Multi-brain organ electron complex and construction method and application thereof

Abstract

The invention discloses a multi-brain organ electron complex as well as a construction method and application thereof. The multi-brain organ electronic complex comprises a microelectrode array and a fusion organ attached to the surface of a chip electrode of the microelectrode array through a PDMS film, and the fusion organ can grow for more than or equal to 120 days through culture. The PDMS film is attached to the surface of the MEA chip electrode, so that the stability of combination of the MEA chip electrode and the organoid is remarkably enhanced, the construction efficiency of a multi-brain organoid electron complex is improved, and the continuity of neural signal detection is improved; a high-performance model platform is provided for multi-brain organ research, and the method has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of brain organoids, and more particularly to a multi-brain organoid electronic complex, a construction method thereof, and an application thereof. Background Art

[0002] The organoid technology is an important research direction in the field of biomedicine in recent years. The three-dimensional organoid structure generated by induced differentiation culture can simulate the structure and function of human tissues and organs, and is widely used in the establishment of disease models, drug screening, and neurobiological research.

[0003] However, there are the following problems in the current research on multi-brain organoids:

[0004] 1. The existing construction methods of multi-brain organoids are carried out in a static state, which easily leads to low organoid fusion efficiency and limited growth and development;

[0005] 2. In the technology of combining organoids with MEA (microelectrode array), simple attachment methods are mostly used, which have problems such as low attachment efficiency, poor stability, and low signal coupling efficiency, and it is difficult to meet the long-term recording and analysis requirements of complex neural signals.

[0006] Therefore, developing a new method that can effectively improve the organoid fusion efficiency and optimize its combination with MEA has important research and application values. Summary of the Invention

[0007] To solve the deficiencies of the above technical solutions, the purpose of the present invention is to provide a construction method of a multi-brain organoid electronic complex.

[0008] Another object of the present invention is to provide a multi-brain organoid electronic complex constructed by the above construction method.

[0009] Another object of the present invention is to provide an application of the multi-brain organoid electronic complex.

[0010] The object of the present invention is achieved by the following technical solutions.

[0011] A construction method of a fused brain organoid electronic complex includes the following steps:

[0012] Step 1, inducing embryonic stem cells expressing green fluorescent protein (GFP) to differentiate into ventral cortical organoids, and inducing embryonic stem cells expressing red fluorescent protein (mCherry) to differentiate into dorsal cortical organoids, and culturing them separately until both types of organoids reach the fusion time point;

[0013] Step 2, dynamic fusion: Mix the ventral cortical organoids and dorsal cortical organoids that have reached the fusion time point obtained in Step 1 and perform fusion in a dynamic environment to obtain fused organoids;

[0014] Step 3, fabricate a PDMS membrane accommodation unit with the same shape as the fused organoids. After cleaning and sterilization, attach the PDMS membrane accommodation unit to the surface of each chip electrode of the microelectrode array (MEA) coated with Matrigel. Replace the Matrigel with a culture medium for culturing the mature stage of the fused organoids, and then attach the fused organoids to the PDMS membrane accommodation unit and perform cell culture until the fused organoids are fully attached to the chip electrodes of the microelectrode array, obtaining a multi-brain organoid electronic complex.

[0015] In the above technical solution, the operations of Step 1, Step 2, and Step 3 are all carried out in a sterile environment.

[0016] In the above technical solution, in Step 3, the PDMS membrane accommodation unit is washed with sterile ultrapure water to remove surface residues, and the PDMS membrane accommodation unit is sterilized using an ultraviolet sterilization lamp.

[0017] In the above technical solution, in Step 3, the chip electrodes need to be soaked in sterile ultrapure water before coating, then taken out and washed with ethanol, and finally subjected to ultraviolet sterilization treatment.

[0018] In the above technical solution, in Step 3, Matrigel is used to coat the chip electrodes of the microelectrode array (MEA): DF12 and Matrigel are thoroughly pipetted and mixed evenly, and the chip electrodes are coated at 0 - 4°C.

[0019] Another aspect of the present invention further includes the multi-brain organoid electronic complex constructed by the construction method. The multi-brain organoid electronic complex includes a microelectrode array and fused organoids attached to the surface of the chip electrodes of the microelectrode array through a PDMS membrane, and the fused organoids can grow to be greater than or equal to 120 days through culture.

[0020] Another aspect of the present invention further includes the application of the multi-brain organoid electronic complex in neural research.

[0021] The advantages and beneficial effects of the present invention are:

[0022] 1. The construction method of the present invention performs the fusion of organoids in a dynamic environment, improving the quality and efficiency of organoid fusion, and ensuring the uniformity and developmental stability of organoid fusion;

[0023] 2. The present invention attaches the PDMS membrane to the surface of the MEA chip electrode, significantly enhancing the stability of the binding between the MEA chip electrode and the organoid, improving the construction efficiency of the multi-brain organoid electronic complex, and enhancing the persistence of neural signal detection; it provides a high-performance model platform for multi-brain organoid research and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a diagram of the induction and differentiation process in Step 1 of Example 1.

[0025] Figure 2 It is a light microscope image of different stages of organoid differentiation in Example 1.

[0026] Figure 3 It is a physical image of the ventral cortical organoid (MGEOs) and dorsal cortical organoid (COs) in Example 1.

[0027] Figure 4 It is a diagram of the dynamic fusion process in Example 1.

[0028] Figure 5 It is a schematic diagram of the processes in Steps 2 and 3 of Example 1.

[0029] Figure 6 It is an electrophysiological signal diagram of 8 channels.

[0030] Figure 7 It is an original spike signal diagram of the 2nd channel among the 8 channels.

[0031] Figure 8 It is a fusion result diagram of static fusion culture and dynamic fusion culture. Among them, A is the 6th day of dynamic fusion, B is the 12th day of dynamic fusion, C is the 6th day of static fusion, D is the 12th day of static fusion, E is the 25th day of dynamic fusion, and F is the 25th day of dynamic fusion. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0032] The technical solution of the present invention will be further described below in conjunction with specific embodiments.

[0033] Example 1

[0034] A method for constructing a multi-brain organoid electronic complex, comprising the following steps:

[0035] As Figures 1-3As shown in the figure, in step 1, the embryonic stem cells expressing green fluorescent protein (ES-G) are induced to differentiate into medial ganglionic eminence organoids (MGE Organoid), and the embryonic stem cells expressing red fluorescent protein (ES-M) are induced to differentiate into cortical organoids (Cortical Organoid, CO). They are cultured separately until the organoids reach the fusion time point, which specifically includes the following steps:

[0036] Step 1.1, the culture stage of embryoid bodies (EB): On the 0th day of organoid culture, all differentiated cells in the embryonic stem cell ESC culture are scraped off under a microscope. The ESC clone clusters are dissociated into single-cell suspensions and digested with Accutase cell digestive solution. Then, 9000 cells in the digested single-cell suspension are seeded in the neural induction medium (the neural induction medium contains DMEM / F12 medium, 15% (v / v) knockout serum replacement, 1% (v / v) MEM non-essential amino acids (MEM NEAA), 1% (v / v) Glutamax, 100 mM β-mercaptoethanol, 100 nM LDN 193189, 10 mM SB431542, and 2 mM XAV939. 50 mM Y 27632 compound and 5% (v / v) heat-inactivated fetal bovine serum FBS are also added to this neural induction medium) in each well of a U-bottom ultra-low attachment 96-well plate, and statically cultured in an incubator at 37 °C and 5% CO2. The neural induction medium is changed every other day and cultured until the 10th day. At the same time, on the 2nd day of culture, 50 mM Y27632 compound is added to the neural induction medium, but without heat-inactivated FBS. Starting from the 4th day of culture, Y27632 compound is no longer added to the neural induction medium, where the embryonic stem cells are embryonic stem cells expressing green fluorescent protein or embryonic stem cells expressing red fluorescent protein.

[0037] Step 1.2, Cortical ventral patterning stage: When the embryonic stem cells in Step 1.1 are green fluorescent protein-expressing embryonic stem cells, transfer the cells after static culture in Step 1.1 to an ultra-low attachment 6-well plate (it should be noted that at most 8 organoids are transferred to each well of the 6-well plate), and use ventral patterning medium (ventral patterning medium contains DMEM F12 medium, 0.15% (w / v) dextrose, 100 mM β-mercaptoethanol, 1% (v / v) N2 supplement, 2% B27 supplement (without vitamin A), 100 ng / ml recombinant sonic hedgehog (SHH), and 1 mM purmorphamine). The 6-well plate is maintained on a horizontal shaker at 80 rpm / min, and the medium is changed every other day. Culture for 18 days to differentiate into cortical ventral organoids (Cortical ventral-G).

[0038] Step 1.3, DCOs neural expansion stage: When the embryonic stem cells in Step 1.1 are red fluorescent protein-expressing embryonic stem cells, transfer the cells after static culture in Step 1.1 to an ultra-low attachment 6-well plate (it should be noted that at most 8 organoids are transferred to each well of the 6-well plate), and use neural differentiation medium without vitamin A (this medium contains a 1:1 mixture of DMEM F12 medium and Neurobasal medium, supplemented with 0.5% (v / v) N2 supplement, 1% (v / v) B27 supplement (without vitamin A), 1% (v / v) Glutamax, 0.5% (v / v) MEM NEAA, 0.025% (v / v) human insulin solution, 50 mM β-mercaptoethanol, and 1% (v / v) penicillin / streptomycin). The 6-well plate is maintained on a shaker at 80 rpm / min, and the medium is changed every other day. Culture for 18 days to differentiate into dorsal cortical organoids (Cortical-M). During the culture process, add 3 μM CHIR to the medium from day 10 to day 14 to effectively promote the formation of the neural tube. From day 14 to day 18, thaw Matrigel and dissolve it in the neural differentiation medium at a dilution of 1:100 to support the emergence of the cortical plate.

[0039] Step 1.4, Maturation stage: The ventral cortical organoids from Step 1.2 and the dorsal cortical organoids from Step 1.3 are respectively cultured with a neural differentiation medium containing vitamin A (this medium contains a 1:1 mixture of DMEM F12 and Neurobasal medium, supplemented with 0.5% (v / v) N2 supplement, 1% (v / v) B27 supplement (containing vitamin A), 1% (v / v) Glutamax, 0.5% (v / v) MEM NEAA, 0.025% (v / v) human insulin solution, 50 mM β-Mercaptoethanol, and 1% (v / v) Penicillin / Streptomycin, 20 ng / ml BDNF, 200 mM cAMP, and 200 mM ascorbic acid), and continue to rotate the culture until day 20 to allow the organoids to reach the fusion time point, and change the medium every 4 days.

[0040] As Figure 4 shown, Step 2, Dynamic fusion: Transfer the ventral cortical organoids and dorsal cortical organoids that have reached the fusion time point obtained in Step 1 to a 50 ml centrifuge tube with a conical round bottom using a large-bore pipette, and place it on a shaker in a cell culture incubator for dynamic culture and fusion at 80 rpm for 48 h to obtain fused organoids. Dynamic culture reduces the impact of mechanical stress and insufficient nutrient supply exchange in the static state on the development of organoids, and improves the fusion efficiency and the quality of the fused organoids.

[0041] Furthermore, the fused organoids can be transferred to a six-well plate using a large-bore pipette for continued dynamic culture for subsequent experiments, such as for studying the developmental function of the fused organoids, or after culturing to 120, for detecting electrical signals.

[0042] As Figure 5 shown, Step 3, Fabricate a PDMS membrane accommodation unit with the same shape as the fused organoids, clean and sterilize the PDMS membrane accommodation unit, attach the PDMS membrane accommodation unit to the surface of each chip electrode of a microelectrode array (MEA) coated with Matrigel, replace the Matrigel with a medium for culturing the fused organoids during the maturation stage, attach the fused organoids obtained in Step 2 to the PDMS membrane accommodation unit, and perform cell culture until the fused organoids are fully attached to the chip electrodes of the microelectrode array to obtain a multi-brain organoid electronic complex. Specifically, it includes the following steps:

[0043] Step 3.1, Prepare the PDMS membrane: Mix 50 g of PDMS prepolymer Sylgard 184 Silicone Base and 5 g of curing agent Sylgard 184 Curing Agent, and stir evenly with a sterile stir bar for 3 minutes to avoid generating excessive bubbles, obtaining a PDMS mixture. Slowly pour the PDMS mixture into a 15-cm cell culture dish and place it in a laminar flow hood to cure for 2 days, ensuring that the thickness of the membrane is uniform during casting to obtain the PDMS membrane.

[0044] Step 3.2, After cleaning and sterilizing the PDMS membrane, make a PDMS membrane accommodating unit with the same shape as the fused organoids. Wash the PDMS membrane accommodating unit with sterile ultrapure water to remove surface residues, and sterilize the PDMS membrane accommodating unit with an ultraviolet sterilization lamp for 30 min.

[0045] Step 3.3, Immerse the MEA chip electrodes in 2 ml of sterile ultrapure water for 24 h, remove the ultrapure water, add 2 ml of 75% ethanol to the culture wells of the MEA chip electrodes, gently pipette and wash 5 - 10 times with a 1-ml pipette, and then thoroughly rinse with sterile ultrapure water to remove alcohol residues, so as to avoid the toxic side effects of alcohol on cells and damage to the chip electrodes. Finally, place the chip electrodes in a laminar flow hood or a biosafety cabinet, turn on the hair dryer and ultraviolet lamp (UV) to irradiate the surface of the chip electrodes for 30 minutes to kill potential microorganisms. After the above treatment, the MEA chip electrodes are convenient for coating and the attachment of organoids. The cleaning is to remove the possible grease, dust and residues on the surface of the chip electrodes.

[0046] Step 3.4, At 4 °C, thoroughly pipette and mix 1 ml of DF12 and 17 ml of Matrigel, add it to each MEA chip electrode and coat it in a cell culture incubator for 2 h to coat each MEA chip electrode with Matrigel, obtaining the chip electrodes of the microelectrode array (MEA) coated with Matrigel.

[0047] Step 3.5, Attach the sterilized PDMS membrane accommodating unit in Step 3.2 to the MEA chip electrodes, use a 200-μl pipette to aspirate the coated DF12 medium and replace it with 2 ml of the medium for the maturation culture of organoids. Gently place the fused organoids obtained in Step 2 on the PDMS membrane accommodating unit attached with a large-bore pipette, and place the chip electrodes in a cell culture incubator for 3 days until the fused organoids are fully attached to the chip electrodes of the microelectrode array, obtaining a multi-brain organoid electronic complex.

[0048] Further, change the medium at a frequency of changing the medium every three days, and continue to culture the fused organoids. When the fused organoids are further cultured in the incubator for 120 days, they are used for neural signal detection, as Figures 6-7 shown, byFigures 6-7 It can be seen that PDMS is a biocompatible material, which is friendly to cells and tissues, does not affect the growth and function of cells, and helps to maintain the activity of brain organoids; the PDMS membrane has good flexibility, can be used to design molds for brain organoids of different shapes and sizes, and reduces the risk of detachment of brain organoids from the chip caused by the shaking of the culture medium. Therefore, relatively stable electrophysiological signals can be collected; the PDMS membrane can be combined with other materials and functional modules to facilitate the integration of optical, chemical, and electrical signal monitoring, forming a multi-functional biosensing platform. For example, it can be combined with microfluidic devices, etc.

[0049] As Figure 8 shown, the ventral cortical organoids and dorsal cortical organoids obtained in step 1 of Example 1 were subjected to dynamic fusion culture or static fusion culture. At the 6th day and the 12th day of culture, fusion comparisons were respectively carried out. It can be seen from Figure 8 that there were cell drops in the statically cultured organoids ( Figure 8 the positions circled in blue in Figure 8 ), and the number of dropped cells increased with time. The white dotted lines in E and F in Figure 8 are the fusion surfaces, and the black dotted lines are the approximate outlines of normal organoids. It can be seen that in a static environment, the organoid fusion is poor, the edges are damaged, and abnormal growth occurs.

[0050] The above is an exemplary description of the present invention. It should be noted that without departing from the core of the present invention, any simple deformation, modification, or equivalent replacement that can be made by those skilled in the art without creative labor falls within the protection scope of the present invention.

Claims

1. A method for constructing a fusion brain organoid electronic complex, characterized in that: The following steps are involved: Step 1, inducing embryonic stem cells expressing green fluorescent protein to differentiate into ventral cortical organoids, inducing embryonic stem cells expressing red fluorescent protein to differentiate into dorsal cortical organoids, and culturing them separately until the organoids reach a fusion time point; Step 2, dynamic fusion: the ventral cortical organoids and dorsal cortical organoids that have reached the fusion time point obtained in step 1 are mixed and fused in a dynamic environment to obtain fused organoids; Step 3, make the PDMS membrane into a PDMS membrane containing unit consistent with the shape of the fused organoid, after cleaning and sterilization, attach the PDMS membrane containing unit to the surface of each chip electrode of the microelectrode array coated with matrix gel, replace the matrix gel with the culture medium for the mature stage culture of the fused organoid, and then attach the fused organoid to the PDMS membrane containing unit for cell culture until the fused organoid is fully fitted with the chip electrode of the microelectrode array to obtain a multi-brain organoid electronic complex.

2. The construction method according to claim 1, characterized in that: The operation processes of step 1, step 2 and step 3 are all carried out under a sterile environment.

3. The construction method according to claim 1, characterized in that: In the step 3, the PDMS membrane containing unit is cleaned with sterilized ultrapure water to remove surface residues, and the PDMS membrane containing unit is sterilized with an ultraviolet sterilization lamp.

4. The construction method according to claim 1, characterized in that: In step 3, the chip electrodes need to be soaked in sterile ultrapure water before coating, then taken out and cleaned with ethanol, and finally sterilized with ultraviolet light.

5. The construction method according to claim 1, characterized in that: In the step 3, the chip electrodes of the microelectrode array are coated with matrix gel: DF12 and matrix gel are thoroughly blown and mixed evenly, and the chip electrodes are coated at 0-4°C.

6. The multi-brain organoid electronic complex constructed by the construction method according to any one of claims 1 to 5, characterized in that: The multi-brain organoid electronic complex includes a microelectrode array and a fused organoid attached to the chip electrode surface of the microelectrode array through a PDMS membrane, and the fused organoid can be grown to more than or equal to 120 days through culture.

7. Use of the multi-brain organoid electronic complex according to claim 6 in neurological research.

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