Human glioma organoid isolated culture kit and human glioma organoid culture method

Abstract

The invention belongs to the technical field of regenerative medicine and biology, and particularly relates to a human glioma organoid isolated culture kit and a human glioma organoid culture method. The human glioma organoid isolated culture kit comprises a tissue activity protection solution, a P1 buffer solution, a human glioma organoid culture medium, an organoid cryopreservation solution and a red blood cell lysis buffer, and the culture method is carried out by adopting the human glioma organoid isolated culture kit. The human glioma organoid can be cultured by adopting the human glioma organoid isolated culture kit and the culture method, and the kit and the culture method have the advantages that the human glioma organoid grows quickly and can be cryopreserved and resuscitated.

Description

Technical Field

[0001] The present invention belongs to the field of regenerative medicine and biological technology, and specifically relates to a human glioma organoid isolation and culture kit and a human glioma organoid culture method. Background Art

[0002] Glioma can be divided into four grades according to its classification, including grade 1, grade 2, grade 3, and grade 4. Except for grade 1, grade 2, grade 3, and grade 4 tumors are incurable. Therefore, even if grade 2, grade 3, and grade 4 tumors are completely removed by surgery, standardized radiotherapy, chemotherapy, or radiotherapy and chemotherapy should be performed after surgery.

[0003] Glioma is the most common primary malignant tumor in the brain, accounting for about 40%-50% of brain tumors, with an annual incidence of 3-8 / 100,000. Glioma is a relatively common primary central nervous system tumor, mainly due to the uncontrollable proliferation of glial cells to form tumors. This tumor is prone to occur in the brain and occasionally in the spinal cord. In general, this tumor grows aggressively and is not easy to be completely removed by surgery during clinical surgery, so it is more likely to recur and progress.

[0004] Glioma is a malignant tumor. It can be said that more than 50% of gliomas are the most malignant glioblastoma, which is clinically called grade IV glioma. Glioma is the most common type of intracranial tumor. The cause and pathogenesis are still unclear. It may be related to genetics, ionizing radiation, viral infection, age and other factors. It can be divided into astrocytoma, oligodendroglioma, pilocytic astrocytoma and other types.

[0005] Organoids are three-dimensional (3D) cell cultures that contain some of the key properties of the organs they represent. Because part of the organoid model system is the presence of an active stem cell population, organoids are extremely amenable to expansion, providing researchers with a highly reliable and scalable platform to study a variety of organs.

[0006] Therefore, in order to conduct more in-depth research on gliomas, providing a method for rapidly culturing human glioma organoids has become a problem that needs to be solved at present. Summary of the invention

[0007] Based on the above technical background, the main purpose of the present invention is to provide a human glioma organoid isolation and culture kit and a human glioma organoid culture method to overcome the shortcomings of the prior art.

[0008] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention includes:

[0009] The first aspect of the present invention is to provide a human glioma organoid isolation and culture kit, which comprises a tissue activity protection solution, a P1 buffer, a human glioma organoid culture medium, an organoid freezing solution and a red blood cell lysis solution.

[0010] Preferably, the volume ratio of the tissue activity protection solution, P1 buffer, human glioma organoid culture medium, organoid freezing solution and red blood cell lysis solution is 50:(3-7):(80-120):(15-25):(40-60).

[0011] The second aspect of the present invention is to provide a method for culturing human glioma organoids using the human glioma organoid isolation and culture kit described in the first aspect of the present invention, wherein the culturing method comprises the following steps:

[0012] Step 1, thawing the human glioma organoid culture medium, adding P1 buffer into PBS to prepare P1 phosphate buffer, and then precooling the solution to precool the tissue activity protection solution;

[0013] Step 2: Cut the tissue sample into pieces, add pre-cooled P1 phosphate buffer to wash, remove necrosis, bleeding and normal brain tissue in the tissue sample, and then cut it into tissue fragments, let the tissue fragments settle by gravity, remove the supernatant, add P1 phosphate buffer at room temperature to wash, remove the supernatant, then add red blood cell lysis solution to shake, aspirate the red blood cell lysis solution, and wash the lysed tissue fragments;

[0014] Step 3, adding human glioma organoid culture medium to the lysed tissue fragments for sterile culture, and during the culture period, continuously removing tissue fragments that cannot grow to obtain human glioma organoids;

[0015] Step 4: Centrifuge the human glioma organoids, aspirate the supernatant, then add tissue activity protection solution, aspirate the supernatant, and then add pre-cooled tissue activity protection solution, cut the human glioma organoids into pieces, wash at room temperature, aspirate the supernatant, add human glioma organoid culture medium to the human glioma organoid fragments for sterile subculture.

[0016] In step 1,

[0017] Preferably, the precooling temperature is 0-4°C.

[0018] In step 2,

[0019] Preferably, the tissue fragments are gravity settled for 1 to 5 minutes.

[0020] Preferably, add room temperature P1 phosphate buffer to wash 2 to 5 times, remove the supernatant, then add red blood cell lysis solution and shake at room temperature for 5 to 15 minutes at a shaking speed of 7 to 15 rpm, aspirate the red blood cell lysis solution, wash the lysed tissue fragments with room temperature DMEM / F12 for 2 to 5 times, and aspirate the supernatant.

[0021] In step 3,

[0022] Preferably, the conditions for sterile culture are: shaking speed 100-150 rpm, 37°C, 5% CO 2 , relative humidity 85~95%.

[0023] Preferably, during the sterile culture process, the human glioma organoid culture medium is replaced every 45 to 50 hours.

[0024] In step 4,

[0025] Preferably, the conditions for sterile subculture are: shaking speed 100-140 rpm, 37°C, 5% CO 2 , relative humidity 85~95%.

[0026] Preferably, during the subculture process, the human glioma organoid culture medium is replaced every 45 to 55 hours.

[0027] The beneficial effects of the present invention are:

[0028] (1) The human glioma organoid isolation and culture kit of the present invention can be used to culture human glioma organoids, and the use of the kit for culturing human glioma organoids has the advantages of rapid growth and cryopreservation and recovery.

[0029] (2) The culture method described in the present invention is carried out using a human glioma organoid isolation and culture kit. The culture method can quickly culture human glioma organoids and can be subcultured, providing strong support for subsequent research. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Showing photos of tissue samples, before culture, and after two weeks of culture using the culture method described in Example 2;

[0031] Figure 2 Microscopic photographs of tissue samples before culture, after culture for one week using the culture method described in Example 2, and after culture for two weeks are shown;

[0032] Figure 3 The figure shows the pathological identification results of tissue samples after two weeks of culture using the culture method described in Example 2. DETAILED DESCRIPTION

[0033] The present invention will be described in detail below, and the characteristics and advantages of the present invention will become clearer and more distinct with the description.

[0034] The first aspect of the present invention is to provide a human glioma organoid isolation and culture kit, which comprises a tissue activity protection solution, a P1 buffer, a human glioma organoid culture medium, an organoid freezing solution and a red blood cell lysis solution.

[0035] The volume ratio of the tissue activity protection solution, P1 buffer, human glioma organoid culture medium, organoid freezing solution and red blood cell lysis solution is 50: (3-7): (80-120): (15-25): (40-60).

[0036] Preferably, the volume ratio of the tissue activity protection solution, P1 buffer, human glioma organoid culture medium, organoid freezing solution and red blood cell lysis solution is 50:5:100:20:50.

[0037] The second aspect of the present invention is to provide a method for culturing human glioma organoids using the human glioma organoid isolation and culture kit described in the first aspect of the present invention, wherein the culturing method comprises the following steps:

[0038] Step 1, thawing the human glioma organoid culture medium, adding P1 buffer into PBS to prepare P1 phosphate buffer, and then precooling the solution to precool the tissue activity protection solution;

[0039] Step 2: Cut the tissue sample into pieces, add pre-cooled P1 phosphate buffer to wash, remove necrosis, bleeding and normal brain tissue in the tissue sample, and then cut it into tissue fragments, let the tissue fragments settle by gravity, remove the supernatant, add P1 phosphate buffer at room temperature to wash, remove the supernatant, then add red blood cell lysis solution to shake, aspirate the red blood cell lysis solution, and wash the lysed tissue fragments;

[0040] Step 3, adding human glioma organoid culture medium to the lysed tissue fragments for sterile culture, and during the culture period, continuously removing tissue fragments that cannot grow to obtain human glioma organoids;

[0041] Step 4: Centrifuge the human glioma organoids, aspirate the supernatant, then add tissue activity protection solution, aspirate the supernatant, and then add pre-cooled tissue activity protection solution, cut the human glioma organoids into pieces, wash at room temperature, aspirate the supernatant, add human glioma organoid culture medium to the human glioma organoid fragments for sterile subculture.

[0042] The above steps are described in detail below.

[0043] In step 1, the precooling temperature is 0-4°C, preferably 4°C.

[0044] In step 2, the tissue sample is cut into blocks of 0.4-0.7 cm wide x 0.4-0.7 cm thick.

[0045] Preferably, the tissue sample is cut into blocks of 0.5 cm wide x 0.5 cm thick.

[0046] Pre-cooled P1 phosphate buffer is added to the block tissue sample for washing 2 to 5 times, preferably 3 times.

[0047] Remove necrotic, hemorrhagic and normal brain tissue from the tissue sample and cut it into pieces of 0.5-1 mm 3 of tissue fragments.

[0048] Allow the tissue fragments to settle by gravity for 1 to 5 minutes, remove the supernatant, add room temperature P1 phosphate buffer to wash 2 to 5 times, remove the supernatant, then add red blood cell lysis buffer and shake at room temperature for 5 to 15 minutes at a shaking speed of 7 to 15 rpm, aspirate the red blood cell lysis buffer, wash the lysed tissue fragments 2 to 5 times with room temperature DMEM / F12, and aspirate the supernatant.

[0049] Preferably, the tissue fragments are gravity settled for 2 to 3 minutes, the supernatant is removed, and the tissue fragments are washed three times with P1 phosphate buffer at room temperature, the supernatant is removed, and then red blood cell lysis solution is added and shaken at room temperature for 10 minutes at a shaking speed of 10 rpm to lyse most of the contaminated red blood cells in the tissue fragments, the red blood cell lysis solution is aspirated, and the lysed tissue fragments are washed three times with room temperature DMEM / F12, and the supernatant is aspirated.

[0050] In step 3, the conditions for sterile culture are: shaking speed 100-150 rpm, 37°C, 5% CO 2 , relative humidity 85~95%.

[0051] Preferably, the conditions for sterile culture are: shaking speed 120 rpm, 37°C, 5% CO 2 , relative humidity 90%.

[0052] During the sterile culture process, the medium is changed every 45 to 50 hours, preferably every 48 hours. When changing the medium, the culture plate is tilted at a 45° angle, 75% of the human glioma organoid culture medium is aspirated, and then 3 mL of new human glioma organoid culture medium is added.

[0053] After one week of sterile culture, some tissue fragments began to aggregate, and the non-aggregated tumor tissue fragments were removed. Most of these fragments came from necrotic tissue and could not form organoids. Continue to culture for 2 to 4 weeks, and continue to remove tissue that cannot grow. At different time points, some glioma organoids were taken out as samples for follow-up research. The successful signs of human glioma organoid culture are: survival for more than 2 weeks, formation of spherical structures, and continued growth.

[0054] In step 4, when the human glioma organoids grow to a diameter of 1 to 2 mm, subculture begins.

[0055] Cut the human glioma organoids into pieces, wash the organoids 2 to 5 times with room temperature DMEM / F12 medium, preferably 3 times, and aspirate the supernatant.

[0056] The conditions for sterile subculture were: shaking speed 100-140 rpm, 37°C, 5% CO 2 , relative humidity 85~95%.

[0057] Preferably, the conditions for sterile subculture are: shaking speed 120 rpm, 37°C, 5% CO 2 , relative humidity 90%.

[0058] During the subculture process, the medium is changed every 45 to 55 hours, preferably every 48 hours. When changing the medium, the culture plate is tilted at a 45° angle, 75% of the human glioma organoid culture medium is aspirated, and then 3 mL of new human glioma organoid culture medium is added.

[0059] Example

[0060] The present invention is further described below by means of specific examples. These embodiments are only intended to illustrate the present invention but are not intended to limit the scope of the present invention.

[0061] Example 1

[0062] A human glioma organoid isolation and culture kit, the kit comprising 50 mL of tissue activity protection solution, 5 mL of P1 buffer, 100 mL of human glioma organoid culture medium, 20 mL of organoid freezing solution, and 50 mL of red blood cell lysis solution. The product information of each substance included in the kit is shown in Table 1:

[0063] Table 1

[0064]

[0065]

[0066] The product information of human glioma organoid culture medium is shown in Table 2:

[0067] Table 2

[0068]

[0069] The formula information of tissue active protection solution is shown in Table 3:

[0070] Table 3

[0071]

[0072] Example 2

[0073] A method for culturing human glioma organoids using the human glioma organoid isolation and culture kit described in Example 1, the culturing method comprising the following steps:

[0074] Pre-preparation:

[0075] 1. Thaw the human glioma organoid culture medium in advance according to the culture demand;

[0076] 2. Add 5 mL of P1 buffer to 500 mL of PBS (without Ca + Mg + ) was prepared into P1 phosphate buffer and pre-cooled in a 4°C refrigerator;

[0077] 3. Pre-cool the tissue activity protection solution in a 4°C refrigerator.

[0078] Human glioma organoid construction procedures:

[0079] 1. If the collected fresh clinical tissue samples cannot be cultured for organoids immediately, please place them in tissue activity protection solution and store them at 4°C ("Tissue activity protection solution" can only delay cell apoptosis and loss of activity in the tissue, but it is still recommended to separate and culture them as soon as possible, and it is generally not recommended to store them for more than 48 hours); the following uses a tissue block of approximately 0.5 cm wide × 0.5 cm thick as an example of operation application;

[0080] 2. Discard the tissue active protection solution and add 10 mL of pre-cooled P1 phosphate buffer to wash three times;

[0081] 3. Add 1 mL of pre-cooled tissue active protection solution to a 35 mm diameter cell culture dish, take out the tissue sample, remove some necrotic, hemorrhagic and normal brain tissue, and cut it into 0.5-1 mm pieces with ophthalmic scissors. 3 of tissue fragments;

[0082] 4. Transfer the tissue fragments into a 15 mL centrifuge tube, let the tissue fragments settle under gravity for 2 to 3 minutes, remove the supernatant, add room temperature P1 phosphate buffer to wash three times, and remove the supernatant;

[0083] 5. Add 10 mL of red blood cell lysis buffer and shake on a seesaw shaker at room temperature for 10 minutes at a speed of about 10 rpm to lyse most of the contaminating red blood cells in the tissue fragments;

[0084] 6. Aspirate the red blood cell lysis solution, wash three times with 10 mL of room temperature DMEM / F12 medium, and aspirate the supernatant;

[0085] 7. Transfer the tumor tissue fragments to a 6-well culture plate, with each well containing 10 to 20 fragments and 4 mL of human glioma organoid culture medium;

[0086] 8. Place the culture plate in a CO2-resistant 2 On an orbital shaker, at 120 rpm, 37°C, 5% CO 2 , cultured in a sterile incubator at a relative humidity of 90%;

[0087] 9. Change the medium every 48 hours. When changing the medium, tilt the culture plate at a 45° angle, aspirate 75% of the human glioma organoid culture medium, and then add 3 mL of new human glioma organoid culture medium;

[0088] 10. After one week of culture, some fragments begin to aggregate, and the non-aggregated tumor tissue fragments are removed. Most of these fragments come from necrotic tissue and cannot form organoids. (In the first week, tumor tissue usually sheds cells and blood fragments, making the culture medium turbid. Generally, within 2 weeks, as the tumor forms organoids, the shedding will stop quickly. Stopping the shedding ultimately depends on the quality of the tissue and the patient's specific tumor growth characteristics).

[0089] 11. Continue to culture for 2 to 4 weeks and continue to remove tissue that cannot grow. Take out some glioma organoids at different time points as samples for follow-up research. Successful signs: survive for more than 2 weeks, form a spherical structure, and continue to grow;

[0090] Passaging of Human Glioma Organoids:

[0091] 1. When the organoids grow to a diameter of 1-2 mm, start subculturing;

[0092] 2. Transfer the organoids to a 15 mL centrifuge tube using a Pasteur pipette, let them sink to the bottom of the tube, and then aspirate the supernatant;

[0093] 3. Wash once with 2 mL of tissue active protection solution, let it sink to the bottom of the centrifuge tube, then aspirate the supernatant, transfer the organoid to a 35 mm diameter cell culture dish, add 1 mL of pre-cooled tissue active protection solution to the cell culture dish, and cut each organoid into 4 small pieces with ophthalmic scissors;

[0094] 4. Wash the organoids three times with 10 mL of room temperature DMEM / F12 and aspirate the supernatant;

[0095] 5. Transfer the tumor organoid fragments to a 6-well culture plate, with each well containing 10 to 20 fragments and 4 mL of human glioma organoid culture medium;

[0096] 6. Place the culture plate in a CO2-resistant 2 On an orbital shaker, at 120 rpm, 37°C, 5% CO 2 , 90% relative humidity in a sterile incubator; change the medium every 48 hours. When changing the medium, tilt the culture plate at a 45° angle, aspirate 75% of the human glioma organoid culture medium, and then add 3 mL of new human glioma organoid culture medium for culture. The cultured human glioma organoids can be stored in organoid cryopreservation medium.

[0097] The tissue samples used in this example, and the photos of organoid tissue samples before and after two weeks of culture are shown in the figure. Figure 1 shown.

[0098] Example 3

[0099] A method for culturing human glioma organoids using the human glioma organoid isolation and culture kit described in Example 1, the culturing method comprising the following steps:

[0100] Pre-preparation:

[0101] 1. Thaw the human glioma organoid culture medium in advance according to the culture demand;

[0102] 2. Add 5 mL of P1 buffer to 500 mL of PBS (without Ca + Mg + ) was prepared into P1 phosphate buffer and pre-cooled in a 4°C refrigerator;

[0103] 3. Pre-cool the tissue activity protection solution in a 4°C refrigerator.

[0104] Human glioma organoid construction procedures:

[0105] 1. If the collected fresh clinical tissue samples cannot be cultured for organoids immediately, please place them in tissue activity protection solution and store them at 4°C ("Tissue activity protection solution" can only delay cell apoptosis and loss of activity in the tissue, but it is still recommended to separate and culture them as soon as possible, and it is generally not recommended to store them for more than 48 hours); the following uses a tissue block of approximately 0.5 cm wide × 0.5 cm thick as an example of operation application;

[0106] 2. Discard the tissue active protection solution and add 10 mL of pre-cooled P1 phosphate buffer to wash three times;

[0107] 3. Add 1 mL of pre-cooled tissue active protection solution to a 35 mm diameter cell culture dish, take out the tissue sample, remove some necrotic, hemorrhagic and normal brain tissue, and cut it into 0.5-1 mm pieces with ophthalmic scissors. 3 of tissue fragments;

[0108] 4. Transfer the tissue fragments into a 15 mL centrifuge tube, let the tissue fragments settle under gravity for 3 to 5 minutes, remove the supernatant, add room temperature P1 phosphate buffer to wash three times, and remove the supernatant;

[0109] 5. Add 10 mL of red blood cell lysis buffer and shake on a seesaw shaker at room temperature for 10 minutes at a speed of about 7 rpm to lyse most of the contaminating red blood cells in the tissue fragments;

[0110] 6. Aspirate the red blood cell lysis solution, wash three times with 10 mL of room temperature DMEM / F12 medium, and aspirate the supernatant;

[0111] 7. Transfer the tumor tissue fragments to a 6-well culture plate, with each well containing 10 to 20 fragments and 4 mL of human glioma organoid culture medium;

[0112] 8. Place the culture plate in a CO2-resistant 2 On an orbital shaker, at 100 rpm, 37°C, 5% CO 2 , cultured in a sterile incubator at 85% relative humidity;

[0113] 9. Change the medium every 48 hours. When changing the medium, tilt the culture plate at a 45° angle, aspirate 75% of the human glioma organoid culture medium, and then add 3 mL of new human glioma organoid culture medium;

[0114] 10. After one week of culture, some fragments begin to aggregate, and the non-aggregated tumor tissue fragments are removed. Most of these fragments come from necrotic tissue and cannot form organoids. (In the first week, tumor tissue usually sheds cells and blood fragments, making the culture medium turbid. Generally, within 2 weeks, as the tumor forms organoids, the shedding will stop quickly. Stopping the shedding ultimately depends on the quality of the tissue and the patient's specific tumor growth characteristics).

[0115] 11. Continue to culture for 2 to 4 weeks and continue to remove tissue that cannot grow. Take out some glioma organoids at different time points as samples for follow-up research. Successful signs: survive for more than 2 weeks, form a spherical structure, and continue to grow;

[0116] Passaging of Human Glioma Organoids:

[0117] 1. When the organoids grow to a diameter of 1-2 mm, start subculturing;

[0118] 2. Transfer the organoids to a 15 mL centrifuge tube using a Pasteur pipette, let them sink to the bottom of the tube, and then aspirate the supernatant;

[0119] 3. Wash once with 2 mL of tissue active protection solution, let it sink to the bottom of the centrifuge tube, then aspirate the supernatant, transfer the organoid to a 35 mm diameter cell culture dish, add 1 mL of pre-cooled tissue active protection solution to the cell culture dish, and cut each organoid into 4 small pieces with ophthalmic scissors;

[0120] 4. Wash the organoids three times with 10 mL of room temperature DMEM / F12 and aspirate the supernatant;

[0121] 5. Transfer the tumor organoid fragments to a 6-well culture plate, with each well containing 10 to 20 fragments and 4 mL of human glioma organoid culture medium;

[0122] 6. Place the culture plate in a CO2-resistant 2 On an orbital shaker, at 100 rpm, 37°C, 5% CO 2 , 85% relative humidity in a sterile incubator; change the medium every 48 hours. When changing the medium, tilt the culture plate at a 45° angle, aspirate 75% of the human glioma organoid culture medium, and then add 3 mL of new human glioma organoid culture medium for culture. The cultured human glioma organoids can be stored in organoid cryopreservation medium.

[0123] Experimental example

[0124] Experimental Example 1 Optical Microscope Test

[0125] The tissue samples were tested under optical microscope before culture, after culture for one week and after culture for two weeks using the culture method described in Example 2. The test results are as follows: Figure 2 shown.

[0126] from Figure 2 It can be seen that after one week (7 days) and two weeks (14 days) of culture, the tissue grows rapidly and can be frozen and revived, indicating that human glioma organoids can be quickly cultured using the human glioma organoid isolation and culture kit and culture method of the present invention.

[0127] Experimental Example 2 Pathological Identification

[0128] The human glioma organoids cultured in Example 2 were pathologically identified, and the identification results were as follows: Figure 3 shown.

[0129] from Figure 3It can be seen that the human glioma organoids cultured in Example 2 have consistent expressions of HE (tumor marker), GFAP (glial fibrillary acidic protein, a marker protein of astrocytes) and NEUN (neuronal core antigen) with those of tissues, indicating that human glioma organoids can be cultured using the human glioma organoid isolation and culture kit and culture method described in the present invention.

[0130] The present invention has been described in detail above in conjunction with specific implementations and exemplary examples, but these descriptions cannot be understood as limiting the present invention. Those skilled in the art understand that, without departing from the spirit and scope of the present invention, a variety of equivalent substitutions, modifications or improvements may be made to the technical solution of the present invention and its implementation methods, all of which fall within the scope of the present invention. The scope of protection of the present invention shall be subject to the attached claims.

Claims

1. A human glioma organoid isolation and culture kit, characterized in that: The human glioma organoid isolation and culture kit comprises a tissue activity protection solution, a P1 buffer solution, a human glioma organoid culture medium, an organoid freezing solution and a red blood cell lysis solution.

2. The human glioma organoid isolation and culture kit according to claim 1, characterized in that: The volume ratio of the tissue activity protection solution, P1 buffer, human glioma organoid culture medium, organoid freezing solution and red blood cell lysis solution is 50: (3-7): (80-120): (15-25): (40-60).

3. A method for culturing human glioma organoids using the human glioma organoid isolation and culture kit according to claim 1 or 2, characterized in that: The method comprises the following steps: Step 1, thawing the human glioma organoid culture medium, adding P1 buffer into PBS to prepare P1 phosphate buffer, and then precooling the solution to precool the tissue activity protection solution; Step 2: Cut the tissue sample into pieces, add pre-cooled P1 phosphate buffer to wash, remove necrosis, bleeding and normal brain tissue in the tissue sample, and then cut it into tissue fragments, let the tissue fragments settle by gravity, remove the supernatant, add P1 phosphate buffer at room temperature to wash, remove the supernatant, then add red blood cell lysis solution to shake, aspirate the red blood cell lysis solution, and wash the lysed tissue fragments; Step 3, adding human glioma organoid culture medium to the lysed tissue fragments for sterile culture, and during the culture period, continuously removing tissue fragments that cannot grow to obtain human glioma organoids; Step 4: Centrifuge the human glioma organoids, aspirate the supernatant, then add tissue activity protection solution, aspirate the supernatant, and then add pre-cooled tissue activity protection solution, cut the human glioma organoids into pieces, wash at room temperature, aspirate the supernatant, add human glioma organoid culture medium to the human glioma organoid fragments for sterile subculture.

4. The method according to claim 3, characterized in that In step 1, The precooling temperature is 0-4℃.

5. The method according to claim 3, characterized in that: In step 2, Allow tissue fragments to settle by gravity for 1 to 5 minutes.

6. The method according to claim 3, characterized in that In step 2, Add room temperature P1 phosphate buffer to wash 2 to 5 times, remove the supernatant, then add red blood cell lysis buffer and shake at room temperature for 5 to 15 minutes at a shaking speed of 7 to 15 rpm, aspirate the red blood cell lysis buffer, wash the lysed tissue fragments 2 to 5 times with room temperature DMEM / F12 culture medium, and aspirate the supernatant.

7. The method according to claim 3, characterized in that In step 3, The conditions for sterile culture are: shaking speed 100-150 rpm, 37°C, 5% CO2, and relative humidity 85-95%.

8. The method according to claim 3, characterized in that In step 3, During the sterile culture process, the human glioma organoid culture medium was replaced every 45 to 50 hours.

9. The method according to claim 3, characterized in that: In step 4, The conditions for sterile subculture are: shaking speed 100-140 rpm, 37°C, 5% CO2, and relative humidity 85-95%.

10. The method according to claim 3, characterized in that In step 4, During the subculture process, the human glioma organoid culture medium was replaced every 45 to 55 hours.

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