CN116622616A - Bile duct organoid culture medium and culture method - Google Patents
Bile duct organoid culture medium and culture method Download PDFInfo
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- CN116622616A CN116622616A CN202310510721.6A CN202310510721A CN116622616A CN 116622616 A CN116622616 A CN 116622616A CN 202310510721 A CN202310510721 A CN 202310510721A CN 116622616 A CN116622616 A CN 116622616A
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- 210000002220 organoid Anatomy 0.000 title claims abstract description 69
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
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Abstract
The invention discloses a bile duct organoid culture medium and a culture method, and belongs to the field of culture mediums. The bile duct organoid medium comprises: advanced DMEM/F12, HEPES, N21, N-acetgl-L-cysteine, glutaMAX, epiregulin, wnt a, R-spondin1, noggin protein, HGF recombinant protein, FGF10, thiazovivin, A8301, forskolin, gastrin1, PGE2, primocin, nicotinamide and P/S. The bile duct organoid culture medium can form organoids after culturing or passaging for 1-2 days, and the organoids have very obvious diameter increase along with the increase of the culturing time, so that the bile duct organoid culture medium can efficiently culture bile duct organoids. The preservation solution and the digestive juice in the culture method can be matched with the bile duct organoid culture medium to efficiently culture the bile duct organoid.
Description
Technical Field
The disclosure relates to the field of culture media, in particular to a bile duct organoid culture medium and a culture method.
Background
Organoids have particular potential in tissue repair because they retain the primary function and characteristics of tissue. Several years ago, scientists have found a method for artificially culturing bile duct organoids and successfully achieved in animal experiments. Cholangiocytes cultured in the experiments were transplanted into the donated human liver, which was maintained in vitro. This experiment performed on human livers eventually demonstrated that cells they cultured did "stitch" into damaged liver tissue. Thus, the organ which is not suitable for transplantation originally can be reused after being repaired; the organ that needs to be replaced may also be returned to an available state.
However, the culture medium for culturing the bile duct organoids adopted at present has long culture time and is not beneficial to the later application of the bile duct organoids.
BRIEF SUMMARY OF THE PRESENT DISCLOSURE
In order to solve the problems in the prior art, embodiments of the present disclosure provide a bile duct organoid medium and a culturing method. The technical scheme is as follows:
in one aspect, the present disclosure provides a bile duct organoid medium comprising: advanced DMEM/F12, HEPES, N21, N-acetgl-L-cysteine, glutaMAX, epiregulin, wnt a, R-spondin1, noggin protein, HGF recombinant protein,
FGF10, thiazovivin, A8301, forskolin, gastrin1, PGE2, primocin, nicotinamide and P/S, advanced DMEM/F12, HEPES, N21, N-acegl-L-cysteine, glutaMAX, epiregulin, wnt3a, R-spondin1, noggin protein, HGF recombinant protein, recombinant protein,
The concentrations of FGF10, thiazovivin, A8301, forskolin, gastrin, PGE2, primocin, nicotinamide and P/S were in the order:
1×、10～20mM、1～5×、0.5～5mM、2～10mM、50～600ng/mL、50～500ng/mL、500-
3000ng/mL, 50-500 ng/mL, 5-100 ng/mL, 10-500 ng/mL, 0.5-5. Mu.M, 1-10. Mu.M, 5-20 nM, 0.5-5. Mu.M, 100-200. Mu.g/mL, 5-20 mM and
100～200U/mL。
in another aspect, the present disclosure provides a method of culturing a bile duct organoid, the method comprising:
digesting the bile duct stored in the preservation solution to obtain digested bile duct cells;
culturing the digested cholangiocytes by using the cholangioceanic culture medium to obtain the cholangioceanic organs.
Specifically, the preservation solution comprises: DMEM/F12, Y-27632, primocin, P/S, glutaMAX and BSA.
Specifically, the bile duct digestion comprises:
cleaning the bile duct with a cleaning liquid, and cutting the bile duct into small pieces to obtain bile duct tissue fragments;
adding digestive juice into the bile duct tissue fragments, and vibrating and digesting for 30-50 min in a 37 ℃ incubator. Blowing once every 15 min;
adding 5mL of cleaning solution, and centrifuging 300g for 5min at 4 ℃ to obtain a first precipitate;
adding 5mL of cleaning solution into the first precipitate for resuspension, filtering with a filter membrane with the aperture of 70 mu m, collecting filtrate, and centrifuging 300g of the filtrate at 4 ℃ for 5min to obtain a second precipitate;
and adding 2mL of erythrocyte lysate into the second precipitate, stopping the lysis with 5mL of cleaning liquid after the lysis for 3min, and centrifuging 300g at 4 ℃ for 5min to obtain a third precipitate, wherein the third precipitate is the bile duct cells after digestion.
Specifically, the digestive juice comprises: DMEM/F12, collagenase, DNaseI, primocin and P/S.
Further, culturing the digested biliary tract tissue with the biliary tract organoid medium comprises:
preparing the third precipitate into a cell suspension;
uniformly mixing the cell suspension with melted matrigel to obtain mixed gel;
dripping the mixed glue into a culture dish;
adding the bile duct organoid culture medium into the culture dish for culture.
The technical scheme provided by the embodiment of the disclosure has the beneficial effects that: the embodiment of the invention provides a bile duct organoid culture medium and a culture method. The preservation solution and the digestive juice in the culture method can be matched with the bile duct organoid culture medium to efficiently culture the bile duct organoid.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the drawings required for the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present disclosure, and other drawings may be obtained according to these drawings without inventive effort for a person of ordinary skill in the art.
FIG. 1 is an enlarged view under a 10X microscope at 1 day of P0-generation culture in biliary organoid medium provided in accordance with one embodiment of the present disclosure;
FIG. 2 is an enlarged view under a 20X microscope at 4 days of P0 generation culture in biliary organoid medium provided in accordance with one embodiment of the present disclosure;
FIG. 3 is an enlarged view under a 10X microscope at 5 days of P0 generation culture in biliary organoid medium provided in accordance with one embodiment of the present disclosure;
FIG. 4 is an enlarged view of a 20X microscope at 5 days of P0 generation culture in biliary organoid medium provided in accordance with one embodiment of the present disclosure, wherein the scale is 100um;
FIG. 5 is a chart showing the culture of the biliary organoid medium according to the first embodiment of the present disclosure at 2 at 6 days of the P0-generation culture1An enlarged view under a 0X microscope, wherein the scale is 100um;
FIG. 6 is an enlarged view of a P0 generation culture of cholangioid media culture under a 20X microscope at 6 days, with a scale of 100um, provided in an embodiment of the present disclosure;
FIG. 7 is an enlarged view under a 10X microscope at day 0 of P1-generation culture in biliary organoid medium provided in accordance with one embodiment of the present disclosure;
FIG. 8 is an enlarged view of a P1-generation culture of cholangioid media according to the first embodiment of the present disclosure under a 20X microscope at 1 day, wherein the scale is 100um;
FIG. 9 is a schematic illustration of a P1-generation culture 1 cultured in a bile duct organoid medium according to an embodiment of the present disclosure2At the time of day 21An enlarged view under a 0X microscope, wherein the scale is 100um;
FIG. 10 is an enlarged view of a P1 generation culture of cholangioid media culture under a 20X microscope at 2 days, with a scale of 100um, provided in an embodiment of the present disclosure;
FIG. 11 is an enlarged view of a 4 day P1 substitution culture under a 20X microscope, with a scale of 100um, in accordance with an embodiment of the present disclosure;
FIG. 12 is an enlarged view under a 10X microscope at 5 days of P1-generation culture in biliary organoid medium provided in accordance with one embodiment of the present disclosure, wherein the scale is 100um;
FIG. 13 is a P1-generation culture 1 of a bile duct organoid medium according to a second embodiment of the present disclosure4Magnification under a 10X microscope at day 1;
FIG. 14 is a P1-generation culture 1 of a biliary organoid culture medium provided in embodiment III of the disclosure4Magnified view under a 10X microscope at day time.
Detailed Description
For the purposes of clarity, technical solutions and advantages of the present disclosure, the following further details the embodiments of the present disclosure with reference to the accompanying drawings.
Example 1
Bile duct organoids medium includes: advanced DMEM/F12 (Dulbecco's modified Eagle Medium/Ham's F-12), HEPES (4-hydroxyethyl piperazine ethanesulfonic acid buffer), N21, N-acetylgl-L-cysteine, glutaMAX (L-alanyl-L-glutamine dipeptide), epiregulin (epidermal regulator), wnt3a protein, R-spondin1 protein, noggin protein, HGF recombinant protein, FGF10 (fibroblast growth factor 10), FGF10,
thiazovivin, A8301, forskolin, gastin 1 (Gastrin), PGE2 (dienoprost), primocin, nicotinamide (nicotinamide) and P/S (diabody (penicillin plus streptomycin)), advanced DMEM/F12, HEPES, N21, N-acegl-L-cysteine, glutaMAX, epiregulin, wnt3a, R-spondin1, noggin protein, HGF recombinant protein, noggin protein,
The concentrations of FGF10, thiazovivin, A8301, forskolin, gastrin, PGE2, primocin, nicotinamide and P/S (penicillin-streptomycin) are shown in Table 1 below.
Table 1 shows the composition and concentration of the bile duct organoid medium according to the first embodiment of the invention
Example two
Bile duct organoids medium includes: advanced DMEM/F12, HEPES, N21, N-acetgl-L-cysteine, glutaMAX, epiregulin, wnt a, R-spondin1, noggin protein, HGF recombinant protein,
FGF10, thiazovivin, A8301, forskolin, gastrin1, PGE2, primocin, nicotinamide and P/S, advanced DMEM/F12, HEPES, N21, N-acegl-L-cysteine, glutaMAX, epiregulin, wnt3a, R-spondin1, noggin protein, HGF recombinant protein, recombinant protein,
The concentrations of FGF10, thiazovivin, A8301, forskolin, gastrin, PGE2, primocin, nicotinamide and P/S are shown in Table 2 below.
Table 2 shows the composition and concentration of the bile duct organoid medium according to the second embodiment of the invention
Example III
Bile duct organoids medium includes: advanced DMEM/F12, HEPES, N21, N-acetgl-L-cysteine, glutaMAX, epiregulin, wnt a, R-spondin1, noggin protein, HGF recombinant protein,
FGF10, thiazovivin, A8301, forskolin, gastrin1, PGE2, primocin, nicotinamide and P/S, advanced DMEM/F12, HEPES, N21, N-acegl-L-cysteine, glutaMAX, epiregulin, wnt3a, R-spondin1, noggin protein, HGF recombinant protein, recombinant protein,
The concentrations of FGF10, thiazovivin, A8301, forskolin, gastrin, PGE2, primocin, nicotinamide and P/S are shown in Table 3 below.
Table 3 shows the composition and concentration of the bile duct organoid medium according to the third embodiment of the invention
| Reagent name | Company name | Goods number | Concentration of |
| Advanced DMEM/F12 | Thermo-Gibco | 12634010 | 1× |
| HEPES | Thermo-Gibco | 15630080 | 20mM |
| N21 | R&D system | AR008 | 5× |
| N-acetgl-L-cysteine | MCE | HY-B0215 | 0.5mM |
| GlutaMAX | Thermo-Gibco | 35050061 | 2mM |
| Epiregulin | MCE | HY-P7011 | 600ng/mL |
| Wnt3a | R&D system | 5036-WN | 50ng/mL |
| R-spondin1 | R&D system | 4645-RS | 3000ng/mL |
| noggin | R&D system | 6057-NG | 50ng/mL |
| HGF | R&D system | 294-HG | 100ng/mL |
| FGF10 | R&D system | 345-FG | 10ng/mL |
| thiazovivin | MCE | HY-13257 | 5μM |
| A8301 | R&D system | 2939 | 1μM |
| forskolin | R&D system | 1099 | 20μM |
| Gastrin1 | R&D system | 3006 | 5nM |
| PGE2 | MCE | HY-101952 | 5μM |
| Primocin | InvivoGen | ant-pm-1 | 200μg/ml |
| Nicotinamide | R&D system | 4106 | 5mM |
| P/S | Thermo-Life | 15140122 | 100U/mL |
Example IV
The embodiment provides a method for culturing biliary tract organoids, which uses the biliary tract of a cynomolgus monkey as a sample for culturing, wherein the liver sample of the cynomolgus monkey is purchased from experimental animals limited company in the department of western mountain in su state, and the specific culturing method is as follows:
bile duct pretreatment method for cynomolgus monkey
The whole liver of the cynomolgus monkey is taken down by adopting a perfusion method, liver parenchymal cells in the liver are used for other purposes in a laboratory, the bile duct obtained in the embodiment comes from residual tissues of perfusion of the liver in the laboratory, specifically, the white bile duct in the liver is sheared off by scissors and put into preservation solution to be preserved at 4 ℃ for 24 hours. Digesting the bile duct stored in the preservation solution to obtain digested bile duct cells;
culturing the digested cholangiocytes by adopting the cholangiocellate culture medium to obtain the cholangiocellate.
Specifically, the preservation solution includes: DMEM/F12 (nutrient cocktail F-12), Y-27632 (dihydrochloride), primocin (primary cell antibiotic), P/S, glutaMAX and BSA (Bovine serum albumin). The components and concentrations of the preservation solution are shown in Table 4.
Table 4 shows the components and concentrations of the preservation solution
| Reagent name | Company name | Goods number | Concentration of |
| DMEM/F12 | biosharp | BL305A | 1× |
| Y-27632 | R&D system | 1254 | 10μM |
| Primocin | InvivoGen | ant-pm-1 | 100μg/mL |
| P/S | Thermo-Life Tech | 15140122 | 1× |
| GlutaMAX | Thermo-Gibco | 35050061 | 1× |
| BSA | Sigma-Aldrich | B2064-50g | 0.1％ |
Specifically, bile duct digestion includes:
cleaning the bile duct with a cleaning liquid, and cutting the bile duct into small blocks to obtain bile duct tissue fragments;
adding digestive juice into bile duct tissue fragments, and shaking and digesting for 30-50 min in a incubator at 37 ℃. Blowing once every 15 min;
adding 5mL of cleaning solution, and centrifuging 300g for 5min at 4 ℃ to obtain a first precipitate;
adding 5mL of cleaning solution into the first precipitate for resuspension, filtering with a filter membrane with the aperture of 70 μm, collecting filtrate, and centrifuging 300g of the filtrate at 4 ℃ for 5min to obtain a second precipitate;
2mL of erythrocyte lysate is added to the second precipitate, after 3min of lysis, 5mL of washing liquid is used for stopping the lysis, 300g of the mixture is centrifuged for 5min at 4 ℃ to obtain a third precipitate, and the third precipitate is digested cholangiocytes.
Specifically, the digestive juice includes: DMEM/F12, collage (collagenase), DNaseI (Deoxyribonuclease I, DNaseI), primocin and P/S. The components and concentrations of the digestive juice are shown in Table 5.
Table 5 shows the components and concentrations of the digestive juice
| Reagent name | Company name | Goods number | Concentration of |
| DMEM/F12 | biosharp | BL305A | 1× |
| collagenase | Sigma-Aldrich | C9407-500MG | 1.5mg/mL |
| DNaseI | Sigma-Aldrich | DN25 | 0.1mg/mL |
| Primocin | InvivoGen | ant-pm-1 | 100μg/mL |
| P/S | Thermo-Life Tech | 15140122 | 1× |
Further, culturing the digested biliary tract tissue with the biliary tract organoid medium described above comprises:
preparing a third precipitate into a cell suspension;
mixing the cell suspension with melted matrigel to obtain mixed gel;
dripping the mixed glue into a culture dish;
the cholangioid culture medium provided in examples one to three was added to each of the plurality of dishes for culture and verification of the results. The bile duct organoid verification results provided in the first embodiment are shown in fig. 1 to 12, and the procedure of organoid growth from 1 day to 6 days of generation P0 can be seen in conjunction with fig. 1 to 6, wherein the diameters of some classified organs can reach about 350um when the organoid is cultured for 5 days, and the diameters of some classified organs can reach about 550um when the organoid is cultured for 6 days.
As can be seen in connection with fig. 7 to 12, fromGeneration P1A process of organoid growth from 0 days to 5 days, wherein 0 days is just digested single cells or small clusters of several cells together; after 1 day of culture, the diameter of a part of organoids can reach about 140 um; after 2 days of culture, the diameter of partial organoids can reach about 250 um; when cultured for 4 days, the diameter of a part of organoids can reach about 300 um; cultivation methodWhen the culture is carried out for 5 days, the diameter of a part of organoids can reach about 650 um.
Comparative example one
This comparative example one differs from example one in that R-spondin1 is absent.
The cynomolgus monkey bile duct organoids were cultured using the ingredients provided in comparative example one according to the culture method provided in example one. After 3 days of culture, the bile duct organoids were found to grow slowly, and after 4 days the cells were collected and then counted with trypan blue staining, the results of the counting are shown in table 6.
Table 6 shows the number of living cells, the total number of cells and the proportion of living cells
As can be seen from Table 6, the total number of cells and the proportion of living cells were significantly smaller than in example one. This suggests that R-spondin1 is beneficial for bile duct organoid cell growth.
Comparative example two
The difference between the second comparative example and the first example is that the bile duct organ of cynomolgus monkey is cultured by the culture method provided in the first example using the components provided in the first comparative example without Wnt3a protein. After 3 days of culture, the number of bile duct organoids was shown to be very small and the size to be very small. This suggests that Wnt3a protein is beneficial for bile duct organoid cell growth.
The embodiment of the invention provides a bile duct organoid culture medium and a culture method, wherein the bile duct organoid culture medium can be used for culturing the bile duct organoidCultivation or cultivationThe organoids are formed after passage for 1-2 days,and the organoid diameter increases very significantly with increasing culture time,the culture medium can be used for efficiently culturing biliary organs. The preservation solution and the digestive juice in the culture method can be matched with the bile duct organoid culture medium to efficiently culture the bile duct organoid. Because the cynomolgus monkey is similar to the human gene, the bile duct organoid medium and the culture method in the embodiment are also applicableCulturing human biliary tract organoids.
The foregoing description of the preferred embodiments of the present disclosure is provided for the purpose of illustration only, and is not intended to limit the disclosure to the particular embodiments disclosed, but on the contrary, the intention is to cover all modifications, equivalents, alternatives, and alternatives falling within the spirit and principles of the disclosure.
Claims (6)
1. A bile duct organoid medium, the bile duct organoid medium comprising: advanced DMEM/F12, HEPES, N21, N-acetgl-L-cysteine, glutaMAX, epiregulin, wnt a, R-spondin1, noggin protein, HGF recombinant protein,
FGF10, thiazovivin, A8301, forskolin, gastrin1, PGE2, primocin, nicotinamide and P/S, advanced DMEM/F12, HEPES, N21, N-acetgl-L-cysteine, glutaMAX, epiregulin, wnt a, R-spondin1, noggin protein, HGF recombinant protein, recombinant protein,
The concentrations of FGF10, thiazovivin, A8301, forskolin, gastrin1, PGE2, primocin, nicotinamide and P/S were in the order:
1×、10～20mM、1～5×、0.5～5mM、2～10mM、50～600ng/mL、50～500ng/mL、500-
3000ng/mL, 50-500 ng/mL, 5-100 ng/mL, 10-500 ng/mL, 0.5-5. Mu.M, 1-10. Mu.M, 5-20 nM, 0.5-5. Mu.M, 100-200. Mu.g/mL, 5-20 mM and 100-200U/mL.
2. A method of culturing a bile duct organoid, the method comprising:
digesting the bile duct stored in the preservation solution to obtain digested bile duct cells;
culturing the digested cholangiocytes by using the cholangiocellular substance culture medium according to claim 1 to obtain the cholangiocellular substance.
3. The culture method according to claim 2, wherein the preservation solution comprises:
DMEM/F12, Y-27632, primocin, P/S, glutaMAX and BSA.
4. The culture method of claim 2, wherein the bile duct digestion comprises:
cleaning the bile duct with a cleaning liquid, and cutting the bile duct into small pieces to obtain bile duct tissue fragments;
adding digestive juice into the bile duct tissue fragments, and vibrating and digesting for 30-50 min in a 37 ℃ incubator. Blowing once every 15 min;
adding 5mL of cleaning solution, and centrifuging 300g for 5min at 4 ℃ to obtain a first precipitate;
adding 5mL of cleaning solution into the first precipitate for resuspension, filtering with a filter membrane with the aperture of 70 mu m, collecting filtrate, and centrifuging 300g of the filtrate at 4 ℃ for 5min to obtain a second precipitate;
and adding 2mL of erythrocyte lysate into the second precipitate, stopping the lysis with 5mL of cleaning liquid after the lysis for 3min, and centrifuging 300g at 4 ℃ for 5min to obtain a third precipitate, wherein the third precipitate is the bile duct cells after digestion.
5. The culture method according to claim 2, wherein the digestive juice comprises: DMEM/F12, collagenase, DNaseI, primocin and P/S.
6. The method of culturing of claim 4, wherein culturing the digested biliary tract tissue with the biliary tract organoid medium of claim 1 comprises:
preparing the third precipitate into a cell suspension;
uniformly mixing the cell suspension with melted matrigel to obtain mixed gel;
dripping the mixed glue into a culture dish;
adding the bile duct organoid medium according to claim 1 to the culture dish for culture.
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