CN1556407A - Cell chip manufacturing method and its device - Google Patents
Cell chip manufacturing method and its device Download PDFInfo
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- CN1556407A CN1556407A CNA2004100228194A CN200410022819A CN1556407A CN 1556407 A CN1556407 A CN 1556407A CN A2004100228194 A CNA2004100228194 A CN A2004100228194A CN 200410022819 A CN200410022819 A CN 200410022819A CN 1556407 A CN1556407 A CN 1556407A
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Abstract
Cell chip is prepared from following steps: fixing cells and carrying treatments of dehydration and transparency; wax dipping treated cells in bar type die to prepare wax bar with cell core; putting the said wax bar with cell core into receptor of wax block; slicing up the block by using adhesive tape transferring auxiliary system to prepare cell chip in different density. Besides application of tissue chip, cell chip possesses applications as target in authenticating drug action, in use for discovering gene product of changing physiological function of cell, transfection of gene of cell, and testing sensitivity of drug etc. The invention provides an economic and simple method for conserving various cells in long period.
Description
Technical field:
The present invention relates to the detection method of cell, be specifically related to a kind of cell chip.
Background technology:
The method that present cultured cell prepares cell chip has 3 kinds: fixing paraffin imbedding, the agarose of cell centrifugation precipitation paper bag is to punching on fixing paraffin imbedding of matrix and the agarose plate, the paraffin imbedding of perfusion cell fixation in the hole.These three kinds of methods respectively have its shortcoming, and preceding two kinds of methods cause too cell disperse, although a kind of method in back can obtain the cell of the higher uniformity of density, technological process is many, complicated operation.In addition, when making cell chip, all need puncture to the acceptor paraffin mass when greatest drawback of above-mentioned three kinds of disposal routes is to make cell chip and make recipient cell core bar by the method for making organization chip, because cultured cell is the matter fiber continuously, the puncture cell causes cell to disperse often, is unfavorable for repeated puncture making cell chip.Along with finishing of the Human Genome Project, functional genome's science study method of genome times afterwards comprehensively just develops towards extensive, high-octane direction.The development of chips such as genetic chip, organization chip, protein-chip provides a series of high-throughout research tools for the research of functional genomics, has greatly accelerated the research steps of functional genome.But various chips in the market can not be satisfied with the various albumen of extensive in situ detection and gene is expressed in the good pernicious research cultured cell strain of immortalization.Therefore research and develop out a kind of more simple and practical, making cell chip method of settling at one go, the research that makes cell chip be used for the gene expressed in situ then is the problem of needs solution.
Summary of the invention:
It is simple that the present invention aims to provide a kind of technology, the new method that does not need to puncture and make, and the cell chip of making can be used for the research of gene expressed in situ.
The present invention is achieved through the following technical solutions goal of the invention.Through cellular incubation, collect fixed cell, cell dehydration, after cell is transparent, waxdip is that cell suspension is moved into the transparent bar-type mould from the Tube pipe, will be in the bar shaped mould cell of uniform deposition and mould upright to immerse molten point be waxdip three times in 58 ℃ the LMP paraffin solution, the making of acceptor paraffin mass and receptor hole is that to make spacing be that the lattice array mould paper of 2mm is affixed on the acceptor paraffin mass of the corresponding specification size of making by the density of cell chip, in grid paper, pass receptor hole with the vacuum lancing pin successively, the making of recipient cell chip paraffin mass is the lower end that cell mineral wax mixture softening in placing 37 ℃ of incubators is clipped the bar shaped mould, extrude bar shaped cell mineral wax mixture from cutting end, and block into section, put into corresponding paraffin mass receptor hole.Waxdip is 1.0mm with a kind of transparent bar-type mould, this mold bottom diameter, and the bottom is plugged with and stops cell on a small quantity and help the cotton that terebinthina flows out through mold bottom.
Be described in further detail the present invention below in conjunction with accompanying drawing.
Description of drawings:
Fig. 1 is a bar shaped mould synoptic diagram; Wherein: 1, base diameter 1mm mould; 2, the cotton of bottom filling
Fig. 2 is a bar shaped cell mineral wax mixture;
Fig. 3 is the paraffin cell core bar that places the Tube pipe;
Fig. 4 is the recipient cell dot chart;
Fig. 5 is the recipient cell dot chart;
Fig. 6 is a SABC, 4 * cell microarray figure;
Fig. 7 is HE, 4 * cell microarray figure;
Fig. 8 is HE, 20 * cell microarray figure;
Fig. 9 is a SABC, 20 * cell microarray figure;
Figure 10 is a SABC, 20 * cell microarray figure.
The concrete grammar of making cell chip is as follows: (1) cultured cell: the immortalization of cultivating various tissues and organ origin is disliked The property tumour cell and optimum immortalized cell line in the 100ml blake bottle, every kind of cell is cultivated 3 bottles. (2) collection is fixing thin Born of the same parents: when treating that cell grows to 90% density, collecting cell is handled as follows: D-Hanker ' s liquid washed cell 4 times, fill Divide clean residual media, add 1ml D-Hanker ' s liquid in the blake bottle, plastics cell sleaker scrapes cell, cell gently Move in 1.0ml～1.5ml Tube pipe, centrifugal 2500 rev/mins, 5min outwells liquid in the Tube pipe, filter paper altogether The exhaustion residual liquid adds 1.0ml～1.5ml 4% paraformaldehyde and also blow and beat gently re-suspended cell (subsequently in the Tube pipe During the no specified otherwise of each step, all carry out in 1.0ml～1.5ml Tube pipe, reagent is 1.0ml～1.5ml), Make it be separated into the individual cells suspension, 4 ℃ of placements are fixedly spent the night. (3) cell dehydration: successively by following 6 step steps Dehydration, after each dehydration, centrifugal 5000 rev/mins, 5min/ time, discard the dehydrating agent of back, 70% (contains in the alcohol 0.5% Yihong indicator) dehydration of alcohol 40min, 80% dehydration of alcohol 40min, 90% dehydration of alcohol 45min, 95% alcohol takes off 45min, 100% dehydration of alcohol 60min totally 2 times. (4) cell is transparent: rear centrifugal 5000 rev/mins of dehydration, and 5min/ time, Filter paper exhausts residual alcohol, and turpentine oil (dimethylbenzene has toxicity, replaces dimethylbenzene with turpentine oil) makes the transparent secondary of cell, Each 60min. (5) waxdip: centrifugal 5000 rev/mins, 5min/ time, suck upper strata 0.8ml turpentine oil, piping and druming is thin repeatedly Born of the same parents form the turpentine oil cell suspension, and then cell suspension being moved into base diameter from the Tube pipe is the transparent bar-type plastics of 1.0mm In the mould (1), mold bottom is plugged with a small amount of cotton (2) to be stopped cell and is conducive to turpentine oil and flow out through mold bottom, Under the Action of Gravity Field, the cell uniform deposition is in strip mould. 3 of the upright immersions of strip mould that cell will be housed successively are equipped with In the LMP paraffin solution beaker (molten point is 58 ℃), be placed with die frame in each beaker, the waxdip time is respectively 30min, 45min, 60min, totally three times. Cell mineral wax mixture numbering is stored in normal temperature or 4 ℃ in the strip mould behind the waxdip For subsequent use in the refrigerator. (6) making of cell chip acceptor paraffin mass and receptor hole: by the density of required making cell chip, make The acceptor paraffin mass of dimension size. Spacing is that the lattice array mould paper of 2mm is affixed on the paraffin mass, uses successively diameter 1.0mm The vacuum lancing pin passes diameter in grid paper be 1.0mm, and the degree of depth is the receptor hole of 4～10mm. (7) recipient cell chip stone The making of wax stone: the strip mould that the cell mineral wax mixture is housed is positioned in 37 ℃ of incubators, 30min, and purpose makes cell stone Wax mixture is softening. Clip the lower end of strip mould, the upper end that haemostatic clamp is clamped strip mould progressively moves down, from cutting end Extrude diameter and be the bar shaped cell mineral wax mixture (Fig. 2) about 1.0mm. Bar shaped cell mineral wax mixture is blocked into The 4-10mm/ section is put into corresponding paraffin mass receptor hole. After treating to fill up in whole receptor hole cell paraffin bar shaped mixture, acceptor 30min in 37 ℃ of baking boxs of paraffin mass, glass section concora crush acceptor wax block surface makes all cells core bar at same plane. (8) The making of cell chip section: carry out earlier rough lumber and repair sheet on the cycle type slicer, HE dyeing Microscopic observation confirms all to cut After going out all recipient cell dot matrix (Fig. 4,5), utilize the section of paraffin adhesive tape transfer system, the thick section of 5 μ m is pasted on carries On the slide, crosslinked 1min under the uviol lamp, section is dipped in 30sec in the adhesive tape lysate, stripping tape, conventional dewaxing aquation, Carry out various researchs, then immerse fast such as the need long preservation and dissolve in the paraffin, slice surface is carried out again cover thin layer paraffin,-20 ℃ of storages. (9) assessment of the quality testing of cell chip and practical application: randomly draw a section and carry out HE dyeing, Image analysis system is gathered the image of each intact cell dot matrix under low power lens, under the stereological analysis module to each cell The dot matrix image of strain is analyzed counting. In situ hybridization and SABC detection LRRC4mRNA and P53 albumen are at cell chip The middle expression.
The cell chip that the inventive method is made has a plurality of for gene functional research provides another kind of new high flux instrument The purposes of aspect. Cell chip with organize chip the same, can be used for the research of mRNA expression in situ such as in situ hybridization, original position PCR, SABC, immunofluorescence etc. In addition, cell chip also has the irreplaceable following purposes of the chip of organizing. (1) The alternative method that can be used as protein microarray confirms the effect target of medicine; (2) be used for sending out as a kind of clonal expression system Now change the gene outcome of cell physiological function. (3) be used for the research that the gene configuration of transfectional cell changes. (4) transfection order Gene cell clone's screening. (5) be used under high flux ground drugs various dose and the different pharmaceutical effect cell mutually The difference of correlation gene or protein expression, the sensitiveness of trial drug is conducive to find novel drugs. (6) paraffin cell mixture Has the same energy of the paraffin mass of organizing long preservation advantage, for the various cells of long preservation provide a kind of economy simple method.
Embodiment:
Embodiment: contain the cell chip preparation of 20 kinds of clones
1.1 cell is handled
(1) kind of clone and cellular incubation: the clone of introducing and preserving comprises following 15 kinds.COS7: African green monkey kidney cell line; 3T3: mouse fibroblast cell system; CNE-1 and HNE1: Chinese's nasopharyngeal carcinoma cell system; Hella: human cervical carcinoma cell system; U251: people's astroglioma clone; C6: rat glioma cell line; HT29 and SW480: CCL188; Soas-2 and MG-63: people's human osteosarcoma cell system; K562: chronic myeloid leukemia cells system; B16: mouse malignant melanoma cell system; 293: human kidney cells system; BHK-21: hamster kidney cell system.Foundation also identifies that stable transfection genes of interest clone has 3 kinds of clones, totally 5 clones.CNE-1-BRD7, CNE-1-LMP1: the KB cell system of transfection BRD7 gene, LMP1 gene; HT29-NGX6-2 and HT29-NGX6-10: the CCL188 clone 2 and the clone 5 of transfection NGX6 gene; U251-LRRC4: people's astroglioma clone of transfection LRRC4 gene.The above-mentioned clone of recovering according to a conventional method also goes down to posterity, every kind of cell 100cm
2Culture flask is cultivated 5 bottles.
(2) collect, fixed cell: treat that cellular morphology is good, when cell grows to 90% density, collecting cell carries out following processing: D-Hanker ' s liquid washed cell 4 times, fully clean residual media, add 1ml D-Hanker ' s liquid in the culture flask, plastics cell sleaker scrapes cell gently, and move in the 1.5ml Tube pipe, centrifugal 2500 rev/mins, be total to 5min, outwell D-Hanker ' s liquid in the Tube pipe, filter paper exhaustion residual liquid adds 1.5ml 4% paraformaldehyde in the Tube pipe, 1.0ml blowing and beating cell gently, suction nozzle make it become even cell suspension (when each step does not have specified otherwise subsequently, all carry out in the 1.5mlTube pipe, reagent is 1.5ml), 4 ℃ of placements are fixedly spent the night.
(3) cell dehydration: successively by following 6 step steps dehydration, after each dehydration, centrifugal 5000 rev/mins, 5min/ time, discard the back dewatering agent, 70% (contain in this step alcohol 0.5% Yihong make cell dye redness be convenient to the operational observations cell) alcohol 40min, 80% alcohol 40min, 90% alcohol 45min, 95% alcohol 45min, 100% alcohol 60min totally 2 times.
(4) cell is transparent: centrifugal 5000 rev/mins of dehydration back, and 5min/ time, filter paper exhausts residual alcohol, and terebinthina makes the transparent secondary of cell, each 60min.
(5) waxdip: centrifugal 5000 rev/mins, 5min/ time, upper strata 1.0ml terebinthina is removed in suction, after 200 μ l suction nozzles are blown and beaten cell formation cell terebinthina suspension repeatedly, it is the transparent bar-type mould of plastics of 1.0mm that cell suspension moves into base diameter from the Tube pipe, mold bottom is plugged with a small amount of cotton and stops cell and help terebinthina and flow out through mold bottom that under action of gravity, the cell uniform deposition is in the bar shaped mould.The bar shaped mould that cell will be housed successively inserts on the die frame, and die frame uprightly immerses 3 successively (come card company, molten point is 58 ℃) in the LMP paraffin solution beaker is housed, difference waxdip 30min, 45min, 60min.The bar shaped mould of cell mineral wax mixture numbering is stored in 4 ℃ of refrigerators standby behind the waxdip.
1.2 the making of cell microarray acceptor paraffin mass and section
(1) blocks 58 ℃ of paraffin of company and beeswax by the acceptor paraffin mass that is mixed and made into 2 * 2 * 2cm at 9: 1, the making spacing is that the grid paper of 2mm is affixed on the paraffin mass, passing diameter with the puncture needle of diameter 1.0mm vacuum in grid paper successively is 1.0mm, and the degree of depth is the receptor hole of 10mm.
(2) the bar shaped mould of cell mineral wax mixture places 37 ℃ of incubator heating 30min, softening cell mineral wax mixture.Scalpel is clipped the lower end of bar shaped mould, and the upper end that hemostatic forceps is clamped the bar shaped mould progressively moves down, and to extrude diameter be 1.0mm bar shaped cell mineral wax mixture from cutting end.Scalpel blocks bar shaped cell mineral wax mixture and is every segment length 10mm, puts into corresponding paraffin mass receptor hole, gently presses cell core bar with microslide, and it is entered in the acceptor wax block receptor hole.Each clone is made 5 recipient cell core bars, and after treating to fill up the paraffin cell mixture in whole receptor hole, 30min during the acceptor paraffin mass is placed horizontally in 37 ℃ of incubators gently presses the acceptor wax block surface with microslide then, makes all cells core bar on same plane.If paraffin cell core bar wouldn't be used to make cell microarray, also can be positioned in the Tube pipe, number 4 ℃ of long preservation.
(3) making of cell microarray section: the acceptor paraffin mass at first carries out rough lumber on the cycle type microtome, HE dyeing mirror is observed down and is confirmed all complete back (Fig. 4 that cuts out of all recipient cell dot matrix, 5), utilize the paraffin adhesive tape to shift backup system and carry out the section of acceptor paraffin mass, during each the section, the adhesive tape that has mark is affixed on the paraffin mass surface, the thick cell adhesive tape section of 5 μ m is pasted on the microslide of special adhering substrate, crosslinked 90sec under the uviol lamp, slide is dipped in 30sec in the adhesive tape lysate, dissolving and stripping tape gently, section is immersed fast and is dissolved in the paraffin then, slice surface is carried out again covered thin layer paraffin, be convenient to long preservation, the section that disposes can be standby-20 ℃ of long term storage.If section is used immediately, behind tape stripping, the aquation that can conventionally dewax is carried out various researchs.After per 20 sections, need carry out HE dyeing again, mirror is observed down, if when finding to have cell dot matrix disappearance, needs to end section.
1.3 the quality testing of cell chip and practical application
(1) quality testing: randomly draw a section and carry out HE dyeing, image analysis system is gathered the image of each intact cell dot matrix under low power lens, under the stereological analysis module dot matrix image of all cells system is analyzed counting.
(2) application of cell microarray in SABC: the EnVision of DAKO company two-step approach immunologic combined detection reagent kit detects P53 and the expression of P16 in this cell microarray.The SABC running program of histotomy is carried out routinely.Section dewaxes to water 3% H
2O
2Blocking-up endogenous peroxydase 30min, section places sodium citrate damping fluid (PH 6.4), carries out antigen retrieval 20min in the micro-wave oven; Drip mouse-anti human monoclonal antibodies P53 respectively, P21, PTEN and P16 (Santa Cruz company) spend the night in 4 ℃ of warm boxes in section, and PBS washes 3 * 5min, two anti-incubated at room 45 minutes, PBS washes 3 * 5min, the DAB colour developing, mirror is the control chromogenic reaction down; Haematoxylin is redyed, the special-purpose mountant mounting of adhesive tape transfer system.
(3) application during cell microarray is hybridized in position: detect BRD7 (Genbank:AF179285), the expression of NGX6 (Genbank:AF188239) gene in cell microarray; BRD7 and NGX6 gene are read design primer in the frame, the PCR 350bp that increases respectively, and 400bp purpose fragment, glue reclaims, and biotin random primer sign kit (KPL company) carries out the probe sign.Hybridization in situ detection kit (there is the TSA signal amplifying system in PE company) detects hybridization signal.Step alcohol is gone in the section dewaxing, DEPC washing, the endogenic peroxidase of 3% hydrogen peroxide deactivation.Proteinase K carries out the cell permeability and handles mRNA in the exposed cell, 37 ℃ 15-20 minute.Prehybridization, each section prehybridization solution 100 μ l/ sheet seals film and covers, 42 ℃ of prehybridizations 3 hours.Do not wash, contain 95 ℃ of sex change 10min of hybridization solution of probe, interrogate fast cooled on ice, drip hybridization solution 100 μ l/ sheets, seal film and cover, 42-45 hybridization 15 hours.Hybridization back 2 * SSC, 0.5 * SSC, 0.2 * SSC washing 15 minutes/time.TNB damping fluid blocking-up unspecific staining.Drip SA-HRP (strepto-avidin-horseradish peroxidase), room temperature 30 minutes.The TNT damping fluid is washed, and 5 minutes/time, plus signal amplified reagent (Biotinyl Tyamid), room temperature 10 minutes.The TNT damping fluid is washed, and 5 minutes/time, drips SA-HRP (strepto-avidin-horseradish peroxidase), room temperature 30 minutes.The TNT damping fluid was washed 5 minutes/time, the DAB colour developing, and mirror is the control chromogenic reaction down; Haematoxylin is redyed, the special-purpose mountant mounting of adhesive tape transfer system
2, result
(1) successfully make the cell microarray of 100 dot matrix, every kind of cell comprises 5 dot matrix, the HE dyeing of all cells mooring points battle array, and observation of cell clear in structure under the mirror, form is neat; The all cells mooring points system of battle formations is looked like to carry out analysis of accounts, and cell number is 1150～1324 a/cell point.Use cell microarray to carry out in situ hybridization and SABC detects, the result shows that cell microarray does not have and falls the sheet phenomenon fully, and testing result consistent with cell smear (Fig. 6-10).
Claims (2)
1, a kind of cell chip method for making, comprise cellular incubation, collect fixed cell, cell dehydration, cell be transparent, the making of the making of the making of waxdip, cell chip acceptor paraffin mass and receptor hole, recipient cell chip paraffin mass and cell chip section is characterized in that:
(1) waxdip is that cell suspension is moved into the transparent bar-type mould (1) from Tube pipe, will be in the bar shaped mould cell and the molten point of the upright immersion of mould of uniform deposition be waxdip three times in 58 ℃ the LMP paraffin solution,
(2) making of acceptor paraffin mass and receptor hole: the making spacing is that the lattice array mould paper of 2mm is affixed on the acceptor paraffin mass of the corresponding specification size of making by the density of cell chip, passes receptor hole with the vacuum lancing pin in grid paper successively,
(3) making of recipient cell chip paraffin mass: the lower end that softening cell mineral wax mixture is clipped the bar shaped mould in placing 37 ℃ of incubators, extrude bar shaped cell mineral wax mixture from cutting end, and block into section, put into corresponding paraffin mass receptor hole.
2, the utensil of a kind of cell chip method for making as claimed in claim 1 special use, it is characterized in that: waxdip is with a kind of transparent bar-type mould (1), this mold bottom diameter is 1.0mm, and the bottom is plugged with and stops cell and help a small amount of cotton (2) that terebinthina flows out through mold bottom.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101158677B (en) * | 2007-10-29 | 2011-08-31 | 浙江大学 | Cell electric physiology integrated chip and preparation method |
| CN105628477A (en) * | 2014-04-23 | 2016-06-01 | 杭州电子科技大学 | Device for automatically and quickly preparing cell wax block |
| CN108593380A (en) * | 2018-04-25 | 2018-09-28 | 复旦大学附属中山医院 | A kind of organization chip volume production production method |
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| CN102511980A (en) * | 2011-12-31 | 2012-06-27 | 福建师范大学 | Preparation method for preparing mementoes storing genetic information of human bodies in direct coating manner |
| CN102511979A (en) * | 2011-12-31 | 2012-06-27 | 福建师范大学 | Preparation method for preparing mementoes storing genetic information of human bodies in indirect coating manner |
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| US3838013A (en) * | 1971-04-01 | 1974-09-24 | Gen Electric | Combined sampling and bacteriological culturing device |
| US4933148A (en) * | 1988-08-04 | 1990-06-12 | Brandeis University | Pipetter barrel extension tube |
| CN2052898U (en) * | 1989-09-05 | 1990-02-14 | 周润华 | Polyester coverture forming mould for biological cut section |
| JP3049537B2 (en) * | 1995-02-03 | 2000-06-05 | 悌二 竹崎 | Fixation support method for biopsy sample, fixation support agent and embedding cassette |
| US6103479A (en) * | 1996-05-30 | 2000-08-15 | Cellomics, Inc. | Miniaturized cell array methods and apparatus for cell-based screening |
| CN2349576Y (en) * | 1998-12-26 | 1999-11-17 | 河南省人民医院 | Pathologic tissue packing die |
| CN1330154A (en) * | 2000-06-27 | 2002-01-09 | 南京益来基因医学有限公司 | Cell microarray chip and its preparing process |
| CN1204405C (en) * | 2001-09-13 | 2005-06-01 | 陕西超英生物医学研究开发有限公司 | Tissue microarray biochip |
| US20050014155A1 (en) * | 2001-11-16 | 2005-01-20 | Xu Wilson C | High-density cell microarrays for parallel functional determinations |
| KR20030068780A (en) * | 2002-02-18 | 2003-08-25 | 서울대학교병원 | Bio-cell chip |
| CN1193101C (en) * | 2002-05-15 | 2005-03-16 | 上海新世界基因技术开发有限公司 | Tissue chip and cellline chip for observation of cell signal conducting path |
| CN2578830Y (en) * | 2002-11-15 | 2003-10-08 | 胡东升 | Micro-array chip configurating device of biological tissue |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101158677B (en) * | 2007-10-29 | 2011-08-31 | 浙江大学 | Cell electric physiology integrated chip and preparation method |
| CN105628477A (en) * | 2014-04-23 | 2016-06-01 | 杭州电子科技大学 | Device for automatically and quickly preparing cell wax block |
| CN105628477B (en) * | 2014-04-23 | 2018-01-16 | 杭州电子科技大学 | The full-automatic device for quickly preparing cell block |
| CN108593380A (en) * | 2018-04-25 | 2018-09-28 | 复旦大学附属中山医院 | A kind of organization chip volume production production method |
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