MACS-W:A modified optical clearing agent for imaging 3D cell cultures

Three-dimensional(3D)cell cultures have contributed to a variety of biological research fields by filling the gap between monolayers and animal models.The modern optical sectioning microscopic methods make it possible to probe the complexity of 3D cell cultures but are limited by the inherent opaqueness.While tissue optical clearing methods have emerged as powerful tools for investigating whole-mount tissues in 3D,they often have limitations,such as being too harsh for fragile 3D cell cultures,requiring complex handling protocols,or inducing tissue deformation with shrinkage or expansion.To address this issue,we proposed a modified optical clearing method for 3D cell cultures,called MACS-W,which is simple,highly efficient,and morphology-preserving.In our evaluation of MACS-W,we found that it exhibits excellent clearing capability in just 10 min,with minimal deformation,and helps drug evaluation on tumor spheroids.In summary,MACS-W is a fast,minimally-deformative and fluorescence compatible clearing method that has the potential to be widely used in the studies of 3D cell cultures.

An Innovative Design of Incubator Structure for Cell Culture

In view of the problems of the traditional cell incubator,such as the small range of cell culture types,the inability to adjust the internal space of the incubator according to needs,and the inconvenient sampling,this study innovatively designed a cell incubator structure.It proposed a new design concept that can solve the above-mentioned shortcomings.The cell incubator after the new structural modification can adjust the internal space structure of cell culture by setting the bolt-fixed connection between the fixed plate and the vessel divider.It realizes the cultivation of various cells through refrigeration modules and heating modules.Through setting a sampling hole in the glass inner door,it is favorable for operators to take samples,making cell culture more convenient and efficient.

In Vitro Invasive Pattern of Hepatocellular Carcinoma Cell Line HCCLM9 Based on Three-dimensional Cell Culture and Quantum Dots Molecular Imaging

Summary： This study aimed to establish a new in vitro three-dimensional （3D） cell culture and use quantum dots （QDs） molecular imaging to examine the invasive behaviors of hepatocellular carcinoma （HCC） cells. Each well of the 24-well cell culture plate was cover-slipped. Matrigel diluted with se- rum-free DMEM was added and HCCLM9 cells were cultured on the Matrigel. The cell morphological and cell growth characteristics were observed by inverted microscopy and laser confocal microscopy at different culture time. Cell invasive features were monitored by QDs-based real-time molecular imaging techniques. The results showed that on this 3D cell culture platform, HCCLM9 cells exhibited typical multi-step invasive behaviors, including reversion of cell senescence, active focal proliferation and dominant clones invasion. During the process, cells under 3D cell culture showed biological behaviors of spatio-temporal characteristics. Cells first merged on the surface of matrix, then gradually infiltrated and migrated into deep part of matrix, presenting polygonal morphology with stretched protrusions, forming tubular, annular and even network structure, which suggested that HCC cells have the morpho- logical basis for vasculogenic mimicry. In addition, small cell clones with their edges well-circumscribed in early stage, progressed into a large irregular clone with ill-defined edge, while the other cells developed invadopodia. And QDs probing showed MT1-MMP was strongly expressed in the invadopodia. These findings indicate that a novel 3D cell culture platform has been successfully estab- lished, which can mimic the in vivo tumor microenvironment, and when combined with QDs-based mo- lecular imaging, it can help to better investigate the invasive behaviors of HCC cells.

Biological Characteristics of Human Bone Marrow Mesenchymal Stem Cell Cultured in Vitro

Some biological characteristics of human bone marrow mesenchymal stem cells (MSCs) cultured in vitro were observed. hMSCs were isolated from bone marrow and purified by density gradient centrifugation method, and then cultured in vitro. The proliferation and growth characteristics of hMSCs were observed in primary and passage culture. MSCs of passage 3 were examined for the purify by positive rate of CD29 and CD44 through flow cytometry. Human bone marrow MSCs showed active proliferation capacity in vitro. The purify of MSCs separated by our method was higher than 90 %. It was concluded that hMSCs have been successfully cultured and expanded effectively. It provided a foundation for further investigation and application of MSCs.

Effect of elicitors, precursors and metabolic inhibitors on paclitaxel production by Taxus cuspidata cell culture

In this study, using Taxus cuspidata as a raw material, we obtained stable high-yielding cell lines by subculturing and quantified paclitaxel content using ultra- sonic extraction combined with TLC-UV spectrophotom- etry. In single factor and multiple factors tests to optimize design and study the effects of elicitors, precursors, and metabolic inhibitors on paclitaxel production by Taxus cuspidata cells, paclitaxel production reached 4.32 mg/L when 100 μmol/L methyl jasmonate, 20 mg/L salicylic acid, 400 mg/L phenylalanine and 2 mg/L gibberellin （GA3） were added to the culture medium of suspension cells. When adding metabolic adjustment factors on the 7th day of culture, extra- and intracellular paclitaxel production was the highest at 4.855 mg/L, paclitaxel release rate was 10.48 %, fresh mass and paclitaxel production of cell increased, respectively, by 6.08 and 11.57 %. By controlling the anabolism of paclitaxel, paclitaxel yield was significantly improved.

Glutathione peroxidase activity in cell cultures from different regions of human epididymis

Aim: To study the secretory activity and androgen regulation of glutathione peroxidase (GPx) in epithelial cell cultures from human epididymis. Methods: Tissue was obtained from patients undergoing therapeutic orchidectomy for prostatic cancer. Epithelial cell cultures were obtained from the caput, corpus and cauda epididymides. Enzymatic activity was measured in conditioned media by colorimetric methods in absence or presence of 1, 10 or 100 nrnoI.L^(-1) testosterone. The effect of 1 μmol.L^(-1) flutamide was also evaluated. Results: GPx activity was higher in cultures from corpus and cauda than caput epididymidis. The presence of different concentrations of testosterone increase enzyme activity in cell cultures from all epididymal regions. Addition of flutamide reverses the androgen dependent increase of GPx activity. Conclusion: GPx activity is secreted from human epididymal cells in a region dependent manner and is regulated by androgens.

PRIMARY CELL CULTURE FOR EMBRYONIC CARDIAC MYOCYTES OF MEXICAN AXOLOTL AND DISTRIBUTION OF DESMIN AND VIMENTIN

Desmin and vimentin are major components of intermediate filament proteins in cardiac myocytes. We developed a primary cell culture method for cardiac myocytes of axolotl embryos. Cardiac myocytes of embryonic stage 39 were cultured for 1-14 days. Myocytes showed spontaneous contractions (15-30 beats/min) after 48-72 hours in culture, round shape and large irregular projections. Desmin and vimentin were observed in the cultured myocytes by means of immunofluorescent staining in combination with immunofluorescent microscopy. Immunofluorescent staining of the cultured cardiac myocytes after different lengths of time in culture(3,6,9 days) showed that vimentin staining was stronger than desmin staining during the early stages of culture (3 days). The myocytes exhibited various forms of staining, including parallel lines and interconnected networks. Some lines showed regular striation; most of the myofibrils were arranged in parallel arrays along the cell's long axis. Both desmin and vimentin in the cell appeared to encirele the Z lines and to link myofibrils laterally at the Z lines.

Microfabrication of Bubbular Cavities in PDMS for Cell Sorting and Microcell Culture Applications

We describe a novel technique, low surface energy Gas Expansion Molding （GEM）, to fabricate microbubble arrays in polydimethylsiloxane （PDMS） which are incorporated into parallel plate flow chambers and tested in cell sorting and microcell cuTture applications. This architecture confers several operational advantages that distinguish this technology approach from currently used methods. Herein we describe the GEM process and the parameters that are used to control microbubble formation and a Vacuum-Assisted Coating （VAC） process developed to selectively and spatially alter the PDMS surface chemistry in the wells and on the microchannel surface. We describe results from microflow image visualization studies conducted to investigate fluid streams above and within microbubble wells and conclude with a discussion of cell culture studies in PDMS.

Cell culture isolation can miss the laboratory diagnosis of HSV ocular infection

AIMWe compared polymerase chain reaction (PCR) to cell culture isolation for the laboratory diagnosis of ocular herpes simplex virus (HSV) disease.

ERK phosphorylation functions in invadopodia formation in tongue cancer cells in a novel silicate fibre-based 3D cell culture system

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 Cellbedwith a structure resembling the loose connective tissue morphology in a novel 3 D culture system. We confirmed that the 3 D system using CellbedTMaccurately mimicked cancer cell morphology in vivo. Furthermore, cell projections were much more apparent in 3 D-cultured tongue cancer cell lines than in 2 D cultures. Typically, under conventional 2 D culture conditions, F-actin and cortactin are colocalized in the form of puncta within cells.However, in the 3 D-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 3 D 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 3 D 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.

Schwann cell cultures from human fetal dorsal root ganglia

BACKGROUND： Previous studies have used many methods for in vitro Schwann cells （SCs） cultures and purification, such as single cell suspension and cytosine arabinoside. However, it has been difficult to obtain sufficient cellular density, and the procedures have been quite tedious. OBJECTIVE： To investigate the feasibility of culturing high-density SCs using fetal human dorsal root ganglion tissue explants. DESIGN, TIME AND SETTING： Cell culture and irnmunohistochernistry were performed at the Central Laboratory of Kunrning General Hospital of Chinese PLA between March 2001 and October 2008. MATERIALS： Culture media containing 10% fetal bovine serum, as well as 0.2% collagenase and 0.25% trypsin were purchased from Gibco, USA; mouse anti-human S-100 monoclonal antibody and goat anti-mouse IgG labeled with horseradish peroxidase were provided by Beijing Institute of Biological Products, China. METHODS： Primarily cultured SCs were dissociated from dorsal root ganglia of human aborted fetuses at 4 6 months pregnancy. Following removal of the dorsal root ganglion perineurium, the ganglia were dissected into tiny pieces and digested with 0.2% collagenase and 0.25% trypsin （volume ratio 1：1）, then explanted and cultured. SC purification was performed with 5 rnL 10% fetal bovine serum added to the culture media, followed by differential adhesion. MAIN OUTCOME MEASURES： SCs morphology was observed under inverted phase contrast light microscopy. SC purity was evaluated according to percentage of S-100 immunostained cells. RESULTS： SCs were primarily cultured for 5 6 days and then subcultured for 4 5 passages. The highly enriched SC population reached 〉 95% purity and presented with normal morphology. CONCLUSION： A high purity of SCs was obtained with culture methods using human fetal dorsal root ganglion tissue explants.

Coscinium fenestratum: Callus and Suspension Cell Culture of the Endangered Medicinal Plant Using Vermicompost Extract and Coelomic Fluid as Plant Tissue Culture Media

In vitro tissue culture of hard woody, endangered, medicinal plant Coscinium fenestratum is most challenging to plant tissue culturists. In the present study, petiole and leaf explants of Coscinium fenestratum were induced to form callus when cultured on vermicompost extract media along with coelomic fluid. Suspension medium was developed using vermicompost extract and coelomic fluid in 3:1 ratio. Phytochemical analysis of the alkaloid berberine was confirmed from callus, suspension cell culture and suspension medium by Thin Layer Chromatography and High Performance Liquid Chromatography. Vermicompost and its extracts with coelomic fluid have shown maximum (100 per cent) response of callus induction. Callus mass enlarged with increasing concentration of coelomic fluid and callus growth was assessed from the biomass. Incubation of culture tubes in dark supported callus development significantly. The Rf value of 0.36 confirmed the presence of berberine by Thin Layer Chromatography. Qualitative analysis confirmed the presence of alkaloid berberine with the retention time of 2.8 minutes similar to that of standard reference sample from Sigma chemicals, USA. The suspension medium turned deep yellow because of the release of the alkaloid. Vermicompost and its extracts along with coelomic fluid have shown the economical approach for micropropagation of economically and medicinally important plants.

Summary of the Frontier Introduction of Preparation of Secondary Metabolites in Plant Cell Culture

Plant cell culture technology is a technology that applies the research results of cell engineering to produce plant biological products at the cellular level.In recent years,the secondary metabolites of plants have attracted more and more attention.The use of plant cell culture technology is a fast and efficient method of producing secondary metabolites.

Three-dimensional cell culture systems as an in vitro platform for cancer and stem cell modeling

Three-dimensional(3D)culture systems are becoming increasingly popular due to their ability to mimic tissue-like structures more effectively than the monolayer cultures.In cancer and stem cell research,the natural cell characteristics and architectures are closely mimicked by the 3D cell models.Thus,the 3D cell cultures are promising and suitable systems for various proposes,ranging from disease modeling to drug target identification as well as potential therapeutic substances that may transform our lives.This review provides a comprehensive compendium of recent advancements in culturing cells,in particular cancer and stem cells,using 3D culture techniques.The major approaches highlighted here include cell spheroids,hydrogel embedding,bioreactors,scaffolds,and bioprinting.In addition,the progress of employing 3D cell culture systems as a platform for cancer and stem cell research was addressed,and the prominent studies of 3D cell culture systems were discussed.

Microfluidic three-dimensional cell culture of stem cells for high-throughput analysis

Although the recent advances in stem cell engineering have gained a great deal of attention due to their high potential in clinical research,the applicability of stem cells for preclinical screening in the drug discovery process is still challenging due to difficulties in controlling the stem cell microenvironment and the limited availability of high-throughput systems.Recently,researchers have been actively developing and evaluating three-dimensional(3D)cell culture-based platforms using microfluidic technologies,such as organ-on-a-chip and organoid-on-a-chip platforms,and they have achieved promising breakthroughs in stem cell engineering.In this review,we start with a comprehensive discussion on the importance of microfluidic 3D cell culture techniques in stem cell research and their technical strategies in the field of drug discovery.In a subsequent section,we discuss microfluidic 3D cell culture techniques for high-throughput analysis for use in stem cell research.In addition,some potential and practical applications of organ-on-a-chip or organoid-on-a-chip platforms using stem cells as drug screening and disease models are highlighted.

Biotransformation of 14-Deacetoxy-13-oxo sinenxan A by Ginkgo Cell Cultures

Deacetoxy-13-oxo sinenxan A (1) was converted to 9a-hydroxy-13-oxo-2a, 5a, 10b-triacetoxy-4(20),11-taxadiene (2) and 10b-hydroxy-13-oxo-2a,5a,9a-triacetoxy- 4(20), 11- taxadiene (3) by Ginkgo cell suspension cultures in 45% and 15% yields, respectively.

Artificial meat? Feasible approach based on the experience from cell culture studies

This short review is to list pros and cons which are based on the literature and personal experience in cell culture studies related to possible commercial production of artificial meat as functional food. The general view of muscle composition and determinants of meat quality are shortly described. Principles of muscle cell propagation in culture and mutual relationships between different cell types present in this organ are briefly discussed. Additionally, the effects of some cytokines and growth factors for muscle cell growth and muscle tissue development are indicated. Finally, conclusion remarks related to detrimental consequences of meat production to natural environment as well as personal opinion of author on the prospects of artificial meat production are declared.

Total Phenolic Content and Antioxidant Activity of Standardized Extracts from Leaves and Cell Cultures of Three Callistemon Species

A comparative study was carried out with ethanolic (80%) extracts from leaves and cell cultures of three Callistemon species, namely C. lanceolatus (CL), C. viridiflorous (CV), and C. comboynensis (CC). Cell suspensions of the three species were grown in liquid Murashige and Skoog (MS) medium (100 ml) supplemented with 0.9 mg·g-1 kinetin in combination with 1.1 mg·g-1 NAA. The CL leaf extract was standardized to contain the highest amount of phenolics (104 ± 2.0 mg·g-1), followed by CC (95.8 ± 1.2 mg·g-1) and CV (79.8 ± 4.6 mg·g-1). On the other hand, cell cultures of CV contained more phenolics (14.9 ± 0.6 mg·g-1) than those of the other two species, CL and CC, which contained 12.2 ± 0.16 and 9.12 ± 0.16 mg·g-1, respectively. Nevertheless, CV leaf extract exhibited the highest antioxidant activity (91.4% ± 0.4%) at a concentration of 1000 μg·ml-1, comparable to 100 μg·ml-1 gallic acid (90.8% ± 1.5%).

Developments in cell culture systems for human pluripotent stem cells

Human pluripotent stem cells(hPSCs)are important resources for cell-based therapies and pharmaceutical applications.In order to realize the potential of hPSCs,it is critical to develop suitable technologies required for specific applications.Most hPSC technologies depend on cell culture,and are critically influenced by culture medium composition,extracellular matrices,handling methods,and culture platforms.This review summarizes the major technological advances in hPSC culture,and highlights the opportunities and challenges in future therapeutic applications.