Culture method of neurofibroma organoid

Abstract

The invention provides a culture method of a neurofibroma organ. The balling culture medium of the neurofibroma organ comprises EGF (epidermal growth factor) and FGF (fibroblast growth factor) with the concentration of 200-250ng The organoid culture method comprises the following steps: digesting neurofibroma tissue blocks to prepare a single-cell suspension; the single-cell suspension is placed in a culture dish treated by polylysine, a Schwann cell culture medium is added to be cultured for 24 hours, the Schwann cell culture medium contains forsklin and beta-heragulin needed by Schwann cell enrichment, and Schwann cell enrichment liquid is obtained; collecting Schwann cell enrichment liquid, adding the balling culture medium and Y-27632, culturing for 6-12 hours, collecting cells, adding the balling culture medium, and culturing for 7-14 days. The tumor ball culture method comprises the following steps: culturing the organoid according to the method, digesting the organoid and culturing. According to the present invention, the Schwann cell screening step is added, the components of the balling culture medium are improved, and the Y-27632 is added, such that the problems of small tumor ball number and small size are solved, and the stable neurofibroma organ can be formed.

Description

Technical Field

[0001] The present disclosure relates to the technical field of organoid culture, and in particular to a method for culturing neurofibroma organoids. Background Art

[0002] Neurofibromatosis type 1 (NF1) is a type of autosomal dominant hereditary disease. Neurofibromas are benign peripheral nerve sheath tumors and are the most common clinical manifestation of neurofibromatosis type 1. 95% of patients suffer from cutaneous neurofibromas, of which 30-50% of NF1 patients also suffer from plexiform neurofibromas (PNF). PNF can grow huge and compress normal tissues and organs around the tumor. At the same time, 10-13% have a tendency to malignant transformation. Due to their close connection with nerves, surgical efficacy is poor. Therefore, it has become a hot spot in current multi-omics and targeted therapy research. However, neurofibromas are benign tumors. In in vitro studies, unlike most malignant tumors, their tumor cells are not immortal and it is difficult to survive for a long time in vitro, making it difficult to conduct research. Organoids have the advantages of being highly consistent with the source tissue in terms of tissue structure, cell type and function. Compared with other cell lines, their drug sensitivity and drug responsiveness are closer to human responses. Therefore, culturing neurofibroma organoids in vitro is of great significance for conducting research and finding treatment options.

[0003] Currently, researchers are using IRB-approved surgery to treat acquired human plexiform neurofibromas, cutting the tumors into 1 mm 3 size, and transferred to the digestion solution for digestion. The digested cells were then inoculated in a low-adsorption 24-well plate, and neurofibroma organoids were obtained after 21 days of culture. However, PNF is composed of Schwann cells, fibroblasts, perineuronal cells and mast cells, among which Schwann cells are considered to be the starting cells / neurofibroma stem cells for the formation of neurofibromas. Cell screening was not performed in the above culture method, and it cannot be guaranteed that Schwann cells account for the vast majority, which will affect the formation and proliferation of spheres. In addition, the number of tumor spheres obtained by the above culture method is scarce and small in size, and the stability of multiple passages is poor, and the number of tumor spheres cannot meet the experimental needs.

[0004] Based on this, it is necessary to provide a method for culturing neurofibroma organoids to solve the problem of the small number of tumor spheres, so that the experiments are not limited to observing the effects of drugs on tumor sphere formation, but can also be studied at the molecular level, giving full play to the advantages of organoids in disease treatment research. Summary of the invention

[0005] The present disclosure provides a method for culturing neurofibroma organoids to at least solve the above technical problems existing in the prior art.

[0006] According to a first aspect of the present disclosure, a spheroid culture medium for neurofibroma organoids is provided, comprising the following components: DMEM / F12, 1-1.5% B27, 1-1.5% N2, 200-250 ng / mL EGF, 200-250 ng / mL FGF, 0.1-0.3 mM β-ME, 2-3 μg / mL heparin, 2.5-5 μg / mL amphotericin B, and 5-7 μg / mL levofloxacin.

[0007] Specifically, two antibiotics, amphotericin B and levofloxacin, are added to the spheroidization culture medium, which can effectively eliminate common bacterial and fungal contamination in cell culture and provide a more stable growth environment for the formation of tumor organoids. In addition, the present disclosure improves the formula of the existing spheroidization culture medium, increases the working concentrations of growth factors EGF and FGF by about 10 times, and can significantly promote cell proliferation, so that the number of tumor spheres of the obtained neurofibroma organoids is greater, each tumor sphere contains more cells, and the volume of the tumor sphere is larger.

[0008] In a preferred embodiment, a spheroid culture medium for neurofibroma organoids comprises the following components: DMEM / F12 (1:1), 1% B27, 1% N2, 200 ng / mL EGF, 200 ng / mL FGF, 0.1 mM β-ME, 2 μg / mL heparin, 2.5 μg / mL amphotericin B, and 5 μg / mL levofloxacin.

[0009] In one embodiment, the neurofibroma organoid is a plexiform neurofibroma organoid.

[0010] According to a second aspect of the present disclosure, a method for culturing neurofibroma organoids is provided, comprising the following steps:

[0011] S1: Digest neurofibroma tissue blocks and prepare single cell suspensions;

[0012] S2: placing the single cell suspension described in step S1 in a culture dish treated with poly-lysine, adding Schwann cell culture medium and culturing for 24 hours to obtain Schwann cell enrichment solution;

[0013] S3: The Schwann cell enrichment fluid is collected, and the spheroidization medium and growth factor Y-27632 are added. After culturing for 6-12 hours, the cells are collected, and the spheroidization medium is added to continue culturing for 7-14 days to obtain the neurofibroma organoid.

[0014] In one embodiment, step S1 of digesting the neurofibroma tissue block comprises the following steps: cleaning and chopping the neurofibroma tissue block, and placing it in a digestion solution for digestion, and after the digestion is completed, adding DMEM+10% Clark medium to terminate the digestion.

[0015] Specifically, cleaning and chopping the neurofibroma tissue block specifically includes the following steps: washing the neurofibroma tissue block with PBS for 2 to 3 times; then chopping the washed neurofibroma tissue block into 1 to 1.5 cm 3 The extra tissue on the tissue block was removed and finally cut into 1-1.5 mm 3 Minced meat tissue mass.

[0016] Specifically, the neurofibroma tissue block is washed with the PBS in order to wash away the culture medium and blood remaining on the surface.

[0017] Specifically, removing the extra tissue on the tissue block refers to removing hair, blood clots, blood vessels, fat and other tissues on the tissue block to prevent contamination.

[0018] Specifically, the usage ratio of the digestive solution and the neurofibroma tissue blocks is: every 20 to 25 mL of the digestive solution is used to digest 5 to 6 neurofibroma tissue blocks.

[0019] Specifically, the digestive fluid includes the following components: L-15 culture medium, 50-80 mg / mL gentamicin, 250-280 μg / mL amphotericin B, 500-520 μg / mL levofloxacin, 10,000 IU penicillin / streptomycin / ampicillin (Pen / Strep / Amp), 155-160 u / mL type I collagenase, and 1.25-1.5 u / mL dispersase II.

[0020] More specifically, the digestion solution includes the following components: L-15 culture medium, 50 mg / mL gentamicin, 250 μg / mL amphotericin B, 500 μg / mL levofloxacin, 10000 IU penicillin / streptomycin / ampicillin (Pen / Strep / Amp), 156 u / mL type I collagenase, and 1.25 u / mL dispersase II.

[0021] Specifically, two antibiotics, amphotericin B and levofloxacin, were added to the digestive fluid, which can effectively eliminate common bacterial and fungal contamination in cell culture and provide a more stable growth environment for tumor organoid formation.

[0022] Specifically, the digestion conditions include: digestion in a constant temperature shaker at 35-37° C. for 3-3.5 hours at a rotation speed of 180-200 rpm.

[0023] Specifically, during the digestion process, the tissue block can be shaken to observe the digestion status, and the digestion can be terminated when the edges of the tissue fragments are unclear and diffuse tailing.

[0024] Specifically, the volume ratio of the DMEM+10% Clark medium to the digestion solution is 1:1.

[0025] In one embodiment, in step S1, after digesting the neurofibroma tissue block, the tissue is blown away, centrifuged, and the supernatant is discarded to obtain a first cell pellet.

[0026] Specifically, the blowing comprises the following steps: first, the tissue is blown away with a sterile Pasteur pipette until there is no resistance and no precipitation is visible to the naked eye, and then the tissue is blown away with a 1000 μL pipette tip for dozens of times until there is no resistance or the resistance becomes smaller.

[0027] During the above-mentioned blowing process, do not blow violently to avoid mechanical cell death and reduce the extraction efficiency of primary cells.

[0028] Specifically, the centrifugal speed is 2000-2500 rpm, and the time is 10-15 min.

[0029] In one embodiment, step S1 further includes the following steps: adding fresh DMEM+10% Clark medium to the first cell pellet, blowing with a 1000 μL pipette tip until no tissue blocks are visible to the naked eye, replacing the glass tube for blowing to blow off the Schwann cells adhered around the nerves; then centrifuging and discarding the supernatant to obtain a second cell pellet; adding Schwann cell culture medium to the second cell pellet to obtain the single cell suspension.

[0030] Specifically, the narrow opening of the glass tube is the size of a pinhole.

[0031] Specifically, the centrifugal speed is 2000-2500 rpm, and the time is 10-15 min.

[0032] In one embodiment, in step S2, the culture dish is treated with 0.05-0.1% poly-lysine for ≥30 min, and then the culture dish is washed with PBS for ≥3 times.

[0033] In one embodiment, step S2 is to screen and enrich in the culture dish for 24 to 26 hours, during which the medium is changed 2 to 3 times to obtain the Schwann cell enrichment medium.

[0034] Specifically, the medium is changed 2 to 3 times to wash away incompletely digested tissues and non-Schwann cells.

[0035] Specifically, through the above screening and enrichment, a relatively single Schwann cell collection can be obtained.

[0036] Specifically, in the Schwann cell enrichment solution, the proportion of Schwann cells is more than 90%.

[0037] In one embodiment, collecting the Schwann cell enriched solution in step S3 specifically comprises the following steps: digesting the Schwann cell enriched solution in step S2, centrifuging after pipetting, discarding the supernatant, and obtaining a third cell precipitate.

[0038] Specifically, the Schwann cell enrichment solution in step S2 is digested with 0.25% trypsin digestion solution for 8 to 10 minutes, and then DMEM+10% Clark medium is added to terminate the digestion.

[0039] Specifically, the cells were blown down from the bottom of the culture dish using a 1000 μL pipette and blown into a single cell suspension to avoid cell damage or death caused by severe blowing.

[0040] Specifically, the centrifugal speed is 1000-1200 rpm, and the time is 5-8 min.

[0041] In one embodiment, step S3 adds the spheroidization medium and the growth factor Y-27632 to the third cell pellet for re-suspending, and transfers the cell suspension to a low-adhesion cell culture plate for suspension culture for 6-12 hours.

[0042] In one embodiment, the concentration of the growth factor Y-27632 is 5-10 μM.

[0043] In one embodiment, in step S3, the cells are collected after culturing for 6-12 hours, and centrifuged at 1000-1500 rpm for 5-8 minutes, the supernatant is discarded to obtain a fourth cell pellet, and the spheroid culture medium is added to the fourth cell pellet to continue culturing for 7-14 days to obtain the neurofibroma organoid.

[0044] Specifically, neurofibroma organoids are miniature organs formed by three-dimensional culture in vitro using special culture media, which have the advantages of being highly consistent with the source tissue in terms of tissue structure, cell type and function. Compared with other cell lines, neurofibroma organoids are more sensitive and responsive to drugs than other cell lines, which can make experimental results more scientific and treatment plans more promising for clinical application.

[0045] According to a third aspect of the present disclosure, a method for culturing neurofibroma tumor spheres is provided, comprising the following steps:

[0046] The above-mentioned culture method is used to culture and form neurofibroma organoids, and the neurofibroma organoids are digested into single cell suspensions, which are transferred to low-adsorption cell culture plates for culture to form the neurofibroma tumor spheres.

[0047] In one embodiment, digesting the neurofibroma organoid into the single cell suspension specifically includes the following steps: centrifuging the cell suspension of the neurofibroma organoid to obtain a fifth cell pellet, and retaining the supernatant separately; digesting the fifth cell pellet, centrifuging it again, discarding the supernatant, and obtaining a sixth cell pellet; adding the supernatant corresponding to the fifth cell pellet and an equal volume of fresh spheroidization culture medium to the sixth cell pellet to form the single cell suspension.

[0048] Specifically, the rotation speed of the above two centrifugations is 1000-1200 rpm, and the time is 5-8 min.

[0049] Specifically, in the above step, 0.25% trypsin digestion solution is added to digest the fifth cell precipitation, and the digestion time is 10 to 12 minutes.

[0050] Specifically, when shaking revealed that the white discs of the neurofibroma organoids had unclear edges, spheroidization medium was added to terminate the digestion.

[0051] According to an implementable embodiment of the present disclosure, at least the following beneficial effects are achieved:

[0052] 1. The present invention improves the existing spheroid culture medium formula, increases the working concentration of growth factors EGF and FGF to about 10 times of the existing technology, and can significantly promote cell proliferation; in addition, in the process of culturing neurofibroma organoids, the growth factor Y-27632 is first added and cultured for a certain period of time (6 hours to 12 hours). The combination of the two can increase the number of neurofibroma tumor spheres obtained, each tumor sphere contains more cells, and the volume of the tumor sphere is larger.

[0053] 2. Since the initiating cells of plexiform neurofibromas are Schwann cells, but plexiform neurofibroma tissues include multiple types of cells such as Schwann cells, fibroblasts and mast cells, this protocol uses Schwann cell culture medium (SCM) and culture dishes treated with poly-lysine to allow the selected Schwann cells to adhere to the bottom of the culture dish, thereby obtaining relatively single Schwann cells.

[0054] 3. The present invention adds antibiotics levofloxacin and amphotericin B to the culture medium and tumor tissue digestion fluid, which can effectively eliminate common bacterial and fungal contamination in cell culture and provide a more stable growth environment for the formation of tumor spheres.

[0055] 4. The neurofibroma organoids cultured according to the method disclosed in the present invention can not only observe the effects of drugs on tumor sphere formation, but also conduct research at the molecular level, giving full play to the advantages of organoids in disease treatment research.

[0056] It should be understood that the content described in this section is not intended to identify the key or important features of the embodiments of the present disclosure, nor is it intended to limit the scope of the present disclosure. Other features of the present disclosure will become easily understood through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] The above and other objects, features and advantages of the exemplary embodiments of the present disclosure will become readily understood by reading the detailed description below with reference to the accompanying drawings. In the accompanying drawings, several embodiments of the present disclosure are shown in an exemplary and non-limiting manner, in which:

[0058] In the drawings, the same or corresponding reference numerals represent the same or corresponding parts.

[0059] Figure 1 A schematic diagram of the process of culturing neurofibroma organoids in Example 1 of the present disclosure is shown;

[0060] Figure 2 The immunofluorescence imaging diagram of the Schwann cell enrichment solution in Example 1 of the present disclosure is shown;

[0061] Figure 3 Optical microscopic images of the neurofibroma tumor spheres cultured in Example 2 and the neurofibroma tumor spheres cultured in Comparative Example 1 in the experimental examples of the present disclosure are shown;

[0062] Figure 4 Optical micrographs of the culture environment of the neurofibroma tumor spheres of Example 2 and Comparative Example 2 in the test examples of the present disclosure are shown;

[0063] Figure 5 Optical micrographs of the culture environment of the neurofibroma tumor spheres of Example 2 and Comparative Example 3 in the test examples of the present disclosure are shown. DETAILED DESCRIPTION

[0064] In order to make the purpose, features, and advantages of the present disclosure more obvious and easy to understand, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below in conjunction with the drawings in the embodiments of the present disclosure. Obviously, the described embodiments are only part of the embodiments of the present disclosure, not all of the embodiments. Based on the embodiments in the present disclosure, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present disclosure.

[0065] The digestion fluid and culture medium used are as follows:

[0066] Digestive juice:

[0067] L-15 medium (4°C, 20 mL)

[0068] 50mg / mL gentamicin (20μL)

[0069] 250 μg / mL amphotericin B (-20°C, 200 μL)

[0070] 500 μg / mL levofloxacin (-20°C, 200 μL)

[0071] 10000 IU of Penicillin / Streptomycin / Ampicillin (Pen / Strep / Amp, -20°C, 200 μL)

[0072] 1.56u / mL collagenase type I (4℃, 10mg)

[0073] 1.25u / mL of Disperse Proteinase II (4°C, 50mg).

[0074] Schwann cell culture medium:

[0075] DMEM, 10% Clark (FBS), 2 μM Forskolin, 10 ng / ml Human heregulin β, 2.5 μg / ml amphotericin B, 5 μg / ml levofloxacin

[0076] Sphere formation medium:

[0077] DMEM / F12 (1:1), 1% B27, 1% N2, 200 ng / mL EGF, 200 ng / mL FGF, 0.1 mM β-ME, 2 μg / mL heparin, 2.5 μg / mL amphotericin B, 5 μg / mL levofloxacin.

[0078] Example 1

[0079] This example cultured neurofibroma organoids. The specific process (see Figure 1 )as follows:

[0080] (1) Preserve the tumor tissue blocks of surgically removed plexiform neurofibromas in sterile L-15 (without double antibody) culture medium, and prepare high-temperature and high-pressure sterilized experimental tools (ophthalmic scissors and tweezers). Transfer the tumor tissue blocks to the cell culture room, take them out from the clean bench, and rinse them twice with pre-cooled sterile PBS to wash away the residual culture medium and blood on the surface. Place the tumor tissue blocks in a 10 cm cell culture dish and use high-pressure sterilized scissors to cut the tumor tissue blocks into 1 cm pieces. 3The tissue blocks were cut into 1mm thick pieces and the hair, blood clots, blood vessels, fat and other tissues on the tissue blocks were removed to prevent contamination. 3 Minced meat tissue mass.

[0081] Use a sterile Pasteur pipette to suck the shredded tissue blocks into the prepared digestion solution, with a ratio of 5 neurofibroma tissue blocks digested per 20 mL digestion solution. Seal the 50 mL centrifuge tube containing the digestion solution and tissue blocks with a sealing film and place it in a 37°C constant temperature shaker. Digest for 3 hours at 180 rpm, take out the centrifuge tube and shake it for observation. When the edges of the tissue fragments are unclear and the tail is diffuse, the digestion can be terminated. Take the centrifuge tube to the clean bench, tear off the sealing film, open the lid, and add 20 mL of DMEM + 10% Clark medium to terminate the digestion (the volume ratio of DMEM + 10% Clark medium to the digestion solution is 1:1).

[0082] First, use a sterile Pasteur pipette to blow the tissue apart until there is no resistance and no precipitation can be seen with the naked eye. Then continue to blow dozens of times with a 1000μL pipette tip until there is no resistance or the resistance becomes smaller (note: do not blow violently during the operation to avoid mechanical cell death and reduce the extraction efficiency of primary cells). Centrifuge at 2000rpm for 10min, observe the supernatant, if there is still tissue floating, centrifuge at the same speed for another 5min, then aspirate most of the supernatant (do not pour it out, as it is easy to lose cells), leave about 5mL of culture medium in the first cell pellet, add 10mL of fresh DMEM+10% Clark culture medium, blow with a 1000μL pipette tip until there are no visible tissue blocks, then replace with a pre-prepared glass tube with a smaller suction port (preparation of glass tube: take out the glass tube sterilized by high temperature and high pressure, light the alcohol lamp, sterilize the glass tube by passing it over the flame, hold the top of the glass tube, aim the suction port of the glass tube at the outer flame of the alcohol lamp, heat it in circles at a uniform speed to shrink the suction port of the glass tube, keep picking it up and observing it during the process, until the suction port of the glass tube becomes the size of a pinhole), insert the electric pipette and blow it, the purpose is to blow down the Schwann cells adhered to the nerves, blow until there is no resistance, centrifuge at 2000rpm for 10min, aspirate most of the supernatant, and leave the white cell pellet. Add 10 mL of fresh DMEM + 10% Clark medium to the white cell pellet, and continue to blow with the above glass tube to facilitate subsequent cell screening. After blowing dozens of times, centrifuge at 2000 rpm for 10 minutes, remove the old medium, and obtain the second cell pellet; add Schwann cell culture medium to the second cell pellet to obtain a single cell suspension.

[0083] (2) Soak a 10 cm cell culture dish in 0.05% poly-lysine for at least 30 min, recover the poly-lysine, and wash with PBS three times. The single cell suspension obtained in step (1) is plated on a 10 cm cell culture dish treated with poly-lysine, and Schwann cell culture medium is added for screening and enrichment for 24 h. During this period, the medium is changed twice to wash away incompletely digested tissues and non-Schwann cells, and obtain a relatively single Schwann cell enrichment solution.

[0084] The Schwann cell enrichment fluid was subjected to immunofluorescence staining using the Schwann cell marker S100. Figure 2 shown. Figure 2 It showed that 90% of the cells were Schwann cells. The nucleus of Schwann cells was long oval, with its long axis parallel to the axon, and a small amount of cytoplasm around the nucleus.

[0085] (3) Use 4 mL of 0.25% trypsin digestion solution to digest the Schwann cell enrichment solution in step (2) for 8 to 10 minutes, and add 4 mL of DMEM + 10% Clark medium to terminate the digestion. Use a 1000 μL pipette to blow the cells down from the bottom of the cell culture dish and blow them into a single cell suspension (avoid vigorous blowing to cause cell damage or death), centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and obtain the third cell pellet. Add the pre-prepared sphere medium and growth factor Y-27632 (working concentration is 10 μM) to the third cell pellet and resuspend it, and transfer the cell suspension to a low-adsorption 24-well plate for suspension culture for 12 hours, add 800 μL of cell suspension to each well, a total of 4 wells; add 200 μL of sphere medium every other day, add along the wall of the well to avoid blowing away the newly formed spheres. After 12 hours of culture, the cells were collected and centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded to obtain the fourth cell pellet. The spheroid culture medium was added to the fourth cell pellet and cultured for 7 to 14 days. During this period, the medium was changed every two and a half days. The single Schwann cell growing in suspension divided and proliferated around itself until a huge sphere was formed, which was the neurofibroma organoid.

[0086] Example 2

[0087] In this example, neurofibroma tumor spheres were cultured, and the specific process was as follows:

[0088] After the neurofibroma organoids of Example 1 are formed, the cell suspensions of the four wells are collected into a 15 mL centrifuge tube and centrifuged at 1200 rpm for 5 min to obtain the fifth cell precipitate. The supernatant (i.e., the old culture medium) obtained at this time is transferred to another new centrifuge tube. 1 mL of 0.25% trypsin digestion solution is added to the fifth cell precipitate, and the precipitate is digested at a constant temperature of about 10 min in a 37°C metal bath pot until the edge of the white disc precipitate of the organoid is found to be unclear by shaking, and 1 mL of spheroidization culture medium is added to terminate the digestion. Gently blow until the small disc precipitate is no longer visible, centrifuge the digested single cell suspension at 1200 rpm for 5 min, discard the supernatant, add the supernatant corresponding to the fifth cell precipitate and an equal volume of fresh spheroidization culture medium, blow and mix, and transfer to a low-adsorption 24-well plate for culture, with 1 mL of cell suspension per well to form a second-generation neurofibroma tumor sphere.

[0089] Comparative Example 1

[0090] This comparative example cultured neurofibroma tumor spheres. The difference between this comparative example and Example 2 is that the concentrations of EGF and FGF in the sphere culture medium used in this comparative example are both 20 ng / mL, and the other components are the same as those in Example 2; and this comparative example did not perform screening and enrichment of Schwann cells when preparing neurofibroma organoids. The rest of the culture process is the same as Example 2.

[0091] Comparative Example 2

[0092] This comparative example cultured neurofibroma tumor spheres. The difference between this comparative example and Example 2 is that the antibiotics amphotericin B and levofloxacin were not added to the digestion solution and culture medium used in the preparation of neurofibroma organoids and neurofibroma tumor spheres in this comparative example. The rest of the process was the same as Examples 1 and 2.

[0093] Comparative Example 3

[0094] This comparative example cultured neurofibroma organoids. The difference between this comparative example and Example 1 is that in step (3), only spheroidization medium was added to the third cell precipitate, and no growth factor Y-27632 was added. The rest of the process was the same as Example 1.

[0095] Test example

[0096] 1. The neurofibroma tumor spheres cultured in Example 2 and the neurofibroma tumor spheres cultured in Comparative Example 1 were observed under an optical microscope. The results are as follows: Figure 3 shown. Figure 3It shows that the number of neurofibroma tumor spheres cultured in Comparative Example 1 is small and the volume is small, and the number of tumor spheres cannot meet the experimental requirements. This is because Comparative Example 1 did not perform cell screening, and it cannot ensure that Schwann cells account for the vast majority, which affects the formation and proliferation of spheres; in addition, the concentrations of EGF and FGF in the sphere formation medium are low, resulting in a small number of tumor spheres. The number of neurofibroma tumor spheres cultured in Example 2 is more, the volume of the tumor spheres is larger, and each tumor sphere contains more cells. This is because Example 2 screened and enriched Schwann cells, so that 90% of the cells are Schwann cells (the starting cells of plexiform neurofibromas), which can ensure the formation and proliferation of spheres; at the same time, the concentrations of EGF and FGF in the sphere formation medium of Example 2 are increased by 10 times compared with those of Comparative Example 1, which can significantly increase the number and volume of tumor spheres.

[0097] 2. The neurofibroma tumor spheres cultured in Example 2 and Comparative Example 2 were observed under an optical microscope. Figure 4 shown. Figure 4 It shows that the fungal and bacterial contamination in the culture environment of Example 2 is significantly less, while the fungal and bacterial contamination area is larger in the culture environment of Comparative Example 2. It shows that the addition of amphotericin B and levofloxacin can effectively eliminate common fungal and bacterial contamination in cell culture and provide a more stable growth environment for tumor sphere formation.

[0098] 3. The neurofibroma organoids cultured in Example 1 and Comparative Example 3 were observed under an optical microscope. Figure 5 shown.

[0099] Figure 5 The results of the culture without adding growth factor Y-27632 (0uM) and adding 5uM for 6h, removing after 12h and adding 10uM for 6h, removing after 12h, and continuing to culture cells for 24h and 48h, and then taking pictures to monitor the number and volume of tumor spheres. Compared with the pure sphere culture medium without Y-27632 (Comparative Example 3), adding Y-27632 5uM or 10uM can significantly promote the growth of tumor spheres. Among them, the treatment of growth factor Y-27632 5uM for 12h or growth factor Y-27632 10uM for 6h or 12h has significant effects.

[0100] It should be understood that the various forms of processes shown above can be used to reorder, add or delete steps. For example, the steps recorded in this disclosure can be executed in parallel, sequentially or in different orders, as long as the desired results of the technical solution of this disclosure can be achieved, and this document is not limited here.

[0101] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of the features. In the description of the present disclosure, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0102] The above is only a specific embodiment of the present disclosure, but the protection scope of the present disclosure is not limited thereto. Any person skilled in the art who is familiar with the technical field can easily think of changes or substitutions within the technical scope disclosed in the present disclosure, which should be included in the protection scope of the present disclosure. Therefore, the protection scope of the present disclosure should be based on the protection scope of the claims.

Claims

1. A sphere-forming culture medium for neurofibroma organoids, characterized in that: The spheroidization culture medium comprises the following components: DMEM / F12, 1-1.5% B27, 1-1.5% N2, 200-250 ng / mL EGF, 200-250 ng / mL FGF, 0.1-0.3 mM β-ME, 2-3 μg / mL heparin, 2.5-5 μg / mL amphotericin B, and 5-7 μg / mL levofloxacin.

2. The spheroidization culture medium according to claim 1, characterized in that: The neurofibroma organoids are plexiform neurofibroma organoids.

3. A method for culturing neurofibroma organoids, characterized in that: The following steps are involved: S1: Digest neurofibroma tissue blocks and prepare single cell suspensions; S2: placing the single cell suspension described in step S1 in a culture dish treated with poly-lysine, adding Schwann cell culture medium and culturing for 24 hours to obtain Schwann cell enriched solution; S3: Collect the Schwann cell enrichment fluid, and add the spheroidization medium and growth factor Y-27632 described in any one of claims 1 to 2, collect the cells after culturing for 6h-12h, then add the spheroidization medium and continue culturing for 7-14d to obtain the neurofibroma organoid.

4. The culture method according to claim 3, characterized in that: Step S1 digesting the neurofibroma tissue block comprises the following steps: cleaning and chopping the neurofibroma tissue block, and placing it in a digestion solution for digestion, and after the digestion is completed, adding DMEM+10% Clark medium to terminate the digestion; Preferably, the usage ratio of the digestive solution and the neurofibroma tissue blocks is: 5 to 6 neurofibroma tissue blocks are digested for every 20 to 25 mL of the digestive solution; Preferably, the digestive fluid comprises the following components: L-15 culture medium, 50-80 mg / mL gentamicin, 250-280 μg / mL amphotericin B, 500-520 μg / mL levofloxacin, 10,000 IU penicillin / streptomycin / ampicillin, 155-160 u / mL type I collagenase, and 1.25-1.5 u / mL dispersase II.

5. The culture method according to claim 3, characterized in that: Step S1: after digesting the neurofibroma tissue block, blowing the tissue apart, centrifuging it, discarding the supernatant, and obtaining a first cell pellet; Preferably, step S1 further comprises the following steps: adding fresh DMEM+10% Clark culture medium to the first cell precipitate, blowing with a 1000 μL pipette tip until no tissue blocks are visible to the naked eye, replacing the glass tube for blowing to blow off the Schwann cells adhered around the nerves; then centrifuging and discarding the supernatant to obtain a second cell precipitate; adding Schwann cell culture medium to the second cell precipitate to obtain the single cell suspension.

6. The culture method according to claim 3, characterized in that: Step S2: treating the culture dish with 0.05-0.1% poly-lysine for ≥30 min, and then washing the culture dish with PBS for 2-3 times; Preferably, step S2 is to screen and enrich in the culture dish for 24 to 26 hours, during which the medium is changed 2 to 3 times to obtain the Schwann cell enrichment medium; Preferably, in the Schwann cell enrichment solution, the proportion of Schwann cells is more than 90%.

7. The culture method according to claim 3, characterized in that: Step S3 of collecting the Schwann cell enriched solution specifically comprises the following steps: digesting the Schwann cell enriched solution in step S2, centrifuging after pipetting, discarding the supernatant, and obtaining a third cell precipitate; Preferably, the Schwann cell enrichment solution in step S2 is digested with 0.25% trypsin digestion solution for 8 to 10 minutes, and then DMEM+10% Clark medium is added to terminate the digestion.

8. The culture method according to claim 7, characterized in that: Step S3: adding the spheroidization medium and the growth factor Y-27632 to the third cell pellet for resuspending, and transferring the cell suspension to a low-adsorption cell culture plate for suspension culture for 6-12 hours; Preferably, the concentration of the growth factor Y-27632 is 5-10 μM; Preferably, in step S3, the cells are collected after culturing for 6-12 hours, and centrifuged at 1000-1500 rpm for 5-8 minutes, the supernatant is discarded to obtain a fourth cell pellet, and the spheroidization medium is added to the fourth cell pellet and culture is continued for 7-14 days to obtain the neurofibroma organoid.

9. A method for culturing neurofibroma tumor spheres, characterized in that: The following steps are involved: The neurofibroma organoid is formed by the culture method according to any one of claims 3 to 8, the neurofibroma organoid is digested into a single cell suspension, and the suspension is transferred to a low-adsorption cell culture plate for culture to form the neurofibroma tumor sphere.

10. The culture method according to claim 9, characterized in that: Digesting the neurofibroma organoid into the single cell suspension specifically includes the following steps: centrifuging the cell suspension of the neurofibroma organoid to obtain a fifth cell pellet, and retaining the supernatant separately; digesting the fifth cell pellet, centrifuging it again, discarding the supernatant, and obtaining a sixth cell pellet; adding the supernatant corresponding to the fifth cell pellet and an equal volume of fresh spheroid culture medium to the sixth cell pellet to form the single cell suspension.

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