CN116769715A - Culture medium and culture method for suspension culture of medulloblastoma organoids and 3D microspheres - Google Patents
Culture medium and culture method for suspension culture of medulloblastoma organoids and 3D microspheres Download PDFInfo
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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 discloses a culture medium and a culture method for suspension culture of medulloblastoma organoids and 3D microspheres. The culture medium consists of the following components: basic culture medium, vitamin A-free B27, N2, N-acetylcysteine, FGFb, noggin, R-spondin, wnt3a, CHIR99021, laminin, sialic acid, NEAA, penicillin streptomycin mixed solution and Primocin; the basic culture medium is formed by mixing Advanced DMEM/F12 and a nerve Basal culture medium according to the volume ratio of 1:10-150:1. The culture medium and the method can be used for rapidly and stably culturing the 3D organoid of the medulloblastoma without Matrigel, greatly reduce the cost of organoid and three-dimensional cell culture, and are beneficial to the mass expansion of cells.
Description
Technical Field
The invention relates to the technical field of tumor organoids, in particular to a culture medium and a culture method for suspension culture of medulloblastoma organoids and 3D microspheres.
Background
How to accurately recognize human diseases is a serious problem for scientists, and many diseases occurring clinically are not effectively treated, mainly because of the lack of understanding of the occurrence cause and mechanism of the diseases. At present, mice are the most important model animals in medical research, the similarity of genes of the mice and human beings reaches 95 percent, the mice have human-like immune systems and are easy to carry out genetic engineering and cell engineering transformation, and the advantages determine the unique advantages of the mouse model in the biological medical science research. However, even if the mouse animal model has many advantages, the characteristics of complicated modeling flow, long modeling time, high operation difficulty, high hardware requirement, high modeling cost and the like still exist.
Organoids are three-dimensional cell mass structures formed by in vitro culture of induced pluripotent stem cells, embryonic stem cells, or adult tissue-resident progenitor cells. Organoids are an important bridge between two-dimensional culture and in vivo models, which are more physiologically relevant than single-layer cell culture models, while manipulating the interest components, signaling pathways, and genome editing more easily than in vivo models. Organoids are valuable in that they are capable of self-organizing into minimal biological units, exhibiting similar functions and complexities as the original tissue. The operability of organoids suggests that organoids will provide an excellent model system for extensive basic research, including expression profiling studies and analysis of rare cell lineages that are difficult to obtain in vivo. Meanwhile, the function evaluation can be carried out in the organoid transplant. In addition, organoids can also be produced from the patient's own tissues or induced pluripotent stem cells and used to study rare diseases lacking animal models. In regenerative medicine, organoid technology is expected to replace severely damaged organs, such as the pancreas of type 1 diabetics.
At present, organoids and other three-dimensional cell culture techniques are mainly performed manually and by using Matrigel as a biological scaffold. The method has the limitation that manual operation can cause experimental errors, and the stability and uniformity of the experiment are reduced.
Disclosure of Invention
The invention aims to provide a suspension culture medium and a culture method for medulloblastoma organoids and 3D microspheres. The culture medium and the culture method can be used for culturing the medulloblastoma 3D organoids rapidly and stably without Matrigel. Can greatly reduce the cost of organoid and three-dimensional cell culture, and is favorable for the mass expansion of cells.
In order to achieve the above purpose, the technical scheme provided by the invention is as follows:
a culture medium for suspension culture of medulloblastoma organoids and 3D microspheres consists of the following components: basic culture medium, B27-5X, N2 1-5X, N-acetylcysteine 0.2-5 mu M, FGFb-100 ng/mL, noggin 5-200ng/mL, R-spondin 1-1000 ng/mL, wnt3a 5-200ng/mL, CHIR990211-10 mu M, laminin 0.5-100ng/mL, sialic acid 5-50 mu M, NEAA 1-10X, penicillin streptomycin mixture 1-5X and Primocin 0.5-5mg/mL.
Furthermore, the basic culture medium is formed by mixing Advanced DMEM/F12 and a biological Basal culture medium according to the volume ratio of 1:10-150:1.
Furthermore, the basic culture medium is formed by mixing Advanced DMEM/F12 and a biological Basal culture medium according to a volume ratio of 2:1.
The invention also provides a suspension culture method of the medulloblastoma organoid and the 3D microsphere, which comprises the following steps:
(1) Separating and obtaining medulloblastoma cells;
(2) The cells were resuspended using the above media, transferred to ultra-low adhesion cell culture dishes or plates, and incubated in a carbon dioxide incubator at 37℃with 5% CO 2 Culturing for 4-7 days at 60-120rpm to obtain the medulloblastoma organoid or medulloblastoma 3D microsphere.
Further, the medulloblastoma cells are obtained by preprocessing a surgical excision specimen or biopsy tissue of the medulloblastoma.
Further, the pretreatment includes the steps of washing, shredding, tissue block digestion, erythrocyte lysis and filtering to remove impurities.
Further, during the culturing in step (2), the liquid medium is replaced every 3 to 5 days.
The invention has the following technical advantages:
1) The culture medium can fully promote self-organization and self-differentiation of cells, simulate the development and formation process of tissues in vivo, and obtain a three-dimensional culture which is more similar to the structure in vivo.
2) The culture medium of the invention can be used for culturing tissues, effusions or cells of animal or human origin.
3) The culture medium and the culture method do not need matrigel, so that potential pollution and influence of the murine matrigel on a personnel sample are avoided, and the cost is greatly saved.
4) The microspheres or organoids cultured by the culture medium can be amplified in vitro for a long time, and the generation number is more than 5.
5) The cell mass with the diameter of 20-40 μm can be cultivated into organs by the culture medium and the culture method.
6) The cell mass with the diameter of 70-100 μm can be cultivated into organoids by the culture medium and the culture method.
7) The culture medium of the invention can be matched with the culture method of the invention to culture up to 5000 cell clusters into organoids.
8) The culture medium is matched with the culture method disclosed by the invention, the operation is simple, the influence of personnel operation is less, and the culture result is stable.
Drawings
FIG. 1 is a bright field image of a medulloblastoma organoid culture in example 1.
FIG. 2 is a photograph of a 3D microsphere culture bright field of a medulloblastoma cell line in example 2.
FIG. 3 is a bright field image of medulloblastoma organoids after removal of Sialic acid in comparative example 1.
FIG. 4 is a bright field image of medulloblastoma organoid culture after adjusting the FGFb concentration to 500ng/mL in comparative example 2.
Detailed Description
The following examples are further illustrative of the invention and are not intended to be limiting thereof.
Example 1
1. The culture medium consists of the following components: basic culture medium, B27 1X, N2 1X, N-acetylcysteine2 mu M, FGFb ng/mL, noggin 100ng/mL, R-spondin 150 ng/mL, wnt3a 15ng/mL, CHIR99021 5 mu M, laminin ng/mL, sialic acid 5 mu M, NEAA X, penicillin streptomycin mixed solution 1X and Primocin2mg/mL, wherein the basic culture medium is formed by mixing Advanced DMEM/F12 and a nerve Basal culture medium according to a volume ratio of 2:1.
2. Organoid culture of medulloblastoma
1) Surgical resection specimens or biopsied tissue from a fresh source of medulloblastoma are pre-treated, the pretreatment being completed within 5 hours after tissue isolation. The pretreatment steps are as follows: (1) the operation tissue is quickly washed 3-5 times by using physiological saline containing antibiotics (penicillin, streptomycin and amphotericin B). (2) Tissue was cut into 1mm using sterilized ophthalmic scissors 3 Is a small block of (a). (3) The tissue was further digested with a mixed enzyme solution of trypsin, collagenase, dnase. (4) The red blood cells are lysed and removed using a red blood cell lysate. (5) Cells were filtered using a 70mm cell filter to remove impurities. After obtaining a cell mass with a cell number of 3-50 cells, the supernatant was removed by centrifugation for use.
2) Cells were resuspended using the medium described in 1 above and transferred into ultra-low adhesion cell culture dishes or plates.
3) The horizontal shaker was placed in a carbon dioxide incubator and the culture plate was placed on the horizontal shaker.
4) The horizontal shaker was started and the rotational speed was set at 120rpm.
5) At 37 ℃,5% CO 2 Culturing under concentration.
6) The liquid culture medium is replaced every 3-5 days, and the medulloblastoma organoids can be obtained after culturing for 4-7 days (figure 1).
EXAMPLE 2 three-dimensional cell pellet culture of medulloblastoma cell lines
1. The culture medium consists of the following components: basic culture medium, B27 1X, N2 1X, N-acetylcysteine5 mu M, FGFb ng/mL, noggin 20ng/mL, R-spondin 110 ng/mL, wnt3a 5ng/mL, CHIR9902110 mu M, laminin ng/mL, sialic acid 15 mu M, NEAA X, penicillin streptomycin mixed solution 1X and Primocin2mg/mL, wherein the basic culture medium is formed by mixing Advanced DMEM/F12 and a nerve Basal culture medium according to a volume ratio of 2:1.
2. Three-dimensional cell ball culture of medulloblastoma cell line
1) And taking out the medulloblastoma cell line frozen by liquid nitrogen, placing the medulloblastoma cell line in a water bath at 37 ℃ for 5min, and taking out.
2) The frozen stock was diluted by adding Advanced DMEM/F12 medium, transferred to a 15mL centrifuge tube, centrifuged at 1500rpm for 5min and the supernatant discarded.
3) Cells were resuspended using the medium described in 1 above and transferred into ultra-low adhesion cell culture dishes or plates.
4) The horizontal shaker was placed in a carbon dioxide incubator and the culture plate was placed on the horizontal shaker.
5) The horizontal shaker was started and the rotational speed was set at 120rpm.
6) At 37 ℃,5% CO 2 Culturing under concentration.
7) The liquid culture medium is replaced every 3-5 days, and the medulloblastoma 3D microsphere can be obtained after culturing for 4-7 days (figure 2).
Comparative example 1: the culture medium of the invention has a comparison of culture effect with that of the culture medium from which the Sialic acid is removed
1) Surgical resection specimens or biopsied tissue from a fresh source of medulloblastoma are pre-treated, the pretreatment being completed within 5 hours after tissue isolation. The pretreatment steps include washing, shredding, tissue mass digestion, erythrocyte lysis and filtration to remove impurities (for specific steps, reference example 1), obtaining a cell mass with a cell number of 3-50 cells, and centrifuging to remove the supernatant for later use.
2) Cells were resuspended using the medium described in example 1 without the addition of Sialic acid and transferred to ultra-low adhesion cell culture dishes or plates.
3) The horizontal shaker was placed in a carbon dioxide incubator and the culture plate was placed on the horizontal shaker.
4) The horizontal shaker was started and the rotational speed was set at 120rpm.
5) At 37 ℃,5% CO 2 Culturing under concentration.
6) The liquid culture medium was changed every 3-5 days, and after 4-7 days of culture, some medulloblastoma organoids were found to be formed, but the culture effect was poor, the cell-line phenomenon was not obvious, the cell-line was loose, and no clear tight connection was formed between cells (fig. 3).
Comparative example 2: the culture medium of the invention has a comparison of culture effect with that of the culture medium of high-concentration FGFb
1) Surgical resection specimens or biopsied tissue from a fresh source of medulloblastoma are pre-treated, the pretreatment being completed within 5 hours after tissue isolation. The pretreatment steps include washing, shredding, tissue mass digestion, erythrocyte lysis and filtration to remove impurities (for specific steps, reference example 1), obtaining a cell mass with a cell number of 3-50 cells, and centrifuging to remove the supernatant for later use.
2) The culture medium described in example 1 was used, and after adjusting the concentration of FGFb to 500ng/mL, cells were resuspended and transferred to a adherent cell culture dish or plate.
3) The horizontal shaker was placed in a carbon dioxide incubator and the culture plate was placed on the horizontal shaker.
4) The horizontal shaker was started and the rotational speed was set at 120rpm.
5) At 37 ℃,5% CO 2 Culturing under concentration.
6) The liquid medium was changed every 3-5 days, and after 4-7 days of culture, no obvious aggregation of cells was found, and no obvious organoids were formed (FIG. 4).
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that the above-mentioned preferred embodiment should not be construed as limiting the invention, and the scope of the invention should be defined by the appended claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and such modifications and adaptations are intended to be comprehended within the scope of the invention.
Claims (9)
1. A culture medium for suspension culture of medulloblastoma organoids and 3D microspheres, which is characterized by comprising the following components: basic culture medium, B27-5X, N2 1-5X, N-acetylcysteine 0.2-5 mu M, FGFb-100 ng/mL, noggin 5-200ng/mL, R-spondin 1-1000 ng/mL, wnt3a 5-200ng/mL, CHIR990211-10 mu M, laminin 0.5-100ng/mL, sialic acid 5-50 mu M, NEAA 1-10X, penicillin streptomycin mixture 1-5X and Primocin 0.5-5mg/mL.
2. The culture medium according to claim 1, wherein the Basal medium is composed of Advanced DMEM/F12 and a Neural Basal medium mixed in a volume ratio of 1:10-150:1.
3. The culture medium according to claim 2, wherein the Basal medium is composed of Advanced DMEM/F12 and a Neural Basal medium mixed in a volume ratio of 2:1.
4. A suspension culture method of medulloblastoma organoids and 3D microspheres, comprising the steps of:
(1) Separating and obtaining medulloblastoma cells;
(2) The cells are resuspended using the medium of claim 1, transferred into an ultra-low adhesion cell culture dish or plate, and cultured in a carbon dioxide incubator until medulloblastoma organoids or medulloblastoma 3D microspheres are obtained.
5. The method of claim 4, wherein the time of culturing is 4-7 days.
6. The method according to claim 4, wherein the conditions for the cultivation are 37℃and 5% CO 2 、60-120rpm。
7. The method of claim 4, wherein the liquid medium is changed every 3-5 days during the culturing.
8. The method of claim 4, wherein the medulloblastoma cells are obtained from a surgical resection specimen or biopsy of the medulloblastoma by pretreatment.
9. The method of claim 8, wherein the pretreatment comprises the steps of washing, shredding, tissue block digestion, erythrocyte lysis, and filtering to remove impurities.
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