CN101294951A - Tissue chip production method - Google Patents
Tissue chip production method Download PDFInfo
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- CN101294951A CN101294951A CNA2007100401651A CN200710040165A CN101294951A CN 101294951 A CN101294951 A CN 101294951A CN A2007100401651 A CNA2007100401651 A CN A2007100401651A CN 200710040165 A CN200710040165 A CN 200710040165A CN 101294951 A CN101294951 A CN 101294951A
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Abstract
The invention discloses a manufacturing method for a tissue chip, which comprises the following steps: the manufacture and perforation of a receptor wax block, the manufacture of the mark of a point to be taken and a donor wax block, the sampling of the donor wax block, recording of array point information, and the processing and slicing of an array wax block; wherein, the manufacture of the mark of the point to be taken and the donor wax block comprises the following steps: (1) when obtaining the materials, a needle is used for perforating at 1/3 position of the left upper quadrant of the wax block so as to mark as the origin a of an XY-axis; (2) the a is taken as the origin, and coordinate distances of the point which needs to obtain materials on the horizontal axis (X-axis) and the vertical axis (Y-axis) are respectively measured in a microscope; (3) marking is carried out when returning the corresponding coordinate point on the wax block; (4) the sampling needle is used for perforating and sampling on the parts to be taken out on the donor wax block; (5) the sample is planted accurately in the array wax block (receptor wax block); the manufacturing method of the invention can achieve the material-obtaining of precise point by point to point, thus avoiding ineffective chip deviation caused by heterogeneity of tumors reported before; the manufacturing method can also record point information of each chip precisely which is convenient for test in future, and can establish a tumor tissue chip library with normal series effectively.
Description
Technical field
The present invention relates to a kind of tissue chip production method, the present invention especially relates to the tissue chip production method of drawing materials in the accurate site of a kind of energy.
Background technology
Organization chip (tissue chip) claims that again (tissue microarray TMA), is another the important biochip technology that occurs to micro-array tissue after genetic chip, protein-chip.This technology equals reported first [1] on Nature Medicine in 1998 by the Kononen of the U.S. state-run cancer research institute.It is fitly to be emitted on the microslide and the histotomy of making according to design to thousands of cells tens of.
Because existing tissue chip production method needs special instrument, the expense costliness, and sampling site is not accurate enough, has deviation, is unfavorable for extensively carrying out of TMA work.
Summary of the invention
The purpose of this invention is to provide a kind of tissue chip production method, solved a series of difficult problems of in the past making in the organization chip, realized the sampling of accurate site first.
The invention provides a kind of tissue chip production method, comprise the making and the punching of acceptor wax block, treat the making of fetch bit point mark and donor wax stone, the sampling of donor wax stone, array site information record, the processing of array wax stone and the step of section, it is characterized in that treating fetch bit point mark and donor wax stone: (a) when drawing materials, make marks in the punching of left upper quadrant 1/3 place of wax stone with pin with following step making, initial point a as the XY axle, (b) with a be initial point, measure the coordinate distance of site on transverse axis (X-axis) and Z-axis (Y-axis) that to draw materials at microscopically respectively, (c) turning back on the wax stone corresponding coordinate site makes marks, (d) take out punching sampling on the position with sampling needle in donor wax stone desire, (e) go up and accurately implant sample at array wax stone (acceptor wax block).
The present invention also provides the sampling procedure of donor wax stone in the described tissue chip production method employed instrument, it is characterized in that engraving exact scale on the rectangle right-angle side empty in two, form a hollow, rectangular with two relative both sides, right angle of rectangle during sampling, distance and direction according to the measurement of front microscopically, with the drilling point is the summit at a right angle of rectangle, is exactly the exact position in the site that will take a sample to angular vertex.Organization chip is because its high flux, collimation and science provide a very outstanding platform can for undoubtedly the leap of scientific development, because this technology is also not really ripe, need special expensive device, and operability is bad, domestic research to it also is in the starting stage, this paper is comprehensive and systematic to have set forth the method for making of organization chip and the making of equipment, all difficult problems of making in the tissue array technology have been solved, can better promote the flourish of China's tissue array technology, set up serial tissue bank in the whole nation rapidly.
The advantage of our technology: (1) relies on the existing equipment of paraffin section, oneself designs and making various tool and equipment, and cost is low, and is workable, is very beneficial for carrying out the work.(2) realize that successfully point-to-point accurate site draws materials, thus avoided former report because the invalid chip deviation that the tumour heterogeneity is caused.(3) accurately write down the information in each chip site, test after being convenient to.Can effectively set up the normal and tumor tissues chip storehouse of series.(4) all unified size specification of our various organization chip wax stones, can with embedding base plate compatibility commonly used, made things convenient for section, also can make technical manualization, standardization, computing machine is read sheet after being convenient to.(5) can make up needed various organization chip very easily in practice.
Owing to contain much information, the efficient height, it is few to consume reagent, the proposition of TMA technology, making with immunohistochemistry and original position PCR is that the molecular pathology TMA of representative has the incomparable superiority of common section: very small (diameter 0.6-2.0mm), quantity many (can reach 1000 dot matrix at present), regular shape, arrange in order, have high flux and collimation, the science and the comparability of testing result are strong, contain much information, the efficient height, it is few to consume reagent.The proposition of this technology, making with immunohistochemistry and original position PCR is further march toward standardization, scientific and hi-techization [3] of the molecular pathology research of representative.To the molecular changes of tumour and getting in touch in the research of clinical pathologic characteristic, TMA will provide huge help, and TMA also can be used for the screening of gene outcome, the test of biological reagent, control and standardization, teaching and examination and the making micro histology and the pathology collection of illustrative plates of pathology quality simultaneously.Continuous development along with the TMA technology, the foundation in tumour and normal structure storehouse, the structure of TMA forms seriation, and pathological effect will face transformation, promptly when the tumour somatotype, no longer lay stress on the histology form changes, but from many aspects, multilevelly provide additional information for clinical treatment.Believe that in the near future the TMA technology will be brought into play more importantly effect in the diagnosis of tumour and research field.
Embodiment
Institute's materials used is commercially available.
Embodiment 1
The making of perforating needle and sampling probe.Perforating needle and sampling probe all can grind with the puncture needle oneself with real core pin, and a trocar has four, two hollow needles and two supporting solid needles.The pin footpath can be 0.6-2.0mm, must notice that the perforating needle footpath is than sampling probe footpath little No. 1 (external diameter that is perforating needle is consistent with the internal diameter of sampling probe), so that the diameter of sampling tissue is consistent with the punching aperture, in case being connected with acceptor wax block, organization chip tightly do not come off, the sampling probe diameter that we use is 1.6mm, the degree of depth that the periphery cover colored plastic film of perforating needle and sampling probe enters wax stone with the mark pin, the degree of depth of perforating needle punching is than the dark 1.0mm of sampling probe.Someone reports it is to adopt the microscopic carvings reference mark technology of windowing, and we think inadvisable, because pin will grind when not sharp for a long time, can influence scale [2].
Embodiment 2
The making of flux locating template.According to the needs of oneself, the template (the most handy transparent material) that can use the pin of different-diameter to make different flux, pitch of holes can arrive 2.0mm for 1.0mm, suggestion has just begun to operate unskilled time interval (2.0mm) more greatly, stay white distance (3.0mm) more greatly all around, punched askew or split, impact effect preventing.Preferably three weeks baffle plate was arranged all around the template, the internal diameter size of template is identical with cured of acceptor, so that template is stuck on the acceptor wax block, it is very light to operate.The flux template that we use has two kinds: (1) flux array 5 * 7, sampling diameter 2.0mm, spacing 2.0mm; (2) the flux array 7 * 9, sampling diameter 1.5mm, spacing 1.5mm; The wax stone unification is 30.0 * 25.0mm, and thickness 15.0mm is convenient to management.
Embodiment 3
The TMA method for making may further comprise the steps
The making of acceptor wax block and punching (1) can use fusing point to make at 60-62 ℃ of common section purified paraffin, and the needs of foundation oneself design and produce the various embedding frames of the consistent size with template of specification; (2) pour the whiteruss of thawing into, box at the bottom of the face of wax stone adds the common plastics embedding, so that when section be wax stone and numbering fixedly, also can be directly with the label paper numbering (end box and wax stone are come off, do not influence section) of sealing with wax but wax stone is still very regular.Pick up counting behind the potting syrup paraffin body, the embedding frame is peeled off in (concrete time may because the difference of room temperature and difference) wax stone moulding and be in solidification state fully behind the 40-50min, begins to punch; Earlier locating template is blocked acceptor wax block when (3) punching and fix, the perforating needle with corresponding model punches by the array hole on the locating template then, and the degree of depth is 6.0mm.Punching need be carried out fast, and easy shattered crack during hardening otherwise wax stone turns cold also can be put into stand sheet machine water tank (46 ℃ of water temperatures) heating 3-5min this moment, makes wax stone be heated deliquescing and is difficult for shattered crack.
Treat fetch bit point mark and donor wax stone making this be the key of technological innovation.Wax stone is tetragonal because majority is drawn materials, seldom be irregular, certain that makes us be difficult to get back to after microscopically is read sheet on the wax stone is a bit precisely drawn materials, common method is the mark of drawing a circle on slide, return to estimate on the wax stone and look for the site, this method need to determine the many places sampling owing to the sampling site quadrant is bad, this has increased our workload and cost greatly.Our solution is that coordinate positioning method is accurately located: (1) makes marks in the punching of left upper quadrant 1/3 place of wax stone (also can with little stylus printer hole mark on the wax stone that has been shaped) with pin when drawing materials, as the initial point of XY axle.Be initial point with this drilling point position when (2) we read sheet, measure the coordinate distance of site on transverse axis (X-axis) and Z-axis (Y-axis) that we will draw materials at microscopically respectively, turn back to again that corresponding coordinate site makes marks sampling on the wax stone.Thereby realize point-to-point accurate site sampling, only once just can be successful.
Need heating before the sampling of the sampling of donor wax stone (1) donor wax stone, method has oven heat and water-bath heating, and we think that water-bath is more convenient, and are easy to control.Water temperature 46-48 ℃, heating 5-10min; (2) coordinate according to preceding planar survey finds the site that need draw materials, take out punching sampling on the position with sampling needle in donor wax stone desire, degree of depth 5.0mm (than the shallow 1.0mm in acceptor wax block hole), the degree of depth can be used the colored plastic membrane marker earlier on pin, need during sampling slowly firmly, prevent that the donor wax stone from splitting, will rotate for two weeks so that nook closing member is full of after the degree of depth that arrival requires.(3) on array wax stone (acceptor wax block), find the site that to insert, slowly release, accurately implant, press and flatly will make the plane of organizing core and the basically identical of wax stone with solid nook closing member.(4) for more convenient sampling accurately, we have made simple sampling orientation tool, method for making: in two, engrave exact scale on the rectangle right-angle side of sky, form a hollow, rectangular with two relative both sides, right angle of rectangle during sampling, distance and direction according to the measurement of front microscopically, with the drilling point is the summit at a right angle of rectangle, is exactly the exact position in the site that will take a sample to angular vertex.We are named as coordinate setting rectangle template sampling method above innovative approach.
Array site information record (1) some sites on the left upper quadrant off-diagonal when the implanting tissue core will be vacated, so that the orientation of identification array.Someone reports that adopt implanting brain tissue locatees, and thinks after our practice and does not still implant conveniently, because 1) array of implanting tissue core not, we just can determine the orientation by naked eyes, and the array of implantation brain tissue will could be determined at microscope; 2) brain tissue scatters when the sheet of stand sometimes easily, determines the orientation difficulty thereby increase.(2) the information record adopts two-dimentional recording mode, accurately writes down the information in each site.Horizontal direction from left to right each point coordinate be labeled as A, B, C ..., each point coordinate of vertical direction is labeled as 1,2,3 ..., each site is all by the coordinate of being made up of letter and number by like this, as (A3), (B5), (G2) or the like.Can on register, write down the information in each site, set up the organization chip storehouse very easily.
After the processing array of array wax stone is made and is finished, the one side that the array wax stone is contained in a organized way lies on the glass plate, places in 56 ℃ of constant temperature ovens, and every 5min observes once, the liquid paraffin of appearance takes out wax stone in the middle for the treatment of wax stone and glass plate, places in the refrigerator and cools off on the ice cube.Someone reports it is to cool off at normal temperatures, our practice and relatively after think that cooling effect is better in the refrigerator because: normal temperature down the cooling wax stone must bottom surface (face of chip in a organized way) up, at this moment whiteruss flows out easily, make the corner rust of wax stone, influence the section of organizing core of corner like this.
The good back of section ice is with the thick section of 4 μ m, and mount is on the microslide of handling through 2%3-aminopropyl three ethoxy silane (APES) acetone solutions.The section of microarray wax stone is cut into slices on come card microtome with the Leca disposable blade, basic identical with the conventional organization section, but because the piece of tissue in the wax stone is many, operation wants light when mounting sheet, speed is grasped suitable, otherwise from disconnected or tissue array skew, in addition, be also noted that water temperature easily, cross low section Zhan Bukai, too high easy elongation is broken, and it is more suitable to remain on about 48 ℃.With disposable slicer than good with normal thick steel knife chipping qualities.
Claims (2)
1. tissue chip production method, the sampling, array site information record, the processing of array wax stone and the step of section that comprise the making and the punching of acceptor wax block, the making for the treatment of fetch bit point mark and donor wax stone, donor wax stone is characterized in that treating fetch bit point mark and donor wax stone with following step making:
(a) when drawing materials, make marks in the punching of left upper quadrant 1/3 place of wax stone with pin, as the initial point a of XY axle,
(b) with a be initial point, measure the coordinate distance of site on transverse axis (X-axis) and Z-axis (Y-axis) that need draw materials at microscopically respectively,
(c) turn back on the wax stone corresponding coordinate site and make marks,
(d) take out punching sampling on the position with sampling needle in donor wax stone desire,
(e) go up accurately implantation sample at array wax stone (acceptor wax block).
2. the employed instrument of sampling procedure of donor wax stone in the tissue chip production method as claimed in claim 1, it is characterized in that engraving exact scale on the rectangle right-angle side empty in two, form a hollow, rectangular with two relative both sides, right angle of rectangle during sampling, distance and direction according to the measurement of front microscopically, with the drilling point is the summit at a right angle of rectangle, is exactly the exact position in the site that will take a sample to angular vertex.
Priority Applications (1)
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CN200710040165A CN101294951B (en) | 2007-04-28 | 2007-04-28 | Tissue chip production method |
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CN200710040165A CN101294951B (en) | 2007-04-28 | 2007-04-28 | Tissue chip production method |
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CN101294951A true CN101294951A (en) | 2008-10-29 |
CN101294951B CN101294951B (en) | 2012-09-12 |
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108088729A (en) * | 2018-01-18 | 2018-05-29 | 熊中堂 | Organization chip producing device and method |
CN108827746A (en) * | 2018-05-18 | 2018-11-16 | 郑州大学第附属医院 | A kind of one-pass molding, exempt from fusion organization chip production method and device |
CN110132702A (en) * | 2019-06-11 | 2019-08-16 | 河南科技学院 | A kind of embedding method for particular pathologies tissue |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1445523A (en) * | 2003-02-20 | 2003-10-01 | 但汉雷 | Method for preparing tissue chip |
FR2860319B1 (en) * | 2003-09-30 | 2006-02-10 | Ct De Lutte Contre Le Cancer F | METHOD FOR DETERMINING THE POSITION OF TISSUE SAMPLER CHIPS ON A BLADE OF TRANSPARENT MATERIAL |
-
2007
- 2007-04-28 CN CN200710040165A patent/CN101294951B/en not_active Expired - Fee Related
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108088729A (en) * | 2018-01-18 | 2018-05-29 | 熊中堂 | Organization chip producing device and method |
CN108827746A (en) * | 2018-05-18 | 2018-11-16 | 郑州大学第附属医院 | A kind of one-pass molding, exempt from fusion organization chip production method and device |
CN110132702A (en) * | 2019-06-11 | 2019-08-16 | 河南科技学院 | A kind of embedding method for particular pathologies tissue |
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CN101294951B (en) | 2012-09-12 |
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