SYSTEM FOR TRACKING AND OBSERVING EXTRACELLULAR VESICLES

The present invention refers to mass transfer for observation of the microfluidic chip and cell-to-cell-to-search using the same observation method relates to mass transfer, the donor cell receptor, extracellular vesicle motion and receiving cells receiving part and, said donor cell receptor extracellular vesicle the mobile portion in said receiving portion receiving said emitted [eyk[eyk] Cow petty slope (gradient) according to move towards a receiving cell generates said cells and communicate respectively with said donor cells by, extracellular cell-vesicle delivery and extracellular vesicle delivery embodiment method is tracked change between observation and Image into the server can be extracellular vesicle tracking are disclosed.

The present invention provides a cell-to-movement of matter in living cells between the microfluidic device and Image embodiment can be quickly and efficiently performing cell-to-mass transfer process based on development of technique are disclosed.

Hereinafter disclosed microfluidic chip has a construction and according to name but for example described for example, which are not limited to the, invention should not interpreted for understanding and interpreting corresponding configuration.

Donor cell receptor part extracellular vesicle tracking a monitoring system of the present invention, the donor and recipient cell receptor extracellular vesicle moveable portion from the upper portion, said donor cell receptor extracellular vesicle the mobile portion in said receiving portion receiving said emitted [eyk[eyk] Cow petty tilt (gradient) according to move towards a receiving cell generates said donor cells and said cells respectively communicate with each other.

Extracellular vesicle (extracellular vesicle, EV) cells is created in cells is emitted to the outside as a vesicle, etc. [eyk[eyk] Cow petty (exosome) and fine vesicle (microvesicle). The signals between which are known to involving in extracellular vesicle cells, cell differentiation, immune cell activation, an alteration in inflammation, of malignant cancer, cancer metastasis in a signal involving in various phenomena such as is known. Extracellular vesicle from a cell number derived proteins and RNA which, particular diseases such as cancer specific proteins and corresponding diseases caused from extracellular vesicle number can be utilized even for diagnosis of illnesses is RNA.

Donor cells (donor cells) cells receiving extracellular vesicle release and the receiving part, the receiving cell receptor on cells of a donor (receiving cells) emitted in extracellular vesicle accept the receiving substrate. On the mobile portion of a donor cell receptor extracellular vesicle formed by each receiving portion communicates with and receiving cells, donor cells received from single cell receptor extracellular vesicle movement of part causes.

According to one in the embodiment, extracellular vesicle tracking viewing system extracellular vesicle unit and is transmitted if they only have to communicate respectively with donor cells receiving portion and generates receiving cells, donor cells receiving part, in the form of a vesicle extracellular receiving portion and moveable portion receiving cells, the number and direction needed can be tube or the like. For example, in the embodiment 1 according to one of a donor cell receptor extracellular vesicle tracking observation system includes two part, both of which communicate respectively with two accommodating cells receiving portion and 1 1 and may be with a extracellular vesicle mobile portions, each receiving portion and the shape of the moveable portion may be in the form of straight line or a curve, donor cells receiving portion, receiving cells receiving portion and are juxtaposed the extracellular vesicle the mobile portion can be parallel to each other. According to another one in the embodiment 2 of a donor cell receptor extracellular vesicle tracking observation system includes two part, 1 and 2 and two accommodating cell receptor of the extracellular vesicle shifting-may be, at this time the first extracellular vesicle the mobile portion generates a first donor cells receiving portion and receiving cells and communicate respectively with, second extracellular vesicle the mobile portion generates the second donor cells receiving portion and receiving cells may be respectively communicate, each receiving portion and the shape of the moveable portion may be in the form of straight line or a curve, donor cell receptor part, receiving cells receiving portion and are juxtaposed the extracellular vesicle the mobile portion can be parallel to each other. In the conventional observation illustrated form of the present invention extracellular vesicle tracking thereover has the limited, and in addition if necessary donor cell receptor part, in the form of a vesicle extracellular receiving portion and receiving cells moving part, such as the number and direction different can be implemented.

Donor cells of the present invention extracellular vesicle tracking communication with the receiving portion in the conventional observation well (well) unit, the communication with the receiving cell receptor portion moveable portion further communication with the vesicle and extracellular well departmentwell department can be. These in well department by feed medium, donor cell receptor part, receiving cells receiving portion and supplied medium extracellular vesicle moving unit can, at this time extracellular vesicle mobile inside while not affecting the production of fine to affect the flow can be continuously repetitive and feed medium, suitable for use in imaging studies observed long-term tracking.

Donor cells receiving portion and receiving cell receptor extracellular vesicle unit and is transmitted and which communicate respectively with certain sizes and are linked to each other can be selectively passing filter material also disclosed. For example, observing [eyk[eyk] Cow petty tracking movement in order to about 0 Å diameter. 4 micro m can be in filter. [eyk[eyk] Cow petty than large order about 1 micro m line filter having fine voids diameter tracking vesicles can be observed.

In donor cell receptor extracellular vesicle inclination (gradient) emitted is formed moves to a portion receiving cell receptor extracellular vesicle mobile along.

There is mass transfer involves the treatment of cells in vivo to finely flow (interstitial flow), it is through this observation (gradient) of the present invention extracellular vesicle tracking by fine particles and to in vivo extracellular vesicle moveable part medium fine flow closely resembles fine particles and substrate. The, derived using such extracellular vesicle moving unit by flowing water can be mobile in vivo extracellular vesicle movement of the sample.

Donor cell receptor portion of the accommodation portion is inclined (gradient) acceptor cells gas inlet pipe difference, difference potential energy, a variety of medium inlet and discharge control method can be formed.

According to one in the embodiment, donor cells of recipient cells to utilize the differential to tilt (gradient) medium inlet pipe receiving portion of the accommodation portion can be formed. For example, in a face of the donor cells in medium (medium) receiving cell receptor medium inlet pipe larger than inlet pipe receiving portion, receiving cell receptor negative pressure formed at donor cell receptor (gradient) is formed larger than the track formed on the pressure emanating from the, along the slope receiving cells from donor cell receptor extracellular vesicle of the moving part can be receiving portion of media flow. Donor cell receptor part, and the supply of medium receiving cell receptor extracellular vesicle the mobile portion communication with the window to each well (well) portion, the receiving portion by a donor cells in number in this case each well department inlet pipe-free medium, receiving cells receiving portion and be differentially number [e[e] it will do extracellular vesicle motion each inlet pipe. More specifically, each in well department medium contained by the difference between the number of the surface area of inlet pipe be differentially [e[e] it will do. For example, each of the second cells receiving portion communicating with the vertical cylindrical well addition both donor cells receiving portion communicating with the gas inlet pipe to well bringing up for discussionwell bringing up for discussion diameter should be less than that number can be differentially [e[e] it will do, wherein the velocity of the flow on a fine two well bringing up for discussion determined by the difference between the opening area (the surface area of the medium contained in). Thus, donor cells receiving portion with respect to the acceptor cell receptor medium inlet pipe of the direction of flow and velocity [e[e] it will do extracellular vesicle of the moving part number be fine-free medium.

According to one another in the embodiment, recipient cells of donor cell receptor portion of the accommodation portion to tilt (gradient) can form a difference in potential energy. For example, donor cell unit is a cellular receptor part is higher than that position receiving medium to flow towards the cell receptor extracellular vesicle of the moving part can be. The recipient cell donor cells receiving portion height of each difference the direction of flow and velocity of the moving part number [e[e] it will do vesicle extracellular to be fine-free medium.

According to one another in the embodiment, donor cells by recipient cells (gradient) internal pressure receiving portion of the accommodation portion to tilt difference can be formed. For example, recipient cells when donor cells receiving portion receiving feed medium, each receiving a separate pump to one medium supply, of different donor cell receptor medium inflow sensor is used for collecting inner opening connected to the donor cell, receiving cell receptor medium each other at different inflow sensor connected to the receiving surface of the accommodation portion cells demonstrates the effect of internal pressure, the, donor cells receiving cell internal pressure greater than the pressure that inner surface of the accommodation portion by receiving cells from donor cell receptor extracellular vesicle mobile along fine flow medium receiving portion to form a can. In this case each of the accommodation portion of the accommodation portion internal pressure medium inlet opening each difference amount can be [e[e] it will do number, recipient cells by the donor cells receiving portion connected to the respective inner surface of the accommodation portion pressure difference the direction of flow and velocity of the moving part number extracellular vesicle number be [e[e] it will do fine-free medium.

In the present invention is not limited to formation of inclination of (gradient) method on an illustrated method has the, donor cell receptor portion of the accommodation portion acceptor cells energy, pressure energy in various energy difference of one like non-roll using various method can be can be.

Donor cells and receiving cells is simplified and a smooth free number cells can be used, as well as a variety of various foreign cells such as bacteria cells to an animal-origin cells can be used. For example, immune cell, connective tissue cells, vascular cell, nerve tissue cells, cancer cells, stem cells, can be foreign cells is used as the alkali. In addition e.g., T cells (T cell) immune cells, B cells (B cell), macrophage colony stimulating factor (macrophage), leukocyte (white blood cell) or dendritic cells (dendritic cell) can have, other immune cell be a. In addition e.g., fibroblast (fibroblast) can have a connective tissue cells, other connective tissue cell can be. In addition e.g., vascular cells have the (endothelial cell) can have, other vascular cell be a. In addition e.g., neural tissue in neuronal cells (neuron) or can have a neural progenitor cells (neuronal progenitor cells), other nerve tissue cells and be a life. In addition e.g., prevention may be foreign cells, other be a foreign cells such as bacteria.

Embodiment of the present invention extracellular vesicle the mobile portion is extracellular vesicle movement to enable tracking observing from a transparent gel with gel or hydrocodone may be loaded into the disclosed. According to one in the embodiment, the alginate (Alginate) on gel or hydrogels, collagen (Collagen), peptide (Peptide), fibrin (Fibrin), hyaluronic acid (Hyaluronic Acid), direct recovery system (Agarose), PHEMA (Polyhydroxyethylmethacrylate), PVA (Polyvinyl alcohol), PEG (Poly (ethylene glycol)), PEO (Poly (ethylene oxide)), (Polyethylene (glycol) diacrylate) PEGDA, gelatin (Gelatin), mat gel (Matrigel), PLLA (poly (L-a lactic acid)), carboxymethylcellulose (Carboxymethylcellulose), SAP, PHEMA-a MMA, dextran (Dextran) and chitosan (Chitosan) comprising at least one can be. For example, extracellular vesicle the mobile portion the metal number 1 collagen (type I collagen) gel with gel or hydrocodone may be loaded into the disclosed.

According to one in the embodiment, of the present invention can be embodied in the form of extracellular vesicle tracking observation system includes, according to one more specific in the embodiment can be embodied in the form of the microfluidic chip. However of the present invention in the form of limited only extracellular vesicle tracking has the observation system includes a substrate or the microfluidic chip, can be implemented in various forms as needed.

Hereinafter, exemplary embodiments of the present invention extracellular vesicle tracking viewing system are described as follows. The contents of the implementation of the present invention be implemented under one yale represented is received, implementation of the present invention and not limited to under the side walls.

In one aspect of the present invention extracellular vesicle tracking viewing system includes the following configuration can be implemented:

Substrate (100); and using the upper external respectively at the both end communicating with said inlet (11, 12) is formed number 1 channel (10); each one of said number 1 adjoining a communication channel to channel said number 1 left and raincoat both sides formed side-by-side, each inlet communicating with the outside at both ends (21, 22, 31, 32) is formed number 2 (20) and number 3 channel (3)); said number 2 and number 3 channel adjoining said number 2 and number 3 side channel communicating with the channel unit is said number 1 to number 4 side face (40) and number 5 channel (50); and said number 4 and number 5 channel end capable of receiving a respective amount of liquid wells (41, 42, 51, 52) which, channel and channel and said number 1 number 2 number 4 channel; said number 1 or number 3 channel and the side channel communication channel and said contact number 5 separated from each other when the filter.

The reference also 1, according to the microfluidic chip is herein rectangular-like substrate (100) according to the channel herein, inlet and well formed. According to the microfluidic substrate there is to implement a number bath material can be disclosed.

Each arrangement includes the disclosed microfluidic chip has a transmissive transparent material, used for the preparation of a bioactive free cells required for thermally processing said features hereinafter to photoresist is removed material or volatile material delivery number preferably being small.

According to the microfluidic chip of solid channel layer can be formed on a substrate of herein. In particular glass, quartz or silicon substrate, or (poly (dimethylsiloxane); PDMS) polydimethylsiloxane, (poly (methyl methacrylate); PMMA) slurry by, polycarbonate (polycarbonate; PC), polystyrene (polystyrene), cellulose acetate (cellulose acetate) and polyethylene terephthalate (such as poly (ethyleneterephthalate; PETP) polymer can be used.

According to the microfluidic chip is herein bottom - up or top - down manner (bottom provided up) number bath 1308. (top a-down). Said bottom - up scheme is very small size such as nanoparticles scale of atoms or molecules by a combination of the large-scale structures and method than means, top - down mode method for forming a structure of certain size desired absorbable fixing means other. These non-number example as little self-assembled, photolithography, imprint, etching, lithography or the like which, for example the number grudge without microfluidic form a channel can be used.

According to the microfluidic chip the cells herein between embodiment can be useful material movement by observation. For example differentiated cells (donor cells, Donor) to (receiving cells, Recipient) in undifferentiated cells, and heat transfer material while culturing cells that can, culture lysis method of embodiment movement of material between between can be observed.

To this end herein according to the microfluidic chip of channel number 1 (10) (recipient) the skeins fold and the receptor cells the lungs. Number 1 channel at both ends mounting two inlet (11, 12) are formed thereof can. Said inlet opening communicating with the channel is, through an inlet channel cells, cell culture, other fluids can be made entrance.

According to number 1 channel types of the scaffold is being injected into the gelling or used herein, refer to cells-derived material movable battery comprises material having a gold.

This scaffold is prepared alginate (Alginate), collagen (Collagen), peptide (Peptide), fibrin (Fibrin), hyaluronic acid (Hyaluronic Acid), direct recovery system (Agarose), PHEMA (Polyhydroxyethylmethacrylate), PVA (Polyvinyl alcohol), PEG (Poly (ethylene glycol)), PEO (Poly (ethylene oxide)), (Polyethylene (glycol) diacrylate) PEGDA, gelatin (Gelatin), mat gel (Matrigel), PLLA (poly (L-a lactic acid)), carboxymethylcellulose (Carboxymethylcellulose), SAP, PHEMA-a MMA, dextran (Dextran), chitosan (Chitosan) or any one material, or a material including mixing two or more thereof, this number are not correct valve timing.

Number 2 channel (20) and number 3 channel (30) and a body part standing adjoining the side face of the left and right sides of each communication channel number 1 can be. The aforementioned channel number 2 channel and number 3 is filled with gel (scaffold) thereof can. As well as mounting two inlet channel and each channel number 2 number 3 at both ends (21, 22; 31, 32) can be formed. Said inlet opening communicating with the channel is, through an inlet channel cells, cell culture, other fluids can be made entrance.

Channel number 4 (40) is channel number 2, number 5 channel (50) to one side of the channel number 3 - number 1 unit is communicating with the one side face of the adjoining channel can be. One of channel number 4 channel and number 5 located donor cells, donor cells located in the number 2 and number 4 formed on the channel, or the number 3 and number 5 channel is in communication through the filter can be contact. Number 5 channel number 4 channel and a mounting end to a respective amount of two well (41, 42; 51, 52) can be formed. Media portions falling within a well cultivating cells cells are disclosed.

According to the microfluidic chip in left or right piece channel is herein about which communication channel number 1 through the filter, the remaining untransmitted mask left or right side column spaced apart small piece channel faces can be formed. I.e., number 1 and number 2 channel and channel number 4 channel; number 5 channel number 1 or number 3 channel and one of the side channel and contact the communication unit can be filters is formed. Filter selectively passing only certain sizes having a core material, for example, order tracking filter having a pore of dimensions suitable passage [eyk[eyk] Cow petty[eyk[eyk] Cow petty observed with the oversize material than [eyk[eyk] Cow petty passing through the can. For example, diameter of the voids observed [eyk[eyk] Cow petty tracking order about 0. 4 micro m line filter is used can be, [eyk[eyk] Cow petty than large voids about 1 m diameter in order micro fine vesicles observed tracking can be filter is used.

In another aspect, of the present invention is a monitoring system of the present invention in one embodiment according to the microfluidic chip and said extracellular vesicle tracking number 1 and number 2 with microfluidic chip cultured in cells, said number 1 or number 2 cells which express one of the detectable signal, said detectors capable of detecting the detectable signal can be. According to cells herein used in the system, has a detection, reference detector can be represented.

With reference to the drawing of the microfluidic chip according to aspect herein used to describe the substrate.

Number 1, number 2 and number 3 channel through an inlet, said channel filled pawl [tu[tu] skeins. Ear number 4 or number 5 of the channels form a vanishing of culture medium through the inlet opening and one channel ion implantation process wherein a corresponding donor cells, donor cells can be further differentiated by adding donor gadd45gamma medium material and n is an integer. Ear number 1 channel form a donor cells not receiving cells or undifferentiated cells seeding channel (channel number 4 or number 5 of the channels not seeding donor cells) is supplied to the paper. Through differentiated donor cells (number 4 or number 5 channel) through channel (or channel number 3) number 2 from undifferentiated mass transfer to the receiving cells can be observing (channel number 1). Sharing the same channel number 1 and number 2 number 4 progenitor cells in contact with a filter is fitted to the side of the channel can be selectively movable only predetermined substance, can be selectively detecting only movement of a specific substance.

According to one embodiment [eyk[eyk] Cow petty mobility [eyk[eyk] Cow petty secreting cells can be observed herein (donor cells) to CD63 non-GFP vector to vector 56 can be fluorescence-labeled specifically only [eyk[eyk] Cow petty intracellular [eyk[eyk] Cow petty[eyk[eyk] Cow petty (recipient receiving cells) cells secreting cells and shifted fluorescence-labeled and receiving each microfluidic for different channels after incubating together at 1) neurons is derived cells concentrated in a high chloride ion secretion in cells using fluorescence microscopy [eyk[eyk] Cow petty differentiation between the substrate after neural cell differentiation by secreted [eyk[eyk] Cow petty embodiment by observation can be observed in the microfluidic chip mobility, guided through the nerves of the mother is evaluated [eyk[eyk] Cow petty differentiation of whole cells by observing [eyk[eyk] Cow petty mediated neural differentiation induction process the microfluidic chip environment can be efficiently for hereinafter.

In yet another aspect disclosed herein according to the microfluidic chip or system using interlayer movement detecting method embodiment to liver cells are disclosed.

Donor and recipient cells according to method steps herein under public affairs number; said donor cells detectable light-emitting or fluorescent protein or gene delivery vector to express 56 including step; and said receiving cells and donor cells in communication the culturing step, said receiving and said centrally located cells culture, said on opposite sides of said receiving cells receiving cells and donor cells derived from material located between movable interconnected cells, said donor cells is arranged on one side of cell populations, cell culture medium and positioned in the one side of the culture, cells cultured in said receiving and said scaffold, said culturing step, said donor that is expressed in tracing paths to move the light emitting protein receiving cells comprising the following steps.

In substances such as organelles is involved in donor according to method herein [eyk[eyk] Cow petty with is labeled with fluorescence or luminescence material, through movement of the material can be between [eyk[eyk] Cow petty embodiment by observation. In one implementation according to [eyk[eyk] Cow petty herein specifically labeled vectors 56 by using a donor cells with anti-neoplastics. In the embodiment described herein with reference to a specific marker and delivery 56 [eyk[eyk] Cow petty associated with bar can in this case.

Donor [eyk[eyk] Cow petty microfluidic chips of about seeding to left or on the right of channel number 1, and comprise a moving in different directions therefrom, moving [eyk[eyk] Cow petty emitted therefrom over a fluorescence or luminescence between embodiment can be detected.

Herein according to the microfluidic chip, the system and method specific filter and skeins [eyk[eyk] Cow petty[eyk[eyk] Cow pettypawl [tu[tu] voids between mobile cells can be specifically by changing the observing, in addition, other cells artificially maintain static conditions in size, different surface tension difference in each reservoir inlet pipe does not use very similar living body using fine flow artificially capable...copyright 2001.

In addition according to the microfluidic chip using cell-herein when detecting method includes 3 dimensional environment interacting with fluorescence-labeled [eyk[eyk] Cow petty lower ambient atmosphere cells in between can be caused by the evaporation of medium to each embodiment [eyk[eyk] Cow petty number [eyk[eyk] Cow petty cells involved in door for movement does not affect the flow as much as possible fine to medium designed to observing the daily supply must even suitable for long study. In particular when differentiation disorders derived neural stem cells secreted [eyk[eyk] Cow petty study treatment in recovery of damaged cells around cell inducing study is to determine the functionality delivered even long-term observation time is converted even study evaluating movement of long intercellular material can be utilized.

Hereinafter, the present invention through a corresponding business are provided in the embodiment as follows. In the embodiment of the present invention for aiding the understanding of the present invention is to however only and not the confined to form in the embodiment.

In the embodiment

Microarray Analysis

Processed using Trizol Ngn1 F11 neurons differentiated from all RNA isolated each other. Controls and experiments for GenoExplorer RNA group^TM Using hybrid coupling of target miRNA miRNALabelingKit synthesis and probe performs. 5 - exposed end of biotin labels of RNA using a gross RNA Enzyme L 5 provided 10 μg in 3 time marker-gate 37 °C. The output signal of the labeled RNA Biotin have the same volume on afferent GenoExplorer Hyb buffer 5 minutes^TM Placed miRNABiochip's desire. The hybrid coupling LifterSlip^TM In time using 16 42 °C performed during the vehicle from the outside. First hybrid coupling reaction after washing and is located on microarrays is equal to dry. Fluorescent dye for dyeing is biotin labeled RNA (streptavidin-a stain) dye SA provided S on hybrid coupled chips 25 °C whereupon on 30 minutes reaction in reaction chamber. Finally hybridized microarray is a stand-alone by non-specific in their number and rolled behind GenePix 4000B scanner scanning the other clothes. Hybridization images can be GenePix Software been to quantification. Correction of the results of the gene change ratio and all of selecting GeneSpringGX 7. 3 enforcing to him. Average percentage of the corrected intensity of a calibrated controls channel experiments group mean average intensity value correction channel dividing the bill.

Gene Annotation Analysis.

For selected miRNA target gene of functional annotations are Database for Annotation, Visualization, and Integrated Discovery (DAVID 6. 7) by irradiating the satisfactorily. For category is on Gene ontology (GO) functional tin in interaction between proteins, molecular function, cell composition, function of protein domain, etc. biological pathway. The program each list enumerating functionalized tin and tin terms associated gene number under public affairs substrate. The method of gene comprising DAVID gene IDs to avoid redundant Fishers exact test is calculated based on the was used.

Cell culture and delivery 56

A number DMEM 10% FBS 1% antibiotics including HeLa cells and mouse neuroblastoma hybrid cell F11 rat dorsal root ganglion cells culture together his. The cells may be 37 °C, 5% CO₂ In engineers. After cultured in 24 well plate HeLa cells using Lipofectamine 2000 on chemically modified miRNA-a 193 pRV provided effluc provided miR provided 193a_3XPT reporter gene on miRNA-a scr been processing such as cells. Vectors has been processed cells 2 is reflected in the culture medium DMEM 10% FBS. 3 having two binding sites for expressing miR provided 193a effLuc F11 cells have been prepared through Retrovirus infection. F11 cells containing DMEM 0 1 mm db-a cAMP for inducing differentiation of neuronal cells. 5% FBS with or without using the Lipofectamine 2000 was miR provided 193a.

Reverse transcriptase Quantitative PCR

Trizol reagent (Invitrogen) using total RNA derived differentiation mirVanaTM miRNA Isolation Kit F11 on separating from him. The concentration using Nanodrop provided 1000 Spectrophotometer to be measured is made. 1 μg/ml total RNA is reverse transcribed for analysis qRT-a PCR process scholarships. QRT-a PCR marker specific to the target gene for nerve specified Table 2 primer using miR provided 193a and conducting. PCR reaction is the ABI TaKaRa SYBR Green Master mix reagents ^{7500 been performed. Each experiment to correct sample correcting structure of beta-actin at night. For miRNA using reverse transcriptase reaction is specific for expression of miRNA 1st-a strand cDNA synthesis kit using the air into the space between the miRNA have irradiated TaKaRa SYBR Green Master mix. U6 snRNA internal control as well as a group. Using have ExoMirTM PLUS Kit Exosomal miRNA is to separate, isolated miRNA is through process by using a reverse transcriptase miRNA 1st-a strand cDNA synthesis kit has been TaKaRa SYBR Green Master mix for miRNA expression visit from the police. U6 snRNA used therein have the whole experiment has been three times experiment was regulated.}

Immune fluorescent Dyeing

F11 cells in 4% paraformaldehyde (PFA) in PBS at room temperature PBS 3 times 15 minutes then was fixed to clothes. In order to enhance cell permeability 0. 4 °C using 5 minutes in 5% Triton X-a 100 in PBS with respect to the reaction. Each samples are to wash to do, PBS, 1% normal goat serum in PBS at room temperature using 1 time the steam to prevent non-specific binding level's Grace. Each dilution in PBS Antirabbit Tuj provided 1 antibodies (1:1300 dilution; Sigma), anti-a rabbit NeuroD (1:500 dilution; Abcam), anti-a rabbit MAP2 (1:1000 dilution; Sigma), antirabbit GAP43 (1:2000 dilution; Sigma), and antirabbit PSD-a 95 (1:100 dilution; MILLIPORE) with respect to the antibodies on in response to the samples in 4 °C next to reaction. 2 difference antibody using Alexa Fluor 488 a-conjugated anti-a rabbit each samples are expected to 1 with respect to the reaction time at room temperature. Using DAPI fluorescent staining nuclei was. F11 cells cultured in 3 dimensional microfluidic device at room temperature PBS 3 times 15 minutes using 4% PFA-a PBS was reacting to main washing-gate. After fixing process cells in order to enhance permeability 0. 3 to 5 minutes in PBS 5% Triton X-a 100 by reacting at wash-gate. And, reacting 1 time 1% normal goat serum in PBS at room temperature level's Grace. Antirabbit Tuj provided 1 (1:1300 dilution; Sigma), antirabbit MAP2 (1:1000 dilution; Sigma) of 4 °C for using such antibodies in next to with respect to the reaction. And, 4 °C using Alexa Fluor 488 a-conjugated anti-a rabbit 2 difference antibody in next to with respect to the reaction. Using DAPI fluorescent staining nuclei was. Fluorescent images are captured by his using confocal laser scanning microscope.

In vitroAs base:00 it ladles, number Analysis

Cells may be PBS to wash and then been dissolved using lysis buffer. Cells are collected into a 96 well plate in which the release of luciferase assay using luciferase assay kit was after a delay. Each cells were measured using a luminometer biopharmaceutical production of light emitting extent. Each assay is quantitative amount when the repetition rate of the Luciferase protein was active degree operation of 3 experiments.

[eyk[eyk] Cow petty A circumferential separation and feature

4 °C [eyk[eyk] Cow petty FBS for reparing over time to 150000 × g in number 16 is planed core through separation of Royal. F11 cells in DMEM 10% dFBS (FBS for reparing over [eyk[eyk] Cow petty number) 2 or 1 mm db-a cAMP liver containing DMEM 0. 5% dFBS 3 in liver engineers. Cells in 10 minutes and 20 minutes to 4 °C debris are 500 × g in a stand-alone been number 3000 × g by centrifuging. And, 150000 × g in 2 to 4 °C [eyk[eyk] Cow petty planed time core isolation is separated from bacillus [eyk[eyk] Cow petty been reconstituted with PBS. Protein is measured through the bicinchoninic acid assay. Using [eyk[eyk] Cow petty Nanoparticle tracking analysis (NTA) system size and number of particles measured.

[eyk[eyk] Cow petty And fluorescent staining of cells

F11 cells (0. 8 × 10⁵ Cellsperwell) 24 a-well plates have the F11 cells using Lipofectamine 2000 CD63 a-GFP vector was prepared. DMEM 10% FBS containing DMEM 0 or 1 mm db-a cAMP speaker's vocal tract cells. 2 to 5% FBS liver engineers. F11 cells in 5 minutes 37 °C CM provided DiI cell labeling solution processing, and additionally 4 °C fluorescent staining cells was 15 minutes in reacting. Fluorescent dyed F11 cells prepared with a few other speaker's vocal tract cells CD63 a-GFP. Fluorescent Image using confocal laser scanning microscope images are captured by other.

Trans well Analysis

Movement [eyk[eyk] Cow petty to apoptosis, 0. 4 a-μm pore size PET membrane having a transwell chambers (CORNING) was used. GFP-a exosome F11 cells producing (0. 8 × 10⁵ Cellsperwell) well above the downward and F11 to denaturalization followed two cells of the cell culture chamber 3 is with respect to the culturing cells in the culture medium. In order to identify movement of the F11 cells mediated [eyk[eyk] Cow petty miR provided 193a or below chamber whereupon the DMEM 10% FBS, 1 mm db-a cAMP added with DMEM 0. With respect to the culture is 2 to 5% FBS. After two days, 1 mm db-a cAMP is processed cells have PBS washing to DMEM 0. 5% FBS abrasive media to shaking. Lower chamber above chamber whereupon the cells F11/effLuc provided miR provided 193_3XPT cells with him as liver 3. Differentiating cells secreted [eyk[eyk] Cow petty glioblastoma cell differentiation or lower chamber F11 cells to apoptosis minced and seasoned DMEM 10% FBS, 1 mm db-a cAMP added with DMEM 0. With respect to the culture is 2 to 5% FBS. PBS washing to DMEM 0 1 mm db-a cAMP is processed cells have. 5% FBS abrasive media to shaking. F11 cells includes a pair 3 and 5 with respect to the cells during each minced and seasoned chamber lower chamber receives a binary code. The whole experiment has been enforcing operation of his 3.

the [wey[wey] the [su[su] it shook off[pul[pul] Analysis

30 minutes after dissolving in RIPA buffer to recruit cells from the cell debris was 12,000 rpm centrifuging the number alone. Protein concentration were measured through the BCA assay. Connected to the same protein have been moving into SDS-a PAGE protein PVDF membranes. To move the membrane protein antibodies anti-a rabbit KRAS 1 difference, PLAU, CCKAR, CYSLTR 1 and GALR1 (1:1000 dilution; Abcam) and exosomal markers such as anti-a mouse TSG101 (1:1000 dilution; Abcam), anti-a rabbit CD63 (1:1000 dilution; Santa Cruz biotechnology), and the ER membrane marker, anti-a rabbit Calnexin (1:1000 dilution; Abcam) in 4 °C to reaction with respect to the next. The HRP is synthesized using a 2 2 then Membranes antibody was the temporal behavior at room temperature. Using chemiluminescence detection system of this protein has been confirmed. Protein band is using his AlphaView Software MRSI. The whole experiment has been enforcing his operation of 3 experiments.

Small microfluidic cell culture device number

Microfluidic Small number of Device: Glass cover slide number was then attached to the microchannel-a patterned PDMS Microfluidic Device is small. Photoresist SU provided 8 used as main form characterized in that it has, been small number along soft-a lithography process. PDMS is cured such as number 1 in 30 minutes and then at a rate of 80 °C 10:1 mix time been baked. Bent PDMS punches to each reservoir separated from the wafer and a perforate me. PDMS and glass cover slip through oxygen plasma processing to denaturalization followed by bonding two 80 °C microchannel surface hydrophobic properties and sterilization storage amount with respect to the recovery.

The microfluidic chip using F11 cells Amount public times: 2 Collagen type 1 hydrogel of a 30 minutes gels pumped behind gel channel or any other. The leash comprises completely filling the microchannel before seeding the growth of cells. F11 (donor) cells prepared right cells left cells placed in a channel filled with media general channel rested. Cells attached to the donor cells after differentiation to receive only differentiation medium DMEM to floor group (0. Comprising 5% FBS and 1 mm db-a cAMP) after a further him as. F11 cells including a type 1 collagen hydrogel as the gel in body board 37 °C in 5% CO (receiving cells) among the injection weight fold² 30 minutes in 3 dimensional secondly reacting one or his. The amount of type 1 collagen solution injection with 1 × 10 cells⁶ Cells/mL min. On the PBS after dilution in an amount of 2 mg/mL with distilled water Hydrogel 3 to 0. 5 N NaOH to pH 7. 4 as well as the cavity is.

Time lapse[eyk[eyk] Cow petty Zoom

F11 (donor) cells into cells of channel number 1 channel left piece about general been filled with unit cells prepared right channel medium. Donor cells using Lipofectamine 2000 (System Biosciences) CD63 a-GFP cell adhesion after the meeting. 2 speaker's vocal tract cells in DMEM supplemented with 10% during him as. F11 (receiving cells) is 1 × 10⁶ Cells/mL collagen solution in an amount of 37 °C in 5% CO pumped behind such as gel channel² Reacting 30 minutes in 3 dimensional environment made me. Embodiment 37 °C in 5% CO for photographing progenitor cells₂ Been maintained. 30 seconds intervals [eyk[eyk] Cow petty been photographing using time-a lapse confocal microscopy. Fluorescent Image is NIS provided Elements Viewer is reconfigured with the him.

Biological light emitting Image (Bioluminescence imaging)

F11 (receiving cells) before or after differentiation (donor) cells cultured with F11 for biological light emitting Image after 1 and 3 and third differentiation were measured. Each cell number after PBS cells from the wash-gate from media wetting ability. A PBS 150 μg/ml D-a luciferin carcinogens by mixing each cell channel of his. Then, with respect to the microfluidic chip located IVIS provided 100 imager. Upon activation Fluc biological light emitting Image after 5 component D-a luciferin were measured. Image photographing number after each specie cells into new media channel from immediately cells D-a luciferin is a stand-alone studies. Cells further 2 is further engineers. The amount of photon per second biological light emitting Image ROI quantitative analysis has been representation.

Statistical analysis

Average ± standard deviation (SD) precursor Students to all results t- Test was assessed by using. The statistical significance P -Values < 0. 05 was assessed to.

In the embodiment 1. D provided F11 cells derived In [eyk[eyk] Cow petty Mi-a RNA concentration rise confirmation

The miRNA is secreted in differentiated cells in the invention through [eyk[eyk] Cow petty can be transported to other cells, neural cells undifferentiated cells adjacent said generating accelerates by hepatitis c viral replicon which two lines and adjacent to him. The first, 0. In DMEM containing 5% depleted non-FBS and 1 mm db-a cAMP F11 during culturing cells differentiated F11 [eyk[eyk] Cow petty in his 3 after separating. As db-a cAMP after being processed recording neural cells is extended protrusion F11 (neurite) up (1a also). Included in the separated [eyk[eyk] Cow petty including CD63 and TSG101 protein marker for irradiating whether [eyk[eyk] Cow petty, the the [wey[wey] the [su[su] it shook off[pul[pul] conducting analysis in the invention. Undifferentiated F11 cells isolated from [eyk[eyk] Cow petty and differentiated F11 (UN provided Exo) isolated from marker protein in cells (D provided Exo) [eyk[eyk] Cow petty[eyk[eyk] Cow petty CD63, re-TSG101 has been detected, this the [wey[wey] the [su[su] it shook off[pul[pul] been verified by analysis result. However ER membrane marker knife neck new (Calnexin) was not detected even generates (1b also). These results indicate that, cell destruction without contamination such as positive number [eyk[eyk] Cow petty spoke substrate. In addition, a separate UD-a Exo herein subject to analysis results and D provided Exo NTA (nanoparticle-a tracking analysis), approximately 150 provided 200 nm with a size distribution peaks of relatively uniform diameter showed (also 1c). The present invention is secreted in the total protein concentration [eyk[eyk] Cow petty[eyk[eyk] Cow petty UD or D positive number when measured, total protein per cell compared to approximately 6 times the same [eyk[eyk] Cow petty[eyk[eyk] Cow and UD - exo D - been increased (also 1d).

MiRNA expression increased D-a F11 cells in order to identify whether its [eyk[eyk] Cow petty miRNA having large, the miRNA [eyk[eyk] Cow petty[eyk[eyk] Cow petty of isolated from a sample reservoir in the invention UD or D provided F11 for conducting RT-a PCR. Quantitative RT-a PCR for quantitative analysis to the miRNA [eyk[eyk] Cow petty separated, UD-a F11 cells (UD-a EXO) compared to isolated from cells isolated from [eyk[eyk] Cow petty with D provided F11 in increased levels of miRNA [eyk[eyk] Cow petty (D provided Exo) as in the picomolar (1e also). These results indicate that, during neural differentiation [eyk[eyk] Cow petty have significantly increased amounts of secreted through the miRNA [eyk[eyk] Cow petty D provided F11 cells contains a number of joined substrate.

In the embodiment 2. Derived from cell differentiation [eyk[eyk] Cow petty Validates to undifferentiated cells

Cellular differentiation by cells around a sophisticated control occurs. Neural progenitor cells (or neural stem cells) cells from neural differentiation is soluble factor exchange directly or subjected dominated by contact (for example, roh [chwi[chwi] - delta reaction (Notch a-delta reaction)). In addition involves the treatment of a neurological factor affecting cell differentiation through [eyk[eyk] Cow petty with exchange is such as EV can be. The heterogeneous cell populations in the invention in general outline in the initial cAMP initially single neurons induced by affecting the undifferentiated cells can be differentiated, by moving the miRNA [eyk[eyk] Cow petty neurological through their neuronal differentiation to facilitate the two lines and adjacent to him.

A first time, the F11 [eyk[eyk] Cow petty involves the treatment of undifferentiated and differentiation for tracking movement, a GFP fusion vector was constructed containing CD63. Basing the cell protein such as GFP fluorescent reporter protein fused [eyk[eyk] Cow petty[eyk[eyk] Cow petty - can be used tracking report in the nanometer range. Fluorescent protein - protein is labeled [eyk[eyk] Cow petty[eyk[eyk] Cow petty movement can be visualizing cells to other cells. When plasmid vector is transferred in-GFP - F11 cells labeled CD63, clear [eyk[eyk] Cow petty been probed by the formula 1 site.

Endogenous [eyk[eyk] Cow petty production in normal conditions or neural differentiation to apoptosis, F11 cells in a medium during normal or inducing differentiation of CD63 non-GFP - trans [pheyk[pheyk] It became the [thu[thu] him as 3. Initial and terminal differentiation marker dyed differentiated F11 endogenous [eyk[eyk] Cow petty Tuj provided 1 been production of nerve in creating (2a also). Differentiated cells derived from undifferentiated [eyk[eyk] Cow petty F11 to move the cells to apoptosis, cells (donor cells) and DiI - F11 cells labeled CD63 non-GFP - trans [pheyk[pheyk] It became the [thu[thu] F11 (receiving cells) by mixing (1:1) him as liver 2. In the case of group differentiation, neural differentiation after receiving cells derive DiI - F11 cells (donor cells) with ball - CD63 non-GFP - trans [pheyk[pheyk] It became the [thu[thu] label (co-a culture) culture was. The F11 cells differentiated in the invention derived from undifferentiated cells with respect to the label receiving [eyk[eyk] Cow petty DiI - CD63 non-GFP - F11 mobile (2b also).

0. 4 m PET (polyethylene terephthalate) membrane micro voids (Transwell chambers) fax (shedding) but most of trans well chamber [eyk[eyk] Cow petty movement in fine vesicle (diameter 0. 1 - 1 micro m) (apoptotic bodies) (diameter > 1 micro m) and apoptosis body insulation property the tropics. F11 insertion chamber above said receiving cells followed by addition of GFP - 2 - producing cells [eyk[eyk] Cow petty bottom for exposing the liver him as well.

F11 [eyk[eyk] Cow pettypublic times donor cells released from formula 1 site (also 2c) has been detected in GFP - F11 during receiving amount 2. The time course and procedure of the present invention listed in fig. 4d disclosed. In order to identify [eyk[eyk] Cow petty - mediated movement of miRNA, trans well receiving cells with respect to the upper insertion chamber F11 effLuc provided miR provided 193_3XPT infected. As the number of undifferentiated donor cells in a group natural base:00 it ladles, receiving signaling and is gradually been increased cell proliferation. The methods for D-a F11, donor cells after GFP - cells producing F11 [eyk[eyk] Cow petty db provided cAMP processing 2 when culturing cells receiving F11/effLuc provided miR provided 193_3XPT - ball, and significantly reduced in number as donor cells only group receiving active base:00 it ladles, D provided F11 (2d also). These results indicate that, during neural differentiation through [eyk[eyk] Cow petty miRNA is differentiated neuronal cells moved in undifferentiated cells that tell other.

In the embodiment 3. Of miRNA[eyk[eyk] Cow petty- Mediated by movements of the neural differentiation induction increased

Then the receiving gate line in the invention F11 in neural differentiation (donor cells) through movement of the miRNA is secreted [eyk[eyk] Cow petty D provided F11 cells in test whether induced by him. F11 - F11 donor cells with ball receiving cells followed by addition of then him as said upper insertion chamber. In the case of group D provided F11 cells, such as donor cells after receiving cells db-a cAMP processing 2 him as (3a also). In phase difference Image (phase contrast image), donor cells cultured nerve cells as 3 and 5 amount public times after receiving elongated protrusion extended - such as neurons appeared similar features. Receiving the change of expression of biological markers for proving the neuronal cell, quantitative RT-a PCR assays using 3 - 5 cell culture after expression of MAP2 Tuj-a 1 and receiving ball and to visit from the police. The MAP2 level is only receiving cell culturing Tuj-a 1 and significantly been increased donor cells D provided F11 (3b also). In addition ball - 3 and 5 of the culture cells after culturing immune dyes contained in neural marker Tuj-a 1 and MAP D provided F11 donor cells are expressed high only (3c also). In order to identify target gene regulation of miRNA [eyk[eyk] Cow petty - mediated by movement of the miRNA, using quantitative RT-a PCR of target gene mRNA level likely to be involved in neural activity - ligand receptor receiving visit from the police. Quantitative analysis by quantitative RT-a PCR device includes a receiving cells cultured with donor cells D provided F11 CCKAR, CYSLTR 1 significantly reduced levels of these markers (also 3d). In addition the miRNA expression of neural cells in the invention known as receiving and using quantitative RT-a PCR generating miRNA miR provided 124a below but, their expression increase at the receiving cells found that culturing D provided F11 cells (also 3e). These results indicate that, differentiated donor cells containing [eyk[eyk] Cow petty miRNA emits F11, they moved receiving cells, cells in which the target gene that exhibits controlled downward - receiving the miRNA.

In the embodiment 4. Customized microfluidic system using differentiated cells released from [eyk[eyk] Cow petty To move the undifferentiated cells observed

In neuronal differentiation for visualizing [eyk[eyk] Cow petty intercellular delivery, the time lapse of a microfluidic device for the device in the invention transport [eyk[eyk] Cow petty live camera was. Trans well and microfluidic cell culture device is compared, by two fluid flow can be directed to a special behavior of scale marks in mobile and molecules [eyk[eyk] Cow petty micro - scale fluid micro environment which includes the barrel number force (controllability) and high - marine embodiment Image number is under public affairs substrate. In the present invention, UD or D provided F11 for visualizing [eyk[eyk] Cow petty transport from UD provided F11 cells inserted microfluidic device (also 4a) hydrogel - are used. Visualizing [eyk[eyk] Cow petty delivery for the next conducting. Cells type 1 collagen extracellular (extracellular) after 3 hydrogel substrate dimensionally number data, ear multi - exclusive to form compartments during antiques. F11 CD63 a-GFP cells left channel 56 at the specified delivery time after receiving donor seeding F 11 of his central channel with gel cells (4b also). 30 seconds intervals using fluorescent Image cone gun knife microscope live - cells are obtained. 4 minutes 30 seconds [eyk[eyk] Cow petty initially derived from donor cells in cell culture have type 1 collagen hydrogel fiber channel between space moved out (also 4c, supp movie 1a, b). Said method and apparatus, then 8 minutes type 1 collagen hydrogel receiving cells through the gel 3 dimensional [eyk[eyk] Cow petty must be mobile that appeared (4d also, supp movie 1c). Of infusing a fluid transported by said [eyk[eyk] Cow petty two fluid-been the microenvironment. Microfluidic device and experimental protocol (Peclet number) of about 30 the lung the [ley[ley] possibility of growing in interstitial (interstitial) transport that mimic the device for peripheral brain having been. Each of the storage medium closes-free medium caused by evaporation surface maintained by some pressure head by, interstitial fluid flow with large storage (reservoir) similar medicines is rolled a central left channel having been produced. Flow the lung the [ley[ley] possibility of growing 21. 5 had calculated, this diffusion and attachment of and peripheral cells on high shear stress (shear stress) can interfere [eyk[eyk] Cow petty without, receiving cells from donor [eyk[eyk] Cow petty active transport to enable other. 4 hereinafter simply [eyk[eyk] Cow pettythe lung the [ley[ley] possibility of growing - the delivery of power flow spreading codes having epilepsy and evenly distribute throughout (interstitum) 2000. 400 or more the lung the [ley[ley] possibility of growing in two fluid flow with minimum diffusion at or wired (streamline) power - the delivery is fast.

CD63 non-GFP - tagged [eyk[eyk] Cow petty in time lapse, received from 1 to 3 minutes [eyk[eyk] Cow petty donor cells transported into cells received within cells that successfully attached at or (also 4e, 4f and 4g) absorption. Receiving cell cone gun knife microscope using Image through Z - stack, - cavity by yellow color by localized [eyk[eyk] Cow petty vary, forty-eight receptor [eyk[eyk] Cow petty absorption. Z - stack 3D Image to the revascularization, UD or D public timesboth grudges[eyk[eyk] Cow petty accumulation of donor cells receiving cells appeared. The phenomenon such as ball receiving cells are cultured in the cell group in addition UD-a F11 - even inhibin receptor. 0 (completely blocked by) [eyk[eyk] Cow petty in 100 micro m/min. (blocked from without, by two fluid by) a wide range of speed distribution, type 1 collagen ECM has been transported through a fibrous spacing. The rate [eyk[eyk] Cow petty 28. 7 the lung the [ley[ley] prisoner who will grow of wheel is calculated, design (21. 5) associated with epilepsy transport on peripheral brain in 2000. In one embodiment of the present invention also includes using the microfluidic chip as 4h [eyk[eyk] Cow petty tracking for monitoring each step also representing flow among others.

In the embodiment 5. In microfluidic During angiogenesis[eyk[eyk] Cow petty Through MiRNA Transport MiRNAIdentifying the second memory - reporter gene specificity

Receiving from being absorbed by cells containing [eyk[eyk] Cow petty miRNA is undifferentiated for proving the whether the, with respect to the F11 effLuc/3xPT_miRNA optical reporter infected cells. The present invention also disclosed to design a path 5a. Donor F11 cells cultured with normal or differentiation medium was left cell culture after number data channel. After 2, donor cells of media db-a cAMP free 0. Changing his DMEM containing 5% FBS. F11 cells (receiving cells) is added to the cell culture channel/effLuc/3xPT_miRNA public times-gate to the right amount. After receiving 1 have reduced light emitting signaling in undifferentiated biological cells, undifferentiated donor cells F11 - F11 cells after differentiation is significantly reduced compared public timesboth grudges donor group 3 (5b also). In addition to numerical ROI public times up significantly reduced amount after light emitting biological quantitative analysis after 5 (5c also). These data, derived from undifferentiated cells which contained [eyk[eyk] Cow petty miRNA is transported to a receiving cell differentiation, effLuc/3xPT_miRNA miRNA binding site 3 reporter system that delivered in two biological fluorescent signal reduced by binding to copy substrate.

For debiting accounts containing miRNA glioblastoma [eyk[eyk] Cow petty differentiation induction, a UD-a F11 (donor cells) cells or D provided F11 cells form a channel with a hydrogel gel of his left cell culture said F11 cells (receiving cells). After public times amount 4, phase difference Image is receiving cells such as the dendritic growth culturing D provided F11 cells morphological change appeared (5d also). The MAP2 nerve in the invention in addition to identify expression of marker Tuj provided 1 - specificity and conducting an immune in various colors. As a result of immune fluorometric, amount of a donor and receiving cells after 4 and 7 after public times D provided F11 cells group of neural marker Tuj-a 1 and MAP2 levels very increased as in the picomolar (5e also). These results indicate that trans well culture research and discovered in a match immune fluorescent, differentiation is induced by neuronal differentiation in [eyk[eyk] Cow petty motions - mediated miRNA, miRNA target gene is increased by a down-proliferative of means that other (also 5f). Also 5g is differentiated neural progenitor cells (neural precursor cell) [eyk[eyk] Cow petty emitted from inducing differentiation of undifferentiated neural progenitor cells on and type indicating mechanisms are disclosed. In one embodiment of the present invention also includes generating (neurogenesis) 5h nerve studies utilizing the microfluidic chip as exhibits for example.

In the embodiment of the present disclosure are described herein but detailed above exemplary rights range and then a received and in the claims herein are not limited to basic general outline of various modified and improved form herein using one skilled in addition range rights are disclosed.

The term used in the present invention all techniques, are not defined or more with, such as typically encountered in the field of the present invention is generally understand sense of one skilled are used. The specification described in the present invention are introduced into the contents of the document on the right side of all Publication.