Cell Preparation Methods Research Articles

Advances in Oral Exfoliative Cytology: From Cancer Diagnosis to Systemic Disease Detection.

Oral exfoliative cytology has emerged as a valuable tool in the early detection of oral cancer and other systemic diseases. This review comprehensively examines the current applications and recent advancements in oral exfoliative cytology techniques. We analyzed published literature from the past decade, focusing on methodological improvements, diagnostic accuracy, and emerging applications. Key findings include: (1) Enhanced cell collection and preparation methods have significantly improved sample quality and diagnostic reliability. (2) Integration of molecular markers and DNA analysis with traditional cytomorphological assessment has increased diagnostic sensitivity and specificity for oral cancer detection. (3) Novel applications in systemic disease detection, including diabetes and iron overload disorders, demonstrate the expanding utility of this technique. (4) Computer-assisted analysis and deep learning algorithms show promise in improving diagnostic accuracy and efficiency. Despite these advancements, challenges remain in standardization and widespread clinical implementation. This review provides a critical evaluation of oral exfoliative cytology's current status and future potential in oral and systemic disease diagnosis.

Human-Induced Pluripotent Stem Cell-Derived Neural Progenitor Cells Showed Neuronal Differentiation, Neurite Extension, and Formation of Synaptic Structures in Rodent Ischemic Stroke Brains.

Ischemic stroke is a major cerebrovascular disease with high morbidity and mortality rates; however, effective treatments for ischemic stroke-related neurological dysfunction have yet to be developed. In this study, we generated neural progenitor cells from human leukocyte antigen major loci gene-homozygous-induced pluripotent stem cells (hiPSC-NPCs) and evaluated their therapeutic effects against ischemic stroke. hiPSC-NPCs were intracerebrally transplanted into rat ischemic brains produced by transient middle cerebral artery occlusion at either the subacute or acute stage, and their in vivo survival, differentiation, and efficacy for functional improvement in neurological dysfunction were evaluated. hiPSC-NPCs were histologically identified in host brain tissues and showed neuronal differentiation into vGLUT-positive glutamatergic neurons, extended neurites into both the ipsilateral infarct and contralateral healthy hemispheres, and synaptic structures formed 12 weeks after both acute and subacute stage transplantation. They also improved neurological function when transplanted at the subacute stage with γ-secretase inhibitor pretreatment. However, their effects were modest and not significant and showed a possible risk of cells remaining in their undifferentiated and immature status in acute-stage transplantation. These results suggest that hiPSC-NPCs show cell replacement effects in ischemic stroke-damaged neural tissues, but their efficacy is insufficient for neurological functional improvement after acute or subacute transplantation. Further optimization of cell preparation methods and the timing of transplantation is required to balance the efficacy and safety of hiPSC-NPC transplantation.

Round‐robin test of all‐solid‐state battery with sulfide electrolyte assembly in coin‐type cell configuration

AbstractThe replacement of conventional lithium‐ion batteries with solid‐state batteries is currently under investigation by many players both from academia and industry. Sulfide‐based electrolytes are among the materials that are regarded as most promising, especially for application in the transport sector. The performance of anode, cathode, and solid electrolyte materials of this type of solid electrolyte is typically evaluated using manually assembled cells such as Swagelok cells, EL‐CELLs, and in‐house built pressure devices. Coin cells, however, are often disregarded. Though coin cells cannot accurately predict how a material will perform in an end‐use application battery cell format, they are easy to assemble and can provide reproducible data compared to the other cell types, which make them an interesting option for testing the materials under conditions more relevant for their envisioned application. The coin cell preparation method presented in this work has been evaluated interlaboratory for reproducibility and, in addition, can be modified depending on the optimization parameters of the solid electrolyte, cathode material, bilayer comprised on cathode and solid electrolyte, lithium metal anode, and cell in general. Besides, an interlab round‐robin test (RRT) is carried out between four laboratories, measuring defined electrochemical tests of sulfide solid‐state batteries in coin cell configuration. This RRT for the preparation of coin cell solid‐state batteries with sulfide solid electrolyte, lithium nickel manganese cobalt oxides cathode, and lithium metal anode is intended for academic researchers and provides guidelines of research in this field.

An Optimized Transformation Protocol for Escherichia coli BW3KD with Supreme DNA Assembly Efficiency.

DNA cloning requires two steps: the assembly of recombinant DNA molecules and the transformation of the product into a host organism for replication. High efficiencies in both processes can increase the success rate. Recently, we developed an Escherichia coli BW3KD strain with higher transformation efficiency than commonly used cloning strains. Here, we further developed a simple method named TSS-HI (transformation storage solution optimized by Hannahan and Inoue method) for competent cell preparation, which combined the advantages of three common methods for operational simplicity and high transformation efficiency. When competent BW3KD cells were prepared using this developed method, the transformation efficiency reached up to (7.21 ± 1.85) × 109 CFU/μg DNA, which exceeded the levels of commercial chemically competent cells and homemade electrocompetent cells. BW3KD cells formed colonies within 7 h on lysogeny broth agar plates, quicker than the well-known fast-growing E. coli cloning strain Mach1. The competent cells worked effectively for the transformation of assembled DNA of 1 to 7 fragments in one step and promoted efficiencies of transformation or cloning with large plasmids. The cloning efficiency of BW3KD cells prepared by this method increased up to 828-fold over that of E. coli XL1-Blue MRF' cells prepared by a common method. Thus, competent cells are suitable for different cloning jobs and should help with the increased demand for DNA assembly in biological studies and biotechnology. IMPORTANCE DNA transformation is commonly used in cloning; however, high transformation efficiency becomes a limiting factor in many applications, such as the construction of CRISPR and DNA libraries, the assembly of multiple fragments, and the transformation of large plasmids. We developed a new competent cell preparation method with unmatched transformation efficiency. When the BW3KD strain, derived from Escherichia coli BW25113 cells, was prepared by this method, its transformation efficiency reached up to (7.21 ± 1.85) × 109 CFU/μg DNA, which broke the record for chemically prepared competent cells. Routine cloning could be completed in 1 day due to the high growth rate of this strain. The competent cells were shown to be highly efficient for transformation or cloning with large plasmids and for the assembly of multiple fragments. The results highlight the effectiveness of the new protocol and the usefulness of the BW3KD strain as the host.

Stem cell transplantation for induction of remission in medically refractory Crohn's disease.

Stem cell transplantation for induction of remission in medically refractory Crohn's disease.

Analysis of adherent cell culture lysates with low metabolite concentrations using the Biocrates AbsoluteIDQ p400 HR kit

The AbsoluteIDQ p400 HR kit is a commercial product for targeted metabolomics. While the kit has been validated for human plasma and serum, adherent cell lysates have not yet been evaluated. We have optimized the detection of polar and lipid metabolites in cell lysates using the kit to enable robust and repeatable analysis of the detected metabolites. Parameters optimized include total cell mass, loading volume and extraction solvent. We present a cell preparation and analytical method and report on the performance of the kit with regard to detectability of the targeted metabolites and their repeatability. The kit can be successfully used for a relative quantification analysis of cell lysates from adherent cells although validated only for human plasma and serum. Most metabolites are below the limit of the Biocrates’ set quantification limits and we confirmed that this relative quantification can be used for further statistical analysis. Using this approach, up to 45% of the total metabolites in the kit can be detected with a reasonable analytical performance (lowest median RSD 9% and 13% for LC and FIA, respectively) dependent on the method used. We recommend using ethanol as the extraction solvent for cell lysates of osteosarcoma cell lines for the broadest metabolite coverage and 25 mg of cell mass with a loading volume of 20 µL per sample.

Human papillomavirus E6E7 mRNA and TERC lncRNA in situ detection in cervical scraped cells and cervical disease progression assessment

BackgroundHuman papillomavirus screen in female cervical cells has demonstrated values in clinical diagnosis of precancerous lesions and cervical cancers. Human papillomavirus tests of cervical cells by utilizing Polymerase Chain Reaction (PCR) method provides human papillomavirus infection status however no further virus in situ information. Although it is well known that the tests of human papillomavirus E6/E7 RNA location in infected cervical cells and cell internal malignancy molecular will provide clues for gynecologists to evaluate disease progression, there are technique difficulties to preserve RNAs in cervical scraped cells for in situ hybridization.MethodsIn current study, after developing a cervical cell collection and preparation method for RNA in situ hybridization, we captured the chance to screen 98 patient cervical cell samples and detected human papillomavirus E6/E7 mRNAs of high-risk subtypes, low-risk subtypes and long non-coding RNA (lncRNA) TERC in the cells.ResultsThere were 69 samples exhibited consistence between human papillomavirus PCR and human papillomavirus RNA in situ hybridization results in cervical collected cells. Among them, 23 were both positive and 46 were both negative. In the rest 29 samples, 8 were HPV RNAscope positive, either high risk or low risk subtypes, however HPV PCR negative. Another 9 samples were HPV PCR results positive whereas RNAscope negative. The last 12 samples were HPV positive detected by both RNAscope and PCR methods, however inconsistent between high-risk and low-risk subtypes. In RNAscope positive samples, viral E6/E7 mRNAs were observed to distribute in cervical scraped cell nucleus and cytoplasm. Moreover, HPV viral RNA gathered clusters were observed outside of cells through human papillomavirus RNA in situ hybridization detection. Varied numbers of human papillomavirus infective cells were detected by RNAscope assay in different patients even though they were all human papillomavirus high-risk subtype positive discovered by human papillomavirus PCR results. A cell malignancy related long non-coding RNA, TERC, has been detected in seven patient samples. The patient follow-up information was further analyzed with RNAscope results which indicated a combination of RNAscope positive signals of TERC and human papillomavirus high risk signals in more than 10 cells (cytoplasm or nucleus) may connect with cervical lesion fast progression which deserves further studies in the future.CConclusionsTaken together, current study has provided an observable clue for gynecologists to evaluate human papillomavirus infection stage and cell malignancy status which may contribute for assessment of cervical disease progression.

Efficient and stable carbon-based CsPbBr<sub>3</sub> solar cells added with PEABr additive

<sec>In recent years, organic-inorganic hybrid perovskite solar cells have become a research hotspot in the photovoltaic field because of their excellent power conversion efficiency. However, this hybrid perovskite material's intrinsic instability and the harsh preparation environment limit its further commercial application. All-inorganic CsPbBr<sub>3</sub> perovskite materials have attracted much attention because of their good stability, low cost and can be prepared in an atmospheric environment, showing great application potential. The controllable preparation and growth kinetics of CsPbBr<sub>3</sub> materials need to be further studied, and the conversion efficiency of photovoltaic devices is still low. Considering the instability caused by traditional organic hole transport materials and their high preparation cost, this work focuses on the systematical studies of CsPbBr<sub>3</sub> all-inorganic perovskite cells without a hole transport layer. Growth kinetics material of CsPbBr<sub>3</sub> is controlled by adding 2-phenylethylamine bromide to precursor solution<sub>.</sub> The main research contents and results are described as follows.</sec><sec>Based on multi-step spin-coating preparation of CsPbBr<sub>3</sub> perovskite films, the perovskite cell preparation method is studied, and the critical process parameters including the spin-coating PbBr<sub>2</sub>, amount and number of spin-coating of CsBr, substrate preheating temperature, and the annealing temperature, are optimized. The optimization tests show that the optimal spin-coating of CsBr is obtained by being optimized five times and the spin-coating PbBr<sub>2</sub> is conducted in the atmospheric environment. The optimal preheating temperature of the substrate is 80 ℃, and the optimal annealing temperature is 100 ℃. The perovskite films prepared under this condition are compact, each with a continuous high phase purity and good crystallization performance.</sec><sec>The PbBr<sub>2</sub> in DMF is first adopted and the 2-phenylethylamine bromide (PEABr) solution is added to regulate the CsPbBr<sub>3</sub> crystalline quality of the film. The effects of PEABr on the perovskite crystallization process and device performance are systematically investigated. The results show that the introduction of PEABr can effectively optimize the CsPbBr<sub>3</sub>. The crystalline properties of the two-dimensional perovskite materials can improve the grain boundaries and improve their transport properties. The prepared perovskite solar cell with PEABr shows the highest power conversion efficiency of 8.25%, and it can maintain the efficiency of more than 90% when being stored for 1500 h under the condition of no encapsulation. Finally, stable, efficient and low-cost all-inorganic CsPbBr<sub>3</sub> solar cells without a hole layer are obtained.</sec>

Generation of Induced Pluripotent Stem Cells from Turner Syndrome (45XO) Fetal Cells for Downstream Modelling of Neurological Deficits Associated with the Syndrome.

Chromosomal aneuploidies cause severe congenital malformations including central nervous system malformations and fetal death. Prenatal genetic screening is purely diagnostic and does not elucidate disease mechanism. Although cells from aneuploid fetuses are valuable biological material bearing the chromosomal aneuploidy, these cells are short lived, limiting their use for downstream research experiments. Generation of induced pluripotent stem cell (iPSC) models is an effective method of cell preparation for perpetual conservation of aneuploid traits. They are self-renewing and differentiate into specialized cells reminiscent of embryonic development. Thus, iPSCs serve as excellent tools to study early developmental events. Turner syndrome (TS) is a rare condition associated with a completely or partially missing X chromosome. The syndrome is characterized by infertility, short stature, endocrine, metabolic, autoimmune and cardiovascular disorders and neurocognitive defects. The following protocol describes isolation and culturing of fibroblasts from TS (45XO) fetal tissue, generation of integration free TSiPSCs through delivery of episomal reprogramming plasmids by nucleofection followed by characterization. The reprogramming TSiPSCs were initially screened by live cell alkaline phosphatase staining followed by extensive probing for pluripotency biomarkers. Selected colonies were mechanically dissected, passaged several times and stable self-renewing cells were used for further experiments. The cells expressed pluripotency transcription factors OCT4, NANOG, SOX2, cell surface markers SSEA 4 and TRA1-81 typical of pluripotent stem cells. The original 45XO karyotype was retained post reprogramming. The TSiPSCs were able to form embryoid bodies and differentiate into cells of endoderm, mesoderm and ectoderm expressing lineage specific biomarkers ((SRY BOX17), (MYOSIN VENTRICULAR HEAVY CHAINα/β), (βIII TUBULIN)). The exogenous episomal plasmids were lost spontaneously and not detected after passage 15 in cells. These TSiPSCs are a valuable cellular resource for modelling defective molecular and cellular neurodevelopment causing neurocognitive deficits associated with Turner syndrome.

Cell Therapy for Anal Sphincter Incontinence: Where Do We Stand?

Anal sphincter incontinence is a chronic disease, which dramatically impairs quality of life and induces high costs for the society. Surgery, considered as the best curative option, shows a disappointing success rate. Stem/progenitor cell therapy is pledging, for anal sphincter incontinence, a substitute to surgery with higher efficacy. However, the published literature is disparate. Our aim was to perform a review on the development of cell therapy for anal sphincter incontinence with critical analyses of its pitfalls. Animal models for anal sphincter incontinence were varied and tried to reproduce distinct clinical situations (acute injury or healed injury with or without surgical reconstruction) but were limited by anatomical considerations. Cell preparations used for treatment, originated, in order of frequency, from skeletal muscle, bone marrow or fat tissue. The characterization of these preparations was often incomplete and stemness not always addressed. Despite a lack of understanding of sphincter healing processes and the exact mechanism of action of cell preparations, this treatment was evaluated in 83 incontinent patients, reporting encouraging results. However, further development is necessary to establish the correct indications, to determine the most-suited cell type, to standardize the cell preparation method and to validate the route and number of cell delivery.

Decomposition of Trace Li2CO3 During Charging Leads to Cathode Interface Degradation with the Solid Electrolyte LLZO

AbstractA major challenge for lithium‐containing electrochemical systems is the formation of lithium carbonates. Solid‐state electrolytes circumvent the use of organic liquids that can generate these species, but they are still susceptible to Li2CO3 formation from exposure to water vapor and carbon dioxide. It is reported here that trace quantities of Li2CO3, which are re‐formed following standard mitigation and handling procedures, can decompose at high charging potentials and degrade the electrolyte–cathode interface. Operando electrochemical mass spectrometry (EC–MS) is employed to monitor the outgassing of solid‐state batteries containing the garnet electrolyte Li7La3Zr2O12 (LLZO) and using appropriate controls CO2 and O2 are identified to emanate from the electrolyte–cathode interface at charging potentials > 3.8 V (vs Li/Li+). The gas evolution is correlated with a large increase in cathode interfacial resistance observed by potential‐resolved impedance spectroscopy. This is the first evidence of electrochemical decomposition of interfacial Li2CO3 in garnet cells and suggests a need to report “time‐to‐assembly” for cell preparation methods.

Fuel cell assemblies and preparation methods, therefore.

Fuel cell assemblies and preparation methods, therefore.

Acellular Wharton's Jelly, Potentials in T-Cell Subtypes Differentiation, Activation and Proliferation.

Purpose: Because of different potentials of T-cell subtypes in T-cell based cellular immunotherapy approaches such as CAR-T cell therapies; Regarding the high cost of the serum-free specific culture media, having distinct control on T-cell subset activation, expansion and differentiation seem crucial in T-cell expansion step of cell preparation methods. By the way, there was no clear data about the effect of acellular Wharton’s Jelly (AWJ) on T-cells expansion, activation or differentiation status. So, we have launched to study the effect of AWJ on T-cell’s immunobiological properties. Methods: CD3+ T-cells were isolated from healthy bone marrow allogeneic donors, sorted by FACS method and cultured on either routine phyto-hemagglutinin complemented and different concentrations of AWJ, lag phase and doubling time of the cells calculated from cell growth curve. After 3, 7 and 14-days T-cell subtypes cell markers and cell activity related genes expression rate have been evaluated by flow cytometry and real-time polymerase chain reaction (PCR) methods respectively. Results: AWJ in a 1:1 ratio compared with contemporary lymphocyte culture media showed significant activating and proliferative capacities. The introduced condition has not affected the frequency of CD4+ subpopulation of T-cells, but significantly increased even CD8+ cells and immune-activator genes in T-cells. The regulatory and memory subsets of T-cells in this study have not affected significantly. Conclusion: the study results revealed that AWJ can be utilized as a supportive substance to increase the memory properties of the T-cells, gives control to design a selective medium for expanding and differentiating memory T-cells, relatively.

Strategy for the anemia parameters demand management in primary care

This chapter will focus on the application of Raman spectroscopy to red blood cell (RBC) research providing a practical approach to methods of cell preparation for Raman spectroscopy and band assignment. The unusual properties of Hb in the highly concentrated environment of the RBC will be discussed and some applications of Raman spectroscopy to RBC analysis will be highlighted.

Preparation of cellular samples using graphene cover and air-plasma treatment for time-of-flight secondary ion mass spectrometry imaging.

We report on sample preparation methods based on plasma treatment for an improvement of multiple molecular ion images of cellular membranes in the ToF-SIMS method. The air-plasma treatment of fixed cellular samples efficiently removed the organic residues of any solutions used during sample preparation and improved the quality of ToF-SIMS images due to the resulting clean surface. We also studied cell preparation methods that combine single-layer graphene covering with air-plasma treatment to achieve a synergistic effect that eliminates background spectra by organic impurities while minimizing morphological cell deformation in a vacuum environmental analysis. When the cellular sample on the glass substrate is completely covered with the single-layer graphene, the cells trapped between the graphene and the substrate can effectively reduce morphological deformation by slow-dehydration. After slow-dehydration of cells is completed inside the graphene-cover, the covered graphene layer can be peeled off by air-plasma treatment, and the unwanted organic residues on the surface of cells and substrate can also be removed by plasma cleaning, thereby much improving ion imaging of cells with the ToF-SIMS method. It is confirmed that the cell samples in which the graphene-cover was removed by air-plasma treatment maintained their morphology well in comparison with the rapid air-dried cells in atomic force microscopy (AFM) and ToF-SIMS images.

Systematic optimization and evaluation of sample pretreatment methods for LC-MS-based metabolomics analysis of adherent mammalian cancer cells

An each-step-matched cell preparation method for LC-MS-based metabolomics analysis of adherent mammalian cancer cells was developed.

Micro-fabrication process of vapor cells for chip-scale atomic clocks

As the key part of chip-scale atomic clocks (CSACs), the vapor cell directly determines the volume, stability, and power consumption of the CSAC. The reduction of the power consumption and CSAC volumes demands the manufacture of corresponding vapor cells. This overview presents the research development of vapor cells of the past few years and analyzes the shortages of the current preparation technology. By comparing several different vapor cell preparation methods, we successfully realized the micro-fabrication of vapor cells using anodic bonding and deep silicon etching. This cell fabrication method is simple and effective in avoiding weak bonding strengths caused by alkali metal volatilization during anodic bonding under high temperatures. Finally, the vapor cell D2 line was characterized via optical-absorption resonance. According to the results, the proposed method is suitable for CSAC.

Isolation and Identification of Innate Lymphoid Cells (ILCs) for Immunotoxicity Testing.

Innate lymphoid cells (ILCs) comprise a family of innate immune cells that orchestrate mucosal immune responses: initiating, sustaining, and even curbing immune responses. ILCs are relatively rare (≤1% of lymphocytes in mucosal tissues), lack classical cell-surface markers, and can be divided into three subsets (type 1-3 ILCs) based on differences in cytokine production, phenotype, and developmental pathway. Because ILCs can only be identified by combinations of cell-surface markers and cytokine production, multicolor flow cytometry is the most reliable method to purify, characterize, and assess the functionality of ILCs. Here, we describe the methods for cell preparation, flow cytometric analysis, and purification of murine ILCs from the lung.

Comparing the invivo and invitro effects of hypoxia (3% O2) on directly derived cells from murine cardiac explants versus murine cardiosphere derived cells

Coronary heart disease (CHD) is still one of the main causes of death in the world, despite significant advances in clinical treatments. Stem cell transplantation methods have the potential to improve cardiac function and patients' outcome following heart attack, but optimal cell types, cell preparation methods and cell delivery routes are yet to be developed. Mammalian hearts contain a small fraction of progenitor cells which, in culture, migrate out of the cardiac explants, known as explant-derived cell (EDCs) and contribute to spheroids known as cardiospheres (Csphs). Following further culture and cell passaging, Csphs give rise to cardiosphere-derived cells (CDCs). EDCs, Csphs and CDCs show in vitro and in vivo angiogenesis and tissue regeneration in myocardial ischemia. However, CDC and Csph formation is time consuming, expensive and not always successful. Therefore, this study aims to compare EDCs with CDCs and assess the effect of hypoxic preconditioning on their pro-angiogenic potential. The data showed that preconditioning EDCs in hypoxic cell culture enhances cell growth, viability and expression of stem cell and pro-angiogenic markers more than CDCs. In vivo experiments using a sub-dermal matrigel plug assay showed that EDCs and CDCs alone have limited pro-angiogenic potential; however, hypoxic preconditioning of EDCs and CDCs significantly enhances this process. Further research will increase our understanding of cardiac stem cell mediated angiogenesis and improve clinical therapies for myocardial infarction (MI) patients.

An efficient dye sensitized solar cells based on SrTiO3 nanoparticles prepared from a new amine-modified sol-gel route

The nanoparticles of SrTiO3 synthesized via a new sol-gel method in the presence of different alkaline agents. In this preparation method used from various amines which play the role of capping agent for preparation of nano-size products. The optimized structures of SrTiO3 used in dye sensitized solar cell (DSSCs) in photo-anode. For improving the efficiency of DSSCs based on SrTiO3, these nanoparticle mixed with TiO2 as the best material in photo-anode of DSSCs. The photovoltaic behavior investigated in the as-prepared SrTiO3 based DSSCs. The best efficiency observed in 1:10 ratio for TiO2:SrTiO3, which improved the photovoltaic behavior of DSSCs about 45% in comparison to pure TiO2 in photo-anode. The composition of TiO2 with SrTiO3 introduces a suitable method for preparation of an efficient solar cell.