Prostate cancer organoid culture medium and preparation method thereof, prostate cancer organoid and culture method thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a prostate cancer organoid culture medium, a preparation method thereof, a prostate cancer organoid and a culture method thereof. The prostate cancer organ culture medium provided by the invention comprises a basic culture medium and a culture additive, and the culture additive comprises FGF-10 (Fibroblast Growth Factor), Zeaxanthin dipalmitate and Polydatin. In the prostate cancer organoid culture medium provided by the invention, the success rate of organoid culture is greatly improved by utilizing the synergistic effect of the FGF-10, the Zeaxanthin dipalmitate and the Polydatin, the cultured prostate cancer organoid is high in growth speed, the passage frequency is remarkably improved, in-vitro culture can be specifically carried out on prostate cancer tissues of people, and the prostate cancer organoid culture medium has the advantages that the prostate cancer tissue culture efficiency is improved; the survival rate and the formation efficiency of the prostatic cancer organs are further improved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a prostate cancer organoid culture medium and a preparation method thereof, a prostate cancer organoid and a culture method thereof. Background Art

[0002] Prostate cancer (PCa) is a malignant tumor caused by the malignant proliferation of prostate epithelial cells. It is one of the most common malignant tumors, with the morbidity and mortality ranking second and fifth among male malignant tumors worldwide, respectively. In clinical diagnosis, the malignancy of prostate cancer is closely related to the patient's treatment effect. Since early prostate cancer often has no obvious symptoms, most clinically diagnosed prostate cancer is in the late stage, resulting in poor treatment effect and prognosis of prostate cancer.

[0003] Tumor organoids are cell cultures derived from patient tumor tissue that can be cultured in three dimensions (3D) in vitro. Tumor organoids preserve the heterogeneity, tissue characteristics, and gene mutation information of tumors. They can simulate the process of cancer occurrence and development in vitro, build disease models, and conduct rapid and accurate cancer drug screening, effectively supplementing the deficiencies of existing animal and two-dimensional (2D) cell culture models, and providing a new technical platform for the rapid and effective development of tumor drugs.

[0004] Zhang et al. (Zhang ZD, Karthaus WR, Lee YS, et al. Tumor microenvironment-derived NRG1 promotes antiandrogen resistance in prostate cancer [J]. Cancer Cell, 2020, 38 (2): 279-96.) demonstrated that fibroblasts near prostate tumor cells can activate the HER3 signaling pathway of tumor cells by secreting NRG1, thereby affecting the cells' response to androgens.

[0005] Chinese invention patent publication number CN116286654A discloses a prostate cancer organoid, culture medium and culture method. The prostate cancer organoid culture medium includes a basal culture medium, basal cytokines, specific additive factors, inhibitors and antibiotics; the basal cytokines include the following components at final concentrations: EGF, 10-100 ng / mL; Noggin, 20-500 ng / mL; R-spondin 1, 20-500 ng / mL; Wnt3a, 20-500 ng / mL; FGF10, 1-50 ng / mL; FGF2, 1-50 ng / mL; the specific additive factors include the following components at final concentrations: Protodioscin, 0.1-50 nM; Dehydroepiandrosterone acetate, 1-10 μM; SCF, 0.5-50 ng / mL. The prostate cancer organoid culture medium provided by the invention can specifically culture human prostate cancer tissue in vitro. The various components in the culture medium act synergistically, and can maintain the same tissue structure and genomic characteristics of the primary tissue after multiple passaging. However, its culture process grows slowly, so it requires increased time investment costs and is not suitable as a clinical drug screening model.

[0006] Another Chinese invention patent publication number CN114958756A discloses a culture medium for culturing prostate cancer organoids and a preparation method thereof. The components of the culture medium include DMEM / F12 culture medium, penicillin-streptomycin, B27, DHT, Prostaglandin E2, FGF2, A8301, R-spondin, Noggin and a functional nucleic acid-based HGF mimetic. The invention uses the above-mentioned culture medium for culturing prostate cancer organoids and a preparation method thereof to increase the rate of organoid formation, but the cultured prostate cancer organoids are small in size and have a long culture cycle.

[0007] In view of this, there is an urgent need in the art to provide a prostate cancer organoid culture medium that improves the survival rate and formation efficiency of prostate cancer organoids. Summary of the invention

[0008] In view of the problems existing in the prior art, the present invention provides a prostate cancer organoid culture medium and a preparation method thereof, a prostate cancer organoid and a culture method thereof.

[0009] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0010] A prostate cancer organoid culture medium comprises a basal culture medium and a culture additive, wherein the culture additive comprises FGF-10, Zeaxanthin dipalmitate and Polydatin.

[0011] Preferably, the culture additives further include IGF-1, R-spondin 1, Noggin, EGF, GlutaminePlus, A83-01, Y-27632, N-Acetylcysteine, Primocin, Nicotinamide and SB202190.

[0012] Preferably, the culture supplement includes the following components at final concentrations: FGF-10 10-40 ng / mL, Zeaxanthin dipalmitate 2-3 mM, Polydatin 200-400 μg / mL, 1-2% penicillin-streptomycin, IGF-1 1-10 ng / mL, R-spondin 1 100-300 ng / mL, Noggin 100-300 ng / mL, EGF 60-90 ng / mL, GlutaminePlus 5-20 ng / mL, A83-01 1-2 μM, Y-27632 10-20 μM, N-Acetylcysteine ​​2-8 μM, Primocin 1-2 μM, Nicotinamide 10-20 μM and SB 202190 200-1000 nM.

[0013] More preferably, the culture supplement includes the following components at final concentrations: FGF-10 20-35 ng / mL, Zeaxanthin dipalmitate 2.5-3 mM, Polydatin 250-350 μg / mL, 1% penicillin-streptomycin, IGF-1 5-8 ng / mL, R-spondin1 150-250 ng / mL, Noggin 150-200 ng / mL, EGF 65-80 ng / mL, GlutaminePlus 10-15 ng / mL, A83-01 1.5-2 μM, Y-27632 10-15 μM, N-Acetylcysteine ​​3-6 μM, Primocin 1.5-2 μM, Nicotinamide 15-20 μM and SB 202190 300-600 nM.

[0014] Preferably, the basic culture medium comprises Advanced DMEM / F12, N21-MAX Media Supplement 50× and 1% HEPES buffer.

[0015] The present invention also provides a method for preparing the above-mentioned prostate cancer organoid culture medium, comprising the following steps: preparing a culture additive into a mother solution with sterile water, and then adding the mother solution into a basal culture medium to obtain the mother solution.

[0016] The present invention also provides a method for culturing prostate cancer organoids, comprising the following steps:

[0017] (1) Pre-treat fresh surgical specimens to obtain cell clusters, centrifuge, and obtain cell cluster sediments;

[0018] (2) Then the cell pellet was precipitated and Basement membrane extracts were mixed to obtain a gel mixed with cells;

[0019] (3) Finally, the prostate cancer organoid culture medium is added to the gel and cultured together to obtain the result.

[0020] Preferably, the pretreatment in step (1) comprises the following steps: firstly, the surgical resection specimen is rinsed 3-5 times with sterile PBS buffer, cut into 1-3 mm 3 Small pieces; then add TrypLE TM Express enzyme, digest at 32-37°C, 40-80rpm for 30-60min; finally filter through a 100-120 mesh filter to obtain a cell pellet.

[0021] Preferably, the centrifugation temperature in step (1) is 4-10°C, the centrifugation speed is 1000-2000 rpm, the centrifugation time is 5-10 min, and the number of centrifugation is 3-5 times.

[0022] Preferably, in step (2) The temperature of the basement membrane extract is 4-10°C, the temperature of the mixture is 23-27°C, and the cell pellet is The volume ratio of basement membrane extract is 1:1-2.

[0023] Preferably, the co-culture conditions in step (3) are at 37°C, 5% CO 2 The cells were cultured in a concentration incubator for 4-7 days.

[0024] The present invention also provides prostate cancer organoids cultured by the above culture method.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) The prostate cancer organoid culture medium provided by the present invention utilizes the synergistic effect of FGF-10, Zeaxanthindipalmitate and Polydatin to greatly improve the success rate of organoid culture. The cultured prostate cancer organoids grow rapidly, the number of passages is significantly increased, and the same tissue structure and genomic characteristics of the primary tissue can be maintained after multiple passages.

[0027] (2) The prostate cancer organoid culture medium provided by the present invention can specifically culture human prostate cancer tissue in vitro, further improving the survival rate and formation efficiency of prostate cancer organoids. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is an optical microscopic image of the prostate cancer organoids on day 3 of culture in Example 1.

[0029] Figure 2 This is an optical microscopic image of the prostate cancer organoids on day 7 of culture in Example 1.

[0030] Figure 3 This is an optical microscopic image of the prostate cancer organoids on day 3 of culture in Example 2.

[0031] Figure 4 This is an optical microscopic image of the prostate cancer organoids on day 7 of culture in Example 2.

[0032] Figure 5 This is an optical microscopic image of the prostate cancer organoids of Comparative Example 1 on the third day of culture.

[0033] Figure 6 This is an optical microscopic image of the prostate cancer organoids of Comparative Example 2 on the third day of culture.

[0034] Figure 7 This is an optical microscopic image of the prostate cancer organoids of Comparative Example 3 on the third day of culture.

[0035] Figure 8 This is an optical microscopic image of the prostate cancer organoids of Comparative Example 4 on the third day of culture.

[0036] Fig. 9 This is an optical microscopic image of the prostate cancer organoids of Comparative Example 5 on the third day of culture.

[0037] Fig.10 This is an optical microscopic image of the prostate cancer organoids of Comparative Example 6 on the third day of culture.

[0038] Fig.11 This is an optical microscopic image of the prostate cancer organoids of Comparative Example 7 on the third day of culture. DETAILED DESCRIPTION

[0039] It is worth noting that the raw materials used in the present invention are all common commercially available products, among which HEPES buffer, Basement membrane extract, FGF-10, R-spondin 1, Noggin, EGF, GlutaminePlus, and N21-MAX MediaSupplement were all purchased from R&Dsystems; A83-01, Y-27632, Nicotinamide, and SB 202190 were all purchased from Tocris bioscience; IGF-1 and N-Acetylcysteine ​​were all purchased from Sigma; Primocin was purchased from InvivoGen; Zeaxanthin dipalmitate and Polydatin were all purchased from MedChemExpress; Penicillin-streptomycin solution, TrypLE TM Express enzyme and Advanced DMEM / F12 were purchased from Thermo Fisher Scientific.

[0040] The steps for prostate cancer organoid morphology identification are as follows:

[0041] (1) Take an appropriate amount of prostate cancer organoids after passage and place them in a 15 mL centrifuge tube. After standing, remove the culture medium by negative pressure aspiration. Add 5 mL of DPBS buffer for washing. Centrifuge at 300 g for 3 min and then remove the DPBS. Add 5 mL of 4% PFA solution to the centrifuge tube and place it on a shaker at room temperature for 30 min.

[0042] (2) After fixation, centrifuge at 1200 rpm for 5 minutes and discard the fixative. Dehydrate and embed in the following order: 85% alcohol for 0.5 hours - 95% alcohol for 0.5 hours - 95% alcohol for 0.5 hours - anhydrous ethanol for 0.5 hours - xylene for 20 minutes - paraffin for 1.5 hours.

[0043] (3) Wrap the prostate cancer organoids with an embedding mold, and then cut them into 4-6μm slices with a slicer and stick them on a slide. Place the slide on a slide rack and put it in an oven at 65°C for 30 minutes to dry the moisture on the slide and melt the paraffin. Dewax with xylene 3 times, 10 minutes each time; then soak in 100% alcohol 3 times, 1 minute each time; finally soak in running water for 1 minute.

[0044] (4) Place the dewaxed and rehydrated sections horizontally in a wet box and stain them in the following order: first stain with hematoxylin for 5 min—rinse with tap water for 5 min—1% hydrochloric acid ethanol for 1 s—reverse blueing with tap water for 10 min—eosin for 5 min—95% alcohol for 5 min.

[0045] (5) Then immerse in 95% alcohol and 100% alcohol, twice for each reagent, 2 minutes each time. Use xylene to make it transparent for 2 minutes, take it out and dry it, then seal it with neutral gum. Observe the tissue morphology and structure under an ordinary optical microscope.

[0046] Example 1

[0047] A prostate cancer organoid culture medium, comprising a basal culture medium and a culture supplement, wherein the basal culture medium comprises Advanced DMEM / F12, N21-MAX Media Supplement 50× and 1% HEPES buffer; the culture supplement is in a mixture of the basal culture medium and sterile water, and comprises the following components at final concentrations: FGF-10 30 ng / mL, Zeaxanthin dipalmitate 3 mM, Polydatin 300 μg / mL, 1% penicillin-streptomycin, IGF-1 6 ng / mL, R-spondin1 200 ng / mL, Noggin 200 ng / mL, EGF 70 ng / mL, GlutaminePlus 12 ng / mL, A83-01 1.8 μM, Y-27632 12 μM, N-Acetylcysteine ​​5 μM, Primocin 1.6 μM, Nicotinamide 18 μM and SB202190 500nM. The preparation method is as follows: add the culture additive to the basic culture medium and mix evenly.

[0048] Prostate cancer organoids were cultured using the above medium as follows:

[0049] (1) Take a fresh tumor tissue sample from a patient who has undergone surgery and wash it four times with sterile PBS buffer for 5 minutes each time. Then cut the washed tissue into approximately 1 mm thick pieces with a scalpel. 3 Transfer the tissue of different size to a 15 mL centrifuge tube and add appropriate amount of TrypLE TM Express enzyme, placed in a 37℃ water bath shaker, speed of 60rpm, time for 40min, gently shake every 5min, after digestion, the digestion status can be observed under a microscope. After stopping digestion, blow the sample evenly, filter with a 100-mesh filter to obtain a cell mass suspension, and finally place it in a pre-cooled 4℃ centrifuge at 1500rpm for 10min, remove the supernatant, repeat 3 times, and obtain the cell mass precipitate;

[0050] (2) Then the cell pellet was mixed with 4°C The basement membrane extracts were mixed at a volume ratio of 1:1.5 at 25°C to obtain a gel mixed with cells; finally, the above-mentioned prostate cancer organoid culture medium was added to the gel and incubated at 37°C and 5% CO2 The cells were cultured in a constant temperature incubator for 7 days, with the culture medium replaced every 3 days.

[0051] Optical microscopy images of prostate cancer organoids on days 3 and 7 of culture are shown below. Figure 1 and Figure 2 shown.

[0052] Example 2

[0053] A prostate cancer organoid culture medium, comprising a basal culture medium and a culture supplement, wherein the basal culture medium comprises Advanced DMEM / F12, N21-MAX Media Supplement 50× and 1% HEPES buffer; the culture supplement is in a mixture of the basal culture medium and sterile water, and comprises the following components at final concentrations: FGF-10 20 ng / mL, Zeaxanthin dipalmitate 2.5 mM, Polydatin 250 μg / mL, 1% penicillin-streptomycin, IGF-1 5 ng / mL, R-spondin 1 150 ng / mL, Noggin 150 ng / mL, EGF 65 ng / mL, GlutaminePlus 10 ng / mL, A83-01 1.5 μM, Y-27632 10 μM, N-Acetylcysteine ​​3 μM, Primocin 1.5 μM, Nicotinamide 15 μM and SB 202190 300nM. The preparation method is as follows: add the culture additive into the basic culture medium and mix evenly.

[0054] Prostate cancer organoids were cultured using the above medium as follows:

[0055] (1) Take a fresh tumor tissue sample from a patient who has undergone surgery and wash it four times with sterile PBS buffer for 5 minutes each time. Then cut the washed tissue into approximately 1 mm thick pieces with a scalpel. 3 Transfer the tissue of different size to a 15 mL centrifuge tube and add appropriate amount of TrypLE TM Express enzyme, placed in a 37℃ water bath shaker, speed of 60rpm, time of 60min, gently shake every 5min, after digestion, the digestion status can be observed under a microscope. After stopping digestion, blow the sample evenly, filter with a 1020-mesh filter to obtain a cell mass suspension, and finally place it in a pre-cooled 4℃ centrifuge at 1800rpm for 10min, remove the supernatant, repeat 5 times, and obtain a cell mass precipitate;

[0056] (2) Then the cell pellet was mixed with 4°C The basement membrane extracts were mixed at a volume ratio of 1:1.6 at 27°C to obtain a gel mixed with cells; finally, the above-mentioned prostate cancer organoid culture medium was added to the gel and incubated at 37°C and 5% CO 2 The cells were cultured in a constant temperature incubator for 7 days, with the culture medium replaced every 3 days.

[0057] Optical microscopy images of prostate cancer organoids on days 3 and 7 of culture are shown below. Figure 3 and Figure 4 shown.

[0058] Example 3

[0059] A prostate cancer organoid culture medium, comprising a basal culture medium and a culture supplement, wherein the basal culture medium comprises Advanced DMEM / F12, N21-MAX Media Supplement 50× and 1% HEPES buffer; the culture supplement is in a mixture of the basal culture medium and sterile water, and comprises the following components at final concentrations: FGF-10 40 ng / mL, Zeaxanthin dipalmitate 2 mM, Polydatin 400 μg / mL, 1% penicillin-streptomycin, IGF-1 10 ng / mL, R-spondin 1 300 ng / mL, Noggin 300 ng / mL, EGF 90 ng / mL, GlutaminePlus 20 ng / mL, A83-012 μM, Y-27632 20 μM, N-Acetylcysteine ​​8 μM, Primocin 2 μM, Nicotinamide 20 μM and SB 2021901000nM. The preparation method is as follows: adding the culture additive into the basic culture medium and mixing evenly.

[0060] Prostate cancer organoids were cultured using the above medium as follows:

[0061] (1) Take a fresh tumor tissue sample from a patient who has undergone surgery and wash it five times with sterile PBS buffer, each time for 5 minutes. Then cut the washed tissue into approximately 1 mm thick pieces with a scalpel. 3 Transfer the tissue of different size to a 15 mL centrifuge tube and add appropriate amount of TrypLE TM Express enzyme, placed in a 37℃ water bath shaker, speed of 80rpm, time for 30min, gently shake every 5min, after digestion, the digestion status can be observed under a microscope. After stopping digestion, blow the sample evenly, filter with a 100-mesh filter to obtain a cell mass suspension, and finally place it in a pre-cooled 4℃ centrifuge at 2000rpm for 5min, remove the supernatant, repeat 3 times, and obtain the cell mass precipitate;

[0062] (2) Then the cell pellet was mixed with 4°C The basement membrane extracts were mixed at a volume ratio of 1:2 at 25°C to obtain a gel mixed with cells; finally, the above-mentioned prostate cancer organoid culture medium was added to the gel and incubated at 37°C and 5% CO 2 The cells were cultured in a constant temperature incubator for 7 days, with the culture medium replaced every 3 days.

[0063] Example 4

[0064] A prostate cancer organoid culture medium consists of a basal culture medium and a culture supplement, wherein the basal culture medium consists of Advanced DMEM / F12, N21-MAX Media Supplement 50× and 1% HEPES buffer; the culture supplement consists of the following components at final concentrations in a mixture of the basal culture medium and sterile water: FGF-10 10 ng / mL, Zeaxanthin dipalmitate 2 mM, Polydatin 200 μg / mL, 2% penicillin-streptomycin, IGF-1 1 ng / mL, R-spondin1 100 ng / mL, Noggin 100 ng / mL, EGF 60 ng / mL, GlutaminePlus 5 ng / mL, A83-01 1 μM, Y-27632 10 μM, N-Acetylcysteine ​​2 μM, Primocin 1 μM, Nicotinamide 10 μM and SB 202190200 nM. The preparation method is as follows: adding the culture additive into the basic culture medium and mixing evenly to obtain the culture additive.

[0065] Prostate cancer organoids were cultured using the above medium as follows:

[0066] (1) Take a fresh tumor tissue sample from a patient who has undergone surgery and wash it three times with sterile PBS buffer, each time for 5 minutes. Then cut the washed tissue into approximately 1 mm thick pieces with a scalpel. 3 Transfer the tissue of different size to a 15 mL centrifuge tube and add appropriate amount of TrypLE TM Express enzyme, placed in a 32℃ water bath shaker, speed of 40rpm, time of 60min, gently shake every 5min, after digestion, the digestion status can be observed under a microscope. After stopping digestion, blow the sample evenly, filter with a 100-mesh filter to obtain a cell mass suspension, and finally place it in a pre-cooled 4℃ centrifuge at 1000rpm for 10min, remove the supernatant, repeat 5 times, and obtain the cell mass precipitate;

[0067] (2) Then the cell pellet was mixed with 4°C The basement membrane extracts were mixed at a volume ratio of 1:1 at 25°C to obtain a gel mixed with cells; finally, the above-mentioned prostate cancer organoid culture medium was added to the gel and incubated at 37°C and 5% CO 2 The cells were cultured in a constant temperature incubator for 7 days, with the culture medium replaced every 3 days.

[0068] Comparative Example 1

[0069] The same as Example 1, the only difference is that the Advanced DMEM / F12 medium is replaced by a high-glucose DMEM medium.

[0070] A prostate cancer organoid culture medium, comprising a basal culture medium and a culture supplement, wherein the basal culture medium comprises a high-glucose DMEM culture medium, N21-MAX Media Supplement 50× and a 1% HEPES buffer; the culture supplement is in a mixture of the basal culture medium and sterile water and comprises the following components at final concentrations: FGF-10 30 ng / mL, Zeaxanthindipalmitate 3 mM, Polydatin 300 μg / mL, 1% penicillin-streptomycin, IGF-1 6 ng / mL, R-spondin1 200 ng / mL, Noggin 200 ng / mL, EGF 70 ng / mL, GlutaminePlus 12 ng / mL, A83-01 1.8 μM, Y-27632 12 μM, N-Acetylcysteine ​​5 μM, Primocin 1.6 μM, Nicotinamide 18 μM and SB 202190 500nM. The preparation method is as follows: add the culture additive to the basic culture medium and mix evenly.

[0071] Prostate cancer organoids were cultured using the above medium as follows:

[0072] (1) Take a fresh tumor tissue sample from a patient who has undergone surgery and wash it four times with sterile PBS buffer for 5 minutes each time. Then cut the washed tissue into approximately 1 mm thick pieces with a scalpel. 3 Transfer the tissue of different size to a 15 mL centrifuge tube and add appropriate amount of TrypLE TM Express enzyme, placed in a 37℃ water bath shaker, speed of 60rpm, time for 40min, gently shake every 5min, after digestion, the digestion status can be observed under a microscope. After stopping digestion, blow the sample evenly, filter with a 100-mesh filter to obtain a cell mass suspension, and finally place it in a pre-cooled 4℃ centrifuge at 1500rpm for 10min, remove the supernatant, repeat 3 times, and obtain the cell mass precipitate;

[0073] (2) Then the cell pellet was mixed with 4°C The basement membrane extracts were mixed at a volume ratio of 1:1.5 at 25°C to obtain a gel mixed with cells; finally, the above-mentioned prostate cancer organoid culture medium was added to the gel and incubated at 37°C and 5% CO 2 The cells were cultured in a constant temperature incubator for 7 days, with the culture medium replaced every 3 days.

[0074] Optical microscopy of prostate cancer organoids on day 3 of culture Figure 5 shown.

[0075] Comparative Example 2

[0076] The same as Example 1, except that the culture supplement does not contain FGF-10.

[0077] A prostate cancer organoid culture medium consists of a basal culture medium and a culture additive, wherein the basal culture medium consists of Advanced DMEM / F12, N21-MAX Media Supplement 50× and 1% HEPES buffer; the culture additive consists of the following components with final concentrations in a mixture of the basal culture medium and sterile water: Zeaxanthindipalmitate 3mM, Polydatin 300μg / mL, 1% penicillin-streptomycin, IGF-1 6ng / mL, R-spondin 1200ng / mL, Noggin 200ng / mL, EGF 70ng / mL, GlutaminePlus 12ng / mL, A83-01 1.8μM, Y-27632 12μM, N-Acetylcysteine ​​5μM, Primocin 1.6μM, Nicotinamide 18μM and SB202190500nM. The preparation method is as follows: adding the culture additive into the basic culture medium and mixing evenly to obtain the culture additive.

[0078] Prostate cancer organoids were cultured using the above medium as follows:

[0079] (1) Take a fresh tumor tissue sample from a patient who has undergone surgery and wash it four times with sterile PBS buffer for 5 minutes each time. Then cut the washed tissue into approximately 1 mm thick pieces with a scalpel. 3 Transfer the tissue of different size to a 15 mL centrifuge tube and add appropriate amount of TrypLE TMExpress enzyme, placed in a 37℃ water bath shaker, speed of 60rpm, time for 40min, gently shake every 5min, after digestion, the digestion status can be observed under a microscope. After stopping digestion, blow the sample evenly, filter with a 100-mesh filter to obtain a cell mass suspension, and finally place it in a pre-cooled 4℃ centrifuge at 1500rpm for 10min, remove the supernatant, repeat 3 times, and obtain the cell mass precipitate;

[0080] (2) Then the cell pellet was mixed with 4°C The basement membrane extracts were mixed at a volume ratio of 1:1.5 at 25°C to obtain a gel mixed with cells; finally, the above-mentioned prostate cancer organoid culture medium was added to the gel and incubated at 37°C and 5% CO 2 The cells were cultured in a constant temperature incubator for 7 days, with the culture medium replaced every 3 days.

[0081] Optical microscopy of prostate cancer organoids on day 3 of culture Figure 6 shown.

[0082] Comparative Example 3

[0083] The same as Example 1, except that the culture supplement does not contain Polydatin and Zeaxanthindipalmitate.

[0084] A prostate cancer organoid culture medium is composed of a basal culture medium and a culture additive, wherein the basal culture medium is composed of Advanced DMEM / F12, N21-MAX Media Supplement 50× and 1% HEPES buffer; the culture additive is composed of the following components at final concentrations in a mixture of the basal culture medium and sterile water: FGF-10 30ng / mL, 1% penicillin-streptomycin, IGF-1 6ng / mL, R-spondin 1 200ng / mL, Noggin 200ng / mL, EGF 70ng / mL, GlutaminePlus 12ng / mL, A83-01 1.8μM, Y-27632 12μM, N-Acetylcysteine ​​5μM, Primocin1.6μM, Nicotinamide 18μM and SB 202190 500nM. The preparation method is as follows: adding the culture additive to the basal culture medium and mixing evenly to obtain the culture additive.

[0085] Prostate cancer organoids were cultured using the above medium as follows:

[0086] (1) Take a fresh tumor tissue sample from a patient who has undergone surgery and wash it four times with sterile PBS buffer for 5 minutes each time. Then cut the washed tissue into approximately 1 mm thick pieces with a scalpel.3 Transfer the tissue of different size to a 15 mL centrifuge tube and add appropriate amount of TrypLE TM Express enzyme, placed in a 37℃ water bath shaker, speed of 60rpm, time for 40min, gently shake every 5min, after digestion, the digestion status can be observed under a microscope. After stopping digestion, blow the sample evenly, filter with a 100-mesh filter to obtain a cell mass suspension, and finally place it in a pre-cooled 4℃ centrifuge at 1500rpm for 10min, remove the supernatant, repeat 3 times, and obtain the cell mass precipitate;

[0087] (2) Then the cell pellet was mixed with 4°C The basement membrane extracts were mixed at a volume ratio of 1:1.5 at 25°C to obtain a gel mixed with cells; finally, the above-mentioned prostate cancer organoid culture medium was added to the gel and incubated at 37°C and 5% CO 2 The cells were cultured in a constant temperature incubator for 7 days, with the culture medium replaced every 3 days.

[0088] Optical microscopy of prostate cancer organoids on day 3 of culture Figure 7 shown.

[0089] Comparative Example 4

[0090] The same as Example 1, the only difference is that the culture supplement does not contain Polydatin.

[0091] A prostate cancer organoid culture medium consists of a basal culture medium and a culture supplement, wherein the basal culture medium consists of Advanced DMEM / F12, N21-MAX Media Supplement 50× and 1% HEPES buffer; the culture supplement consists of the following components at the following final concentrations in a mixture of the basal culture medium and sterile water: FGF-10 30 ng / mL, Zeaxanthin dipalmitate 3 mM, 1% penicillin-streptomycin, IGF-1 6 ng / mL, R-spondin 1 200 ng / mL, Noggin 200 ng / mL, EGF 70 ng / mL, Glutamine Plus 12 ng / mL, A83-01 1.8 μM, Y-27632 12 μM, N-Acetylcysteine ​​5 μM, Primocin 1.6 μM, Nicotinamide 18 μM and SB 202190 500 nM. The preparation method is as follows: adding the culture additive into the basic culture medium and mixing evenly to obtain the culture additive.

[0092] Prostate cancer organoids were cultured using the above medium as follows:

[0093] (1) Take a fresh tumor tissue sample from a patient who has undergone surgery and wash it four times with sterile PBS buffer for 5 minutes each time. Then cut the washed tissue into approximately 1 mm thick pieces with a scalpel. 3 Transfer the tissue of different size to a 15 mL centrifuge tube and add appropriate amount of TrypLE TM Express enzyme, placed in a 37℃ water bath shaker, speed of 60rpm, time for 40min, gently shake every 5min, after digestion, the digestion status can be observed under a microscope. After stopping digestion, blow the sample evenly, filter with a 100-mesh filter to obtain a cell mass suspension, and finally place it in a pre-cooled 4℃ centrifuge at 1500rpm for 10min, remove the supernatant, repeat 3 times, and obtain the cell mass precipitate;

[0094] (2) Then the cell pellet was mixed with 4°C The basement membrane extracts were mixed at a volume ratio of 1:1.5 at 25°C to obtain a gel mixed with cells; finally, the above-mentioned prostate cancer organoid culture medium was added to the gel and incubated at 37°C and 5% CO 2 The cells were cultured in a constant temperature incubator for 7 days, with the culture medium replaced every 3 days.

[0095] Optical microscopy of prostate cancer organoids on day 3 of culture Figure 8 shown.

[0096] Comparative Example 5

[0097] The same as Example 1, except that the culture supplement does not contain Zeaxanthin dipalmitate.

[0098] A prostate cancer organoid culture medium consists of a basal culture medium and a culture additive, wherein the basal culture medium consists of Advanced DMEM / F12, N21-MAX Media Supplement 50× and 1% HEPES buffer; the culture additive consists of the following components at the following final concentrations in a mixture of the basal culture medium and sterile water: FGF-10 30 ng / mL, Polydatin 300 μg / mL, 1% penicillin-streptomycin, IGF-1 6 ng / mL, R-spondin 1 200 ng / mL, Noggin 200 ng / mL, EGF 70 ng / mL, Glutamine Plus 12 ng / mL, A83-01 1.8 μM, Y-27632 12 μM, N-Acetylcysteine ​​5 μM, Primocin 1.6 μM, Nicotinamide 18 μM and SB 202190 500 nM. The preparation method is as follows: adding the culture additive into the basic culture medium and mixing evenly to obtain the culture additive.

[0099] Prostate cancer organoids were cultured using the above medium as follows:

[0100] (1) Take a fresh tumor tissue sample from a patient who has undergone surgery and wash it four times with sterile PBS buffer for 5 minutes each time. Then cut the washed tissue into approximately 1 mm thick pieces with a scalpel. 3 Transfer the tissue of different size to a 15 mL centrifuge tube and add appropriate amount of TrypLE TM Express enzyme, placed in a 37℃ water bath shaker, speed of 60rpm, time for 40min, gently shake every 5min, after digestion, the digestion status can be observed under a microscope. After stopping digestion, blow the sample evenly, filter with a 100-mesh filter to obtain a cell mass suspension, and finally place it in a pre-cooled 4℃ centrifuge at 1500rpm for 10min, remove the supernatant, repeat 3 times, and obtain the cell mass precipitate;

[0101] (2) Then the cell pellet was mixed with 4°C The basement membrane extracts were mixed at a volume ratio of 1:1.5 at 25°C to obtain a gel mixed with cells; finally, the above-mentioned prostate cancer organoid culture medium was added to the gel and incubated at 37°C and 5% CO 2 The cells were cultured in a constant temperature incubator for 7 days, with the culture medium replaced every 3 days.

[0102] Optical microscopy of prostate cancer organoids on day 3 of culture Fig. 9 shown.

[0103] Comparative Example 6

[0104] The same as Example 1, except that the final concentration of R-spondin 1 is 500 ng / mL.

[0105] A prostate cancer organoid culture medium, comprising a basal culture medium and a culture supplement, wherein the basal culture medium comprises Advanced DMEM / F12, N21-MAX Media Supplement 50× and 1% HEPES buffer; the culture supplement is in a mixture of the basal culture medium and sterile water, and comprises the following components at the following final concentrations: FGF-10 30 ng / mL, Zeaxanthin dipalmitate 3 mM, Polydatin 300 μg / mL, 1% penicillin-streptomycin, IGF-1 6 ng / mL, R-spondin1 500 ng / mL, Noggin 200 ng / mL, EGF 70 ng / mL, GlutaminePlus 12 ng / mL, A83-01 1.8 μM, Y-27632 12 μM, N-Acetylcysteine ​​5 μM, Primocin 1.6 μM, Nicotinamide 18 μM and SB202190 500nM. The preparation method is as follows: add the culture additive to the basic culture medium and mix evenly.

[0106] Prostate cancer organoids were cultured using the above medium as follows:

[0107] (1) Take a fresh tumor tissue sample from a patient who has undergone surgery and wash it four times with sterile PBS buffer for 5 minutes each time. Then cut the washed tissue into approximately 1 mm thick pieces with a scalpel. 3 Transfer the tissue of different size to a 15 mL centrifuge tube and add appropriate amount of TrypLE TM Express enzyme, placed in a 37℃ water bath shaker, speed of 60rpm, time for 40min, gently shake every 5min, after digestion, the digestion status can be observed under a microscope. After stopping digestion, blow the sample evenly, filter with a 100-mesh filter to obtain a cell mass suspension, and finally place it in a pre-cooled 4℃ centrifuge at 1500rpm for 10min, remove the supernatant, repeat 3 times, and obtain the cell mass precipitate;

[0108] (2) Then the cell pellet was mixed with 4°C The basement membrane extracts were mixed at a volume ratio of 1:1.5 at 25°C to obtain a gel mixed with cells; finally, the above-mentioned prostate cancer organoid culture medium was added to the gel and incubated at 37°C and 5% CO 2 The cells were cultured in a constant temperature incubator for 7 days, with the culture medium replaced every 3 days.

[0109] Optical microscopy of prostate cancer organoids on day 3 of culture Fig.10 shown.

[0110] Comparative Example 7

[0111] Same as Example 1, except that the final concentration of Y-27632 is 50 μM.

[0112] A prostate cancer organoid culture medium, comprising a basal culture medium and a culture supplement, wherein the basal culture medium comprises Advanced DMEM / F12, N21-MAX Media Supplement 50× and 1% HEPES buffer; the culture supplement is in a mixture of the basal culture medium and sterile water, and comprises the following components at final concentrations: FGF-10 30 ng / mL, Zeaxanthin dipalmitate 3 mM, Polydatin 300 μg / mL, 1% penicillin-streptomycin, IGF-1 6 ng / mL, R-spondin1 200 ng / mL, Noggin 200 ng / mL, EGF 70 ng / mL, GlutaminePlus 12 ng / mL, A83-01 1.8 μM, Y-27632 50 μM, N-Acetylcysteine ​​5 μM, Primocin 1.6 μM, Nicotinamide 18 μM and SB202190 500nM. The preparation method is as follows: add the culture additive to the basic culture medium and mix evenly.

[0113] Prostate cancer organoids were cultured using the above medium as follows:

[0114] (1) Take a fresh tumor tissue sample from a patient who has undergone surgery and wash it four times with sterile PBS buffer for 5 minutes each time. Then cut the washed tissue into approximately 1 mm thick pieces with a scalpel. 3 Transfer the tissue of different size to a 15 mL centrifuge tube and add appropriate amount of TrypLE TM Express enzyme, placed in a 37℃ water bath shaker, speed of 60rpm, time for 40min, gently shake every 5min, after digestion, the digestion status can be observed under a microscope. After stopping digestion, blow the sample evenly, filter with a 100-mesh filter to obtain a cell mass suspension, and finally place it in a pre-cooled 4℃ centrifuge at 1500rpm for 10min, remove the supernatant, repeat 3 times, and obtain the cell mass precipitate;

[0115] (2) Then the cell pellet was mixed with 4°C The basement membrane extracts were mixed at a volume ratio of 1:1.5 at 25°C to obtain a gel mixed with cells; finally, the above-mentioned prostate cancer organoid culture medium was added to the gel and incubated at 37°C and 5% CO 2 The cells were cultured in a constant temperature incubator for 7 days, with the culture medium replaced every 3 days.

[0116] Optical microscopy of prostate cancer organoids on day 3 of culture Fig.11 shown.

[0117] Test Example 1

[0118] During the prostate cancer organoid culture process of Examples 1-4 and Comparative Examples 1-7, live cell counts were performed on day 0, day 3, and day 7, respectively. The results are shown in Table 1.

[0119] Table 1 Number of living cells

[0120]

[0121]

[0122] As shown in Table 1, the number of viable cells of prostate cancer organoids cultured in Examples 1-4 is significantly higher than that in Comparative Examples 1-7. Compared with Example 1, when the components of the basal culture medium are changed in Comparative Examples 1, 6 and 7, the number of viable cells is significantly reduced; compared with Comparative Examples 2-5, Example 1 shows that FGF-10, Zeaxanthin dipalmitate and Polydatin in the culture medium have a synergistic effect, which can significantly increase the number of viable cells.

[0123] Test Example 2

[0124] After the prostate cancer organoids of Examples 1-4 and Comparative Examples 1-7 were cultured for 7 days, 5 visual fields in the middle area were randomly selected under an ordinary optical microscope to count the number, average size and viability of the organoids. The results are shown in Table 2.

[0125] Table 2 Number and size of prostate cancer organoids

[0126] serial number Average number of Average size (μm) Organoid viability (%) Example 1 132 70-75 97.5 Example 2 128 65-70 96.4 Example 3 119 70-75 93.7 Example 4 121 68-72 95.8 Comparative Example 1 <![CDATA[88 # ]]> 42-47 <![CDATA[62.8 # ]]> Comparative Example 2 <![CDATA[82 # ]]> 41-45 <![CDATA[60.5 # ]]> Comparative Example 3 <![CDATA[60 ## ]]> 35-38 <![CDATA[51.3 ## ]]> Comparative Example 4 <![CDATA[75 # ]]> 37-42 <![CDATA[63.4 # ]]> Comparative Example 5 <![CDATA[63 ## ]]> 36-40 <![CDATA[53.8 ## ]]> Comparative Example 6 <![CDATA[74 # ]]> 37-43 <![CDATA[58.7 # ]]> Comparative Example 7 <![CDATA[76 # ]]> 38-42 <![CDATA[60.2 # ]]>

[0127] Note: Compared with Example 1, # indicates P < 0.05, ## Indicates P<0.01.

[0128] As shown in Table 2, the organoids of Examples 1-4 of the present invention are significantly better than those of Comparative Examples 1-7 in terms of quantity, average size and organoid viability, indicating that the culture medium of Examples 1-4 of the present invention is more suitable for organoid growth. Comparative Examples 2-5 also show that FGF-10, Zeaxanthin dipalmitate and Polydatin have a synergistic effect in promoting the growth of prostate cancer organoids.

[0129] Test Example 3

[0130] The prostate cancer organoids obtained by Example 1 and Comparative Examples 1-7 were tested for passage. The prostate cancer organoids cultured by Example 1 of the present invention can be passaged to 5-10 generations, at which time the organoids can still grow well; while Comparative Examples 1-7 apoptosis within 3 generations and the culture fails.

[0131] In summary, the prostate cancer organoid culture medium provided by the present invention utilizes the synergistic effect of FGF-10, Zeaxanthindipalmitate and Polydatin to greatly improve the success rate of organoid culture. The cultured prostate cancer organoids grow quickly, and the number of passages is significantly increased. Human prostate cancer tissue can be specifically cultured in vitro, further improving the survival rate and formation efficiency of prostate cancer organoids.

[0132] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A prostate cancer organoid culture medium, characterized in that The invention comprises a basic culture medium and a culture supplement, wherein the culture supplement comprises FGF-10, Zeaxanthin dipalmitate and Polydatin.

2. The prostate cancer organoid culture medium according to claim 1, characterized in that The culture supplements also include IGF-1, R-spondin 1, Noggin, EGF, GlutaminePlus, A83-01, Y-27632, N-Acetylcysteine, Primocin, Nicotinamide and SB 202190.

3. The prostate cancer organoid culture medium according to claim 2, characterized in that The culture supplement includes the following components at final concentrations: FGF-10 10-40 ng / mL, Zeaxanthin dipalmitate 2-3 mM, Polydatin 200-400 μg / mL, 1-2% penicillin-streptomycin, IGF-1 1-10 ng / mL, R-spondin 1 100-300 ng / mL, Noggin 100-300 ng / mL, EGF 60-90 ng / mL, GlutaminePlus 5-20 ng / mL, A83-01 1-2 μM, Y-27632 10-20 μM, N-Acetylcysteine ​​2-8 μM, Primocin 1-2 μM, Nicotinamide 10-20 μM, and SB 202190 200-1000 nM.

4. The prostate cancer organoid culture medium according to claim 3, characterized in that The culture supplement includes the following components at final concentrations: FGF-10 20-35 ng / mL, Zeaxanthin dipalmitate 2.5-3 mM, Polydatin 250-350 μg / mL, 1% penicillin-streptomycin, IGF-1 5-8 ng / mL, R-spondin 1 150-250 ng / mL, Noggin 150-200 ng / mL, EGF 65-80 ng / mL, Glutamine Plus 10-15 ng / mL, A83-01 1.5-2 μM, Y-27632 10-15 μM, N-Acetylcysteine ​​3-6 μM, Primocin 1.5-2 μM, Nicotinamide 15-20 μM, and SB 202190 300-600 nM.

5. The prostate cancer organoid culture medium according to claim 1, characterized in that The basic culture medium includes Advanced DMEM / F12, N21-MAX Media Supplement 50× and 1% HEPES buffer.

6. A method for preparing a prostate cancer organoid culture medium according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: preparing a culture additive into a mother solution with sterile water, and then adding the mother solution into a basic culture medium to obtain the mother solution.

7. A method for culturing prostate cancer organoids, characterized in that: The steps include: (1) Pre-treat fresh surgical specimens to obtain cell clusters, centrifuge, and obtain cell cluster sediments; (2) Then the cell pellet was precipitated and Basement membrane extracts were mixed to obtain a gel mixed with cells; (3) Finally, add the prostate cancer organoid culture medium described in any one of claims 1 to 5 to the gel and culture together to obtain the prostate cancer organoid.

8. The culture method according to claim 7, characterized in that: The pretreatment in step (1) includes the following steps: first, the surgical specimen is rinsed 3-5 times with sterile PBS buffer, cut into 1-3 mm 3 Small pieces; then add TrypLE TM Express enzyme, digest at 32-37°C, 40-80rpm for 30-60min; finally filter through a 100-120 mesh filter to obtain a cell pellet.

9. The culture method according to claim 7, characterized in that: The centrifugal temperature in step (1) is 20-25°C, the centrifugal speed is 1000-2000 rpm, and the centrifugal time is 5-10 min; The temperature of the basement membrane extract is 4-10°C, the temperature of the mixture is 23-27°C, and the cell pellet is The volume ratio of the basement membrane extract is 1:1-2; the co-culture conditions in step (3) are cultured in an incubator at 37° C. and 5% CO 2 concentration for 4-7 days.

10. A prostate cancer organoid cultured using the culture method according to any one of claims 7 to 9.

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