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.

3D Collagen Gels:A Promising Platform for Dendritic Cell Culture in Biomaterials Research

The three-dimensional(3D)cell culture system has garnered significant attention in recent years as a means of studying cell behavior and tissue development,as opposed to traditional two-dimensional cultures.These systems can induce specific cell reactions,promote specific tissue functions,and serve as valuable tools for research in tissue engineering,regenerative medicine,and drug discovery.This paper discusses current developments in the field of three-dimensional cell culture and the potential applications of 3D type 1 collagen gels to enhance the growth and maturation of dendritic cells.

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.

Chemical Constituents of the Suspension Cell Cultures of Maytenus hookeri

Suspension cell cultures of Maytenus hookeri Loos. (Celastraceae) in SH media were established from the calli induced from the leaves and young steins of M. hookeri on MS media with the supplement of 2 mg/L 2,4-D and 0.1 mg/L KIN (kinetin). Ethyl acetate extract of the cultures showed inhibitory activities against Penicillium avellaneum UC-4376 which was sensitive to maytansinoids. Exhaustive isolation of natural products from a large scale of suspension cell cultures did not yield maytansine instead of affording nine compounds including one novel triterpenoid, named 2, 3-diacetoxyl maytenusone (1), and eight known ones including squalene (2), beta-sitosterol (3), 2', 3', 4-triacetyl-sitoindoside I (4), salaspermic acid (5), maytenonic acid (6), 2alpha-hydroxy-maytenonic acid (7), 6, 11,12-trihydroxy-8, 11, 13-abietrien-7-one (8) and 11, 12-dihydroxy-8, 11, 13-abietatrien-7-one (9) elucidated on the basis of 1D and 2D NMR data. The H-1-NMR and C-13-NMR assignments were made for 1, 5, 6 and 7, while the C-13-NMR assignments for 5 and 6 were revised. The chemical results suggested that the suspension cell cultures of M. hookeri did not produce maytansinoids under the reported experiment conditions.

Detection of Mycoplasma Contamination in Animal Cell Cultures

[ Objective ] To develop a rapid efficient method for detecting mycoplasma contamination in cell cultures. [ Method] A pair of primers was designed according to two highly conserved nucleotide sequences of the 16S RNA from six kinds of mycoplasma that commonly contaminated cells. Then the mycoplasma contamination of 25 cell samples was defected by PCR and DNA fluorescence staining. EResultl When these cell samples were detected by DNA fluorescence staining, the positive rate and probable positive rate were respectively 24% and 16%. And when they were detected by PCR, the positive rate was 36%. [ Condusion] The PCR method is more sensitive and specific than the DNA fluorescence staining, and combining these two methods is the optimal way to detect mycoplasma contamination in cell cultures.

Influence of Bioelectret Materials on cell Culture

The aim of this work is to study the effects of the electret property of natural biopolymers (such as collagen) on tissue repair. Type I collagen was prepared from pigskin,and polarized by using the TSDC (thermally stimulated depolarization current) technique. The polarized materials are used for cell culture. and then the cell growth curve is drawn. It was found that the polarized biomaterials accelerated cell differentiation. which indicates that they may be applied in the biomedical field.

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.

Evaluation of diffusion in gel entrapment cell culture within hollow fibers

AIM: To investigate diffusion in mammalian cell culture by gel entrapment within hollow fibers. METHODS: Freshly isolated rat hepatocytes or human oral epidermoid carcinoma (KB) cells were entrapped in type I collagen solutions and statically cultured inside microporous and ultrafiltration hollow fibers. During the culture time collagen gel contraction, cell viability and specific function were assessed. Effective diffusion coefficients of glucose in cell-matrix gels were determined by lag time analysis in a diffusion cell. RESULTS: Significant gel contractions occurred in the collagen gels by entrapment of either viable hepatocytes or KB cells. And the gel contraction caused a significant reduction on effective diffusion coefficient of glucose. The cell viability assay of both hepatocytes and KB cells statically cultured in hollow fibers by collagen entrapment further confirmed the existence of the inhibited mass transfer by diffusion. Urea was secreted about 50% more by hepatocytes entrapped in hollow fibers with pore size of 0.1 μm than that in hollow fibers with MWCO of 100 ku. CONCLUSION: Cell-matrix gel and membrane pore size are the two factors relevant to the limited mass transfer by diffusion in such gel entrapment of mammalian cell culture.

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.

Complete Genomic Sequence of Transmissible Gastroenteritis Virus TS and 3' End Sequence Characterization Following Cell Culture

The complete genome sequence of transmissible Gastroenteritis virus (TGEV) strain TS, previously isolated from Gansu province, was cloned and compared with published sequence data from other TGEV strains. Phylogenetic tree analysis based on the amino acid and nucleotide sequences of the S gene showed that the TGEV strains were divided into 3 clusters. TGEV TS showed a close evolutionary relationship to the American Miller cluster but had a 5' non-translated region (NTR) sequence closely related to the American Purdue cluster. Continued culture in different cell types indicated that TGEV TS virulence could be attenuated after fifty passages in Porcine kidney (PK-15) cells, and that the Porcine kidney cell line IB-RS-2 (IBRS) was not suitable for culture of the TGEV strain TS.

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.

Increased production of recombinant prourokinase with porous microcarrier cell culture by periodic pressure oscillation in a stirred tank reactor

An rCHO cell line expressing recombinant human prourokinase （pro-UK） at the level of 5μg/ 10^6cells/d was cultivated on Cytopore cellulose porous microcarriers in a 7.5L Biostat CT stirred tank reactor. A periodic pressure oscillation of 0.04 MPa and 0.04 Hz was adopted to introduce a physical stimulus on the rCHO cells and to improve mass transfer characteristic between cells and medium in the process of porous microcarrier CHO cell culture. Compared to constant pressure culture, the oscillation culture didn＇t influence specific cell growth rate significantly, but could enhance the pro-UK specific production by 10% - 40%, and reduce production of lactate by 10% - 30%. In the perfusion culture of recombinant CHO cell with serum-free medium for 67 days, cell density could reach 2.64×10^7/ml, the maximal prourokinase concentration in harvested supernatant was about 118mg/L, a total of 21.1 grams of prourokinase was produced in 313 liters of supernatant. In conclusion, the perfusion cell culture with periodic pressure oscillation can enhance the production of recombinant protein and increase the reactor specific productivity.

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.

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.

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.

Effects of medium nutrition on cell growth and isocamptothecin A and B production by suspension cell culture of Camptotheca acuminata

The effects of initial sucrose concentration, nitrate to ammonium ratio, total N concentration and phosphate concentration in medium on cell growth and isocamptothecin A and B synthesis by suspension call culture of Camptotheca acuminata were investigated in 250 mL shake flasks. 30 g L^-1 sucrose concentration was beneficial to secondary metabolites synthesis. The cell growth and metabolites synthesis were also affected by the ratio of NO3^-/NH4^＋ , and nitrate was tavourable for cell growth. The maximum dry weight was achieved when nitrate was used as the sole N souree. The effect of total initial N on the cell cultures was also investigated with NO3^-/NH4^＋ ratio of 1 ： 2. The final dry cell weight was similar throughout culture period and 50 mM initial N was favourable for secondary metabolite synthesis. 50 mM initial phosphate concentration facilitated both cell growth and secondary metabolites synthesis.

3D bioprinting for cell culture and tissue fabrication

Three-dimensional （3D） bioprinting is a computer-assisted technology which precisely controls spatial position of biomaterials, growth factors and living cells, offering unprecedented possibility to bridge the gap between structurally mimic tissue constructs and functional tissues or organoids. We briefly focus on diverse bioinks used in the recent progresses of biofabrication and 3D bioprinting of various tissue architectures including blood vessel, bone, cartilage, skin, heart, liver and nerve systems. This paper provides readers a guideline with the conjunction between bioinks and the targeted tissue or organ types in structuration and final functionalization of these tissue analogues. The challenges and perspectives in 3D bioprinting field are also illustrated.

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.