Using a developed co-culture device to evaluate the proliferation of bone marrow stem cells by stimulation with platelet-rich plasma and electromagnetic field.

BACKGROUNDS: Bone marrow stem cell can differentiate to osteoblast by growth factors, pulsed low-intensity ultrasound and electric magnetic field. In the research, bone marrow stem cells were cultured; bone marrow stem cells in culture can be stimulated by platelet-rich plasma and electric field. METHODS: The culture well of the co-cultivation device has a radius of 7.5 mm and a depth of 7 mm. It is divided into two sub-chambers separated by a 3 mm high and 1 mm wide barrier. The bone marrow stem cells were seeded at a density of 2 × 104 cells and the medium volume was 120µl. Platelet-rich plasma (PRP) or platelet-poor plasma (PPP) was added to the other sub-chamber at a volume of 10µl. The bone marrow stem cells were subjected to different electric fields (0 ~ 1 V/cm) at a frequency of 70 kHz for 60 min. RESULTS: The highest osteogenic capacity of bone marrow stem cells was achieved by addition of PRP to electric field stimulation (0.25 V/cm) resulted in a proliferation rate of 599.78%. In electric field stimulation (0.75 V/cm) with PPP, the proliferation rate was only 10.46%. CONCLUSIONS: Bone marrow stem cell with PRP in the co-culture device combined with electric field at 0.25 V/cm strength significantly promoted the growth of bone marrow stem cells.

Hypoxia Promotes Cartilage Regeneration in Cell-Seeded 3D-Printed Bioscaffolds Cultured with a Bespoke 3D Culture Device.

In this study, we investigated the effect of oxygen tension on the expansion of ADMSCs and on their differentiation toward their chondrocytic phenotype, regenerating a lab-based cartilaginous tissue with superior characteristics. Controversial results with reference to MSCs that were cultured under different hypoxic levels, mainly in 2D culturing settings combined with or without other biochemical stimulus factors, prompted our team to study the role of hypoxia on MSCs chondrogenic differentiation within an absolute 3D environment. Specifically, we used 3D-printed honeycomb-like PCL matrices seeded with ADMSCs in the presence or absence of TGF and cultured with a prototype 3D cell culture device, which was previously shown to favor nutrient/oxygen supply, cell adhesion, and infiltration within scaffolds. These conditions resulted in high-quality hyaline cartilage that was distributed uniformly within scaffolds. The presence of the TGF medium was necessary to successfully produce cartilaginous tissues with superior molecular and increased biomechanical properties. Despite hypoxia's beneficial effect, it was overall not enough to fully differentiate ADMSCs or even promote cell expansion within 3D scaffolds alone.

In Vitro Assessment of Antimicrobial Resistance Dissemination Dynamics during Multidrug-Resistant-Bacterium Invasion Events by Using a Continuous-Culture Device.

Antimicrobial-resistant pathogens display significant public health threats by causing difficulties in clinical treatment of bacterial infection. Antimicrobial resistance (AMR) is transmissible between bacteria, significantly increasing the appearance of antimicrobial-resistant pathogens and aggravating the AMR problem. In this work, the dissemination dynamics of AMR from invading multidrug-resistant (MDR) Escherichia coli to a community of pathogenic Salmonella enterica was investigated using a continuous-culture device, and the behaviors of dissemination dynamics under different levels of antibiotic stress were investigated. Three MDR E. coli invasion events were analyzed in this work: MDR E. coli-S. enterica cocolonization, MDR E. coli invasion after antibiotic treatment of S. enterica, and MDR E. coli invasion before antibiotic treatment of S. enterica It was found that both horizontal gene transfer (HGT) and vertical gene transfer (VGT) play significant roles in AMR dissemination, although different processes contribute differently under different circumstances, that environmental levels of antibiotics promote AMR dissemination by enhancing HGT rather than leading to selective advantage for resistant bacteria, and that early invasion of MDR E. coli completely and quickly sabotages the effectiveness of antibiotic treatment. These findings contribute to understanding the drivers of AMR dissemination under different antibiotic stresses, the detrimental impact of environmental tetracycline contamination, and the danger of nosocomial presence and dissemination of MDR nonpathogens.IMPORTANCE Antimicrobial resistance poses a grave threat to public health and reduces the effectiveness of antimicrobial drugs in treating bacterial infections. Antimicrobial resistance is transmissible, either by horizontal gene transfer between bacteria or by vertical gene transfer following inheritance of genetic traits. The dissemination dynamics and behaviors of this threat, however, have not been rigorously investigated. In this work, with a continuous-culture device, we studied antimicrobial resistance dissemination processes by simulating antimicrobial-resistant Escherichia coli invasion to a pathogenic Salmonella enterica community. Using this novel tool, we provide evidence on the drivers of antimicrobial resistance dissemination, on the detrimental impact of environmental antibiotic contamination, and on the danger of antimicrobial resistance in hospitals, even if what harbors the antimicrobial resistance is not a pathogen. This work furthers our understanding of antimicrobial resistance and its dissemination between bacteria and of antibiotic therapy, our most powerful tool against bacterial infection.

A cell culture device equipped with a micro-needle electrode array fabricated using backside exposure mold and resin casting.

A cell culture device equipped with a micro-needle electrode array was fabricated for the signal analysis of cell spheroids, cell masses, and cell sheets. For the analysis, sharp needle electrodes with a high aspect ratio for facilitating easy penetration into the cell mass and a small pitch for fine spatial resolution were required. Microelectromechanical systems (MEMS) technology is one of the common solutions for the fabrication of devices. However, an additional process, such as anisotropic etching or electro-polishing, is required for fabricating sharp needles. Tapered needles were fabricated using backside exposure for coating a layer of thick resist film on a glass substrate. The incident beam from mask apertures were diffracted and attenuated in the medium, resulting in tapered intensity distribution. A needle-like shape was obtained after performing resist development without using additional MEMS process. In this study, the theoretical analysis of optical intensity distribution and design and fabrication process of the device were described. Finally, the effectiveness of the device was evaluated by adding cultured cell mass on the needle array. Signals with spikes and fluctuations were observed in the electrode covered with cell mass, whereas only noise was observed on the non-covered electrode, demonstrating the signal pick-up ability of the device during cell culture.

Temporal relation between neural activity and neurite pruning on a numerical model and a microchannel device with micro electrode array.

Synapse elimination and neurite pruning are essential processes for the formation of neuronal circuits. These regressive events depend on neural activity and occur in the early postnatal days known as the critical period, but what makes this temporal specificity is not well understood. One possibility is that the neural activities during the developmentally regulated shift of action of GABA inhibitory transmission lead to the critical period. Moreover, it has been reported that the shifting action of the inhibitory transmission on immature neurons overlaps with synapse elimination and neurite pruning and that increased inhibitory transmission by drug treatment could induce temporal shift of the critical period. However, the relationship among these phenomena remains unclear because it is difficult to experimentally show how the developmental shift of inhibitory transmission influences neural activities and whether the activities promote synapse elimination and neurite pruning. In this study, we modeled synapse elimination in neuronal circuits using the modified Izhikevich's model with functional shifting of GABAergic transmission. The simulation results show that synaptic pruning within a specified period like the critical period is spontaneously generated as a function of the developmentally shifting inhibitory transmission and that the specific firing rate and increasing synchronization of neural circuits are seen at the initial stage of the critical period. This temporal relationship was experimentally supported by an in vitro primary culture of rat cortical neurons in a microchannel on a multi-electrode array (MEA). The firing rate decreased remarkably between the 18-25 days in vitro (DIV), and following these changes in the firing rate, the neurite density was slightly reduced. Our simulation and experimental results suggest that decreasing neural activity due to developing inhibitory synaptic transmission could induce synapse elimination and neurite pruning at particular time such as the critical period. Additionally, these findings indicate that we can estimate the maturity level of inhibitory transmission and the critical period by measuring the firing rate and the degree of synchronization in engineered neural networks.

Hydrogel microfluidic co-culture device for photothermal therapy and cancer migration.

We developed the photo-crosslinkable hydrogel microfluidic co-culture device to study photothermal therapy and cancer cell migration. To culture MCF7 human breast carcinoma cells and metastatic U87MG human glioblastoma in the microfluidic device, we used 10 w/v% gelatin methacrylate (GelMA) hydrogels as a semi-permeable physical barrier. We demonstrated the effect of gold nanorod on photothermal therapy of cancer cells in the microfluidic co-culture device. Interestingly, we observed that metastatic U87MG human glioblastoma largely migrated toward vascular endothelial growth factor (VEGF)-treated GelMA hydrogel-embedding microchannels. The main advantage of this hydrogel microfluidic co-culture device is to simultaneously analyze the physiological migration behaviors of two cancer cells with different physiochemical motilities and study gold nanorod-mediated photothermal therapy effect. Therefore, this hydrogel microfluidic co-culture device could be a potentially powerful tool for photothermal therapy and cancer cell migration applications.

A compartmentalized culture device for studying the axons of CNS neurons.

We report here the development of a compartmentalized culture device that allows the spatial separation of the somatodendrites and axons of central nervous system (CNS) neurons. The device consists of two compartments separated by a septum constructed by attaching a porous polycarbonate track etch (PCTE) filter on top of a microchannel-filled polydimethylsiloxane (PDMS) membrane. The surface and microchannels of the septum are coated and filled, respectively, with materials that support neuron growth and neurite migration. When rat hippocampal neurons are cultured in the top compartment, axons are the only processes that can migrate through the septum to the bottom compartment. The axons in the bottom compartment can be studied directly in real-time or through immunofluorescence staining after fixation. Axons containing ∼3 µg protein can be isolated from each device for biochemical analyses. In addition, the septum also impedes the movement of small molecules between the top and bottom compartments. This feature allows the somatodendrites and axons of neurons, which occupy the top and bottom compartments of the device, respectively, to be manipulated independently. The potential applications of the device as a tool in diverse studies concerning neuronal axons and in screening reagents that regulate axonal functions have also been discussed.

Development of a simple cultured cell-anaerobic microbial co-culture system using liquid paraffin.

Conventional co-culture systems are complicated, lack versatility, and do not adequately replicate the intestinal lumen environment. This study aimed to devise a system that allows for (i) arbitrary sampling of the culture medium, (ii) monitoring the growth of co-cultured cells, (iii) aerobic-anaerobic co-culture, (iv) simple operation, and (v) evaluation of multiple samples. We built a simple cell-anaerobic microorganism co-culture system using liquid paraffin to separate growth spaces for aerobic cells and anaerobic bacteria. Mineral oil was added to the top of the anaerobic bacterial cultivation space to seal the space and reduce gas exchange. Co-culture of anaerobic, Bifidobacterium bifidum and aerobic, epithelial Madin-Darby canine kidney (MDCK) cells demonstrated that the barrier function and viability of co-cultured MDCK cells were comparable to those of a pure MDCK culture after 24 h, and the growth curve of co-cultured B. bifidum was similar to that of pure B. bifidum. Furthermore, the growth of B. bifidum pure culture under sealed conditions was approximately 1.5 times greater than that under non-sealed conditions at 24 h. Glucose consumption at 24 h of co-culture under sealed conditions was 10%-15% higher than that under non-sealed conditions. This highly versatile culture method enabled the quantitative characterisation of B. bifidum and MDCK cells upon co-culture. The newly established co-culture system could be applied to various aerobic cell-anaerobic bacteria co-culture which will provide a strategy for basic and applied research on host-microbe interactions.

Gelatin-based cell culture device for construction and X-ray irradiation of a three-dimensional oral cancer model.

Bioassays using three-dimensional (3D) tissue models offer several advantages over 2D culture assays because they can reproduce the structure and function of native tissues. In this study, we used our newly designed gelatin device to generate a miniature 3D model of human oral squamous cell carcinoma with stroma and blood vessels. To enable air-liquid interface culture, we conceived a new device structure in which three wells were lined up and separated by a dividing thread; the wells could be connected by removing the dividing thread. Cells were seeded in the center well with the dividing thread to form a multilayer, followed by the supply of media from the side wells after thread removal. Human oral squamous cell carcinoma (HSC-4) cells, human umbilical vein endothelial cells (HUVECs), and normal human dermal fibroblasts (NHDFs) were successfully cocultured, resulting in structures that mimicked 3D-cancer tissues. This 3D-cancer model was subjected to an X-ray sensitivity assay, followed by the evaluation of DNA damage using confocal microscopy and section-scanning electron microscopy.

A practical guide to promote informatics-driven efficient biotopographic material development.

Micro/nano topographic structures have shown great utility in many biomedical areas including cell therapies, tissue engineering, and implantable devices. Computer-assisted informatics methods hold great promise for the design of topographic structures with targeted properties for a specific medical application. To benefit from these methods, researchers and engineers require a highly reusable "one structural parameter - one set of cell responses" database. However, existing confounding factors in topographic cell culture devices seriously impede the acquisition of this kind of data. Through carefully dissecting the confounding factors and their possible reasons for emergence, we developed corresponding guideline requirements for topographic cell culture device development to remove or control the influence of such factors. Based on these requirements, we then suggested potential strategies to meet them. In this work, we also experimentally demonstrated a topographic cell culture device with controlled confounding factors based on these guideline requirements and corresponding strategies. A "guideline for the development of topographic cell culture devices" was summarized to instruct researchers to develop topographic cell culture devices with the confounding factors removed or well controlled. This guideline aims to promote the establishment of a highly reusable "one structural parameter - one set of cell responses" database that could facilitate the application of informatics methods, such as artificial intelligence, in the rational design of future biotopographic structures with high efficacy.

Development of a device for cultivation and isolation of microbes using a specialized cellulose film.

A device was developed for the cultivation and isolation of microbes using a specialized cellulose film (SCF). This method can solve problems associated with the agar plate method. It can also be used for other culture-related operations, which is not possible with the traditional agar plate culture method.

Hepatocytes and Endothelial Networks in a Fluid-Based In Vitro Model of Liver Drug Metabolism.

Drug-induced liver toxicity remains a major cause of drug withdrawal from animal testing and human clinical trials. A functional liver culture model corresponding to the liver is urgently required; however, in previous liver models, it has proven difficult to stably maintain multiple liver functions. Previously reported fluid-based systems have some advantages for hepatocyte culture, but have insufficient liver-specific functions because they simply involve moving conventional hepatocyte cultures from a dish into a fluid-based system. Importantly, these cultures have no liver tissue-specific structures that construct liver-specific cellular polarities, such as apical, basolateral, and basal faces. In this study, we developed a fluid-based system for our liver tissue culture models. The liver tissues that were constructed in our originally designed fluid-based systems represent a tissue culture model for studying hepatic functions. Together, our findings show that by mimicking the structure of the liver in the body, our system effectively maintains multiple liver-specific functions. Impact statement A functional liver culture model corresponding to the liver is urgently required; however, in previous liver models, it has proven difficult to stably maintain multiple liver functions. In this study, we developed a fluid-based system for our liver tissue culture models. The liver tissues that were constructed in our originally designed fluid-based systems represent a tissue culture model for studying hepatic functions. Together, our findings show that by mimicking the structure of the liver in the body, our system effectively maintains multiple liver-specific functions.

Integrated On-Chip 3D Vascular Network Culture under Hypoxia.

We developed a portable device made of poly(dimethylsiloxane) (PDMS)/polymethylmethacrylate (PMMA) for long-term 3D cell culture of vascular endothelial cells for the development of a vascular network and evaluated the device under different transitions between normoxia and hypoxia with good optical accessibility. The combination of a nested reservoir device and a bicarbonate/ascorbate buffer system accomplished on-chip incubation with 4.91 ± 0.86% pO2 and 5.19 ± 1.70% pCO2 for up to 10 days. Seventy-two hours of normoxic incubation preceding hypoxic culture increased the cell viability, network formation, and size and stability of the resulting lumens compared with those completely maintained in normoxia for the same total duration. We employed different parameters of the network (e.g., total mesh area, total length, number of branches, among others) for the comparison of different oxygen treatments in the device. The differential effect of hypoxic conditions based on the maturity of the vessels may be used as an external factor to improve vascular development in vitro.

Long-Term Retinal Differentiation of Human Induced Pluripotent Stem Cells in a Continuously Perfused Microfluidic Culture Device.

Understanding how microenvironmental cues influence cellular behavior will enable development of efficient and robust pluripotent stem cell differentiation protocols. Unlike traditional cell culture dishes, microfluidic bioreactors can provide stable microenvironmental conditions by continuous medium perfusion at a controlled rate. The aim of this study is to investigate whether a microfluidic culture device could be used as a perfused platform for long-term cell culture processes such as the retinal differentiation of human induced pluripotent stem cells. The perfusion flow rate is established based on the degradation and consumption of growth factors (DKK-1, Noggin, IGF-1, and bFGF) and utilizing the Péclet number. The device's performance analyzed by qRT-PCR show improvements compared to the well-plate control as characterized by significantly higher expression of the markers Pax6, Chx10, and Crx on Day 5, Nrl on day 10, Crx, and Rhodopsin on day 21. Optimization of perfusion rate is an important operating variable in development of robust processes for differentiation cultures. Result demonstrates convective delivery of nutrients via perfusion has a significant impact upon the expression of key retinal markers. This study is the first continuously perfused long-term (21 days) retinal differentiation of hiPSCs in a microfluidic device.

Culture of hybrid spheroids composed of calcium phosphate materials and mesenchymal stem cells on an oxygen-permeable culture device to predict in vivo bone forming capability.

Three-dimensional (3-D) cell culture can better mimic physiological conditions in which cells interact with adjacent cells and the extracellular matrix than monolayer culture. We have developed a 3-D cell culture device, the Oxy chip, which can be used to generate and supply oxygen to cell spheroids to prevent hypoxia. Here, we used the Oxy chip to generate hybrid spheroids comprising calcium phosphate (CaP) particles (hydroxyapatite (HA), ß-tricalcium phosphate (ß-TCP) or octacalcium phosphate (OCP)) and mesenchymal stem cells (MSCs, C3H10T1/2 cells or D1 cells) that can be used to analyze cell differentiation mechanisms. We showed that the 3-D cell-cell and cell-material interactions and oxygenation offered by the Oxy chip promoted osteoblastic differentiation of MSCs. We also used histomorphometric analysis of hematoxylin and eosin staining, quality analyses by µCT and collagen orientation observation with picrosirius red staining in bone regeneration following implantation of three CaPs in a critical-sized defect in mouse calvaria. The in vivo bone formation capacity of the three tested CaP materials was OCP ≥ ß-TCP > HA: the newly formed bone by OCP had a structure relatively close to that of the calvaria intact bone. When MSCs were 3-D cultured with the CaP materials using the Oxy chip, the in vitro osteogenic capacity of these materials was highly similar to trends observed in vivo. The in vitro alkaline phosphatase activity of D1 cells had the highest correlation with in vivo bone volume (R = 0.900). Chemical and FTIR spectroscopic analyses confirmed that differentiation of D1 cells could be associated with amorphous calcium phosphate (ACP) precipitation concomitant with OCP hydrolysis. Taken together, hybrid spheroid cultures using the Oxy chip can be used to screen and predict bone forming potential of bone substitute materials. STATEMENT OF SIGNIFICANCE: An oxygen permeable-culture chip (Oxy chip) can be used to induce formation of cell spheroids by mesenchymal stem cells (MSCs). Use of the Oxy chip avoids hypoxia in the spheroid core and enhances MSC osteoblastic differentiation relative to conventional spheroid culture methods. The present study showed that the Oxy chip mimics the in vivo environment associated with bone formation and can be used to generate hybrid spheroids consisting of calcium phosphates and MSCs that are useful for analyzing cell differentiation mechanisms. Bone formation analysis following implantation of calcium phosphate materials in mouse calvaria defects showed positive correlation with the in vitro results. We propose that hybrid spheroids cultured on the Oxy chip can be used to screen and predict the bone forming potential of bone substitute materials.

Paper-based 3D culture device integrated with electrochemical sensor for the on-line cell viability evaluation of amyloid-beta peptide induced damage in PC12 cells.

In this communication, a paper-based 3D cell culture device integrated with electrochemical biosensor was applied to on-line monitoring of dopamine release from PC12 cell damage models induced by amyloid-beta peptide (Aß25-35) and cell intervene models protected by curcumin (Cur) and marrow mesenchymal stem cells (MSC) supernatant. The adhesion and proliferation of PC12 cells cultured on the paper scaffold was characterized by scanning electron microscopy and laser scanning confocal microscopy, which verify unique biocompatibility and 3D microarchitecture similar to human body microenvironment of paper substrate, so an artificial model simulating 3D microenvironment in vivo was constructed easily. The PC12 cells in paper-based devices consisted of four groups containing control group, Aß25-35 group, Aß25-35+Cur group and Aß25-35+MSC supernatant group. Under optimal conditions, this proposed device displayed a wide linear range from 0.05 to 1 µmol/L with a detection limit of 0.009 µmol/L (S/N = 3) and exhibited high sensitivity, good selectivity and excellent reproducibility. Furtherly, electrochemcial analysis and MTT assay gave a clue that the cell viability of Aß25-35+MSCs supernatant group was higher than that of Aß25-35+Cur group. Therefore, the detachable paper-based 3D device paves the way to a direct detection of exocytosis DA from neuron cells for on-line cell viability evaluation of neurodegenerative disease cell damage models.

Development of a 3D printed device to support long term intestinal culture as an alternative to hyperoxic chamber methods.

BACKGROUND: Most interactions between pathogenic microorganisms and their target host occur on mucosal surfaces of internal organs such as the intestine. In vitro organ culture (IVOC) provides an unique tool for studying host-pathogen interactions in a controlled environment. However, this technique requires a complex laboratory setup and specialized apparatus. In addition, issues arise when anaerobic pathogens are exposed to the hyperoxic environment required for intestinal culture. The objective of this study was to develop an accessible 3D-printed device that would allow manipulation of the gas mixture used to supply the tissue culture media separately from the gas mixture exposed to the mucosal side of explants. RESULTS: Porcine colon explants from 2 pigs were prepared (n = 20) and cultured for 0h, 8h, 18h and 24h using the device. After the culture period, explants were fixed in formalin and H&E stained sections were evaluated for histological defects of the mucosa. At 8h, 66% of samples displayed no histological abnormalities, whereas samples collected at 18h and 24h displayed progressively increasing rates of superficial epithelial erosion and epithelial metaplasia. CONCLUSIONS: The 3D-design reported here allows investigators to setup intestinal culture explants while manipulating the gas media explants are exposed to, to support tissue viability for a minimal of 8h. The amount of media necessary and tissue contamination are potential issues associated with this apparatus.

Real-time monitoring of specific oxygen uptake rates of embryonic stem cells in a microfluidic cell culture device.

Oxygen plays a key role in stem cell biology as a signaling molecule and as an indicator of cell energy metabolism. Quantification of cellular oxygen kinetics, i.e. the determination of specific oxygen uptake rates (sOURs), is routinely used to understand metabolic shifts. However current methods to determine sOUR in adherent cell cultures rely on cell sampling, which impacts on cellular phenotype. We present real-time monitoring of cell growth from phase contrast microscopy images, and of respiration using optical sensors for dissolved oxygen. Time-course data for bulk and peri-cellular oxygen concentrations obtained for Chinese hamster ovary (CHO) and mouse embryonic stem cell (mESCs) cultures successfully demonstrated this non-invasive and label-free approach. Additionally, we confirmed non-invasive detection of cellular responses to rapidly changing culture conditions by exposing the cells to mitochondrial inhibiting and uncoupling agents. For the CHO and mESCs, sOUR values between 8 and 60 amol cell(-1) s(-1) , and 5 and 35 amol cell(-1) s(-1) were obtained, respectively. These values compare favorably with literature data. The capability to monitor oxygen tensions, cell growth, and sOUR, of adherent stem cell cultures, non-invasively and in real time, will be of significant benefit for future studies in stem cell biology and stem cell-based therapies.

Polymeric inserts differing in their chemical composition as substrates for dendritic cell cultivation.

Dendritic cells (DC) contribute to immunity by presenting antigens to T cells and shape the immune response by the secretion of cytokines. Due to their immune stimulatory potential DC-based therapies are promising approaches to overcome tolerance e.g. against tumors. In order to enforce the immunogenicity of DCs, they have to be matured and activated in vitro, which requires an appropriate cell culture substrate, supporting their survival expansion and activation.Since most cell culture devices are not optimized for DC growth, it is hypothesized that polymers with certain physicochemical properties can positively influence the DC cultures. With the aim to evaluate the effects that polymers with different chemical compositions have on the survival, the activation status, and the cytokine/chemokine secretion profile of DC, their interaction with polystyrene (PS), polycarbonate (PC), poly(ether imide) (PEI), and poly(styrene-co-acrylonitrile) (PSAN)-based cell culture inserts was investigated. By using this insert system, which fits exactly into 24 well cell culture plates, effects induced from the culture dish material can be excluded. The viability of untreated DC after incubation with the different inserts was not influenced by the different inserts, whereas LPS-activated DC showed an increased survival after cultivation on PC, PS, and PSAN compared to tissue culture polystyrene (TCP). The activation status of DC estimated by the expression of CD40, CD80, CD83, CD86 and HLA-DR expression was not altered by the different inserts in untreated DC but slightly reduced when LPS-activated DC were cultivated on PC, PS, PSAN, and PEI compared to TCP. For each polymeric cell culture insert a distinct cytokine profile could be observed.Since inserts with different chemical compositions of the inserts did not substantially alter the behavior of DC all insert systems could be considered as alternative substrate. The observed increased survival on some polymers, which showed in contrast to TCP a hydrophobic surface, could be beneficial for certain applications such as T cell expansion and activation.

A novel closed cell culture device for fabrication of corneal epithelial cell sheets.

Automation technology for cell sheet-based tissue engineering would need to optimize the cell sheet fabrication process, stabilize cell sheet quality and reduce biological contamination risks. Biological contamination must be avoided in clinical settings. A closed culture system provides a solution for this. In the present study, we developed a closed culture device called a cell cartridge, to be used in a closed cell culture system for fabricating corneal epithelial cell sheets. Rabbit limbal epithelial cells were cultured on the surface of a porous membrane with 3T3 feeder cells, which are separate from the epithelial cells in the cell cartridges and in the cell-culture inserts as a control. To fabricate the stratified cell sheets, five different thicknesses of the membranes which were welded to the cell cartridge, were examined. Multilayered corneal epithelial cell sheets were fabricated in cell cartridges that were welded to a 25 µm-thick gas-permeable membrane, which was similar to the results with the cell-culture inserts. However, stratification of corneal epithelial cell sheets did not occur with cell cartridges that were welded to 100-300 µm-thick gas-permeable membranes. The fabricated cell sheets were evaluated by histological analyses to examine the expression of corneal epithelial-specific markers. Immunohistochemical analyses showed that a putative stem cell marker, p63, a corneal epithelial differentiation maker, CK3, and a barrier function marker, Claudin-1, were expressed in the appropriate position in the cell sheets. These results suggest that the cell cartridge is effective for fabricating corneal epithelial cell sheets.