Rotating Wall Vessel Research Articles

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.

Reduced gravity contributes to Neutrophil to Lymphocyte Ratio shifting and promotion of the Oxidative Stress Response

Abstract Spaceflight can cause immune system dysfunction, such as elevated white blood cells (WBC) and polymorphonuclear neutrophils (PMN), along with unchanged or reduced lymphocyte counts. A high PMN to lymphocyte ratio (NLR) can acts as a poor prognosis in cancer and a biomarker for subclinical inflammation however, the NLR has not been identified as a predictor of astronaut health during spaceflight. CBC data collected on board the International Space Station (ISS) was repurposed to determine the granulocyte to lymphocyte ratio (GLR) in humans and the NLR in rodents. The results displayed a progressive increase in GLR and NLR during spaceflight and at landing. The mechanism for increased NLR was assessed in vitro using the microgravity-analog, rotating wall vessel (RWV), with human WBCs. The results indicated that simulated microgravity led to increased GLR and NLR profiles, and production of reactive oxygen species (ROS) and myeloperoxidase (MPO). Interestingly, simulated microgravity increased the number of matured PMNs that showed impaired phagocytic function, while treatment with tert-Butyl hydroperoxide (TBHP), also reduced PMN phagocytosis. In addition, 30-days of simulated microgravity (hindlimb unloading) in mice, indicated an increased NLR and MPO gene expression, which were mitigated in mitochondrial catalase overexpressing transgenic mice, suggesting ROS scavenging is essential for maintaining homeostatic immunity. Collectively, we propose that the health status of astronauts during future short- and long-term space missions can be monitored by their NLR profile, in addition to utilizing this measurement as a tool for oxidative stress response countermeasure development to restore homeostatic immunity.

Effect of Weightlessness on the 3D Structure Formation and Physiologic Function of Human Cancer Cells.

With the rapid development of modern medical technology and the deterioration of living environments, cancer, the most important disease that threatens human health, has attracted increasing concerns. Although remarkable achievements have been made in tumor research during the past several decades, a series of problems such as tumor metastasis and drug resistance still need to be solved. Recently, relevant physiological changes during space exploration have attracted much attention. Thus, space exploration might provide some inspiration for cancer research. Using on ground different methods in order to simulate microgravity, structure and function of cancer cells undergo many unique changes, such as cell aggregation to form 3D spheroids, cell-cycle inhibition, and changes in migration ability and apoptosis. Although numerous better experiments have been conducted on this subject, the results are not consistent. The reason might be that different methods for simulation have been used, including clinostats, random positioning machine (RPM) and rotating wall vessel (RWV) and so on. Therefore, we review the relevant research and try to explain novel mechanisms underlying tumor cell changes under weightlessness.

Treatment with hydrogen sulfide donor attenuates bone loss induced by modeled microgravity

The present study was undertaken to explore the therapeutic potential of hydrogen sulfide against bone loss induced by modeled microgravity. Hindlimb suspension (HLS) and rotary wall vessel bioreactor were applied to model microgravity in vivo and in vitro, respectively. Treatment of rats with GYY4137 (a water soluble donor of hydrogen sulfide, 25 mg/kg per day, i.p.) attenuated HLS-induced reduction of bone mineral density in tibiae, and preserved bone structure in tibiae and mechanical strength in femurs. In HLS group, GYY4137 treatment significantly increased levels of osteocalcin in sera. Interestingly, treatment of HLS rats with GYY4137 enhanced osteoblast surface, but had no significant effect on osteoclast surface of proximal tibiae. In MC3T3-E1 cells exposed to modeled microgravity, GYY4137 stimulated transcriptional levels of runt-related transcription factor 2 and enhanced osteoblastic differentiation, as evidenced by increased mRNA expression and activity of alkaline phosphatase. HLS in rats led to enhanced levels of interleukin 6 in sera, skeletal muscle, and tibiae, which could be attenuated by GYY4137 treatment. Our study showed that GYY4137 preserved bone structure in rats exposed to HLS and promoted osteoblastic differentiation in MC3T3-E1 cells under modeled microgravity.

The dynamic adaptation of primary human endothelial cells to simulated microgravity.

Culture of human endothelial cells for 10 d in real microgravity onboard the International Space Station modulated more than 1000 genes, some of which are involved in stress response. On Earth, 24 h after exposure to simulated microgravity, endothelial cells up-regulate heat shock protein (HSP) 70. To capture a broad view of endothelial stress response to gravitational unloading, we cultured primary human endothelial cells for 4 and 10 d in the rotating wall vessel, a U.S. National Aeronautics and Space Administration-developed surrogate system for benchtop microgravity research on Earth. We highlight the crucial role of the early increase of HSP70 because its silencing markedly impairs cell survival. Once HSP70 up-regulation fades away after 4 d of simulated microgravity, a complex and articulated increase of various stress proteins (sirtuin 2, paraoxonase 2, superoxide dismutase 2, p21, HSP27, and phosphorylated HSP27, all endowed with cytoprotective properties) occurs and counterbalances the up-regulation of the pro-oxidant thioredoxin interacting protein (TXNIP). Interestingly, TXNIP was the most overexpressed transcript in endothelial cells after spaceflight. We conclude that HSP70 up-regulation sustains the initial adaptive response of endothelial cells to mechanical unloading and drives them toward the acquisition of a novel phenotype that maintains cell viability and function through the sequential involvement of different stress proteins.-Cazzaniga, A., Locatelli, L., Castiglioni, S., Maier, J. A. M. The dynamic adaptation of primary human endothelial cells to simulated microgravity.

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.

Analysis of Host Responses to Neisseria gonorrhoeae Using a Human Three-Dimensional Endometrial Epithelial Cell Model.

Neisseria gonorrhoeae infections have been associated with complications including chronic endometritis and pelvic inflammatory disease. Robust in vitro models of the female reproductive tract are urgently needed to better understand the biological mechanisms leading to these pathophysiological changes. Our human three-dimensional (3D) endometrial epithelial cell (EEC) model, which is generated using the HEC-1A cell line and rotating wall vessel (RWV) bioreactor technology, replicates several hallmarks of endometrial tissue in vivo. Studying the interactions of N. gonorrhoeae with the host using this newly characterized human 3D EEC model allows for the investigation of unique mechanisms of gonococcal pathogenesis in the upper female reproductive tract. In this chapter, we describe methodologies that can be used to investigate the interactions of N. gonorrhoeae with the human 3D endometrial epithelium. Protocols for generating the human 3D EEC model using the RWV technology and assessing the host response (including morphological/ultrastructural changes to the epithelial cells; cytokine/chemokine secretion or gene expression changes) following infection with N. gonorrhoeae are presented.

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.

Synergistic Effects of Weightlessness, Isoproterenol, and Radiation on DNA Damage Response and Cytokine Production in Immune Cells.

The implementation of rotating-wall vessels (RWVs) for studying the effect of lack of gravity has attracted attention, especially in the fields of stem cells, tissue regeneration, and cancer research. Immune cells incubated in RWVs exhibit several features of immunosuppression including impaired leukocyte proliferation, cytokine responses, and antibody production. Interestingly, stress hormones influence cellular immune pathways affected by microgravity, such as cell proliferation, apoptosis, DNA repair, and T cell activation. These pathways are crucial defense mechanisms that protect the cell from toxins, pathogens, and radiation. Despite the importance of the adrenergic receptor in regulating the immune system, the effect of microgravity on the adrenergic system has been poorly studied. Thus, we elected to investigate the synergistic effects of isoproterenol (a sympathomimetic drug), radiation, and microgravity in nonstimulated immune cells. Peripheral blood mononuclear cells were treated with the sympathomimetic drug isoproterenol, exposed to 0.8 or 2 Gy γ-radiation, and incubated in RWVs. Mixed model regression analyses showed significant synergistic effects on the expression of the β2-adrenergic receptor gene (ADRB2). Radiation alone increased ADRB2 expression, and cells incubated in microgravity had more DNA strand breaks than cells incubated in normal gravity. We observed radiation-induced cytokine production only in microgravity. Prior treatment with isoproterenol clearly prevents most of the microgravity-mediated effects. RWVs may be a useful tool to provide insight into novel regulatory pathways, providing benefit not only to astronauts but also to patients suffering from immune disorders or undergoing radiotherapy.

An integrated biomanufacturing platform for the large-scale expansion and neuronal differentiation of human pluripotent stem cell-derived neural progenitor cells

Human pluripotent stem cell derived neural progenitor cells (hNPCs) have the unique properties of long-term in vitro expansion as well as differentiation into the various neurons and supporting cell types of the central nervous system (CNS). Because of these characteristics, hNPCs have tremendous potential in the modeling and treatment of various CNS diseases and disorders. However, expansion and neuronal differentiation of hNPCs in quantities necessary for these applications is not possible with current two dimensional (2-D) approaches. Here, we used a fully defined peptide substrate as the basis for a microcarrier (MC)-based suspension culture system. Several independently derived hNPC lines were cultured on MCs for multiple passages as well as efficiently differentiated to neurons. Finally, this MC-based system was used in conjunction with a low shear rotating wall vessel (RWV) bioreactor for the integrated, large-scale expansion and neuronal differentiation of hNPCs. Overall, this fully defined and scalable biomanufacturing system will facilitate the generation of hNPCs and their neuronal derivatives in quantities necessary for basic and translational applications. Statement of SignificanceIn this work, we developed a microcarrier (MC)-based culture system that allows for the expansion and neuronal differentiation of human pluripotent stem cell-derived neural progenitor cells (hNPCs) under defined conditions. In turn, this MC approach was implemented in a rotating wall vessel (RWV) bioreactor for the large-scale expansion and neuronal differentiation of hNPCs. This work is of significance as it overcomes current limitations of conventional two dimensional (2-D) culture systems to enable the generation of hNPCs and their neuronal derivatives in quantities required for downstream applications in disease modeling, drug screening, and regenerative medicine.

Tissue Engineering Under Microgravity Conditions-Use of Stem Cells and Specialized Cells.

Experimental cell research studying three-dimensional (3D) tissues in space and on Earth using new techniques to simulate microgravity is currently a hot topic in Gravitational Biology and Biomedicine. This review will focus on the current knowledge of the use of stem cells and specialized cells for tissue engineering under simulated microgravity conditions. We will report on recent advancements in the ability to construct 3D aggregates from various cell types using devices originally created to prepare for spaceflights such as the random positioning machine (RPM), the clinostat, or the NASA-developed rotating wall vessel (RWV) bioreactor, to engineer various tissues such as preliminary vessels, eye tissue, bone, cartilage, multicellular cancer spheroids, and others from different cells. In addition, stem cells had been investigated under microgravity for the purpose to engineer adipose tissue, cartilage, or bone. Recent publications have discussed different changes of stem cells when exposed to microgravity and the relevant pathways involved in these biological processes. Tissue engineering in microgravity is a new technique to produce organoids, spheroids, or tissues with and without scaffolds. These 3D aggregates can be used for drug testing studies or for coculture models. Multicellular tumor spheroids may be interesting for radiation experiments in the future and to reduce the need for in vivo experiments. Current achievements using cells from patients engineered on the RWV or on the RPM represent an important step in the advancement of techniques that may be applied in translational Regenerative Medicine.

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.

Combined Effects of Simulated Microgravity and Radiation Exposure on Osteoclast Cell Fusion.

The loss of bone mass and alteration in bone physiology during space flight are one of the major health risks for astronauts. Although the lack of weight bearing in microgravity is considered a risk factor for bone loss and possible osteoporosis, organisms living in space are also exposed to cosmic radiation and other environmental stress factors. As such, it is still unclear as to whether and by how much radiation exposure contributes to bone loss during space travel, and whether the effects of microgravity and radiation exposure are additive or synergistic. Bone is continuously renewed through the resorption of old bone by osteoclast cells and the formation of new bone by osteoblast cells. In this study, we investigated the combined effects of microgravity and radiation by evaluating the maturation of a hematopoietic cell line to mature osteoclasts. RAW 264.7 monocyte/macrophage cells were cultured in rotating wall vessels that simulate microgravity on the ground. Cells under static 1g or simulated microgravity were exposed to γ rays of varying doses, and then cultured in receptor activator of nuclear factor-κB ligand (RANKL) for the formation of osteoclast giant multinucleated cells (GMCs) and for gene expression analysis. Results of the study showed that radiation alone at doses as low as 0.1 Gy may stimulate osteoclast cell fusion as assessed by GMCs and the expression of signature genes such as tartrate resistant acid phosphatase (Trap) and dendritic cell-specific transmembrane protein (Dcstamp). However, osteoclast cell fusion decreased for doses greater than 0.5 Gy. In comparison to radiation exposure, simulated microgravity induced higher levels of cell fusion, and the effects of these two environmental factors appeared additive. Interestingly, the microgravity effect on osteoclast stimulatory transmembrane protein (Ocstamp) and Dcstamp expressions was significantly higher than the radiation effect, suggesting that radiation may not increase the synthesis of adhesion molecules as much as microgravity.

Simulated microgravity ‘disarms’ human Natural Killer cells and suppresses cytotoxic activity against tumor target cells

Maintaining immune system integrity is of paramount importance for astronauts embarking on long-duration spaceflight missions. We have shown that Natural killer (NK) cell function is impaired during spaceflight; however, the mechanisms responsible for this effect are unknown. We exposed primary human blood NK cells to 12 h of simulated microgravity (SMG) using a rotating wall vessel zero gravity cell culture analog. Compared to static and vertical axis rotational controls, SMG (horizontal axis rotation) was found to decrease subsequent NK-cell cytotoxic function in 1G against allogeneic target cell lines of leukemia (K562), multiple myeloma (U266), B-lymphoma (721.221) and HLA-E transfected lymphoma (221.AEH) origin. Flow cytometric analysis revealed that SMG had little effect on the surface expression of a wide-range of NK-cell activating and inhibitory receptors, but did decrease intracellular perforin expression. Using cytometry by time of flight (CyTOF), SMG was found to lower NK-cell degranulation as determined by CD107a expression following co-incubation with K562 target cells in 1G. Moreover, SMG reduced NK-cell expression of the pro-inflammatory cytokines TNF-alpha and IFN-gamma in response to K562 co-culture at 1G. These results indicate that microgravity may be involved in spaceflight-associated reductions in NK cell function. Specifically, pre-exposure to SMG appears to disrupt NK-cell killing by ‘disarming’ them of cytolytic granules and impairing their ability to secrete effector cytokines when confronted with tumor target cells in the 1G environment.

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.

Mesenchymal stem cells support growth and organization of host-liver colorectal-tumor organoids and possibly resistance to chemotherapy

Despite having yielded extensive breakthroughs in cancer research, traditional 2D cell cultures have limitations in studying cancer progression and metastasis and screening therapeutic candidates. 3D systems can allow cells to grow, migrate, and interact with each other and the surrounding matrix, resulting in more realistic constructs. Furthermore, interactions between host tissue and developing tumors influence the susceptibility of tumors to drug treatments. Host-liver colorectal-tumor spheroids composed of primary human hepatocytes, mesenchymal stem cells (MSC) and colon carcinoma HCT116 cells were created in simulated microgravity rotating wall vessel (RWV) bioreactors. The cells were seeded on hyaluronic acid-based microcarriers, loaded with liver-specific growth factors and ECM components. Only in the presence of MSC, large tumor foci rapidly formed inside the spheroids and increased in size steadily over time, while not greatly impacting albumin secretion from hepatocytes. The presence of MSC appeared to drive self-organization and formation of a stroma-like tissue surrounding the tumor foci and hepatocytes. Exposure to a commonly used chemotherapeutic 5-FU showed a dose-dependent cytotoxicity. However, if tumor organoids were allowed to mature in the RWV, they were less sensitive to the drug treatment. These data demonstrate the potential utility of liver tumor organoids for cancer progression and drug response modeling.

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.

Human Three-Dimensional Endometrial Epithelial Cell Model To Study Host Interactions with Vaginal Bacteria and Neisseria gonorrhoeae.

Colonization of the endometrium by pathogenic bacteria ascending from the lower female reproductive tract (FRT) is associated with many gynecologic and obstetric health complications. To study these host-microbe interactions in vitro, we developed a human three-dimensional (3-D) endometrial epithelial cell (EEC) model using the HEC-1A cell line and the rotating wall vessel (RWV) bioreactor technology. Our model, composed of 3-D EEC aggregates, recapitulates several functional/structural characteristics of human endometrial epithelial tissue, including cell differentiation, the presence of junctional complexes/desmosomes and microvilli, and the production of membrane-associated mucins and Toll-like receptors (TLRs). TLR function was evaluated by exposing the EEC aggregates to viral and bacterial products. Treatment with poly(I·C) and flagellin but not with synthetic lipoprotein (fibroblast-stimulating lipoprotein 1 [FSL-1]) or lipopolysaccharide (LPS) significantly induced proinflammatory mediators in a dose-dependent manner. To simulate ascending infection, we infected EEC aggregates with commensal and pathogenic bacteria: Lactobacillus crispatus, Gardnerella vaginalis, and Neisseria gonorrhoeae All vaginal microbiota and N. gonorrhoeae efficiently colonized the 3-D surface, localizing to crevices of the EEC model and interacting with multiple adjacent cells simultaneously. However, only infection with pathogenic N. gonorrhoeae and not infection with the other bacteria tested significantly induced proinflammatory mediators and significant ultrastructural changes to the host cells. The latter observation is consistent with clinical findings and illustrated the functional specificity of our system. Additionally, we highlighted the utility of the 3-D EEC model for the study of the pathogenesis of N. gonorrhoeae using a well-characterized ΔpilT mutant. Overall, this study demonstrates that the human 3-D EEC model is a robust tool for studying host-microbe interactions and bacterial pathogenesis in the upper FRT.

Aligned Nanofibrous Cell-Derived Extracellular Matrix for Anisotropic Vascular Graft Construction.

There is a large demand for tissue engineered vascular grafts for the application of vascular reconstruction surgery or in vitro drug screening tissue model. The extracellular matrix (ECM) composition along with the structural and mechanical anisotropy of native blood vessels is critical to their functional performance. The objective of this study is to develop a biomimetic vascular graft recapitulating the anisotropic features of native blood vessels by employing nanofibrous aligned fibroblast-derived ECM and human mesenchymal stem cells (hMSCs). The nanotopographic cues of aligned ECM direct the initial cell orientation. The subsequent maturation under circumferential stress generated by a rotating wall vessel (RWV) bioreactor further promotes anisotropic structural and mechanical properties in the graft. The circumferential tensile strength is significantly higher than longitudinal strength in bioreactor samples. Expression of smooth muscle cell specific genes, α-smooth muscle actin and calponin, in hMSCs is greatly enhanced in bioreactor samples without any biochemical stimulation. In addition, employment of premade ECM and RWV bioreactor significantly reduces the graft fabrication time to three weeks. Mimicking the ECM composition, cell phenotype, structural and mechanical anisotropy, the vascular graft presented in this study is promising for vascular reconstruction surgery or in vitro tissue model applications.