KR20110035213A - Cell signaling analysis method of single-cell - Google Patents
Cell signaling analysis method of single-cell Download PDFInfo
- Publication number
- KR20110035213A KR20110035213A KR1020090092836A KR20090092836A KR20110035213A KR 20110035213 A KR20110035213 A KR 20110035213A KR 1020090092836 A KR1020090092836 A KR 1020090092836A KR 20090092836 A KR20090092836 A KR 20090092836A KR 20110035213 A KR20110035213 A KR 20110035213A
- Authority
- KR
- South Korea
- Prior art keywords
- cell
- single cell
- cells
- signal
- jurkat
- Prior art date
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2527/00—Culture process characterised by the use of mechanical forces, e.g. strain, vibration
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2535/00—Supports or coatings for cell culture characterised by topography
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Hematology (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Urology & Nephrology (AREA)
- Wood Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Genetics & Genomics (AREA)
- Toxicology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Cell Biology (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to a signal analysis method of a single cell, and more particularly, preparing a fluorescent mammalian cell (Jurkat) for single cell signal analysis; And analyzing a single cell by a signal response from the single cell of the fluorescent mammalian cell (Jurkat). By using the signal analysis method of a single cell of the present invention, it is possible to understand the signal control mechanism of the cell through real-time response analysis.
Signal Analysis, Fluorescent Proteins, Nanoenvironments, Fluorescent Mammalian Cells (Jurkat)
Description
The present invention relates to a single cell signal analysis method.
Portable nano-based platform devices have been developed that integrate live single-cell isolation, capture, analysis, and diagnostics into small chips, and individual specificity to disease, in addition to the level of DNA or protein analysis, provides a single A direct correlation can be established through analysis of cell and cell ensemble, which can be quickly and accurately diagnosed in the medical field, which can be expected to prolong the lifespan of humans and greatly improve national health. (MC Park, JY Hur, KW Kwon, S.-H. Park and KY Suh.Pumpless, selective docking of yeast cells inside a microfluidic channel induced by receding meniscus.Lab Chip (2006), 6: 988-994) .
Cells in vivo constantly receive various stimuli from the external environment. Accurate detection of changing environmental stimuli and control of their corresponding cellular responses are the most important biological mechanisms for sustaining life. The detection of these stimuli and the control of the corresponding bioreactions take place through a number of signaling pathways consisting of protein interaction networks. The characteristics of biosignal systems are their diversity and selective specificity. Specific stimuli always give rise to corresponding specific biological responses, and the selective specificity of these signaling systems is maintained by the organic linkage of numerous protein interactions (see FIG. 1) (S.-H. Park, A. Zarrinpar and WA). Lim.Rewiring MAP kinase pathways using alternative scaffold assembly mechanisms.Science (2003), 299: 1061-1064; A. Zarrinpar, S.-H. Park and WA Lim. Optimization of specificity in a cellular protein interaction network by negative selection. Nature (2003), 426: 676-680). Therefore, in many cases, a malfunction of signal transmission may appear as a disease phenomenon such as cancer. In light of this importance, understanding the mechanisms by which cell signaling is regulated is very important.
For most cell-based methodologies used in most biological studies, data analysis is based on the assumption that individual cells in the cell population produce a uniform cellular response. In practice, however, if individual cells do not produce a uniform response, results analysis based on average measurements may lead to errors (see Figure 3). This error is caused by the differentiation of individual cells due to the mixing of the cytoplasm of each cell during the hemolysis process of the cell population. Indeed, whether or not an individual cell produces a uniform response is very rarely proved experimentally due to various technical problems.
Accordingly, the present inventors have been trying to integrate at the single cell level whether the signaling of individual cells in a cell group using nanobiotechnology is differentiated, and in the individual cells using nanotechnology, cell biology and molecular biological research techniques. The present invention was completed by observing flux (see FIG. 2), such as activity of signal transduction and signal adaption, over time, and variously clarifying the mechanism of control and regulation of signal transduction.
An object of the present invention is to provide a stochasticity of the cell signaling network by external stimulation by modeling the MAP kinase signaling system through a single cell analysis using a microwell structure.
The present invention comprises the steps of: i) preparing a fluorescent mammalian cell (Jurkat) for single cell signal analysis; And ii) analyzing a single cell by a signal response from the single cell of the fluorescent mammalian cell (Jurkat).
Hereinafter, the present invention will be described in detail.
In the single cell signal analysis method of the present invention, after the step of preparing the fluorescent mammalian cell (Jurkat), further comprising: building a nanoenvironment for cell capture using the fluorescent mammalian cell (Jurkat); And capturing a single cell from the nanoenvironment, wherein the construction of the nanoenvironment is more preferably using a microwell based on microfluidics, and the use of the microwell is photolithography. PDMS (polydimethyl siloxane) polymer is cured on the intaglio silicon wafer master fabricated by the process to produce embossed PDMS stamp, biocompatible UV curable polymer is coated on glass coverslip and embossed PDMS stamp is contacted with the polymer It is even more desirable to harden with a negative polymer microwell structure to form on the glass coverslip.
In addition, in the single cell signal analysis method of the present invention, the single cell analysis of the signal response is preferably to determine the stochasticity of the cell signal response by quantitative time-course analysis of the signal response of the living single cells, Single cell analysis of the signal response is most preferably analyzed by processing the PMA, measuring the brightness of the fluorescent protein.
Isolates single cells from cell populations, captures them at the single cell level on surfaces designed to resemble in vivo environments, and supplies isolated single cells with chemicals that can stimulate mechanical stress and various signaling systems Therefore, the construction of a nanoenvironmental system that can control and measure secretion, shape change, fluorescence, etc. of a single cell is essential for identifying pathologies and discovering therapeutic methods through single cell research (see FIG. 4).
The present invention models the ERK MAP kinase pathway, which mediates the cell proliferative response of Jurkat, a mammalian cell, in real time to observe the flux of signal transduction in individual cells and to identify various mechanisms of control and regulation of signal transduction.
MAP kinase signaling systems are well conserved in most eukaryotic life systems, from yeast to humans. In particular, the ERK MAP kinase signal transduction system is strictly regulated as a pathway deeply involved in cell growth, differentiation and proliferation. The mammalian cell Jurkat's ERK MAP kinase signaling system consists of Raf, which is a MAP kinase kinase (MAPKKK), MEK, which is a MAP kinase kinase (MAPKK), and ERK, a MAP kinase (MAPK). These signals are phosphorylated sequentially from cell surface receptors, and Raf, MEK, and ERK are also phosphorylated sequentially, and phosphorylated ERK activates lower transcription factors, and cell surface signals are transmitted to the nucleus. (See Figure 5).
The present inventors produced mammalian cells (Jurkat) expressing signaling activator protein and report fluorescent protein, in particular fluorescent mammalian cells (Jurkat) for single cell signal analysis. Specifically, plasmids encoding activator proteins and fluorescent proteins and antibiotic resistance genes (selection markers) were prepared and inserted into the chromosomes of mammalian cells (Jurkat). As a result, when intracellular signaling is initiated by external stimulation, the activator protein is phosphorylated, and the phosphorylated activator protein activates the transcription of the reporter fluorescent protein, thereby expressing the fluorescent protein. The expressed fluorescence can be visually observed using a fluorescence microscope.
In addition, the present inventors construct a new concept of precision nanoenvironment and capture single cells into the nanoenvironment platform using ultra-microfluidic network technology (see FIGS. 7 and 8), thereby living biosignal response by various signal transmissions. Single cells were measured at high speed and in real time. Compared to the existing DEP, sedimentation, hydrodynamic focusing, and optical tweezer methods, the method can capture a large area with a high probability and can minimize non-invasive impact on cells. In addition, the signal response of the living single cells was quantitatively time-course analyzed to determine whether stochasticity of the cell signal response (see FIG. 9).
We have prepared a microfluidics platform capable of capturing single cells in large areas through which a cell has been captured and analyzed at the single cell level with a greater than 90% probability. First, the cells were treated with PMA, which mediates the proliferative response of mammalian cell Jurkat, and then 3 hours later, the PMA-treated cells were captured on a microfluidic platform, and then the cell responses were analyzed after 10 hours (Fig. 10).
The inventors built a new concept of precise nanoenvironment and captured single cells into the nanoenvironment platform using ultra-fluid network technology. The present inventors measured high-speed, real-time measurements on living single cells of biosignal responses by various signal transductions. That is, the surface tension of the fluid in the microchannel was controlled to capture the cells at the desired position at the single cell level (see FIG. 8). In addition, the present inventors confirmed the stochasticity of the cell signal response by time-course analysis of the signal responses of living single cells quantitatively every 20 minutes (see FIG. 9). In addition, the inventors have prepared a microfluidics platform capable of capturing single cells in large areas, thereby capturing cells in large areas and analyzing them at the single cell level with a greater than 90% probability (see FIG. 10).
Through the real-time response analysis of single cells of the present invention, the signal flux mechanism and dynamics can be understood, and the activity, adaptation, and stochasticity of signal transduction are identified.
Hereinafter, the present invention will be described in detail by way of examples.
However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.
Example 1 Preparation of Signaling activator Protein and Report (Fluorescent) Protein Expressing Mammalian Cell (Fluorescent Mammalian Cell for Single Cell Signal Analysis)
The present inventors have investigated the ensemble average problem inherent in observing biological phenomena by modeling the ERK MAP kinase pathway of mammalian cell Jurkat through a new concept of single cell analysis, and whether the signaling of individual cells in the cell population is different. It was clarified.
To this end, we first inserted each plasmid encoding the activator protein and fluorescent protein into the chromosome of mammalian cell Jurkat for readout of the ERK MAP kinase pathway of mammalian cell Jurkat (FIG. 6).
Specifically, a plasmid encoding a Gal4 UAS-TATA promoter-fluorescent protein and a puromycin resistance gene (selection marker) was prepared. Plasmids pFR-Luc, pEGFP-N1, and pCDNA3.1 (+)-puro were used by those produced by others.
The vector pFR-Luc was cut with restriction enzymes Sac I and Pac I for 2 hours at 37 ° C. EGFP gene is a 'antisense primer 5'-tttttaattaattacttgtacagctcgtcca-3 described in the sequence number 2' sense primer 5'-aaagagctcaattctgcagtcgacggtac-3 is used as a template the plasmid pEGFP-N1 using the Pfu DNA polymerase and described in SEQ ID NO: 1 Amplification was performed by PCR. PCR temperature and time conditions were 95 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 1 minutes and performed 30 cycles. The PCR product was cut for 2 hours at 37 ° C. with restriction enzymes Sac I and Pac I, which was treated with vector and ligase enzyme for 15 minutes at room temperature to prepare pFR-EGFP plasmid.
Then, antisense primer 5'-aaagaattcttacttgtacagctcgtcca-3 are of the pFR-EGFP plasmid as a template and substrate with sense primer 5'-aaaagatctttctaattgtggtttgtccaa-3 'and SEQ ID NO: 4 described in SEQ ID NO: 3 by using a Pfu DNA polymerase Was amplified by PCR. PCR temperature and time conditions were 94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 1 minutes and performed 30 cycles. This PCR product was digested with restriction enzymes Bgl II and EcoR I at 37 ° C. for 2 hours. The vector PCDNA3.1 (+)-puro was also digested with restriction enzymes Bgl II and EcoR I at 37 ° C. for 2 hours. The cleaved vector and the PCR product were treated with ligase enzyme for 15 minutes at 25 ° C. to prepare PCDNA3.1 (+)-Gal4 UAS-TATA promoter-EGFP-puro plasmid.
Cells were transformed by injecting 10 μg of pFA2-Elk1 plasmid into mammalian cell Jurkat via electroporation. Electroporation conditions are 1400 V, 30 ms, once. In order to select stably transformed cells, G418 was incubated for about 2 weeks in a culture solution treated with a concentration of 600 µg / ml. The surviving cells were transformed by injecting 10 µg of the prepared plasmid PCDNA3.1 (+)-Gal4 UAS-TATA promoter-EGFP-puro under the electroporation conditions. In order to select stably transformed cells, the cells were cultured for one week in a culture solution treated with 600 μg / ml of G418 and 0.5 μg / ml of puromycin. At this time, the surviving cells were treated with PMA, and the cells fluorescing after 15 hours were sorted by FACS. Sorted cells were incubated in 96 well plates by diluting 0.5 cells into one well of a 96 well plate. After about two weeks, the cells cultured in each well of a 96 well plate were treated with PMA and after 15 hours, cells were selected to fluoresce and cultured these cells.
Example 2 Construction of Nanoenvironment for Cell Capture
The inventors built a new concept of precise nanoenvironment and captured single cells into the nanoenvironment platform using ultra-fluid network technology.
Specifically, in order to capture living cells at the level of single cells, a microwell structure suitable for the size of cells should be manufactured in an environment suitable for survival of cells. To this end, a capillary molding process was first used to form a microwell structure with a nanoliter capacity. In the above process, first, a PDMS stamp was fabricated by curing a PDMS polymer on an intaglio silicon wafer master fabricated by photolithography. Next, a biocompatible UV curable polymer was coated on the glass coverslip, an embossed PDMS stamp was contacted with the polymer, and cured with UV to form a negative polymer microwell structure on the laser coverslip.
Example 3 Single Cell Capture
The inventors of the present invention have attempted to measure a high-speed, real-time measurement of a biosignal response by various signal transductions in living single cells. Specifically, the surface tension of the fluid in the microchannels was controlled to capture the cells at desired locations on a single cell level. As shown in FIG. 8, it can be seen that cells are trapped in the micro wells with a probability of 90% or more. Compared to the DEP, sedimentation, hydrodynamic focusing, and optical tweezer methods, the method can capture a large area with high probability and can minimize non-invasive impact on the cells.
Example 4 Single Cell Analysis of Signal Response
The present inventors confirmed the stochasticity of the cell signal response by time-course analysis of the signal responses of living single cells quantitatively every 20 minutes. Specifically, the cells were treated with PMA, captured in the microwell, and the fluorescent protein expressed in the reporter gene was observed in real time using an fluorescence microscope (automated fluorescence microscopy) and photographed by a CCD camera. The photographed image was analyzed by measuring the brightness of the fluorescent protein expressed in a single cell using an image analysis computer program. Quantitative analysis of environmental stimuli at various concentrations and signal responses at different times of day based on the amount of expression of each fluorescent protein confirms stochasticity of the signal responses and whether each single cell exhibits a homogeneous response to a given stimulus. It was. On the basis of this, it was confirmed whether a uniform response to a specific signal response at the single cell level and whether an actual ensemble average problem exists. 9 shows a schematic flowchart for analyzing signal responses of single cells.
Example 5 Cell Capture and Analysis
We have prepared a microfluidics platform capable of capturing single cells in large areas through which a cell has been captured and analyzed at the single cell level with a greater than 90% probability. First, cells were treated with PMA and captured and analyzed after 10 hours of cell response (FIG. 10).
As shown in FIG. 10, the intensity of fluorescence varies from cell to cell, indicating that the cell's response is very non-uniform at the single cell level. The present inventors need further studies to find out whether this is an intrinsic problem of cells or due to external culture and capture conditions.
1 is a schematic diagram showing cell signaling using a biochemical network of signaling proteins,
2 is a kinetic model of signal response,
3 is an Ensemble average problem; In the case of having a heterogeneous population at a single cell level, the readout in the study of the bulk population is a measure of the average of the real response of each population as the readout.
4 are nanoenvironmental parameters important for single cell control and analysis,
5 is the ERK MAP kinase pathway of mammalian cells Jurkat,
Figure 6 is a schematic diagram showing the production of fluorescent mammalian cells (Jurkat) for single cell signal analysis,
7 is a process of manufacturing a nano-environmental platform having a microwell structure,
8 is a photograph of mammalian cells Jurkat captured in a single cell unit in a microwell having a diameter of 24 μm and a depth of 12 μm.
9 is a single cell signal response analysis flow chart,
10 is an example of cell capture and analysis at the single cell level.
<110> Seoul National University industry Foundation <120> Cell Signaling Analysis Method of Single-Cell <130> 2009p-9-202 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> pEGFP-N1 sense primer <400> 1 aaagagctca attctgcagt cgacggtac 29 <210> 2 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> pEGFP-N1 antisense primer <400> 2 tttttaatta attacttgta cagctcgtcc a 31 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> pFR-EGFP sense primer <400> 3 aaaagatctt tctaattgtg gtttgtccaa 30 <210> 4 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> pFR-EGFP antisense primer <400> 4 aaagaattct tacttgtaca gctcgtcca 29
Claims (6)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020090092836A KR20110035213A (en) | 2009-09-30 | 2009-09-30 | Cell signaling analysis method of single-cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020090092836A KR20110035213A (en) | 2009-09-30 | 2009-09-30 | Cell signaling analysis method of single-cell |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| KR20110035213A true KR20110035213A (en) | 2011-04-06 |
Family
ID=44043585
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020090092836A KR20110035213A (en) | 2009-09-30 | 2009-09-30 | Cell signaling analysis method of single-cell |
Country Status (1)
| Country | Link |
|---|---|
| KR (1) | KR20110035213A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20150143031A (en) * | 2014-06-13 | 2015-12-23 | 한국과학기술원 | Bio material capturing structure with polymer layer, and apparatus and method for collecting bio material carrier sellectively using the same |
| WO2016099207A1 (en) * | 2014-12-18 | 2016-06-23 | Unist (Ulsan National Institute Of Science And Technology | Device and method for single cell screening based on inter-cellular communication |
| WO2024187710A1 (en) * | 2023-03-14 | 2024-09-19 | 常州大学 | Method for simulating sensitive response of jurkat cells in mechanical microenvironments |
-
2009
- 2009-09-30 KR KR1020090092836A patent/KR20110035213A/en not_active Application Discontinuation
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20150143031A (en) * | 2014-06-13 | 2015-12-23 | 한국과학기술원 | Bio material capturing structure with polymer layer, and apparatus and method for collecting bio material carrier sellectively using the same |
| WO2016099207A1 (en) * | 2014-12-18 | 2016-06-23 | Unist (Ulsan National Institute Of Science And Technology | Device and method for single cell screening based on inter-cellular communication |
| US10385306B2 (en) | 2014-12-18 | 2019-08-20 | Unist(Ulsan National Institute Of Science And Technology) | Device and method for single cell screening based on inter-cellular communication |
| WO2024187710A1 (en) * | 2023-03-14 | 2024-09-19 | 常州大学 | Method for simulating sensitive response of jurkat cells in mechanical microenvironments |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Sivagnanam et al. | Exploring living multicellular organisms, organs, and tissues using microfluidic systems | |
| Xiong et al. | Recent developments in microfluidics for cell studies | |
| CN103502424B (en) | For the nanopipette device of manipulating cells | |
| CN105164531B (en) | Nanopippettes device and method for subcellular analysis | |
| Unal et al. | Micro and nano-scale technologies for cell mechanics | |
| JP6858769B2 (en) | Cell culture, transport, and investigation | |
| Roper | Cellular analysis using microfluidics | |
| Li et al. | Exclusive liquid repellency: an open multi-liquid-phase technology for rare cell culture and single-cell processing | |
| Liu et al. | Microfluidics in systems biology—hype or truly useful? | |
| Agrawal et al. | Devices and techniques used to obtain and analyze three‐dimensional cell cultures | |
| Sima et al. | 3D biomimetic chips for cancer cell migration in nanometer-sized spaces using “Ship-in-a-Bottle” femtosecond laser processing | |
| Chen et al. | Dynamic microfluidic cytometry for single-cell cellomics: High-throughput probing single-cell-resolution signaling | |
| Corbin et al. | Measuring physical properties of neuronal and glial cells with resonant microsensors | |
| Lee et al. | Dynamic culture and selective extraction of target microbial cells in self-assembled particle membrane-integrated microfluidic bioreactor array | |
| Charles et al. | High-temporal-resolution smFISH method for gene expression studies in Caenorhabditis elegans embryos | |
| KR20110035213A (en) | Cell signaling analysis method of single-cell | |
| Zhao et al. | Rapid generation of hybrid biochemical/mechanical cues in heterogeneous droplets for high-throughput screening of cellular responses | |
| Dolan et al. | Techniques for studying mechanobiology | |
| KR100968640B1 (en) | Cell Signaling Analysis Method of Single-Cell | |
| Lin et al. | Microfluidic platform for omics analysis on single cells with diverse morphology and size: A review | |
| Kerk et al. | Recent advances of integrated microfluidic suspension cell culture system | |
| KR20110035212A (en) | Fluorescent mammalian stable cell for single-cell signal analysis, and preparation method thereof | |
| Fakhoury et al. | Microsystems for controlled genetic perturbation of live Drosophila embryos: RNA interference, development robustness and drug screening | |
| JP6176703B2 (en) | Hepatocyte culture substrate and hepatocyte culture method | |
| Grünberger | Single cell analysis of microbial production strains in microfluidic bioreactors |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A201 | Request for examination | ||
| E902 | Notification of reason for refusal | ||
| E601 | Decision to refuse application | ||
| J201 | Request for trial against refusal decision | ||
| J301 | Trial decision |
Free format text: TRIAL DECISION FOR APPEAL AGAINST DECISION TO DECLINE REFUSAL REQUESTED 20120525 Effective date: 20131125 |