CN117551613A - Serum-free special culture medium for esophageal squamous carcinoma organoids - Google Patents
Serum-free special culture medium for esophageal squamous carcinoma organoids Download PDFInfo
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- CN117551613A CN117551613A CN202310777313.7A CN202310777313A CN117551613A CN 117551613 A CN117551613 A CN 117551613A CN 202310777313 A CN202310777313 A CN 202310777313A CN 117551613 A CN117551613 A CN 117551613A
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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Abstract
The invention provides a culture medium, which comprises a basal culture medium and an additive, wherein the additive comprises N-acetylcysteine, nicotinamide, Y27632, A83-01, noggin protein and serum substitute B plus and at least one of pleiotropic protein and midkine. The inventor finds that when the culture medium contains the additive, the additive has obvious promotion effect on the growth of the esophageal squamous carcinoma organoids and improves the cell activity of the esophageal squamous carcinoma organoids.
Description
Technical Field
The invention relates to the technical field of tumor organoids, in particular to a serum-free special culture medium and a culture method for esophageal squamous carcinoma organoids.
Background
Esophageal squamous carcinoma is the sixth leading lethal cancer species worldwide, and is caused by a number of complex causes, differing by histological typing and population differences. Esophageal squamous cell carcinoma is generally classified into Esophageal Squamous Cell Carcinoma (ESCC) and Esophageal Adenocarcinoma (EADC), and has almost completely different geographical distribution, event trend and main etiology, and the disease has poor prognosis regardless of the etiology.
Organoids refer to cultures of stem cells or precursor cells, formed by cell self-proliferation, directed differentiation and lineage commitment and self-assembly, in vitro in three dimensions, with organ-specific cellular composition, key structural and functional properties. The organoid function can partially or even completely reduce the cell constitution and the structural function of organs in the body, maintain the stability of inheritance and phenotype while expanding for a long time, solve the difficulty that the traditional biological model can not simulate tissue organs, lead the research of the tissue organs in the body to be changed from the surface level of two-dimensional cells to the deep level of three-dimensional tissues, provide a good model for basic research of organ development and the like, and open up an innovative approach for medical transformation and disease treatment.
However, the construction and research of esophageal squamous carcinoma organoids in the world are very limited, and the esophageal squamous carcinoma organoids are related to the culture of esophageal squamous carcinoma, and all the published data at present show that the primary culture has low power and cannot be effectively passaged for a long time. Thus, there is a need to develop a culture method suitable for the growth of esophageal squamous carcinoma organoids.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems existing in the prior art to at least some extent.
In order to improve the success rate of primary culture of the esophageal squamous carcinoma organoids and meet the nutritional requirements of the esophageal squamous carcinoma organoids, the invention provides a culture medium of the esophageal squamous carcinoma organoids, which contains PTN (pleiotropic protein) and MDK (midkine) factors. Experiments prove that the two factors are particularly important for the growth of esophageal squamous carcinoma organoids, wherein PTN is a secreted growth factor which mediates signals of the PTN through cell surface proteoglycan and non-proteoglycan receptors, can bind ALK (anaplastic lymphoma kinase) and promote cell survival and cell proliferation through MAPK pathway activation; MDK binds to cell surface proteoglycan receptors via the Chondroitin Sulfate (CS) group, thereby regulating many life processes and promoting tumor cell abnormal proliferation. These factors and their corresponding functions are critical to the self-organizing ability of tumor cells and the reduction of the tumor tissue microenvironment. In addition, experiments prove that PTN and MDK have better promotion effect on the growth of organoids when used singly and cooperatively.
Thus, in one aspect of the invention, the invention provides a culture medium. According to an embodiment of the invention, the medium comprises a basal medium and an additive comprising N-acetylcysteine, nicotinamide, ROCK anti-apoptotic factor, ALK 5 inhibitor, noggin protein and serum replacement B plus and at least one of pleiotrophin and midkine. The inventor finds that when the culture medium contains the additive, the additive has obvious promotion effect on the growth of the esophageal squamous carcinoma organoids and improves the cell activity of the esophageal squamous carcinoma organoids.
It should be noted that "pleiotropic protein" referred to in the present application is PTN, which is a secreted growth factor mediating its signal through cell surface proteoglycan and non-proteoglycan receptors, capable of binding ALK (anaplastic lymphoma kinase) and promoting cell survival and cell proliferation through MAPK pathway activation; the term "midkine" referred to in this application is MDK, which binds to cell surface proteoglycan receptors via the Chondroitin Sulfate (CS) group, thereby regulating many life processes and promoting tumor cell abnormal proliferation; "ROCK anti-apoptotic factor" referred to in this application is Y27632 (ROCK is a serine/threonine protein kinase, a key role for ROCK protein in the development and progression of tumors); the ALK 5 inhibitor is A83-01 (A83-01 is a powerful TGF-beta I type receptor ALK-5 (IC 50:12 nm)/ALK 4 ((IC 50:45 nm)/ALK 7 (IC 50:7.5 nm)) kinase inhibitor, the inhibiting effect of A83-01 on ALK-5 is strong), the Noggin protein is an inhibitor of BMP signal channel, and the serum substitute Bplus is platelet lysate, and can promote the growth and reproduction of cells like serum.
According to an embodiment of the invention, the additives include pleiotropic proteins, N-acetylcysteine, nicotinamide, ROCK anti-apoptotic factors, ALK 5 inhibitors, noggin proteins and serum substitutes B plus. The inventor finds that when the additive is contained in the culture medium, the additive has obvious promotion effect on the growth of the esophageal squamous carcinoma organoids and improves the cell activity of the esophageal squamous carcinoma organoids.
According to an embodiment of the invention, the additives include midkine, N-acetylcysteine, nicotinamide, ROCK anti-apoptotic factor, ALK 5 inhibitor, noggin protein and serum replacement B plus. The inventor finds that when the additive is contained in the culture medium, the additive has obvious promotion effect on the growth of the esophageal squamous carcinoma organoids and improves the cell activity of the esophageal squamous carcinoma organoids.
According to an embodiment of the invention, the additives include pleiotropic proteins, metaphase factors, N-acetylcysteine, nicotinamide, ROCK anti-apoptotic factors, ALK 5 inhibitors, noggin proteins, and serum substitutes B plus. The inventor finds that when the additive is contained in the culture medium, the additive has obvious promotion effect on the growth of the esophageal squamous carcinoma organoids and improves the cell activity of the esophageal squamous carcinoma organoids.
According to an embodiment of the invention, the basal medium is DMEM/F12 medium.
According to an embodiment of the invention, the pleiotropic protein is PTN. Thus, the medium is capable of promoting cell survival and cell proliferation.
According to an embodiment of the present invention, the midkine is MDK. Thus, the culture medium is capable of regulating many life processes and promoting abnormal proliferation of tumor cells.
According to an embodiment of the invention, the ROCK anti-apoptosis factor is Y27632.
According to an embodiment of the invention, the ALK 5 inhibitor is A83-01.
According to an embodiment of the invention, the basal medium further comprises a hydrogen ion buffer and GlutaMax. Thus, the medium is capable of meeting the basic nutritional requirements of cell growth.
According to an embodiment of the invention, the hydrogen ion buffer is 4-hydroxyethylpiperazine ethanesulfonic acid.
According to an embodiment of the present invention, the hydrogen ion buffer is present in the medium in a volume fraction of 0.5 to 2%.
According to a specific embodiment of the invention, the volume fraction of the hydrogen ion buffer in the medium is 1%.
The component in the stock solution of "hydrogen ion buffer" described herein was 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), and the concentration of the component was 1M.
According to an embodiment of the present invention, the volume fraction of the GlutaMax in the medium is 0.5 to 2%.
According to a specific embodiment of the present invention, the volume fraction of the GlutaMax in the medium is 1%.
The concentration of the "GlutaMax" stock solution described herein was 200mM.
According to an embodiment of the invention, the basal medium further comprises an antibacterial ingredient. Therefore, the culture medium is further ensured to be beneficial to culturing the esophageal squamous carcinoma organoids and avoid the esophageal squamous carcinoma organoids from being polluted.
According to an embodiment of the invention, the antibacterial component is a double antibody to green streptomycin.
According to the embodiment of the invention, the concentration of the sinostreptomycin diabody in the culture medium is 90-110 IU/mL.
According to an embodiment of the invention, the concentration of the neomycin biantide in the culture medium is 100IU/mL.
According to an embodiment of the present invention, the concentration of the N-acetylcysteine in the medium is 1 to 1.5mM, the concentration of the nicotinamide in the medium is 0.05 to 1. Mu.g/mL, the concentration of the ROCK anti-apoptosis factor in the medium is 5 to 20. Mu.M, the concentration of the ALK 5 inhibitor in the medium is 0.1 to 1. Mu.M, the concentration of the Noggin protein in the medium is 0.05 to 1. Mu.g/mL, and the volume fraction of the serum replacement B plus in the medium is 1 to 3%. The inventor finds through continuous experiments that when the components in the culture medium can ensure that the growth state of the esophageal squamous carcinoma organoids is better and the cell activity is higher in the concentration range, the foundation is laid for the subsequent research of the esophageal squamous carcinoma by researchers.
According to an embodiment of the present invention, the concentration of the N-acetylcysteine in the medium is 1 to 1.5mM, the concentration of the nicotinamide in the medium is 0.05 to 0.2. Mu.g/mL, the concentration of the ROCK anti-apoptosis factor in the medium is 5 to 20. Mu.M, the concentration of the ALK 5 inhibitor in the medium is 0.2 to 0.8. Mu.M, the concentration of the Noggin protein in the medium is 0.05 to 0.2. Mu.g/mL, and the volume fraction of the serum replacement B plus in the medium is 1 to 3%. The inventor finds through continuous experiments that when the components in the culture medium can ensure that the growth state of the esophageal squamous carcinoma organoids is better and the cell activity is higher in the concentration range, the foundation is laid for the subsequent research of the esophageal squamous carcinoma by researchers.
According to an embodiment of the present invention, the concentration of N-acetylcysteine in the medium is 1 to 1.5mM, the concentration of nicotinamide in the medium is 0.05 to 1. Mu.g/mL, the concentration of Y27632 in the medium is 5 to 20. Mu.M, the concentration of A83-01 in the medium is 0.1 to 1. Mu.M, the concentration of Noggin protein in the medium is 0.05 to 1. Mu.g/mL, and the concentration of serum replacement B plus in the medium is 1 to 3%. The inventor finds through continuous experiments that when the components in the culture medium can ensure that the growth state of the esophageal squamous carcinoma organoids is better and the cell activity is higher in the concentration range, the foundation is laid for the subsequent research of the esophageal squamous carcinoma by researchers.
According to an embodiment of the present invention, the concentration of N-acetylcysteine in the medium is 1 to 1.5mM, the concentration of nicotinamide in the medium is 0.05 to 0.2. Mu.g/mL, the concentration of Y27632 in the medium is 5 to 20. Mu.M, the concentration of A83-01 in the medium is 0.2 to 0.8. Mu.M, the concentration of Noggin protein in the medium is 0.05 to 0.2. Mu.g/mL, and the concentration of serum replacement B plus in the medium is 1 to 3%. The inventor finds through continuous experiments that when the components in the culture medium can ensure that the growth state of the esophageal squamous carcinoma organoids is better and the cell activity is higher in the concentration range, the foundation is laid for the subsequent research of the esophageal squamous carcinoma by researchers.
According to an embodiment of the invention, the concentration of the pleiotropic protein in the culture medium is 0.05-1 μg/mL. Preferably, the concentration of the pleiotropic protein in the culture medium is 0.05-0.2 μg/mL. When the pleiotropic protein in the culture medium is in the concentration range, the growth state of the esophageal squamous carcinoma organoids can be ensured to be better, and the cell survival and the cell proliferation are promoted.
According to an embodiment of the present invention, the medium factor has a concentration of 0.05 to 1. Mu.g/mL in the medium. Preferably, the concentration of the midkine in the medium is 0.05-0.2. Mu.g/mL. When the midkine in the culture medium can be combined with the cell surface proteoglycan receptor in the concentration range, the growth state of the esophageal squamous carcinoma organoid is ensured to be better, and the abnormal proliferation of cells is promoted.
According to an embodiment of the present invention, the concentration of the pleiotropic protein in the medium is 0.05 to 1. Mu.g/mL, and the concentration of the midkine in the medium is 0.05 to 1. Mu.g/mL. Preferably, the concentration of the pleiotropic protein in the culture medium is 0.05-0.2 mug/mL, and the concentration of the midkine in the culture medium is 0.05-0.2 mug/mL. When pleiotropic proteins and midkine in the culture medium are in the concentration range, the growth state of the esophageal squamous carcinoma organoids can be guaranteed to be good, and the cell activity is high.
In another aspect of the invention, the invention provides the use of the medium as described above for culturing an esophageal squamous carcinoma organoid.
In yet another aspect of the invention, the invention provides a method of preparing an esophageal squamous carcinoma organoid. According to an embodiment of the invention, the method comprises: the esophageal squamous carcinoma cells are subjected to culture treatment in the aforementioned medium to obtain esophageal squamous carcinoma organoids. The esophageal squamous carcinoma organoids cultured by the method have good morphology, can promote the rapid growth of the esophageal squamous carcinoma organoids, greatly increase the quantity of the organoids and greatly improve the cell survival rate.
According to an embodiment of the invention, the esophageal squamous carcinoma cells are isolated from esophageal squamous carcinoma tissue.
The invention has the beneficial effects that:
(1) The culture medium for the esophageal squamous carcinoma organoids contains pleiotropic Protein (PTN), can maintain cell viability, promote cell proliferation and angiogenesis;
(2) The medium for the esophageal squamous carcinoma organoid contains Midkine (MDK) in the components, so that the medium can maintain the cell activity and promote the cell proliferation;
(3) The culture medium of the invention has the advantages of short culture period, fast proliferation, high cell number and cell survival rate, and long-term stable passage.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 is a culture chart of esophageal squamous carcinoma organoids according to example 1 of the invention, at 4-fold magnification, for seven days;
FIG. 2 is a culture chart showing the organ culture of esophageal squamous carcinoma organoids for seven days at 4-fold magnification according to example 2 of the invention;
FIG. 3 is a culture chart showing the organ culture of esophageal squamous carcinoma organoids for seven days at 4-fold magnification according to example 3 of the invention;
FIG. 4 is a culture chart of esophageal squamous carcinoma organoids according to comparative example 4 of the invention, at 4-fold magnification, for seven days;
FIG. 5 is a culture chart of esophageal squamous carcinoma organoids according to comparative example 5 of the invention, at 4-fold magnification, for seven days;
FIG. 6 is a graph of organoid number comparisons according to an embodiment of the invention.
Detailed Description
Embodiments of the present invention are described in detail below. The following examples are illustrative only and are not to be construed as limiting the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The relative reagent numbers involved are as follows:
DMEM/F12：Gibco TM Advanced DMEM/F-12，12634010；
HEPES：Gibco TM HEPES(1M)，15630130；
GlutaMax：Gibco TM GlutaMAX TM additive (200 mM), 35050079;
green streptomycin diabody (Penicillin): gibco TM Penicillin-streptomycin (10,000U/mL), 15140163;
nicotinamide (Nicotinamid): sigma-Aldrich TM Nicotinamide, 72340;
N-Acetylcysteine (N-Acetylcysteine): sigma-Aldrich TM N-acetylcysteine amide, A0737;
Y27632：R&D SYSTEMS TM Y-27632dihydrochloride，TB1254-GMP；
A83-01：R&D SYSTEMS TM A 83-01，2939；
noggin protein: r is R&D SYSTEMS TM Recombinant Human Noggin Fc Chimera Protein,CF，3344-NG；
Serum replacement B plus: gibco TM B-27 TM Plus supplement, a3582801;
PTN：R&D SYSTEMS TM Recombinant Human Pleiotrophin/PTN Protein,CF，252-PL；
MDK：R&D SYSTEMS TM Recombinant Human Midkine Protein,CF，258-MD。
example 1
Media for esophageal squamous carcinoma organoids were prepared according to the formulation in table 1:
TABLE 1
The esophageal squamous carcinoma organoid is cultured by adopting the culture medium, and the specific culture method is as follows:
1. transferring the esophageal squamous carcinoma tissue sample to be cultured into a 3.5cm or 10cm culture dish with DPBS (DPBS-PS) or tumor tissue basal medium added with 1% of streptomycin, removing adipose tissue and normal tissue as much as possible, only retaining the esophageal squamous carcinoma tissue sample, and repeatedly cleaning the esophageal squamous carcinoma tissue sample by using the DPBS-PS until the cleaning liquid is clear, wherein the cleaning liquid is generally cleaned for 3-5 times.
2. After removing residual DPBS-PS in the centrifuge tube, the esophageal squamous carcinoma tissue sample is sheared into 3mm 3 Transferring the tissue fragments to a 15mL centrifuge tube containing 5-10 mL of primary digestion liquid of tumor tissue (the main components of the digestion liquid are collagenase I and nuclease), and performing digestion in a horizontal shaker at 37 ℃ and a rotating speed of 100rpm for 20-40min, and then adding FBS with the volume ratio of 4% to terminate the digestion. And (3) passing the cell fluid through a 100 mu M cell screen, centrifuging 300g for 3min, discarding the supernatant, adding 1mL of erythrocyte lysate into the container, blowing up and down by using a pipetting gun, incubating at room temperature for 3min, centrifuging 300g for 3min after incubation is finished, and discarding the supernatant to obtain cell sediment which is the esophageal squamous cell carcinoma cells.
3. Fully mixing the cell sediment obtained in the step 2 with a tumor tissue basal medium, re-suspending, centrifuging, discarding supernatant, repeating for a plurality of times to obtain the cell sediment, and mixing the cell sediment and matrigel (ECM) in a volume ratio of 3:7; after mixing, performing glue dripping and seeding, wherein each glue drop is 20 mu L, and the cell quantity in each glue drop is more than or equal to 1 ten thousand; after inoculation is completed, the mixture is kept stand at 37 ℃ until the glue drops are fully solidified.
4. Taking the esophageal squamous carcinoma organoid culture medium, preheating at 37 ℃, adding 500 mu L of preheated esophageal squamous carcinoma organoid culture medium on each glue drop, and carrying out edge sealing treatment by using DPBS added with 1% of streptomycin double antibody; and replacing the esophageal squamous carcinoma organoid culture medium on each glue drop every 2 days, and culturing for 5 days to obtain the esophageal squamous carcinoma organoid.
As shown in FIG. 1, a large number of organoid clones, 100 μm in average diameter, and having a good compact sphere shape, were obtained on day 7 by the culture medium and the culture method of this example.
Example 2
Media for esophageal squamous carcinoma organoids were prepared according to the formulation in table 2:
TABLE 2
| Component name | Final concentration |
| DMEM/F12 | Basal Medium |
| HEPES (4-hydroxyethyl piperazine ethane sulfonic acid) | 1:100 |
| GlutaMax | 1:100 |
| Penicillin/Streptomycin (Green Streptomycin double antibody) | 1:100 |
| Nicotinamid (nicotinamide) | 0.1μg/mL |
| N-Acetylcysteine (N-Acetylcysteine) | 1.25mM |
| Y27632 | 10μM |
| A83-01 | 0.5μM |
| Noggin | 100ng/mL |
| Bplus (serum replacement) | 1:50 |
| PTN (pleiotropic protein) | 0.1μg/mL |
The esophageal squamous carcinoma organoid is cultured by adopting the culture medium, and the specific culture method is as follows:
1. transferring the esophageal squamous carcinoma tissue sample to be cultured into a 3.5cm or 10cm culture dish with DPBS (DPBS-PS) or tumor tissue basal medium added with 1% of streptomycin, removing adipose tissue and normal tissue as much as possible, only retaining the esophageal squamous carcinoma tissue sample, and repeatedly cleaning the esophageal squamous carcinoma tissue sample by using the DPBS-PS until the cleaning liquid is clear, wherein the cleaning liquid is generally cleaned for 3-5 times.
2. After removing residual DPBS-PS in the centrifuge tube, the esophageal squamous carcinoma tissue sample is sheared into 3mm 3 Transferring the tissue fragments to a 15mL centrifuge tube containing 5-10 mL of primary digestion liquid of tumor tissue (the main components of the digestion liquid are collagenase I and nuclease), and performing digestion in a horizontal shaker at 37 ℃ and a rotating speed of 100rpm for 20-40min, and then adding FBS with the volume ratio of 4% to terminate the digestion. And (3) passing the cell fluid through a 100 mu M cell screen, centrifuging 300g for 3min, discarding the supernatant, adding 1mL of erythrocyte lysate into the container, blowing up and down by using a pipetting gun, incubating at room temperature for 3min, centrifuging 300g for 3min after incubation is finished, and discarding the supernatant to obtain cell sediment which is the esophageal squamous cell carcinoma cells.
3. Fully mixing the cell sediment obtained in the step 2 with a tumor tissue basal medium, re-suspending, centrifuging, discarding supernatant, repeating for a plurality of times to obtain the cell sediment, and mixing the cell sediment and matrigel (ECM) in a volume ratio of 3:7; after mixing, performing glue dripping and seeding, wherein each glue drop is 20 mu L, and the cell quantity in each glue drop is more than or equal to 1 ten thousand; after inoculation is completed, the mixture is kept stand at 37 ℃ until the glue drops are fully solidified.
4. Taking the esophageal squamous carcinoma organoid culture medium, preheating at 37 ℃, adding 500 mu L of preheated esophageal squamous carcinoma organoid culture medium on each glue drop, and carrying out edge sealing treatment by using DPBS added with 1% of streptomycin double antibody; and replacing the esophageal squamous carcinoma organoid culture medium on each glue drop every 2 days, and culturing for 5 days to obtain the esophageal squamous carcinoma organoid.
As shown in FIG. 2, organoid clones having an average diameter of 100 μm and a good compact sphere shape were obtained by culturing on day 7 using the medium and the culture method of this example.
Example 3
Media for esophageal squamous carcinoma organoids were prepared according to the formulation in table 3:
TABLE 3 Table 3
The esophageal squamous carcinoma organoid is cultured by adopting the culture medium, and the specific culture method is as follows:
1. transferring the esophageal squamous carcinoma tissue sample to be cultured into a 3.5cm or 10cm culture dish with DPBS (DPBS-PS) or tumor tissue basal medium added with 1% of streptomycin, removing adipose tissue and normal tissue as much as possible, only retaining the esophageal squamous carcinoma tissue sample, and repeatedly cleaning the esophageal squamous carcinoma tissue sample by using the DPBS-PS until the cleaning liquid is clear, wherein the cleaning liquid is generally cleaned for 3-5 times.
2. After removing residual DPBS-PS in the centrifuge tube, the esophageal squamous carcinoma tissue sample is sheared into 3mm 3 Transferring the tissue fragments into a 15mL centrifuge tube containing 5-10 mL of primary digestion solution of tumor tissue (the main components of the digestion solution are collagenase I and nuclease), and heating at 37deg.CDigestion is carried out in a horizontal shaker at 100rpm for a period of typically 20-40 minutes, after which digestion is terminated by the addition of 4% by volume FBS. And (3) passing the cell fluid through a 100 mu M cell screen, centrifuging 300g for 3min, discarding the supernatant, adding 1mL of erythrocyte lysate into the container, blowing up and down by using a pipetting gun, incubating at room temperature for 3min, centrifuging 300g for 3min after incubation is finished, and discarding the supernatant to obtain cell sediment which is the esophageal squamous cell carcinoma cells.
3. Fully mixing the cell sediment obtained in the step 2 with a tumor tissue basal medium, re-suspending, centrifuging, discarding supernatant, repeating for a plurality of times to obtain the cell sediment, and mixing the cell sediment and matrigel (ECM) in a volume ratio of 3:7; after mixing, performing glue dripping and seeding, wherein each glue drop is 20 mu L, and the cell quantity in each glue drop is more than or equal to 1 ten thousand; after inoculation is completed, the mixture is kept stand at 37 ℃ until the glue drops are fully solidified.
4. Taking the esophageal squamous carcinoma organoid culture medium, preheating at 37 ℃, adding 500 mu L of preheated esophageal squamous carcinoma organoid culture medium on each glue drop, and carrying out edge sealing treatment by using DPBS added with 1% of streptomycin double antibody; and replacing the esophageal squamous carcinoma organoid culture medium on each glue drop every 2 days, and culturing for 5 days to obtain the esophageal squamous carcinoma organoid.
As shown in FIG. 3, organoid clones having an average diameter of 100 μm and a good compact sphere shape were obtained by culturing on day 7 using the medium and the culture method of this example.
Comparative example 4
Media for esophageal squamous carcinoma organoids were prepared according to the formulation in table 4:
TABLE 4 Table 4
| Component name | Final concentration |
| DMEM/F12 | Basal Medium |
| HEPES (4-hydroxyethyl piperazine ethane sulfonic acid) | 1:100 |
| GlutaMax | 1:100 |
| Penicillin/Streptomycin (Green Streptomycin double antibody) | 1:100 |
| Nicotinamid (nicotinamide) | 0.1μg/mL |
| N-Acetylcysteine (N-Acetylcysteine) | 1.25mM |
| Y27632 | 10μM |
| A83-01 | 0.5μM |
| Noggin | 100ng/mL |
| Bplus (serum replacement) | 1:50 |
The esophageal squamous carcinoma organoid is cultured by adopting the culture medium, and the specific culture method is as follows:
1. transferring the esophageal squamous carcinoma tissue sample to be cultured into a 3.5cm or 10cm culture dish with DPBS (DPBS-PS) or tumor tissue basal medium added with 1% of streptomycin, removing adipose tissue and normal tissue as much as possible, only retaining the esophageal squamous carcinoma tissue sample, and repeatedly cleaning the esophageal squamous carcinoma tissue sample by using the DPBS-PS until the cleaning liquid is clear, wherein the cleaning liquid is generally cleaned for 3-5 times.
2. After removing residual DPBS-PS in the centrifuge tube, the esophageal squamous carcinoma tissue sample is sheared into 3mm 3 Transferring the tissue fragments to a 15mL centrifuge tube containing 5-10 mL of primary digestion liquid of tumor tissue (the main components of the digestion liquid are collagenase I and nuclease), and performing digestion in a horizontal shaker at 37 ℃ and a rotating speed of 100rpm for 20-40min, and then adding FBS with the volume ratio of 4% to terminate the digestion. And (3) passing the cell fluid through a 100 mu M cell screen, centrifuging 300g for 3min, discarding the supernatant, adding 1mL of erythrocyte lysate into the container, blowing up and down by using a pipetting gun, incubating at room temperature for 3min, centrifuging 300g for 3min after incubation is finished, and discarding the supernatant to obtain cell sediment which is the esophageal squamous cell carcinoma cells.
3. Fully mixing the cell sediment obtained in the step 2 with a tumor tissue basal medium, re-suspending, centrifuging, discarding supernatant, repeating for a plurality of times to obtain the cell sediment, and mixing the cell sediment and matrigel (ECM) in a volume ratio of 3:7; after mixing, performing glue dripping and seeding, wherein each glue drop is 20 mu L, and the cell quantity in each glue drop is more than or equal to 1 ten thousand; after inoculation is completed, the mixture is kept stand at 37 ℃ until the glue drops are fully solidified.
4. Taking the esophageal squamous carcinoma organoid culture medium, preheating at 37 ℃, adding 500 mu L of preheated esophageal squamous carcinoma organoid culture medium on each glue drop, and carrying out edge sealing treatment by using DPBS added with 1% of streptomycin double antibody; and replacing the esophageal squamous carcinoma organoid culture medium on each glue drop every 2 days, and culturing for 5 days to obtain the esophageal squamous carcinoma organoid.
The culture medium provided in this comparative example was the same as in example 1 except that pleiotropic protein and midkine were subtracted from the culture medium provided in example 1. As shown in FIG. 4, the esophageal squamous carcinoma organoids cultured with this medium grew more slowly and in smaller numbers. Compared with examples 1,2 and 3, the number of esophageal squamous carcinoma organoids grown in the culture medium is obviously reduced, and the activity is reduced.
Comparative example 5
Media for esophageal squamous carcinoma organoids were prepared according to the formulation in table 5:
TABLE 5
The esophageal squamous carcinoma organoid is cultured by adopting the culture medium, and the specific culture method is as follows:
1. transferring the esophageal squamous carcinoma tissue sample to be cultured into a 3.5cm or 10cm culture dish with DPBS (DPBS-PS) or tumor tissue basal medium added with 1% of streptomycin, removing adipose tissue and normal tissue as much as possible, only retaining the esophageal squamous carcinoma tissue sample, and repeatedly cleaning the esophageal squamous carcinoma tissue sample by using the DPBS-PS until the cleaning liquid is clear, wherein the cleaning liquid is generally cleaned for 3-5 times.
2. After removing residual DPBS-PS in the centrifuge tube, the esophageal squamous carcinoma tissue sample is sheared into 3mm 3 Transferring the tissue fragments to a 15mL centrifuge tube containing 5-10 mL of primary digestion liquid of tumor tissue (the main components of the digestion liquid are collagenase I and nuclease), and performing digestion in a horizontal shaker at 37 ℃ and a rotating speed of 100rpm for 20-40min, and then adding FBS with the volume ratio of 4% to terminate the digestion. And (3) passing the cell fluid through a 100 mu M cell screen, centrifuging 300g for 3min, discarding the supernatant, adding 1mL of erythrocyte lysate into the container, blowing up and down by using a pipetting gun, incubating at room temperature for 3min, centrifuging 300g for 3min after incubation is finished, and discarding the supernatant to obtain cell sediment which is the esophageal squamous cell carcinoma cells.
3. Fully mixing the cell sediment obtained in the step 2 with a tumor tissue basal medium, re-suspending, centrifuging, discarding supernatant, repeating for a plurality of times to obtain the cell sediment, and mixing the cell sediment and matrigel (ECM) in a volume ratio of 3:7; after mixing, performing glue dripping and seeding, wherein each glue drop is 20 mu L, and the cell quantity in each glue drop is more than or equal to 1 ten thousand; after inoculation is completed, the mixture is kept stand at 37 ℃ until the glue drops are fully solidified.
4. Taking the esophageal squamous carcinoma organoid culture medium, preheating at 37 ℃, adding 500 mu L of preheated esophageal squamous carcinoma organoid culture medium on each glue drop, and carrying out edge sealing treatment by using DPBS added with 1% of streptomycin double antibody; and replacing the esophageal squamous carcinoma organoid culture medium on each glue drop every 2 days, and culturing for 5 days to obtain the esophageal squamous carcinoma organoid.
The culture medium provided in this comparative example has reduced concentration of pleiotropic proteins and metaphase factors as compared to the culture medium of example 1, and the culture process is the same as in example 1. As shown in FIG. 5, the esophageal squamous carcinoma organoids cultured with this medium grew more slowly and in smaller numbers. Compared with example 1, the number of esophageal squamous carcinoma organoids grown in the culture medium is obviously reduced, and the activity is reduced.
The following conclusions can be drawn from figures 1 to 6 and tables 1 to 5:
(1) The medium of example 2 was not supplemented with midkine, and the organoid number was reduced compared to example 1 with the other components and amounts being the same, so that midkine had an important role in the culture of esophageal squamous carcinoma organoids, whereas the use of pleiotropic proteins alone was able to support the growth of esophageal squamous carcinoma organoids.
(2) The culture medium of example 3 was free of pleiotropic protein, and the organoid number was reduced compared to example 1 with the same other components and content, so that pleiotropic protein was also important in the culture of pancreatic cancer organoids, whereas the use of midkine alone was able to support the growth of esophageal squamous carcinoma organoids.
(3) The culture medium of comparative example 4 was free of pleiotropic proteins and metaphase factors, and the organoid numbers were greatly reduced and cell viability was also greatly reduced compared to example 1, with the other components and amounts being the same, so pleiotropic proteins and metaphase factors have an important role in the cultivation of esophageal squamous cell carcinoma organoids.
(4) The medium of comparative example 5 added small amounts of midkine and pleiotropic protein, the organoid number was reduced compared to example 1, and thus 0.1 μg/ml was the lowest effective concentration of midkine and pleiotropic protein in esophageal squamous carcinoma organoid culture.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (14)
1. A culture medium comprising a basal medium and an additive comprising N-acetylcysteine, nicotinamide, ROCK anti-apoptotic factor, ALK-5 inhibitor, noggin protein and serum replacement B plus, and at least one of pleiotrophin and midkine.
2. The medium of claim 1, wherein the additives comprise pleiotropic proteins, N-acetylcysteine, nicotinamide, ROCK anti-apoptotic factors, ALK-5 inhibitors, noggin proteins, and serum substitutes B plus.
3. The medium of claim 1, wherein the additives comprise midkine, N-acetylcysteine, nicotinamide, ROCK anti-apoptotic factor, ALK-5 inhibitor, noggin protein, and serum replacement B plus.
4. The medium of claim 1, wherein the additives comprise pleiotropic proteins, metaphase factors, N-acetylcysteine, nicotinamide, ROCK anti-apoptotic factors, ALK 5 inhibitors, noggin proteins, and serum substitutes B plus.
5. The culture medium according to any one of claims 1 to 4, wherein the basal medium is DMEM/F12 medium;
optionally, the pleiotropic protein is PTN;
optionally, the midkine is MDK;
optionally, the ROCK anti-apoptotic factor is Y27632;
optionally, the ALK 5 inhibitor is a83-01.
6. The medium of claim 5, wherein the basal medium further comprises a hydrogen buffer and GlutaMax;
optionally, the hydrogen ion buffer is 4-hydroxyethyl piperazine ethane sulfonic acid;
optionally, the hydrogen ion buffer is present in the medium in a volume fraction of 0.5 to 2%;
optionally, the volume fraction of the GlutaMax in the culture medium is 0.5-2%.
7. The medium of claim 6, wherein the basal medium further comprises an antimicrobial component;
optionally, the antibacterial component is a dual resistance to green streptomycin;
optionally, the concentration of the sinostreptomycin diabody in the culture medium is 90-110 IU/mL.
8. The medium of claim 1, wherein the concentration of N-acetylcysteine in the medium is 1-1.5 mM, the concentration of nicotinamide in the medium is 0.05-1 μg/mL, the concentration of ROCK anti-apoptotic factor in the medium is 5-20 μΜ, the concentration of ALK 5 inhibitor in the medium is 0.1-1 μΜ, the concentration of Noggin protein in the medium is 0.05-1 μg/mL, and the volume fraction of serum replacement Bplus in the medium is 1-3%.
9. The medium according to claim 2 or 8, wherein the concentration of the pleiotropic protein in the medium is 0.05-1 μg/mL.
10. The medium according to claim 3 or 8, wherein the medium factor has a concentration of 0.05 to 1 μg/mL in the medium.
11. The medium according to claim 4 or 8, wherein the concentration of the pleiotropic protein in the medium is 0.05-1 μg/mL and the concentration of the metaphase factor in the medium is 0.05-1 μg/mL.
12. Use of a medium according to any one of claims 1 to 11 for culturing an esophageal squamous carcinoma organoid.
13. A method of preparing an esophageal squamous carcinoma organoid comprising:
culturing esophageal squamous carcinoma cells in the medium of any of claims 1-11 to obtain an esophageal squamous carcinoma organoid.
14. The method of claim 13, wherein the esophageal squamous carcinoma cells are isolated from esophageal squamous carcinoma tissue.
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