Method for large-scale preparation and extraction of sheep embryonic cells

Abstract

The invention provides a method for large-scale preparation and extraction of sheep embryonic cells, which belongs to the technical field of embryonic cell extraction, and comprises the following steps: collecting blood from the carcasses of newly-developed pregnant ewes, separating and preparing serum, and subsequently adding the serum into a culture solution for separation and multiplication culture of the sheep embryonic cells; meanwhile, the uterus is taken out from the corpse, the sheep embryo is taken out in a sterile environment, sheep embryo cells are separated out, then through in-vitro continuous passage and mass amplification, the sheep embryo cells can be used as raw material components for medical beauty and health care on a large scale, and the sheep embryo cells have the advantages of being easy to operate and capable of achieving mass amplification and are suitable for large-scale production in the industrial circle.

Description

Technical Field

[0001] The invention belongs to the technical field of embryonic cell extraction, and in particular relates to a method for large-scale preparation and extraction of sheep embryonic cells. Background Art

[0002] Extracts of perinatal tissues such as umbilical cord and placenta contain many nutrients and active factors, and are excellent raw materials for the development of medical beauty, health care and cosmetic products. However, perinatal tissues have limitations in efficacy and application due to their complex and diverse internal components, poor breeding environment and sanitation, and are susceptible to microbial contamination, as well as high hormone content.

[0003] Sheep embryos contain many young cells such as stem cells and progenitor cells, which contain more abundant and comprehensive active nutrients, and have more guaranteed sanitary conditions and biosafety, which have more obvious advantages than perinatal tissues. However, the separation and extraction of sheep embryo cells is still at the scientific research level due to the high technical difficulty and high cost, and has not been mass-produced in the industry. For example, the invention patent with publication number CN107365739B provides a method for culturing mammalian embryonic cells, which includes the extraction and cultivation of sheep embryos, but the proliferation activity of the embryonic cells cultured is poor, which is not enough to meet the mass production needs of the industry. Summary of the invention

[0004] Based on the above problems in the prior art, the present invention provides a method for large-scale preparation and extraction of sheep embryo cells, which comprises the following detailed steps:

[0005] Step S1, collecting blood from the body of a pregnant ewe that has just died, separating and preparing serum for later use;

[0006] Step S2, performing laparotomy on the ewe corpse to remove the uterus, ligating the uterus and transferring it to a sterile experimental environment;

[0007] Step S3, taking out the sheep fetus from the uterus, rinsing the sheep fetus, and then cutting it into tissue blocks;

[0008] Step S4, washing the tissue block with physiological saline for 1-2 times, removing the washing supernatant by centrifugation, resuspending the tissue block with complete culture medium, and inoculating it in a culture bottle for culture;

[0009] Step S5, after the tissue block adheres to the wall, new culture medium is added to continue culturing until the cell clones migrated out of the tissue block grow to confluence, and then passage and amplification are performed to obtain a large number of sheep embryonic cells.

[0010] Preferably, the pregnant ewe is a pregnant goat with a 2.5-month-old fetus.

[0011] Preferably, the pregnant ewe is a twin pregnant ewe, so that more primary embryonic cells can be obtained.

[0012] Preferably, the whole culture medium is a basic culture medium DM / F2, supplemented with 10% serum from pregnant ewe blood.

[0013] Preferably, the step S3 further comprises: step S31, after taking out the sheep fetus, removing the head, limbs and internal organs, and then rinsing the body part.

[0014] Preferably, in step S4, the tissue block is inoculated into a T-175 culture flask, and the inoculation volume is 1 ml of tissue block / flask.

[0015] The beneficial effects of the present invention are:

[0016] 1. The natural adherent culture method is simpler to operate than the traditional enzymatic digestion method, which requires strict control of the selection of digestive enzymes, digestion time and digestion degree, but has similar harvesting efficiency. A large number of sheep embryonic cells with high purity and strong proliferation activity can be obtained in a relatively short period of time, and can be continuously passaged for a long time, which can meet the application requirements of subsequent production transformation and is suitable for large-scale production in the industry.

[0017] 2. The culture medium adopts ordinary basic culture medium DM / F2, and adds 10% serum from pregnant ewe blood, which reduces the cost of culture medium and does not introduce the risks brought by exogenous traditional bovine serum. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a picture of cells (P0 generation) observed 4-5 days after goat embryo tissue blocks were adhered to the wall.

[0019] Figure 2 This is an observation picture of P1 goat embryo cells.

[0020] Figure 3 This is an observation picture of P5 goat embryo cells.

[0021] Figure 4 This is a curve chart of goat embryo cell proliferation quantity.

[0022] Figure 5 This is a curve chart of the number of cell proliferation and division in goat embryos.

[0023] Figure 6 This is a statistical chart of goat embryo cell viability.

[0024] Figure 7 This is a statistical chart of goat embryo cell size. DETAILED DESCRIPTION

[0025] The technical solution of the present invention is described below in conjunction with the accompanying drawings and specific embodiments.

[0026] A method for large-scale preparation and extraction of sheep embryo cells, comprising the following detailed steps:

[0027] Step S1, collecting blood from the corpse of a pregnant ewe that has just died, separating and preparing serum for standby use, wherein the pregnant ewe is a pregnant goat with a 2.5-month-old fetus, preferably a pregnant goat with twins, which can obtain more primary embryonic cells than the 1-1.5-month-old fetus in traditional research, and the obtained cells have better proliferation ability and activity, which can meet the needs of later continuous expansion;

[0028] Step S2, performing laparotomy on the ewe corpse to remove the uterus, placing the uterus in a sterile beaker after ligation, sending it to a clean laboratory, and transferring it to a sterile biosafety cabinet;

[0029] Step S3, taking out the sheep fetus from the uterus in a sterile biological safety cabinet, removing the head, limbs, internal organs and other organs, then rinsing the body parts of the sheep fetus, and fully chopping them into tissue blocks in a small beaker;

[0030] Step S4, transferring the tissue block into a 50 ml centrifuge tube, washing with physiological saline for 1-2 times, centrifuging to remove the washing supernatant, resuspending the tissue block with a small amount of complete culture medium, and inoculating the tissue block into a T-175 culture bottle for culture, the inoculation volume is 1 ml tissue block / bottle, the complete culture medium is a basic culture medium DM / F2, supplemented with 10% serum from pregnant ewe blood;

[0031] Step S5, after the tissue block adheres to the wall, new culture medium is added to continue culturing until the cell clones migrated out of the tissue block grow to confluence, and then passage and amplification are performed to obtain a large number of sheep embryonic cells.

[0032] After the goat embryonic tissue pieces are cultured on the wall, a large number of cells can be seen migrating out in 2-3 days, and they can grow to confluence in 4-5 days. Figure 1 As shown, the cells are mostly fibrous, with extremely high migration efficiency and strong proliferation activity.

[0033] After subculture, embryonic-derived cells have a strong ability to proliferate continuously. As the number of generations increases, the proportion of fibroblasts increases continuously, and the cell purity continues to improve. Figure 2 As shown, P5 cells Figure 3 shown.

[0034] like Figure 4 As shown in Figure 2, goat embryonic cells have a good ability to continue to proliferate after passage. By the tenth passage, the total number of cells in the passage can reach 10 18 Order of magnitude.

[0035] According to the statistical results of cell doubling times, by the 10th generation, the number of cell divisions had reached 35-37 times, and the division and proliferation were still linear, such as Figure 5As shown in the figure, it shows that the cells have a strong ability to continue to proliferate and can provide sufficient cell raw materials for the application end. Figure 6 As shown in the figure, the viability of the harvested cells can reach more than 95% up to the 10th generation of embryonic cells, and the cell viability of each generation is good; Figure 7 As shown, up to the 10th generation of embryonic cells, the particle size of the harvested cells was relatively uniform between 15-17 μM, and the cells of each generation were relatively uniform.

[0036] The present invention provides the above embodiments only to illustrate rather than limit the technical solutions of the present invention. Although the above embodiments describe the present invention in detail, relevant technical personnel in the field should understand that the present invention can be modified or equivalently replaced, but any modification and partial replacement that does not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A method for large-scale preparation and extraction of sheep embryo cells, characterized in that: It includes the following detailed steps: Step S1, collecting blood from the body of a pregnant ewe that has just died, separating and preparing serum for later use; Step S2, performing laparotomy on the ewe corpse to remove the uterus, ligating the uterus and transferring it to a sterile experimental environment; Step S3, taking out the sheep fetus from the uterus, rinsing the sheep fetus, and then cutting it into tissue blocks; Step S4, washing the tissue block with physiological saline for 1-2 times, removing the washing supernatant by centrifugation, resuspending the tissue block with complete culture medium, and inoculating it in a culture bottle for culture; Step S5, after the tissue block adheres to the wall, new culture medium is added to continue culturing until the cell clones migrated out of the tissue block grow to confluence, and then passage and amplification are performed to obtain a large number of sheep embryonic cells.

2. The method for large-scale preparation and extraction of sheep embryo cells according to claim 1, characterized in that: The pregnant ewe is a pregnant goat with a 2.5-month-old fetus.

3. The method for large-scale preparation and extraction of sheep embryo cells according to claim 2, characterized in that: The pregnant ewe is a twin pregnant ewe.

4. The method for large-scale preparation and extraction of sheep embryo cells according to claim 1, characterized in that: The whole culture medium is a basic culture medium DM / F2, supplemented with 10% serum from pregnant ewe blood.

5. The method for large-scale preparation and extraction of sheep embryo cells according to claim 1, characterized in that: The step S3 also includes: step S31, after taking out the sheep fetus, removing the head, limbs and internal organs, and then rinsing the body part.

6. The method for large-scale preparation and extraction of sheep embryo cells according to claim 1, characterized in that: In the step S4, the tissue block is inoculated into a T-175 culture bottle, and the inoculation volume is 1 ml of tissue block / bottle.

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