OBJECTIVE: This study optimizes three-dimensional (3D) culture conditions of HepG2 using response surface methodology (RSM) based on the VitroGel system to facilitate the cell model in vitro for liver tissues. METHOD: HepG2 cell was 3D cultured on the VitroGel system. Cell viability was detected using Cell Counting Kit-8 (CCK-8) assay of HepG2 lived cell numbers. The proliferation of HepG2 cell and clustering performance was measured via fluorescence staining test. Albumin concentration in the culture medium supernatant as an index of HepG2 cell biological function was measured with ELISA kit. Independent factor tests were conducted with three key factors: inoculated cell concentration, cultured time, and dilution degree of the hydrogel. The preliminary results of independent factor tests were used to determine the levels of factors for RSM. RESULT: The selected optimal culture conditions are as follows: concentration of inoculated cells was 4.44 × 10 ⁵/mL, culture time was 4.86 days, and hydrogel dilution degree was 1:2.23. The result shows that under optimal conditions, the predicted optical density (OD) value of cell viability was 3.10 and measured 2.978 with a relative error of 3.94%. CONCLUSION This study serves as a reference for the 3D HepG2 culture and constructs liver tissues in vitro. Additionally, it provides the foundation for repeated dose high-throughput toxicity studies and other scientific research work.
Albumins ; Cell Culture Techniques/methods* ; Hep G2 Cells ; Humans ; Hydrogels

PURPOSE: The incidence of acute lung injury (ALI) in severe trauma patients is 48% and the mortality rate following acute respiratory distress syndrome evolved from ALI is up to 68.5%. Alveolar epithelial type 1 cells (AEC1s) and type 2 cells (AEC2s) are the key cells in the repair of injured lungs as well as fetal lung development. Therefore, the purification and culture of AEC1s and AEC2s play an important role in the research of repair and regeneration of lung tissue. METHODS: Sprague-Dawley rats (3-4 weeks, 120-150 g) were purchased for experiment. Dispase and DNase I were jointly used to digest lung tissue to obtain a single-cell suspension of whole lung cells, and then magnetic bead cell sorting was performed to isolate T1α positive cells as AEC1s from the single-cell suspension by using polyclonal rabbit anti-T1a (a specific AEC1s membrane protein) antibodies combined with anti-rabbit IgG microbeads. Afterwards, alveolar epithelial cell membrane marker protein EpCAM was designed as a key label to sort AEC2s from the remaining T1α-neg cells by another positive immunomagnetic selection using monoclonal mouse anti-EpCAM antibodies and anti-mouse IgG microbeads. Cell purity was identified by immunofluorescence staining and flow cytometry. RESULTS: The purity of AEC1s and AEC2s was 88.3% ± 3.8% and 92.6% ± 2.7%, respectively. The cell growth was observed as follows: AEC1s stretched within the 12-16 h, but the cells proliferated slowly; while AEC2s began to stretch after 24 h and proliferated rapidly from the 2nd day and began to differentiate after 3 days. CONCLUSION AEC1s and AEC2s sorted by this method have high purity and good viability. Therefore, our method provides a new approach for the isolation and culture of AEC1s and AEC2s as well as a new strategy for the research of lung repair and regeneration.
Alveolar Epithelial Cells/cytology* ; Animals ; Cell Culture Techniques ; Cell Separation/methods* ; Immunoglobulin G/metabolism* ; Lung ; Magnetic Phenomena ; Rats ; Rats, Sprague-Dawley

Bioreactors have been central in monoclonal antibodies and vaccines manufacturing by mammalian cells in suspension culture. Numerical simulation of five impeller combinations in a stirred bioreactor was conducted, and characteristics of velocity vectors, distributions of gas hold-up, distributions of shear rate in the bioreactor using 5 impeller combinations were numerically elucidated. In addition, genetically engineered CHO cells were cultivated in bioreactor installed with 5 different impeller combinations in fed-batch culture mode. The cell growth and antibody level were directly related to the maximum shear rate in the bioreactor, and the highest viable cell density and the peak antibody level were achieved in FBMI3 impeller combination, indicating that CHO cells are sensitive to shear force produced by impeller movement when cells were cultivated in bioreactor at large scale, and the maximum shear rate would play key roles in scaling-up of bioreactor at industrial scale.
Animals ; Batch Cell Culture Techniques ; Bioreactors ; standards ; CHO Cells ; Cell Count ; Computer Simulation ; Cricetinae ; Cricetulus ; Industrial Microbiology ; instrumentation ; methods

In this study, we improved the culture method of mouse hippocampal primary microglia to obtain hippocampal ramified microglia with high activity and purity, which were resemble to the resting status of normal microglia in healthy brain in vivo. Hippocampal tissue was excised from 2-4-week-old SPF C57BL/6J mice and cut into pieces after PBS perfusion, and then manually dissociated into the single-cell suspension by using Miltenyi Biotec's Adult Brain Dissociation Kit. The tissue fragments such as myelin in the supernatant were removed by debris removal solution in the kit. The cell suspension was incubated with CD11b immunomagnetic beads for 15 min at 4 °C. To obtain high-purity microglia, we used two consecutive cell-sorting steps by magnetic activated cell sorting (MACS). After centrifugation, the cells were resuspended and seeded in a 24-well culture plate. The primary microglia were cultured with complete medium (CM) or TIC medium (a serum-free medium with TGF-β, IL-34 and cholesterol as the main nutritional components) for 4 days, and then were used for further experiments. The results showed that: (1) The cell viability was (56.03 ± 2.10)% by manual dissociation of hippocampus; (2) Compared with immunopanning, two-step MACS sorting allowed for efficient enrichment of microglia with higher purity of (86.20 ± 0.68)%; (3) After being incubated in TIC medium for 4 d, microglia exhibited branching, quiescent morphology; (4) The results from qRT-PCR assay showed that the levels of TNF-α, IL-1β and CCL2 mRNA in TIC cultured-microglia were similar to freshly isolated microglia, while those were much higher in CM cultured-microglia after incubation for 4 d and 7 d (P < 0.05). Taken together, compared to the conventional approaches, this modified protocol of mouse hippocampal primary microglia culture by using MACS and TIC medium enables the increased yield and purity of microglia in the quiescent state, which is similar to normal ramified microglia in healthy brain in vivo.
Animals ; Cell Culture Techniques ; methods ; Cell Separation ; methods ; Cells, Cultured ; Hippocampus ; Magnetics ; Mice ; Mice, Inbred C57BL ; Microglia ; cytology

Emerging antibiotic resistance is a major global health threat. The analysis of nucleic acid sequences linked to susceptibility phenotypes facilitates the study of genetic antibiotic resistance determinants to inform molecular diagnostics and drug development. We collected genetic data (11,087 newly-sequenced whole genomes) and culture-based resistance profiles (10,991 out of the 11,087 isolates comprehensively tested against 22 antibiotics in total) of clinical isolates including 18 main species spanning a time period of 30 years. Species and drug specific resistance patterns were observed including increased resistance rates for Acinetobacter baumannii to carbapenems and for Escherichia coli to fluoroquinolones. Species-level pan-genomes were constructed to reflect the genetic repertoire of the respective species, including conserved essential genes and known resistance factors. Integrating phenotypes and genotypes through species-level pan-genomes allowed to infer gene-drug resistance associations using statistical testing. The isolate collection and the analysis results have been integrated into GEAR-base, a resource available for academic research use free of charge at https://gear-base.com.
Acinetobacter baumannii ; genetics ; isolation & purification ; Bacteria ; genetics ; isolation & purification ; Cell Culture Techniques ; methods ; Drug Resistance, Microbial ; genetics ; Escherichia coli ; genetics ; isolation & purification ; Genome, Bacterial ; Genotype ; Humans ; Internet ; Microbial Sensitivity Tests ; Phenotype ; Whole Genome Sequencing

PURPOSE: The reaction of cells to a titanium implant depends on the surface characteristics of the implant which are affected by decontamination. The aim of this study was to evaluate the cytocompatibility of titanium disks treated with various decontamination methods, using salivary bacterial contamination with dental pellicle formation as an in vitro model. METHODS: Sand-blasted and acid-etched (SA) titanium disks were used. Three control groups (pristine SA disks [SA group]; salivary pellicle-coated SA disks [pellicle group]; and biofilm-coated, untreated SA disks [NT group]) were not subjected to any decontamination treatments. Decontamination of the biofilm-coated disks was performed by 14 methods, including ultrasonic instruments, rotating instruments, an air-powder abrasive system, a laser, and chemical agents. MG63 cells were cultured in the presence of the treated disks. Cell proliferation assays were performed on days 2 and 5 of cell culture, and cell morphology was analyzed by immunofluorescence and scanning electron microscopy (SEM). A vascular endothelial growth factor (VEGF) assay was performed on day 5 of culture. RESULTS: The cell proliferation assay revealed that all decontaminated disks, except for the 2 groups treated using a plastic tip, showed significantly less cell proliferation than the SA group. The immunofluorescence and SEM analyses revealed that most groups showed comparable cell density, with the exception of the NT group, in which the cell density was lower and bacterial residue was observed. Furthermore, the cells grown with tetracycline-treated titanium disks showed significantly lower VEGF production than those in the SA group. CONCLUSIONS: None of the decontamination methods resulted in cytocompatibility similar to that of pristine SA titanium. However, many methods caused improvement in the biocompatibility of the titanium disks in comparison with the biofilm-coated, untreated titanium disks. This suggests that decontamination is indispensable for the treatment of peri-implantitis, even if the original biocompatibility cannot be restored.
Biocompatible Materials ; Cell Count ; Cell Culture Techniques ; Cell Proliferation ; Decontamination ; Dental Implants ; Dental Pellicle ; Fluorescent Antibody Technique ; In Vitro Techniques ; Methods ; Microscopy, Electron, Scanning ; Peri-Implantitis ; Plastics ; Titanium ; Ultrasonics ; Vascular Endothelial Growth Factor A

To screen for additional treatment targets against tongue cancer, we evaluated the contributions of extracellular signal-related kinase (ERK), AKT and ezrin in cancer development. Immunohistochemical staining showed that ERK and ezrin expressions were significantly higher in invasive squamous cell carcinoma than in carcinoma in situ. To investigate the roles of ERK and ezrin in cancer development, we used the non-woven silica fibre sheet Cellbed with a structure resembling the loose connective tissue morphology in a novel 3D culture system. We confirmed that the 3D system using Cellbed accurately mimicked cancer cell morphology in vivo. Furthermore, cell projections were much more apparent in 3D-cultured tongue cancer cell lines than in 2D cultures. Typically, under conventional 2D culture conditions, F-actin and cortactin are colocalized in the form of puncta within cells. However, in the 3D-cultured cells, colocalization was mainly observed at the cell margins, including the projections. Projections containing F-actin and cortactin colocalization were predicted to be invadopodia. Although suppressing ezrin expression with small interfering RNA transfection caused no marked changes in morphology, cell projection formation was decreased, and the tumour thickness in vertical sections after 3D culture was markedly decreased after suppressing ERK activity because both the invasion ability and proliferation were inhibited. An association between cortactin activation as well as ERK activity and invadopodia formation was detected. Our novel 3D culture systems using Cellbed™ are simple and useful for in vitro studies before conducting animal experiments. ERK contributes to tongue cancer development by increasing both cancer cell proliferation and migration via cortactin activation.
Carcinoma in Situ ; metabolism ; pathology ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Cell Culture Techniques ; methods ; Cell Movement ; Cell Proliferation ; Cytoskeletal Proteins ; metabolism ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Humans ; Neoplasm Invasiveness ; pathology ; Phosphorylation ; Podosomes ; pathology ; Proto-Oncogene Proteins c-akt ; metabolism ; Silicon Dioxide ; Tongue Neoplasms ; metabolism ; pathology ; Tumor Cells, Cultured

The laboratory-based diagnosis of viral infection has been evolving over the years, to increase objectivity, accuracy, and sensitivity via the continuous development of various technologies. Cell culture, which is one of the methods used for the diagnosis of viral infection, is a “gold-standard” approach; however, it is time consuming and is associated with a high risk of contamination. To overcome these shortcomings, molecular biology methods, such as conventional polymerase chain reaction (cPCR), real-time PCR, and sequencing, have been used recently for virus diagnosis. Realtime PCR has higher accuracy and sensitivity compared with cPCR. Moreover, realtime PCR can quantify viral nucleic acids by confirming the amplification using the threshold cycle, which is the initial amplification point. Real-time PCR applications for the detection of various types of viruses in clinical settings should be based on the use of appropriate samples, nucleic acid extraction according to virus characteristics, and selection of diagnostic methods using sensitivity and specificity targets. In addition, the implementation of real-time PCR requires to evaluate the performance of the test protocol by measuring sensitivity, specificity, accuracy, and reproducibility. The verified real-time PCR method is an easy, fast, and accurate method for monitoring the diagnosis and treatment outcomes in a clinical setting. In this review, we summarize the characteristics of the typical diagnostic methods for viral infection, especially of the advanced real-time PCR method, to detect human pathogenic viruses.
Cell Culture Techniques ; Diagnosis* ; Humans ; Methods ; Molecular Biology ; Nucleic Acids ; Polymerase Chain Reaction ; Real-Time Polymerase Chain Reaction* ; Sensitivity and Specificity

The optimal cell culture method of autologous oral mucosal epithelial cell sheet is not well established for a safe transplantation on to the patients' ocular surface. Animal serum and 3T3 mouse feeder cells are currently being used to stimulate the growth of the epithelial cells. However, the use of animal compounds can have potential side effects for the patient after transplantation of the engineered cell sheet. In the present study, we focused on engineering a rabbit oral mucosal epithelial cell sheet without 3T3 mouse feeder cells using a mix of Dulbecco's Modified Eagle Medium/Bronchial Epithelial Cell Growth Medium culture media (DMEM/BEGM). Autologous oral mucosal epithelial cell sheets, engineered with DMEM/BEGM feeder cell free culture media, were compared to those cultured in presence of serum and feeder cells. Using a DMEM/BEGM mix culture media, feeder cell free culture condition, autologous oral mucosal epithelial cells reached confluence and formed a multilayered sheet. The phenotype of engineered cell sheets cultured with DMEM/BEGM were characterized and compared to those cultured with serum and feeder. Hematoxylin and eosin staining showed the formation of a similar stratified multilayer cell sheets, in both culture conditions. The expression of deltaN-p63, ABCG2, PCNA, E-cadherin, Beta-catenin, CK3, CK4, CK13, Muc5AC, was similar in both culture conditions. We demonstrated that rabbit autologous oral mucosal epithelial cell sheet can be engineered, in feeder cell free conditions. The use of the DMEM/BEGM culture media to engineer culture autologous oral mucosa epithelial cell sheet will help to identify key factors involved in the growth and differentiation of oral mucosal epithelial cells.
Animals ; beta Catenin ; Cadherins ; Cell Culture Techniques ; Culture Media ; Eagles ; Eosine Yellowish-(YS) ; Epithelial Cells* ; Feeder Cells* ; Hematoxylin ; Humans ; Methods ; Mice ; Mouth Mucosa* ; Phenotype ; Proliferating Cell Nuclear Antigen

BACKGROUND AND OBJECTIVES: Salivary hypofunction is one of the common side effects after radioiodine therapy, and its pathophysiology is salivary ductal stenosis resulting from ductal cell injury. This study aimed to develop the functional culture environment of human parotid gland ductal cells in in vitro three-dimensional perfusion culture system. MATERIALS AND METHODS: We compared plastic dish culture method and three-dimensional culture system containing Matrigel and nanofiber. Morphogenesis of reconstituted salivary structures was assessed by histomorphometry. Functional characteristics were assessed by immunohistochemistry and reverse transcription polymerase chain reaction (aquaporin 5, CK7, CK18, connexin 43, and p21). In addition, we designed the media perfusion culture system and identified higher rate of cell proliferation and expression of connexin 43 in perfusion system comparing to dish. RESULTS: Human parotid ductal cells were well proliferated with the ductal cell characters under environment with Matrigel. In the presence of Matrigel, aquaporin 5, CK18 and connexin 43 were more expressed than 2D dish and 3D nanofiber setting. In the media perfusion culture system, ductal cells in 3D culture media showed higher cells count and connexin 43 expression compared to 2D dish. CONCLUSION: This in vitro ductal cell perfusion culture system using Matrigel could be used to study for radioiodine induced sialadenitis model in vivo.
Aquaporin 5 ; Cell Proliferation ; Connexin 43 ; Constriction, Pathologic ; Culture Media ; Humans ; Immunohistochemistry ; In Vitro Techniques ; Methods ; Morphogenesis ; Nanofibers ; Parotid Gland ; Perfusion ; Plastics ; Polymerase Chain Reaction ; Reverse Transcription ; Salivary Ducts ; Salivary Glands ; Sialadenitis ; Thyroid Neoplasms