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Cytotechnology Apr 2019Low intensity (< 2 V/cm (peak to peak voltage/cm)), high frequency (10-30 MHz), and 10 min alternating electric fields (sine wave with no DC component) induce...
Low intensity (< 2 V/cm (peak to peak voltage/cm)), high frequency (10-30 MHz), and 10 min alternating electric fields (sine wave with no DC component) induce non-contact and enzyme-free cell detachment of anchorage-dependent cells directly from commercially available cell culture flasks and stack plates. 0.25 V/cm, 20 MHz alternating electric field for 10 min at room temperature (RT) induced maximum detachment and separated 99.5 ± 0.1% (mean ± SEM, n = 6) of CHO-K1 and 99.8 ± 0.2% of BALB/3T3 cells from the culture flasks. Both vertical and lateral alternating electric field applications for 10 min at RT detach the CHO-K1 cells from 25 cm culture flasks. The alternating electric field application induced cell detachment is almost noncytotoxic, and over 90% of the detached cells remained alive. The alternating electric field applied CHO-K1 cells for 90 min showed little or no lag phase and immediately enter exponential phase in cell growth. Combination of the 20 MHz alternating electric field and enzymatic treatment for 4 min at 37 °C showed synergetic effect and quickly detached human induced pluripotent stem cells from a laminin-coated culture flask compared with the only enzymatic treatment. These results indicate that the rapid cell detachment with both the electric field application and the enzymatic treatment could be applied to subcultures of cells that are susceptible to prolonged enzymatic digestion damage for mass culture of sustainable clinical use.
PubMed: 30783819
DOI: 10.1007/s10616-019-00307-4 -
International Journal of Retina and... 2019Retinoschisis and retinal detachment are distinguished based on features in clinical examination. Even to skilled examiners, some cases may be diagnostic challenges.... (Review)
Review
BACKGROUND
Retinoschisis and retinal detachment are distinguished based on features in clinical examination. Even to skilled examiners, some cases may be diagnostic challenges. Infrared and wide-angle infrared reflectance imaging are relatively new modalities that can provide additional diagnostic information. Non-contact infrared reflectance imaging (also described as near-infrared imaging) highlights sub-retinal features which may otherwise be obscured by standard retinal photography. It is non-invasive and uses the retina's ability to absorb, reflect or scatter infrared light to produce high quality images.
MAIN BODY
The aim of this review is to describe the role of wide-field infrared imaging in screening, diagnosing, and monitoring structural peripheral retinal disorders including retinoschisis, retinal detachment or combined retinoschisis rhegmatogenous detachments. Infrared imaging can also be used to monitor anterior segment inflammation. Heidelberg Wide-Field Module lens and Heidelberg Spectralis HRA + OCT machine (Heidelberg Engineering, Heidelberg, Germany) were used to obtain noncontact, wide-field infrared images on each study eye. Pseudocolor photos were captured by Optos Optomap (Optos, Inc, Massachusetts, USA).
CONCLUSION
Wide angle infrared imaging offers a quick, noncontact, and noninvasive way to help specialists accurately diagnose, monitor for progression, and educate patients about retinal detachment, retinoschisis and even anterior segment inflammation.
PubMed: 31890288
DOI: 10.1186/s40942-019-0188-5 -
Frontiers in Cellular Neuroscience 2022Retinal detachment is a sight-threatening disorder, which occurs when the photoreceptors are separated from their vascular supply. The aim of the present study was to...
Retinal detachment is a sight-threatening disorder, which occurs when the photoreceptors are separated from their vascular supply. The aim of the present study was to shed light on photoreceptor energy metabolism during experimental detachment in rats. Retinal detachment was induced in the eyes of rats subretinal injection of sodium hyaluronate. Initially, we investigated whether detachment caused hypoxia within photoreceptors, as evaluated by the exogenous and endogenous biomarkers pimonidazole and HIF-1α, as well as by qPCR analysis of HIF target genes. The results showed no unequivocal staining for pimonidazole or HIF-1α within any detached retina, nor upregulation of HIF target genes, suggesting that any reduction in pO is of insufficient magnitude to produce hypoxia-induced covalent protein adducts or HIF-1α stabilisation. Subsequently, we analysed expression of cellular bioenergetic enzymes in photoreceptors during detachment. We documented loss of mitochondrial, and downregulation of glycolytic enzymes during detachment, indicating that photoreceptors have reduced energetic requirements and/or capacity. Given that detachment did not cause widespread hypoxia, but did result in downregulated expression of bioenergetic enzymes, we hypothesised that substrate insufficiency may be critical in terms of pathogenesis, and that boosting metabolic inputs may preserve photoreceptor bioenergetic production and, protect against their degeneration. Thus, we tested whether supplementation with the bioavailable energy substrate pyruvate mitigated rod and cone injury and degeneration. Despite protecting photoreceptors in culture from nutrient deprivation, pyruvate failed to protect against apoptotic death of rods, loss of cone opsins, and loss of inner segment mitochondria, , when evaluated at 3 days after detachment. The regimen was also ineffective against cumulative photoreceptor deconstruction and degeneration when evaluated after 4 weeks. Retinal metabolism, particularly the bioenergetic profiles and pathological responses of the various cellular subtypes still presents a considerable knowledge gap that has important clinical consequences. While our data do not support the use of pyruvate supplementation as a means of protecting detached photoreceptors, they do provide a foundation and motivation for future research in this area.
PubMed: 36467607
DOI: 10.3389/fncel.2022.1036834 -
Scientific Reports Feb 2021During cancer, a major challenge faced by oncologists is the treatment of metastasis; a leading cause of cancer-related deaths around the world. Metastasis involves a...
During cancer, a major challenge faced by oncologists is the treatment of metastasis; a leading cause of cancer-related deaths around the world. Metastasis involves a highly ordered sequence of events starting with the detachment of tumor cells from the extracellular matrix (E.C.M.). In normal cells, detachment from E.C.M. triggers programmed cell death, termed anoikis. However, tumor cells dodge their way to anoikis and spread to distant sites for initiating the metastatic program. In this work, we explored the impact of E.C.M. detachment on the expression of some major oncogenic histone methyltransferases. Results showed both EZH2 expression and its enzymatic activity were significantly increased in E.C.M. detached cancer cells when compared to the attached cells. Inhibition of EZH2 results in a significant reduction in cell proliferation, spheroids size, and induction in apoptosis in E.C.M. detached cells. Furthermore, we observed a reduction in EZH2 expression levels in single cells when compared to clusters of E.C.M. detached cells. Finally, we combined the EZH2 inhibition with AMPK, known to be highly expressed in E.C.M. detached cancer cells and observed antagonistic effects between the two pathways. The observed results clearly showed that E.C.M. detached cancer cells require oncogenic EZH2 and can be targeted by EZH2 inhibitors.
Topics: Cell Adhesion; Cell Movement; Enhancer of Zeste Homolog 2 Protein; Extracellular Matrix; HCT116 Cells; HeLa Cells; Humans; Neoplasms
PubMed: 33531586
DOI: 10.1038/s41598-021-82431-w -
International Journal of Molecular... Nov 2019Cell sheet engineering has evolved rapidly in recent years as a new approach for cell-based therapy. Cell sheet harvest technology is important for producing viable,... (Review)
Review
Cell sheet engineering has evolved rapidly in recent years as a new approach for cell-based therapy. Cell sheet harvest technology is important for producing viable, transplantable cell sheets and applying them to tissue engineering. To date, most cell sheet studies use thermo-responsive systems to detach cell sheets. However, other approaches have been reported. This review provides the progress in cell sheet detachment techniques, particularly reactive oxygen species (ROS)-responsive strategies. Therefore, we present a comprehensive introduction to ROS, their application in regenerative medicine, and considerations on how to use ROS in cell detachment. The review also discusses current limitations and challenges for clarifying the mechanism of the ROS-responsive cell sheet detachment.
Topics: Animals; Humans; Reactive Oxygen Species; Tissue Engineering
PubMed: 31726692
DOI: 10.3390/ijms20225656 -
BioRxiv : the Preprint Server For... Nov 2023Biofilm formation is an important virulence factor for methicillin-resistant (MRSA). The extracellular matrix of MRSA biofilms contains significant amounts of...
Biofilm formation is an important virulence factor for methicillin-resistant (MRSA). The extracellular matrix of MRSA biofilms contains significant amounts of double-stranded DNA. MRSA cells also secrete micrococcal nuclease (Nuc1) which degrades double-stranded DNA. In this study we used a mutant strain to investigate the role of Nuc1 in MRSA biofilm formation and dispersal. Biofilm was quantitated in microplates using a crystal violet binding assay. Extracellular DNA (eDNA) was isolated from colony biofilms and analyzed by agarose gel electrophoresis. In some experiments, broth or agar was supplemented with sub-MIC amoxicillin to induce biofilm formation. Biofilm erosion was quantitated by culturing biofilms on rods, transferring the rods to fresh broth, and enumerating CFUs that detached from the rods. Biofilm sloughing was investigated by culturing biofilms in glass tubes perfused with broth and measuring the sizes of the detached cell aggregates. We found that a mutant strain produced significantly more biofilm and more eDNA than a wild-type strain in both the absence and presence of sub-MIC amoxicillin, mutant biofilms grown on rods detached significantly less than wild-type biofilms. Detachment was restored by exogenous DNase or a wild-type gene on a plasmid. In the sloughing assay, mutant biofilms released cell aggregates that were significantly larger than those released by wild-type biofilms. Our results suggest that Nuc1 modulates biofilm formation, biofilm detachment, and the sizes of detached cell aggregates. These processes may play a role in the spread and subsequent survival of MRSA biofilms during biofilm-related infections.
PubMed: 37961602
DOI: 10.1101/2023.11.05.565664 -
PloS One 2019Retinal detachment (RD) is one of the most frequently diagnosed ophthalmologic conditions requiring prompt surgical intervention. Combination of proper surgical...
PURPOSE
Retinal detachment (RD) is one of the most frequently diagnosed ophthalmologic conditions requiring prompt surgical intervention. Combination of proper surgical technique and new diagnostic markers, both clinical and molecular, can help improve the diagnosis and prognosis of RD treatment.
METHODS
12 patients with rhegmatogenous RD (rRD) were included into the study after obtaining patient consent and Regional Ethical Approval (average age: 58.1 ± 17.4 years). OCT was performed before and after 23G vitrectomy for RD. Pure subretinal fluid (SRF) was collected during surgery and analyzed by protein array profiling on a panel of 105 inflammatory cytokines (Human XL Cytokine Array), while the effect of SRF upon human macrophages-driven phagocytosis of apoptotic retinal pigment epithelial (RPE) cells ex vivo was quantified by flow cytometry. Immunohistochemistry (IHC) of retinectomized tissue due to PVR caused by RD was performed to determine presence of markers for microglial cells (CD34), macrophages and activated microglia (CD68), regulator of the immune response to infection (NFkB), progenitor and stem cell marker (Sox2), pluripotency marker (Oct4) and intermediate filament markers (GFAP and Nestin).
RESULTS
OCT of fresh RD patients contained pre-operatively hyper reflective points (HRPs) at the detached neuroretina border and proximal to the RPE layer-their size and number decreased following successful reattachment surgery. IHC of the retinectomized tissue from detached retina due to severe PVR showed presence of cell conglomerates at the detached neuroretina border which were positive for CD68, NFkB, Sox2 and GFAP, less positive for CD47 and Nestin and negative for Oct4 and CD34. The SRF contained at least 37 cytokines with higher, and 4 cytokine with lower concentration compared to that in vitreous from non-RD pathology; when used as conditional medium to human macrophages ex vivo, the SRF doubled their capacity for engulfing dying RPEs.
CONCLUSIONS
Fresh RD can be hallmarked by presence of HRPs at the detached neuroretina border on OCT; the HRPs decrease in size and number after successful reattachment surgery, and likely resemble the macrophage conglomerates seen by IHC. The neuroretina in RD contains progenitor/stem-like cells and signs of inflammatory reaction, while the SRF contains inflammatory cytokines and other factors which increase the ability of professional phagocytes to engulf dying RPE, or for that matter, other dying cells in the retina.
Topics: Adult; Aged; Antigens, Differentiation; Apoptosis; Epithelial Cells; Eye Proteins; Female; Humans; Inflammation; Macrophages; Male; Microglia; Middle Aged; Phagocytosis; Retinal Detachment; Retinal Pigment Epithelium; Stem Cells
PubMed: 31185026
DOI: 10.1371/journal.pone.0217548 -
Ulusal Travma Ve Acil Cerrahi Dergisi =... May 2020Tick is among the important ectoparasites of humans and animals. Ticks may transmit disease-causing pathogens to humans. Tick contact may be resulted in several viral...
BACKGROUND
Tick is among the important ectoparasites of humans and animals. Ticks may transmit disease-causing pathogens to humans. Tick contact may be resulted in several viral and bacterial infections, including Crimean-Congo Hemorrhagic Fever. Timely removal of ticks with appropriate methods is important in prevention of disease transmission. There are many methods reported for tick detachment. In this study, we aimed to evaluate two of them, suture lassoing and freezing and to compare both methods and to examine technical mistakes with these techniques.
METHODS
This study was designed as a prospective cross-sectional study, and included the ticks detached by healthcare professionals or directly by patients who presented to the emergency department due to tick contact. The ticks were recorded as larvae, nymphs, and adults according to their growth period. Ticks detachment types with surgical sutures and removal mistakes were recorded.
RESULTS
The majority (77.4%) of the ticks were removed by healthcare professionals and a lower rate by patients themselves with hand (22.6%). No technical mistake was found in 72 (77.4%) patients, and the tick was detached as a whole, while detached broken in 15 (16.1%) patients, and the tick was detached as a whole, but the sutures were attached wrong in six (6.5%) patients. Tick broken off due to technical mistakes was most commonly seen in the ticks removed by the individuals themselves.
CONCLUSION
The results of this study suggest that when appropriately and correctly used, both suture lassoing and tweezers are effective in tick removal. Public awareness-raising and training programs should be increased on this issue.
Topics: Animals; Bites and Stings; Cross-Sectional Studies; Humans; Prospective Studies; Surgical Instruments; Ticks
PubMed: 32436976
DOI: 10.14744/tjtes.2020.59680 -
Frontiers in Immunology 2022The nature of the culture dish surface and the technique used to detach adherent cells could very likely influence the cell viability and cell membrane protein integrity...
The nature of the culture dish surface and the technique used to detach adherent cells could very likely influence the cell viability and cell membrane protein integrity of harvested macrophages. Several previous studies assessed the detachment efficacies of enzymatic and non-enzymatic methods for harvesting the single cell suspensions of macrophages, but a comprehensive study assessing different dissociation methods and culture conditions for detaching functionally different macrophage populations has not yet been reported. In this study, the well-established GM-CSF and M-CSF differentiated bone marrow derived macrophage models (GM-BMDMs and M-BMDMs), we compared four commonly used enzymatic (trypsin and accutase) and non-enzymatic (PBS and EDTA) dissociation methods along with necessary mechanical detaching steps (scraping and pipetting) to evaluate the viable cell recovery and cell surface marker integrality of GM-BMDMs and M-BMDMs cultured on standard cell culture dish (TC dish), or on culture dish (noTC dish) that was not conditioned to enhance adherence. The data showed that accutase yielded a better recovery of viable cells comparing with PBS and EDTA, especially for tightly adherent GM-BMDMs on TC dishes, with a relatively higher level of detected cell membrane marker F4/80 than trypsin. An additional gradient centrifugation-based dead cell removal approach could increase the proportion of viable cells for TC cultured GM-BMDMs after accutase dissociation. Furthermore, transcriptome analysis was performed to evaluate the putative influence of culture dishes. At steady state, BMDMs cultured on noTC dishes exhibited more proinflammatory gene expression signatures (e.g. IL6, CXCL2 and ILlβ) and functions (e.g. TNF and IL17 signaling pathways). Similar inflammatory responses were observed upon LPS challenge regardless of culture conditions and differentiation factors. However, in LPS treated samples, the difference of gene expression patterns, signaling pathways and molecular functions between TC and noTC cultured BMDMs were largely dependent on the types of growth factors (M-CSF and GM-CSF). This observation might provide valuable information for macrophage studies.
Topics: Animals; Cell Culture Techniques; Edetic Acid; Granulocyte-Macrophage Colony-Stimulating Factor; Lipopolysaccharides; Macrophage Colony-Stimulating Factor; Macrophages; Mice; Phenotype; Trypsin
PubMed: 35874686
DOI: 10.3389/fimmu.2022.920232 -
Journal of Ophthalmic & Vision Research 2018To determine 1) which components of retinal function are impaired after rhegmatogenous retinal detachment, 2) which outer retinal pathways (rod- or cone-driven) are more...
PURPOSE
To determine 1) which components of retinal function are impaired after rhegmatogenous retinal detachment, 2) which outer retinal pathways (rod- or cone-driven) are more severely affected, and 3) whether there is concomitant inner retinal dysfunction.
METHODS
We conducted a prospective observational study in a large academic institution. We performed preoperative electroretinography on eight patients to assess outer and inner retinal function. In all cases, a comparison between the eye with the detached retina and the control fellow eye was made.
RESULTS
Eyes with a detached retina had significantly lower a-wave and b-wave amplitudes with respect to both rod- and cone-dominated testing parameters ( < 0.05) and reduced 30 Hz flicker responses compared to fellow eyes ( < 0.05); the effect size was similar for all significantly reduced parameters (r~0.6). There were no significant differences between eyes with detached retinas and control fellow eyes with respect to b/a-wave ratios, a-wave latencies, or b-wave latencies.
CONCLUSION
Patients with rhegmatogenous retinal detachment have preoperative outer retinal dysfunction equally affecting both rod- and cone-driven pathways, and they have minimal inner retinal dysfunction.
PubMed: 30090179
DOI: 10.4103/jovr.jovr_161_17