Single-cell Isolation Technique Research Articles

Single-Cell Isolation Microfluidic Chip Based on Thermal Bubble Micropump Technology

The isolation of single cells is essential for the development of single cell analysis methods, such as single-cell sequencing, monoclonal antibodies, and drug development. Traditional single-cell isolation techniques include flow cytometry (FACS), laser capture microdissection (LCM), micromanipulation, etc., but their operations are complex and have low throughput. Here, we present a microfluidic chip that can isolate individual cells from cell suspension and release them onto a well plate. It uses thermal bubble micropump technology to drive the fluid flow, and single-cell isolation is achieved by matching the flow resistance of the flow channel. Therefore, injection pumps and peristaltic pumps are not required for cell loading. Because of its small size, we can integrate hundreds of single-cell functional modules, which makes high-throughput single-cell isolation possible. For polystyrene beads, the capture rate of the single bead is close to 100%. Finally, the method has been applied to cells, and the capture rate of the single cell is also about 75%. This is a promising method for single-cell isolation.

Single-cell RNA-sequencing of retrieved human oocytes and eggs in clinical practice and for human ovarian cell atlasing.

With the advancement of single-cell separation techniques and high-throughput sequencing platforms, single-cell RNA-sequencing (scRNA-seq) has emerged as a vital technology for understanding tissue and organ systems at cellular resolution. Through transcriptional analysis, it is possible to characterize unique or rare cell types, interpret their interactions, and reveal novel functional states or shifts in developmental stages. As such, this technology is uniquely suited for studying the cells within the human ovary. The ovary is a cellularly heterogeneous organ that houses follicles, the reproductive and endocrine unit that consists of an oocyte surrounded by hormone-producing support cells, as well as many other cell populations constituting stroma, vasculature, lymphatic, and immune components. Here we review studies that have utilized scRNA-seq technology to analyze cells from healthy human ovaries and discuss the single-cell isolation techniques used. We identified two overarching applications for scRNA-seq in the human ovary. The first applies this technology to investigate transcriptional differences in oocytes/eggs from patients undergoing in vitro fertilizationtreatments to ultimately improve clinical outcomes. The second utilizes scRNA-seq for the pursuit of creating a comprehensive single-cell atlas of the human ovary. The knowledge gained from these studies underscores the importance of scRNA-seq technologies in unlocking a new biological understanding of the human ovary.

In vivo single microglial cell isolation after intracerebral hemorrhage in mice

Failure to translate promising potential therapeutics for intracerebral hemorrhage (ICH) partially results from limited understanding of cellular mechanisms underlying brain injury and repair. Understanding neural repair mechanisms after brain injury requires intricate comprehension of microglial behavior; however, studying individual microglial cell behavior is challenging. Further single cell isolation techniques may be an excellent means to expand known differences in male and female microglial cell response to ICH. In this study, 24 h after intrastriatal collagenase injection, one male and one female CX3CR1-GFP mouse underwent ex vivo microglial cell isolation via micropipette from perihematomal regions and equivalent location of contralateral striata. After cell collection, individual and grouped cell samples underwent reverse transcription and analyses for gene expression using Fluidigm RT-PCR technology. Data were analyzed by t-tests and visualized as a heatmap of the log2 Ct values. Gene expression assays were chosen for target-specific amplification, including markers of M1 pro-inflammatory microglial phenotype (i.e., Tnf, Il6, Fcgr3/CD16), M2 anti-inflammatory markers (i.e., Mrc1/CD206, Arg1, Tgfb1), and genes involved in the toll-like receptor pathway (i.e., Tlr2, Tlr4 and Myd88). Greater number of individual microglia cells expressed Mcr1, Tlr2, and Arg1 in perihematomal tissue than in contralateral hemispheres. Additionally, more male microglia expressed Myd88, Tlr2, Il6, and Arg1 than did female microglia. Single cell microglial isolation is feasible after in vivo rodent ICH. Differential gene expression can be detected between individual cells from different brain regions and experimental conditions. Cell-specific analyses will contribute to improved understanding of microglial roles in both post-ICH pathogenesis and recovery.

Transcriptomic profiling of single circulating tumor cells provides insight into human metastatic gastric cancer

Transcriptome analysis of circulating tumor cells (CTCs), which migrate into blood vessels from primary tumor tissues, at the single-cell level offers critical insights into the biology of metastasis and contributes to drug discovery. However, transcriptome analysis of single CTCs has only been reported for a limited number of cancer types, such as multiple myeloma, breast, hepatocellular, and prostate cancer. Herein, we report the transcriptome analysis of gastric cancer single-CTCs. We utilized an antigen-independent strategy for CTC isolation from metastatic gastric cancer patients involving a size-dependent recovery of CTCs and a single cell isolation technique. The transcriptomic profile of single-CTCs revealed that a majority of gastric CTCs had undergone epithelial-mesenchymal transition (EMT), and indicated the contribution of platelet adhesion toward EMT progression and acquisition of chemoresistance. Taken together, this study serves to employ CTC characterization to elucidate the mechanisms of chemoresistance and metastasis in gastric cancer.

Advances in Single-Cell Printing.

Single-cell analysis is becoming an indispensable tool in modern biological and medical research. Single-cell isolation is the key step for single-cell analysis. Single-cell printing shows several distinct advantages among the single-cell isolation techniques, such as precise deposition, high encapsulation efficiency, and easy recovery. Therefore, recent developments in single-cell printing have attracted extensive attention. We review herein the recently developed bioprinting strategies with single-cell resolution, with a special focus on inkjet-like single-cell printing. First, we discuss the common cell printing strategies and introduce several typical and advanced printing strategies. Then, we introduce several typical applications based on single-cell printing, from single-cell array screening and mass spectrometry-based single-cell analysis to three-dimensional tissue formation. In the last part, we discuss the pros and cons of the single-cell strategies and provide a brief outlook for single-cell printing.

Target pollen isolation using automated infrared laser-mediated cell disruption

Single-cell analysis is important to understand how individual cells work and respond at the cell population level. Experimental single-cell isolation techniques, including dilution, fluorescence-activated cell sorting, microfluidics, and micromanipulation, have been developed in recent decades. However, such applications typically require large cell populations and skilled professionals. Additionally, these methods are unsuitable for sequential analysis before and after cell isolation. In this study, we propose a method for target cell isolation using automated infrared laser-mediated disruption of pollen grains in pollen populations. Germination of the target pollen was observed at the same location as that before laser irradiation, and germinated pollen grains were enriched in the cell population. Pollination of laser-irradiated bulk pollen populations also showed that the target pollen preferentially germinated on the stigma. This method is expected to facilitate physiological analyses of target cells at the single-cell level and effectively produce seeds derived from target pollen.

The Isolation and Deep Sequencing of Mitochondrial DNA.

In recent years, next-generation sequencing (NGS) has become a powerful tool for studying both inherited and somatic heteroplasmic mitochondrial DNA (mtDNA) variation. NGS has proved particularly powerful when combined with single-cell isolation techniques, allowing the investigation of low-level heteroplasmic variants both between cells and within tissues. Nevertheless, there remain significant challenges, especially around the selective enrichment of mtDNA from total cellular DNA and the avoidance of nuclear pseudogenes. This chapter summarizes the techniques needed to enrich, amplify, sequence, and analyse mtDNA using NGS .

Methods for mammalian single cell research - a review

Basic research in life science and medicine has dug into single cell level in recent years. Single-cell analysis offers to understand life from diverse perspectives and is used to profile cell heterogeneity to investigate mechanism of diseases. Single cell technologies have also found applications in forensic medicine and clinical reproductive medicine, while the techniques are rapidly evolving and have become more and more sophisticated. In this article, we reviewed various single cell isolation techniques and their pros and cons, including manual cell picking, laser capture microdissection and microfluidics, as well as analysis methods for DNA, RNA and protein in single cell. In addition, we summarized major up-to-date single cell research achievements and their potential applications.

Mitochondrial dysfunction in dopaminergic neurons differentiated from exfoliated deciduous tooth-derived pulp stem cells of a child with Rett syndrome

Rett syndrome is an X-linked neurodevelopmental disorder associated with psychomotor impairments, autonomic dysfunctions and autism. Patients with Rett syndrome have loss-of-function mutations in MECP2, the gene encoding methyl-CpG-binding protein 2 (MeCP2). Abnormal biogenic amine signaling and mitochondrial function have been found in patients with Rett syndrome; however, few studies have analyzed the association between these factors. This study investigated the functional relationships between mitochondria and the neuronal differentiation of the MeCP2-deficient stem cells from the exfoliated deciduous teeth of a child with Rett syndrome. An enrolled subject in this study was a 5-year-old girl carrying a large deletion that included the methyl-CpG-binding domain, transcriptional repression domain, and nuclear localization signal of MECP2. Using the single-cell isolation technique, we found that the two populations of MeCP2-expressing and MeCP2-deficient stem cells kept their MECP2 expression profiles throughout the stages of cell proliferation and neuronal differentiation in vitro. Neurite outgrowth and branching were attenuated in MeCP2-deficient dopaminergic neurons. MeCP2-deficient cells showed reduced mitochondrial membrane potential, ATP production, restricted mitochondrial distribution in neurites, and lower expression of a central mitochondrial fission factor, dynamin-related protein 1 than MeCP2-expressing cells. These data indicated that MeCP2-deficiency dysregulates the expression of mitochondrial factors required for the maturation of dopaminergic neurons. This study also provides insight into the pathogenic mechanism underlying dysfunction of the intracerebral dopaminergic signaling pathway in Rett syndrome.

Abstract 2052: Chemotherapy sensitivity, cytokine IL-8 level, and genetic expression mRNA IL-8 in gastric cancer treatment response prediction

Abstract Aims: To determine chemotherapy sensitivity in primary gastric cancer cell isolated from Thai gastric cancer patients against anticancer drugs. To search for the correlation of interleukin-6 and interleukin-8 levels in primary gastric cancer cell culture in comparison with tissue mRNA IL-8 gene expression and drug response or resistance. Method: The control background of corpus normal epithelial and gastric adenocarcinoma biopsy tissues were treated for primary cell culture with single cell isolation technique. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was performed to ex vivo examine the chemotherapy sensitivity to three anticancer drug, cisplatin, oxaliplatin, 5-Fluorouracil , and Irinotecan. Biopsy tissues from tumor site were collected before chemotherapy treatment in 63 patient who received neo-adjuvant chemotherapy by FOLFOX IV regimen due to advance stage III and IV in both resectable and un-resectable cases. The IL-8 mRNA gene expression was reported by relative quantitation real time RT-PCR. ELISA technique was performed to investigate the cytokine level from primary cultures taken from 41 gastric cancer patients who undergone biopsy from Maharaj Nakorn Chiang Mai Hospital during year 2014 to 2016. The clinical data base was parallel collected and analysed in correlation with clinical response and ex-vivo response. Results: Each patient has different sensitivity pattern to the standard anticancer drugs. There are no significantly direct correlations or difference between cytokine IL-8 levels or mRNA IL-8 expression, and the chemotherapy sensitivity to the actual drugs that are being used for treatment of gastric adenocarcinoma in individual patients. However, the chemotherapy resistance to all drugs in primary gastric cancer cell culture has trend to increase after the third cycle of chemotherapy treatment when compared to prior cell treatment with fluctuation of cytokine level. Different cytotoxic drug gives the same response result for chemrsensitivity test in this population. The author observed the down regulation of tissue mRNA IL-8 expression and cytokine level in the good responders. Conclusion: The results provide an additional basis of information for primary chemotherapy selection in the individualised treatment for advance gastric adenocarcinoma patients. Therefore, the author suggest to select the less toxicity cytotoxic drug for being primary chemotherapy of choice. Cytokine gene level and mRNA IL-8 gene expression show individually significantly change after chemotherapy treatment in good responder. This result may lead us to find some new targeted therapy or additional treatment for its suppression. Citation Format: Sirikan Limpakan (Yamada). Chemotherapy sensitivity, cytokine IL-8 level, and genetic expression mRNA IL-8 in gastric cancer treatment response prediction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2052. doi:10.1158/1538-7445.AM2017-2052

Advances in single-cell RNA sequencing and its applications in cancer research.

Unlike population-level approaches, single-cell RNA sequencing enables transcriptomic analysis of an individual cell. Through the combination of high-throughput sequencing and bioinformatic tools, single-cell RNA-seq can detect more than 10,000 transcripts in one cell to distinguish cell subsets and dynamic cellular changes. After several years’ development, single-cell RNA-seq can now achieve massively parallel, full-length mRNA sequencing as well as in situ sequencing and even has potential for multi-omic detection. One appealing area of single-cell RNA-seq is cancer research, and it is regarded as a promising way to enhance prognosis and provide more precise target therapy by identifying druggable subclones. Indeed, progresses have been made regarding solid tumor analysis to reveal intratumoral heterogeneity, correlations between signaling pathways, stemness, drug resistance, and tumor architecture shaping the microenvironment. Furthermore, through investigation into circulating tumor cells, many genes have been shown to promote a propensity toward stemness and the epithelial-mesenchymal transition, to enhance anchoring and adhesion, and to be involved in mechanisms of anoikis resistance and drug resistance. This review focuses on advances and progresses of single-cell RNA-seq with regard to the following aspects:1. Methodologies of single-cell RNA-seq2. Single-cell isolation techniques3. Single-cell RNA-seq in solid tumor research4. Single-cell RNA-seq in circulating tumor cell research5. Perspectives

Abstract 2091: IL-8 mRNA gene expression up-regulated level in both primary culture cell and tissue biopsy from gastric cancer patient without Helicobacter pylori infection and down- regulated level predict clinical response after chemo

Abstract Background: There is some recent in vitro study reported that IL-8 mRNA gene had over expression when Helicobacter pylori was co- infected. In the Northern Thai population, we found independently IL-8 mRNA gene expression significantly rising level in gastric tissue biopsies when comparing to AGS cell line, and mainly in non-cag A H. pylori infection or without H. pylori infection. Aim: The author aim to demonstrate the IL-8 mRNA gene expression level in vitro and in vivo study in the same host in order to see the change of its level in primary culture cell in same host gastric cancer patients, tissue biopsy, and AGS cell line. The comparative level before and after chemotherapy treatment were targeted for clinical response prediction. Material and Methods: The control background of corpus normal epithelial and gastric adenocarcinoma biopsy tissues were treated for primary cell culture with single cell isolation technique. The histopathology, background atrophy, and H. pylori infection was confirmed. Biopsy tissues from tumor site also were collected at the same time before and after chemotherapy treatment in patient who received neo-adjuvant chemotherapy by FOLFOX IV regimen due to locally advance stage and unresectable tumor. The IL-8 mRNA gene expression was tested by relative quantitation real time RT-PCR. Results: IL-8 mRNA gene expression can be significantly higher up-regulated level to 1.5-2.2 folds in gastric cancer tissue biopsy comparing to from 0.35-1.16 fold of primary culture cell without H. pylori infection when comparing to normal background of gastric epithelial tissue, and AGS cell line. The IL-8 mRNA gene expression down-regulated level is also remark decreased and/or undetected after chemotherapy treatment in correlation with patients who had well clinical response as a preliminary result. Conclusion: We first demonstrated that the IL-8 mRNA gene expression is detected in primary gastric adenocarcinoma cell and its up-regulated level in tissue biopsies independently related to H. pylori infection in the same gastric cancer host background. The pilot sample size is needed to confirm in further clinical trial on chemotherapy as a new biomarker for advance gastric cancer treatment. Citation Format: Sirikan Limpakan (Yamada), ML.Bandhuphat Chakrabandhu, Shunji Kato, Pattama Wongsirisin. IL-8 mRNA gene expression up-regulated level in both primary culture cell and tissue biopsy from gastric cancer patient without Helicobacter pylori infection and down- regulated level predict clinical response after chemo [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2091. doi:10.1158/1538-7445.AM2015-2091

Abstract P1-04-03: Genome-wide analysis of copy number variations and mutation profiles of single circulating tumour cells using massively parallel paired-end sequencing

Abstract INTRODUCTION Recent advances in single cell isolation techniques and next generation sequencing (NGS) have paved the way for the genome-wide molecular analysis of individual circulating tumour cells (CTCs) in patients with metastatic carcinomas. Here we present the results of a pilot study evaluating the feasibility and reliability of NGS of single CTC from whole blood samples. MATERIALS & METHODS Single cells of the human breast cancer cell line HCC38 were harvested from spiked blood samples in a semi-automated workflow consisting of immunomagnetic enrichment using the CellSearch system and dielectrophoretic cell sorting using the DEPArray system. DNA was isolated and amplified using the Ampli1 whole genome amplification (WGA) kit and subjected to low-coverage genome-wide paired-end sequencing for copy number variation (CNV) analysis and targeted re-sequencing of 200 cancer-related genes for somatic mutation analysis. RESULTS Single-cell WGA products of four HCC38 cells were subjected to whole genome sequencing for CNV analysis. Average coverage depth was 0,68x. At a binning window of 50 kb, detection results of CNVs in single-cell samples were highly consistent (>81% copy number concordance per bin genome wide) with CNV profiles from non-amplified multi-cell samples of the same cell line. We could demonstrate that part of the discordance was due to the acquisition of novel DNA-rearrangements in the single cells. Three of the single-cell WGA products were additionally subjected to targeted re-sequencing for mutation analysis of 200 selected genes, of which the analysis is currently ongoing. DISCUSSION Our study demonstrates the feasibility of a comprehensive genome-wide CNV analysis and targeted mutation analysis using NGS of single tumour cells isolated from whole blood samples in a highly automated isolation workflow. This approach provides a robust framework for the study of intercellular heterogeneity within the CTC population in blood samples of patients with (metastatic) breast cancer. In addition, our results document the extent of WGA-induced bias of a recently commercialized PCR-based WGA kit. These authors contributed equally to the data presented in this abstract. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-04-03.

Identification and isolation of active N2O reducers in rice paddy soil

Dissolved N(2)O is occasionally detected in surface and ground water in rice paddy fields, whereas little or no N(2)O is emitted to the atmosphere above these fields. This indicates the occurrence of N(2)O reduction in rice paddy fields; however, identity of the N(2)O reducers is largely unknown. In this study, we employed both culture-dependent and culture-independent approaches to identify N(2)O reducers in rice paddy soil. In a soil microcosm, N(2)O and succinate were added as the electron acceptor and donor, respectively, for N(2)O reduction. For the stable isotope probing (SIP) experiment, (13)C-labeled succinate was used to identify succinate-assimilating microbes under N(2)O-reducing conditions. DNA was extracted 24 h after incubation, and heavy and light DNA fractions were separated by density gradient ultracentrifugation. Denaturing gradient gel electrophoresis and clone library analysis targeting the 16S rRNA and the N(2)O reductase gene were performed. For culture-dependent analysis, the microbes that elongated under N(2)O-reducing conditions in the presence of cell-division inhibitors were individually captured by a micromanipulator and transferred to a low-nutrient medium. The N(2)O-reducing ability of these strains was examined by gas chromatography/mass spectrometry. Results of the SIP analysis suggested that Burkholderiales and Rhodospirillales bacteria dominated the population under N(2)O-reducing conditions, in contrast to the control sample (soil incubated with only (13)C-succinate). Results of the single-cell isolation technique also indicated that the majority of the N(2)O-reducing strains belonged to the genera Herbaspirillum (Burkholderiales) and Azospirillum (Rhodospirillales). In addition, Herbaspirillum strains reduced N(2)O faster than Azospirillum strains. These results suggest that Herbaspirillum spp. may have an important role in N(2)O reduction in rice paddy soils.

Isolation of Oligotrophic Denitrifiers Carrying Previously Uncharacterized Functional Gene Sequences

Oligotrophic denitrifying bacteria, including those belonging to the genera Herbaspirillum, Azospirillum, and Bradyrhizobium, were obtained using a single-cell isolation technique. The taxonomic composition of the denitrifier population was similar to those assessed by previous culture-independent studies. The sequencing of nitrite reductase and N(2)O reductase genes of these strains revealed previously unknown links between 16S rRNA and the denitrification-functional gene phylogenies. In particular, we identified Bradyrhizobium strains that harbor nirS sequences previously detected only in culture-independent studies.

Single-cell analysis and isolation for microbiology and biotechnology: methods and applications

Various single-cell isolation techniques, including dilution, micromanipulation, flow cytometry, microfluidics, and compartmentalization, have been developed. These techniques can be used to cultivate previously uncultured microbes, to assess and monitor cell physiology and function, and to screen for novel microbiological products. Various other techniques, such as viable staining, in situ hybridization, and those using autofluorescence proteins, are frequently combined with these single-cell isolation techniques depending on the purpose of the study. In this review article, we summarize currently available single-cell isolation techniques and their applications, when used in combination with other techniques, in microbiological and biotechnological studies.

Isolating Human Islets of Langerhans Causes Loss of Decay Accelerating Factor (CD55) on β-Cells

It has previously been reported that human decay accelerating factor (DAF; CD55) is not expressed on cells isolated from human islets. We have investigated if this absence is caused by the islet isolation procedure and/or the single cell isolation technique. We focused on loss of DAF expression on beta-cells within the intact islet and on isolated individual beta-cells. We established that DAF was expressed in islets and on beta-cells prior to isolation by in situ analysis in the intact pancreas. In situ immunohistochemistry (IHC) was used to examine DAF expression on human pancreatic islets and isolated islets. A reverse transcriptase-polymerase chain reaction (RT-PCR) specific for human DAF mRNA was developed to measure mRNA levels in situ in islets within the intact pancreas, isolated islets, and purified beta-cells. beta-Cells were purified by fluorescence-activated cell sorting. DAF protein expression on these purified cells was measured using flow cytometry. Expression of DAF protein was present on the islets, including beta-cells within the human pancreas; however, comparative data from IHC and flow cytometry revealed the absence of DAF protein on beta-cells in both isolated islets and single cell preparations. Furthermore, compared to mRNA levels detected by in situ RT-PCR in the intact pancreas and in human HEK 293 cells, isolated islets, and purified human beta-cells showed downregulation of DAF mRNA. mRNA was detectable in both of these preparations by RT-PCR; levels were lower following both the islet isolation process (53%) and single cell preparation (a further 62%) compared to HEK 293 controls. Human islet allotransplantation might be more successful if either de novo transfer of DAF onto the isolated islets or novel techniques for islet isolation preserving DAF could be developed.

Dual variant of Epstein-Barr virus in Hodgkin/Reed-Sternberg cells: single-cell PCR study on latent membrane protein-1 gene.

Isolation of single cells permits analysis of DNA or RNA from individual cells among heterogeneous populations. This technique is particularly useful in the study of classical Hodgkin's lymphoma (cHL) due to the scarcity of H/RS tumor cells among large numbers of reactive leukocytes. In a previous study, we found a high frequency of dual LMP-1 variant (concurrent presence of deleted and nondeleted variants) in cHL from whole-tissue sections. For the present study, we applied a single-cell isolation technique to determine the LMP-1 oncogene variant in EBV-associated H/RS cells. Five cases of EBV-infected cHL, containing nondeleted (n=1), deleted (n=1) and dual infection (n=3) based on whole-tissue section analysis, were selected for study. Paraffin-embedded tissue sections were stained with antibody to LMP-1 and positively stained H/RS cells isolated using a semiautomated micromanipulator. Each isolated single cell was subjected to PCR for amplification of the LMP-1 gene flanking the 30 bp deletion region and Xho1 restriction site. Cases with either nondeleted variant or the deleted variant showed similar LMP-1 variant expression in isolated single H/RS cells. However, 1 of the 3 cases with dual variants showed only the deleted variant in H/RS cells. The other 2 cases showed mixed patterns of deleted, nondeleted and dual LMP-1 variants in isolated single H/RS cells. All cases showed loss of the Xho1 restriction site, with the exception of the case with nondeleted LMP-1. Results of single-H/RS cell analysis of the Xho1 restriction site concur with those of whole-tissue section amplification. A mixed pattern of LMP-1 variants was observed in isolated H/RS cells, and it is speculated that this is due to the accumulation of mutation and deletion events.

Y specific sequence gene analysis of single fetal nucleated erythroblasts from the peripheral blood of pregnant women.

The single cell isolation technique was used to detect fetal nucleated erythroblasts (FNRBCs) at a single cell level from the peripheral blood of pregnant women in order to investigate the feasibility of this method for noninvasive prenatal diagnosis. Single fetal nucleated erythroblasts were isolated from the peripheral blood samples from 51 pregnant women (14 to 26 weeks of gestation) by micromanipulation techniques after density gradient centrifugation. Nested polymerase chain reaction method was used to amplify the SRY gene. It was found that the concordance rate of amplification results with real fetal sex was 82.61%. The sensitivity and specificity were 80% and 87.50% respectively. It was suggested that it is feasible and promising in non invasive prenatal diagnosis to detect fetal nucleated erythroblasts at a single cell level by using micromanipulation techniques.

Membrane currents in cardiac pacemaker tissue

The present work is a brief survey of the mechanism of the cardiac pacemaker in sinoatrial node cells. Information on the pacemaker mechanism in cardiac tissue has been greatly enhanced by the development of the single cell isolation technique and the patch clamp technique. These methods circumvent to a large extent the difficulties involved in voltage clamping multicellular preparations. The calcium current (ICa), delayed rectifier potassium current (IK), transient outward current (Ito;IA), and the hyperpolarization activated inward current (Ih or If) were found both in whole cell preparations and in single channel analysis. The physiological significance of these currents, together with the exchange current systems for the pacemaker depolarization are discussed.