Three-dimensional brain organoid culture method based on low-intensity ultrasound stimulation

Abstract

A three-dimensional brain organoid culture method based on low-intensity ultrasound stimulation. The method comprises: first, culturing a brain organoid; and then intervening the development of the br

Description

TECHNICAL FIELD

[01] The invention belongs to the field of biomedical technology, in particular to a three-dimensional brain organoid culture method based on low-intensity ultrasonic stimulation. BACKGROUND ART

[02] The development of the nervous system undergoes processes such as nerve induction, pattern formation, cell fate determination and specialization, nerve cell polarity establishment and migration, and neural circuit formation. It is of great significance to carry out in-depth research on the mechanism of neurodevelopment to guide the prevention and treatment of such major neurodevelopmental defects. Microcephaly is a neurological developmental disorder in which the head of a child is significantly smaller due to abnormal brain development in the fetus or infancy. The patient's brain is underdeveloped, and the patients cannot take care of themselves in their entire life. At present, the treatment is limited and the curative effect is not ideal.

[03] Brain organoids are a good three-dimensional model to simulate the development of an infant's brain due to their high diversity of cell composition, high complexity of structure and tissue, relatively mature electrophysiological functions, and similar gene expression patterns to humans. ASPM is the most common autosomal recessive microcephaly gene. When the ASPM gene is mutated, the human brain size will be reduced by 50 %. However, ASPM gene inactivation can only cause 10 % brain atrophy in mice, and ASPM gene knockout mice can only provide a very limited reference in the study of human cortical development (Nature.2018; 556:370-375). Studies have shown that the ASPM knockout human brain organoid model can better reproduce the phenotype of microcephaly (ProteinCell.2017; 8 (11):823-833). 104] However, the application of low-intensity ultrasound to the development of three-dimensional brain organoids has not been reported. SUMMARY

[05] In view of this, the purpose of the invention is to provide a three-dimensional brain organoid culture method based on low-intensity ultrasound stimulation.

[06] The purpose of the invention is realized by the following:

[07] The invention discloses a three-dimensional brain organoid culture method based on low-intensity ultrasonic stimulation, comprising the following steps:

[08] (1) Construction of a three-dimensional brain organoid: culturing embryonic stem cells or induced pluripotent stem cells and differentiating them into a three-dimensional brain organoid;

[09] (2) Optimization of a development of the brain organoid by low-intensity ultrasound: using low-intensity ultrasound to carry out a stimulation intervention for the development of the brain organoid, and recording structural changes of the three-dimensional brain organoid at different time points.

[10] Based on the above technical solution, furthermore, a specific process of the construction of the three-dimensional brain organoid in Step (1) is as follows: inoculating a single cell suspension of embryonic stem cells or induced pluripotent stem cells in an ultra-low adhesion U-shaped bottom orifice plate, and adding a neural induction medium and culturing to form embryoid bodies, after culturing to an 8th day -a 12th day, transferring it to a low adhesion orifice plate, after adding a neural differentiation medium, and culturing it in a shaker, after culturing to a 16th day -a 20th day, replacing with a mature medium to continue culturing in the shaker.

[11] Based on the above technical solution, furthermore, the neural induction medium is DMEM / F-12 medium containing 15 % Knockout serumreplacement, 1 % MEM-NEAA, 1 % Glutamaxsupplement, 100 pM P-Mercaptoethanol, 100 nM LDN-193189, 10 uM SB431542, 2 pMXAV939.

[12] Based on the above technical solution, furthermore, the neural differentiation medium is 50 % DMEM / F-12 medium containing 50 % Neurobasal medium, 0.025 % Insulin, 0.5 % MEM-NEAA and 1 % Glutamax supplement, 1 % Penicillin / Streptomycin, 0.5 % N2supplement, 1 % B27supplement without vitamin A, 50pM P-Mercaptoethanol.

[13] Based on the above technical solution, furthermore, the mature medium is 50 % DMEM / F-12 medium containing 50 % Neurobasal medium, 0.025 % Insulin, 0.5 % MEM-NEAA, 1 % Glutamax supplement, 1 % Penicillin / Streptomycin, 0.5 % N2 supplement, 1 % B27supplement, 50pM P-Mercaptoethanol. 200pM Ascorbic acid, 20ng / ml BDNF.

[14] Based on the above technical solution, furthermore, parameters of low-intensity ultrasound described in Step (2) are as follows: a pulse intensity of 30-50mW / cm2, a rated frequency of l-5MHz, 5-15 % duty cycle, and processing l-5min per day.

[15] Based on the above technical solution, furthermore, a start time of stimulation intervention in step (2) is a 15th day-the 20th day of the development of the brain organoid.

[16] The invention also discloses an application of low-intensity ultrasonic stimulation in a construction of a microcephaly brain organoid disease model.

[17] Based on the above technical solution, the human abnormal spindle-like microcephaly-associated protein gene (ASPM) is further knocked out in the microcephaly brain organoid disease model.

[18] The beneficial effects of the invention relative to the existing technology are as follows:

[19] The invention is the first to find that low-intensity ultrasound stimulation optimizes the development of the brain organoid and discloses a three-dimensional brain organoid culture method based on low-intensity ultrasound stimulation, it provides a reliable and effective in vitro research model for studying the development process of the nervous system and develops a new path for studying the treatment of neurodevelopmental related diseases such as microcephaly. BRIEF DESCRIPTION OF THE DRAWINGS

[20] In order to explain the embodiments of the invention more clearly, the attached drawings involved in the embodiment are briefly introduced in the following.

[21] Fig. 1 is a time axis of the low-intensity ultrasound intervention in brain organoid development;

[22] Fig. 2 is a light microscope comparison of brain organoids treated with low-intensity ultrasound stimulation at different times;

[23] Fig. 3 is an immunofluorescence comparison of brain organoids treated with low-intensity ultrasound stimulation at different times, where (A): The proportion of proliferating cells (ki67) and neural progenitor cells (SOX2) in the ultrasound group increases significantly; (B): The proportion of cortical neurons (TBR1) in the ultrasound group increases significantly; (C): The proportion of apoptotic cells (TUNEL) in the ultrasound group decreases significantly;

[24] Fig. 4 is an integration map of low-intensity ultrasound-treated brain organoids transplanted into the host primary sensory cortex at different time points;

[25] Fig. 5 shows the light microscope images of ASPM knockout brain organoids and ASPM brain organoids after ultrasound treatment. (A) are light microscope images of the ASPM knockout group and control group; (B) is a light microscope comparison of brain organoids in the ASPM knockout group, control group, and ASPM knockout group after ultrasound treatment. DETAILED DESCRIPTION OF THE EMBODIMENTS

[26] The following is a detailed description of the invention in combination with the embodiment, but the implementation method of the invention is not limited to those embodiments. Obviously, the embodiments described below are only a part of the embodiments of the invention. For the technical personnel in this field, other similar embodiments obtained without paying creative labor fall into the protection range of the invention.

[27] Embodiment 1

[28] Construction of brain organoids: When the H9 human embryonic stem cells grew to 80 % confluence, the cells were digested with accutase for 10 min to form a single cell suspension, after the cells were resuspended in a neural induction medium containing 50pM Y27632 (DMEM / F-12, 15 % Knockout serum replacement, 1 % MEM-NEAA, 1 % Glutamaxsupplement, IOOliM P-Mercaptoethanol, lOOnMLDN-193189, lOpM SB431542, 2pM AV939), they were seeded in an ultra-low adhesion U-shaped bottom 96 orifice plate at a ratio of 9000 cells / hole and 150pL per hole, and the embryoid bodies were formed. Half of the medium was changed every 2 days using a neural induction medium without Y27632. On the 10th day of culture, they were transferred to a low adhesion 6 orifice plate using a 5 mL bus pipette, with 6-8 embryoids per hole and 3mL neurodifferentiation medium per hole(50 % DMEM / F-12,50 % Neurobasalmedium, 0.025 % Insulin, 0.5 % MEM-NEAA, 1 % Glutamaxsupplement, 1 % Penicillin / Streptomycin, 0.5 % N2supplement, 1 % B27supplement withoutvitaminA, 50pM P-Mercaptoethanol)l. it was cultured in a shaker at 80 rpm, and the medium was changed every 2 days. On the 18th day of culture, the medium was replaced with mature medium (50 % DMEM / F-12,50 % Neurobasal medium, 0.025 % Insulin, 0.5 % MEM-NEAA, 1 % Glutamaxsupplement, 1 % Penicillin / Streptomycin, 0.5 % N2supplement, 1 % B27supplement, 50liM P-Mercaptoethanol, 200pM Ascorbic acid, 20ng / ml BDNF), the liquid was replaced once every 3-4 days.

[29] Embodiment 2

[30] low-intensity ultrasound was used to carry out a stimulation intervention for the development of the brain organoids: Starting from the 18th day of the development of the brain organoids, about 6-8 brain organoids were cultured in each hole of a low adhesion 6 orifice plate containing 3mL mature medium, and sterile coupling agent (< 1mm thick) was applied at the bottom of each hole of the low adhesion 6 orifice plate. The ultrasonic probe (MESonicator740, 5 square centimeters) was placed under the orifice plate, they were in close contact with no gap. The parameters of the ultrasonic therapeutic instrument were adjusted: the pulse intensity was 40mW / cm2, the rated frequency was 3MHz, 10 % duty cycle, and it was processed 2min per day. After treatment, the coupling agent of the bottom plate was wiped, and the orifice plate was put back into the 80 rpm shaker for continuous culture. The proportion of neural progenitor cells (SOX2), cortical neurons (TBR1), proliferating cells (ki67), and apoptotic cells (TUNEL) were detected on the 35th, 55th, and 75th day of brain organoid development. The results are shown in Fig. 3, it can be seen that the brain organoids after low-intensity ultrasound stimulation show increased size, increased proportion of neural progenitor cells and cortical neurons, significantly increased proportion of proliferating cells, and significantly reduced proportion of apoptotic cells, which effectively promoted the structure of brain organoids.

[31] Embodiment 3

[32] Detection of brain organoid integration after low-intensity ultrasound stimulation intervention: The brain organoids carrying GFP treated by ultrasound on the 40th day were transplanted into the primary sensory cortex of Nod-SCID mice. The survival ability of brain organoids in the host and the integration ability with the host were detected in the 2nd month and the 5th month after transplantation. The results are shown in Fig. 4, the survival ability of brain organoids treated with low-intensity ultrasound stimulation after transplantation in the host was significantly increased, and the ability to integrate with the host was significantly improved.

[33] Embodiment 4

[34] Construction of ASPM knockout microcephaly brain organoid model and ultrasonic treatment: Embryonic stem cells were infected with Crispr-cas9-mediated ASPM knockout lentivirus, after two weeks of drug screening, single cell clones were selected for gene fragment verification and knockout effect identification, and ASPM knockout embryonic stem cell lines were constructed.

[35] Subsequently, the culture and identification were carried out according to the method of human brain organoid construction in Embodiment 1. After the 30th day, according to the parameter scheme of low-intensity ultrasound intervention in ordinary brain organoids in Embodiment 2, the specific results are shown in Fig. 5. It can be seen that the brain organoids of microcephaly after low-intensity ultrasound treatment showed a significant increase in size.

[36] Finally, it should be stated that the above embodiments are only used to illustrate the technical solution of the invention and not to restrict it; although the invention is described in detail concerning the aforementioned embodiments, the general technical personnel in this field should understand that they can still modify the technical solution recorded in the aforementioned embodiments, or replace some or all of the technical features with equivalents; these modifications or replacements do not make the essence of the corresponding technical solution out of the scope of the technical solution of each embodiment of the invention.

Claims

1. A three-dimensional brain organoid culture method based on low-intensity ultrasonic stimulation, comprising the following steps:(1) construction of a three-dimensional brain organoid: culturing embryonic stem cells or induced pluripotent stem cells and differentiating them into a three-dimensional brain organoid;(2) optimization of a development of the brain organoid by low-intensity ultrasound: using low-intensity ultrasound to carry out a stimulation intervention for the development of the brain organoid, and recording structural changes of the three-dimensional brain organoid at different time points.

2. The culture method according to claim 1, wherein a specific process of the construction of the three-dimensional brain organoid in Step (1) is as follows: inoculating a single cell suspension of embryonic stem cells or induced pluripotent stem cells in an ultra-low adhesion U-shaped bottom orifice plate, and adding a neural induction medium and culturing to form embryoid bodies, after culturing to a 8th day -a 12th day, transferring it to a low adhesion orifice plate, after adding a neural differentiation medium, and culturing it in a shaker, after culturing to a 16th day -a 20th day, replacing with a mature medium to continue culturing in the shaker.

3. The culture method according to claim 2, wherein the neural induction medium is DMEM / F-12 medium containing 15 % Knockout serumreplacement, 1 % MEM-NEAA, 1 % Glutamaxsupplement, 100 pM p-Mercaptoethanol, 100 nM LDN-193189, 10 uM SB431542, 2 pMXAV939.

4. The culture method according to claim 2, wherein the neural differentiation medium is 50 % DMEM / F-12 medium containing 50 % Neurobasal medium, 0.025 % Insulin, 0.5 % MEM-NEAA and 1 % Glutamax supplement, 1 %Penicillin / Streptomycin, 0.5 % N2supplement, 1 % B27supplement without vitamin A, 50pM P-Mercaptoethanol.

5. The culture method according to claim 2, wherein the mature medium is 50 % DMEM / F-12 medium containing 50 % Neurobasal medium, 0.025 % Insulin, 0.5 % MEM-NEAA, 1 % Glutamax supplement, 1 % Penicillin / Streptomycin, 0.5 % N2 supplement, 1 % B27supplement, 50pM P-Mercaptoethanol. 200pM Ascorbic acid, 20ng / ml BDNF.

6. The culture method according to claim 1, wherein parameters of low-intensity ultrasound described in Step (2) are as follows: a pulse intensity of 30-50mW / cm2, a rated frequency of l-5MHz, 5-15 % duty cycle, and processing l-5min per day.

7. The culture method according to claim 1, wherein a start time of stimulation intervention in step (2) is a 15th day-the 20th day of the development of brain organoid.

8. An application of low-intensity ultrasonic stimulation according to claim 1 in a construction of a microcephaly brain organoid disease model.

9. The application according to claim 8, wherein the human abnormal spindle-like microcephaly-associated protein gene (ASPM) is knocked out in the microcephaly brain organoid disease model.A. CLASSIFICATION OF SUBJECT MATTERC12N 5 / 10(2006.01)i; C12N 15 / 12(2006.01)iAccording to International Patent Classification (IPC) or to both national classification and IPCB. FIELDS SEARCHEDMinimum documentation searched (classification system followed by classification symbols) IPC: C12NDocumentation searched other than minimum documentation to the extent that such documents are included in the fields searchedElectronic data base consulted during the international search (name of data base and, where practicable, search terms used)CNTXT, ENTXT, ENTXTC, CNKI, EE WANFANG DATABASE, Pubmed, Baidu: jgE, JlJft, HHtf'l’, LDN193189, SB431542, XAV939, ASPM, organoid,ffi, ESC, iPSC, embryonic stem cell, induced pluripotent stem cell, brain, organoid, neuralinduction medium, neural differentiation medium, low-intensity, ultrasound, B27, N2, mercaptoethanol, ascorbic acid, ASPM,DOCUMENTS CONSIDERED TO BE RELEVANTCategory*Citation of document, with indication, where appropriate, of the relevant passagesRelevant to claim No.PX1-9CN 116376840 A (TIANJIN UNIVERSITY) 04 July 2023 (2023-07-04) claims 1-9Y XIANG, Yangfei et al. "Fusion of Regionally-specified hPSC-derived Organoids Models Human Brain Development and Interneuron Migration" Cell Stem Cell. Vol. 21, No. 3, 07 September 2017 (2017-09-07), pages 14-15, "Method details" 1-7 Y HOU, Xuandi et al. "Precise Ultrasound Neuromodulation in a Deep Brain Region Using Nano Gas Vesicles as Actuators" Advanced Science, Vol. 8, 21 September 2021 (2021-09-21), article 2101934, abstract, page 1, right-hand column, paragraph 1, and sections 2.2 and 2.4 1-9 Y LI, Rui et al. "Recapitulating Cortical Development with Organoid Culture in Vitro and Modeling Abnormal Spindle-like (ASPM related primary) Microcephaly Disease" Protein Cell. Vol. 8, No. 11, 31 December 2017 (2017-12-31), abstract, and page 828, left-hand column 8-9| | Further documents are listed in the continuation of Box C.annex.* Special categories of cited documents:“A” document defining the general state of the art which is not considered to be of particular relevance“D” document cited by the applicant in tire international application“E” earlier application orpatent but published on or after the international“T”“O”“P”filing datedocument which may throw doubts on priority claim(s) or which is cited to establish the publication date of another citation or other special reason (as specified)document referring to an oral disclosure, use, exhibition or other meansdocument published prior to the international filing date but later than the priority date claimed‘Y’later document published after the international filing date or priority date and not in conflict with the application but cited to understand the principle or theory underlying the inventiondocument of particular relevance; the claimed invention cannot be considered novel or cannot be considered to involve an inventive step when the document is taken alonedocument of particular relevance; the claimed invention cannot be considered to involve an inventive step when the document is combined with one or more other such documents, such combination being obvious to a person skilled in the artdocument member of the same patent familyDate of the actual completion of the international searchDate of mailing of the international search report06 March 2024Name and mailing address of the ISA / CNChina National Intellectual Property Administration (ISA / CN)China No. 6, Xitucheng Road, Jimenqiao, Haidian District,Beijing 100088Authorized officer15 March 2024Telephone No.C. DOCUMENTS CONSIDERED TO BE RELEVANTCategory*Citation of document, with indication, where appropriate, of the relevant passagesRelevant to claim No.CAKIR, B. et al. "Development of Human Brain Organoids with Functional Vascular-like System"Nature Methods, Vol. 16, No. 11, 30 November 2019 (2019-11-30),pages 1169-1175CN 112226410 A (GUANGZHOU WOMEN AND CHILDREN'S MEDICAL CENTER (GUANGZHOU MATERNAL AND CHILD HEALTH HOSPITAL, GUANGZHOU CHILDREN'S HOSPITAL, GUANGZHOU MATERNITY AND INFANT HOSPITAL, GUANGZHOU MATERNAL AND CHILD HEALTH AND FAMILY PLANNING SERVICE CENTER)) 15 January 2021 (2021-01-15) claims 1-10WO 2022134229 Al (WUHAN IREGENE THERAPEUTICS CO., LTD.) 30 June 2022 (2022-06-30)description, embodiment 5COTERO, V, et al. "Noninvasive Sub-organ Ultrasound Stimulation for Targeted Neuromodulation"Nature Communications, Vol. 10, 12 March 2019 (2019-03-12),pages 1-12WH) (XU, Lei et al.). "A^3DW®j^Sli’CT?LjS® (Research Progresses of 3D BrainOrganoids Derived from Pluripotent Stem Cells)"(144444^4414) (Journal of Nanjing Medical University (Natural Sciences)), Vol. 40, No. 1, 31 January 2020 (2020-01-31), pages 133-1401-71-91-91-91-9PCT / CN2023 / 139394Patent document cited in search report Publication date (day / month / year) Patent family member)s) Publication date (day / month / year) CN 116376840 A 04 July 2023 None CN 112226410 A 15 January 2021 None WO 2022134229 Al 30 June 2022 None

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