CN111548986A - Simple and efficient method for preparing embryo culture liquid drops - Google Patents
Simple and efficient method for preparing embryo culture liquid drops Download PDFInfo
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- CN111548986A CN111548986A CN202010471808.3A CN202010471808A CN111548986A CN 111548986 A CN111548986 A CN 111548986A CN 202010471808 A CN202010471808 A CN 202010471808A CN 111548986 A CN111548986 A CN 111548986A
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- 239000007788 liquid Substances 0.000 title claims abstract description 69
- 210000001161 mammalian embryo Anatomy 0.000 title claims abstract description 47
- 238000000034 method Methods 0.000 title claims abstract description 26
- 238000012546 transfer Methods 0.000 claims abstract description 21
- 239000005662 Paraffin oil Substances 0.000 claims abstract description 11
- 238000012258 culturing Methods 0.000 claims abstract description 3
- 238000004519 manufacturing process Methods 0.000 claims description 12
- 238000009281 ultraviolet germicidal irradiation Methods 0.000 claims 1
- 238000005516 engineering process Methods 0.000 description 11
- 238000000338 in vitro Methods 0.000 description 9
- 230000001850 reproductive effect Effects 0.000 description 5
- 244000144972 livestock Species 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 230000004720 fertilization Effects 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000011161 development Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 210000002257 embryonic structure Anatomy 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000010374 somatic cell nuclear transfer Methods 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 230000003068 static effect Effects 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 1
- 235000009161 Espostoa lanata Nutrition 0.000 description 1
- 240000001624 Espostoa lanata Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 208000000509 infertility Diseases 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 231100000535 infertility Toxicity 0.000 description 1
- 230000009027 insemination Effects 0.000 description 1
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- 210000000287 oocyte Anatomy 0.000 description 1
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- 210000004681 ovum Anatomy 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 210000004291 uterus Anatomy 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0604—Whole embryos; Culture medium therefor
Abstract
The invention relates to a simple and efficient method for preparing embryo culture liquid drops, which comprises the following steps: placing the culture dish, the pipettor, the suction head and the annular channel pipetting device on a super clean bench for ultraviolet irradiation treatment; preheating the embryo culture solution; placing the preheated embryo culture solution and paraffin oil in a super clean bench; adjusting the range of the pipettor to the required range, connecting the pipettor with an annular pipetting device, installing a suction head and sucking the embryo culture solution; pushing out liquid from the middle position of the culture dish by the liquid transfer device, adding paraffin oil to cover the culture liquid drop, covering the culture dish cover, and taking in CO2Culturing in an incubator; the annular channel liquid transfer device comprises a liquid transfer device combination part, a cavity and a plurality of sucker seats, wherein each sucker seat is sleeved with a sucker. The method for preparing the embryo culture liquid drop is easy to operate, simple and efficient.
Description
Technical Field
The invention relates to the field of biology, in particular to a simple and efficient method for preparing embryo culture liquid drops.
Background
With the continuous progress of embryo engineering technology in recent years, the vigorous development of related industries is promoted, such as assisted reproduction technology, which is a technology for assisting the combination of sperms and ova by an artificial method and mainly comprises artificial insemination, in vitro fertilization, somatic cell nuclear transfer, embryo transfer and the like. The assisted reproduction technology has been widely applied to the production of common livestock such as pigs, cattle, sheep and the like. The auxiliary reproduction technology is used in animal husbandry, so that the genetic improvement process of excellent livestock varieties can be accelerated, and the breeding of excellent populations is promoted; producing precious medical protein; protecting endangered animals and the like. For example, the combined application of the in vitro fertilization technology and the embryo transplantation technology in livestock can fully exert the reproductive capacity of female excellent individuals, shorten the reproductive cycle of the excellent individuals and increase the reproductive quantity. The application of somatic cell nuclear transfer technology in livestock can produce precious medical protein; cloning animal tissue and organ as transplant donor; protecting endangered species, increasing the number of surviving species, etc. After the first test-tube baby is born, the assisted reproduction technology is widely applied to human reproductive medicine, mainly used as an important means for treating human infertility, and secondly, the diseases of human can be further studied by preparing an animal model of the diseases of human. Embryo in vitro production is an indispensable link for assisted reproductive technologies, and the main contents of embryo in vitro production include in vitro maturation of oocytes, in vitro fertilization, and in vitro culture of early embryos. Since embryo in vitro culture is an artificial uterus supporting embryo pre-implantation development, in vitro culture of isolated embryos is increasingly regarded as a key link.
In recent years, although embryo culture systems are gradually developed from static micro-drop methods to dynamic micro-fluid culture with better effect, the dynamic culture systems are more complex to operate and limit the wide application of the dynamic culture systems, and static culture systems have the advantages of greatly improving the efficiency and quality along with the continuous improvement of embryo culture solution and the gradual improvement of culture conditions, along with stable culture effect and easy operation, wherein the micro-drop method is the most common culture method at present. A pipette is often used to make the droplets. At present, the mode of using a micro-drop method to manufacture liquid drops in a laboratory is that a liquid shifter is used for dropping liquid drop by drop, the method for manufacturing the liquid drops is still tedious, the efficiency is still to be improved, a large amount of exercises are needed to ensure that the made liquid drops do not cross, and the time difference of liquid drop manufacturing exists. The invention designs a better method for making the embryo culture liquid drop by improving on the basis of the traditional method for making the embryo culture liquid drop, thereby eliminating the time difference of liquid drop making, simplifying the fussy operation process, reducing the operation difficulty and improving the efficiency.
Disclosure of Invention
The invention aims to: provides a simple and efficient method for preparing embryo culture liquid drops to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme:
a simple and efficient method for preparing embryo culture liquid drops comprises the following steps:
s1: placing the culture dish, the pipettor, the suction head and the annular channel pipetting device on a super clean bench for ultraviolet irradiation treatment;
s2: preheating the embryo culture solution;
s3: placing the preheated embryo culture solution and paraffin oil in a super clean bench;
s4: adjusting the range of the pipettor to the required range, connecting the pipettor with an annular pipetting device, installing a suction head and sucking the embryo culture solution; the liquid transfer device is positioned in the middle position of the culture dish to push out liquid,
s5: adding paraffin oil to cover the culture liquid drop, covering the culture dish, and adding CO2Culturing in an incubator;
the annular channel liquid transfer device comprises a liquid transfer device combination part, a cavity and a plurality of sucker seats, wherein each sucker seat is provided with a sucker in a sleeved mode.
Preferably, the ultraviolet irradiation treatment time in step S1 is 25 to 35 min.
Preferably, the ultraviolet irradiation treatment time in step S1 is 30 min.
Preferably, the sucker bases are conical shells and are communicated with the cavity, 12 sucker bases are arranged and are arranged along two concentric circles, and 9 sucker bases are uniformly distributed on the outer ring; 3 suction cup bases are uniformly distributed on the inner ring.
Preferably, the included angle between each suction head seat in the outer ring is 45 degrees, and the distance between the suction head seat and the side, close to the edge of the cavity, of the suction head seat is 1.5mm-2 mm.
Preferably, the included angle between each suction head seat in the inner ring is 120 degrees, and the distance between two adjacent suction head seats is 1.5mm-2 mm.
Preferably, the preheating temperature of the embryo culture solution in the step S2 is 35-40 ℃, and the preheating time is 3-8 minutes.
Compared with the prior art, the invention has the beneficial effects that:
the method for preparing the embryo culture liquid drop is simple and easy to operate, can ensure the quality of the prepared liquid drop, and eliminates the time difference between the liquid drops; the annular liquid transfer device can convert a single channel into multiple channels, so that the conventional method for manufacturing one drop of liquid is converted into the method for manufacturing all the liquid drops of the whole culture dish at one time, and the manufacturing efficiency is greatly improved; through annular pipetting device's distance setting, can set up the distance between every liquid drop and the distance of liquid drop and culture dish edge into a safe distance, can guarantee like this that the liquid drop of making can not appear the string drop phenomenon. The method can make the embryo culture liquid drop more simple and efficient for operators, and the process that the operators can master the skill only by practicing a large amount is omitted.
Drawings
FIG. 1 is a front view of a circular channel pipetting device;
FIG. 2 is a bottom view of the annular channel pipetting device;
in the reference symbols: 1-pipette joint, 2-cavity, 3-pipette seat.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
Example 1: making porcine embryo culture droplets
Materials, reagents and sources thereof used in the following examples
1. Material
The materials used in this study were: yellow suction head, blue suction head, pipette 1000 microlitre, 3.5cm import culture dish, annular liquid-transfering device.
2. Reagent
The reagents used in this study were: pig embryo culture solution and paraffin oil.
A simple and efficient method for preparing embryo culture liquid drops comprises the following steps:
(1) preparation of embryo culture liquid drop making related material
Placing a 3.5cm inlet culture dish, a pipettor of 1000 mu L, a yellow suction head, a blue suction head and a circular channel pipetting device on a super clean bench, and carrying out ultraviolet irradiation for 30 minutes;
secondly, taking out the pig embryo culture solution from a refrigerator at 4 ℃ and preheating the pig embryo culture solution on a hot table at 37 ℃ for 5 minutes;
placing the preheated pig embryo culture solution and paraffin oil in a test tube rack on an ultra-clean bench after ultraviolet irradiation is finished;
(2) embryo culture droplet fabrication
Firstly, a piece of alcohol cotton ball is clamped by tweezers to wipe the desktop of a super clean bench, the range of a 1000-mu-L pipettor is adjusted to 600 mu L (12 liquid drops can be made in a 3.5cm culture dish, and each liquid drop is 50 mu L), and the pipettor is connected with an annular pipetting device;
② placing a culture dish with 3.5cm inlet in front of the body, installing a suction head on the annular liquid transfer device and sucking embryo culture liquid, placing a liquid transfer device in the middle of the culture dish and pushing out the liquid, replacing the liquid transfer device, slowly adding 3mL of paraffin oil to cover the culture liquid drop along the inner wall of the culture dish, covering the culture dish cover, and taking in CO2And (5) an incubator.
As shown in fig. 1 and 2: the annular channel liquid transfer device comprises a liquid transfer device combination part (the liquid transfer device combination part is a cylindrical shell and can be directly inserted onto the liquid transfer device), a cavity and a plurality of sucker seats, wherein each sucker seat is provided with a sucker in a sleeved mode; the sucker seats are 12 and are arranged along two concentric circles, and 9 sucker seats are uniformly distributed on the outer ring; 3 suction head seats are uniformly distributed on the inner ring; the included angle between each suction head seat in the outer ring is 45 degrees, and the distance between the suction head seat and the side close to the edge of the cavity is 1.5mm-2 mm; the included angle between each suction head seat in the inner ring is 120 degrees, and the distance between every two adjacent suction head seats is 1.5mm-2 mm.
Attention points in the operation
(1) When the liquid is transferred, the suction head and the liquid are kept at the same temperature so as to ensure the accuracy of the liquid transfer.
(2) When liquid is sucked, attention should be paid to the liquid transfer device above the suction head so as to ensure the service life of the liquid transfer device.
(3) Before the liquid is dripped, the sucker with one circle of the outer circle is kept at the middle position of the culture dish as much as possible.
(4) The petri dish should be opened with a minimum of hand passing over it.
(5) When paraffin oil is added, the paraffin oil is slowly stuck to the edge to prevent the liquid drops from being flushed and dripped.
(6) The dripping device is always arranged in the ultraviolet operation cabinet.
Claims (7)
1. A simple and efficient method for making embryo culture liquid drops is characterized by comprising the following steps:
s1: placing the culture dish, the pipettor, the suction head and the annular channel pipetting device on a super clean bench for ultraviolet irradiation treatment;
s2: preheating the embryo culture solution;
s3: placing the preheated embryo culture solution and paraffin oil in a super clean bench;
s4: adjusting the range of the pipettor to the required range, connecting the pipettor with an annular pipetting device, installing a suction head and sucking the embryo culture solution; pushing the liquid out of the culture dish by the liquid transfer device at the middle position of the culture dish;
s5: adding paraffin oil to cover the culture liquid drop, covering the culture dish, and adding CO2Culturing in an incubator;
the annular channel liquid transfer device comprises a liquid transfer device combination part, a cavity and a plurality of sucker seats, wherein each sucker seat is provided with a sucker in a sleeved mode.
2. The simple and efficient method for producing embryo culture droplet as claimed in claim 1, wherein the UV irradiation treatment time in step S1 is 25-35 min.
3. The simple and efficient method for producing an embryo culture droplet as claimed in claim 2, wherein the ultraviolet irradiation treatment time in step S1 is 30 min.
4. The simple and efficient method for making liquid drops for embryo culture according to claim 1, wherein the pipette tip seat is a conical shell and is communicated with the chamber, 12 pipette tip seats are arranged along two concentric circles, and 9 pipette tip seats are uniformly distributed on the outer circle; 3 suction cup bases are uniformly distributed on the inner ring.
5. A simple and efficient method for making liquid drops for embryo culture according to claim 4, wherein the included angle between each two pipette tip seats in the outer ring is 45 ° and the distance between the pipette tip seats and the side closer to the edge of the chamber is 1.5mm-2 mm.
6. The simple and efficient method for making the embryo culture liquid drop according to claim 4, wherein the included angle between each two pipette seat in the inner ring is 120 ° and the distance between two adjacent pipette seats is 1.5mm-2 mm.
7. The simple and efficient method for making embryo culture liquid drop according to claim 1, wherein the preheating temperature of the embryo culture liquid in step S2 is 35-40 ℃ and the preheating time is 3-8 minutes.
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| CN202010471808.3A CN111548986A (en) | 2020-05-29 | 2020-05-29 | Simple and efficient method for preparing embryo culture liquid drops |
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| CN202010471808.3A CN111548986A (en) | 2020-05-29 | 2020-05-29 | Simple and efficient method for preparing embryo culture liquid drops |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108165524A (en) * | 2018-02-05 | 2018-06-15 | 苏州大学 | It is a kind of to tear the liquid droplet distribution system of egg and liquid droplet distribution method open for egg cell |
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| AUPP421298A0 (en) * | 1998-06-19 | 1998-07-09 | Fertilitescentrum Ab | Method and medium for in vitro culture of human embryos |
| WO2012002500A1 (en) * | 2010-06-30 | 2012-01-05 | 大日本印刷株式会社 | Method for producing embryo by in vitro culture, and method, device, and system for selecting embryo |
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2020
- 2020-05-29 CN CN202010471808.3A patent/CN111548986A/en active Pending
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108165524A (en) * | 2018-02-05 | 2018-06-15 | 苏州大学 | It is a kind of to tear the liquid droplet distribution system of egg and liquid droplet distribution method open for egg cell |
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Application publication date: 20200818 |
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