Comparison of biophysical properties characterized for microtissues cultured using microencapsulation and liquid crystal based 3D cell culture techniques

Growing three dimensional (3D) cells is an emerging research in tissue engineering. Biophysical properties of the 3D cells regulate the cells growth, drug diffusion dynamics and gene expressions. Scaffold based or scaffoldless techniques for 3D cell cultures are rarely being compared in terms of the physical features of the microtissues produced. The biophysical properties of the microtissues cultured using scaffold based microencapsulation by flicking and scaffoldless liquid crystal (LC) based techniques were characterized. Flicking technique produced high yield and highly reproducible microtissues of keratinocyte cell lines in alginate microcapsules at approximately 350 ± 12 pieces per culture. However, microtissues grown on the LC substrates yielded at lower quantity of 58 ± 21 pieces per culture. The sizes of the microtissues produced using alginate microcapsules and LC substrates were 250 ± 25 μm and 141 ± 70 μm, respectively. In both techniques, cells remodeled into microtissues via different growth phases and showed good integrity of cells in field-emission scanning microscopy (FE-SEM). Microencapsulation packed the cells in alginate scaffolds of polysaccharides with limited spaces for motility. Whereas, LC substrates allowed the cells to migrate and self-stacking into multilayered structures as revealed by the nuclei stainings. The cells cultured using both techniques were found viable based on the live and dead cell stainings. Stained histological sections showed that both techniques produced cell models that closely replicate the intrinsic physiological conditions. Alginate microcapsulation and LC based techniques produced microtissues containing similar bio-macromolecules but they did not alter the main absorption bands of microtissues as revealed by the Fourier transform infrared spectroscopy. Cell growth, structural organization, morphology and surface structures for 3D microtissues cultured using both techniques appeared to be different and might be suitable for different applications.     

In vivo therapeutic applications of cell spheroids

Spheroids are increasingly being employed to answer a wide range of clinical and biomedical inquiries ranging from pharmacology to disease pathophysiology, with the ultimate goal of using spheroids for tissue engineering and regeneration. When compared to traditional two-dimensional cell culture, spheroids have the advantage of better replicating the 3D extracellular microenvironment and its associated growth factors and signaling cascades. As knowledge about the preparation and maintenance of spheroids has improved, there has been a plethora of translational experiments investigating in vivo implantation of spheroids into various animal models studying tissue regeneration.We review methods for spheroid delivery and how they have been utilized in tissue engineering experiments. We break down efforts in this field by organ systems, discussing applications of spheroids to various animal models of disease processes and their potential clinical implications. These breakthroughs have been made possible by advancements in spheroid formation, in vivo delivery and assessment. There is unexplored potential and room for further research and development in spheroid-based tissue engineering approaches. Regenerative medicine and other clinical applications ensure this exciting area of research remains relevant for patient care.     

Copper complex derived from <Emphasis Type="Italic">S</Emphasis>-benzyldithiocarbazate and 3-acetylcoumarin induced apoptosis in breast cancer cell

Copper complexes have been widely studied for the anti-tumour application as cancer cells are reported to take up greater amounts of copper than normal cells. Preliminary study revealed that the newly synthesised copper complex [Cu(SBCM)₂] displayed marked anti-proliferative towards triple-negative MDA-MB-231 breast cancer cells. Therefore, Cu(SBCM)₂ has great potential to be developed as an agent for the management of breast cancer. The present study was carried out to investigate the mode of cell death induced by Cu(SBCM)₂ towards MDA-MB-231 breast cancer cells. The inhibitory and morphological changes of MDA-MB-231 cells treated with Cu(SBCM)₂ was determined by using MTT assay and inverted light microscope, respectively. The safety profile of Cu(SBCM)₂ was also evaluated towards human dermal fibroblast (HDF) normal cells. Confirmation of apoptosis and cell cycle arrest were determined by flow cytometry analysis. The expression of p53, Bax, Bcl-2 and MMP2 protein were detected with western blot analysis. Cu(SBCM)₂ significantly inhibited the growth of MDA-MB-231 cells in a dose-dependent manner with GI₅₀ 18.7?±?3.06 µM. Indeed, Cu(SBCM)₂ was less toxic towards HDF normal cells with GI₅₀ 31.8?±?4.0 µM. Morphological study revealed that Cu(SBCM)₂-treated MDA-MB-231 cells experienced cellular shrinkage, membrane blebbing, chromatin condensation and formation of apoptotic bodies, suggesting that Cu(SBCM)₂ induced apoptosis in the cells, which was confirmed by Annexin-V/PI flow cytometry analysis. It was also found that Cu(SBCM)₂ induced G₂/M phase cell cycle arrest towards MDA-MB-231 cells. The induction of apoptosis and cell cycle arrest in the present study is possibly due to the down-regulation of the mutant p53 and MMP2 protein. In conclusion, Cu(SBCM)₂ can be developed as a targeted therapy for the treatment of triple-negative breast cancer.     

Functional expression of monomeric streptavidin and fusion proteins in Escherichia coli: applications in flow cytometry and ELISA

Monomeric streptavidin (mSA) offers a combination of structural and binding properties that are useful in many applications, including a small size and monovalent biotin binding. Because mSA contains a structurally important disulfide bond, the molecule does not fold correctly when expressed inside the cell. We show that mSA can be expressed in a functional form in Escherichia coli by fusing the OmpA signal sequence at the amino terminus. Expressed mSA is exported to the periplasm, from which the molecule leaks to the medium under vigorous shaking. Purified mSA can be conjugated with FITC and used to label microbeads and yeast cells for analysis by flow cytometry, further expanding the scope of mSA-based applications. Some applications require recombinant fusion of mSA with another protein. mSA fused to EGFP cannot be secreted to the medium but was successfully expressed in an engineered cell line that supports oxidative folding in the cytoplasm. Purified mSA-EGFP and mSA-mCherry bound biotin with high affinity and were successfully used in conventional flow cytometry and imaging flow cytometry. Finally, we demonstrate the use of mSA in ELISA, in which horseradish peroxidase-conjugated mSA and biotinylated secondary antibody are used together to detect primary antibody captured on an ELISA plate. Engineering mSA to introduce additional lysine residues can increase the reporter signal above that of wild-type streptavidin. Together, these examples establish mSA as a convenient reagent with a potentially unique role in biotechnology.

     

Abscisic acid and salinity stress induced somaclonal variation and increased histone deacetylase (HDAC) activity in <Emphasis Type="Italic">Ananas comosus</Emphasis> var. MD2

Somaclonal and phenotypic variation caused by genetic and/or epigenetic modifications, are a valuable source of genetic variation to improve desirable polygenetic traits in crops. In this study, we induced somaclonal variation in vitro pineapple (Ananas comosus var. MD2) through hormonal induction, NaCl, and abscisic acid (ABA) supplementation. Our results showed that supplementation of high concentration of 6-benzylaminopurine (4.0 mg/L BAP) alone or combined with indole-butyric acid (IBA) produced the highest percentage of dwarf variants (100%). Murashige and Skoog (MS) media containing 4.0 mg/L BAP plus 2.0 mg/L IBA produced the shortest plantlets (1.9?±?0.1 cm). In comparison, MS media containing 1.0% NaCl induced formation of dwarf plantlets with a mean plantlet height of 1.4?±?0.3 cm, whereas 1.0 mg/L ABA generated plantlets with a mean plantlet height of 1.7?±?0.1 cm. We then analyzed the histone deacetylase (HDAC) enzyme activity for dwarf and non-dwarf plantlets. In general, dwarf plantlets exhibited higher HDAC activity than non-dwarf plantlets. The highest HDAC activity (109, 333.33?±?4.40 ng/min/mg) was recorded for dwarf plantlets grown on media supplemented with 1.0 mg/L ABA. The dwarf variants also underwent phenotypic recovery to normal phenotype within 8 months after transferred to MS basal media. No ploidy alteration was detected in these dwarf plantlets after analyzed by flow cytometry. Taken together, although the generated dwarf plantlets showed higher HDAC activity compared to non-dwarf plantlets, their capability of reverting to non-dwarf phenotype suggested that it might be due to epigenetic modulation.     

Genetic Analysis of Adult-Specific Surface Antigenic Differences between Varieties of the Nematode Caenorhabditis elegans

We have studied developmental stage-specificity and genetic specification of surface antigens in the nematode Caenorhabditis elegans. Rabbit antisera directed against the adult C. elegans cuticle were used in conjunction with antiserum adsorption experiments to obtain antibody reagents with specificity for the adult surface. Adult-specific antibodies were used to identify several varietal strains of C. elegans that display antigen-negative phenotypes as adults. Genetic mapping results using the surface antigen phenotype as a marker indicated that a single gene (designated srf-1) or cluster of genes on linkage group II determines the adult surface antigen phenotype.     

A monoclonal anti-HEBFPP antibody with specificity for lymphocyte surface molecules mediating adhesion to Peyer's patch high endothelium of the rat

Cell surface molecules involved in lymphocyte adhesion to high endothelial cell venules (HEV) of Peyer's patches (PP) have been studied in the rat by using a mouse monoclonal anti-HEBFPP (1B.2) antibody. We previously showed that rat thoracic duct lymph contains a high endothelial cell binding factor termed HEBFPP, which in vitro blocks lymphocyte binding sites of HEVPP but not HEVLN. Monoclonal 1B.2 antibody was produced by fusing P3U1 myeloma cells with spleen cells of a mouse immunized with this material. Immunoprecipitation studies with 125I surface-labeled rat thoracic duct lymphocytes (TDL) showed that the antibody recognized an 80-kilodalton protein. This antigen was present in the majority of TDL, spleen, LN, and PP cells but was found on few (5 to 10%) thymus and bone marrow cells (indirect immunofluorescence). Treatment of TDL with 1B.2 antibody blocked their ability to bind in vitro to HEVPP; antibody treatment did not interfere with TDL adhesion to HEVLN. Analysis of 1B.2 antigen isolated from lymph and detergent lysates of TDL by antibody-affinity chromatography showed that this material had the capacity to block lymphocyte binding sites of HEVPP but not HEVLN. In contrast, material with such blocking activity was not isolated from detergent lysates of thymocyte, a population deficient in HEV-binding cells. The results indicate that the 1B.2 antigen is a component of the lymphocyte surface recognition structure mediating adhesion to HEVPP and provide further evidence that distinct adhesion molecules of rat TDL mediate interaction with high endothelium of LN and PP.     

Multidrug resistance of DNA-mediated transformants is linked to transfer of the human mdr1 gene.

Mouse NIH 3T3 cells were transformed to multidrug resistance with high-molecular-weight DNA from multidrug-resistant human KB carcinoma cells. The patterns of cross resistance to colchicine, vinblastine, and doxorubicin hydrochloride (Adriamycin; Adria Laboratories Inc.) of the human donor cell line and mouse recipients were similar. The multidrug-resistant human donor cell line contains amplified sequences of the mdr1 gene which are expressed at high levels. Both primary and secondary NIH 3T3 transformants contained and expressed these amplified human mdr1 sequences. Amplification and expression of the human mdr1 sequences and amplification of cotransferred human Alu sequences in the mouse cells correlated with the degree of multidrug resistance. These data suggest that the mdr1 gene is likely to be responsible for multidrug resistance in cultured cells.     

Structure and expression of the human MDR (P-glycoprotein) gene family.

The human MDR (P-glycoprotein) gene family is known to include two members, MDR1 and MDR2. The product of the MDR1 gene, which is responsible for resistance to different cytotoxic drugs (multidrug resistance), appears to serve as an energy-dependent efflux pump for various lipophilic compounds. The function of the MDR2 gene remains unknown. We have examined the structure of the human MDR gene family by Southern hybridization of DNA from different multidrug-resistant cell lines with subfragments of MDR1 cDNA and by cloning and sequencing of genomic fragments. We have found no evidence for any other cross-hybridizing MDR genes. The sequence of two exons of the MDR2 gene was determined from genomic clones. Hybridization with single-exon probes showed that the human MDR1 gene is closely related to two genes in mouse and hamster DNA, whereas MDR2 corresponds to one rodent gene. The human MDR locus was mapped by field-inversion gel electrophoresis, and both MDR genes were found to be linked within 330 kilobases. The expression patterns of the human MDR genes were examined by enzymatic amplification of cDNA. In multidrug-resistant cell lines, increased expression of MDR1 mRNA was paralleled by a smaller increase in the levels of MDR2 mRNA. In normal human tissues, MDR2 was coexpressed with MDR1 in the liver, kidney, adrenal gland, and spleen. MDR1 expression was also detected in colon, lung, stomach, esophagus, muscle, breast, and bladder.     

Retroviral transfer of a chimeric multidrug resistance-adenosine deaminase gene

A fusion between a selectable multidrug resistance (MDR1) cDNA and an adenosine deaminase (ADA) cDNA concomitantly confers multidrug resistance and ADA activity on transfected cells. We have produced a Harvey murine sarcoma virus-derived, replication-defective, recombinant retrovirus to transduce this chimeric MDR-ADA gene efficiently into a great variety of cells. Infection with the MDR-ADA retrovirus conferred the multidrug resistance phenotype on drug-sensitive cells, therefore allowing selection in the presence of colchicine. Colchicine-resistant cells synthesized large amounts of a membrane-associated 210-kDa MDR-ADA fusion protein that preserved both MDR and ADA functional activities. To monitor expression of the chimeric gene in vivo, Kirsten virus-transformed NIH cells were infected with the MDR-ADA retrovirus, and after drug-selection, injected into athymic nude mice. Tumors developed that contained the bifunctionally active MDR-ADA fusion protein. When these mouse tumor cells were placed in tissue culture without the selecting drug, they did not lose the bifunctionally active MDR-ADA fusion protein. The replication-defective, recombinant MDR-ADA retrovirus should be useful to stably introduce the chimeric MDR-ADA gene into a variety of cell types for biological experiments in vitro and in vivo.