Swift Formation of Optimal Single Spheroids towards In-Vitro 3-Dimensional Tumour Models

Monolayer cell culture, while useful for basic in vitro studies, are not physiologically relevant. Spheroids, on the other hand provide a more complex 3-dimensional (3D) structure which more resemble the in vivo tumour growth thereby allowing results obtained with those on proliferation, cell death, differentiation, metabolism, and various anti-tumour therapies to be more predictive of in vivo outcomes. However, the cost associated with their generation often involve expensive, plate, media, and growth supplements, which have limited their use for high throughput experiments. The protocol herein presents a novel and rapid generation for single spheroids of various cancer cell lines, U87 MG; SEBTA-027; SF188, brain cancer cells, DU-145, TRAMP-C1, prostate cancer cells, in 96-round bottom well plates. Cells are washed with anti-adherent solution, and the homogeneous compact spheroid morphology was evidenced as early as 24 hours after 10 minutes centrifugation for the seeded cells. By using confocal microscopy, the proliferating cells were traced in the rim and the dead cells were found inside the core region of the spheroid. The H&E stain of spheroid slices and the western blotting were utilised to investigate the tightness of the cell packaging by adhesion proteins. Carnosine was used as an example of treatment for U87 single spheroids. The protocol allows the rapid generation of spheroids, which will help towards reducing the number of tests performed on animals.

Experimental and Theoretical Analysis of Metal Complex Diffusion through Cell Monolayer

The study of drugs diffusion through different biological membranes constitutes an essential step in the development of new pharmaceuticals. In this study, the method based on the monolayer cell culture of CHO-K1 cells has been developed in order to emulate the epithelial cells barrier in permeability studies by laser interferometry. Laser interferometry was employed for the experimental analysis of nickel(II) and cobalt(II) complexes with 1-allylimidazole or their chlorides’ diffusion through eukaryotic cell monolayers. The amount (mol) of nickel(II) and cobalt(II) chlorides transported through the monolayer was greater than that of metals complexed with 1-allylimidazole by 4.34-fold and 1.45-fold, respectively, after 60 min. Thus, laser interferometry can be used for the quantitative analysis of the transport of compounds through eukaryotic cell monolayers, and the resulting parameters can be used to formulate a mathematical description of this process.

The Role of Organoids as a Novel Platform for Modeling of Inflammatory Bowel Disease

Inflammatory bowel disease (IBD) is a chronic relapsing-remitting immune-mediated disorder affecting the gut. It is common in Westernized regions and is increasing in incidence in developing countries. At a molecular level, intrinsic deficiencies in epithelial integrity, mucosal barrier function, and mechanisms of immune response and resolution contribute to the development of IBD. Traditionally two platforms have been utilized for disease modeling of IBD;in-vitromonolayer cell culture andin-vivoanimal models. Both models have limitations, including cost, lack of representative cell types, lack of complexity of cellular interactions in a living organism, and xenogeneity. Organoids, three-dimensional cellular structures which recapitulate the basic architecture and functional processes of the organ of origin, hold potential as a third platform with which to investigate the pathogenesis and molecular defects which give rise to IBD. Organoids retain the genetic and transcriptomic profile of the tissue of origin over time and unlike monolayer cell culture can be induced to differentiate into most adult intestinal cell types. They may be used to model intestinal host-microbe interactions occurring at the mucosal barrier, are amenable to genetic manipulation and can be co-cultured with other cell lines of interest. Bioengineering approaches may be applied to render a more faithful representation of the intestinal epithelial niche. In this review, we outline the concept of intestinal organoids, discuss the advantages and disadvantages of the platform comparative to alternative models, and describe the translational applications of organoids in IBD.

Surface charge modulates the internalization vs. penetration of gold nanoparticles: comprehensive scrutiny on monolayer cancer cells, multicellular spheroids and solid tumors by SERS modality

Differential distribution of gold nanoparticles with respect to surface charges on monolayer cell culture, multicellular spheroids and in mouse models.

EXPRESSION OF TUMOR ASSOSIATED AND EPITHELIAL-MESENCHYMAL TRANSITION MARKERS IN 2D AND 3D CELL CULTURES OF MCF-7

The target effects on the expression of epithelial-mesenchymal transition regulation molecules are promising for cancer therapy, including breast cancer. 3D cell culture is a model for studying epithelial-mesenchymal transition in vitro and may become a test system for anticancer therapy. Aim of research. The aim of this research was to evaluate and compare the expression of tumor associated and epithelial-mesenchymal transition markers in tumor cells of breast adenocarcinoma (MCF-7 cell line) in 2D and 3D cell culture. Methods. For realization of the aim MCF-7 cell line (breast adenocarcinoma) was chosen as an experimental model in vitro. The monolayer cell culture was cultured in standard conditions (37 0C, 5 % CO2, humidity 95 %). The initial density of inoculated cells was 2 x 104 cells/cm2. The cells were incubated for two days before their use in the experiment. For the initial generation of spheroids the monolayer cell culture was removed off the substrate after the four days of incubation, using 0,25 % Trypsin-EDTA, and placed in nutrient medium with 5 % carboxymethyl cellulose (Bio-Rad, USA) at concentration of 5 x 105 cells/ml. Then the plates were incubated on an orbital shaker (Orbital shaker, PSU-10i, Biosan, Latvia) at 50 rpm for 3–5 hours. Half of culture medium was replenished every 3 days. A spheroid culture was maintained for 14 days. Detection of markers (ER, p53, EpCAM, vim, AE1/AE3, panCK, EGFR) in 2D and 3D cell culture was performed using immunohistochemistry method with primary monoclonal antibodies. Histological samples of cells were photographed to compare the morphological characteristics and the expression of proteins in monolayer and spheroid culture Results. The results demonstrated that the percentage of tumor marker positive cells (ER+, EGFR+, EpCAM+, panCK+, AE1/AE3+) in monolayer culture is 1.25–2 times than more in spheroid culture. In contrast, tumor spheroids consist of fewer cells with the expression of epithelial markers such as EpCAM and AE1/AE3, but they contain a large number of cells that expressed mesenchymal marker vimentin by 5 % and p53 by 10 %. This may indicate that the cells acquire a mesenchymal phenotype. However, tumor cells of monolayer cell culture were not expressed vimentin. Conclusions. Our results demonstrated the differences of expression of tumor associated and epithelial-mesenchymal transition markers in 2D and 3D breast cancer cell cultures. Thus, the percentage of epithelial markers (Cytokeratines and epithelial cell adhesion molecule) in tumor spheroids is less than in cells of monolayer however spheroids cells begin expressing a mesenchymal marker – vimentin. In 3D cell culture only the outer cell layers expressed tumor associated proteins unlike 2D cell culture in which all of cells showed equally expression. Reduced of manifestation of tumor associated markers in 3D cell culture may indicate an increase of stem properties. These data showed that 3D cell culture more than 2D cell culture characterized processes of epithelial-mesenchymal transition.

The expression of the Candida albicans gene SAP4 during hyphal formation in human serum and in adhesion to monolayer cell culture of colorectal carcinoma Caco-2 (ATCC)

Candida albicans SAP4 gene encodes secretory aspartyl protease Sap4 which is involved in hyphae formation and virulence. Transcriptional factors Cph1 and Efg1 govern the expression of several C. albicans genes and contribute to morphogenesis. We investigated the expression of SAP4 in C. albicans clinical isolate and mutants lacking Efg1 or/ and Cph1 grown in human serum and during contact with Caco-2 cell line. mRNA was analyzed with the use of RT-PCR; relative quantification was normalized against an ACT1 in cells after 18-h growth either in serum or on monolayer as well as in their counterparts in YEPD medium. We assessed the role of Sap4, Efg1 and Cph1 in adhesion of C. albicans to epithelial cells. Additionally, adherence assay was performed with sap4/sap4. Adhesion was expressed as a percent of adherent cells to monolayer at 90 min vs. total cells added (100%). No differences were observed in adhesion of efg1/efg1 and sap4/sap4 compared with SC5314 (P≥0.05 statisitically insignificant). SAP4 expression indicated that it is not involved in adapting to the tested conditions. SAP4 expression can be strainspecific and is not solely controlled by the Efg1 pathway but also by the Cph1 pathway. Neither Efg1 nor Sap4 can influence adhesion.