Preparation method of bionic senescence brain-like organ for simulating senescence brain

Abstract

The invention provides a preparation method of a bionic senescence brain-like organ for simulating the brain of the elderly, and the preparation method comprises the following steps: inducing human induced pluripotent stem cells into embryoid bodies, and inducing and differentiating the embryoid bodies EB into brain-like organs; the brain-like organ is placed in a culture medium containing bleomycin, the brain-like organ is induced to differentiate the senescence phenotype, and the bionic senescence brain-like organ with senescence characterization is prepared, so that the physiological condition of senescence brains of people is simulated. According to the preparation method of the senescence brain-like organ, the time required by the senescence of the brain-like organ in a natural state is greatly shortened, the preparation method is simple and easy to implement, the physiological condition of the brain of the elderly people can be simulated more truly, related basic research, disease modeling and drug screening are carried out, and the preparation method is suitable for popularization and application. The method has the advantages which cannot be achieved by traditional in-vitro culture and animal experiments, and is wide in application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of bionic brain organoids, and in particular relates to a method for preparing a bionic aging brain organoid for simulating an aged brain. Background Art

[0002] As the most complex organ in the human body in terms of structure and function, the human brain not only regulates body functions, but also serves as the material basis for higher-level neural activities such as consciousness, spirit, language, learning, memory, and intelligence. Brain aging can lead to the occurrence of neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). With the increasing aging of the global population and the aging process in my country, neurodegenerative diseases are increasingly endangering human health. However, the complexity of brain structure and function has largely limited the relevant research in brain science. Therefore, establishing a real and reliable in vitro model of brain aging is difficult and in great demand for basic biological research, disease treatment, and targeted drug testing.

[0003] In recent years, research on brain organoids (BO) has become a hot topic in the field of neuroscience. Brain organoids can form three-dimensional neural tissues similar to the human brain, and can well simulate in vivo processes such as neurogenesis, neuronal migration, cortical stratification, and neural circuit establishment. Aging brain organoids can reproduce brain aging to a certain extent, but there are currently few related models and it takes a long time, up to 8-9 months, with a long cycle and low efficiency.

[0004] Therefore, the present invention provides a method for preparing a bionic aging brain organoid for simulating the aged brain to solve the above problems. The bionic aging brain organoid has both the physiological phenotype of the aged human brain and the aging phenotype, and can be used to study the effects of drugs and inflammatory stimulation on the aged brain. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing a bionic aging brain organoid for simulating the aged brain, by inducing human induced pluripotent stem cells into brain organoids, adding bleomycin, so that the brain organoids have the characteristics of aging, and the brain conditions of the aged human brain can be simulated in vitro and further research can be carried out.

[0006] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0007] The present invention provides a method for preparing a bionic aging brain organoid for simulating an aged brain, comprising the following steps:

[0008] S1. Induce human induced pluripotent stem cells (hiPSCs) into embryoid bodies (EBs), and then induce the embryoid bodies EBs to differentiate into brain organoids (BOs);

[0009] S2. Place the brain organoids in a culture medium containing Bleomycin (BLM) to prepare biomimetic aging brain organoids (Aging-BO) with aging characteristics.

[0010] Furthermore, in step S1, the method for inducing human induced pluripotent stem cells into embryoid bodies is as follows: culturing human induced pluripotent stem cells, and when the confluence density reaches 80%, aspirating excess culture medium, adding trypsin for digestion and then aspirating trypsin, adding inoculation culture medium containing Y-27632, and gently blowing the cells to form a single cell suspension; adding the single cell suspension to a transparent round-bottom ultra-low adsorption microplate, culturing in a cell culture incubator, and observing under a microscope that the cells aggregate into round cell clusters, i.e., embryoid bodies.

[0011] Furthermore, the single cell suspension is diluted to a concentration of 90,000 cells / ml before culture.

[0012] Furthermore, the culture temperature in the cell culture incubator was 37° C. and the culture time was 48 h.

[0013] Furthermore, the step S1 also includes: after observing that the cells aggregate into round cell clusters under a microscope, adding an organoid formation culture medium with the same volume as the single cell suspension every two days until the culture ends on the fifth day.

[0014] Furthermore, in step S1, the method for inducing the differentiation of embryoid bodies into brain organoids is as follows: adding the embryoid bodies to a transparent round-bottom ultra-low adsorption microplate, adding induction culture medium for culturing; coating the embryoid bodies after induced culture with matrix gel, placing them in an incubator to solidify the matrix gel, and then placing the solidified matrix gel containing the embryoid bodies in a transparent round-bottom ultra-low adsorption microplate, adding expansion culture medium for culturing; after the expansion culture is completed, aspirating the culture medium in the well plate, adding maturation culture medium, and placing them on a horizontal cell shaker for culturing to obtain mature brain organoids.

[0015] Furthermore, after the embryoid bodies were added to a transparent round-bottom ultra-low adsorption microplate and induction medium was added, the culture temperature was 37° C. and the culture time was 48 h.

[0016] Furthermore, the rotation speed of the horizontal cell shaker is 65 rpm; after being placed on the horizontal cell shaker, the maturation culture medium is replaced every 3 days until the 60th day of cultivation.

[0017] Furthermore, the step S2 specifically includes: adding a maturation culture medium containing bleomycin to the mature brain organoid from which the maturation culture medium has been removed by suction, and culturing the mature brain organoid to obtain a bionic aging brain organoid with aging characteristics.

[0018] Furthermore, in the bleomycin-containing maturation culture medium, the concentration of bleomycin is 4 μM; the culture time is 14 days, and the culture medium is replaced every three days.

[0019] Beneficial Effects

[0020] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0021] 1) The method for preparing bionic aging brain organoids for simulating the aged brain provided by the present invention comprises preparing brain organoids by using human induced pluripotent stem cells hiPSC; adding bleomycin to the culture medium of the brain organoids, and the brain organoids age after 14 days to become induced aging brain organoids. The preparation method is simple, reliable and has a high success rate;

[0022] 2) The bionic aging brain organoids provided by the present invention have the aging phenotype of the human aging brain, can better simulate the conditions of the elderly brain in vitro, and can be used for basic biological research, disease treatment, and targeted drug testing;

[0023] 3) The bionic aged brain organoids provided by the present invention greatly shorten the culture time of traditional aged brain organoids from 8-9 months to 2.5 months, making it more convenient and rapid to study the effects of various stresses on aged brain organoids. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The figure is a schematic diagram of the preparation process of the bionic aging brain organoid for simulating the aged brain according to the present invention;

[0025] Figure 2 for Figure 1 Changes in brain organoid preparation;

[0026] Figure 3 This is the SA β-gal staining image of aged brain organoids;

[0027] Figure 4 This is the immunofluorescence staining of Lamin B1 in aged brain organoids;

[0028] Figure 5 Immunofluorescence staining of p21 in aged brain organoids. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] The experimental methods used in the following examples are conventional methods unless otherwise specified, and the reagents, methods and equipment used are conventional reagents, methods and equipment in the art unless otherwise specified.

[0031] The present invention provides a method for preparing a bionic aging brain organoid for simulating an aged brain, comprising the following steps:

[0032] S1. Inducing human induced pluripotent stem cells into embryoid bodies, and then inducing the embryoid bodies to differentiate into brain organoids:

[0033] Culture human induced pluripotent stem cells. When the confluence density reaches 80%, remove the excess culture medium, add trypsin for digestion, remove the trypsin, add inoculation medium containing Y-27632, gently blow the cells to form a single cell suspension, and dilute the single cell suspension to a concentration of 90,000 cells / ml; add the diluted single cell suspension to a transparent round-bottom ultra-low adsorption microplate, culture in a cell culture incubator at 37°C for 48 hours, and observe the cells aggregate into round cell clusters, i.e., embryoid bodies, under a microscope. Add the same volume of organoid formation medium as the single cell suspension every two days until the end of the culture on the 5th day.

[0034] On the 5th day, the embryoid bodies were added to a transparent round-bottom ultra-low adsorption microplate, and induction medium was added to culture at 37°C for 48 hours; on the 7th day, the embryoid bodies after induced culture were coated with matrix gel and placed in a 37°C incubator for 30 minutes to solidify the matrix gel, and then the solidified matrix gel containing the embryoid bodies was placed in a transparent round-bottom ultra-low adsorption microplate, and expansion medium was added for culture; on the 10th day, the culture medium in the well plate was aspirated without disturbing the matrix gel containing the embryoid bodies, and then the maturation medium was added, and the plate was placed on a horizontal cell shaker, and the speed was adjusted to 65 rpm for culture. The maturation medium was replaced every 3 days, and mature brain organoids were obtained after the 60th day of culture.

[0035] S2. Placing the brain organoids in a culture medium containing bleomycin to prepare biomimetic aging brain organoids with aging characteristics:

[0036] Bleomycin was added to the maturation medium to prepare a medium containing 4 μM bleomycin; the maturation medium was added to the mature brain organoids from which the maturation medium was removed and cultured for 14 days to obtain bionic aging brain organoids with aging characteristics.

[0037] The culture medium used in this example comes from the kit (STEMdiff TM Cerebral Organoid Kit), catalog number 08570. The culture medium used in this example (including the inoculation medium of Y-27632, the organoid formation medium, the induction medium, the expansion medium and the maturation medium) was prepared according to the instructions of the above kit.

[0038] Example 1

[0039] This embodiment provides a bionic aging brain organoid for simulating the elderly brain, and the preparation method thereof is as follows: Figure 1 and Figure 2 As shown, the specific steps include:

[0040] (1) Preparation of embryoid bodies:

[0041] Human induced pluripotent stem cells with a confluence density of 80% were digested and diluted into a single cell suspension at a concentration of 90,000 cells / ml. 100 μl of the diluted single cell suspension was added to each well of a 96-well transparent round-bottom ultra-low adsorption microplate, and cultured in a cell culture incubator at 37°C for 48 hours. Under a microscope, the cells in each well aggregated into round cell clusters, i.e., embryoid bodies. After that, 100 μl of organoid formation medium was added to each well every two days until the 5th day.

[0042] (2) Preparation of mature brain organoids:

[0043] On the 5th day, the embryoid bodies were transferred to a 24-well transparent round-bottom ultra-low adsorption microplate, 0.5 ml of induction medium was added to each well, and cultured at 37°C for 48 hours; on the 7th day, each embryoid body after induction culture was coated with 15 μl of matrix gel, placed in a 37°C incubator for 30 minutes to solidify the matrix gel, and then the solidified matrix gel containing the embryoid bodies was placed in a 6-well transparent round-bottom ultra-low adsorption microplate, and 3 ml of fresh expansion medium was added to each well for culture. On the 10th day, the medium in the well plate was aspirated without disturbing the matrix gel containing the embryoid bodies, and 3 ml of fresh maturation medium was added, and the well plate was placed on a horizontal cell shaker, and the speed was adjusted to 65 rpm for culture. After that, the maturation medium was replaced every 3 days, and mature brain organoids were obtained after the 60th day of culture.

[0044] (3) Preparation of bionic aging brain organoids with aging characteristics:

[0045] Bleomycin was added to the maturation medium to prepare a medium containing bleomycin at a concentration of 4 μM.

[0046] On the 60th day, the culture medium in the 6-well transparent round-bottom ultra-low adsorption microplate was discarded, and the mature brain organoids were cultured with a maturation culture medium containing 4 μM bleomycin. The medium was changed every three days, and the mature brain organoids were treated with a maturation culture medium containing BLM for 14 days to induce the aging of the mature brain organoids to become bionic aging brain organoids Aging-BO, simulating the human brain in an aging state.

[0047] Example 2: Senescence-associated β-galactosidase (SA-β-Gal) staining test of bionic aging brain organoids.

[0048] The biomimetic aging brain organoid Aging-BO prepared in Example 1 was transferred to a 50ml centrifuge tube using a wide-bore 1ml pipette tip, added to a 4% paraformaldehyde solution and placed in a 4°C refrigerator overnight, and then taken out. After removing the paraformaldehyde solution, the Aging-BO was immersed in 10%, 20%, and 30% sucrose solutions for 2 hours each using the sucrose gradient dehydration method. The Aging-BO was transferred to a new centrifuge tube, 5ml of a 7.5% gelatin solution was added, and it was placed in a 37°C water bath for 2 hours to allow the gelatin to replace the sucrose and immerse into the organoid. The Aging-BO was taken out to a frozen section embedding box, and 1ml of a 7.5% gelatin solution was added to cover the Aging-BO. The organoid was gently moved to the center of the embedding box with a pipette tip. Add anhydrous ethanol to dry ice. When the "boiling" phenomenon disappears, place the embedding box containing Aging-BO on the surface of the anhydrous ethanol / dry ice mixture and let it stand for 2 minutes until the gelatin cools and solidifies. Store it in a -80°C refrigerator. Use dry ice to transfer the frozen embedded organoids, and then use a freezing microtome to cut the organoids into 10μm thick frozen sections, and bake the slices in a 37°C oven for 2 hours.

[0049] Wash the sections 3 times with PBS, 5 min each time. Add 100 μl of β-galactosidase staining fixative to each tissue to fully cover the tissue and fix at room temperature for 15 min. Wash the sections 3 times with PBS, 5 min each time. Remove the PBS, add 100 μl of β-galactosidase staining working solution, and incubate at 37°C overnight.

[0050] Figure 3As observed under an ordinary optical microscope, compared with the brain organoids without bleomycin (BLM), most areas of the Aging-BO in Example 1 after 14 days of culture with 4 μM BLM turned blue-green, indicating that the activity level of SA-β-Gal was significantly upregulated, showing the characteristics of aging.

[0051] Example 3: Immunofluorescence staining test of frozen sections of bionic aging brain organoids.

[0052] The bionic aging brain organoid prepared in Example 1 was cryosectioned, and the specific method was the same as that in Example 2.

[0053] Place the frozen sections in a staining jar containing 2.94‰ (w / v) sodium citrate dihydrate solution at pH 6, steam the staining jar in a steamer for 20 minutes, take it out and soak and wash the sections in PBS-T solution 3 times, 10 minutes each time. Add sheep serum blocking solution, remove the blocking solution after 1 hour at room temperature, add 100μl of 1:100 Lamin B1 and p21 primary antibody solutions respectively, put them in a wet box with water at the bottom of the section, and then place the wet box in a 37℃ oven for 2 hours. Take out the sections and soak and wash them in PBS-T solution 3 times, 10 minutes each time. Add 100μl of the corresponding secondary antibody solutions of Lamin B1 and p21 diluted in PBS-T at a concentration of 1:200 on the sections, respectively, and soak and wash them in PBS-T solution 3 times after 2 hours at room temperature, 30 minutes each time. After taking out the sections, add 100μl of DAPI working solution on the sections, and soak and wash them in PBS-T solution 3 times after 8 minutes, 5 minutes each time. Take out the slices, lay them flat for 5 minutes until they are dry, and then add anti-fluorescence quenching sealing agent to seal the slices. Observe the slices with a laser scanning confocal microscope.

[0054] like Figure 4 and Figure 5 As shown, compared with the brain organoids without bleomycin (BLM), the expression of Lamin B1 in Aging-BO after 14 days of culture with 4 μM BLM in Example 1 was reduced ( Figure 4 ), p21 expression increased ( Figure 5 ), showing characteristics of aging.

[0055] The preparation method of the bionic aging brain organoid for simulating the aged brain provided by the present invention greatly shortens the time required for the brain organoid to reach aging under natural conditions. The preparation method is simple and easy, and can more realistically simulate the pathophysiological conditions of the aged human brain, and conduct related basic research, disease modeling and drug screening. It has advantages that traditional in vitro culture and animal experiments cannot have, and has broad application prospects.

[0056] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by a technician familiar with the technical field within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention.

Claims

1. A method for preparing a bionic aging brain organoid for simulating an aged brain, characterized in that: The following steps are involved: S1. Inducing human induced pluripotent stem cells into embryoid bodies, and then inducing the embryoid bodies to differentiate into brain organoids; S2. Place the brain organoid in a culture medium containing bleomycin to prepare a bionic aging brain organoid with aging characteristics.

2. The method for preparing a bionic aging brain organoid for simulating an aged brain according to claim 1, characterized in that: In step S1, the method for inducing human induced pluripotent stem cells into embryoid bodies is: Human induced pluripotent stem cells were cultured. When the confluence density reached 80%, the excess medium was removed, trypsin was added for digestion, and then the trypsin was removed. Inoculation medium containing Y-27632 was added, and the cells were gently pipetted to form a single cell suspension; The single cell suspension was added to a transparent round-bottom ultra-low attachment microplate and cultured in a cell culture incubator. Under a microscope, the cells were observed to aggregate into round cell clusters, namely embryoid bodies.

3. The method for preparing a bionic aging brain organoid for simulating an aged brain according to claim 2, characterized in that: The single cell suspension was diluted to a concentration of 90,000 cells / ml before culturing.

4. The method for preparing a bionic aging brain organoid for simulating an aged brain according to claim 2, characterized in that: The culture temperature in the cell culture incubator was 37°C and the culture time was 48 h.

5. The method for preparing a bionic aging brain organoid for simulating an aged brain according to claim 2, characterized in that: The step S1 further comprises: After the cells were observed to aggregate into round cell clusters under a microscope, organoid formation medium with the same volume as the single-cell suspension was added every two days until the end of the culture on the 5th day.

6. The method for preparing a bionic aging brain organoid for simulating an aged brain according to claim 1, characterized in that: In step S1, the method of inducing embryoid bodies to differentiate into brain organoids is: The embryoid bodies were added into a transparent round-bottom ultra-low attachment microplate and induction medium was added for culture; The induced embryoid bodies are coated with matrix gel, placed in an incubator to solidify the matrix gel, and then the solidified matrix gel containing the embryoid bodies is placed in a transparent round-bottom ultra-low adsorption microplate, and expansion culture medium is added for culture; After the expansion culture is completed, the culture medium in the well plate is aspirated, and then the maturation culture medium is added, and the well plate is placed on a horizontal cell shaker for culture to obtain mature brain organoids.

7. The method for preparing a bionic aging brain organoid for simulating an aged brain according to claim 6, characterized in that: After the embryoid bodies were added to a transparent round-bottom ultra-low attachment microplate and induction medium was added, the culture temperature was 37°C and the culture time was 48 hours.

8. The method for preparing a bionic aging brain organoid for simulating an aged brain according to claim 6, characterized in that: The horizontal cell shaker has a rotation speed of 65 rpm; After being placed on a horizontal cell shaker, the maturation medium was replaced every 3 days until the 60th day of culture.

9. The method for preparing a bionic aging brain organoid for simulating an aged brain according to claim 1, characterized in that: The step S2 is specifically as follows: The maturation medium containing bleomycin is added to the mature brain organoid from which the maturation medium has been removed for culturing, thereby obtaining a bionic aging brain organoid with aging characteristics.

10. The method for preparing a bionic aging brain organoid for simulating an aged brain according to claim 9, characterized in that: In the bleomycin-containing maturation culture medium, the concentration of bleomycin is 4 μM; and the culture time is 14 days.

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