CN111548986A - Simple and efficient method for preparing embryo culture liquid drops - Google Patents

Abstract

The invention relates to a simple and efficient method for producing embryo culture droplets, which comprises the following steps: placing a culture dish, a pipette, a suction head and a ring channel pipetting device on an ultra-clean table for ultraviolet irradiation treatment; Preheat the liquid; place the preheated embryo culture medium and paraffin oil in the ultra-clean bench; adjust the range of the pipette to the required range, connect the pipette to the ring pipetting device, install the tip and suck the embryos for culture Put the pipette in the middle of the petri dish to push out the liquid, add paraffin oil to cover the culture droplets, cover the petri dish, and put it into the CO ₂ incubator; the annular channel pipetting device includes a pipette combined with A part, a chamber and a suction head seat are provided, and a plurality of the suction head seats are arranged, and each suction head seat is sleeved with a suction head. The preparation method of the embryo culture droplet of the present invention is not only easy to operate, but also simple and efficient.

Description

A simple and efficient method for making embryo culture droplets

technical field

The invention relates to the biological field, in particular to a simple and efficient method for making embryo culture droplets.

Background technique

In recent years, with the continuous progress of embryo engineering technology, it has promoted the vigorous development of related industries, such as assisted reproductive technology. Assisted reproductive technology is a technology that uses artificial methods to help sperm and eggs combine with each other. It mainly includes artificial insemination, in vitro fertilization, Somatic cell nuclear transfer, embryo transfer, etc. Assisted reproductive technology has long been widely used in the production of general livestock such as pigs, cattle and sheep. Through the use of assisted reproductive technology in animal husbandry, the process of genetic improvement of excellent livestock breeds can be accelerated, the breeding of excellent populations can be promoted, precious medical proteins can be produced, and endangered animals can be protected. For example, the combined application of in vitro fertilization and embryo transfer technology in livestock can fully exert the reproductive ability of excellent female individuals, shorten the reproductive cycle of excellent individuals, and increase the number of reproductions. The application of somatic cell nuclear transfer technology in livestock can produce precious medical proteins; clone animal tissues and organs as donors for transplantation; protect endangered species and increase the number of species alive. Since the birth of the first test-tube baby, assisted reproductive technology has also been widely used in human reproductive medicine, mainly as an important means of human infertility treatment, and secondly, it can also be used to prepare animal models of human diseases. disease. For assisted reproductive technology, in vitro production of embryos is an indispensable link. The main contents of in vitro production of embryos include in vitro maturation of oocytes, in vitro fertilization and in vitro culture of early embryos. Since embryo culture in vitro is an artificial uterus that supports the development of embryos before implantation, the in vitro culture of embryos after in vitro has been paid more and more attention as a key link.

Embryos are cultured in vitro by droplet method, group culture, microfluidics and other methods. In recent years, although the embryo culture system has gradually developed from the static droplet method to the dynamic microfluidic culture with better effect, the operation of the dynamic culture system is more complicated. , which limits its wide application, while the static culture system has greatly improved its efficiency and quality with the continuous improvement of embryo culture medium and the gradual improvement of culture conditions, and the culture effect is stable and easy to operate. It is the most commonly used cultivation method. A pipette is often used to make droplets. At present, the laboratory uses the droplet method to make droplets drop by drop with a pipette. It is still a bit cumbersome to make droplets by this method, and the efficiency needs to be improved, and it takes a lot of practice to ensure that the The droplets are not strung together, and there is also a time difference in the production of droplets. The present invention designs a better production method for embryo culture droplets by improving the traditional embryo culture droplet production method, so as to eliminate the time difference of droplet production, simplify the tedious operation process and reduce the operation time. difficulty and increased efficiency.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a simple and efficient method for producing embryo culture droplets, so as to solve the problems raised in the above-mentioned background art.

To achieve the above object, the present invention provides the following technical solutions:

A simple and efficient method for making embryo culture droplets, comprising the following steps:

S1: Place the petri dish, pipette, tip, and ring channel pipetting device on the ultra-clean bench for ultraviolet irradiation treatment;

S2: Preheat the embryo culture medium;

S3: Place the preheated embryo culture medium and paraffin oil in a clean bench;

S4: Adjust the range of the pipette to the required range, connect the pipette to the ring pipetting device, install the tip and suck the embryo culture medium; put the pipette in the middle of the petri dish to push out the liquid,

S5: Add paraffin oil to cover the culture droplets, cover the petri dish, and put it into the CO ₂ incubator;

Wherein, the annular channel pipetting device includes a pipette joint part, a chamber and a suction head seat, a plurality of the suction head seats are arranged, and a suction head is sleeved on each suction head seat.

Preferably, the ultraviolet irradiation treatment time in step S1 is 25-35 min.

Preferably, the ultraviolet irradiation treatment time in step S1 is 30min.

Preferably, the suction head seat is a conical shell and communicates with the chamber, there are 12 suction head seats arranged along two concentric circles, and 9 suction head seats are evenly distributed in the outer ring; Evenly distribute the 3 tip holders.

Preferably, the included angle between each suction head seat in the outer ring is 45°, and the distance between the suction head seat and the side closer to the edge of the chamber is 1.5mm-2mm.

Preferably, the included angle between each suction head seat in the inner ring is 120°, and the distance between two adjacent suction head seats is 1.5mm-2mm.

Preferably, in step S2, the preheating temperature of the embryo culture medium is 35-40° C., and the preheating time is 3-8 minutes.

Compared with the prior art, the beneficial effects of the present invention are:

The method for producing embryo culture droplets of the present invention is simple and easy to operate, can ensure the quality of the produced droplets, and eliminates the time difference between the production of droplets; single channel can be converted into multi-channel through the annular pipetting device, so that the The previous method of making one drop by one drop has been transformed into a method of making all the droplets of the entire petri dish at one time, which greatly improves the production efficiency; by setting the distance of the circular pipetting device, the distance between each droplet and the The distance between the droplet and the edge of the petri dish is set to a safe distance, so as to ensure that the produced droplet will not have a string dripping phenomenon. This method makes it easier and more efficient for the operator to make embryo culture droplets, and the process of mastering this skill is reduced by a lot of practice by the operator.

Description of drawings

Fig. 1 is the front view of the ring channel pipetting device;

Figure 2 is a bottom view of the annular channel pipetting device;

In the reference numbers: 1-pipette joint, 2-chamber, 3-tip holder.

specific embodiment

Example 1: Making pig embryo culture droplets

Materials, reagents and sources used in the following examples

1. Materials

The materials used in this study are: yellow tips, blue tips, 1000 μL pipette, 3.5cm imported petri dish, and ring pipetting device.

2. Reagents

The reagents used in this study were: porcine embryo culture medium and paraffin oil.

A simple and efficient method for making embryo culture droplets, comprising the following steps:

(1) Preparation of materials related to embryo culture droplet production

①Place 3.5cm imported petri dish, 1000μL pipette, yellow tip, blue tip and ring channel pipetting device on the ultra-clean bench, and irradiate with UV light for 30 minutes;

② Take the pig embryo culture medium out of the 4°C refrigerator and put it on a 37°C hot stage to preheat for 5 minutes;

③ After the ultraviolet irradiation, place the preheated pig embryo culture medium and paraffin oil in the test tube rack on the ultra-clean bench;

(2) Preparation of embryo culture droplets

①Pull out a piece of alcohol cotton ball with tweezers to wipe the tabletop of the ultra-clean table, adjust the volume of the 1000μL pipette to 600μL (a 3.5cm petri dish can make 12 droplets, each droplet is 50μL), connect the pipette to the ring The pipetting device is connected;

②Place the 3.5cm imported petri dish in front of you, install a tip on the ring pipetting device and suck the embryo culture medium, place the pipette in the middle of the petri dish to push out the liquid, replace the pipette, and stick to the inner wall of the petri dish Slowly add 3 mL of paraffin oil to cover the culture drop; cover the petri dish and put it into the _CO2 incubator.

As shown in Figures 1 and 2: the annular channel pipetting device includes a pipette joint (the pipette joint is a cylindrical shell, which can be directly inserted into the pipette), a chamber and a tip holder , the suction head seat is provided with multiple, and each suction head seat is sleeved with a suction head; the suction head seat is a conical shell and communicates with the chamber, and the suction head seat is provided with 12, Arranged along two concentric circles, the outer ring is evenly distributed with 9 suction head holders; the inner ring is evenly distributed with 3 suction head holders; the angle between each suction head holder in the outer ring is 45° and the suction head The distance between the seat and the side closer to the edge of the chamber is 1.5mm-2mm; the included angle between each tip seat in the inner ring is 120°, and the distance between two adjacent tip seats is 1.5mm-2mm.

Precautions during operation

(1) When pipetting, the tip and the liquid should be kept at the same temperature to ensure the accuracy of pipetting.

(2) When sucking liquid, be careful not to let the liquid enter the inside of the pipette above the tip to ensure the service life of the pipette.

(3) Before dripping the liquid, pay attention to keeping the tip of the outer circle as far as possible in the middle of the petri dish.

(4) After the petri dish is opened, the hand passing over it should be minimized.

(5) When adding paraffin oil, it should be driven in slowly on the side to prevent dripping.

(6) This dripping device should always be placed in the UV 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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