Rotating Wall Vessel Bioreactor Research Articles

Recreating chronic respiratory infections in vitro using physiologically relevant models.

Despite the need for effective treatments against chronic respiratory infections (often caused by pathogenic biofilms), only a few new antimicrobials have been introduced to the market in recent decades. Although different factors impede the successful advancement of antimicrobial candidates from the bench to the clinic, a major driver is the use of poorly predictive model systems in preclinical research. To bridge this translational gap, significant efforts have been made to develop physiologically relevant models capable of recapitulating the key aspects of the airway microenvironment that are known to influence infection dynamics and antimicrobial activity in vivo In this review, we provide an overview of state-of-the-art cell culture platforms and ex vivo models that have been used to model chronic (biofilm-associated) airway infections, including air-liquid interfaces, three-dimensional cultures obtained with rotating-wall vessel bioreactors, lung-on-a-chips and ex vivo pig lungs. Our focus is on highlighting the advantages of these infection models over standard (abiotic) biofilm methods by describing studies that have benefited from these platforms to investigate chronic bacterial infections and explore novel antibiofilm strategies. Furthermore, we discuss the challenges that still need to be overcome to ensure the widespread application of in vivo-like infection models in antimicrobial drug development, suggesting possible directions for future research. Bearing in mind that no single model is able to faithfully capture the full complexity of the (infected) airways, we emphasise the importance of informed model selection in order to generate clinically relevant experimental data.

Structural and Molecular Changes of Human Chondrocytes Exposed to the Rotating Wall Vessel Bioreactor.

Over the last 30 years, the prevalence of osteoarthritis (OA), a disease characterized by a loss of articular cartilage, has more than doubled worldwide. Patients suffer from pain and progressive loss of joint function. Cartilage is an avascular tissue mostly consisting of extracellular matrix with embedded chondrocytes. As such, it does not regenerate naturally, which makes an early onset of OA prevention and treatment a necessity to sustain the patients' quality of life. In recent years, tissue engineering strategies for the regeneration of cartilage lesions have gained more and more momentum. In this study, we aimed to investigate the scaffold-free 3D cartilage tissue formation under simulated microgravity in the NASA-developed rotating wall vessel (RWV) bioreactor. For this purpose, we cultured both primary human chondrocytes as well as cells from the immortalized line C28/I2 for up to 14 days on the RWV and analyzed tissue morphology, development of apoptosis, and expression of cartilage-specific proteins and genes by histological staining, TUNEL-assays, immunohistochemical detection of collagen species, and quantitative real-time PCR, respectively. We observed spheroid formation in both cell types starting on day 3. After 14 days, constructs from C28/I2 cells had diameters of up to 5 mm, while primary chondrocyte spheroids were slightly smaller with 3 mm. Further inspection of the 14-day-old C28/I2 spheroids revealed a characteristic cartilage morphology with collagen-type 1, -type 2, and -type 10 positivity. Interestingly, these tissues were less susceptible to RWV-induced differential gene expression than those formed from primary chondrocytes, which showed significant changes in the regulation of IL6, ACTB, TUBB, VIM, COL1A1, COL10A1, MMP1, MMP3, MMP13, ITGB1, LAMA1, RUNX3, SOX9, and CASP3 gene expression. These diverging findings might reflect the differences between primary and immortalized cells. Taken together, this study shows that simulated microgravity using the RWV bioreactor is suitable to engineer dense 3D cartilage-like tissue without addition of scaffolds or any other artificial materials. Both primary articular cells and the stable chondrocyte cell line C28/I2 formed 3D neocartilage when exposed for 14 days to an RWV.

Effects of short-term exposure to simulated microgravity on the physiology of Bacillus subtilis and multiomic analysis.

In our study, Bacillus subtiliswas disposed to a simulated microgravity (SMG) environment in high-aspect ratio rotating-wall vessel bioreactors for 14 days, while the control group was disposed to the same bioreactors in a normal gravity (NG) environment for 14 days. The B. subtilisstrain exposed to the SMG (labeled BSS) showed an enhanced growth ability, increased biofilm formation ability, increased sensitivity to ampicillin sulbactam and cefotaxime, and some metabolic alterations compared with theB. subtilisstrain under NG conditions (labeled BSN) and the original strain ofB. subtilis(labeled BSO). The differentially expressed proteins (DEPs) associated with an increased growth rate, such as DNA strand exchange activity, oxidoreductase activity, proton-transporting ATP synthase complex, and biosynthetic process, were significantly upregulated in BSS. The enhanced biofilm formation ability may be related with the DEPs of spore germination and protein processing in BSS, and differentially expressed genes involved in protein localization and peptide secretion were also significantly enriched. The results revealed that SMG may increase the level of related functional proteins by upregulating or downregulating affiliated genes to change physiological characteristics and modulate growth ability, biofilm formation ability (epsB, epsC, epsN), antibiotic sensitivity (penP) and metabolism. Our experiment may gives new ideas for the study of space microbiology.

Development of a Novel Perfusion Rotating Wall Vessel Bioreactor with Ultrasound Stimulation for Mass-Production of Mineralized Tissue Constructs.

As stem cells are considered a promising cell source for tissue engineering, many culture strategies have been extensively studied to generate in vitro stem cell-based tissue constructs. However, most approaches using conventional tissue culture plates are limited by the lack of biological relevance in stem cell microenvironments required for neotissue formation. In this study, a novel perfusion rotating wall vessel (RWV) bioreactor was developed for mass-production of stem cell-based 3D tissue constructs. An automated RWV bioreactor was fabricated, which is capable of controlling continuous medium perfusion, highly efficient gas exchange with surrounding air, as well as low-intensity pulsed ultrasound (LIPUS) stimulation. Embryonic stem cells encapsulated in alginate/gelatin hydrogel were cultured in the osteogenic medium by using our bioreactor system. Cellular viability, growth kinetics, and osteogenesis/mineralization were thoroughly evaluated, and culture media were profiled at real time. The in vivo efficacy was examined by a rabbit cranial defect model. Our bioreactor successfully maintained the optimal culture environments for stem cell proliferation, osteogenic differentiation, and mineralized tissue formation during the culture period. The mineralized tissue constructs produced by our bioreactor demonstrated higher void filling efficacy in the large bone defects compared to the group implanted with hydrogel beads only. In addition, the LIPUS modules mounted on our bioreactor successfully reached higher mineralization of the tissue constructs compared to the groups without LIPUS stimulation. This study suggests an effective biomanufacturing strategy for mass-production of implantable mineralized tissue constructs from stem cells that could be applicable to future clinical practice.

CAMDLES: CFD-DEM Simulation of Microbial Communities in Spaceflight and Artificial Microgravity.

We present CAMDLES (CFD-DEM Artificial Microgravity Developments for Living Ecosystem Simulation), an extension of CFDEM®Coupling to model biological flows, growth, and mass transfer in artificial microgravity devices. For microbes that accompany humans into space, microgravity-induced alterations in the fluid environment are likely to be a major factor in the microbial experience of spaceflight. Computational modeling is needed to investigate how well ground-based microgravity simulation methods replicate that experience. CAMDLES incorporates agent-based modeling to study inter-species metabolite transport within microbial communities in rotating wall vessel bioreactors (RWVs). Preexisting CFD modeling of RWVs has not yet incorporated growth; CAMDLES employs the simultaneous modeling of biological, chemical, and mechanical processes in a micro-scale rotating reference frame environment. Simulation mass transfer calculations were correlated with Monod dynamic parameters to predict relative growth rates between artificial microgravity, spaceflight microgravity, and 1 g conditions. By simulating a microbial model community of metabolically cooperative strains of Escherichia coli and Salmonella enterica, we found that the greatest difference between microgravity and an RWV or 1 g gravity was when species colocalized in dense aggregates. We also investigated the influence of other features of the system on growth, such as spatial distribution, product yields, and diffusivity. Our simulation provides a basis for future laboratory experiments using this community for investigation in artificial microgravity and spaceflight microgravity. More broadly, our development of these models creates a framework for novel hypothesis generation and design of biological experiments with RWVs, coupling the effects of RWV size, rotation rate, and mass transport directly to bacterial growth in microbial communities.

A summary of previous investigations of the microgravity effects on bacterial virulence, infection and antibiotic resistance

In the treatment of bacterial infections acquired during spaceflight, changes in bacterial virulence, infection process and antibiotic resistance under microgravity need to be taken into consideration. We therefore summarized previous investigations of the microgravity effects on bacterial virulence, infection and antibiotic resistance in this article. Some of the previous studies were carried out in microgravity environment during spaceflight, while others were carried out in simulated microgravity environment using rotating-wall vessel bioreactor or Rotary Cell Culture System. The results were that microgravity enhanced the virulence of certain bacterial strains and attenuated the virulence of certain bacterial strains, mitigated the severity of infection by certain bacterial strains, and enhanced the antibiotic resistance of certain bacterial strains and attenuated the antibiotic resistance of certain bacterial strains. Variation factors, such as bacterial strains (of same species) and exposure time, were discussed. The limitation of most of the previous spaceflight studies was a lack of an in-flight 1-g centrifuge control. In the investigation of the microgravity effects on bacterial virulence and infection, using non-mammalian animals is not as good as using mammalian animals to model human infection because there are many differences between mammals and non-mammalian animals. Our knowledge about the microgravity effects on bacterial infection and antibiotic resistance is far from completeness and future studies are needed.

Engineered three-dimensional cardiac tissues maturing in a rotating wall vessel bioreactor remodel diseased hearts in rats with myocardial infarction.

SummaryA rotating wall vessel (RWV) bioreactor was constructed for growing massive functional cardiac constructs to recover the function of a distressed rat heart. Three-dimensional cardiac tissues were engineered by seeding human-induced pluripotent stem cell-derived cardiomyocytes on poly(lactic-co-glycolic acid) fiber sheets (3D-hiPSC-CTs) and cultured in the RWV bioreactor (RWV group) or under static conditions (control group). The tissues were transplanted into a myocardial infarction nude rat model, and cardiac performance was evaluated. In the RWV group, cell viability and contractile and electrical properties significantly improved, mature cardiomyocytes were observed, and mechanical stress-related mediators of mammalian target of rapamycin signaling were upregulated compared with those of the control. Four weeks post-transplantation, tissue survival and left ventricular ejection fraction significantly improved in the RWV group. Hence, dynamic culture in an RWV bioreactor could provide a superior culture environment for improved performance of 3D-hiPSC-CTs, providing a means for functional cardiomyogenesis in myocyte-loss heart failure.

Effects of Simulated Microgravity on the Physiology of Stenotrophomonas maltophilia and Multiomic Analysis.

Many studies have shown that the space environment plays a pivotal role in changing the characteristics of conditional pathogens, especially their pathogenicity and virulence. However, Stenotrophomonas maltophilia, a type of conditional pathogen that has shown to a gradual increase in clinical morbidity in recent years, has rarely been reported for its impact in space. In this study, S. maltophilia was exposed to a simulated microgravity (SMG) environment in high-aspect ratio rotating-wall vessel bioreactors for 14days, while the control group was exposed to the same bioreactors in a normal gravity (NG) environment. Then, combined phenotypic, genomic, transcriptomic, and proteomic analyses were conducted to compare the influence of the SMG and NG on S. maltophilia. The results showed that S. maltophilia in simulated microgravity displayed an increased growth rate, enhanced biofilm formation ability, increased swimming motility, and metabolic alterations compared with those of S. maltophilia in normal gravity and the original strain of S. maltophilia. Clusters of Orthologous Groups (COG) annotation analysis indicated that the increased growth rate might be related to the upregulation of differentially expressed genes (DEGs) involved in energy metabolism and conversion, secondary metabolite biosynthesis, transport and catabolism, intracellular trafficking, secretion, and vesicular transport. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that the increased motility might be associated the upregulation of differentially expressed proteins (DEPs) involved in locomotion, localization, biological adhesion, and binding, in accordance with the upregulated DEGs in cell motility according to COG classification, including pilP, pilM, flgE, flgG, and ronN. Additionally, the increased biofilm formation ability might be associated with the upregulation of DEPs involved in biofilm formation, the bacterial secretion system, biological adhesion, and cell adhesion, which were shown to be regulated by the differentially expressed genes (chpB, chpC, rpoN, pilA, pilG, pilH, and pilJ) through the integration of transcriptomic and proteomic analyses. These results suggested that simulated microgravity might increase the level of corresponding functional proteins by upregulating related genes to alter physiological characteristics and modulate growth rate, motility, biofilm formation, and metabolism. In conclusion, this study is the first general analysis of the phenotypic, genomic, transcriptomic, and proteomic changes in S. maltophilia under simulated microgravity and provides some suggestions for future studies of space microbiology.

Artificial tissue creation under microgravity conditions: Considerations and future applications.

Traditional tissue engineering methods often fail to promote robust cell growth and differentiation, limiting the development of functioning tissues. However, the microgravity conditions created by rotating wall vessel bioreactors minimize shear stress and unload the gravitational force usually placed on cells. In a microgravity environment, cell proliferation, cell differentiation, and the 3D organization of cells are altered, potentially encouraging the formation of more biosimilar artificial tissues for certain cell types. Additionally, cells in these engineered tissues display lowered immunogenicity, pointing to the transplantation potential of tissues engineered in microgravity conditions. However, these benefits are not consistent across all cell types, and the long-term impact of microgravity on tissue development and stability remains an unanswered question. Even so, there is potential that with further research, microgravity tissue engineering will have productive clinical applications for medical and pharmaceutical purposes.

3D Tissue Models as an Effective Tool for Studying Viruses and Vaccine Development

The recent SARS-CoV-2 outbreak has researchers working tirelessly to understand the virus' pathogenesis and develop an effective vaccine. The urgent need for rapid development and deployment of such a vaccine has illustrated the limitations of current practices, and it has highlighted the need for alternative models for early screening of such technologies. Traditional 2D cell culture does not accurately capture the effects of a physiologically relevant environment as they fail to promote appropriate cell-cell and cell-environment interactions. This inability to capture the intricacies of the in vivo microenvironment prevents 2D cell cultures from demonstrating the necessary properties of native tissues required for the standard infection mechanisms of the virus, thus contributing the high failure rate of drug discovery and vaccine development. 3D cell culture models can bridge the gap between conventional cell culture and in vivo models. Methods such as 3D bioprinting, spheroids, organoids, organ-on-chip platform, and rotating wall vessel bioreactors offer ways to produce physiologically relevant models by mimicking in vivo microarchitecture, chemical gradients, cell–cell interactions and cell–environment interactions. The field of viral biology currently uses 3D cell culture models to understand the interactions between viruses and host cells, which is crucial knowledge for vaccine development. In this review, we discuss how 3D cell culture models have been used to investigate disease pathologies for coronaviruses and other viruses such as Zika Virus, Hepatitis, and Influenza, and how they may apply to drug discovery and vaccine development.

3D Oral and Cervical Tissue Models for Studying Papillomavirus Host-Pathogen Interactions.

Human papillomavirus (HPV) infection occurs in differentiating epithelial tissues. Cancers caused by high-risk types (e.g., HPV16 and HPV18) typically occur at oropharyngeal and anogenital anatomical sites. The HPV life cycle is differentiation-dependent, requiring tissue culture methodology that is able to recapitulate the three-dimensional (3D) stratified epithelium. Here we report two distinct and complementary methods for growing differentiating epithelial tissues that mimic many critical morphological and biochemical aspects of in vivo tissue. The first approach involves growing primary human epithelial cells on top of a dermal equivalent consisting of collagen fibers and living fibroblast cells. When these cells are grown at the liquid-air interface, differentiation occurs and allows for epithelial stratification. The second approach uses a rotating wall vessel bioreactor. The low-fluid-shear microgravity environment inside the bioreactor allows the cells to use collagen-coated microbeads as a growth scaffold and self-assemble into 3D cellular aggregates. These approaches are applied to epithelial cells derived from HPV-positive and HPV-negative oral and cervical tissues. The second part of the article introduces potential downstream applications for these 3D tissue models. We describe methods that will allow readers to start successfully culturing 3D tissues from oral and cervical cells. These tissues have been used for microscopic visualization, scanning electron microscopy, and large omics-based studies to gain insights into epithelial biology, the HPV life cycle, and host-pathogen interactions. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Establishing human primary cell-derived 3D organotypic raft cultures Support Protocol 1: Isolation of epithelial cells from patient-derived tissues Support Protocol 2: Growth and maintenance of primary human epithelial cells in monolayer culture Support Protocol 3: PCR-based HPV screening of primary cell cultures Basic Protocol 2: Establishing human 3D cervical tissues using the rotating wall vessel bioreactor Support Protocol 4: Growth and maintenance of human A2EN cells in monolayer culture Support Protocol 5: Preparation of the slow-turning lateral vessel bioreactor Support Protocol 6: Preparation of Cytodex-3 microcarrier beads Basic Protocol 3: Histological assessment of 3D organotypic raft tissues Basic Protocol 4: Spatial analysis of protein expression in 3D organotypic raft cultures Basic Protocol 5: Immunofluorescence imaging of RWV-derived 3D tissues Basic Protocol 6: Ultrastructural visualization and imaging of RWV-derived 3D tissues Basic Protocol 7: Characterization of gene expression by RT-qPCR.

Abstract 13559: Engineered Three-dimensional Cardiac Tissues Maturated by Dynamic Culture in a Rotating Wall Vessel Bioreactor Remodel the Diseased Heart in the Rat Infarction Model

Introduction: To completely repair the distressed heart with few myocytes it may be crucial how to construct massive engineered three-dimensional cardiac tissues (E3DCTs) and integrate them to myocardium after transplant. We hypothesized that E3DCTs maturated by dynamic culture could enhance to remodel diseased left ventricle (LV) in the distressed rat heart. Methods: We made E3DCT by seeding 2.0 х 10 6 human induced pluripotent stem cell derived cardiomyocytes on the 6mm х 6mm PLGA fiber sheet. It was cultured in a rotating wall vessel bioreactor for seven days (RWV group) or culture dishes for the control. After in vitro assessment, these tissues were transplanted to myocardial infarction model nude rats, and 4 weeks after transplantation, we evaluated functional recovery and histology. Results: In the RWV group, thickness of E3DCTs were around 400μm, and cardiomyocytes showed mature phenotype evidenced by significantly higher expression of Troponin T (TnT), sarcomeric α actinin (SAA), connexin 43 (Cx43) and myosin heavy chain 7 (MYH7) with upregulation of mammalian target of rapamycin by Western blots (TnT; 2.1±0.2 vs. 1.0±0.2, SAA; 2.0±0.2 vs. 1.0±0.2, Cx 43; 2.0±0.3 vs. 1.0±0.1, MYH7; 2.0±0.2 vs. 1.0±0.2, p<0.05, respectively) (Figure 1A). Moreover, the concentration of angiogenic cytokines was significantly higher in the RWV group. Four weeks after transplantation, the LV ejection fraction was significantly improved in the RWV group than in the control (47±4.9 vs. 38±6.9%, p<0.01). On histological analysis, transplanted E3DCT reached a thickness of 1mm and vascular-like structures were partially observed inside (Figure 1B). In the RWV group, LV remodeling was significantly attenuated and neovascularization was significantly noted (Figure 1C, 1D). Conclusions: E3DCTs maturated by dynamic culture in a RWV bioreactor could remodel the diseased LV in rat infarction model, proposing potent armamentarium for cardiomyogenesis therapy in failed heart.

Functional engineered heart tissue cultured in a rotating wall vessel bioreactor improve cardiac function in the distressed rat heart

Abstract Introduction How to construct massive cardiac tissue and culture it with functional improvement may be crucial as cardiomyogenesis in failed heart. We previously presented that dynamic culture in a rotating wall vessel (RWV) bioreactor could provide a better culture environment for maintenance of the engineered 3D cardiac tissue. However, it is unknown about the effect of the tissue cultured in a RWV bioreactor on engraftment and improvement of function in the distressed rat heart. Hypothesis We hypothesized that the engineered 3D cardiac tissue cultured in a RWV bioreactor could improve its engraftment and lead recovery of cardiac function in rat infarction model. Methods We made engineered cardiac tissue by seeding 2.0 × 106 human induced pluripotent stem cell derived cardiomyocytes on the PLGA fiber sheet. It was cultured in the RWV bioreactor for seven days (RWV group). For the control, static culture has been done. After in vitro assessment, these tissues were transplanted to myocardial infarction model nude rats (sham, control, and RWV group, n=10, respectively) and cardiac performance was evaluated by ultrasonography. Four weeks after transplantation, we evaluated their hearts by histological analysis. Results The RWV group demonstrated maturation of cardiomyocytes evidenced by significantly higher expression of Troponin T (TnT), sarcomeric α actinin (SAA), connexin 43 (Cx43) and myosin heavy chain 7 (MYH7) than the control by Western blots (TnT; 2.7±1.0 vs. 1.0±0.4, p<0.01, SAA; 2.1±0.7 vs. 1.0±0.2, p<0.01, Cx 43; 2.0±0.6 vs. 1.0±0.1, p<0.05, MYH7; 10.9±2.7 vs. 1.0±0.1, p<0.01). In the culture supernatant, the concentration of cytokines related to angiogenesis was significantly higher in the RWV group than in the control (VEGF; 29.6±7.4 vs. 12.2±4.3pg/ml, p<0.01, HGF; 72.7±9.9 vs. 42.6±5.9pg/ml, p<0.01). Four weeks after transplantation, the left ventricular ejection fraction was significantly improved in the RWV group than in the control (RWV vs. control; 47±4.9 vs. 38±6.9%, p<0.01). On histological analysis, more engineered cardiac tissue survived in the RWV group than in the control (RWV vs. control; 7/10 vs. 3/10, p=0.18). A vascular-like structure double-stained with isolectin B4 and smooth muscle actin was partially observed in the transplanted tissue. LV remodeling exhibiting extracellular collagen deposition (fibrotic area, RWV vs. control; 17±4.3 vs. 24±5.2%, p<0.05) and cardiomyocyte hypertrophy (RWV vs. control; 16±1.7 vs. 18±2.1μm, p<0.05) was significantly attenuated in RWV group than in the control. Neovascularization was significantly noted in the RWV group compared with the control (capillary density, RWV vs. control; 545±113 vs. 356±92, p<0.01). Conclusion Functional engineered 3D cardiac tissue cultured in a RWV bioreactor could induce angiogenesis and improved its engraftment, leading significant improvement of cardiac function in rat infarction model. Dynamic culture in a RWV bioreactor Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Japan Society for the Promotion of Science

An Air Bubble-Isolating Rotating Wall Vessel Bioreactor for Improved Spheroid/Organoid Formation.

The rotating wall vessel (RWV) bioreactor is a powerful tool for the generation of sizeable, faster-growing organoids. However, the ideal, low-shear, modeled microgravity environment in the RWV is frequently disrupted by the formation of bubbles, a critical but understated failure mode. To address this, we have designed and fabricated a novel, modified RWV bioreactor capable of continuously removing bubbles while providing optimal fluid dynamics. We validated the capacity of this device with computational and empirical studies. We anticipate that our novel bioreactor will be more consistent and easier to use and may fill a unique and unmet niche in the burgeoning field of organoids.

Knockdown of CD44 inhibits the alteration of osteoclast function induced by simulated microgravity

Mechanical forces are essential to maintain skeletal homeostasis, and microgravity exposure leads to bone loss. During space flight, bone mineral density is decreased because of the inactivation of osteoblast and activation of osteoclast. However, the underlying molecular mechanism is still not clear. CD44 acts as a cellular surface adhesion molecule, which plays an important role in signal transduction between cells. Numerous studies have shown that CD44 plays diverse roles in promoting pre-osteoclast fusion. However, the role of CD44 in the alteration of osteoclast function induced by simulated microgravity remains to be fully elucidated. In this work, we utilized a ground-based, microgravity-simulation system, the Rotating Wall Vessel Bioreactor (RWVB). Using the RWVB, we demonstrated that simulated microgravity enhanced osteoclast function, meantime CD44 mRNA and protein levels were significantly increased. To verify the effects of CD44 on alteration of osteoclast function induced by simulated microgravity, specific siRNAs targeting CD44 were transfected. The results showed knockdown of CD44 inhibited the alteration of osteoclast function induced by simulated microgravity. Moreover, we found that clinorotation activated the NF-κB/NFATc1-mediated signaling pathway, which was downregulated after knockdown of CD44. Our study provided evidence that CD44 positively regulated osteoclast function and therapeutic suppression of CD44 may counteract bone loss induced by simulated microgravity.

Three-dimensional in vitro modeling of malignant bone disease recapitulates experimentally accessible mechanisms of osteoinhibition

Malignant bone disease (MBD) occurs when tumors establish in bone, causing catastrophic tissue damage as a result of accelerated bone destruction and inhibition of repair. The resultant so-called osteolytic lesions (OL) take the form of tumor-filled cavities in bone that cause pain, fractures, and associated morbidity. Furthermore, the OL microenvironment can support survival of tumor cells and resistance to chemotherapy. Therefore, a deeper understanding of OL formation and MBD progression is imperative for the development of future therapeutic strategies. Herein, we describe a novel in vitro platform to study bone–tumor interactions based on three-dimensional co-culture of osteogenically enhanced human mesenchymal stem cells (OEhMSCs) in a rotating wall vessel bioreactor (RWV) while attached to micro-carrier beads coated with extracellular matrix (ECM) composed of factors found in anabolic bone tissue. Osteoinhibition was recapitulated in this model by co-culturing the OEhMSCs with a bone–tumor cell line (MOSJ-Dkk1) that secretes the canonical Wnt (cWnt) inhibitor Dkk-1, a tumor-borne osteoinhibitory factor widely associated with several forms of MBD, or intact tumor fragments from Dkk-1 positive patient-derived xenografts (PDX). Using the model, we observed that depending on the conditions of growth, tumor cells can biochemically inhibit osteogenesis by disrupting cWnt activity in OEhMSCs, while simultaneously co-engrafting with OEhMSCs, displacing them from the niche, perturbing their activity, and promoting cell death. In the absence of detectable co-engraftment with OEhMSCs, Dkk-1 positive PDX fragments had the capacity to enhance OEhMSC proliferation while inhibiting their osteogenic differentiation. The model described has the capacity to provide new and quantifiable insights into the multiple pathological mechanisms of MBD that are not readily measured using monolayer culture or animal models.

Accelerated and Improved Differentiation of Retinal Organoids from Pluripotent Stem Cells in Rotating-Wall Vessel Bioreactors.

SummaryPluripotent stem cells can be differentiated into 3D retinal organoids, with major cell types self-patterning into a polarized, laminated architecture. In static cultures, organoid development may be hindered by limitations in diffusion of oxygen and nutrients. Herein, we report a bioprocess using rotating-wall vessel (RWV) bioreactors to culture retinal organoids derived from mouse pluripotent stem cells. Organoids in RWV demonstrate enhanced proliferation, with well-defined morphology and improved differentiation of neurons including ganglion cells and S-cone photoreceptors. Furthermore, RWV organoids at day 25 (D25) reveal similar maturation and transcriptome profile as those at D32 in static culture, closely recapitulating spatiotemporal development of postnatal day 6 mouse retina in vivo. Interestingly, however, retinal organoids do not differentiate further under any in vitro condition tested here, suggesting additional requirements for functional maturation. Our studies demonstrate that bioreactors can accelerate and improve organoid growth and differentiation for modeling retinal disease and evaluation of therapies.

Emergence of two distinct subpopulations from Klebsiella pneumoniae grown in the stimulated microgravity environment.

To isolate and characterize the two phenotypically distinct subpopulations from Klebsiella pneumoniae clonal cultures grown in the simulate microgravity environment. Here clonal culture of K. pneumoniae strain ATCC BAA-1705 was grown within a vertically rotating wall vessel bioreactor. Microscopic, colony staining, biofilm assays and quantitative proteomics were used to define the features of subpopulations. Two subpopulations were isolated based on colony appearance and bacterial morphology and indicated the different capability of biofilm formation and antibiotics resistance. These findings would raise a possibility of understanding the adaptive roles of bacterial subpopulations formed under certain conditions from the viewpoint of population variation.

Effect of Rotation on Scaffold Motion and Cell Growth in Rotating Bioreactors.

Efficient use of different bioreactor designs to improve cell growth in three-dimensional scaffolds requires an understanding of their mechanism of action. To address this for rotating wall vessel bioreactors, fluid and scaffold motion were investigated experimentally at different rotation speeds and vessel fill volumes. Low cost bioreactors with single and dual axis rotation were developed to investigate the effect of these systems on human osteoblast proliferation in free floating and constrained collagen-glycosaminoglycan porous scaffolds. A range of scaffold motions (free fall, periodic oscillation, and orbital motion) were observed at the rotation speeds and vessel fluid/air ratios used, with 85% fluid fill and an outer vessel wall velocity of ∼14 mm s−1 producing a scaffold in a free fall state. The cell proliferation results showed that after 14 and 21 days of culture, this combination of fluid fill and speed of rotation produced significantly greater cell numbers in the scaffolds than when lower or higher rotation speeds (p < 0.002) or when the chamber was 60% or 100% full (p < 0.01). The fluid flow and scaffold motion experiments show that biaxial rotation would not improve the mass transfer of medium into the scaffold as the second axis of rotation can only transition the scaffold toward oscillatory or orbital motion and, hence, reduce mass transport to the scaffold. The cell culture results confirmed that there was no benefit to the second axis of rotation with no significant difference in cell proliferation either when the scaffolds were free floating or constrained (p > 0.05).

Three-dimensional organotypic co-culture model of intestinal epithelial cells and macrophages to study Salmonella enterica colonization patterns

Three-dimensional models of human intestinal epithelium mimic the differentiated form and function of parental tissues often not exhibited by two-dimensional monolayers and respond to Salmonella in key ways that reflect in vivo infections. To further enhance the physiological relevance of three-dimensional models to more closely approximate in vivo intestinal microenvironments encountered by Salmonella, we developed and validated a novel three-dimensional co-culture infection model of colonic epithelial cells and macrophages using the NASA Rotating Wall Vessel bioreactor. First, U937 cells were activated upon collagen-coated scaffolds. HT-29 epithelial cells were then added and the three-dimensional model was cultured in the bioreactor until optimal differentiation was reached, as assessed by immunohistochemical profiling and bead uptake assays. The new co-culture model exhibited in vivo-like structural and phenotypic characteristics, including three-dimensional architecture, apical-basolateral polarity, well-formed tight/adherens junctions, mucin, multiple epithelial cell types, and functional macrophages. Phagocytic activity of macrophages was confirmed by uptake of inert, bacteria-sized beads. Contribution of macrophages to infection was assessed by colonization studies of Salmonella pathovars with different host adaptations and disease phenotypes (Typhimurium ST19 strain SL1344 and ST313 strain D23580; Typhi Ty2). In addition, Salmonella were cultured aerobically or microaerobically, recapitulating environments encountered prior to and during intestinal infection, respectively. All Salmonella strains exhibited decreased colonization in co-culture (HT-29-U937) relative to epithelial (HT-29) models, indicating antimicrobial function of macrophages. Interestingly, D23580 exhibited enhanced replication/survival in both models following invasion. Pathovar-specific differences in colonization and intracellular co-localization patterns were observed. These findings emphasize the power of incorporating a series of related three-dimensional models within a study to identify microenvironmental factors important for regulating infection.