CN114958746A - Method and kit for inducing pluripotent stem cells to generate 3D brain bodies - Google Patents
Method and kit for inducing pluripotent stem cells to generate 3D brain bodies Download PDFInfo
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Abstract
The invention discloses a method and a kit for inducing pluripotent stem cells to generate 3D brain bodies. The method for inducing the pluripotent stem cells to generate the 3D brain bodies comprises the following steps: and sequentially culturing the pluripotent stem cells in a culture medium I, a culture medium II, a culture medium III, a culture medium IV and a culture medium V to obtain the 3D brain. The method for generating the 3D-like brain body by inducing the pluripotent stem cells can successfully induce and differentiate the pluripotent stem cells into mature 3D-like brain bodies which can generate a large number of specific-form expression specific markers, does not need a co-culture technology and a gene recombination technology, is simple and easy to operate, makes the research on the development of human cerebral cortex, the discovery of key nodes and new treatment targets in the development of the brain-like body possible, and provides a foundation for constructing the brain-like body with specific disease prevention and treatment functions.
Description
Technical Field
The invention relates to a method and a kit for generating a 3D brain body by inducing pluripotent stem cells in the field of stem cell biology.
Background
Human pluripotent stem cells (hpscs), including human embryonic stem cells (hescs) and induced pluripotent stem cells (hipscs), theoretically have the ability to differentiate into all cell types in humans. The hPSC gene targeting technology and reverse genetics mode based on hPSC introduces disease specific mutation to hPSC, differentiates hPSC carrying human disease genetic gene into specific cell type, theoretically can simulate human disease generation in vitro, research human disease generation mechanism, and establish in vitro screening platform to search therapeutic medicine. The development of stem cell technology will bring revolutionary breakthrough to the mechanism research and regenerative medicine treatment of human diseases.
In hPSC differentiation, the physiological relevance of 2D tissue culture systems is limited by environmental rigidity, limited movement of secreted factors in the extracellular space, and spatially restricted cell-cell interactions. Differences between 2D culture and in vivo environment may be responsible for the abnormal characteristics observed in cells grown in adherent culture, which may lead to differences in the results of drug screening assays performed in 2D culture and 3D tissues. While the brain-like bodies are 3D cultures maintained in vitro (also referred to as 3D brain-like bodies) and can be differentiated directly from hpscs. Brain-like bodies resembling neural tissue are likely to create a more realistic cellular environment for simulating the cellular biology of the nervous system due to the more diverse cellular components, lack of cellular interaction with artificial substrates, and the disorientation of complex 3D structures seen in the developing brain. In addition, brain-like bodies can be cultured in vitro for extended periods of time, allowing for the study of prolonged human cortiogenesis. Also, cell types from different brain regions and other tissue organoids can be combined into assemblies to study interactions between cell types of different origin.
The pluripotent stem cells are differentiated in an in vitro differentiation system to form 3D brain bodies by the following procedure: embryoid bodies, neural stem cells, cortical neurons, astrocytes. Although 3D-like somatic differentiation protocols are rapidly evolving, there are few differentiated cell types, heterogeneity in differentiation results, difficulty in control, and long time spent, perhaps even over 200 days.
Therefore, there is a need to provide a simple and convenient method to differentiate and obtain 3D brain bodies.
Disclosure of Invention
The technical problem to be solved by the present invention is how to induce the generation of 3D-like brains of various cell types by a simple and convenient method.
In order to solve the technical problems, the invention firstly provides a method for preparing a 3D brain body, which comprises the following steps:
1) culturing the pluripotent stem cells in a culture medium I to obtain a tissue (namely an Embryoid Body (EB)) cultured by the culture medium I;
the culture medium I is prepared by adding KnockOut into DMEM/F12 culture medium TM Serum Replacement, non-essential amino acids, 2-mercaptoethanol, Y-27632, dorsomorphin, and SB 431542; wherein, KnockOut TM The volume percentage content of the Serum Replacement in the culture medium I is 20 percent; the content of non-essential amino acid in the culture medium I is 1 mM; the content of the 2-mercaptoethanol in the culture medium I is 0.1 mM; the content of Y-27632 in the culture medium I is 20 mu M; the content of dorsomorphin in the medium I is 5 μ M, and the content of SB431542 in the medium I is 5 μ M;
2) culturing the tissue cultured by the culture medium I in a culture medium II to obtain the tissue cultured by the culture medium II (namely an Embroid Body (EB)) with smooth edge;
the culture medium II is prepared by adding KnockOut into DMEM/F12 culture medium TM Serum Replacement, non-essential amino acids, 2-mercaptoethanol, Y-27632, dorsomorphin, and SB 431542; wherein, KnockOut TM The volume percentage content of the Serum Replacement in the culture medium II is 20 percent; the content of non-essential amino acids in the medium II is 1 mM; the content of 2-mercaptoethanol in the culture medium II is 0.1 mM; the content of Y-27632 in the culture medium I is 5 mu M; the content of dorsomorphin in the culture medium II is 5 mu M; the content of SB431542 in the medium II is 5. mu.M;
3) culturing the tissue cultured by the culture medium II in a culture medium III to obtain the tissue cultured by the culture medium III (namely immature brain);
the culture medium III is obtained by adding GlutaMAX additive, B27 additive without vitamin A, epidermal cell growth factor and human fibroblast growth factor-2 into neurobasal A culture medium; wherein, the volume percentage content of the GlutaMAX additive in the culture medium III is 1%, the volume percentage content of the B27 additive without vitamin A in the culture medium III is 2%, and the content of the epidermal growth factor and the content of the human fibroblast growth factor-2 in the culture medium III are both 20 ng/ml;
4) culturing the tissue cultured by the culture medium III in a culture medium IV to obtain a tissue (namely a brain-like body) cultured by the culture medium IV;
the culture medium IV is obtained by adding a GlutaMAX additive, a brain-derived neurotrophic factor, a neurotrophic factor 3 and a glial cell-derived neurotrophic factor into a neurobasal A culture medium; wherein, the volume percentage content of the GlutaMAX additive in the culture medium IV is 1 percent, and the content of the brain-derived neurotrophic factor in the culture medium IV is 20 ng/ml; the content of the neurotrophic factor 3 in the culture medium IV is 20 ng/ml; the content of the glial cell line-derived neurotrophic factor in the culture medium IV is 20 ng/ml;
5) culturing the tissue cultured by the culture medium IV in a culture medium V to obtain a 3D brain body;
the culture medium V is obtained by adding a GlutaMAX additive, a brain-derived neurotrophic factor, a neurotrophic factor 3 and a glial cell-derived neurotrophic factor into a neurobasal A culture medium; wherein, the volume percentage content of the GlutaMAX additive in the culture medium V is 1 percent, and the content of the brain-derived neurotrophic factor in the culture medium V is 10 ng/ml; the content of the neurotrophic factor 3 in the culture medium V is 10 ng/ml; the content of glial cell line-derived neurotrophic factor in the culture medium V is 10 ng/ml.
Wherein the 3D-like brain body can be a brain-like body containing PAX 6-positive neural progenitor cells, SOX 1-positive and SOX 2-positive radial glial-like cells, TUJ 1-positive immature neuronal cells, TBR 1-positive and CTIP 2-positive cortical neurons and/or KI67 and pH3S 10-positive proliferating cells.
In step 1) of the method, the method may further comprise a step of digesting the stem cells before culturing the medium I.
The digestion may be performed using proteolytic enzymes and collagenases. The digestion can be carried out by using ACCUTASE cell digestive juice.
The culture in steps 1) -5) of the method can be carried out at 37 ℃ and 5% CO 2 Under the conditions of (1).
The culture in steps 1) -5) was carried out at 37 ℃ and 5% CO 2 Under the conditions of an incubator.
The cultivation in step 3) can be carried out in a shaker.
In the above method, the time for the culture in step 1) may be 2 days.
The time for the culture in step 2) may be 4 days.
The time for the culture in step 3) may be 19 days.
The time for the culture in step 4) may be 18 days.
The culture time in the step 5) can be determined according to the tissue growth condition, so as to obtain the 3D brain body successfully.
In the above method, the cells may be centrifuged before the culturing in step 1).
The culturing in steps 1) -5) can be performed in a low adsorption cell culture plate.
The steps 2) to 3) may further include a step of replacing the medium.
In the above method, the pluripotent stem cell may be an induced pluripotent stem cell (hiPSC), an adult (adult) stem cell, a somatic (somatic) stem cell, a cancer stem cell, or any other pluripotent stem cell having a differentiation ability.
The induced pluripotent stem cell may be a human induced pluripotent stem cell. In one embodiment of the invention, the human induced pluripotent stem cell is a product of biotechnology limited beijing seebeck, cat # CA 4027106.
The invention also provides a kit, which comprises the culture medium III, the culture medium IV and the culture medium V.
The kit can also comprise the culture medium I and the culture medium II.
The application of the kit in the preparation of 3D brain bodies also belongs to the protection scope of the invention.
The application of the kit in preparing a product for inducing and generating the 3D brain also belongs to the protection scope of the invention.
In the present invention, the DMEM/F12 medium may be Life Technologies, cat # 11330032.
The neurobasal A medium can be Life Technologies, cat # 10888.
KnockOut TM The Serum Replacement can be Invitrogen, and the product is 10828-028.
The nonessential amino acid can be the product of Gibco, cat # 11140050.
Y-27632 may be a Selleck product, cat # S1049.
Dorsomorphin may be a product of SigmaAldrich, cat # P5499.
SB431542 may be the product of Selleck, cat # S1067.
The GlutaMAX additive may be a product of Gibco-BRL, cat.no. 35050.
The B27 additive, without vitamin A, may be a product of Gibco-BRL, cat.no. 12587-010.
The method for preparing the 3D brain body can successfully induce and differentiate the pluripotent stem cells into mature 3D brain bodies which can generate a large amount of specific expression markers with special forms, does not need a co-culture technology and a gene recombination technology, is simple and easy to operate, and the differentiation process does not need the wrapping of matrigel, can effectively reduce the operation on the brain-like body, is beneficial to the uniformity of differentiation and the protection of the brain-like body, can be directionally differentiated into PAX6 positive nerve progenitor cells, SOX2 and SOX1 positive radial glial cells, TUJ1 positive immature neurons, TBR1 positive and CTIP2 positive cortical neurons and other multi-nerve cell type brain-like bodies, the method makes the research on the development of human cerebral cortex, the development of key nodes in the development process of similar brains and new therapeutic targets possible, and also provides a basis for constructing a similar brains with specific disease prevention and treatment functions.
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
Drawings
FIG. 1 is a flow chart of the induction of 3D brain body by human pluripotent stem cells.
FIG. 2 shows the 18 th day immunofluorescence staining results of 3D brains induced by pluripotent stem cells.
FIG. 3 shows the results of 30-day immunofluorescence staining of 3D-like brains induced by pluripotent stem cells.
FIG. 4 shows the 60 th day immunofluorescence staining results of 3D-like brains induced by pluripotent stem cells.
FIG. 5 shows the results of the culture using the medium I'.
Detailed Description
The experiments in the following examples, each set up three replicates.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products.
In the following examples, the conditions of the humidified incubator were: humidity 95%, 37 deg.C, 5% CO 2 。
Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified.
1. Reagents used in the following examples:
(1) human pluripotent stem cell medium (PGM1 medium) (beijing seebeck biotechnology limited, # CA 1007500).
(2) B27 additive without vitamin A (Gibco-BRL, cat. No. 12587-010).
(3) ACCUTASE cell digest (BD, # 561527).
(4) Brain-derived neurotrophic factor (BDNF) (PeproTech, cat. No. 450-02).
(5) GlutaMAX additive (Gibco-BRL, cat. No. 35050).
(6) Epidermal growth factor (Peprotech, AF-100-15).
(7) Matrigel (Matrigel, Corning, # 356234).
(8) Human fibroblast growth factor-2 (Human FGF-basic, peprotech, # AF-100-18B).
(9) neurobasal A medium (Life Technologies, 10888).
(10)dorsomorphin(SigmaAldrich,P5499)。
(11)SB431542(Selleck，S1067)。
(12) Glial cell line-derived neurotrophic factor (Peprotech, 450-10).
(13) Neurotrophic factor 3(Peprotech, 45003).
(14) DMEM/F12 medium (Life Technologies, 11330).
(15)Y-27632(Selleck,#S1049)。
(16) 4% paraformaldehyde solution (Beijing Runzukang Biotech limited, # B1057-100).
(17) Bovine serum albumin (BSA, EASYBIO, # BE 6254).
(18)Tritonx-100(SIGMA-ALDRICH,#HPA014518)。
(19) Sodium azide (SIGMA-ALDRICH, # S2002).
(20) Rabbit anti-KI 67 antibody (ThemoFisher, # RM-9106-S).
(21) Murine anti-TUJ 1 antibody (801202, # 801202).
(22) Rabbit anti-PAX 6 antibody (Biolegend, # 901301).
(23) Rabbit anti-pH 3S10 antibody (Abcam, # Ab 41548).
(24) Rabbit anti-TBR 1 antibody (Abcam, ab 31940).
(25) Rat anti-CTIP 2 antibody (Abcam, # ab 18465).
(26) Rabbit anti-SOX 2 antibody (Cell Signaling Technology, 3728S).
(27) Sheep anti-SOX 1 antibody (RD System, AF 3369).
(28) Alexa Fluor 488 labeled donkey anti-rabbit IgG (H + L) secondary antibody (Jackson, # 711-545-152).
(29) Alexa Fluor 488 labeled donkey anti-mouse IgG (H + L) secondary antibody (Jackson, # 711-545-once 150).
(30) Alexa Fluor 594 labeled donkey anti-rabbit IgG (H + L) secondary antibody (Jackson, # 711-.
(31) The donkey anti-goat IgG (H + L) secondary antibody (Invitrogen, a11057) was labeled with Alexa Fluor 568.
(32) Alexa Fluor 568 labeled goat anti-rat IgG (H + L) secondary antibody (Life, cat. No. a 11077).
(33) Human TruStainFcX TM Blocking reagent (BioLegent, # 422301).
(34) Human induced pluripotent stem cells (Beijing seebeck Biotechnology Co., Ltd. # CA 4027106).
(35) DPBS (duchenne phosphate buffer) (Gibco, C14190500 CP).
(36)OCT(Sakura,4583)。
(37)KnockOut TM Serum Replacement(Invitrogen，10828-028)。
(38) Non-essential amino acids (Gibco, 11140050).
2. The media used in the examples below:
culture medium I: medium I prepared by adding KnockOut to DMEM/F12 medium TM Serum Replacement, non-essential amino acids, 2-mercaptoethanol, Y-27632, dorsomorphin, and SB 431542; wherein, KnockOut TM The volume percentage content of the Serum Replacement in the culture medium I is 20 percent; the content of non-essential amino acid in the culture medium I is 1 mM; the content of the 2-mercaptoethanol in the culture medium I is 0.1 mM; the content of Y-27632 in the culture medium I is 20 mu M; the content of dorsomorphin in the culture medium I is 5 mu M; the content of SB431542 in Medium I was 5. mu.M.
And (3) a culture medium II: medium II by adding KnockOut to DMEM/F12 medium TM Serum Replacement, non-essential amino acids, 2-mercaptoethanol, Y-27632, dorsomorphin, and SB 431542; wherein, KnockOut TM The volume percentage content of the Serum Replacement in the culture medium II is 20 percent; the content of non-essential amino acid in the culture medium II is 1 mM; the content of the 2-mercaptoethanol in the culture medium II is 0.1 mM; the content of Y-27632 in the culture medium II is 5 mu M; the content of dorsomorphin in the culture medium II is 5 mu M; the content of SB431542 in Medium II was 5. mu.M.
And (3) culture medium III: the culture medium III is obtained by adding GlutaMAX additive, B27 additive without vitamin A, epidermal growth factor and human fibroblast growth factor-2 into neurobasal A culture medium; wherein, the volume percentage content of the GlutaMAX additive in the culture medium III is 1%; the volume percentage content of the B27 additive withoutvitamin A in the culture medium III is 2 percent; the contents of the epidermal growth factor and the human fibroblast growth factor-2 in the culture medium III are both 20 ng/ml.
Culture medium IV: the culture medium IV is obtained by adding GlutaMAX additive, brain-derived neurotrophic factor, neurotrophic factor 3 and glial cell line-derived neurotrophic factor into neurobasal A culture medium; wherein, the volume percentage content of the GlutaMAX additive in the culture medium IV is 1 percent; the content of the brain-derived neurotrophic factor in the culture medium IV is 20 ng/ml; the content of the neurotrophic factor 3 in the culture medium IV is 20 ng/ml; the content of glial cell line-derived neurotrophic factor in culture medium IV was 20 ng/ml.
And (3) a culture medium V: the culture medium V is obtained by adding GlutaMAX additive, brain-derived neurotrophic factor, neurotrophic factor 3 and glial cell line-derived neurotrophic factor into neurobasal A culture medium; wherein, the volume percentage content of the GlutaMAX additive in the culture medium V is 1 percent; the content of the brain-derived neurotrophic factor in the culture medium V is 10 ng/ml; the content of the neurotrophic factor 3 in the culture medium V is 10 ng/ml; the content of glial cell line-derived neurotrophic factor in the culture medium V was 10 ng/ml.
Sealing liquid: the confining liquid is obtained by adding BSA, Triton x-100 and sodium azide into DPBS; wherein, the concentration of BSA in the blocking solution is 2g/100mL, the volume percentage content of Triton x-100 in the blocking solution is 0.3 percent, and the concentration of sodium azide in the blocking solution is 0.3g/100 mL.
Example 1 method for inducing the production of 3D-like brains by pluripotent Stem cells
Inducing human induced pluripotent stem cells to 3D brain
The flow chart is shown in FIG. 1, and the steps are as follows:
1. culturing human induced pluripotent stem cells in Matrigel-coated hexagonsOn the well plate, human pluripotent stem cell maintenance medium (PGM1 medium) was used at 37 ℃ with 5% CO 2 And culturing in a humidifying culture box with the humidity of 95% until the adherent growth of the cells reaches 50% -70% (culturing for 4-5 days), and replacing the culture medium once a day, wherein 2mL of the culture medium is used each time.
2. Aspirating the medium, washing DPBS 2 times to remove dead cells, adding 0.5 mM ACCUTASE cell digest at 37 deg.C, 5% CO per well 2 The humidified incubator with a humidity of 95% was left to stand for 3 minutes, and then 1ml of GM1 medium was added to stop the digestion, and 200g was centrifuged for 5 minutes to collect cells.
3. The supernatant was aspirated and resuspended in Medium I, 9000 cells/well, cells were seeded in a low adsorption 96-well plate, 300g immediately, centrifuged for 3 min, 37 ℃, 5% CO 2 And culturing in a humidified incubator with the humidity of 95% for 2 days. The day of starting the culture using the medium I was regarded as the day of 0 culture for 2 days.
4. The supernatant was aspirated off and medium II was added slowly at 37 ℃ with 5% CO 2 Culturing for 4 days in the humidified incubator to obtain 400-micron embryoid bodies, and replacing the culture medium II every day by 150 μ l/hole. The day of starting the culture using the medium II was 2 days in total for 4 days.
5. Sucking out embryoid body with 200 μ l low-adsorption wide-hole pipette tip, transferring to low-adsorption 24-well plate, discarding culture medium, adding culture medium III, and culturing at 37 deg.C with 5% CO 2 The humidified incubator of (1) was incubated for 18 days in a shaker, medium III was changed every two days at 400. mu.l/well. The day of starting the culture using the medium III was 6 days, and the culture was continued for 19 days.
6. Sucking supernatant, adding culture medium IV, standing at 37 deg.C and 5% CO 2 The culture was carried out in a humidified incubator for 18 days, and the medium IV was changed every two days at 500. mu.l/well. The day of starting the culture using the medium IV was 25 days, and the culture was continued for 18 days.
7. Sucking supernatant, adding culture medium V, standing at 37 deg.C and 5% CO 2 The culture was carried out in a humidified incubator, and the medium was changed every 4 days at a rate of 500. mu.l/well, and the day of starting the culture with the medium V was 43 days of the culture, and 3D brains were obtained after the completion of the culture.
Second, induced generation of 3D brain body by pluripotent stem cells immunofluorescence staining at 18 th and 30 th days
Taking the brain-like body cultured for 18 th or 30 th day in the step one, abandoning the culture medium, adding 4% paraformaldehyde solution for fixing for 4-6 hours, abandoning the 4% paraformaldehyde solution, adding 30g/100mL sucrose aqueous solution for dehydration for 2 days, freezing and embedding the brain-like body by using OCT, and slicing the brain-like body by using a freezing microtome (Lyca, CM950) for 20-40 mu m.
Immunofluorescent staining of the sectioned brain-like bodies: rinse three times with 500 μ L DPBS for 5 minutes each time and discard the supernatant with suction. Add 500. mu.L of blocking solution and block for 1 hour at room temperature. The blocking solution was aspirated away, 200. mu.L of fresh blocking solution was added, and the mixture was mixed at 1: 1000 adding one of goat anti-SOX 1 antibody, rabbit anti-SOX 2 antibody, rabbit anti-KI 67 antibody, rabbit anti-pH 3S10 antibody, mouse anti-TUJ 1 antibody and rabbit anti-PAX 6 antibody, and incubating at 4 ℃ for 12 hours. The supernatant was aspirated and rinsed three times with 500 μ L DPBS for 5 minutes each. The supernatant was aspirated off, 200 μ L of fresh blocking solution was added, and the volume ratio of 1: DAPI was added 2000, 1: 500 adding secondary antibodies corresponding to the antibodies, wherein the primary antibodies respectively correspond to the following secondary antibodies: the donkey anti-goat IgG (H + L) secondary antibody was labeled with Alexa Fluor 568, the donkey anti-rabbit IgG (H + L) secondary antibody was labeled with Alexa Fluor 488, the Alexa Fluor 488 donkey anti-mouse IgG (H + L) secondary antibody was labeled with Alexa Fluor 594, and the donkey anti-rabbit IgG (H + L) secondary antibody was labeled with Alexa Fluor 594, and after incubation at 37 ℃ for 1.5 hours in the dark place, the supernatant was discarded, and washed three times with 500 μ LDPBS in the dark place for 5 minutes each. Immunofluorescent-stained brain-like body treated with anti-quenching blocking agent (i.e., human TruStainFcX) TM Sealer) and then observed for staining using a fluorescence microscope.
The immunofluorescence staining results of the 18 th day brains (labeled as D18 brains) cultured in step 5 in step one are shown in FIG. 2: the D18 brains have radial glial-like cells positive for SOX1 and SOX2, PAX6 positive neural progenitor cells, and TUJ1 positive immature neuronal cells, indicating that the brains have neural stem/progenitor cells and neurons.
The immunofluorescence staining results of the brain-like bodies (labeled as D30 brain-like bodies) cultured at the 6 th day of the step I are shown in FIG. 3: the D30 brain has KI67 and pH3S10 positive proliferating cells, PAX6 positive neural progenitor cells and TUJ1 positive immature neuronal cells, indicating that the brain-like body has neural stem/progenitor cells and a plurality of cell types of neurons at the moment.
Third, induced generation of 3D brain body by pluripotent stem cells immunofluorescence staining at day 60
Taking the brain-like body cultured in the step 7 on the 60 th day in the step one, abandoning the culture medium, adding 4% paraformaldehyde solution for fixing for 4-6 hours, abandoning 4% paraformaldehyde solution, adding 30% sucrose solution for dehydration for 2 days, freezing and embedding the brain-like body by using OCT, and slicing the brain-like body by using a freezing microtome (Lyca, CM950) for 20-40 mu m.
Immunofluorescence staining was performed on the sectioned brain-like bodies: rinse three times with 500 μ L DPBS for 5 minutes each time and discard the supernatant with suction. Add 500. mu.L of blocking solution and block for 1 hour at room temperature. The blocking solution was aspirated off, 200. mu.L of fresh blocking solution was added, and the volume ratio of 1: 1000 adding one of rabbit anti-KI 67 antibody, rabbit anti-pH 3S10 antibody, mouse anti-TUJ 1 antibody, rabbit anti-PAX 6 antibody, rabbit anti-TBR 1 antibody and rat anti-CTIP 2 antibody, and incubating at 4 ℃ for 12 hours. The supernatant was aspirated and rinsed three times with 500 μ L DPBS for 5 minutes each. The supernatant was aspirated off, 200 μ L of fresh blocking solution was added, and the volume ratio of 1: 2000 addition of DAPI, 1: 500 adding secondary antibodies corresponding to the antibodies, wherein the primary antibodies respectively correspond to the following secondary antibodies: the sample was incubated at 37 ℃ for 1.5 hours in the dark, and then the supernatant was aspirated and rinsed three times with 500. mu.L of DPBS for 5 minutes each time. Immunofluorescent-stained brain-like body treated with anti-quenching blocking agent (i.e., human TruStainFcX) TM Sealer) and then observed for staining using a fluorescence microscope.
The immunofluorescence staining results of the 60 th day brains (labeled as D60 brains) cultured in step 7 in step one are shown in FIG. 4: d60 brain has KI67 and pH3S10 positive proliferation cells, PAX6 positive neural progenitor cells and TUJ1 positive immature neurons, TBR1 positive and CTIP2 positive cortical deep neurons (i.e. neurons in 5-6 layers of cerebral cortex), which indicates that the brain-like body has multiple cell types of neural stem/progenitor cells and cortical neurons.
Comparative examples 1,
Culture was carried out in the same manner as in example 1 except that the medium I 'was replaced with the medium I', and it was revealed that no embryoid bodies could be formed by this method.
The difference between the medium I 'and the medium I is that the content of dorsomorphin and SB431542 in the medium I' is 10. mu.M, and the other components and concentrations are the same as those in the medium I.
The results (FIG. 5) show that no embryoid bodies could be successfully obtained with medium I'.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific examples, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Claims (10)
1. A method of preparing a 3D brain body, comprising:
1) culturing the pluripotent stem cells in a culture medium I to obtain a tissue cultured by the culture medium I;
the culture medium I is prepared by adding KnockOut into DMEM/F12 culture medium TM Serum Replacement, non-essential amino acids, 2-mercaptoethanol, Y-27632, dorsomorphin, and SB 431542; wherein, KnockOut TM The volume percentage content of the Serum Replacement in the culture medium I is 20 percent; the content of non-essential amino acid in the culture medium I is 1 mM; the content of the 2-mercaptoethanol in the culture medium I is 0.1 mM; the content of Y-27632 in the medium IThe amount was 20. mu.M; the content of dorsomorphin in the culture medium I is 5 mu M, and the content of SB431542 in the culture medium I is 5 mu M;
2) culturing the tissue cultured by the culture medium I in a culture medium II to obtain the tissue cultured by the culture medium II;
the culture medium II is prepared by adding KnockOut into DMEM/F12 culture medium TM Serum Replacement, non-essential amino acids, 2-mercaptoethanol, Y-27632, dorsomorphin, and SB 431542; wherein, KnockOut TM The volume percentage content of the Serum Replacement in the culture medium II is 20 percent; the content of non-essential amino acids in the medium II is 1 mM; the content of 2-mercaptoethanol in the culture medium II is 0.1 mM; the content of Y-27632 in the culture medium I is 5 mu M; the content of dorsomorphin in the culture medium II is 5 mu M; the content of SB431542 in the medium II is 5 μ M;
3) culturing the tissue cultured by the culture medium II in a culture medium III to obtain the tissue cultured by the culture medium III;
the culture medium III is obtained by adding GlutaMAX additive, B27 additive without vitamin A, epidermal cell growth factor and human fibroblast growth factor-2 into neurobasal A culture medium; wherein, the volume percentage content of the GlutaMAX additive in the culture medium III is 1%, the volume percentage content of the B27 additive without vitamin A in the culture medium III is 2%, and the content of the epidermal growth factor and the content of the human fibroblast growth factor-2 in the culture medium III are both 20 ng/ml;
4) culturing the tissue cultured by the culture medium III in a culture medium IV to obtain a tissue cultured by the culture medium IV;
the culture medium IV is obtained by adding a GlutaMAX additive, a brain-derived neurotrophic factor, a neurotrophic factor 3 and a glial cell-derived neurotrophic factor into a neurobasal A culture medium; wherein, the volume percentage content of the GlutaMAX additive in the culture medium IV is 1 percent, and the content of the brain-derived neurotrophic factor in the culture medium IV is 20 ng/ml; the content of the neurotrophic factor 3 in the culture medium IV is 20 ng/ml; the content of the glial cell line-derived neurotrophic factor in the culture medium IV is 20 ng/ml;
5) culturing the tissue cultured by the culture medium IV in a culture medium V to obtain a 3D brain body;
the culture medium V is obtained by adding a GlutaMAX additive, a brain-derived neurotrophic factor, a neurotrophic factor 3 and a glial cell-derived neurotrophic factor into a neurobasal A culture medium; wherein, the volume percentage content of the GlutaMAX additive in the culture medium V is 1 percent, and the content of the brain-derived neurotrophic factor in the culture medium V is 10 ng/ml; the content of the neurotrophic factor 3 in the culture medium V is 10 ng/ml; the content of glial cell line-derived neurotrophic factor in the culture medium V is 10 ng/ml.
2. The method of claim 1, wherein: in the step 1), before the culture of the medium I, the method further comprises the step of digesting the stem cells.
3. The method according to claim 1 or 2, characterized in that: the culture in steps 1) -5) was carried out at 37 ℃ and 5% CO 2 Under the conditions of (1).
4. A method according to any one of claims 1-3, characterized in that: the culture time in the step 1) is 2 days;
and/or, the culturing time in the step 2) is 4 days;
and/or, the time of the culture in the step 3) is 19 days;
and/or, the culturing time in the step 4) is 18 days.
5. The method according to any one of claims 1-4, wherein: the pluripotent stem cell is an induced pluripotent stem cell, an adult stem cell, a somatic stem cell, a cancer stem cell, or any other pluripotent stem cell having the ability to differentiate.
6. The method of claim 5, wherein: the induced pluripotent stem cell is a human induced pluripotent stem cell.
7. A kit comprising medium III, medium IV, and medium V of claim 1.
8. The kit of claim 7, wherein: the kit further comprises the medium I and the medium II as described in claim 1.
9. Use of the kit of claim 7 or 8 for the preparation of a 3D brain.
10. Use of the kit of claim 7 or 8 for the preparation of a product for inducing the production of 3D brain-like bodies.
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