CN117343904A - Keloid organoid culture medium and culture method - Google Patents
Keloid organoid culture medium and culture method Download PDFInfo
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- CN117343904A CN117343904A CN202311338983.5A CN202311338983A CN117343904A CN 117343904 A CN117343904 A CN 117343904A CN 202311338983 A CN202311338983 A CN 202311338983A CN 117343904 A CN117343904 A CN 117343904A
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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
- C12N5/0693—Tumour cells; Cancer cells
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0278—Physical preservation processes
- A01N1/0284—Temperature processes, i.e. using a designated change in temperature over time
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- C12N2500/00—Specific components of cell culture medium
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- C12N2500/32—Amino acids
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Abstract
The invention discloses a keloid organoid culture medium and a culture method, which relate to the organoid culture field, wherein the keloid organoid culture medium comprises a basal culture medium advanced DMEM/F12 and specific additive factors, and the specific additive factors comprise the following components: primocin, HEPES, glutaMAX, B-27 serum free additive, N-acetyl-L-cysteine, mEGF, hFGF, hR-spondin1, afamin-Wnt3A and Nicotinamide; the keloid organoid culture medium can successfully culture keloid organoids aiming at the growth characteristics of keloid tissue-derived cells, and can obtain sufficient organoid development scientific research and sample preservation in a limited time by combining organoid passage and cryopreservation technology.
Description
Technical Field
The invention relates to the field of organoid culture, in particular to a keloid organoid culture medium and a culture method.
Background
Organoids (Organoids) are complex three-dimensional structural systems composed of multiple cells, which exhibit similar genetic characteristics and structural stability in vitro as cells in vivo, and which can be smoothly induced into functional tissue cells. The tumor organoid is a living tissue which is obtained by 3D culture of fresh tissue from a patient in vitro, has similar pathological characteristics and genetic background as the tumor tissue in vivo and can be stably passaged. Compared with animal models, the tumor organoid culture system has the characteristics of simpler and more convenient, low cost, short period, high flux and the like, has wide application and development prospects in clinical tumor accurate drug administration, new drug research and development, regenerative medicine and basic research, and fundamentally leads to implementation of a tumor accurate treatment concept.
Keloids are benign tumors characterized by invasive growth due to excessive proliferation of fibroblasts, excessive deposition of collagen in the extracellular matrix during the healing process of skin wounds, the most common good hair sites being the anterior chest, shoulders, upper back and earlobe, representing hard, shiny, elastic lesions, often accompanied by itching, pain, disfigurement and joint contractures. Keloids may lead to serious functional and psychological sequelae, affecting the quality of life of the patient. The primary treatment of keloids is the injection of corticosteroids within the lesions, other treatments including cryotherapy, 5-fluorouracil, radiation therapy, laser therapy, surgical excision or silicone occlusive dressings, which can be used as monotherapy or in combination with other treatments. Because the pathogenesis of keloids is quite complex, although various treatment schemes exist, the treatment effect is often unsatisfactory, and the recurrence rate is up to 45% -100%. Therefore, the establishment of culture of keloid organoids is of great significance to therapeutic drug screening and scientific research.
Although there are a variety of organoid media and organoid culture methods, such as gastric organoid media and culture methods, the media required and the applicable culture methods for different organoids are not the same and these existing media and culture methods cannot be taken directly for keloid organoid culture.
Disclosure of Invention
The invention aims to: the invention provides a culture medium and a culture method for keloid organoids. Aiming at the growth characteristics of keloid tissue source cells, the culture medium can successfully culture keloid organoids; the culture method provided by the invention comprises a keloid organoid establishment method, a passage method, freezing storage and resuscitation, and the method is good in culture operability, can successfully culture the keloid organoid and passage, and can obtain sufficient organoid development scientific research and save samples in a limited time.
The technical content is as follows: a keloid organoid medium comprising basal medium advanced DMEM/F12 and specific additive factors comprising the following components: primocin, HEPES, glutaMAX, B-27 serum free additives, N-acetyl-L-cysteine, mEGF, hFGF, hR-spondin1, afamin-Wnt3A and Nicotinamide.
Preferably, the final concentrations of the components of the specific additive factor in the keloid organoid medium are as follows:
primocin，0.5～5×；
HEPES，1～100mM；
GlutaMAX，0.2～20mM；
b-27 is free of serum additives, 0.5 to 10X;
N-acetyl-L-cysteine，0.1～200mM；
mEGF，1～300ng/ml；
hFGF10，1～500ng/ml；
hR-spondin 1，0.1～50mg/ml；
Afamin-Wnt3A, 5-50% volume concentration;
Nicotinamide，0.1～200mM。
preferably, the concentrations of the components of the specific additive factor in the keloid organoid medium are as follows:
primocin，1×；
HEPES，10mM；
GlutaMAX，2mM；
b-27 was free of serum additives, 1×;
N-acetyl-L-cysteine，1mM；
mEGF，50ng/ml；
hFGF10，200ng/ml；
hR-spondin 1，1mg/ml；
Afamin-Wnt3A,25% by volume;
Nicotinamide，10mM。
the invention provides a method for establishing keloid organoids, which comprises the following steps:
step one, pretreating keloid tissues to obtain keloid cells;
step two, establishing keloid organoids; mixing keloid cells with matrigel, standing at 37deg.C, and 5% CO 2 Adding keloid organoid culture medium after matrix gel is solidified in cell incubator, again placing at 37deg.C and 5% CO 2 Culturing in a cell culture box, changing the culture medium every 4 days, and culturing for 4-28 days to obtain keloid organoids.
Further, in the first step, the pretreatment of keloid tissues is as follows:
1) Collecting keloid tissue, washing with PBS, removing fat, cutting mung bean tissue block, washing with PBS for at least 2 times, soaking in PBS for 3-5min, and discarding PBS;
2) Soaking the tissue block with the double antibody for 3-5min, and discarding the double antibody;
3) Moistening the tissue blocks in a plate with tissue digestion liquid, cutting up, transferring all the tissue blocks into a centrifuge tube with the tissue digestion liquid, and carrying out shaking table digestion for 15-60 min at 37 ℃;
4) Blowing and mixing uniformly by a pipetting gun, standing, taking supernatant into a centrifuge tube, adding a wash to stop digestion, centrifuging, and discarding the supernatant to obtain cell sediment, namely keloid cells.
Further, the specific procedure for creating keloid organoids in step two is as follows:
1) Taking out matrigel, placing on ice, mixing matrigel with keloid cells with a pipette, and placing on ice;
2) Before experiment, the well plate is placed in a 37 ℃ cell incubator to raise the temperature for at least 30min, the well plate which is preheated is taken out before inoculation, 50 mu l of matrigel mixed with cells is dripped in the center of each well, and the well plate is placed in 37 ℃ and 5% CO 2 Placing in cell incubator for 15min, adding 500 μl of keloid organoid culture medium into each well after matrix gel is solidified, and placing again at 37deg.C and 5% CO 2 Culturing in a cell culture box, changing the culture medium once for 4 days, and culturing for 4-28 days to obtain keloid organoids.
The invention provides a keloid organoid subculture method, which comprises the following steps:
1) Taking keloid organoid pore plates obtained after 4-28 days of culture, sucking and removing culture medium in the pores, blowing with matrigel digestive juice, transferring into a centrifuge tube after matrigel is completely melted, performing water bath digestion at 37 ℃ for 5min, centrifuging at 1400rpm for 5min, and sucking and removing supernatant;
2) Adding TrypLE Express enzyme to the centrifuge tube to resuspend cells; placing the centrifuge tube in a water bath at 37 ℃ for digestion for 5min, adding a wash with the same volume as TrypLE Express enzyme after digestion is finished, stopping digestion, centrifuging at 1400rpm for 5min, and discarding the supernatant to obtain a cell precipitate;
3) Taking out matrigel, placing on ice, sucking matrigel, adding into cell sediment, blowing and mixing to obtain matrigel mixed with cells;
4) Placing the pore plate in a 37 ℃ cell incubator for heating for at least 30min before the experiment, taking out the preheated pore plate before inoculation, dripping 50 μl of matrigel mixed with cells into the center of the pore plate, and solidifying for 15min at 37 ℃; after the matrigel had solidified, 500. Mu.l of keloid organoid medium was added to the wells and placed at 37℃in 5% CO 2 The cells were cultured in an incubator with medium changed every 4 days.
The invention provides a keloid organoid cryopreservation method, which comprises the following steps:
1) Collecting keloid organoid pore plate obtained after culturing for 4-28 days, sucking and removing the culture medium in the pore, blowing with matrigel digestive juice, transferring into centrifuge tube after matrigel is completely melted, digesting for 5min in water bath at 37deg.C, centrifuging at 1400rpm for 5min, and sucking and removing supernatant;
2) Adding TrypLE Express enzyme to the centrifuge tube to resuspend cells; placing the centrifuge tube in a 37 ℃ water bath for digestion for 5min, adding a wash with the same volume as TrypLE Express enzyme to terminate digestion after digestion is finished, centrifuging at 1400rpm for 5min, and discarding the supernatant to obtain a cell precipitate;
3) Adding frozen stock solution into cell sediment, blowing and mixing, transferring into frozen stock tube, placing into gradient cooling frozen stock box, placing at-80deg.C for 1-2 weeks, and transferring into liquid nitrogen for preservation.
The invention provides a keloid organoid resuscitation method, which is characterized by comprising the following steps:
1) Taking out a freezing tube containing keloid organoids, wherein the cell freezing solution in the freezing tube is 300 μl, melting at 37 ℃ for 1min, and transferring into a centrifuge tube;
2) Adding 10ml wash,1400rpm into a centrifuge tube, centrifuging for 5min, and absorbing and discarding the supernatant to obtain a cell precipitate;
3) Placing matrigel on ice, sucking matrigel and cell sediment by a pipetting gun, and blowing and mixing uniformly;
4) Placing the pore plate in a cell incubator at 37 ℃ for heating for at least 30min before experiments, taking out the preheated pore plate before inoculation, dripping 50 μl of matrigel mixed with cell sediment at the center of each pore, and solidifying for 15min at 37 ℃; after solidification, 500. Mu.l of keloid organoid medium was added to each well and placed at 37℃in 5% CO 2 The cells were cultured in an incubator with medium changed every 4 days.
The beneficial effects are that:
1) The keloid organoid culture medium can successfully culture keloid organoids aiming at the growth characteristics of keloid tissue-derived cells, and can obtain sufficient organoid development scientific research and sample preservation in a limited time by combining organoid passage and cryopreservation technology.
2) The culture medium can effectively maintain the specificity of tissue cells, and the cultured keloid organoids are highly consistent with the primary tissues in histopathology.
3) The keloid external organoid cultured by the culture medium can meet the requirements of scientific research and provide more choices for the medication guidance of patients.
Drawings
FIG. 1 is a photograph of a keloid organoid obtained in example 5 under a plain light microscope;
FIG. 2 shows HE staining of keloid primary tissue;
FIG. 3 shows the results of HE staining of keloid organoids cultured in example 5;
FIG. 4 shows immunofluorescence of keloid primary tissue;
FIG. 5 shows the immunofluorescence of the keloid organoids cultured in example 5;
FIG. 6 is a photograph of comparative example 1 under a general optical microscope;
FIG. 7 is a photograph of comparative example 2 under a general optical microscope;
FIG. 8 is a photograph of comparative example 3 under a general optical microscope;
fig. 9 is a photograph of comparative example 4 under a general optical microscope.
Detailed Description
The invention is further described below with reference to the accompanying drawings, without limiting the invention in any way, and any modifications based on the teachings of the invention fall within the scope of the invention.
The sources of the raw materials involved in the embodiment of the invention are as follows:
advanced DMEM/F12: purchased from gibco company and stored at 4 ℃.
Primocin: purchased from the company invivoGen, -20 ℃ for storage.
HEPES: purchased from gibco company and stored at 4 ℃.
GlutaMAX: purchased from gibco company and stored at room temperature.
B-27 serum-free additive: purchased from Yeasen company, -20℃for storage.
N-acetyl-L-cysteine: purchased from sigma company and stored at 4 ℃.
recombinant mouse EGF: purchased from Novoprotein Inc., stored at-20 ℃.
FGF10 Protein, human, recommant, HPLC-verify: purchased from Yiqiao shenzhou corporation and stored at-20 ℃.
hR-spondin 1: purchased from Yeasen corporation, stored at-80 ℃.
Afamin-Wnt3A: purchased from Beijing Boehmeria biotechnology Co., ltd., -20℃for storage.
Nicotinamide: purchased from sigma company and stored at 4 ℃.
Recombinant DNase I: purchased from Roche, inc., stored at 4 ℃.
collageenase II: purchased from gibco company and stored at 4 ℃.
Ceturegel Matrix LDEV-Free matrigel (abbreviated matrigel in the examples of the invention): purchased from Yeasen company, -20℃for storage.
TrypLE Express enzyme (1×) (abbreviated as TrypLE in the examples of the present invention), phenol red: purchased from gibco company and stored at room temperature.
Dispersion enzyme II was purchased from gibco and stored at 4 ℃.
PBS (phosphate buffer 1×): purchased from seville and stored at room temperature.
Green streptomycin mixture (double antibody 100×): purchased from Seville and stored at 4 ℃.
Cryopreservation solution (cell culture cryopreservation medium): purchased from thermo, stored at 4 ℃.
Example 1
The keloid organoid culture medium comprises a basal culture medium advanced DMEM/F12 and specific additive factors, wherein the concentrations of each component in the specific additive factors in the keloid organoid culture medium are as follows: primocin,1×; HEPES,10mM; glutamax,2mM; b-27 was free of serum additives, 1×; N-acetyl-L-cysteine,1mM; mEGF,50ng/ml; hFGF10, 200ng/ml; hR-spondin1, 1mg/ml; afamin-Wnt3A,25% by volume; nicotinamide,10mM. The culture medium of this example is shown in the following table:
example 2
The tissue digestion liquid digest medium is prepared by using a wash, and comprises the following components in percentage by weight: 1000U of Dnase I was mixed with 1g of collageenase II in 200ml of wash and filtered at-80℃at 10 ml/tube. The tissue digests referred to in examples 5-8 and comparative examples 1-4 were prepared in this example.
Example 3
A matrigel digestive juice dispase II is prepared by the following steps: 0.5g of Dispersion enzyme II+25ml advanced DMEM/F12, filtered through a 0.22 μm filter and stored at-80℃in 2 ml/tube. Diluted 20-fold with advanced DMEM/F12 before use and stored at 4 ℃.
The matrigel digests referred to in examples 5-8 and comparative examples 1-4 were prepared in this example.
Example 4
A wave, comprising the following components in percentage by weight: advanced DMEM/F12:500ml,HEPES:5ml,Gluta Max:5ml, primocin:1ml. The washings referred to in examples 2, 5-8 and comparative examples 1-4 were all prepared in this example.
Example 5
A method for establishing keloid organoids comprises the following specific steps:
1) Keloid tissue is taken and placed in a 6cm dish, 2ml PBS is added for cleaning for 2 times, obvious fat is removed by a surgical knife, mung bean-sized tissue blocks are cut off, the tissue blocks are cleaned for 2 times by PBS again and then soaked in PBS for 3min, and the PBS is discarded.
2) Soaking the tissue block with the mixture of green streptomycin and double antibody for 3min, and discarding the double antibody.
3) Adding a small amount of tissue digestion solution digest medium to keep the tissue moist, chopping the tissue blocks by a surgical knife, transferring the tissue blocks to a 15ml centrifuge tube by using 2ml of tissue digestion solution digest medium, and placing the centrifuge tube in a shaking table at 37 ℃ for 15min in an inclined manner.
4) The tissue mass is blown by a pipetting gun (wide-mouth gun head), kept stand for 1min, the supernatant is taken into a new 15ml centrifuge tube, 2ml of wash is added to stop digestion, centrifugation is carried out at 1400rpm for 5min, and the supernatant is discarded, thus obtaining cell sediment, namely keloid cells.
5) Taking out matrigel, placing on ice, mixing with cell sediment by special gun head 200 μl matrigel, and placing on ice without bubbling.
6) Before experiment, the well plate is heated in a 37 deg.c cell incubator for at least 30min, the preheated well plate is taken out before inoculation, 50 mu l of matrigel mixed with keloid cell is dropped into the center of each hole, and the mixture is placed in 37 deg.c and 5% CO 2 Curing in a cell incubator for 15min, adding 500 μl of the culture medium prepared in example 1 into each well after the matrigel is solidified, and placing at 37deg.C and 5% CO 2 Culturing in a cell culture box, changing the culture medium once for 4 days, and culturing for 4-28 days to obtain keloid organoids.
In this example, keloid organoids formed up to 4 days and the keloid organoids formed in 4 days reached the size of the passage. FIG. 1 shows organoids formed by keloids cultured for 4 days. FIGS. 2 and 3 show that comparison of HE staining results of the keloid organoids cultured in example 5 with primary tissue shows that both are highly consistent in histopathological structure. FIGS. 4 and 5 show that comparison of the immunofluorescence results of the keloid organoids cultured in example 5 with the primary tissue shows that both express COLI and alpha-SMA.
Example 6
The steps of subculturing keloid organoids are as follows:
1) Taking the 24-well plate cultured for 4 days in the step 6) of the example 5, and sucking out the medium in the well; blowing matrigel mixed with keloid organoids into holes by matrigel digestive juice dispase II (1 mg/ml), wherein each hole is 500 mu l;
transferring the melted matrigel into a 15ml centrifuge tube, flushing all holes with 1ml dispase II (1 mg/ml) and transferring into the same 15ml centrifuge tube, labeling the tubes, performing water bath digestion at 37 ℃ for 5min, centrifuging at 1400rpm for 5min after digestion is finished, and sucking off the supernatant;
2) Adding a proper amount of TrypLE (500 μl per well) to the centrifuge tube, placing the centrifuge tube in a water bath at 37deg.C for digestion for 5min, adding a wash equal to TrypLE in volume after digestion, centrifuging at 1400rpm for 5min, and discarding the supernatant to obtain cell precipitate.
3) Taking out matrigel, placing on ice, adding appropriate amount of matrigel (50 μl per hole) into cell sediment with a pipette (wide-mouth gun head), and blowing and mixing to obtain matrigel mixed with cells without blowing bubbles;
4) Taking a 24-pore plate, placing the 24-pore plate in a 37 ℃ incubator, heating for 30min, taking out the 24-pore plate, dripping 50 μl of matrigel mixed with cells into the 24-pore plate, and solidifying the matrigel at Kong Zhongyang ℃ for 15min at 37 ℃; after the matrigel had solidified, 500. Mu.l of the medium prepared in example 1 were added to each well and placed at 37℃in 5% CO 2 The cells were cultured in an incubator with medium changed every 4 days. The state was observed daily, and after one week the organoids grew significantly, indicating successful passage.
Example 7
The steps of freezing and preserving keloid organoids are as follows:
1) Taking cultured organoids: taking a 24-well plate for culturing for 4-10 days in the step 6) in the example 5, and sucking out the medium in the well;
blowing matrigel containing keloid organoids in holes by matrigel digestive juice dispase II (1 mg/ml), transferring 500 mu l of matrigel into a 15ml centrifuge tube after all matrigel is melted, flushing all holes by 1ml dispase II (1 mg/ml) and transferring the holes into the same 15ml centrifuge tube, labeling on the tube, digesting for 5min in a 37 ℃ water bath, centrifuging for 5min at 1400rpm after digestion is finished, and sucking and discarding supernatant;
2) Adding a proper amount of TrypLE (500 μl per well) to the centrifuge tube, placing the centrifuge tube in a water bath at 37deg.C for digestion for 5min, adding a wash equal to TrypLE in volume after digestion, centrifuging at 1400rpm for 5min, and discarding the supernatant to obtain cell precipitate.
3) Adding frozen solution into the cell sediment obtained in the step 2), blowing and mixing uniformly, freezing with frozen tube with volume of 2ml, freezing cell frozen solution in each tube of 300 μl, freezing 2-3 tubes in each hole, placing into gradient cooling frozen box, placing at-80deg.C, and transferring into liquid nitrogen for preservation after 1-2 weeks.
Example 8
The steps of the keloid organoid resuscitation are as follows:
1) Taking out one freezing tube in the example 7, melting for 1min at 37 ℃, transferring all liquid in the freezing tube into a new 15ml centrifuge tube, adding 10ml of the wash prepared in the example 4 into the centrifuge tube, centrifuging at 1400rpm for 5min, and absorbing and removing the supernatant to obtain cell sediment.
2) Taking out matrigel, placing on ice, sucking appropriate amount of matrigel with a pipette (wide-mouth gun head), and mixing with cell pellet by blowing, taking care of not to blow bubbles.
3) Placing 24 pore plates in a 37 ℃ incubator, heating for 30min, taking out, dripping 50 μl of the matrigel mixed with cell precipitate obtained in step 2) at the center of each pore, solidifying at 37 ℃ for 15min, adding 500 μl of the culture medium in example 1 into each pore after matrigel solidification, placing at 37 ℃ and 5% CO 2 Culturing in a cell incubator, observing the state every day, and obviously growing the organoids after 1 week, which indicates that the resuscitation is successful.
Comparative example 1
The culture medium provided in this comparative example is derived from the document Divergent Routes toward Wnt and R-spondin Niche Independency during Human Gastric Carcinogenesis, and has the ingredients closest to those of the culture medium of the present invention, and the ingredients and contents are as follows: adDMEM/F12; penicillin/streptomyin, 1×; HEPES,10mM; glutamax,2mM; b27,1×; gastrin,10nM; n-acetylcysteine,1mM; mEGF,50ng/ml; hFGF10, 50ng/ml; mnoggin,100ng/ml; hR-spondin1, 100. Mu.g/ml; afamin-Wnt-3A,25%; a83-01, 500nM. The culture medium was used to establish keloid organoids and the growth was compared to example 5.
The establishment of keloid organoids was carried out in the same manner as in example 5 using the above-described culture medium. After 8 days of culture, organoids were not observed, and the results are shown in FIG. 6.
Comparative example 2
The culture medium provided in this comparative example differs from that of example 1 in that mEGF was removed and the establishment of a keloid organoid was performed in accordance with the method of example 5. After 8 days of culture, the organoids formed were small in size and grew slowly, and the results are shown in FIG. 7.
Comparative example 3
The culture medium provided in this comparative example differs from that of example 1 in that hFGF10 was removed and the establishment of keloid organoids was performed as in example 5. After 8 days of culture, only small cell clusters were formed, no organoids were formed, and the results are shown in fig. 8.
Comparative example 4
The culture medium provided in this comparative example differs from that provided in example 1 in that Nicotinamide was removed and the establishment of a keloid organoid was performed as in example 5. After 8 days of culture, organoids formed, but were smaller in size and grew slowly, the results are shown in FIG. 9.
The foregoing is merely illustrative of several embodiments of the present invention and it is not to be construed that the invention is limited to these specific embodiments. It will be apparent to those skilled in the art that several simple deductions or substitutions may be made without departing from the spirit of the invention, and any modifications, equivalent substitutions, improvements etc. made by these are intended to be included in the scope of the present invention.
Claims (9)
1. A keloid organoid medium, characterized in that it comprises basal medium advanced DMEM/F12 and specific additive factors comprising the following components: primocin, HEPES, glutaMAX, B-27 serum free additives, N-acetyl-L-cysteine, mEGF, hFGF, hR-spondin1, afamin-Wnt3A and Nicotinamide.
2. A keloid organoid medium according to claim 1, wherein the final concentrations of the components of the specific additive factor in the keloid organoid medium are as follows:
primocin，0.5～5×；
HEPES，1～100mM；
GlutaMAX，0.2～20mM；
b-27 is free of serum additives, 0.5 to 10X;
N-acetyl-L-cysteine，0.1～200mM；
mEGF，1～300ng/ml；
hFGF10，1～500ng/ml；
hR-spondin 1，0.1～50mg/ml；
Afamin-Wnt3A, 5-50% volume concentration;
Nicotinamide，0.1～200mM。
3. a keloid organoid medium according to claim 1, wherein the concentration of each component of the specific additive factor in the keloid organoid medium is as follows:
primocin，1×；
HEPES，10mM；
GlutaMAX，2mM；
b-27 was free of serum additives, 1×;
N-acetyl-L-cysteine，1mM；
mEGF，50ng/ml；
hFGF10，200ng/ml；
hR-spondin 1，1mg/ml；
Afamin-Wnt3A,25% by volume;
Nicotinamide，10mM。
4. a method of establishing a keloid organoid comprising the steps of:
step one, pretreating keloid tissues to obtain keloid cells;
step two, establishing keloid organoids;
mixing keloid cells with matrigel, standing at 37deg.C, and 5% CO 2 Adding keloid organoid culture medium after matrix gel is solidified in cell incubator, again placing at 37deg.C and 5% CO 2 Culturing in a cell culture box, changing the culture medium every 4 days, and culturing for 4-28 days to obtain keloid organoids.
5. A method of establishing a keloid organoid according to claim 4, wherein in step one, the keloid tissue pretreatment step is as follows:
1) Collecting keloid tissue, washing with PBS, removing fat, cutting mung bean tissue block, washing with PBS for at least 2 times, soaking in PBS for 3-5min, and discarding PBS;
2) Soaking the tissue block with the double antibody for 3-5min, and discarding the double antibody;
3) Moistening the tissue blocks in a plate with tissue digestion liquid, cutting up, transferring all the tissue blocks into a centrifuge tube with the tissue digestion liquid, and carrying out shaking table digestion for 15-60 min at 37 ℃;
4) Blowing and mixing uniformly by a pipetting gun, standing, taking supernatant into a centrifuge tube, adding a wash to stop digestion, centrifuging, and discarding the supernatant to obtain cell sediment, namely keloid cells.
6. A method of establishing a keloid organoid according to claim 5, wherein in step two, the specific procedure for establishing a keloid organoid is as follows:
1) Taking out matrigel, placing on ice, mixing matrigel with keloid cells with a pipette, and placing on ice;
2) Before experiment, the well plate is placed in a 37 ℃ cell incubator to raise the temperature for at least 30min, the well plate which is preheated is taken out before inoculation, 50 mu l of matrigel mixed with cells is dripped in the center of each well, and the well plate is placed in 37 ℃ and 5% CO 2 Placing in cell incubator for 15min, adding 500 μl of keloid organoid culture medium into each well after matrix gel is solidified, and placing again at 37deg.C and 5% CO 2 Culturing in a cell culture box, changing the culture medium once for 4 days, and culturing for 4-28 days to obtain keloid organoids.
7. A keloid organoid subculturing method, comprising the steps of:
1) Taking keloid organoid pore plates obtained after 4-28 days of culture, sucking and removing culture medium in the pores, blowing with matrigel digestive juice, transferring into a centrifuge tube after matrigel is completely melted, performing water bath digestion at 37 ℃ for 5min, centrifuging at 1400rpm for 5min, and sucking and removing supernatant;
2) Adding TrypLE Express enzyme to the centrifuge tube to resuspend cells; placing the centrifuge tube in a water bath at 37 ℃ for digestion for 5min, adding a wash with the same volume as TrypLE Express enzyme after digestion is finished, stopping digestion, centrifuging at 1400rpm for 5min, and discarding the supernatant to obtain a cell precipitate;
3) Taking out matrigel, placing on ice, sucking matrigel, adding into cell sediment, blowing and mixing to obtain matrigel mixed with cells;
4) Placing the pore plate in a 37 ℃ cell incubator for heating for at least 30min before the experiment, taking out the preheated pore plate before inoculation, dripping 50 μl of matrigel mixed with cells into the center of the pore plate, and solidifying for 15min at 37 ℃; after the matrigel had solidified, 500. Mu.l of keloid organoid medium was added to the wells and placed at 37℃in 5% CO 2 The cells were cultured in an incubator with medium changed every 4 days.
8. A keloid organoid cryopreservation method, comprising the steps of:
1) Collecting keloid organoid pore plate obtained after culturing for 4-28 days, sucking and removing the culture medium in the pore, blowing with matrigel digestive juice, transferring into centrifuge tube after matrigel is completely melted, digesting for 5min in water bath at 37deg.C, centrifuging at 1400rpm for 5min, and sucking and removing supernatant;
2) Adding TrypLE Express enzyme to the centrifuge tube to resuspend cells; placing the centrifuge tube in a 37 ℃ water bath for digestion for 5min, adding a wash with the same volume as TrypLE Express enzyme to terminate digestion after digestion is finished, centrifuging at 1400rpm for 5min, and discarding the supernatant to obtain a cell precipitate;
3) Adding frozen stock solution into cell sediment, blowing and mixing, transferring into frozen stock tube, placing into gradient cooling frozen stock box, placing at-80deg.C for 1-2 weeks, and transferring into liquid nitrogen for preservation.
9. A method of resuscitation of keloid organoids comprising the steps of:
1) Taking out a freezing tube containing keloid organoids, wherein the cell freezing solution in the freezing tube is 300 mu l, melting for 1min at 37 ℃, and transferring into a centrifuge tube;
2) Adding 10ml of wash into a centrifuge tube, centrifuging at 1400rpm for 5min, and sucking off the supernatant to obtain a cell precipitate;
3) Placing matrigel on ice, sucking matrigel and cell sediment by a pipetting gun, and blowing and mixing uniformly;
4) Placing the pore plate in a cell incubator at 37 ℃ for heating for at least 30min before experiments, taking out the preheated pore plate before inoculation, dripping 50 μl of matrigel mixed with cell sediment at the center of each pore, and solidifying for 15min at 37 ℃; to be treatedAfter solidification, 500. Mu.l of keloid organoid medium was added to each well and placed at 37℃in 5% CO 2 The cells were cultured in an incubator with medium changed every 4 days.
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