CN117384844A - Method for preparing midbrain organoids suitable for screening nerve drugs in one-step mode with high flux - Google Patents
Method for preparing midbrain organoids suitable for screening nerve drugs in one-step mode with high flux Download PDFInfo
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Abstract
The invention provides a method for preparing a midbrain organoid suitable for screening nerve drugs in one step with high flux, belonging to the organoid culture field; specifically comprises cell culture, embryogenesis, neuroectodermal induction, midbrain specific region induction, tissue growth and organoid maturation stage; the midbrain organoids prepared by the method have higher flux, and the organoid consistency meets the requirement of the midbrain organoids for drug screening.
Description
Technical Field
The invention belongs to the field of organoid culture, and particularly relates to a method for preparing a midbrain organoid suitable for screening nerve drugs in one step with high flux.
Background
The brain is the most complex organ in the human body, and has a blood brain barrier, which makes basic research and drug development of nerve-related diseases very challenging. In addition, since neuronal tissue cannot be obtained from the brain, it is highly dependent on immortalized cancer cells or animal models to explore the structure of the brain and the nature of neural interactions. Moreover, there are often species differences between human neurons and cell types from other species, which makes it difficult to reasonably interpret the results of the study. Researchers are able to access human brain tissue, typically from necropsy or surgical samples of large cell loss, which are of relatively limited origin, of low quality, and often associated with specific diseases. Therefore, to truly understand the brain, there is a need to develop a new model of human brain to address the limitations of the study subject.
The discovery of Yamanaka factor in 2006 has led to the ability to generate induced pluripotent stem cells (ipscs) from the blood of any individual opening up an unlimited possibility to generate customizable physiological human cell types. iPSCs have self-renewing ability and potential to differentiate into disease-related cells, providing a unique research platform in disease-related human cell types. Based on these findings, brain-like organs were created by Madeline Lancaster and Juergen a. Knoblich et al in succession in 2013 and 2014. To date, 3D neuronal structures have been used to mimic early brain development, neurodevelopment and neurodegenerative diseases, and developed techniques include: midbrain organoids for parkinsonism studies, forebrain organoids for autism, and also whole brain organoids and vascularization of brain organoids, etc.
However, the methods for producing midbrain organoids tend to be labor intensive techniques, requiring high technical demands on the operator, the required coating of matrigel is expensive, time consuming to operate, and limit their massive use when high throughput drug screening is required.
Furthermore, in the existing brain organoid culture method, cells are generally inoculated into a 96-well plate to form Embryoid Bodies (EBs), then the EBs are embedded into matrigel, and finally the cells are transferred to a 6-well plate, so that the steps are complicated and the operation is difficult.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for preparing midbrain organoids suitable for screening nerve drugs in one step with high flux.
Noun interpretation:
embryoid Body (EB): embryoid bodies.
Embryoid body forming medium (EBM): embryoid body formation medium.
Brain organoid generation medium (BGM): brain organogenesis medium.
Brain organoid maturation medium (BMM): brain organoid maturation medium.
The technical scheme of the invention is as follows:
a method for preparing a midbrain organoid suitable for neurological drug screening in one step with high throughput, comprising the steps of:
step 1: cell culture
Culturing hiPSC in cell culture plate, adding appropriate amount of iPSC culture medium into each well, resuscitating for the first day, and keeping the culture dish area per cm 2 0.25 μg of iMatrix-511silk matrigel and ROCK inhibitor at a final concentration of 10 μM/mL were added;
step 2: embryoid body formation
Culturing the cells in the step 1 until the fusion degree is 70-80%, adding ReLeSR digestive juice into each hole, and placing at 37 ℃ and 5% CO 2 Incubating in the environment for 5-7 minutes, sucking out ReLeSR, cleaning with iPSC culture medium, sucking the iPSC culture medium, adding embryoid body formation medium (EBM), and adding ROCK inhibitor with final concentration of 100uM and bFGF with final concentration of 4 ng/ml; placing the culture plate on a horizontal orbital shaker, and continuously vibrating at 200rpm, wherein the culture plate is recorded as in-vitro induction day 0;
embryogenic medium comprises a volume fraction of 75% DMEM/F12 basal medium, 20% KnockOut serum replacement, 3% fetal bovine serum, 1% NEAA (optional amino acids), 1% glutamine supplement (Glutamax), 55uM/mL beta-mercaptoethanol, and 10 μg/mL heparin;
step 3: neuroectodermal induction
After the embryoid body in step 2 was cultured for 24 hours, the embryoid body formation medium was replaced with brain organoid formation medium (BGM) in total, while early neuroectodermal development was performed by adding Noggin, 2 μm TGF- β inhibitor, 2 μm M A83-01 and 0.8 μm CHIR 99021 thereto at a final concentration of 200 ng/ml; placing the culture plate on a horizontal orbital shaking table, continuously shaking at 200rpm, continuously culturing for 3 days, and recording as 1 st to 3 rd days of in-vitro induction;
brain organogenesis medium (BGM) contained volume fractions of 47.5% dmem/F12 and 47.5% neural basal medium (Nerobasal), and 1% neaa (optional amino acids), 1% glutamine supplement, 1% n2 supplement, 2% vitamin a free B27, 100U/mL penicillin & streptomycin, 55 μg β -mercaptoethanol, 1 μg/mL heparin;
step 4: midbrain specific region induction
Carrying out in vitro induction culture of the organoid of the step 3 until the 4 th day, changing the total amount of the culture medium of the step 3 into brain organoid production medium (BGM), and simultaneously adding 200ng/ml Noggin, 2 mu M A-01, 0.8 mu M CHIR 99021, 100ng/ml FGF8b and 100ng/ml SHH to induce the organoid to develop to the midbrain basal plate region from the 4 th day to the 6 th day; placing the culture plate on a horizontal orbital shaker, and continuously vibrating at 200 rpm;
step 5: tissue growth
Carrying out in vitro induction culture of the organoid of the step 4 until the 7 th day, changing the total amount of the culture medium of the step 4 into brain organoid generation culture medium (BGM), and simultaneously adding 100ng/ml FGF8b,100ng/ml SHH,200ng/ml laminin and 2.5 mu g/ml insulin, and inducing the organoid to grow to the midbrain organoid tissue from the 7 th day to the 9 th day; placing the culture plate on a horizontal orbital shaker, and continuously vibrating at 200 rpm;
step 6: organoid maturation
The BMM culture medium is half-replaced every other day in the middle of the organoid maturation stage from the 10 th day to more than 30 th day of in vitro induction;
the maturation of brain organoids adopts brain organoid maturation medium; brain organoid maturation medium (BMM) contained volume fractions 94%Neurobasal Medium,1%100x N2 supplement, 2%50x B27, 1% PS, 1% glutamine supplement, 1% NEAA, final concentration of 55. Mu.M beta. -mercaptoethanol, 1. Mu.g/ml heparin, 10ng/ml BDNF, 10ng/ml GDNF, 200. Mu.M ascorbic acid, 125. Mu.M cAMP.
According to a preferred embodiment of the invention, in step 1, the conditions of the cell culture are 37℃and 5% CO 2 Incubating in the environment.
According to a preferred embodiment of the invention, in step 1, hiPSC is cultured in 6-well plates.
According to a preferred embodiment of the invention, the iPSC medium in step 1 is Stemfit 04CT medium.
According to the invention, the iPSC culture matrigel in step 1 is iMatrix-511silk matrigel.
According to the invention, the cleaning medium in step 2 is preferably a Stemfit 04CT medium.
According to a preferred embodiment of the present invention, in step 6, the induction is performed from day 10 to more than day 70 in vitro.
The beneficial effects of the invention are as follows:
1. the invention provides a novel high-flux method for preparing a midbrain organoid, which has higher flux and organoid consistency and meets the requirement of the midbrain organoid for drug screening.
2. The method provided by the invention does not need to embed EB into matrigel, reduces the cost and reduces the technical operation difficulty.
3. The mesoencephalic organoids produced by the invention have a rose ring-shaped ganglion structure produced by the traditional method for preparing the mesoencephalic organoids, express specific nerve-related marker proteins of the mesoencephalic organoids, and provide a reliable high-throughput screening organoid model for researching pathogenesis and molecular mechanism of mesoencephalic development and parkinsonism and individual drug screening.
4. The 3D midbrain organoid model established by the invention comprises a large number of mature dopaminergic neurons, has good functional activity, has higher consistency for drug screening, and is an ideal humanized in vitro model.
5. The method provided by the invention is to culture in the same culture vessel, and is simple to operate, so that the method is called a one-step preparation method.
6. The midbrain organoids cultured by the method provided by the invention can generate sensitivity to medicines after being induced in vitro for 30 days, and are suitable for medicine screening.
Drawings
FIG. 1 is a time-axis diagram of brain organoid culture according to the present invention.
FIG. 2 is a diagram of cell states observed under a 40-fold mirror;
in the figure: a is the cell state of the example on day 0 of induced differentiation, and B is the EB formation state of the example on day 1 of induced differentiation.
FIG. 3 is a diagram of the open field and diameter growth curves of an organoid cultivated using the method described in the present invention.
FIG. 4 is a graph of the detection results of the example induced day 14 immunofluorescence.
FIG. 5 is a graph of the detection results of day 30 immunofluorescence induced by the examples.
FIG. 6 is a graph showing the addition of H after 30 days of example induction 2 O 2 Immunofluorescence results after 6-OHDA and MPTP broad and specific neurotoxic drugs.
Detailed Description
The invention is further described with reference to the drawings and examples, but the scope of the invention is not limited thereto.
The following examples, unless otherwise indicated, are all procedures and procedures routine in the art.
hiPSC purchase in the middle and flourishing sourceScientific grade hiPSC cell line-male.
Example 1
A method for preparing a midbrain organoid suitable for screening a nerve drug in high throughput, comprising the following steps:
1) hiPSC was cultured in 6-well plates, 2mL of Stemfit 04CT medium was added to each well, and the first day was resuscitated, per cm of dish area 2 0.25 μg of iMatrix-511silk matrigel and a final concentration of 10. Mu.M/mL of ROCK inhibitor were added. The iMatrix-511silk matrigel and ROCK inhibitor were added only the first day of passaging or resuscitation, after which the total daily change was made to Stemfit 04CT medium.
2) After the cells are cultured until the fusion degree is 70-80%, adding 1mL of ReLeSR into each hole, and placing at 37 ℃ and 5% of CO 2 Incubation in incubator for 5min, then blotted ReLeSR, washed with Stemfit 04CT medium, then blotted Stemfit 04CT medium, and performed as follows.
3) Day 0 induction: 2mL embryogenic Medium (EBM) was added to each well of the cells of 2) above, followed by 100uM of ROCK inhibitor and 4ng/mL bFGF. Placing the culture plate on a horizontal orbital shaker for continuous shaking at 200rpm, and recording as in-vitro induction day 0;
embryogenic Medium (EBM) contains a volume fraction of 75% DMEM/F12 basal medium, 20% KnockOut serum replacement, 3% fetal bovine serum, 1% NEAA (optional amino acids), 1% glutamine supplement (Glutamax), 55uM/mL beta-mercaptoethanol (Gibco) and 10 μg/mL heparin (Sigma).
4) After 24 hours, the EB formation state was observed, the 6-well plate was rotated counterclockwise to uniformly concentrate the organoids in the center of the well plate, the embryoid body formation medium was aspirated with a negative pressure aspiration system, and the culture medium was replaced with brain organoid formation medium (BGM), and 200ng/ml Noggin (Peprotech), 2. Mu. M A83-01 (Peprotech), and 0.8. Mu.M CHIR 99021 were added thereto for early neuroectodermal development and midbrain differentiation. Day 1 to day 3 of induction in vitro. And continuing shake culture on a horizontal orbital shaker at 200 rpm.
Brain organogenesis medium (BGM) contained volume fractions of 47.5% dmem/F12 and 47.5% neural basal medium (Nerobasal), and 1% neaa (optional amino acids), 1% glutamine supplement (Glutamax), 1% n2 supplement, 2% vitamin a free B27, 100U/mL penicillin & streptomycin, 55 μΜ β -mercaptoethanol, 1 μg/mL heparin were added.
5) After the organoids were differentiated to day 4, the brain organoid medium was replaced in full volume and inducer concentrations of 200ng/mL Noggin (Peprotech), 2 μ M A-83-01 (Peprotech), and 0.8 μM CHIR 99021, 100ng/mL FGF8b and 100ng/mL SHH were added to the medium and shaking was continued on a horizontal orbital shaker at 200 rpm.
6) On day 7 of differentiation, full-scale exchange was performed, and FGF8b,100ng/mL SHH,200ng/mL laminin (BD science) and 2.5. Mu.g/mL insulin (Thermo) were added to the brain organogenesis medium at a final concentration of 100ng/mL, with shaking at 200rpm on a horizontal orbital shaker lasting from day 7 to day 9 of induction.
7) The total amount of the culture medium is changed into brain organoid mature culture medium (BMM) on 10 th day of differentiation, BMM is changed once every other day, and the culture medium is continuously oscillated on a horizontal orbital shaker at 200rpm from 10 th day to 70 th day of in vitro induction;
brain organoid maturation medium (BMM) contained volume fractions 94%Neurobasal Medium,1%100xN2 supplement, 2%50x B27, 1% PS, 1% glutamine supplement, 1% NEAA, final concentration of 55. Mu.M beta. -mercaptoethanol, 1. Mu.g/ml heparin, 10ng/ml BDNF, 10ng/ml GDNF, 200. Mu.M ascorbic acid, 125. Mu.M cAMP.
Effect example 1
Sample collection and section staining of cultures from example 1
8) Tissues were fixed on days 14 and 30 of the differentiation induced in example 1, respectively, and were identified by tissue immunofluorescent staining. The method comprises the following specific steps: sucking out midbrain organoids by using a 1mL gun head, placing the midbrain organoids in a 1.5mL centrifuge tube, adding Polyformaldehyde (PFA), fixing for 20min, sucking away, adding PBS, cleaning for 3 times for 5min each time, adding 30% sucrose for dehydration, and standing overnight at 4 ℃ until the organoids sink into the bottom of the centrifuge tube. Thereafter, organoids were embedded in OCT and sectioned. Further, washing organoid sections 3 times with PBS for 5min each; blocking for 1 hour by adding PBS solution with final concentration of 1% BSA; the primary antibody was diluted in proportion using 1% BSA in PBS and placed in a wet box overnight at 4 ℃. Washing with PBS for 3 times and 5min each time, adding secondary antibody, preparing with PBS, incubating for 1h at room temperature, and washing with PBS for 3 times and 5min each time. Nuclei were stained with Hoechs, incubated at room temperature for 5min, and washed 3 times with PBS for 5min each. The tablet was sealed with a sealing tablet, and the fluorescence result was observed.
Effect example 2
Drug sensitivity test on cultures of example 1
Differentiation was induced to day 30 in example 1, and all midbrain organoids were divided into four groups, set upIs a control group, H 2 O 2 Group, 6-OHDA group and MPTP group, 100. Mu. M H were added respectively 2 O 2 BMM of 500 mu M6-OHDA and 100 mu M MPTP was cultured with shaking for 48 hours, and then sampled and analyzed for immunofluorescence according to the following procedure, and the results are shown in the figure:
FIG. 1 is a schematic diagram showing the flow of induced hiPSC differentiation into midbrain organoids and shake culture. In the oscillation frequency range of 200rpm, EB formation works best.
FIG. 2A shows hiPSC before digestion on day 0 is induced, and shows a uniform adherence state; panel B shows that the volume of the formed spherical embryoid structure (EB) is relatively uniform after 24 hours of vibration induced differentiation.
As shown in FIG. 3, the average diameter of the midbrain organoids increased continuously from day 1 to day 50 of induction differentiation, and showed positive correlation with the induction days, and the midbrain organoids could reach 1.2mm in diameter at day 50 of induction.
As shown in fig. 4, DIVs 14, midbrain organoids can express the midbrain marker OTX2 (red), neural stem cell marker netin (green) in large amounts.
As shown in fig. 5, DIVs 30, midbrain organoids can express the dopaminergic neuron markers TH (green), and the midbrain marker OTX2 (red), with a scale = 100 μm.
As shown in fig. 6, after 24h of addition of neurotoxic drugs, organoid apoptosis was examined by TUNEL method, and the midbrain organoids expressed apoptosis signals (green) to different extents except the control group, and the dopaminergic neuron markers TH (red) showed damage to different extents. The toxicity of MPTP and 6-OHDA to organoids is global, dopamine nerve damage is also manifested in organoid whole spheres, and H 2 O 2 Toxicity to organoids is only apparent (green part) and internal dopamine nerves are less damaged.
Experimental results prove that the direct induction differentiation method can construct a midbrain organoid containing a large amount of dopaminergic neurons, the organoid can show sensitivity to neurotoxic drugs after being induced and differentiated for 30 days, and the organoid provided by the invention is still good in growth state and free from dead cell generation after being cultured for 100 days.
The inventors have also conducted experimental studies on other conditions, and the inventors have found that Embryoid Bodies (EBs) cannot be formed and midbrain organoids cannot be obtained by using the method provided by the present invention, but without shaking during the culture. Meanwhile, if the oscillation frequency is lower than 200rpm in the culture process, the formed organoids are adhered to each other, and finally, the proper-sized midbrain organoids cannot be obtained. If a specific medium is not changed at a specific time node, the organoids cannot develop in the order of neurodevelopmental, meaning that the midbrain organoids cannot be formed. If embryoid forming medium (EBM) is not used at the stage of induction of embryoid formation on day 0, EB cannot be formed even under shaking conditions, and finally, a midbrain organoid cannot be formed. If FGF8b and SHH are not added to the organogenesis medium on day 4 of organodifferentiation, the resulting organoids cannot differentiate to form dopamine neurons, meaning that they cannot develop in the midbrain direction. If the organoids are in the maturation stage, no neurotrophic factors such as BDNF, GDNF, ascorbic acid, cAMP, etc. are added to the culture medium, the mature nerves cannot be detected by the formed organoids, which means that the mature midbrain organoids cannot be formed, and thus the midbrain organoids function. The cultured midbrain organoids lack cell diversity if the cells are not cultured with hipscs as an initiation.
The invention provides a novel high-flux method for preparing a midbrain organoid, which has higher flux and organoid consistency and meets the requirement of the midbrain organoid for drug screening. The mesoencephalic organoids produced by the invention have a rose ring-shaped ganglion structure produced by the traditional method for preparing the mesoencephalic organoids, express specific nerve-related marker proteins of the mesoencephalic organoids, and provide a reliable high-throughput screening organoid model for researching pathogenesis and molecular mechanism of mesoencephalic development and parkinsonism and individual drug screening.
Claims (7)
1. A method for preparing a midbrain organoid suitable for screening a neurological drug in one step with high throughput, comprising the steps of:
step 1: cell culture
Culturing hiPSC in cell culture plate, adding appropriate amount of iPSC culture medium into each well, resuscitating for the first day, and keeping the culture dish area per cm 2 0.25 μg of iMatrix-511silk matrigel and ROCK inhibitor at a final concentration of 10 μM/mL were added;
step 2: embryoid body formation
Culturing the cells in the step 1 until the fusion degree is 70-80%, adding ReLeSR digestive juice into each hole, and placing at 37 ℃ and 5% CO 2 Incubating in the environment for 5-7 minutes, sucking out ReLeSR, cleaning with an iPSC culture medium, sucking the iPSC culture medium, adding an embryoid body forming culture medium, and adding a ROCK inhibitor with a final concentration of 100uM and bFGF with a final concentration of 4 ng/ml; placing the culture plate on a horizontal orbital shaker, and continuously vibrating at 200rpm, wherein the culture plate is recorded as in-vitro induction day 0;
embryoid body forming medium comprises 75% DMEM/F12 basal medium by volume fraction, 20%
KnockOut serum replacement, 3% fetal bovine serum, 1% NEAA, 1% glutamine supplement,
55uM/mL beta-mercaptoethanol and 10 μg/mL heparin;
step 3: neuroectodermal induction
After the embryoid in step 2 was cultured for 24 hours, the embryoid formation medium was replaced with brain organoid formation medium in total, and early neuroectodermal development was performed by adding Noggin, 2 μm TGF- β inhibitor, 2 μm M A-01 and 0.8 μm CHIR 99021 to the medium at a final concentration of 200 ng/ml; placing the culture plate on a horizontal orbital shaking table, continuously shaking at 200rpm, continuously culturing for 3 days, and recording as 1 st to 3 rd days of in-vitro induction;
brain organogenesis medium contained volume fractions of 47.5% dmem/F12 and 47.5% neural basal medium, and 1% neaa, 1% glutamine supplement, 1% n2 supplement, 2% vitamin a free B27, 100U/mL penicillin & streptomycin, 55 μΜ β -mercaptoethanol, 1 μg/mL heparin;
step 4: midbrain specific region induction
Carrying out in vitro induction culture of the organoid of the step 3 until the 4 th day, changing the total amount of the culture medium of the step 3 into a brain organoid generation culture medium, and simultaneously adding 200ng/ml Noggin, 2 mu M A-83-01, 0.8 mu M CHIR 99021, 100ng/ml FGF8b and 100ng/ml SHH to induce the organoid to develop to a midbrain basal plate region from the 4 th day to the 6 th day; placing the culture plate on a horizontal orbital shaker, and continuously vibrating at 200 rpm;
step 5: tissue growth
Carrying out in vitro induction culture of the organoid of the step 4 until the 7 th day, changing the total amount of the culture medium of the step 4 into a brain organoid generation culture medium, and simultaneously adding 100ng/ml of FGF8b,100ng/ml of SHH,200ng/ml of laminin and 2.5 mu g/ml of insulin, and inducing the organoid to grow to midbrain organoid tissues from the 7 th day to the 9 th day; placing the culture plate on a horizontal orbital shaker, and continuously vibrating at 200 rpm;
step 6: organoid maturation
The method comprises the steps of in vitro induction from 10 days to more than 30 days, changing a brain organoid maturation medium in half every other day in the organoid maturation stage;
the maturation of brain organoids adopts brain organoid maturation medium; brain organoid maturation medium contained volume fractions 94%Neurobasal Medium,1%100x N2 supplement, 2%50x B27, 1% PS, 1% glutamine supplement, 1% NEAA, final concentration of 55. Mu.M beta. -mercaptoethanol, 1. Mu.g/ml heparin, 10ng/ml BDNF, 10ng/ml GDNF, 200. Mu.M ascorbic acid, 125. Mu.M cAMP.
2. The method of claim 1, wherein in step 1, the cell culture conditions are 37℃and 5% CO 2 Incubating in the environment.
3. The method of claim 1, wherein in step 1, hipscs are cultured in 6-well plates.
4. The method of claim 1, wherein the iPSC medium in step 1 is Stemfit 04CT medium.
5. The method of claim 1, wherein the iPSC culture matrigel in step 1 is an iMatrix-511silk matrigel.
6. The method of claim 1, wherein the medium for washing in step 2 is Stemfit 04CT medium.
7. The method of claim 1, wherein in step 6, the induction is from day 10 to more than day 70 in vitro.
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