Prostate cancer organoid culture medium and culture method thereof

Abstract

The invention discloses a prostate cancer organoid culture medium and a culture method thereof, and particularly relates to the technical field of prostate cancer organoid culture, and the prostate cancer organoid culture medium comprises a basic culture medium and the following components: 20-100ng / ml of epidermal growth factors, 20-100ng / ml of prostate cancer organoid and 20-100ng / ml of The concentration of Noggin is 50 to 500 ng / ml; the concentration of R-spondin is 50 to 500 ng / ml; the weight of Wnt3a is 50 to 500 ng / ml; the concentration of FGF is 101 to 50ng / ml; 1 to 50 ng / ml of FGF (fibroblast growth factor); stem cell factors are 1 to 50 ng / mL; the concentration of antibiotics is 50-500 ng / ml; dHT is 1 to 50 ng / ml; the method comprises the following steps: 1, obtaining a prostate cancer tissue sample, placing the prostate cancer tissue in a sampling bottle containing a tissue preservation solution at 2-8 DEG C, and rapidly transferring the prostate cancer tissue to a clean laboratory for subsequent treatment; 2, preparing a plurality of culture dishes, adding a 4 DEG C precooled prostate cancer organoid culture buffer solution for later use, disinfecting a sampling bottle, putting the tissue into the culture dishes, and cleaning the tissue for three times by using the prostate cancer organoid culture buffer solution; the culture medium and the culture method provided by the invention can promote the growth of organs with prostatic cancer, so that the culture medium has a better actual use effect and is convenient to popularize and use.

Description

Technical Field

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

[0002] Prostate cancer organoid culture involves using tumor tissue obtained from prostate cancer patients to culture three-dimensional cell aggregates with prostate cancer characteristics in vitro through specific culture conditions and methods. These organoids can simulate the growth environment of prostate cancer in the body, providing a powerful tool for studying the pathogenesis, drug screening and treatment strategies of prostate cancer.

[0003] The prostate cancer organoid culture medium in the existing technology is not effective in promoting growth, resulting in slow culture of prostate cancer organoids. At the same time, there is no culture method to promote growth, resulting in the existing prostate cancer organoid culture effect being not good enough. Therefore, there is an urgent need for a prostate cancer organoid culture medium and a culture method thereof. Summary of the Invention

[0004] In order to overcome the above-mentioned defects of the prior art, the embodiments of the present invention provide a prostate cancer organoid culture medium and a culture method thereof. The culture medium and culture method of the present invention can promote the growth of prostate cancer organoids, so that the actual use effect of the present invention is better and it is easy to promote and use, so as to solve the problems raised in the above-mentioned background technology.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a prostate cancer organoid culture medium comprising a basal culture medium and components at the following concentrations: Epidermal growth factor 20-100 ng / ml; Noggin 50-500ng / ml; R-spondin 50-500ng / ml; Wnt3a 50-500ng / ml; FGF10 1-50ng / ml; FGF2 1-50ng / ml; Stem cell factor 1-50 ng / mL; Antibiotics 50-500 ng / ml; DHT 1-50ng / ml; Nicotinamide 20-100ng / ml.

[0006] In a preferred embodiment, the basal culture medium includes F12 culture medium.

[0007] A method for culturing prostate cancer organoids, comprising the above-mentioned prostate cancer organoid culture medium and the following steps: Step 1: Obtain a prostate cancer tissue sample and place it in a sampling bottle containing tissue preservation solution at 2-8°C. Rapidly transport the sample to a clean laboratory for subsequent processing. Step 2: Prepare several culture dishes and add 4°C pre-cooled prostate cancer organoid culture buffer for later use. Sterilize the sampling bottle and place the tissue in the culture dish. Wash three times with prostate cancer organoid culture buffer. Use ophthalmic scissors or a scalpel to cut the tumor tissue into tissue pieces of approximately 1-3 mm³. Digest the tissue with prostate cancer primary tissue digestion solution at 37°C with shaking for 10-20 minutes. Observe the digestion process at any time. When a large number of single cells are observed under the microscope, add three volumes of prostate cancer organoid culture buffer to terminate the digestion. Step 3: Filter through a 100 μm pore size mesh, collect the filtrate, centrifuge the filtrate at 300 g for 5 minutes, and remove the supernatant; Step 4: Add prostate cancer organoid culture buffer to resuspend the centrifuged cells, observe the volume of collected tissue, and add 25 times the volume of tissue to Matrigel to resuspend and plate; Step 5: Place the plate in a 37°C incubator for 10-15 minutes to allow the Matrigel to solidify. Add 500 μL of prostate cancer organoid culture medium and culture at room temperature. The culture medium should cover the Matrigel, but avoid adding it directly onto the gel drop. Step 6: Change the culture medium completely every 2-3 days to keep the culture medium fresh and sufficient in nutrients. Observe the culture of organoids under a microscope and record the growth status and morphological changes of the organoids.

[0008] The technical effects and advantages of the present invention are as follows: The culture medium of the present invention includes epidermal growth factor 20-100ng / ml, Noggin 50-500ng / ml, R-spondin 50-500ng / ml, Wnt3a 50-500ng / ml, FGF101-50ng / ml, FGF21-50ng / ml, stem cell factor 1-50ng / mL, antibiotics 50-500ng / ml, DHT1-50ng / ml, and nicotinamide 20-100ng / ml. The present invention first obtains a prostate cancer tissue sample, places the prostate cancer tissue in a sampling bottle containing tissue preservation fluid at 2-8°C, and quickly transports it to a clean laboratory for subsequent processing. Then, several culture dishes are prepared, and a 4°C pre-cooled prostate cancer organoid culture buffer is added for standby use. The sampling bottle is disinfected, the tissue is placed in a culture dish, and the prostate cancer organoid is used to store the tissue. Wash three times with organ culture buffer, use ophthalmic scissors or a scalpel to cut the tumor tissue into tissue blocks of approximately 1-3 mm³ in volume, digest the tissue with prostate cancer primary tissue digestion solution, and digest at 37°C with shaking for 10-20 minutes. Observe the digestion progress at any time during the digestion process. When a large number of single cells are observed under the microscope, add three times the volume of prostate cancer organoid culture buffer to terminate the digestion, filter through a 100μm pore size mesh, collect the filtrate, centrifuge the filtrate, centrifuge at 300g for 5 minutes, remove the supernatant, add prostate cancer organoid culture buffer to resuspend the centrifuged cells, observe the collected tissue volume, add 25 times the volume of tissue matrix gel to resuspend and plate, place the plated culture plate in a 37°C incubator for 10-15 minutes to allow the matrix gel to solidify, and add 500μL prostate cancer organoid culture medium for culture at room temperature. The culture medium should be covered on top of the matrix gel, but avoid adding it directly to the gel droplets. The medium should be completely replaced every 2-3 days to maintain the freshness of the culture medium and the sufficiency of nutrients. The culture of the organoids should be observed under a microscope, and the growth status and morphological changes of the organoids should be recorded. The culture medium and culture method of the present invention can promote the growth of prostate cancer organoids, making the actual use of the present invention better and facilitating its promotion and use. DETAILED DESCRIPTION

[0009] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0010] The present invention provides a prostate cancer organoid culture medium, comprising a basal culture medium and components at the following concentrations: Epidermal growth factor 20-100 ng / ml; Noggin 50-500ng / ml; R-spondin 50-500ng / ml; Wnt3a 50-500ng / ml; FGF10 1-50ng / ml; FGF2 1-50ng / ml; Stem cell factor 1-50 ng / mL; Antibiotics 50-500 ng / ml; DHT 1-50ng / ml; Nicotinamide 20-100ng / ml.

[0011] In a preferred embodiment, the basal culture medium includes F12 culture medium.

[0012] A method for culturing prostate cancer organoids, comprising the above-mentioned prostate cancer organoid culture medium and the following steps: Step 1: Obtain a prostate cancer tissue sample and place it in a sampling bottle containing tissue preservation solution at 2-8°C. Rapidly transport the sample to a clean laboratory for subsequent processing. Step 2: Prepare several culture dishes and add 4°C pre-cooled prostate cancer organoid culture buffer for later use. Sterilize the sampling bottle and place the tissue in the culture dish. Wash three times with prostate cancer organoid culture buffer. Use ophthalmic scissors or a scalpel to cut the tumor tissue into tissue pieces of approximately 1-3 mm³. Digest the tissue with prostate cancer primary tissue digestion solution at 37°C with shaking for 10-20 minutes. Observe the digestion process at any time. When a large number of single cells are observed under the microscope, add three volumes of prostate cancer organoid culture buffer to terminate the digestion. Step 3: Filter through a 100 μm pore size mesh, collect the filtrate, centrifuge the filtrate at 300 g for 5 minutes, and remove the supernatant; Step 4: Add prostate cancer organoid culture buffer to resuspend the centrifuged cells, observe the volume of collected tissue, and add 25 times the volume of tissue to Matrigel to resuspend and plate; Step 5: Place the plate in a 37°C incubator for 10-15 minutes to allow the Matrigel to solidify. Add 500 μL of prostate cancer organoid culture medium and culture at room temperature. The culture medium should cover the Matrigel, but avoid adding it directly onto the gel drop. Step 6: Change the culture medium completely every 2-3 days to keep the culture medium fresh and sufficient in nutrients. Observe the culture of organoids under a microscope and record the growth status and morphological changes of the organoids.

[0013] The culture medium of the present invention comprises a basal culture medium, epidermal growth factor 20-100 ng / ml, Noggin 50-500 ng / ml, R-spondin 50-500 ng / ml, Wnt3a 50-500 ng / ml, FGF101-50 ng / ml, FGF21-50 ng / ml, stem cell factor 1-50 ng / mL, antibiotics 50-500 ng / ml, DHT 1-50 ng / ml, and nicotinamide 20-100 ng / ml. The present invention first obtains a prostate cancer tissue sample, places the prostate cancer tissue in a sampling bottle containing tissue preservation solution at 2-8°C, and quickly transports it to a clean laboratory for subsequent processing. Then, several culture dishes are prepared, and a prostate cancer organoid culture buffer solution pre-cooled at 4°C is added for standby use. The sampling bottle is sterilized, and the tissue is placed in a culture dish. Wash the adenocarcinoma organoid culture buffer three times, use ophthalmic scissors or a scalpel to cut the tumor tissue into tissue blocks of approximately 1-3 mm³ in volume, digest the tissue with prostate cancer primary tissue digestion buffer, and digest at 37°C with shaking for 10-20 minutes. Observe the digestion progress at any time during the digestion process. When a large number of single cells are observed under the microscope, add three volumes of prostate cancer organoid culture buffer to stop the digestion, filter through a 100μm pore size mesh, collect the filtrate, centrifuge the filtrate, centrifuge at 300g for 5 minutes, remove the supernatant, add prostate cancer organoid culture buffer to resuspend the centrifuged cells, observe the collected tissue volume, add 25 times the tissue volume of matrigel to resuspend and plate, place the plated culture plate in a 37°C incubator for 10-15 minutes to allow the matrigel to solidify, and add 500μL prostate cancer organoid culture medium for culture at room temperature. The culture medium should be covered on top of the matrix gel, but avoid adding it directly to the gel droplets. The medium should be completely replaced every 2-3 days to maintain the freshness of the culture medium and the sufficiency of nutrients. The culture of the organoids should be observed under a microscope, and the growth status and morphological changes of the organoids should be recorded. The culture medium and culture method of the present invention can promote the growth of prostate cancer organoids, making the actual use of the present invention better and facilitating its promotion and use.

[0014] Finally: The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A prostate cancer organoid culture medium, characterized in that Includes basal medium and the following components at the following concentrations: Epidermal growth factor 20-100 ng / ml; Noggin 50-500ng / ml; R-spondin 50-500ng / ml; Wnt3a 50-500ng / ml; FGF10 1-50ng / ml; FGF2 1-50ng / ml; Stem cell factor 1-50 ng / mL; Antibiotics 50-500 ng / ml; DHT 1-50ng / ml; Nicotinamide 20-100ng / ml.

2. A prostate cancer organoid culture medium according to claim 1, characterized in that: The basic culture medium includes F12 culture medium.

3. A method for culturing prostate cancer organoids, characterized by: The method comprises the prostate cancer organoid culture medium according to claim 1 and the following steps: Step 1: Obtain a prostate cancer tissue sample and place it in a sampling bottle containing tissue preservation solution at 2-8°C. Rapidly transport the sample to a clean laboratory for subsequent processing. Step 2: Prepare several culture dishes and add 4°C pre-cooled prostate cancer organoid culture buffer for later use. Sterilize the sampling bottle and place the tissue in the culture dish. Wash three times with prostate cancer organoid culture buffer. Use ophthalmic scissors or a scalpel to cut the tumor tissue into tissue pieces of approximately 1-3 mm³. Digest the tissue with prostate cancer primary tissue digestion solution at 37°C with shaking for 10-20 minutes. Observe the digestion process at any time. When a large number of single cells are observed under the microscope, add three volumes of prostate cancer organoid culture buffer to terminate the digestion. Step 3: Filter through a 100 μm pore size mesh, collect the filtrate, centrifuge the filtrate at 300 g for 5 minutes, and remove the supernatant; Step 4: Add prostate cancer organoid culture buffer to resuspend the centrifuged cells, observe the volume of collected tissue, and add 25 times the volume of tissue to Matrigel to resuspend and plate; Step 5: Place the plate in a 37°C incubator for 10-15 minutes to allow the Matrigel to solidify. Add 500 μL of prostate cancer organoid culture medium and culture at room temperature. The culture medium should cover the Matrigel, but avoid adding it directly onto the gel drop. Step 6: Change the culture medium completely every 2-3 days to keep the culture medium fresh and nutrient-rich. Observe the culture of organoids under a microscope and record their growth status and morphological changes.