Lymphoma organoid culture medium and culture method

Abstract

The invention belongs to the technical field of biology, and particularly relates to a lymphoma organoid culture medium and a culture method. The culture medium comprises the following components: 0.5 to 2 X of a GlutaMAX additive, 0.2 to 2 X of B-27, 0.5 to 2 X of N-2, 0.8 to 1.5 X of ITS-A, 1 to 10 mmol / L of sodium pyruvate, 50 to 300 ng / ml of WNT-3A, 2.5 to 10 mmol / L of nicotinamide, and the like which are dissolved in an RPMI 1640 culture medium. According to the culture method provided by the invention, in-vitro culture can be specifically carried out on the human lymphoma tissue, and in the culture process, an organoid which is similar to an in-vivo tumor from cell constitution and space structure is formed; the culture medium provided by the invention has clear components, is suitable for culturing different types of lymphoma organoid, and can provide a favorable culture method and research model for an in-vitro research platform in the disease field.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a lymphoma organoid culture medium and a culture method. BACKGROUND

[0002] Lymphoma is a group of highly heterogeneous blood cancers involving complex subtypes and variable clinical outcomes, which poses significant challenges to its diagnosis and treatment. The diversity of lymphoma is not only reflected in its histological characteristics, but also in its response to different treatments, which requires a more personalized approach to treatment strategies. Lymphoma, also known as malignant lymphoma, is a malignant tumor originating from lymph nodes and lymphoid tissues, and is one of the earliest discovered hematological malignancies. Its specific cause is not clear, and may be related to infection, immune factors, physical and chemical factors, and genetic factors. The tumor can occur in any part of the body, and the clinical manifestations are diverse, usually characterized by painless progressive lymphadenopathy, often accompanied by fever, weight loss, night sweats and other systemic symptoms. Sometimes, it can invade the tonsils, nasopharynx, gastrointestinal tract, bones or skin, causing symptoms of damage to the corresponding tissues and organs. The classification and treatment of lymphoma not only depend on pathological characteristics, but also need to consider individual differences in patients. Especially in the case of relapsed / refractory aggressive B-cell non-Hodgkin lymphoma in children and adolescents, due to the diversity of cell characteristics and gene expression, it often faces the complexity of diagnosis and treatment.

[0003] The rise of organoid technology brings new opportunities for lymphoma research. Compared with traditional cell lines and mouse models, organoids have higher biological similarity and can better reflect the biological characteristics of tumors. In addition, organoids can also be used for high-throughput drug screening, which is of great significance for individualized treatment. By analyzing patient-derived tumor organoids, the therapeutic response of different drugs can be evaluated, and targeted treatment plans can be developed. Therefore, if an organoid model that can simulate the microenvironment of lymphoma can be established, it will provide a new research path. Because organoids not only retain the cell diversity and gene characteristics of tumors, but also reproduce the tumor microenvironment, they provide possible solutions for precision treatment.

[0004] However, despite the great potential of organoid technology in tumor research, there are still many challenges in its practical application. For example, how to efficiently and stably establish organoids with high purity, and how to overcome the heterogeneity problem in cell culture, are difficult problems that need to be solved. In addition, the standardized culture method of organoids has not been fully established, which makes the reproducibility of research results between different laboratories a problem. SUMMARY

[0005] In order to overcome the above-mentioned deficiencies in the prior art, the present application provides a lymphoma organoid culture medium and a culture method.

[0006] According to a first aspect of the present application, the present application provides a lymphoma organoid culture medium, which comprises, in terms of final concentration: 0.5-2X GlutaMAX additive (100X stock concentration), 0.5-2X MEM non-essential amino acids (100X stock concentration), 0.2-2X B-27 (50X stock concentration), 0.5-2X N-2 (100X stock concentration), 0.8-1.5X ITS-A (100X stock concentration), 50-300 ng / ml IL-6, 1-10 mmol / L sodium pyruvate, 50-300 ng / ml WNT-3A, 50-300 ng / ml R-Spondin 1, 2.5-10 mmol / L nicotinamide, 1-10 μM ROCK, 0.5-1.5X penicillin-streptomycin antibiotic solution, all dissolved in RPMI 1640 medium. X represents the concentration multiple.

[0007] In some embodiments, the lymphoma organoid culture medium comprises, in terms of final concentration: 0.5-2X GlutaMAX additive (100X stock concentration), 0.5-2X MEM non-essential amino acids (100X stock concentration), 0.2-2X B-27 (50X stock concentration), 0.5-2X N-2 (100X stock concentration), 0.8-1.5X ITS-A (100X stock concentration), 50-300 ng / ml IL-6, 1-10 mmol / L sodium pyruvate, 50-300 ng / ml WNT-3A, 50-300 ng / ml R-Spondin 1, 2.5-10 mmol / L nicotinamide, 1-10 μM ROCK, 1X penicillin-streptomycin antibiotic solution, all dissolved in RPMI 1640 medium.

[0008] In some embodiments, the final concentration of penicillin in the lymphoma organoid culture medium is 10-100 μg / ml.

[0009] In some embodiments, the final concentration of penicillin in the culture medium of the lymphoma organoid is 100 μg / ml.

[0010] In some embodiments, the final concentration of streptomycin in the culture medium of the lymphoma organoid is 10-100 U / ML.

[0011] In some embodiments, the final concentration of streptomycin in the culture medium of the lymphoma organoid is 100 U / ML.

[0012] In some embodiments, in the penicillin-streptomycin antibiotic solution, the concentration of penicillin is 40-60 μg / ml, and the concentration of streptomycin is 40-60 U / ml.

[0013] In some embodiments, in the penicillin-streptomycin antibiotic solution, the concentration of penicillin is 50 μg / ml, and the concentration of streptomycin is 50 U / ml.

[0014] In some embodiments, the MEM non-essential amino acids (MEM non-essential amino acid solution, mother liquor concentration is 100X) contains 7 non-essential amino acids of L-alanine, L-glutamic acid, L-asparagine, L-aspartic acid, L-proline, L-serine and glycine.

[0015] According to the second aspect of the present application, the present application provides a culture method of a lymphoma organoid, which uses the culture medium of the lymphoma organoid described above.

[0016] The culture method of the lymphoma organoid provided by the present application specifically comprises the following steps: The lymphoma tissue is pretreated, cut into pieces, and cell clusters are obtained. The culture medium of the lymphoma organoid is added, the cells are resuspended to obtain a cell suspension, mixed with Matrigel, plated, and cultured to obtain the lymphoma organoid.

[0017] In some embodiments, the pretreatment of the lymphoma tissue comprises the following steps: The lymphoma tissue is washed with antibiotic-containing normal saline to remove impurities; the antibiotic is penicillin and streptomycin; in the antibiotic-containing normal saline, the concentration of penicillin is 40-60 μg / ml, and the concentration of streptomycin is 40-60 U / ml.

[0018] In some embodiments, in the antibiotic-containing normal saline, the concentration of penicillin is 50 μg / ml, and the concentration of streptomycin is 50 U / ml.

[0019] In some embodiments, the cutting and obtaining cell clusters comprise: The fresh lymphoma tissue is carefully removed from the surface of the fat, connective tissue and necrotic part, trimmed into small pieces of 1-2mm3, filtered by a 220 mesh filter, and the filtrate is collected as the cell cluster suspension.

[0020] In some embodiments, after obtaining the filtrate (cell cluster suspension), the first centrifugation is performed at a centrifugal rate of 12000rpm for 5min, and the first precipitate is obtained. At this time, it is observed whether the first precipitate presents red color. If the first precipitate presents red color, it indicates that there are residual red blood cells. To further remove the red blood cells, the first precipitate can be mixed with red blood cell lysis solution (preferably 6ml) uniformly, lysed at room temperature for 3min, and then the lysis is terminated by adding the lymphoma organoid culture medium. Then the second centrifugation is performed at a centrifugal rate of 12000rpm for 5min, and the second precipitate is obtained. The second precipitate is suspended in the lymphoma organoid culture medium to obtain the cell suspension. If the first precipitate does not present red color, it indicates that there are no residual red blood cells. The first precipitate can be directly suspended in the lymphoma organoid culture medium to obtain the cell suspension.

[0021] In some embodiments, after obtaining the cell suspension, the cell viability can be detected by trypan blue staining method to ensure that the viable cell rate is ≥80%.

[0022] In some embodiments, after obtaining the cell suspension, cell counting is performed.

[0023] In some embodiments, the cell concentration of the cell suspension is 3-7.5 million cells per milliliter of cell suspension.

[0024] In some embodiments, the Matrigel is one of Matrigel, Accumatrix (without phenol red, low factor), Accumatrix Y102.

[0025] In some embodiments, the Matrigel is Matrigel. In some embodiments, the volume ratio of the cell suspension to the Matrigel is 5:1-20:1.

[0026] In some embodiments, the volume ratio of the cell suspension to the Matrigel is 6:1-10:1.

[0027] In some embodiments, the plating uses a 24-well ultra-low attachment flat-bottom culture plate. Preferably, the 24-well ultra-low attachment flat-bottom culture plate produced by Corning Company.

[0028] In some embodiments, the plating includes: adding the mixed solution of the cell suspension and the Matrigel into the ultra-low attachment 24-well plate, and horizontally shaking the ultra-low attachment 24-well plate to make the mixed solution uniformly distributed in the wells.

[0029] In some embodiments, the cell density is adjusted to 6x10 6 cells / ml before culture, and the final volume of each well is 500ul (i.e. 3x10 6 live cells per well).

[0030] In some embodiments, the culture temperature is 37℃, the culture atmosphere is sterile air containing 5% CO2, and the culture is performed by half-volume replacement every 24-48 hours (i.e. 50% new medium is added every 24-48 hours).

[0031] In some embodiments, the culture is performed by half-volume replacement every 36 hours (i.e. 50% new medium is added every 36 hours).

[0032] The culture medium provided by the application, in combination with the corresponding culture method, can efficiently and rapidly culture lymphoma cells derived from tumor tissues of patients into organoids.

[0033] According to a third aspect of the application, the application provides a lymphoma organoid obtained by the culture method described above.

[0034] According to a fourth aspect of the application, the application provides the use of a lymphoma organoid in screening drugs for preventing or treating lymphoma.

[0035] Compared with the prior art, the application has the following beneficial effects and advantages: (1) The culture method provided by the application can specifically culture human lymphoma tissues in vitro, and during the culture process, lymphoma organoids are formed, which are similar to tumors in vivo in terms of cell composition and spatial structure. (2) The culture medium provided by the application is the first lymphoma organoid culture medium disclosed, and the components of the culture medium are clear, which is suitable for the culture of lymphoma organoids of different types. The advantages of the culture medium include the removal of serum components and the influence of batch instability, the determination of the suspension culture method, and the provision of an advantageous culture method and research model for the in vitro research platform of the disease field. (3) At present, there are few reports on lymphoma organoid culture methods, and in vitro modeling faces a certain degree of cell survival and expansion bottleneck. The lymphoma organoid culture medium provided by the application is beneficial to the long-term stable organoid culture and expansion of related organoids in vitro, and can greatly retain the stemness of tumor cells. (4) The culture medium provided by the application, in combination with the corresponding culture method, can obtain a lymphoma organoid, which is simple to operate, has simple and clear components, has less influence on personnel operation, and has stable culture results. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 Figure for microscopic observation of the primary culture result of the lymphoma in Example 4, P0 generation 0 days; Figure 2 Figure for microscopic observation of the primary culture result of the lymphoma in Example 4, P0 generation 7 days; Figure 3 Figure for microscopic observation of the primary culture result of the lymphoma in Example 5, P0 generation 0 days; Figure 4 Figure for microscopic observation of the primary culture result of the lymphoma in Example 5, P0 generation 7 days; Figure 5 Figure for microscopic observation of the primary culture result of the lymphoma in Example 6, P0 generation 0 days; Figure 6 Figure for microscopic observation of the primary culture result of the lymphoma in Example 6, P0 generation 7 days; Figure 7 Figure for microscopic observation of the primary culture result of the lymphoma in Comparative Example 1, P0 generation 2 days; Figure 8 Figure for microscopic observation of the primary culture result of the lymphoma in Comparative Example 1, P0 generation 6 days; Figure 9 Figure for microscopic observation of the primary culture result of the lymphoma in Comparative Example 2, P0 generation 0 days; Figure 10 Figure for microscopic observation of the primary culture result of the lymphoma in Comparative Example 2, P0 generation 7 days; Figure 11 Figure for microscopic observation of the primary culture result of the lymphoma in Comparative Example 3, P0 generation 0 days; Figure 12 Figure for microscopic observation of the primary culture result of the lymphoma in Comparative Example 3, P0 generation 7 days. DETAILED DESCRIPTION

[0037] The application will be further described in conjunction with the examples below, but the embodiments of the application are not limited thereto. It should be noted that if there are processes or parameters not specifically described below, they can be understood or implemented by those skilled in the art with reference to the prior art.

[0038] The main reagents and consumables used in the following examples are shown in Table 1 below.

[0039] Table 1

[0040] Example 1 A medium for culturing a lymphoma organoid comprises, in terms of final concentration: 0.5X GlutaMAX supplement (100X), 0.5X MEM non-essential amino acids (100X), 0.2X B-27 serum-free supplement (50X), 0.5X N-2 serum-free supplement (100X), 0.8X Insulin- transferrin-selenium-sodium pyruvate (ITS-A) (100X), 50 ng / ml IL-6 (Recombinant Human IL-6), 1 mmol / L sodium pyruvate, 50 ng / ml WNT-3A (Recombinat Human Wnt-3a Protein), 50 ng / ml R-Spondin 1, 2.5 mmol / L Nicotinamide, IX Penicillin-Streptomycin antibiotic solution, 10 mM ROCK inhibitor (Y-27632 Dihydrochloride), all dissolved in RPMI 1640 medium.

[0041] Example 2 A medium for lymphoma organoids, consisting of, in final concentrations: 1X GlutaMAX supplement (100X), 1X MEM non-essential amino acids (100X), 1X B-27 serum-free supplement (50X), 1X N-2 serum-free supplement (100X), 1X Insulin- transferrin-selenium-sodium pyruvate (ITS-A) (100X), 100 ng / ml IL-6 (Recombinant Human IL-6), 2 mmol / L sodium pyruvate, 100 ng / ml WNT-3A (Recombinat Human Wnt-3a Protein), 100 ng / ml R-Spondin 1, 5 mmol / L Nicotinamide, IX Penicillin-Streptomycin antibiotic solution, 10 mM ROCK inhibitor (Y27632), all dissolved in RPMI 1640 medium.

[0042] Example 3 A medium for lymphoma organoids, consisting of, in final concentrations: 2X of GlutaMAX additive (100X), 2X of MEM non-essential amino acids (100X), 2X of B-27 serum-free additive (50X), 2X of N-2 serum-free additive (100X), 1.5X of insulin-transferrin-selenium-pyruvate sodium (ITS-A) (100X), 300 ng / ml of IL-6 (Recombinant Human IL-6), 10 mmol / L of sodium pyruvate, 300 ng / ml of WNT-3A (Recombinant Human Wnt-3a Protein), 300 ng / ml of R-Spondin 1, 10 mmol / L of nicotinamide, 1X of penicillin-streptomycin antibiotic solution, and 10 μM of ROCK inhibitor (Y27632) were all dissolved in RPMI 1640 medium.

[0043] Example 4 The culture medium used for lymphoma organoids in Example 4 was the culture medium prepared in Example 1.

[0044] A method for culturing lymphoma organoids, comprising the following steps: Fresh lymphoma tissue was carefully cleaned to remove surface fat, connective tissue, and necrotic tissue. The cleaned tissue was cut into 1-2 mm³ pieces and filtered through a 220-mesh sieve. The filtrate was collected as a cell suspension. The cell suspension was centrifuged at 1200 rpm for 5 minutes at room temperature. The first precipitate was collected and observed for a red color. Since the first precipitate was red, it was mixed thoroughly with 6 ml of erythrocyte lysis buffer and lysed at room temperature for 3 minutes. Lysis was then terminated by adding lymphoma organoid culture medium. A second centrifugation was performed at 1200 rpm for 5 minutes, and the second precipitate was collected. This precipitate was then resuspended in lymphoma organoid culture medium to obtain the first cell suspension. Cell viability was assessed using trypan blue staining to ensure a viable cell rate ≥80%. Cell counting was also performed for later use. The obtained first cell suspension was added to the culture medium for lymphoma organoids in Example 1, and the cells were resuspended to obtain a second cell suspension (containing 6 million cells per milliliter). This second cell suspension was mixed with Matrigel (Matrigel was selected) to obtain a mixture, wherein the volume ratio of the second cell suspension to Matrigel was 19:1. The mixture was then plated (the mixture was added to a 24-well ultra-low adsorption plate, and the plate was shaken horizontally to ensure that the second cell suspension was evenly distributed in the wells). The volume of the mixture added to each well was 500 μl (final cell density was...). Lymphoma organoids were cultured at 37°C in a sterile atmosphere containing 5% CO2, with half the medium replaced every 36 hours during the culture process. Figure 1 and Figure 2 As shown, Figure 1 The cell state at P0-0DAY is characterized by round and bright cell shape, indicating that the cells are in good condition. Figure 2 The cells are in the P0 generation, cultured for 7 days. They are in good viability, and some cells have formed spherical structures.

[0045] Example 5 The culture medium for lymphoma organoids used in Example 5 was the culture medium prepared in Example 2.

[0046] A method for culturing lymphoma organoids, comprising the following steps: Fresh lymphoma tissue was carefully cleaned to remove surface fat, connective tissue, and necrotic tissue. The cleaned tissue was cut into 1-2 mm³ pieces and filtered through a 220-mesh sieve. The filtrate was collected as a cell suspension. The cell suspension was centrifuged at 1200 rpm for 5 minutes at room temperature. The first precipitate was collected and observed for a red color. Since the first precipitate was red, it was mixed thoroughly with 6 ml of erythrocyte lysis buffer and lysed at room temperature for 3 minutes. Lysis was then terminated by adding lymphoma organoid culture medium. A second centrifugation was performed at 1200 rpm for 5 minutes, and the second precipitate was collected. This precipitate was then resuspended in lymphoma organoid culture medium to obtain the first cell suspension. The cell viability of the first cell suspension was assessed using trypan blue staining to ensure a viable cell rate ≥80%. Cell counting was also performed for later use. The obtained first cell suspension was added to the culture medium for lymphoma organoids in Example 2, and the cells were resuspended to obtain a second cell suspension (containing 6 million cells per milliliter). This second cell suspension was mixed with Matrigel (using Matrigel) to obtain a mixture with a volume ratio of 10:1. The mixture was then plated (the mixture was added to a 24-well ultra-low adsorption plate, and the plate was horizontally shaken at 80-120 rpm to evenly distribute the cell suspension in the wells) and cultured (at a temperature of 37°C in a sterile atmosphere containing 5% CO2). During the culture process, half of the medium was replaced every 36 hours (i.e., 50% of the culture medium was replaced every 36 hours) to obtain lymphoma organoids. The mixture was then mixed with Matrigel and seeded into 24-well ultra-low adsorption flat-bottom culture plates (Corning Corporation), and the final cell density was adjusted to [value missing]. The concentration of cells / ml was 500 μl per well (i.e., each well contained 100 cells / ml). (a number of live cells) were cultured, and the results were as follows: Figure 3 and Figure 4 As shown, Figure 3 The results are from primary culture at day 0. The cells are small, round, and bright, and exhibit good cell viability. Figure 4 The results of primary culture for 7 days show good cell viability and the formation of numerous spheroids with a diameter of 60-80 μm, indicating that lymphoma organoids have been formed.

[0047] Example 6 The culture medium for lymphoma organoids used in Example 6 was the culture medium prepared in Example 3.

[0048] A method for culturing lymphoma organoids, comprising the following steps: Fresh lymphoma tissue was carefully cleaned to remove surface fat, connective tissue, and necrotic tissue. The cleaned tissue was cut into 1-2 mm³ pieces and filtered through a 220-mesh sieve. The filtrate was collected as a cell suspension. The cell suspension was centrifuged at 1200 rpm for 5 minutes at room temperature. The first precipitate was collected and observed for a red color. Since the first precipitate was red, it was mixed thoroughly with 6 ml of erythrocyte lysis buffer and lysed at room temperature for 3 minutes. Lysis was then terminated by adding lymphoma organoid culture medium. A second centrifugation was performed at 1200 rpm for 5 minutes, and the second precipitate was collected. This precipitate was then resuspended in lymphoma organoid culture medium to obtain the first cell suspension. Cell viability was assessed using trypan blue staining to ensure a viable cell rate ≥80%. Cell counting was also performed for later use. The first cell suspension was added to the culture medium for lymphoma organoids from Example 3, and the cells were resuspended to obtain a second cell suspension (containing 6 million cells per milliliter). This second cell suspension was mixed with Matrigel (using Matrigel) to obtain a mixture with a cell suspension to Matrigel volume ratio of 19:1. The mixture was then plated (the mixture was added to a 24-well ultra-low adsorption plate, and the plate was horizontally agitated to ensure even distribution of the cell suspension in the wells). The volume of the mixture added to each well was 500 μl (final cell density was...). Lymphoma organoids were cultured at 37°C in a sterile atmosphere containing 5% CO2, with half the medium replaced every 36 hours during the culture process. Figure 5 and Figure 7 As shown, Figure 5 The results are from primary culture day 0, and the cells show good viability. Figure 6 The results of primary culture after 7 days show the formation of small spheres, but other cell morphologies have changed, mostly to spindle-shaped structures, which may be due to morphological changes in the cells themselves.

[0049] Comparative Example 1 A method for culturing lymphoma organoids, comprising the following steps: The culture medium used for lymphoma organoids in Comparative Example 1 consisted of RPMI 1640 medium supplemented with 10% fetal bovine serum. Lymphoma organoids were cultured using the culture medium from Comparative Example 1 according to the culture method described in Example 5. The culture results are as follows... Figure 7 and Figure 8 As shown: Figure 7 The results after 4 days of culture showed that most of the cells had vacuolated structures and the center of the cells was black, indicating that the cells had undergone apoptosis and lysis. Figure 8 After 6 days of primary culture, most cells died and the tumor stem cell activity could not be maintained, indicating that this culture medium is not suitable for lymphoma organoid culture.

[0050] Comparative Example 2 A method for culturing lymphoma organoids, comprising the following steps: The culture medium used for lymphoma organoids in Comparative Example 2 consisted of: 1X B-27 serum-free additive (50X), 2X N-2 serum-free additive (100X), 1X insulin-transferrin-selenium-pyruvate sodium (ITS-A) (100X), 100 ng / ml Noggin, 50 ng / ml EGF, 50 ng / ml FGFB, 100 ng / ml WNT-3A, 100 ng / ml R-Spondin 1, 10 mmol / L nicotinamide, 1X penicillin-streptomycin antibiotic solution, 10 μM ROCK inhibitor (Y-27632 Dihydrochloride), and 10 μM A8301. All of the above components were dissolved in RPMI 1640 medium. Lymphoma organoids were cultured using the culture medium from Comparative Example 2 according to the culture method described in Example 5. The culture results are as follows: Figure 9 and Figure 10 As shown: Figure 9 Results for primary culture after 0 days. Figure 10 Organoid formation is rare in 7-day culture results.

[0051] Comparative Example 3 A method for culturing lymphoma organoids, comprising the following steps: Comparative Example 3 used a commercially available T lymphocyte culture medium. Lymphoma organoids were cultured using the same culture medium as in Comparative Example 3, following the culture method described in Example 5. The results are as follows: Figure 11 and Figure 12 As shown; Figure 11 The cells were in good condition after 2 days of primary culture. Figure 12This is the result of 7 days of culture for P0 generation; small spheroids were formed, but the morphology of other cells changed. It is possible that the culture medium in Comparative Ratio 3 activated T lymphocytes and caused cell morphology changes. This culture medium is also not suitable for culturing lymphoma organoids.

[0052] The above embodiments are merely preferred embodiments of the present invention and are only used to explain the present invention, not to limit the present invention. Any changes, substitutions, modifications, etc., made by those skilled in the art without departing from the spirit and essence of the present invention should be within the protection scope of the present invention.

Claims

1. A culture medium for lymphoma organoids, characterized in that, Based on the final concentration, the composition includes: 0.5-2X GlutaMAX additive, 0.5-2X MEM non-essential amino acids, 0.2-2X B-27, 0.5-2X N-2, 0.8-1.5X ITS-A, 50-300 ng / ml IL-6, 1-10 mmol / L sodium pyruvate, 50-300 ng / ml WNT-3A, 50-300 ng / ml R-Spondin 1, 2.5-10 mmol / L nicotinamide, 1-10 μM ROCK, and 0.5-1.5X penicillin-streptomycin antibiotic solution were all dissolved in RPMI 1640 medium.

2. The culture medium for lymphoma organoids according to claim 1, characterized in that, In the penicillin-streptomycin antibiotic solution, the concentration of penicillin is 40-60 μg / ml, and the concentration of streptomycin is 40-60 U / ml.

3. A method for culturing lymphoma organoids, characterized in that, The culture medium for lymphoma organoids according to any one of claims 1-2.

4. The method for culturing lymphoma organoids according to claim 3, characterized in that, Includes the following steps: Lymphoma tissue was pretreated, minced, and cell clusters were obtained. The culture medium of the lymphoma organoids was added, the cells were resuspended, and the cell suspension was mixed with matrix gel, plated, and cultured to obtain the lymphoma organoids.

5. The method for culturing lymphoma organoids according to claim 4, characterized in that, The pretreatment of lymphoma tissue includes the following steps: The lymphoma tissue was washed with saline containing antibiotics to remove impurities; the antibiotics were penicillin and streptomycin; the concentration of penicillin in the saline containing antibiotics was 40-100 μg / ml and the concentration of streptomycin was 40-100 U / ml.

6. The method for culturing lymphoma organoids according to claim 4, characterized in that, The cell concentration of the cell suspension is 1-4 million cells per milliliter.

7. The method for culturing lymphoma organoids according to claim 4, characterized in that, The matrix gel is one of Matrigel, Accumatrix, and Y102; the volume ratio of the cell suspension to the matrix gel is 5:1-20:

1.

8. The method for culturing lymphoma organoids according to claim 4, characterized in that, The culture temperature was 37°C, the culture atmosphere contained 5% CO2, and half of the medium was replaced every 24-48 hours during the culture process.

9. A lymphoma organoid obtained by the culture method according to any one of claims 3-8.

10. The use of the lymphoma organoids of claim 9 in screening drugs for the prevention or treatment of lymphoma.

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