An Evaluation of a Simplified Impression Membrane Sampling Method for the Diagnosis of Microbial Keratitis

The purpose of this study was to compare bacterial isolation rate using a corneal impression membrane (CIM) and a sharp instrument for obtaining corneal samples from patients with suspected microbial keratitis (MK). Data was retrospectively collected for all patients that had corneal samples taken for presumed MK between May 2014 and May 2020. Prior to May 2017 samples were collected by scraping the edges of the ulcer with a blade. From May 2017, samples were collected by placing a CIM (Millicell cell culture insert) against the ulcer. All corneal samples were processed using the same conventional diagnostic culture method. A total of 3099 corneal samples were included, of which 1214 (39.2%) were corneal scrapes and 1885 (60.9%) CIMs. Microorganisms were isolated from 235 (19.4%) and 1229 (65.2%) cases using a corneal scrape and CIM, respectively (p < 0.001). Of routinely described pathogenic microorganisms, there were significant increases in the isolations of S. aureus (2.4% to 11.3%) and Serratia (0.5% to 1.7%) using the CIM and no significant changes in the isolations of S. pneumoniae and P. aeruginosa. No significant differences were seen between the isolation rates of fungi or Acanthamoeba species. There was a significant increase in the isolation rates of other Streptococcal species (0.7% to 6.9%) and CNS species, specifically, S. epidermidis (2.1% to 26.2%), S. capitis (0.4% to 2.6%) and S. warneri (0.3% to 1.6%) using the CIM. The simplified CIM sampling method is an effective method for collecting corneal samples from patients with presumed MK in clinical practice.

Novel Platform for Regulation of Extracellular Vesicles and Metabolites Secretion from Cells Using a Multi-Linkable Horizontal Co-Culture Plate

Microfluidics is applied in biotechnology research via the creation of microfluidic channels and reaction vessels. Filters are considered to be able to simulate microfluidics. A typical example is the cell culture insert, which comprises two vessels connected by a filter. Cell culture inserts have been used for years to study cell-to-cell communication. These systems generally have a bucket-in-bucket structure and are hereafter referred to as a vertical-type co-culture plate (VTCP). However, VTCPs have several disadvantages, such as the inability to simultaneously observe samples in both containers and the inability of cell-to-cell communication through the filters at high cell densities. In this study, we developed a novel horizontal-type co-culture plate (HTCP) to overcome these disadvantages and confirm its performance. In addition, we clarified the migration characteristics of substances secreted from cells in horizontal co-culture vessels. It is generally assumed that less material is exchanged between the horizontal vessels. However, the extracellular vesicle (EV) transfer was found to be twice as high when using HTCP. Other merits include control of the degree of co-culture via the placement of cells. We believe that this novel HTCP container will facilitate research on cell-to-cell communication in various fields.

Designer Cell Culture Insert with Nanofibrous Membrane toward Engineering an Epithelial Tissue Model Validated by Cellular Nanomechanics

Engineered platforms for culturing cells of the skin and other epithelial tissues are useful for the regeneration and development of in vitro tissue models used in drug screening. Recapitulating the...

A CELL-CULTURING SYSTEM FOR THE STUDY OF INTERACTION BETWEEN MACROPHAGES INFECTED WITH Leishmania amazonensis

The cell culture insert system is a culturing system for the study of contact-independent cellular communication. Leishmaniasis is a neglect tropical disease with no vaccines and the availabledrugs present toxic side effects. Studies on Leishmania interaction with host macrophages aim to develop strategies for parasite control and drug development. The purpose of this study was to evaluate the effects of interaction between non-infected and L. amazonensis-infected human macrophages, by using the cell culture system. The results showed that the infection index was reduced by 56.2% as compared to controls only when infected macrophages were inserted on both sides of the Transwell membranes. An improvement in macrophage viability was also observed in this cell culture. The levels of interleukin-1β, an inflammatory cytokine, and nitric oxide, a microbicidal molecule, did not increase in L. amazonensis-infected macrophagecultures in the Transwell system; thus other soluble factors were responsible for parasite control.

Enucleation of Human Erythroblasts Is a Process of Asymmetric Cytokinesis.

Background. During erythroblast enucleation, nuclei surrounded by plasma membrane separate from erythroblast cytoplasm. Enucleation has been thought to be a process similar to cytokinesis. However, more concrete evidence has been difficult to obtain because of a lack of an ex vivo experimental system capable of confirming cytokinesis. Focusing on the mechanism of cell division, we investigated the redistribution of cytoplasmic proteins and integral membrane proteins during enucleation, using ex vivo generation system of mature human blood cells from hematopoietic stem cells. Materials and Methods. The highly purified human CD34+ cells were grown in the presence of interleukin-3, stem cell factor and erythropoietin (EPO) in a liquid phase. After 7 days of culture, the generated cells (day 7 cells) were replaced in a medium with EPO alone. The cells matured and terminally differentiated into reticulocytes during a 13–15-day culture period. We mainly used non-gravity and non-pipetting system to avoid physical stress that may disrupt the connection between the nucleus and reticulocyte. Day 9 cells, predominantly consisted of polychromatophilic erythroblasts and expressed glycophorin A (GPA) at a purity of 97%, were labeled with DNA-staining dye SYTO21 for the direct monitoring of the enucleation process, using differential interference contrast microscopy. We also cultured day 9 cells until day 14, on 4-well culture slides or on the membrane of a cell culture insert system, and removed culture medium by aspiration without centrifugation and pipetting. The day 14 cells on the slide were analyzed using immunohistochemical staining, whereas the cells on the membrane were embedded in O.C.T. compound for confocal microscopy. Results. Approximately a half of erythroblasts enucleated until day 14. The monitoring of the enucleation process at day 13 showed autonomous extrusion of SYTO21 positive nucleus from single erythroblast. Some of the expelled nuclei were still connected with reticulocyte through strings that were positive for antibody against tubulin, actin, GPA, band 3 and glycophorin C (GPC). The expelled nuclei were covered by lamin, a protein specific for nuclear membrane, which were surrounded by a substance positive for GPA, band 3, GPC, p55, 4.1R80, actin, tubulin, b-spectrin, calnexin and cytochrome C, although the distribution of each proteins were asymmetric between nuclei and reticulocytes. An intense area of GPA, GPC, band 3, 4.1R80, actin, tubulin, myosin and b-spectrin was found in the region of the constriction between the extruding nucleus and incipient reticulocyte in enucleating cells. In cells just before enucleation, tubulin and actin formed a radial array around the nucleus. The center of the radial array was positive for centrin and NuMA, indicating that the cenriole formation occurred during an enucleation process. Conclusion. Our investigations show that a part of human erythroblasts enucleate independent of an interaction with accessory cells. The appearance of cenriole and the asymmetric redistribution of cytoplasmic and integral membrane proteins during enucleation strongly suggest that enucleation of human erythroblasts is a process of asymmetric cytokinesis.

Mammary specific function of a bovine mammary epithelial cell (ABERMEC) clone cultured on collagen I coated inserts in the presence and absence of foetal bovine serum

The aim was to establish a representative model of the bovine mammary gland in order to underpin applied research in mammary gland development and lactation. Cell culture insert methodology is currently being utilized in place of a three dimensional culture system, the shortcomings of which have been discussed elsewhere (McConochie et al., 2004). Cell culture insert methodology offers a promising alternative, with the potential to recreate in vitro a polarised epithelial layer. Previously it has been shown that on collagen I coated inserts, ABERMEC are able to synthesise and secrete mammary specific proteins in the apparent absence of the key mediators laminin and prolactin. It was hypothesised that undefined factors in serum were a possible cause for this phenomenon.