阅读说明：本技术 一种标准化肿瘤动物模型的建立方法及培养装置 (Establishment method and culture device of standardized tumor animal model ) 是由 应巧红 于 2021-09-30 设计创作，主要内容包括：本发明公开了一种标准化肿瘤动物模型的建立方法,包括以下操作步骤：S1：取对数生长期增殖旺盛的肿瘤细胞预处理后进行冷冻保存,S2：将冷冻保存的肿瘤细胞取出加入培养液并转入培养瓶中进行培养,S3：将培养后的肿瘤细胞从培养瓶取出并植入瘤源动物体内,S4：选取接种后7至12天且生长状态良好的瘤源动物颈椎脱臼处死,消毒操作部位的皮肤,切开皮肤,剥离出接种用的瘤块。该标准化肿瘤动物模型的建立方法,通过将生长期增殖旺盛的肿瘤细胞预处理后进行冷冻保存,可以使细胞暂时脱离生长状态而将其细胞特性保存起来,而且可以防止因正在培养的细胞被污染或其他意外事件而使细胞丢种,起到了细胞保种的作用。(The invention discloses a method for establishing a standardized tumor animal model, which comprises the following operation steps: s1: and (3) pretreating tumor cells with vigorous proliferation in logarithmic growth phase, and then performing cryopreservation, wherein S2: the tumor cells preserved by freezing are taken out, added with culture solution and transferred into a culture bottle for culture, S3: and (3) taking out the cultured tumor cells from the culture bottle and implanting the tumor cells into the tumor source animal body, wherein S4: selecting tumor source animals which grow well 7 to 12 days after inoculation, dislocating and killing cervical vertebrae, sterilizing the skin of an operation part, cutting the skin, and stripping off tumor blocks for inoculation. The establishment method of the standardized tumor animal model can ensure that the cells are temporarily separated from the growth state and the cell characteristics are preserved by freezing and storing the tumor cells which are proliferated vigorously in the growth period after pretreatment, and can prevent the cells from losing seeds due to the pollution or other unexpected events of the cells which are cultured, thereby playing the role of protecting the seeds of the cells.)

1. A method for establishing a standardized tumor animal model is characterized by comprising the following steps: the method comprises the following operation steps:

s1: pretreating tumor cells with vigorous proliferation in logarithmic growth phase, and freezing and storing;

s2: taking out the tumor cells which are frozen and preserved, adding a culture solution into the tumor cells, and transferring the tumor cells into a culture bottle for culture;

s3: taking out the cultured tumor cells from the culture bottle and implanting the tumor cells into a tumor source animal body;

s4: selecting a tumor source animal which grows well 7 to 12 days after inoculation and is dislocated in cervical vertebra to be killed, disinfecting the skin of an operation part, cutting the skin, and stripping off tumor blocks for inoculation;

s5: removing non-tumor tissues and necrotic tissues from the stripped tumor mass for inoculation, selecting tumor tissues which grow well and have no degeneration necrosis and light red color and are filled with fish meat, placing the tumor tissues in an aseptic plate, shearing the tumor tissues into small blocks with the size of 2 cubic millimeters, placing the aseptic plate on ice cubes, and adding nutrient solution to cultivate the small blocks for a period of time;

s6: sucking the tumor mass after being cultured for a period of time by using a sterile trocar, and then inoculating the tumor mass under the axillary skin of the same receptor animal;

s7: observing the growth of the tumor cells in the recipient animal.

2. The method of claim 1, wherein the standardized animal model is a tumor model comprising: the step of carrying out freezing preservation after pretreatment on the tumor cells with vigorous proliferation in the logarithmic growth phase comprises the steps of carrying out trypsin digestion, centrifugation and washing on the tumor cells with vigorous proliferation in the logarithmic growth phase, preparing a cell suspension by using a culture solution containing 7 parts, calf serum containing 2 parts and DMSO containing 1 part, placing the cell suspension into a cell freezing tube, placing the cell freezing tube at 4 ℃ for 30 minutes, placing the cell freezing tube at-20 ℃ for 4 hours, placing the cell freezing tube at-70 ℃ overnight, and then placing the cell freezing tube into liquid nitrogen for freezing preservation.

3. The method of claim 1, wherein the standardized animal model is a tumor model comprising: the tumor cells which are frozen and preserved need to be rapidly placed into a water bath at 38-40 ℃ and continuously shaken to be completely melted in one minute, then supernatant is removed in 5 minutes of centrifugation, then culture solution is added, the culture bottle is turned into, the sealed heat insulation cover is rotated and opened through a handle after the tumor cells are turned into the culture bottle, then the culture bottle is lifted into an inner cavity of the sealed culture cylinder body with constant temperature and humidity through two convenient pull rings, two insertion columns are respectively inserted into the inner cavities of the two slots, then the sealed heat insulation cover is sealed through the handle, finally the base is shaken to drive the culture bottle to shake in the inner cavity of the sealed culture cylinder body, so that the liquid in the inner cavity of the culture bottle is uniformly mixed, and then the culture is carried out for 48 hours.

4. The method of claim 1, wherein the standardized animal model is a tumor model comprising: the observation of the growth condition of the tumor cells in the body of the receptor animal refers to the observation of the size and the change of the tumor mass at the axillary skin of the receptor animal starting one week after the operation, the drawing of a tumor growth curve, the in vivo imaging observation of the tumor formation condition 2 weeks after the operation, the death of the receptor animal 4 weeks after the operation, the micro CT examination of the tumor tissue and the observation of the size condition of the tumor mass by the case section, and the verification of the tumor tissue.

5. A culture device for a standardized animal model of a tumour according to any one of claims 1 to 4, comprising a base (1) characterized in that: the top of the base (1) is provided with a sealed culture cylinder body (2), the inner side wall body of the sealed culture cylinder body (2) is provided with a heat insulation layer (8), the inner side wall body of the heat insulation layer (8) is provided with an inner layer (9), the top of the sealed culture cylinder body (2) is provided with a sealed heat insulation cover (6), a handle (7) is arranged in the middle of the top of the sealed heat insulation cover (6), an inner cavity of the sealed culture cylinder body (2) is provided with a culture bottle (3), the top of the culture bottle (3) is provided with a sealed bottle opening (5), the front middle wall body of the culture bottle (3) is longitudinally provided with scale marks (4), the front wall body of the sealed culture cylinder body (2) is embedded with a transparent observation piece (17), the left side wall body and the right side wall body in the middle of the sealed culture cylinder body (2) are symmetrically and fixedly connected with fixed blocks (13), and slots (14) are symmetrically arranged in the middle of the top of the two fixed blocks (13), two the vertical fixedly connected with spring (15) of inner chamber bottom symmetry of slot (14), the symmetrical fixedly connected with connecting block (12), two of the lateral wall symmetry about the upper end of blake bottle (3) the vertical fixedly connected with spliced pole (10) of top symmetry of connecting block (12), two the convenient pull ring (11) of top difference fixedly connected with of spliced pole (10), two post (16) is inserted to the centre symmetry fixedly connected with in the bottom of connecting block (12).

6. A culture device according to claim 5, wherein the animal model comprises: the bottom of the culture bottle (3) is different from the bottom of the inner cavity of the sealed culture cylinder body (2) and is not contacted.

7. A culture device according to claim 5, wherein the animal model comprises: the two inserting columns (16) are respectively inserted into the inner cavities of the two inserting grooves (14), and the bottoms of the two inserting columns (16) are in contact with the tops of the two springs (15).

8. A culture device according to claim 5, wherein the animal model comprises: the bottoms of the two connecting blocks (12) are not in contact with the top wall bodies of the two fixing blocks (13).

9. A culture device according to claim 5, wherein the animal model comprises: the two connecting columns (10) are cylindrical, and the upper ends of the two connecting columns (10) are respectively positioned at the left side and the right side of the upper end of the inner cavity of the culture bottle (3).

10. A culture device according to claim 5, wherein the animal model comprises: the side wall of the inner cavity of the sealed heat insulation cover (6) is connected with the side wall of the upper end of the sealed culture cylinder body (2) through threads.

Technical Field

The invention relates to the field of establishment of tumor animal models, in particular to an establishment method and a culture device of a standardized tumor animal model.

Background

Malignant tumors are the biggest killer to human health, and scientists throughout the world have been trying to find the best therapeutic approach to combat malignant tumors. Tumor research has been the area of greatest concern worldwide. The existing tumor animal model establishing methods also have the defects of low cell survival rate, more cell loss, inconsistent tumor formation rate, large required cell quantity, unstable tumor formation time, inconvenience in using a culture device for culturing a more smooth culture solution, incapability of ensuring a constant-temperature constant-humidity sealed environment and the like, and thus the establishment method and the culture device of the standardized tumor animal model are needed.

Disclosure of Invention

The invention mainly aims to provide a method for establishing a standardized tumor animal model and a culture device, which can effectively solve the problems in the background technology.

In order to achieve the purpose, the invention adopts the technical scheme that:

a method for establishing a standardized tumor animal model comprises the following operation steps:

s1: pretreating tumor cells with vigorous proliferation in logarithmic growth phase, and freezing and storing;

s2: taking out the tumor cells which are frozen and preserved, adding a culture solution into the tumor cells, and transferring the tumor cells into a culture bottle for culture;

s3: taking out the cultured tumor cells from the culture bottle and implanting the tumor cells into a tumor source animal body;

s4: selecting a tumor source animal which grows well 7 to 12 days after inoculation and is dislocated in cervical vertebra to be killed, disinfecting the skin of an operation part, cutting the skin, and stripping off tumor blocks for inoculation;

s5: removing non-tumor tissues and necrotic tissues from the stripped tumor mass for inoculation, selecting tumor tissues which grow well and have no degeneration necrosis and light red color and are filled with fish meat, placing the tumor tissues in an aseptic plate, shearing the tumor tissues into small blocks with the size of 2 cubic millimeters, placing the aseptic plate on ice cubes, and adding nutrient solution to cultivate the small blocks for a period of time;

s6: sucking the tumor mass after being cultured for a period of time by using a sterile trocar, and then inoculating the tumor mass under the axillary skin of the same receptor animal;

s7: observing the growth of the tumor cells in the recipient animal.

Preferably, the step of performing cryopreservation after pretreating the tumor cells proliferating vigorously in the logarithmic growth phase comprises the steps of performing trypsin digestion, centrifugation and washing on the tumor cells proliferating vigorously in the logarithmic growth phase, preparing a cell suspension by using a culture solution containing 7 parts, calf serum containing 2 parts and DMSO containing 1 part, placing the cell suspension into a cell cryopreservation tube, placing the cell cryopreservation tube at 4 ℃ for 30 minutes, 20 ℃ for 4 hours and 70 ℃ overnight, and then placing the tube into liquid nitrogen for cryopreservation.

Preferably, the tumor cells which are subjected to cryopreservation are taken out and required to be rapidly placed into a water bath at 38-40 ℃ and continuously shaken to be completely melted in one minute, then the supernatant is removed after centrifugation for 5 minutes, then culture solution is added, the culture bottle is transferred into the culture bottle, then the sealed heat insulation cover is rotated and opened through a handle after the culture bottle is transferred into the culture bottle, then the culture bottle is lifted into the inner cavity of the sealed culture cylinder body with constant temperature and humidity through two convenient pull rings, two insertion columns are respectively inserted into the inner cavities of the two slots, then the sealed heat insulation cover is sealed through the handle, finally the base is shaken to drive the culture bottle to shake in the inner cavity of the sealed culture cylinder body, the liquid in the inner cavity of the culture bottle is uniformly mixed, and then the culture is carried out for 48 hours.

Preferably, the observation of the growth of the tumor cells in the recipient animal body is to begin to observe the size and change of the tumor mass at the axillary skin of the recipient animal one week after the operation, draw a tumor growth curve, perform in vivo imaging for 2 weeks after the operation to observe the tumor formation, kill the recipient animal 4 weeks after the operation, take the tumor tissue to perform micro-CT examination and observe the tumor size by case section to verify the tumor tissue.

A culture device of a standardized tumor animal model comprises a base, wherein a sealed culture cylinder body is arranged at the top of the base, a heat insulation layer is arranged on the inner side wall body of the sealed culture cylinder body, an inner layer is arranged on the inner side wall body of the heat insulation layer, a sealed heat insulation cover is arranged at the top of the sealed culture cylinder body, a handle is arranged in the center of the top of the sealed heat insulation cover, a culture bottle is arranged in the inner cavity of the sealed culture cylinder body, a sealed bottle opening is arranged at the top of the culture bottle, scale marks are longitudinally arranged on the wall body in the middle of the front of the culture bottle, a transparent observation sheet is embedded in the wall body in the front of the sealed culture cylinder body, fixed blocks are symmetrically and fixedly connected to the left side wall and the right side wall in the middle of the sealed culture cylinder body, slots are symmetrically formed in the center wall body at the tops of the two fixed blocks, and springs are symmetrically and longitudinally and fixedly connected to the bottoms of the inner cavities of the two slots, the symmetrical fixedly connected with connecting block of the left and right sides wall of blake bottle upper end, two the vertical fixedly connected with spliced pole of the top symmetry of connecting block, two the convenient pull ring of fixedly connected with respectively in the top of spliced pole, two the post is inserted to the centre symmetry fixedly connected with in the bottom of connecting block.

Preferably, the bottom of the culture bottle is not in contact with the bottom of the inner cavity of the sealed culture cylinder body.

Preferably, the two inserting columns are respectively inserted into the inner cavities of the two inserting grooves, and the bottoms of the two inserting columns are contacted with the tops of the two springs.

Preferably, the bottoms of the two connecting blocks are not in contact with the top wall bodies of the two fixing blocks.

Preferably, the two connecting columns are cylindrical, and the upper ends of the two connecting columns are respectively positioned at the left side and the right side of the upper end of the inner cavity of the culture bottle.

Preferably, the side wall of the inner cavity of the sealed heat insulation cover is in threaded connection with the side wall of the upper end of the sealed culture cylinder body.

Advantageous effects

Compared with the prior art, the invention provides a method for establishing a standardized tumor animal model and a culture device, and the method has the following beneficial effects:

1. the establishment method of the standardized tumor animal model can ensure that the cells are temporarily separated from the growth state and the cell characteristics are preserved by freezing and preserving the tumor cells which are proliferated vigorously in the growth period after pretreatment, so the cells are recovered for experiments when needed, a certain amount of cells are preserved moderately, the cells can be prevented from being lost due to pollution or other accidents of the cultured cells, the function of cell preservation is achieved, the tumor growth can be allowed to be larger because the skin of the axilla part is loose by inoculating the axilla subcutaneous part of a receptor animal, the service life of the host animal can be prolonged, the inoculation time can be reduced, the tumor tissues which grow well without degeneration necrosis, light red color and fish flesh are selected in a sterile flat dish and cut into small blocks of 2 cubic millimeters and placed on ice cubes for culture, the cell loss is less, the tumor formation rate is high, the tumors are uniform and stable, and the cell number and the modeling cost required by modeling are greatly reduced.

2. This culture apparatus of standardized tumour animal model's method of establishing, sealed culture cylinder body through setting up, sealed temperature-isolating cover and heat insulating layer can provide a constant temperature and humidity's sealed environment for cultivateing work, convenient pull ring, the spliced pole, the connecting block, insert the post, the slot, the setting of fixed block makes things convenient for getting of blake bottle to put, thereby conveniently change the culture solution, the setting of spring not only can play the buffering guard action to the device body, the blake bottle of being convenient for simultaneously rocks, the misce bene of the solution of being convenient for, the setting of transparent observation piece is convenient for observe the inside condition.

Drawings

FIG. 1 is a schematic view of the structure of a culture apparatus of the present invention;

FIG. 2 is an enlarged view of the culture apparatus of the present invention at A in FIG. 1;

FIG. 3 is a schematic view showing the structure of a sealed culture cylinder of the culture apparatus of the present invention.

In the figure: 1. a base; 2. sealing the culture cylinder; 3. a culture bottle; 4. scale lines; 5. sealing the bottle mouth; 6. sealing the heat insulation cover; 7. a handle; 8. a thermal insulation layer; 9. an inner layer; 10. connecting columns; 11. a convenient pull ring; 12. connecting blocks; 13. a fixed block; 14. a slot; 15. a spring; 16. inserting a column; 17. a transparent viewing sheet.

Detailed Description

In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.

The first embodiment is as follows:

a method for establishing a standardized tumor animal model comprises the following operation steps:

s1: pretreating tumor cells which are proliferated vigorously in a logarithmic growth phase, and then performing cryopreservation, wherein the pretreating of the tumor cells which are proliferated vigorously in the logarithmic growth phase is to perform trypsin digestion, centrifugation and washing on the tumor cells which are proliferated vigorously in the logarithmic growth phase, prepare a cell suspension by using a culture solution containing 7 parts, calf serum containing 2 parts and DMSO containing 1 part, then place the cell suspension into a cell cryopreservation tube, place the cell cryopreservation tube at 4 ℃ for 30 minutes, place the cell cryopreservation tube at-20 ℃ for 4 hours and place the cell cryopreservation tube at-70 ℃ overnight, and then place the cell cryopreservation tube into liquid nitrogen for cryopreservation;

s2: taking out the tumor cells which are subjected to cryopreservation, adding a culture solution into the tumor cells, transferring the tumor cells into a culture bottle for culture, taking out the tumor cells which are subjected to cryopreservation, rapidly putting the tumor cells into a water bath at 38-40 ℃ and continuously shaking the tumor cells to completely melt the tumor cells within one minute, centrifuging the cells for 5 minutes to remove supernatant, adding the culture solution into the culture bottle, transferring the tumor cells into the culture bottle, rotating a sealing heat insulation cover through a handle and opening the sealing heat insulation cover, lifting the culture bottle into an inner cavity of a sealed culture cylinder body with constant temperature and humidity through two convenient pull rings, respectively inserting two insertion columns into inner cavities of two slots, sealing the sealing heat insulation cover through the handle, finally shaking a base to drive the culture bottle to shake in the inner cavity of the sealed culture cylinder body, uniformly mixing the liquid in the inner cavity of the culture bottle, and then culturing the tumor cells for 48 hours;

s3: taking out the cultured tumor cells from the culture bottle and implanting the tumor cells into a tumor source animal body;

s4: selecting a tumor source animal which grows well 7 to 12 days after inoculation and is dislocated in cervical vertebra to be killed, disinfecting the skin of an operation part, cutting the skin, and stripping off tumor blocks for inoculation;

s5: removing non-tumor tissues and necrotic tissues from the stripped tumor mass for inoculation, selecting tumor tissues which grow well and have no degeneration necrosis and light red color and are filled with fish meat, placing the tumor tissues in an aseptic plate, shearing the tumor tissues into small blocks with the size of 2 cubic millimeters, placing the aseptic plate on ice cubes, and adding nutrient solution to cultivate the small blocks for a period of time;

s6: sucking the tumor mass after being cultured for a period of time by using a sterile trocar, and then inoculating the tumor mass under the axillary skin of the same receptor animal;

s7: observing the growth condition of tumor cells in a receptor animal body, wherein the observation of the growth condition of the tumor cells in the receptor animal body means that the size and the change of a lump at the axillary skin of the receptor animal are observed starting one week after operation, a tumor growth curve is drawn, living body imaging is carried out 2 weeks after operation to observe the condition of the lump formation, the receptor animal is killed 4 weeks after operation, and the tumor tissue is taken for micro CT examination and case section observation to verify the tumor tissue.

The establishment method of the standardized tumor animal model can ensure that the cells are temporarily separated from the growth state and the cell characteristics are preserved by freezing and preserving the tumor cells which are proliferated vigorously in the growth period after pretreatment, so the cells are recovered for experiments when needed, a certain amount of cells are preserved moderately, the cells can be prevented from being lost due to pollution or other accidents of the cultured cells, the function of cell preservation is achieved, the tumor growth can be allowed to be larger because the skin of the axilla part is loose by inoculating the axilla subcutaneous part of a receptor animal, the service life of the host animal can be prolonged, the inoculation time can be reduced, the tumor tissues which grow well without degeneration necrosis, light red color and fish flesh are selected in a sterile flat dish and cut into small blocks of 2 cubic millimeters and placed on ice cubes for culture, the cell loss is less, the tumor formation rate is high, the tumors are uniform and stable, and the cell number and the modeling cost required by modeling are greatly reduced.

The second embodiment is as follows:

as shown in figures 1-3, a culture device of a standardized tumor animal model comprises a base 1, a sealed culture cylinder 2 is arranged on the top of the base 1, a thermal insulation layer 8 is arranged on the inner side wall body of the sealed culture cylinder 2, an inner layer 9 is arranged on the inner side wall body of the thermal insulation layer 8, a sealed thermal insulation cover 6 is arranged on the top of the sealed culture cylinder 2, the inner cavity side wall of the sealed thermal insulation cover 6 is connected with the upper end side wall of the sealed culture cylinder 2 through threads, a handle 7 is arranged in the middle of the top of the sealed thermal insulation cover 6, a culture bottle 3 is arranged in the inner cavity of the sealed culture cylinder 2, the bottom of the culture bottle 3 is not in contact with the bottom of the inner cavity of the sealed culture cylinder 2, a sealed bottle opening 5 is arranged on the top of the culture bottle 3, a scale mark 4 is longitudinally arranged on the middle wall body of the front of the culture bottle 3, a transparent observation sheet 17 is embedded on the front wall body of the sealed culture cylinder 2, the left side wall and the right side wall of the middle part of the sealed culture cylinder body 2 are symmetrically and fixedly connected with fixed blocks 13, the top middle wall bodies of the two fixed blocks 13 are symmetrically provided with slots 14, the bottoms of the inner cavities of the two slots 14 are symmetrically and longitudinally and fixedly connected with springs 15, the left side wall and the right side wall of the upper end of the culture bottle 3 are symmetrically and fixedly connected with connecting blocks 12, the bottoms of the two connecting blocks 12 are not contacted with the top wall bodies of the two fixed blocks 13, the tops of the two connecting blocks 12 are symmetrically and longitudinally and fixedly connected with connecting columns 10, the two connecting columns 10 are cylindrical, the upper ends of the two connecting columns 10 are respectively positioned at the left side and the right side of the upper end of the inner cavity of the culture bottle 3, the tops of the two connecting columns 10 are respectively and fixedly connected with a convenient pull ring 11, the bottoms of the two connecting blocks 12 are respectively and symmetrically and fixedly connected with inserting columns 16, the two inserting columns 16 are respectively inserted into the inner cavities of the two slots 14, and the bottoms of the two inserting columns 16 are in contact with the tops of the two springs 15.

This culture apparatus of standardized tumour animal model's method of establishing, sealed culture cylinder 2 through setting up, sealed heat insulation cover 6 and heat insulation layer 8 can provide a constant temperature and humidity's sealed environment for cultivateing work, convenient pull ring 11, spliced pole 10, connecting block 12, insert post 16, slot 14, the setting of fixed block 13 makes things convenient for getting of blake bottle 3 to put, thereby conveniently change the culture solution, spring 15's setting not only can play the buffer protection effect to the device body, be convenient for rocking and rocking of blake bottle 3 simultaneously, the misce bene of solution of being convenient for, the setting of transparent observation piece 17 is convenient for observe the inside condition.

The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.