CN112903395A - In-situ slide culture method for villus tissue and chromosome preparation method - Google Patents
In-situ slide culture method for villus tissue and chromosome preparation method Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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Abstract
The invention belongs to the technical field of villus tissue culture and detection, and particularly discloses a villus tissue in-situ glass slide culture method and a chromosome preparation method. The villus tissue in-situ slide culture method comprises the steps of pretreatment, digestion, inoculation and culture, villus tissue cells are successfully cultured in situ on a slide, and the slide after culture can be directly used for preparing chromosomes. The chromosome preparation method is based on an in-situ glass slide culture method, the cell loss is reduced to the minimum degree by the method, the obtained chromosome has good dispersity and complete karyotype, and the accuracy of karyotype analysis is effectively improved.
Description
Technical Field
The invention belongs to the technical field of villus tissue culture and detection, and particularly relates to a villus tissue in-situ glass slide culture method and a chromosome preparation method.
Background
According to statistics, the incidence rate of birth defects in China is about 4% -6%, and nearly millions of defective births are born each year. In order to reduce the birth defect rate, in prenatal diagnosis of fetal diseases, the examination is mainly carried out by adopting methods such as amniocentesis, umbilical vein puncture and the like in the middle pregnancy and late pregnancy. The methods have the disadvantages that the pregnancy week is large, the time for finding the serious abnormality of the fetus and terminating the pregnancy is late, the incidence rate of induced labor complications is high, and the physiological and psychological damage to the pregnant women can be caused. With the development of the medical technology level, the villus biopsy can be adopted at the early pregnancy stage, and the villus biopsy refers to the ultrasonic guided downward puncture, and the villus tissue in the placenta is taken out for chromosome diagnosis, so that the problem can be found out in advance, and the damage can be reduced.
The method for obtaining chromosome in the wool biopsy operation in the early pregnancy stage at present comprises a villus direct method and a culture flask method. The method for directly preparing chromosome by using villus tissue is characterized by that it utilizes the characteristics of that the villus tissue cell is relatively exuberant in division, but the chromosome obtained by using cell which is not cultured is less in division phase and low in quality. The culture bottle is prepared by cleaning villus tissue under aseptic condition, cutting, placing in conventional plastic slant-opening cell culture bottle, adding culture medium, and adding CO2Culturing for several days at 37 ℃ in an incubator, then processing the cells growing adherent to the wall into single cell suspension by pancreatin or a scraper, dripping the cell suspension to a glass slide, and then performing slice-making and staining to obtain the chromosome. Compared with the villus cell direct method, the culture flask method has good quality and a large quantity, but in the cell collection process, the trypsinization method or the scraping method is adopted, so that the cell loss is large, the complete chromosome karyotype is less, and the accuracy of karyotype analysis is reduced.
Disclosure of Invention
One of the purposes of the invention is to provide an in-situ slide glass culture method of villus histiocyte, which directly cultures villus cells on a slide glass, and has a plurality of cell division phases, good shape and good adherence; after the cultivation, the slide glass can be directly subjected to slide preparation and dyeing to obtain the chromosome without pancreatin digestion or scraping, so that the cell loss is effectively reduced, and the accuracy of subsequent karyotype analysis is improved.
In order to achieve the purpose, the invention adopts the following specific technical scheme:
an in-situ slide culture method for villus histiocyte comprises the following steps:
pretreatment: taking a villus tissue sample under an aseptic condition, flushing the villus tissue sample until no visible blood red exists, removing other tissues, adding a double-antibody solution (namely a streptomycin mixed solution), and then incubating for 0.5-1.5 hours at 36-38 ℃ in a carbon dioxide incubator;
secondly, digestion: shearing a villus tissue sample, centrifuging and removing a supernatant; then adding fetal bovine serum culture medium for washing, centrifuging and removing supernatant; then adding tissue digestive juice, and incubating for 0.5-1.5 hours in a carbon dioxide incubator at 36-38 ℃;
inoculating: centrifuging the digested sample, and discarding the supernatant; adding fetal bovine serum culture medium for washing, centrifuging and removing supernatant; adding fetal calf serum culture medium, mixing to obtain suspension, sucking the suspension, adding onto a glass slide in an in-situ culture box, and placing the in-situ culture box in a carbon dioxide incubator at 36-38 deg.C for pre-culture;
fourthly, culturing: adding fetal bovine serum culture medium into the in-situ culture box after pre-culturing for 20-25h, and changing the culture solution after culturing for 20-25 h; and (5) changing the liquid again after culturing for 20-25h, and culturing for 20-25h again to obtain the slide glass with the villus cells.
Preferably, in step (i), the villus tissue sample is at least 10mg of villus branch sample.
Preferably, in step (i), the villus tissue sample is washed with sterile physiological saline.
Preferably, in the second step, the tissue digestive fluid is a trypsin solution or a collagenase ii solution.
Preferably, the slide glass comprises a clamping area and a culture area, and the surface of the culture area is subjected to silicification treatment, polylysine treatment or collagen treatment. The clamping area is used for clamping the glass slide by tweezers, the arrangement of the clamping area can avoid damage to cells when the glass slide is taken and placed, the cell adhesion can be promoted by the special treatment of the culture area, the success rate of cell culture on the glass slide is improved, and the cell shedding phenomenon of the glass slide in the subsequent chromosome preparation process is avoided. More preferably, the surface of the clamping area is a frosted surface so as to facilitate clamping; the surface of the culture area is a smooth surface so as to accelerate the removal speed of the solution (culture medium, colchicine solution, hypotonic solution, pre-fixing solution and fixing solution) used in the chromosome preparation process, reduce the residue of the solution, especially the hypotonic solution, and further ensure good chromosome preparation effect.
Another object of the present invention is to provide a method for preparing chromosomes from villus histiocytes, which comprises the steps of
The method for obtaining the slide with the villus cells by the villus histiocyte in-situ slide culture method and directly flaking and dyeing the slide to obtain the chromosome specifically comprises the following steps:
(1) preparing a glass slide with villus cells according to the villus histiocyte in-situ glass slide culture method;
(2) adding colchicine solution into the in-situ culture box filled with the glass slide in the step (1), uniformly mixing with the culture medium in the box, and standing for 25-35 minutes at 36-38 ℃;
(3) removing liquid in the in-situ culture box, adding the low-permeability liquid into the low-permeability liquid chamber, and standing for 25-30 minutes at the room temperature;
(4) removing liquid in the in-situ culture box, adding a pre-fixing liquid, and standing for 5-10 minutes;
(5) removing liquid in the in-situ culture box, adding a fixing liquid for fixation, removing the liquid in the in-situ culture box, and repeating the fixation for three times;
(6) taking out the glass slide, and placing the glass slide into a chromosome dispersion instrument for dispersion;
(7) baking the glass slide at 85-95 ℃ for 2.5-3.5h, and then aging overnight at 36-38 ℃;
(8) and (3) carrying out chromosome staining on the cells on the glass slide by adopting a G banding staining method.
Preferably, in the step (5), in the first fixing, the fixing solution is added and then is kept still for no more than 1 minute, and preferably, the liquid in the in-situ culture box is removed immediately after the fixing solution is added and mixed uniformly to remove residual hypotonic solution, and the residual hypotonic solution can cause the cells to be over-swelled and ruptured; and when the second fixation and the third fixation are carried out, the fixation solution is added and then the mixture is kept stand for 10 to 20 minutes, the fixation time is insufficient, the fixation is insufficient, and hypotonic expanded cells are easy to break, so that chromosome loss is caused.
Preferably, in the step (6), the chromosome dispersion instrument is set to dispersion conditions of 23-26 ℃ and 48-53% humidity. More preferably, the dispersion conditions are a temperature of 25 ℃ and a humidity of 50%, under which the chromosomes are dispersed to the most suitable extent in the hypotonic cells.
The hypotonic, pre-fixing and fixing solutions may be reagents used in conventional chromosome preparation. Preferably, the hypotonic solution is trisodium citrate solution, the pre-fixing solution is glacial acetic acid, and the fixing solution is methanol-glacial acetic acid solution. More preferably, the hypotonic solution is 1mg/ml trisodium citrate solution, the pre-fixing solution is 5% glacial acetic acid, and the volume ratio of methanol to glacial acetic acid in the fixing solution is 3: 1. The invention has the following beneficial effects:
1. the method successfully cultures the villus histiocyte on the glass slide in situ, the glass slide can be directly used for preparing the chromosome after the culture, compared with the prior culture flask method, the method does not need to harvest the cell by pancreatin digestion or scraping transfer, simplifies the operation, avoids chromosome loss, cell loss and the like caused by experimental operation, and provides high-quality raw materials for the subsequent chromosome preparation.
2. The invention also provides a chromosome preparation method based on the in-situ slide culture method, which reduces the cell loss to the minimum degree, and the obtained chromosome has good dispersity and complete karyotype, and effectively improves the accuracy of karyotype analysis.
3. The method for culturing the villus tissue in-situ slide glass and the method for preparing the chromosome have good stability, simple operation and nearly 100 percent success rate; after the chromosome is prepared, the slide glass can be directly loaded on a GSL-120 chromosome full-automatic scanner for image collection and analysis, and the working efficiency is high.
4. The in-situ culture of the villus histiocyte is a bottleneck that prenatal diagnosis in the early pregnancy can not be realized, the invention breaks through the bottleneck, effectively improves the diagnosis timeliness, can discover the defects as early as possible, and reduces the harm to the pregnant woman to the minimum.
Drawings
FIG. 1: schematic of an in situ culture cassette in an embodiment of the invention;
FIG. 2: a schematic view of a stained slide in an embodiment of the invention;
FIG. 3: the chromosome karyotype of the villus cells in the examples of the present invention.
Detailed Description
The invention is further described below with reference to the accompanying drawings and specific embodiments.
Examples
This example provides a method for preparing chromosomes from villus histiocytes, which comprises the following steps:
(1) in situ slide culture of villus histiocyte
Pretreatment: taking a 15mg villus branch sample as a villus tissue sample under an aseptic condition, putting the villus tissue sample into an aseptic culture dish, and washing the villus tissue sample with aseptic normal saline until no visible blood red exists; using tissue scissors and tweezers to inspect the villus, and removing other tissues to ensure that a villus branch sample is remained; adding 5ml of double-antibody solution into the sample, and then incubating for 1 hour at 37 ℃ in a carbon dioxide incubator;
secondly, digestion: taking out the culture dish, shearing the villus tissue sample by using small tissue scissors and tweezers to break the villus tissue sample as much as possible, then transferring the villus into a sterile centrifuge tube by using a sterile disposable pipette, centrifuging (1500 rpm for 8 minutes), and discarding the supernatant; adding 4ml fetal bovine serum culture medium (BI), mixing uniformly, washing the villus once, centrifuging (1500 rpm for 8 minutes), and removing the supernatant; adding 3mL of collagenase II solution, uniformly mixing, placing in a carbon dioxide incubator, incubating for 1 hour at 37 ℃, and uniformly mixing once every 20 minutes until most villi are digested;
inoculating: centrifuging the digested sample, and discarding the supernatant; adding fetal bovine serum culture medium, washing twice, centrifuging and removing supernatant; adding fetal bovine serum culture medium, mixing to obtain suspension, sucking 1ml of suspension, adding onto a glass slide in an in-situ culture box, respectively inoculating two, synchronously culturing two lines, and placing the in-situ culture box in a carbon dioxide incubator at 37 ℃ for pre-culture;
fourthly, culturing: after the preculture is carried out for 24h, 3ml of fetal calf serum culture medium is added into the in-situ culture box for long-term culture; culturing for 24h, and then carrying out liquid change; after 24h of culture, changing the liquid again, and culturing for 24h again;
(2) adding 40ul of 0.1mg/ml colchicine solution into an in-situ culture box, uniformly mixing with a culture medium in the box, and then placing the mixture into an incubator at 37 ℃ for standing for 30 minutes;
(3) hypotonic: sucking off the liquid in the in-situ culture box by using a suction pipe, adding 5ml of hypotonic solution (1 mg/ml trisodium citrate solution), and standing for 28 minutes at room temperature;
(4) pre-fixing: absorbing the liquid in the in-situ culture box by using a suction pipe, adding 5ml of pre-fixing liquid (5% glacial acetic acid), and standing for 7 minutes;
(5) fixing: sucking off liquid in the in-situ culture box by using a suction pipe, adding 5ml of stationary liquid (methanol-glacial acetic acid solution with the volume ratio of methanol to glacial acetic acid being 3: 1), mixing uniformly, and immediately sucking off the liquid in the in-situ culture box; adding 5ml of fixing solution for second fixing, standing for 15min, and sucking off the liquid in the in-situ culture box; then 5ml of fixing solution is added for third fixing, standing is carried out for 15min, and liquid in the in-situ culture box is sucked out;
(6) dispersing: adjusting the temperature of a THERMOTRON chromosome disperser CDS-5 to 25 ℃ and the humidity of 50%, clamping the glass slide in the in-situ culture box by using forceps, placing the glass slide in the disperser for dispersing, and taking out the glass slide after the dispersion is finished;
(7) baking slices: putting the glass slide into an oven at 90 ℃ for baking for 3 hours, and then transferring the glass slide into an incubator at 37 ℃ for overnight aging;
(8) and (3) band display and dyeing: and (3) carrying out chromosome staining on the cells on the glass slide by adopting a G banding staining method, wherein the specific operation refers to the conventional G banding staining of the amniotic fluid chromosomes.
The in-situ culture box is shown in figure 1 and comprises a box body and an upper cover, wherein a glass slide is arranged in the box body, liquid can be added into the box body and the glass slide can be taken and placed in the box body by opening the upper cover, and the glass slide comprises a clamping area and a culture area; the surface of the clamping area is a frosted surface, and the culture area is a smooth surface and is treated by polylysine.
As shown in figure 2, the slide glass after the banding staining is finished has a plurality of fixed cells, has good slide making effect and embodies the success of in-situ cell culture; the slide glass is put into a GSL-120 chromosome full-automatic scanner for image collection and analysis to obtain a nuclear type image of the villus cell chromosome shown in figure 3, and the nuclear type image has the advantages of good chromosome dispersibility, complete nuclear type, clear display and high analysis reliability.
This detailed description is to be construed as illustrative only and is not to be taken as limiting the invention, as any changes that may be made by a person skilled in the art after reading the present specification will be protected by the patent laws within the scope of the appended claims.
Claims (10)
1. An in-situ slide culture method of villus histiocyte is characterized in that: the method comprises the following steps:
pretreatment: taking a villus tissue sample under an aseptic condition, flushing the villus tissue sample until no visible blood red exists, removing other tissues, adding a double-antibody solution, and then incubating for 0.5-1.5 hours in a carbon dioxide incubator at 36-38 ℃;
secondly, digestion: shearing a villus tissue sample, centrifuging and removing a supernatant; then adding fetal bovine serum culture medium for washing, centrifuging and removing supernatant; then adding tissue digestive juice, and incubating for 0.5-1.5 hours in a carbon dioxide incubator at 36-38 ℃;
inoculating: centrifuging the digested sample, and discarding the supernatant; adding fetal bovine serum culture medium for washing, centrifuging and removing supernatant; adding fetal calf serum culture medium, mixing to obtain suspension, sucking the suspension, adding onto a glass slide in an in-situ culture box, and placing the in-situ culture box in a carbon dioxide incubator at 36-38 deg.C for pre-culture;
fourthly, culturing: adding fetal bovine serum culture medium into the in-situ culture box after pre-culturing for 20-25h, and changing the culture solution after culturing for 20-25 h; and (5) changing the liquid again after culturing for 20-25h, and culturing for 20-25h again to obtain the slide glass with the villus cells.
2. The method of claim 1, wherein the culturing of the villus tissue cells on the slide in situ comprises: in the step (i), the villus tissue sample is at least 10mg of villus branch sample.
3. The method of claim 1, wherein the culturing of the villus tissue cells on the slide in situ comprises: in the step I, the villus tissue sample is washed by sterile normal saline.
4. The method of claim 1, wherein the culturing of the villus tissue cells on the slide in situ comprises: in the second step, the tissue digestive juice is trypsin solution or collagenase II solution.
5. The method of claim 1, wherein the culturing of the villus tissue cells on the slide in situ comprises: the glass slide comprises a clamping area and a culture area, wherein the surface of the culture area is subjected to silicification treatment, polylysine treatment or collagen treatment.
6. The method of claim 5, wherein: the surface of the culture area is smooth.
7. A method for preparing chromosomes of villus histiocytes, which is characterized in that: the method comprises the following steps:
(1) preparing a slide with villous cells by the villous tissue cell in situ slide culture method according to any one of claims 1 to 6;
(2) adding colchicine solution into the in-situ culture box filled with the glass slide in the step (1), uniformly mixing with the culture medium in the box, and standing for 25-35 minutes at 36-38 ℃;
(3) removing liquid in the in-situ culture box, adding the low-permeability liquid into the low-permeability liquid chamber, and standing for 25-30 minutes at the room temperature;
(4) removing liquid in the in-situ culture box, adding a pre-fixing liquid, and standing for 5-10 minutes;
(5) removing liquid in the in-situ culture box, adding a fixing liquid for fixation, removing the liquid in the in-situ culture box, and repeating the fixation for three times;
(6) taking out the glass slide, and placing the glass slide into a chromosome dispersion instrument for dispersion;
(7) baking the glass slide at 85-95 ℃ for 2.5-3.5h, and then aging overnight at 36-38 ℃;
(8) and (3) carrying out chromosome staining on the cells on the glass slide by adopting a G banding staining method.
8. The method for producing a chromosome from a villus tissue cell according to claim 7, wherein: in the step (5), when the fixation is repeated for three times, the standing time after the fixation liquid is added is not more than 1 minute during the first fixation, and the standing time after the fixation liquid is added during the second and third fixation is 10-20 minutes.
9. The method for producing a chromosome from a villus tissue cell according to claim 7, wherein: in the step (6), the chromosome disperser is set at a temperature of 23-26 ℃ and a humidity of 48-53%.
10. The method for producing a chromosome from a villus tissue cell according to claim 7, wherein: the hypotonic solution is trisodium citrate solution, the pre-fixing solution is glacial acetic acid, and the fixing solution is methanol-glacial acetic acid solution.
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| CN113337581A (en) * | 2021-06-21 | 2021-09-03 | 中国医学科学院北京协和医院 | Method for detecting cytogenetic abnormality by using buckle type slide culture box |
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