

Effect of change in cell substrate on the critical quality attributes of L-Zagreb Mumps vaccine manufactured using parallel plate bioreactor

Leningrad-Zagreb strain of mumps vaccine virus was grown on two different cell substrates viz. MRC-5 cells and Vero cells besides its original cell substrate i.e. Chicken Embryo Cells. Homogeneous virus pools prepared from each set of experiments were then lyophilized as per standard in-house protocol.Critical Quality Attributes (CQAs) such as the titer of the bulk vaccine and potency and stability of the lyophilized vaccine were then estimated using the CCID50 method to understand the lyophilization losses and thermal losses respectively in the vaccine. Another CQA viz. the genetic homogeneity of the vaccine was also tested using the single base extension method for identifying the nucleotides present at the three known locations of single nucleotide polymorphism (SNP).Comparison of CQA results across different cell substrates indicated encouraging results for Vero cell grown L-Zagreb virus compared to the MRC-5 cells grown L-Zagreb mumps virus. Significant improvement in productivity was also observed in the dynamic culture conditions compared to the static culture conditions.Progressive work in this research area can lead to development of a cGMP manufacturing process for mumps vaccine with easy scale up potential in future.

Ammonia-nitrogen and orthophosphate removal by immobilized Chlorella sp. isolated from municipal wastewater for potential use in tertiary treatment

Chlorella sp. isolated from municipal wastewater and entrapped in calcium alginate as algal sheets was employed to remove inorganic nutrients from domestic secondary effluents in parallel-plate bioreactor after starvation. The key factors affecting the nutrient removal efficiency, system stability and reuse efficiency of screens were discussed. The results show that cell density and starvation time significantly affected the nutrient uptake. A complete removal of NH 4 + -N and PO 4 3- -P was achieved within 4 h of treatment with the optimal cell density in the mixture of algal and 3 mm gel sheets after third cycle. Six cycles of wastewater treatment were accomplished in 18 days, achieving higher removal efficiency. NH 4 + -N removal efficiency was 81% after 1.25 h and 98.81% after 4 h, while PO 4 3- -P removal efficiency was 77.4% after 1.25 h and 100% after 4 h. It was confirmed that the immobilized Chlorella sp. has great potentialities in nutrient removal. Key words: Immobilized Chlorella, starvation, wastewater treatment, nitrogen removal, phosphorus removal.

Insights into interstitial flow, shear stress, and mass transport effects on ECM heterogeneity in bioreactor-cultivated engineered cartilage hydrogels

Interstitial flow in articular cartilage is secondary to compressive and shear deformations during joint motion and has been linked with the well-characterized heterogeneity in structure and composition of its extracellular matrix. In this study, we investigated the effects of introducing gradients of interstitial flow on the evolution of compositional heterogeneity in engineered cartilage. Using a parallel-plate bioreactor, we observed that Poiseuille flow stimulation of chondrocyte-seeded agarose hydrogels led to an increase in glycosaminoglycan and type II collagen deposition in the surface region of the hydrogel exposed to flow. Experimental measurements of the interstitial flow fields based on the fluorescence recovery after photobleaching technique suggested that the observed heterogeneity in composition is associated with gradients in interstitial flow in a boundary layer at the hydrogel surface. Interestingly, the interstitial flow velocity profiles were nonlinearly influenced by flow rate, which upon closer examination led us to the original observation that the apparent hydrogel permeability decreased exponentially with increased interfacial shear stress. We also observed that interstitial flow enhances convective mass transport irrespective of molecular size within the boundary layer near the hydrogel surface and that the convective contribution to transport diminishes with depth in association with interstitial flow gradients. The implications of the nonlinearly inverse relationship between the interfacial shear stress and the interstitial flux and permeability and its consequences for convective transport are important for tissue engineering, since porous scaffolds comprise networks of Poiseuille channels (pores) through which interstitial flow must navigate under mechanical stimulation or direct perfusion.

Theoretical analysis of engineered cartilage oxygenation: influence of construct thickness and media flow rate

A novel parallel-plate bioreactor has been shown to modulate the mechanical and biochemical properties of engineered cartilage by the application of fluid-induced shear stress. Flow or perfusion bioreactors may improve tissue development via enhanced transport of nutrients or gases as well as the application of mechanical stimuli, or a combination of these factors. The goal of this study was to complement observed experimental responses to flow by simulating oxygen transport within cartilage constructs of different thicknesses (250 microm or 1 mm). Using numerical computation of convection-diffusion equations, the evaluation of the tissue oxygenation is performed. Four culture conditions are defined based on tissue thickness and flow rates ranging from 0 to approximately 25 mL min(-1). Under these experimental conditions results show a mean oxygen concentration within the tissue varying from 0.01 to 0.19 mol m(-3) as a function of the tissue thickness and the magnitude of the applied shear stress. More generally, the influence of shear stress varying (via flow rate modification) from 10(-3) to 10 dynes cm(-2) on the tissue oxygenation is studied. The influence on the results of important physical parameters such as the maximal oxygen consumption rate of cells is discussed. Lastly, the importance of oxygen concentration in the lower chamber and its relevance to tissue oxygenation are highlighted by the model results.

Inhibition of Apoptosis Prevents Shear-Induced Detachment of Endothelial Cells

Biomaterials placed into the vasculature in man fail to develop an endothelial lining. Attempts to seed endothelial cells (ECs) on prosthetic vascular grafts have failed due to flow-induced detachment. The mechanism of flow-induced detachment of ECs from biomaterials is undefined. We hypothesize that endothelial detachment from biomaterials is caused by flow-induced apoptosis related to the inability of human ECs to adapt to and withstand mechanical loading. Human aortic endothelial cells were cultured on Dacron membranes, incubated in the presence or absence of a caspase inhibitor (Z-VAD-FMK), and exposed to 0, 1, 10, 20, or 30 dynes/cm(2) of shear stress for 2, 6, 12, and 24 h in a parallel plate bioreactor. The percent of ECs detached was determined and compared with no-flow controls. Apoptosis was determined by analyzing nuclear morphology and identifying cells with caspase activity using FAM-VAD-FMK. The actin cytoskeleton of cells was visualized with fluorescein phalloidin. Increasing shear stress resulted in detachment of ECs from the Dacron membranes, which was associated with increased apoptosis of the residual cells adherent to the membrane determined by both nuclear morphology and caspase activity. Significantly, treatment with the caspase inhibitor Z-VAD-FMK resulted in improved EC retention following exposure to high shear stress (20 and 30 dynes/cm(2)). The majority of apoptosis and detachment were determined to occur after 6 h. We have demonstrated that high shear stress-induced EC detachment from Dacron is an apoptosis-dependent phenomenon that can be pharmacologically inhibited by a pan-caspase inhibitor.

Ammonia–nitrogen and orthophosphate removal by immobilized Scenedesmus sp. isolated from municipal wastewater for potential use in tertiary treatment

Scendesmus sp. isolated from municipal wastewater, entrapped in calcium alginate as algal sheets was employed to remove inorganic nutrients (N and P) from artificial and real domestic secondary effluents in parallel-plate bioreactor after starvation. The key factors affecting the removal efficiency ( NH 4 + – N and PO 4 3 - – P ), system stability and reuse efficiency of screens were studied and discussed. It has been shown that cell density in the mixture of algal gel was the key factor compared with the thickness of the gel and the cell density of the reactor. A complete removal of NH 4 + – N and PO 4 3 - – P was achieved within 4 h of treatment in parallel bioreactors with the optimal cell density in the mixture of algal (2 × 10 8 algae mL −1) and 3 mm gel sheets after second cycle. Nine cycles of wastewater treatment in 21 days were accomplished, holding higher removal efficiency. NH 4 + – N removal efficiency was 99.1% after 105 min, 100% after 135 min, PO 4 3 - – P removal efficiency was 100% after 15 min in domestic secondary effluents. Immobilized Scendesmus sp. is shown to have great potentialities for removal of inorganic nitrogen and phosphorus from treated effluents.

P290: Shear-induced detachment of endothelial cells is mediated by extrinsic apoptosis

Introduction: Human endothelial cells (ECs) fail to remain adherent to prosthetic biomaterials in the vasculature. We have previously demonstrated that shear stress induces apoptosis of ECs adherent to Dacron, causing detachment. However, it is unclear through what pathway mechanical loading is inducing cell death. In this study, we test the hypothesis that shear-induced apoptosis is mediated through the extrinsic rather than the intrinsic pathway of apoptosis. Methods: Human aortic ECs were established on Dacron membranes and incubated in the presence or absence of inhibitors of the extrinsic [Z-IETD-FMK (25μM), a specific caspase-8 inhibitor] or intrinsic [Z-LEHD-FMK (25μM), a caspase-9 inhibitor] pathways of apoptosis. Membranes were inserted into a parallel plate bioreactor and exposed to 0 or 30 dynes/cm2 of shear stress for 24 hours. Following flow, cells were stained with ethidium bromide and hoechst 33258. Through image analysis, EC retention was calculated as a percentage of the cells remaining relative to the no flow controls. Percentage of remaining ECs that were apoptotic was identified by characteristic nuclear morphology. Results: Exposure to 30 dynes/cm2 of shear stress induced EC apoptosis and detachment. Inhibiting the extrinsic pathway of apoptosis with the caspase-8 inhibitor significantly decreased apoptosis and improved EC retention. However, inhibiting caspase-9, specific to the intrinsic pathway of apoptosis was ineffective at preventing cell detachment and apoptosis. Conclusion: These data demonstrate that shear stress-induced detachment of ECs seeded on Dacron is mediated through the extrinsic pathway of apoptosis. These findings implicate shear induced Fas activation or integrin disengagement as the initiating event leading to apoptosis of ECs. Future work will focus on elucidating further the mechanism of this extrinsic mediated apoptosis, which will guide tissue engineering approaches in the development of endothelialized prosthetic vascular grafts.

Fluid Flow Increases Type II Collagen Deposition and Tensile Mechanical Properties in Bioreactor-Grown Tissue-Engineered Cartilage

A novel parallel-plate bioreactor has been designed to apply a consistent level of fluid flow-induced shear stress to tissue-engineered articular cartilage in order to improve the matrix composition and mechanical properties and more nearly approximate to that of native tissue. Primary bovine articular chondrocytes were seeded into the bioreactor at high densities (1.7 x 10(6) cell/cm2) without a scaffold and cultured for two weeks under static, no-flow conditions. A mean fluid flow-induced shear stress of 1 dyne/cm2 was then applied continuously for 3 days. The application of flow produced constructs with significantly (p < 0.05) higher amounts of total collagen (via hydroxyproline) and specifically type II collagen (via ELISA) (25.3 +/- 2.5% and 22.1 +/- 4.7% of native tissue, respectively) compared to static controls (22.4 +/- 1.7% and 9.5 +/- 2.3%, respectively). Concurrently, the tensile Young's modulus and ultimate strength were significantly increased in flow samples (2.28 +/- 0.19 MPa and 0.81 +/- 0.07 MPa, respectively) compared to static controls (1.55 +/- 0.10 MPa and 0.62 +/- 0.05 MPa, respectively). This study suggests that flow-induced shear stresses and/or enhanced mass transport associated with the hydrodynamic environment of our novel bioreactor may be an effective functional tissue-engineering strategy for improving matrix composition and mechanical properties in vitro.

In Vitro Zonation and Toxicity in a Hepatocyte Bioreactor

The complex architecture of the liver is intertwined with its response to xenobiotic compounds. In particular, hepatocyte subpopulations are distributed along the sinusoid in zones 1 to 3, leading to prototypical "periportal" and "centrilobular" patterns of cell death in response to a toxic insult. In vitro models that more closely represent these zones of sub-specialization may therefore be valuable for the investigation of hepatic physiology and pathophysiology. We have established a perfused hepatocyte bioreactor that imposes physiologic oxygen gradients on co-cultures of rat hepatocytes and non-parenchymal cells, thereby producing an in vitro model of zonation. In order to predict and control oxygen gradients, oxygen transport in a parallel-plate bioreactor containing co-cultures was first mathematically modeled and experimentally validated. Co-cultures exposed to these physiologic oxygen gradients demonstrated regionally heterogeneity of CYP2B and CYP3A protein that mimics the distribution seen in the zonated liver. The distribution of CYP expression in the bioreactor was shown to vary with exposure to different chemical inducers and growth factors, providing a potential platform to study physiologic zonal responses. In order to explore zonal hepatotoxicity, bioreactors were perfused with APAP (acetominophen) for 24 h, resulting in maximal cell death at the low-oxygen outlet region similar to centrilobular necrotic patterns observed in vivo. This hepatocyte bioreactor system enables further in vitro investigation into zonation-dependent phenomena involving drug metabolism and toxicity.

Inhibition of apoptosis prevents shear induced detachment of endothelial cells

Introduction. The mechanism of shear-induced detachment of endothelial cells (ECs) from biomaterials is undefined. Either healthy ECs are forcibly detached by flow, or mechanical forces (shear stress and pressure) induce apoptosis leading to cell loss. Previously published work by others has identified that shear stress can “rescue” endothelial cells from apoptosis, utilizing a model in which apoptosis is first induced by serum starvation. We question the relevance of that model. We hypothesized that shear-induced apoptosis is a critical event leading to EC detachment and if apoptosis could be prevented, detachment from a biomaterial would be inhibited. Methods. Human Aortic ECs (passages 5–8) were established on Dacron membranes and subsequently were cultured in the presence or absence of Z-VAD-FMK (25 μM), a pan-caspase inhibitor, in medium for 2 h. Membranes were placed in a parallel plate bioreactor and exposed to 0, 1, 10, and 30 dyn/cm 2 of shear stress for 24 h. Following flow, cells were stained with ethidium bromide and Hoeschst 33258. The ECs remaining on the membrane, percentage of cell retention on the membrane (normalized to no flow controls), and percentage of cells undergoing apoptosis by identification of nuclear morphology were determined by image analysis ( n = 7–13 per group). Results. TABLE—ABSTRACT 54 Mean ± SEM Dynes/cm 2 EC Retention (%) Apoptosis (%) Z-VAD+ Z-VAD− Z-VAD+ Z-VAD− 1 96.7 ± 6.0 97.1 ± 1.1 2.6 ± 0.9 1.7 ± .03 10 †65.5 ± 8.3 †69.3 ± 7.4 ∗2.5 ± 0.5 †8.9 ± 2.2 30 ∗†78.1 ± 2.8 †45.5 ± 7.9 ∗2.3 ± 0.4 †9.0 ± 0.6 ∗ P < 0.05 for Z-VAD+ versus Z-VAD−, † P < .05 as compared with 1 Dyne/cm 2 Increasing shear stress in the absence of Z-VAD-FMK induced apoptosis and detachment as compared with the 1 dyn/cm 2 group. However, treatment with Z-VAD-FMK effectively inhibited apoptosis and resulted in significantly improved EC retention following exposure to high shear stress of 30 dyn/cm 2. Conclusion. These data demonstrate that apoptosis induced by mechanical loading is a critical element responsible for flow-induced cell detachment from biomaterials, and can be modulated pharmacologically.

Cell growth and differentiation on feeder layers is predicted to be influenced by bioreactor geometry

Tissue function is comprised of a complex interplay between biological and physicochemical rate processes. The design of bioreactors for tissue engineering must account for these processes simultaneously in order to obtain a bioreactor that provides a uniform environment for tissue growth and development. In the present study we consider the effects of fluid flow and mass transfer on the growth of a tissue in a parallel-plate bioreactor configuration. The parenchymal cells grow on a preformed stromal (feeder) layer that secretes a growth factor that stimulates parenchymal stem cell replication and differentiation. The biological dynamics are described by a unilineage model that describes the replication and differentiation of the tissue stem cell. The physicochemical rates are described by the Navier-Stokes and convective-diffusion equations. The model equations are solved by a finite element method. Two dimensionless groups govern the behavior of the solution. One is the Graetz number (Gz) that describes the relative rates of convection and diffusion, and the other a new dimensionless ratio (designated by P) that describes the interplay of the growth factor production, diffusion, and stimulation. Four geometries (slab, gondola, diamond, and radial shapes) for the parallel-plate bioreactor are analyzed. The uniformity of cell growth is measured by a two-dimensional coefficient of variance. The concentration distribution of the stroma-derived growth factor was computed first based on fluid flow and bioreactor geometry. Then the concomitant cell density distribution was obtained by integrating the calculated growth factor concentration with the parenchymal cell growth and unilineage differentiation process. The spatiotemporal cell growth patterns in four different bioreactor configurations were investigated under a variety of combinations of Gz (10(-1), 10(0), and 10(1)) and P(10(-2), 10(-1), 10(0), 10(1), and 10(2)). The results indicate high cell density and uniformity can be achieved for parameter values of P = 0.01, ..., 0.1 and Gz = 0.1, ..., 1.0. Among the four geometries investigated the radial-flow-type bioreactor provides the most uniform environment in which parenchymal cells can grow and differentiate ex vivo due to the absence of walls that are parallel to the flow paths creating slow flowing regions.