Rotary Cell Culture System Bioreactor Research Articles

RCCS Bioreactor-Based Modeled Microgravity Affects Gastric Cancer Cells and Improves the Chemotherapeutic Effect

(1) Background: The main purpose of the study was to determine whether altered gravity might alter cell viability, improve drug delivery and modulate the expression of drug resistance-related genes. (2) Methods: This study investigated the intracellular mechanisms activated by microgravity in human resistant and sensitive gastric cancer cells (EPG85-257 RDB) and (EPG85-257 P). We used a rotary cell culture system (RCCS) developed by NASA to expose cells to altered gravity. The antitumor potential of microgravity was simulated by the RCCS bioreactor, and its effectiveness was evaluated in sensitive cell lines compared to chemotherapy-resistant cells concerning drug-sensitive cancer cells. Microgravity with chemotherapy was estimated by the viability assay, cytoskeleton imaging, MDR (multidrug resistance) gene expression analysis, MTCO-1 (mitochondrially encoded cytochrome C oxidase I), and 8-OHdG immunocytochemical analysis. (3) Results: We found that altered gravity combined with doxorubicin was cytotoxic to cancer cells. Cells following simulated microgravity revealed decreased expression of genes related to drug resistance and increased DNA/RNA damage marker expression. Cytoskeleton evaluation demonstrated significant reorganization of F-actin fibers after exposure to changed gravity conditions. (4) Conclusions: Intracellular alterations caused by simulated microgravity can increase gastric cancer cells’ sensitivity to chemotherapy. We have obtained satisfactory results showing the correlation between altered gravity and MDR phenomena which seems promising in future therapeutic applications.

Effect of simulated microgravity on metabolism of HGC‑27 gastric cancer cells

The understanding into the pathogenesis and treatment of gastric cancer has improved in recent years; however, a number of limitations have delayed the development of effective treatment. Cancer cells can undergo glycolysis and inhibit oxidative phosphorylation in the presence of oxygen (Warburg effect). Previous studies have demonstrated that a rotary cell culture system (RCCS) can induce glycolytic metabolism. In addition, the potential of regulating cancer cells by targeting their metabolites has led to the rapid development of metabolomics. In the present study, human HGC-27 gastric cancer cells were cultured in a RCCS bioreactor, simulating weightlessness. Subsequently, liquid chromatography-mass spectrometry was used to examine the effects of simulated microgravity (SMG) on the metabolism of HGC-27 cells. A total of 67 differentially regulated metabolites were identified, including upregulated and downregulated metabolites. Compared with the normal gravity group, phosphatidyl ethanolamine, phosphatidyl choline, arachidonic acid and sphinganine were significantly upregulated in SMG conditions, whereas sphingomyelin, phosphatidyl serine, phosphatidic acid, L-proline, creatine, pantothenic acid, oxidized glutathione, adenosine diphosphate and adenosine triphosphate were significantly downregulated. The Human Metabolome Database compound analysis revealed that lipids and lipid-like metabolites were primarily affected in an SMG environment in the present study. Overall, the findings of the present study may aid our understanding of gastric cancer by identifying the underlying mechanisms of metabolism of the disease under SMG.

Effects of rotary cell culture system‐simulated microgravity on the ultrastructure and biological behavior of human MDA‐MB‐231 breast cancer cells

AbstractObjectiveThis study aimed to investigate changes in the ultrastructure, apoptosis, cycle progression, and migration ability of human MDA‐MB‐231 breast cancer cells under simulated microgravity conditions.MethodsMDA‐MB‐231 breast cancer cells were cultured in a rotary cell culture system bioreactor to simulate microgravity for 7 days. The cells were randomly divided into a simulated microgravity (SMG) group and normal gravity control (NG) group. Changes in apoptosis, ultrastructure, cell cycle, and migration ability were evaluated.ResultsTransmission electron microscopy showed that cells in the SMG group had more secondary lysosomes than the NG groups. The flow cytometry assay showed that the proportion of apoptotic cells in the SMG group was significantly increased compared with the NG group; additionally, the median and interquartile spacing of the proportion of cells in the G0/G1 phase in the SMG group was lower than in the NG group, but the proportion of cells in the S phase was increased in the SMG group compared with the NG group. The transwell migration assay showed that cell migration in the SMG group was significantly reduced compared with the NG group. The expression of BCL‐2 and MMP9 in the SMG group was decreased compared with the NG group, whereas the expression of cyclin D3 in the SMG group was increased.ConclusionSimulated microgravity through rotary cell culture system had significant effects on human MDA‐MB‐231 breast cancer cells: the cell cycle and ultrastructure were impaired, apoptosis increased, migration ability decreased, and the expression of BCL‐2, cyclin D3, and MMP9 were affected.

Functional tissue-engineered microtissue derived from cartilage extracellular matrix for articular cartilage regeneration

We developed a promising cell carrier prepared from articular cartilage slices, designated cartilage extracellular matrix (ECM)-derived particles (CEDPs), through processes involving physical pulverization, size screening, and chemical decellularization. Rabbit articular chondrocytes (ACs) or adipose-derived stem cells (ASCs) rapidly attached to the surface of the CEDPs and proliferated with high cell viability under microgravity (MG) condition in a rotary cell culture system (RCCS) or static condition. Gene profiling results demonstrated that ACs expanded on CEDPs exhibited significantly enhanced chondrogenic phenotypes compared with monolayer culture, and that ASCs differentiated into a chondrogenic phenotype without the use of exogenous growth factors. Moreover, MG culture conditions in a RCCS bioreactor were superior to static culture conditions in terms of maintaining the chondrogenic phenotype of ACs and inducing ACS chondrogenesis. With prolonged expansion, functional microtissue aggregates of AC- or ASC-laden CEDPs were formed. Further, AC- or ASC-based microtissues were directly implanted in vivo to repair articular osteochondral defects in a rabbit model. Histological results, biomechanical evaluations, and radiographic assessments indicated that AC- and ASC-based microtissues displayed equal levels of superior hyaline cartilage repair, whereas the other two treatment groups, in which osteochondral defects were treated with CEDPs alone or fibrin glue, exhibited primarily fibrous tissue repair. These findings provide an alternative method for cell culture and stem cell differentiation and a promising strategy for constructing tissue-engineered cartilage microtissues for cartilage regeneration. Statement of SignificanceDespite the remarkable progress in cartilage tissue engineering, cartilage repair still remains elusive. In the present study, we developed a cell carrier, namely cartilage extracellular matrix-derived particles (CEDPs), for cell proliferation of articular chondrocytes (ACs) and adipose-derived stem cells (ASCs), which improved the maintenance of chondrogenic phenotype of ACs, and induced chondrogenesis of ASCs. Moreover, the functional microtissue aggregates of AC- or ASC-laden CEDPs induced equal levels of superior hyaline cartilage repair in a rabbit model. Therefore, our study demonstrated an alternative method for chondrocyte culture and stem cell differentiation, and a promising strategy for constructing tissue-engineered cartilage microtissues for in vivo articular cartilage repair and regeneration.

Modeling multiple myeloma-bone marrow interactions and response to drugs in a 3D surrogate microenvironment.

Multiple myeloma develops primarily inside the bone marrow microenvironment, that confers pro-survival signals and drug resistance. 3D cultures that reproduce multiple myeloma-bone marrow interactions are needed to fully investigate multiple myeloma pathogenesis and response to drugs. To this purpose, we exploited the 3D Rotary Cell Culture System bioreactor technology for myeloma-bone marrow co-cultures in gelatin scaffolds. The model was validated with myeloma cell lines that, as assessed by histochemical and electron-microscopic analyses, engaged contacts with stromal cells and endothelial cells. Consistently, pro-survival signaling and also cell adhesion-mediated drug resistance were significantly higher in 3D than in 2D parallel co-cultures. The contribution of the VLA-4/VCAM1 pathway to resistance to bortezomib was modeled by the use of VCAM1 transfectants. Soluble factor-mediated drug resistance could be also demonstrated in both 2D and 3D co-cultures. The system was then successfully applied to co-cultures of primary myeloma cells-primary myeloma bone marrow stromal cells from patients and endothelial cells, allowing the development of functional myeloma-stroma interactions and MM cell long-term survival. Significantly, genomic analysis performed in a high-risk myeloma patient demonstrated that culture in bioreactor paralleled the expansion of the clone that ultimately dominated in vivo. Finally, the impact of bortezomib on myeloma cells and on specialized functions of the microenvironment could be evaluated. Our findings indicate that 3D dynamic culture of reconstructed human multiple myeloma microenvironments in bioreactor may represent a useful platform for drug testing and for studying tumor-stroma molecular interactions.

A mesoscale study of the degradation of bone structural properties in modeled microgravity conditions

One of the most important alterations that occur in man and experimental animals during spaceflight concerns the skeletal system, and entails important bone loss and degradation of mechanical properties. In the present work we investigate ex vivo the long-term effects of weightlessness (simulated microgravity) on bone tissue, by comparing the mesoscale structural properties of weight-bearing rat tibial epiphyseal cancellous structures of healthy animals (ground controls) with those of identical bone explants maintained ex vivo in the Rotary Cell Culture System (RCCS) bioreactor, used to model, on ground, microgravity conditions. Bone structures were reconstructed by synchrotron radiation micro-CT, morphometric analyses were performed, and the apparent elastic properties were computed by means of a numerical model based on the Cell Method. Two novel results were achieved in this study. First of all, the skeletal modifications found in bone explants after 3–4 weeks of culture in the RCCS bioreactor are in perfect agreement with those observed in vivo after a long-term spaceflight (Mice Drawer System mission, 2009), thus confirming the relevance of our model in reproducing the effects of microgravity on whole bone tissue. Secondly, but not less importantly, our study points out that the degradation in bone structural performance (apparent mechanical properties) must be considered in order to achieve an accurate representation of trabecular bone modifications not only in osteoporotic bone diseases, but also in the microgravity-induced bone alterations. In conclusion, our findings, by proving that the association of the RCCS bioreactor-based culture method, used to model microgravity conditions, with numerical simulations able to quantify bone quality, represents the first ground-based reliable model for investigating, ex vivo, some of the spaceflight effects on bone tissue, and open new perspectives to basic research and clinical applications.

Experimental study on effect of simulated microgravity on structural chromosome instability of human peripheral blood lymphocytes.

Experimental study was made by keeping human peripheral blood lymphocytes under simulated microgravity in a Rotary Cell Culture System bioreactor to investigate the changes that occur in the number of chromosomes, the expression rate of chromosome fragile site, and the expressions of DNA replication- and repair-related genes. Experimental results indicate simulated microgravity has no effect on the numerical chromosome instability of human peripheral blood lymphocytes, but it enhances the structural chromosome instability of human peripheral blood lymphocytes through the inhibition of DNA replication and the reduction of DNA repair. So, the mechanism of chromosome fragile site induced by simulated microgravity can be explained using the changes that occur in the chromosome structure of human peripheral blood lymphocytes, the DNA replication and repair under the effect of simulated microgravity.

THU0544 Preclinical Drug Testing in Ex Vivo 3-D Dynamic Culture in Erdheim-Chester Disease

BackgroundErdheim-Chester disease (ECD) is a non-Langerhans cell histiocytosis of unknown etiology and poor prognosis. The disease is characterized by the accumulation of foamy CD68+ histiocytes in bones and soft tissues,...

Tissue Engineering of Human Septal Cartilage Using a Rotary Bioreactor

Tissue engineering of human septal neocartilage offers the potential to produce large quantities of autologous material for use in the repair of cartilaginous craniofacial defects. Culture of septal neocartilage constructs in a rotary cell culture system bioreactor may improve the biochemical and biomechanical properties of engineered tissue. The objective of this study was to characterize the morphological, compositional and mechanical properties of engineered human septal cartilage constructs when cultured in three different environments. Human septal cartilage constructs were cultured in transwell plates for 6 weeks and subsequently cultured for an additional 4 weeks in transwell plates, free-floating in a sterile media bottle, or in a rotary cell culture system bioreactor. The histologic, biochemical, and biomechanical properties of the constructs were examined. All constructs maintained their radial dimensions throughout the culture period. Qualitatively, the constructs cultured in the rotary cell culture system bioreactor were firmer and more resilient compared with the constructs cultured under free-floating static and transwell plate conditions. Culture of human septal neocartilage constructs in a rotary cell culture system bioreactor augmented cell proliferation and extracellular matrix production when compared with constructs cultured in transwell plates and free-floating static conditions. Additionally, rotary cell culture system bioreactor culture enhanced total and type II collagen production. The rotary cell culture system bioreactor constructs possessed improved biomechanical properties compared with the other conditions and this is reflected in their superior resistance to manipulation. These findings suggest that culture in a rotary cell culture bioreactor can modulate the composition and mechanical function of engineered scaffold-free human septal cartilage constructs to produce constructs better suited for reconstructive surgery.

Beta-adrenoceptor pathway enhances mitochondrial function in human neural stem cells via rotary cell culture system

The structure and function of the human nervous system are altered in space when compared with their state on earth. To investigate directly the influence of simulated microgravity conditions which may be beneficial for cultivation and proliferation of human neural stem cells (hNSCs), the rotary cell culture system (RCCS) developed at the National Aeronautics and Space Administration (NASA) was used. RCCS allows the creation of a unique microgravity environment of low shear force, high-mass transfer and enables three-dimensional (3D) cell culture of dissimilar cell types. The results show that simulated microgravity using an RCCS would induce β-adrenoceptor, upregulate cAMP formation and activate both PKA and CREB (cAMP response element binding protein) pathways. The expression of intracellular mitochondrial genes, including PGC1α (PPAR coactivator 1α), nuclear respiratory factors 1 and 2 (NRF1 and NRF2) and mitochondrial transcription factor A (Tfam), regulated by CREB, were all significantly increased at 72h after the onset of microgravity. Accordingly and importantly, the ATP level and amount of mitochondrial mass were also increased. These results suggest that exposure to simulated microgravity using an RCCS would induce cellular proliferation in hNSCs via an increased mitochondrial function. In addition, the RCCS bioreactor would support hNSCs growth, which may have the potential for cell replacement therapy in neurological disorders.