CN109706210B - Biopsy method of trophoblast cells of 6-stage blastula - Google Patents
Biopsy method of trophoblast cells of 6-stage blastula Download PDFInfo
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- CN109706210B CN109706210B CN201811554863.8A CN201811554863A CN109706210B CN 109706210 B CN109706210 B CN 109706210B CN 201811554863 A CN201811554863 A CN 201811554863A CN 109706210 B CN109706210 B CN 109706210B
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Abstract
The invention belongs to the field of biological medical treatment, and provides a method for biopsy of trophoblast cells of a 6-stage blastocyst. The method provided by the invention is simple and stable to operate, greatly improves the detection success rate of the blastocyst, and reduces the damage to the blastocyst.
Description
Technical Field
The invention belongs to the field of biological medical treatment, and relates to biopsy of blastocyst trophoblast cells.
Background
Genetic diagnosis is required prior to embryo implantation, provided that a sample of a small number of cells of the embryo is obtained for chromosome analysis. Biopsy of blastocyst trophoblast cells has gained wider and wider use due to its significant accuracy and convenience advantages. During the process of blastocyst culture, more or less 6 stage blastocysts (fully hatched blastocysts) appear. The blastula in the six stage is the stage from the fertilized egg to the blastula, and the blastula is classified to the six stage, namely the blastula breaks through the limitation of the zona pellucida, completely hatches and falls off the zona pellucida. Because the stage 6 blastocyst lacks the protection and support of the zona pellucida, the blastocyst lacks the position that the fixed needle can hold during the biopsy process, so that the fixed needle directly acts on the trophoblast cells, which brings much inconvenience to the biopsy, and operation failure occurs frequently during the biopsy process.
Disclosure of Invention
Aiming at the defects of the prior art, in order to ensure that the stage 6 blastocyst obtains better control in the biopsy process, the invention provides a method for biopsy of trophoblastic cells of the stage 6 blastocyst, which is mainly characterized by comprising the following steps: the incision of the zona pellucida, the penetration of the biopsy needle from the incision, the opening of the zona pellucida, the penetration of the biopsy needle from the opening, the stretching of trophoblast cells, and the cutting of trophoblast cells by laser.
Specifically, the method of the present invention comprises the steps of:
(1) Slotting of the transparent belt: fixing the transparent belt with the blastocyst falling off in the 6 th stage, preferably fixing by using a fixing needle, and forming a seam on the transparent belt by using a laser or a microdissection needle to ensure that the transparent belt is internally and externally communicated for penetrating through a biopsy needle; preferably, the width of the slits is in the range of 1-300 microns, preferably 2-10 microns, more preferably 5 microns; and the slotting position is positioned at the opposite side of the fixed position of the fixed needle;
(2) The biopsy needle passes through the slit: passing the biopsy needle along the slit position in the step (1) to stay in the transparent belt;
(3) Opening of the transparent tape: a laser or a micro-cutting needle is used for forming a larger opening above the transparent belt, and part of the transparent belt is removed; preferably, the length of the openings is in the range of 1-200 microns, preferably 80 microns;
(4) The biopsy needle penetrates from the opening of the zona pellucida: penetrating the biopsy needle from the inner part of the transparent belt to the outer part along the position of the opening in the step (3) so that the biopsy needle stays at the outer part of the transparent belt;
(5) Suction and stretching of blastula trophoblast cells: aiming the biopsy needle at one side of the 6-stage blastocyst, which is far away from the inner cell mass, preferably the opposite side of the inner cell mass, sucking a small part of trophoblast cells into the biopsy needle, withdrawing the biopsy needle from the opening of the transparent band in the step (3) and the step (1) in sequence to return to the outside of the transparent band, and clamping and stretching the trophoblast cells by utilizing the seam opened in the step (1); preferably, the number of aspirated trophoblast cells ranges from 1 to 30, preferably 10;
(6) Cutting the trophoblast cells by laser: cutting the thinnest part of the stretched trophoblast cells from top to bottom by using laser, and separating a small part of the trophoblast cells sucked by the biopsy needle from the 6-stage blastocyst; preferably, the thinnest point is in the range of 1 to 50 microns, preferably 15 microns.
All blastocysts contemplated by the present invention are derived from non-human mammals, such as mice, rabbits, rats, etc.
The fixing needle, the micro-cutting needle and the biopsy needle in the steps (1) to (6) are made of plastic, metal or glass, preferably glass. Wherein the metal material is one of copper, iron, stainless steel, aluminum magnesium alloy, aluminum, tin and the like; the plastic material is one of Polyethylene (PE), polypropylene (PP), polyvinyl chloride (PVC), polystyrene (PS), ABS, polymethyl methacrylate (PMMA), polyamide (PA) and the like; the glass material is one of the glass materials such as chalcogenide glass, halide glass, silicate glass, borate glass, and phosphate glass.
In the fixed needle, the micro-cutting needle or the biopsy needle in the steps (1) to (6), the front section of the fixed needle, the micro-cutting needle or the biopsy needle has a certain angle with the whole body, and the angle ranges from 0 to 90 degrees, preferably 35 degrees.
The needle point parts of the fixing needle, the microdissection needle and the biopsy needle in the steps (1) - (6) can be flat openings or sharp openings, the fixing needle and the biopsy needle are preferably flat openings, the fixing needle and the biopsy needle are hollow inside, the fixing needle and the biopsy needle can be matched with a microinjector to suction and blowing operations, and the microdissection needle is preferably sharp openings.
The laser in the steps (1), (3) and (6) can be laser with various wavelengths and colors, and is preferably 1480nm infrared laser.
The zona pellucida in the steps (1) - (5) is the zona pellucida of the blastocyst falling off at the stage 6.
On one hand, the invention utilizes the zona pellucida which falls off the blastocyst to be biopsied, so that the material is convenient to obtain; on the other hand, the restraint and the fixation function of the fallen zona pellucida are fully utilized, so that the fixation needle does not directly act on trophoblast cells, thereby reducing the damage to the blastocyst in the biopsy process and improving the stability of the 6-stage blastocyst biopsy. The method provided by the invention is simple and stable to operate, greatly improves the detection success rate of the blastula, and reduces damage to the blastula.
Drawings
FIG. 1 is a schematic representation of one embodiment of the present invention showing a seam in the transparent tape after fixation of the blastocyst; in figure 1 a 5 mm slit is made in a transparent tape by means of a laser.
FIG. 2 is a schematic view of an embodiment of the present invention showing a biopsy needle penetrating the slit of FIG. 1 to reside within the zona pellucida; in fig. 2 a biopsy needle is shown.
FIG. 3 is a schematic view of an embodiment of the present invention showing a larger opening above the transparent band; and 3 is an opening.
FIG. 4 is a schematic view of an embodiment of the present invention showing a biopsy needle being passed along the opening of FIG. 3 to rest outside the zona pellucida; wherein: 4 is the front end of the biopsy needle.
FIG. 5 is a schematic representation of one embodiment of the present invention showing the biopsy needle being aimed at the side of the stage 6 blastocyst distal from the inner cell mass, and a small portion of trophoblast cells being aspirated into the biopsy needle; wherein 5 is the aspirated trophoblast cells.
FIG. 6 is a schematic representation of one embodiment of the present invention showing the separation of a small portion of trophoblast cells from the stage 6 blastocyst aspirated by a biopsy needle; wherein 6 is a cut.
Detailed Description
The present invention will be described with reference to the accompanying drawings and specific embodiments, but the present invention is not limited thereto.
In the following examples, reagents and instruments used are conventional in the art and are commercially available, unless otherwise specified; the basic operations used are all conventional in the art, and those skilled in the art can implement the operations and obtain the corresponding results according to the description.
The rabbit, mouse, and rat blastocysts used in the examples below were derived from cells stored and cultured in this laboratory, and any commercially available rabbit, mouse, or rat blastocysts may be used.
Example 1: trophoblast cell biopsy method of mouse 6-stage blastocyst:
1. the zona pellucida of the mouse blastocyst at stage 6 was fixed with a fixing needle, and a 5 μm seam was formed on the zona pellucida with a microdissection needle.
2. The biopsy needle is passed along the place where the slit was made in step 1, so that it stays inside the zona pellucida.
3. A laser was used to open a relatively large opening, 80 microns in length, above the transparent band.
4. The biopsy needle is passed along the place where the opening was made in step 3, leaving it outside the zona pellucida.
5. And (3) aiming the biopsy needle at one side of the 6-stage blastocyst, far away from the inner cell mass, sucking 10 trophoblast cells into the biopsy needle, withdrawing the biopsy needle to the outer side of the transparent band from the opening of the transparent band in the steps (3) and (1) in sequence, and clamping and stretching the trophoblast cells by utilizing the seam opened in the step (1).
6. A small portion of trophoblast cells aspirated by the biopsy needle were separated from the stage 6 blastocyst by cutting from top to bottom at the thinnest (15 microns) stretched trophoblast cells using a 1480nm infrared laser.
7. Transferring a few parts of the trophoblast cells sucked by the biopsy needle into a culture solution to be detected.
The experiment detects 18 blastocysts of the mouse at the 6 th stage, 17 blastocysts successfully complete the detection, and the detection success rate is obviously improved.
Example 2: trophoblast cell biopsy of stage 6 rabbit blastocysts:
1. the transparent belt with the blastocyst falling off in the stage 6 of the rabbit is fixed by a fixing needle, a slit of about 10 microns is formed on the transparent belt by 1480nm infrared laser, and the slit position is located on the opposite side of the fixing position of the fixing needle.
2. The biopsy needle is passed along the place where the slit was made in step 1, so that it stays inside the zona pellucida.
3. A 1480nm laser is used to open a relatively large opening, on the order of about 70 microns, above the clear band.
4. The biopsy needle is passed from the inside of the zona pellucida to the outside along the place where step 3 was opened, so that it stays outside the zona pellucida.
5. The biopsy needle is aimed at the side of the stage 6 blastocyst far away from the inner cell mass (i.e., the opposite side of the inner cell mass), 30 trophoblast cells are sucked into the biopsy needle, the biopsy needle is withdrawn to the outer side of the transparent band from the opening of the transparent band in the steps 3 and 1 in sequence, and the trophoblast cells are clamped and stretched by the seam opened in the step 1.
6. A small portion of trophoblasts aspirated by the biopsy needle were separated from the stage 6 blastocyst using a laser to cut from top to bottom at the thinnest (45 microns) where the trophoblasts stretched.
7. Transferring the trophoblast cells which are partially cut off by the suction of the biopsy needle into a culture solution, and detecting.
The total number of 6-stage blastula of the rabbits is 10 in the detection, and the detection is completed smoothly.
Example 3: trophoblast cell biopsy method of rat 6-stage blastocyst
1. The transparent belt with the blastocyst falling off at the stage 6 of the rat is fixed by a fixing needle, a slit of about 20 microns is formed on the transparent belt by a micro-cutting needle, and the slit position is positioned at the opposite side of the fixing position of the fixing needle.
2. The biopsy needle is passed along the place where the slit was made in step 1, so that it stays inside the zona pellucida.
3. A micro-cutting needle is used to open a relatively large opening, about 50 microns, above the transparent band, and a portion of the transparent band is removed.
4. The biopsy needle is passed from the inside of the zona pellucida to the outside along the place opened in step 3, so that it stays outside the zona pellucida.
5. And (3) aiming the biopsy needle at the opposite side of the cell mass in the 6-stage blastocyst, sucking 20 trophoblast cells into the biopsy needle, withdrawing the biopsy needle from the opening of the transparent band in the step (3) and the step (1) to the outer side of the transparent band in sequence, and clamping and stretching the trophoblast cells by utilizing the seam opened in the step (1).
6. A small portion of trophoblast cells aspirated by the biopsy needle were separated from the stage 6 blastocyst by cutting from top to bottom at the thinnest (20 microns) stretched trophoblast cells using a 1480nm infrared laser.
7. Transferring a few parts of the trophoblast cells sucked by the biopsy needle into a culture solution to be detected.
The 6-stage blastula of 15 rats are detected at this time, all blastula are detected smoothly, and the detection success rate is greatly improved.
Claims (9)
1. A method of stage 6 blastocyst trophoblast cell biopsy comprising:
(1) Slotting the transparent belt: fixing the transparent belt with the blastocyst falling off at the 6 th stage, and forming a seam on the transparent belt by using laser or a micro-cutting needle, wherein the length range of the seam is 2-10 microns;
(2) The biopsy needle passes through the slit: passing the biopsy needle along the slit position in the step (1) to stay in the transparent belt;
(3) Opening of the transparent tape: a laser or a micro-cutting needle is used for forming a larger opening above the transparent belt, part of the transparent belt is removed, and the length range of the opening is 1-80 microns;
(4) The biopsy needle penetrates from the opening of the zona pellucida: penetrating the biopsy needle from the inner part of the transparent belt to the outer part along the position opened in the step (3) to enable the biopsy needle to stay at the outer part of the transparent belt;
(5) Suction and stretching of blastocyst trophoblast cells: aligning the biopsy needle to one side of the 6-stage blastocyst, which is far away from the inner cell mass, sucking a small part of trophoblast cells into the biopsy needle, withdrawing the biopsy needle from the opening of the transparent band in the step (3) and the step (1) in sequence to return to the outside of the transparent band, and clamping and stretching the trophoblast cells by utilizing the seam opened in the step (1); wherein the number of the sucked trophoblast cells ranges from 1 to 30;
(6) Cutting trophoblast cells by laser: cutting the thinnest part of the stretched trophoblast cells from top to bottom by using laser, and separating a small part of the trophoblast cells sucked by the biopsy needle from the 6-stage blastocyst;
wherein the stage 6 blastocyst is derived from a mouse, rabbit or rat;
wherein the method is a non-disease diagnostic or therapeutic method.
2. The method of claim 1, wherein in step (1), the length of the slot is 5 microns.
3. The method according to claim 1, wherein the number of the sucked trophoblast cells in the step (5) is 10.
4. The method of claim 1, wherein in step (6), the thinnest point thickness is in the range of 1 to 50 microns.
5. The method of claim 1, wherein in step (6), the thinnest point is 15 microns thick.
6. The method according to any one of claims 1 to 5, wherein the laser in steps (1), (3) and (6) is an infrared laser at 1480 nm.
7. The method according to any one of claims 1 to 5, wherein the front section of the fixed needle, micro-cutting needle or biopsy needle in steps (1) to (6) is at an angle to the whole, the angle being in the range of 0 to 90 °.
8. The method of claim 7, wherein the angle is 35 °.
9. The method according to any one of claims 1 to 5, wherein the tip portions of the fixed needle, the micro-dissection needle and the biopsy needle in steps (1) to (6) are flat or sharp, and the fixed needle and the biopsy needle are hollow.
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| CN201811554863.8A CN109706210B (en) | 2018-12-18 | 2018-12-18 | Biopsy method of trophoblast cells of 6-stage blastula |
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| CN109706210B true CN109706210B (en) | 2022-11-18 |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN205974511U (en) * | 2016-07-28 | 2017-02-22 | 柳州市妇幼保健院 | Spilting of an egg phase and embryo's biopsy blastula stage and biopsy specimen tubulature integrated device |
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| US20160355781A1 (en) * | 2015-06-03 | 2016-12-08 | Texas Health Biomedical Advancement Center, Inc. | Systems, methods, and cellular compositions thereof, involving introduction, attachment and proliferation of trophectoderm cells in a blastocyst |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN205974511U (en) * | 2016-07-28 | 2017-02-22 | 柳州市妇幼保健院 | Spilting of an egg phase and embryo's biopsy blastula stage and biopsy specimen tubulature integrated device |
Non-Patent Citations (1)
| Title |
|---|
| 新的囊胚活检方法—囊胚活检不再纠结和繁琐;吴克良;《生殖医学杂志》;20170430;第26卷(第4期);摘要、第325页右栏第3段 * |
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