Culture medium for long-term culture of primary neurons, application and long-term culture method of primary neurons

Abstract

The invention provides a culture medium for long-term culture of primary neurons, application of the culture medium and a long-term culture method of the primary neurons, and relates to the technical field of cell biology. According to the invention, by improving the components of the culture solution and improving the operation of the culture method, the culture medium is ensured to provide sufficient nutrient substances, the long-time culture technology of the in-vitro neurons is explored, and the in-vitro survival of the neurons in continuous 83 days for the longest time under the condition of no glial cell co-culture is realized, so that the related life science problems are solved.

Description

Technical Field

[0001] The present invention relates to the field of cell biotechnology, and more specifically, to a culture medium for long-term culture of primary neurons, its application, and a long-term culture method of primary neurons. Background Art

[0002] Primary neuron in vitro culture refers to the isolation of neuronal cells from the brain or spinal cord tissue of embryos or newborn animals and their culture in vitro in a simulated in vivo environment. This culture method can preserve the basic biological characteristics of neurons and provide an important tool for studying the function, development and disease mechanism of neurons. Primary neurons are highly similar to neurons in vivo in morphology, synaptic connections and physiological functions, and are suitable for neurodevelopmental research, disease modeling, neural function research and other contents. For example, in neurodevelopmental research, in vitro culture can be used to study the synapse formation and maturation process of neurons under in vitro conditions and reveal the molecular mechanism of synaptic plasticity; the extension and branching of neurites can also be observed to study their regulatory mechanisms, providing a theoretical basis for the treatment of nervous system diseases. For example, in disease modeling, primary neuron culture can be used to construct in vitro models of neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease, and to study the molecular mechanisms of disease occurrence and development; in vitro cultured neurons can also be used to evaluate the protective effect or toxicity of drugs on neurons, providing an experimental basis for drug development. For example, in the field of neural function research, electrophysiological recording technology is used to study the excitatory and inhibitory synaptic transmission of neurons under in vitro culture conditions, revealing the functional characteristics of neural circuits; it is also possible to study the repair mechanism of neurons after injury, providing new ideas for the treatment of neural injuries.

[0003] At present, primary neurons can usually survive for 2-3 weeks in three-dimensional culture (such as the report on significantly improving the survival rate of neurons derived from primary cells and stem cells). Under the condition of optimizing culture conditions, it is still challenging to culture primary neurons in vitro for more than 2 months under current experimental conditions. However, biological neural networks that can survive for a long time and be cultured stably are the key foundation for promoting breakthroughs in neuroscience research, brain disease treatment, brain-like intelligence development, and biomedical engineering.

[0004] Neuron in vitro culture is an important means to study neuronal function. This technology was reported as early as the beginning of the last century. However, after decades of development, the current in vitro neuron culture technology still has great limitations. First, the types of neurons that can be cultured in vitro are limited. In addition to glutamatergic neurons, which are easy to culture in large quantities, other excitatory neurons are not easy to culture on a large scale. Second, the survival time of primary cultured neurons is limited, generally not exceeding 1 month. Second, in conventional primary neuron culture, in order to prevent the continuous proliferation of astrocytes from affecting the purity and survival of neurons, cytosine arabinoside AraC is usually added to the culture medium to reduce the contamination of glial cells by inhibiting the DNA synthesis of dividing cells. But in fact, high concentrations of AraC can induce axonal degeneration and programmed cell death of mature neurons, which has an adverse effect on the growth of neurons. Third, in the brain, astrocytes play an important supporting role in neuronal survival and information processing ability by releasing nutrients. However, this nutritional support is lacking in in vitro culture. However, if co-culture is carried out directly, the continuous proliferation of glial cells in the later stage of culture will affect the activity of neurons and the extension of axons and dendrites. The rapid proliferation of glial cells consumes key metabolites such as glucose and glutamine in the culture medium, causing neurons to become less active due to insufficient energy and nutrient supply. At the same time, the proliferating glial cells form a dense monolayer on the bottom of the culture medium, which may hinder the normal extension of neuronal axons and dendrites. Neuronal synapse formation requires a certain space, and excessive proliferation of glial cells will compress this space, thereby inhibiting the construction of synaptic networks. Excessive proliferation of glial cells interferes with neuronal activity, and cell purity and coexistence ratio are difficult to control. Because the human brain is protected by the skull, it supplies nutrients and oxygen and discharges waste through the blood circulation, lymphatic circulation and other systems, and neurons cultured in vitro lack these mechanisms. Therefore, how to achieve stable culture for more than 2 months, or even longer, is one of the key concerns and urgent issues to be solved. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides a culture medium for long-term culture of primary neurons, an application thereof, and a long-term culture method of primary neurons; the present invention improves the composition of the culture solution and the operation of the culture method to ensure that the culture medium provides sufficient nutrients, explores the long-term culture technology of isolated neurons, and achieves the longest continuous survival of neurons in vitro for 83 days without co-culture of glial cells, so as to solve related life science problems.

[0006] In a first aspect, the present application discloses a culture medium for long-term culture of primary neurons, the culture medium comprising a basal culture medium and a neuron culture supplement factor; the neuron culture supplement factor comprises: 35-45 mM Na+, 20-30 mM Glucose, 0.3-0.5 mM L-serine, and 9-11 mM KCl.

[0007] In some embodiments, the Na+ content is 40 mM; Optionally, the content of Glucose is 25 mM; Optionally, the content of L-serine is 0.4 mM; Optionally, the content of KCl is 10 mM; Optionally, the basal culture medium is selected from any one of the following: commercial neuronal culture medium NeurobasalPlus / B27 Plus, commercial culture medium BrainPhys TM ; Preferably it is the commercial neuronal culture medium Neurobasal Plus / B27 Plus.

[0008] In some embodiments, the neuron culture supplement factor further comprises astrocyte inhibitors 0.5 μM cytarabine Ara-C and 0.5 μM fluorouridine FUDR; Optionally, the neuron culture supplement factor also includes astrocyte supernatant culture fluid, which is treated with high temperature inactivated complement and then mixed with neuron culture medium at a ratio of 1:10; Optionally, the oxygen content in each liter of the culture medium composition ranges from 8 to 12%, preferably 10%; Optionally, the osmotic pressure of the culture medium is in the range of 265-275 mOsm / L; preferably 270 mOsm / L; Optionally, a semipermeable membrane without pores but permeable to gas is arranged on the culture medium.

[0009] The second aspect of the present application discloses the use of the culture medium for long-term culture of primary neurons according to the first aspect of the present application in preparing primary neurons, wherein the use comprises preparing neurons after culturing neurons in the culture medium for long-term culture of primary neurons.

[0010] The third aspect of the present application discloses a neuron prepared by the application described in the second aspect of the present application.

[0011] A fourth aspect of the present application provides a method for long-term culture of primary neurons, the method comprising: S101, extracting and separating neurons and astrocytes from experimental animals and culturing them separately, and obtaining neuronal cell suspensions and astrocyte cell suspensions after treatment, and culturing them separately; when extracting from animals, the neurons and astrocytes extracted are separated; S102, recording the inoculation time as day 0, and culturing primary neurons using the culture medium described in the first aspect of the present application; S103, when the inoculation time is day N, the supernatant culture fluid produced during the astrocyte culture process is taken, treated with high temperature to inactivate complement, and then mixed with neuron culture medium at a ratio of 1:10 to form a modified culture medium; S104: Thereafter, half the volume of neurons was replaced with the modified culture medium every M days and culture was continued.

[0012] In some embodiments, the method further comprises: installing a non-porous but gas-permeable semi-permeable membrane on the culture medium; Optionally, the semipermeable membrane is made of FEP material; Optionally, the semipermeable membrane is cut according to the size of the culture dish outside the culture medium; Optionally, the method further comprises: regularly monitoring the osmotic pressure in the culture medium, and providing a risk warning when the osmotic pressure is less than 270 or greater than 330 mOsm / L.

[0013] This application has the following beneficial effects: 1. The present application innovatively discloses a culture medium for long-term culture of primary neurons and a method for long-term culture of primary neurons using the culture medium. By improving the composition of the culture medium, the goals of high purity, survival and long-term culture can be achieved.

[0014] Specifically, 1) In the brain, the most numerous cells are actually astrocytes. Astrocytes play an important role in supporting the survival of neurons and the realization of information processing capabilities by releasing nutrients. However, this nutritional support is lacking in in vitro culture. In order to supplement the nutrients provided by astrocytes, we cultured astrocytes separately, took their supernatant culture fluid, and after attenuation treatment, mixed it with neuronal culture medium in a certain proportion for long-term culture of primary neurons, which can significantly promote the survival of neurons.

[0015] 2) The problem of reduced stability of the culture medium. In vitro culture may encounter sudden death. This is mainly due to changes in osmotic pressure caused by repeated rehydration and fluid replacement. The osmotic pressure in normal culture medium is 270~330 (mOsm / L). Long-term culture will cause the osmotic pressure to increase with water evaporation and increased salt content, resulting in poor survival of neurons or even sudden death. Our measurement data showed that under normal culture conditions, after 20 days of culture, the osmotic pressure of the culture medium has reached the theoretical tolerance limit of nerve cells, and reached 365 mOsm / L after 35 days of culture. These data show that we need to monitor the evaporation of the solution and the change of osmotic pressure. In order to minimize evaporation, a FEP semipermeable membrane was customized. It can pass gases such as oxygen, but has no holes, thereby reducing water evaporation and blocking microorganisms.

[0016] 3) The firing frequency of long-term cultured neural networks decreases, and the proportion of firing neurons decreases. Biological speculation is that this is because the changes in the morphology of the neural network and the intrinsic gene expression tend to minimize energy consumption. At present, the team believes that the information processing capacity of this network is significantly reduced. This study found that by adjusting the composition of the culture medium, the activity of the neural network can be improved: the original culture medium contains four types of components, "activating neurons", "assisting in activating neurons", "pH adjustment" and "energy supply". We adjusted 4 of the key substances and controlled the oxygen content, osmotic pressure and evaporation in the culture medium to ensure the culture environment of primary neurons, thereby significantly affecting the activity of the neural network. This is the main component of the new culture medium we developed, which can achieve the goal of keeping alive, keeping for a long time, and discharging more.

[0017] 2. This application has achieved unexpected results by improving the composition of the culture medium and the process of long-term culture of primary neurons using the culture medium. Specifically, a batch of neurons cultured for a long time were photographed and displayed, and photos were taken every 6 days. These are photos from 6 to 62 days of culture. It can be seen that the axonal dendrites of neurons in the first two weeks of culture are relatively thin, and they become thicker after long-term culture, and the cell body is more swollen.

[0018] In short, by controlling nutrient conditions and culture methods, it is now possible to culture mouse neurons in vitro for 2 months, improve the culture medium to increase the purity and activity of neurons, and further extend the in vitro neuron culture time, achieving the longest continuous survival of neurons in vitro for 83 days without glial cell co-culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0020] Figure 1 is a schematic diagram of a method flow chart provided by the fourth aspect of an embodiment of the present invention; Figure 2 Schematic diagram of a long-term culture system of primary neurons provided in an embodiment of the present invention; Figure 3 is a schematic diagram of a computer device provided by an embodiment of the present invention; Figure 4 is a schematic diagram of the architecture of an exemplary computing device provided by an embodiment of the present invention; Figure 5 is a schematic diagram of a storage medium provided by an embodiment of the present invention; Figure 6 is a schematic diagram of the preparation process of mouse primary neurons provided by an embodiment of the present invention; wherein, Figure 6 A is a mouse lying in supine position. Figure 6 B is to cut open the mouse abdominal skin to expose the uterus. Figure 6 C is the transfer of the mouse embryo head into the culture dish. Figure 6 D is in the dissection under stereoscope, Figure 6 E is the collected centrifuge tube filtrate; Figure 7 is a schematic diagram of the state of neural network activity after long-term culture of primary cortical neurons of mice for several days provided by an embodiment of the present invention; wherein, Figure 7 A is 6 days after culture, Figure 7 B is 12 days after culture. Figure 7 C is 18 days after culture, Figure 7 D is 24 days after culture. Figure 7 E is 30 days after culture, Figure 7 F is 36 days after culture, Figure 7 G is 42 days after culture. Figure 7 H is 48 days after culture, Figure 7 I is 54 days after culture, Figure 7 J is 62 days after culture; Figure 8 This is an identification diagram of DIV83 primary cortical neurons provided in an embodiment of the present invention; Fig. 9 It is a schematic diagram of cells in a bright field state under a microscope using the original culture medium provided by an embodiment of the present invention, without adding an astrocyte inhibitor, without adding additional nutrients, and without containing astrocyte supernatant culture fluid; Fig.10 1 is a schematic diagram of the state of neural network activity after long-term culture of primary cortical neurons of mice for several days provided by an embodiment of the present invention, wherein the left side shows that 470 active neurons were detected after 23 days of culture, and the right side shows that 240 active neurons were detected after 45 days of culture; Fig.11 The results show that the discharge activity of neurons in the improved culture medium provided by the embodiment of the present invention is doubled compared with that of neurons in the conventional culture medium. DETAILED DESCRIPTION

[0021] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0022] In some of the processes described in the specification and claims of the present invention and the above-mentioned figures, multiple operations that appear in a specific order are included, but it should be clearly understood that these operations may not be executed in the order in which they appear in this article or executed in parallel. The serial numbers of the operations, such as 101, 102, etc., are only used to distinguish different operations, and the serial numbers themselves do not represent any execution order. In addition, these processes may include more or fewer operations, and these operations may be executed in sequence or in parallel. It should be noted that the descriptions of "first", "second", etc. in this article are used to distinguish different messages, devices, modules, etc., do not represent the order of precedence, and do not limit the "first" and "second" to be different types.

[0023] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only 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 those skilled in the art without creative work are within the scope of protection of the present invention.

[0024] Table 1 shows the main components of the culture medium and conventional culture medium for long-term culture of primary neurons disclosed in the first aspect of the present application. The use of the culture medium in the present application can achieve the goals of keeping the neurons alive, keeping them for a long time, and maintaining high purity. This embodiment discloses a culture medium for long-term culture of primary neurons, the culture medium comprising a basal culture medium and a neuron culture supplement factor; the neuron culture supplement factor comprises: 35-45 mM Na+, 20-30 mM Glucose, 0.3-0.5 mM L-serine, 9-11 mM KCl. The components of this culture medium are additional improvements added on the basis of the commercial neuron culture medium Neurobasal Plus / B27Plus. In this embodiment, the corresponding concentration unit "nM" of the component is nmol / L, which means that each liter of culture medium contains ×× nmol.

[0025] In some embodiments, the content of Na+ is 40 mM; optionally, the content of Glucose is 25 mM; optionally, the content of L-serine is 0.4 mM; optionally, the content of KCl is 10 mM.

[0026] In some embodiments, the basal culture medium is selected from any one of the following: commercial neuronal culture medium Neurobasal Plus / B27 Plus, commercial culture medium BrainPhys TM ; Preferably it is the commercial neuronal culture medium Neurobasal Plus / B27 Plus.

[0027] In some embodiments, the neuron culture supplement factor further comprises astrocyte inhibitors 0.5 μM cytarabine Ara-C and 0.5 μM fluorouridine FUDR; Optionally, the neuron culture supplement factor also includes astrocyte supernatant culture fluid, which is treated with high temperature inactivated complement and then mixed with neuron culture medium at a ratio of 1:10; Optionally, the oxygen content in each liter of the culture medium composition ranges from 8 to 12%, preferably 10%; Optionally, the osmotic pressure of the culture medium is in the range of 265-275 mOsm / L; preferably 270 mOsm / L; Optionally, a semipermeable membrane without pores but permeable to gas is arranged on the culture medium.

[0028] Table 1

[0029] The second aspect of the present application discloses the use of the culture medium for long-term culture of primary neurons according to the first aspect of the present application in preparing primary neurons, wherein the use comprises preparing neurons after culturing neurons in the culture medium for long-term culture of primary neurons.

[0030] The third aspect of the present application discloses a neuron prepared by the application described in the second aspect of the present application.

[0031] Figure 1 1 is a schematic flow chart of a method for long-term culture of primary neurons provided in a fourth aspect of an embodiment of the present invention. Specifically, the method comprises the following steps: S101, extracting and separating neurons and astrocytes from experimental animals and culturing them separately, and obtaining neuronal cell suspensions and astrocyte cell suspensions after treatment, and culturing them separately; when extracting from animals, the neurons and astrocytes extracted are separated; S102, recording the inoculation time as day 0, and culturing the primary neurons using the culture medium described in the first aspect of the present application; the method adopted here is to take the supernatant and add it to the primary neuron culture medium, and add it at a ratio of 1:10 after high-temperature inactivation of complement; S103, when the inoculation time is day N, the supernatant culture fluid produced during the astrocyte culture process is taken, treated with high temperature to inactivate complement, and then mixed with neuron culture medium at a ratio of 1:10 to form a modified culture medium; S104: Thereafter, half the volume of neurons was replaced with the modified culture medium every M days and culture was continued.

[0032] In some embodiments, both N and M are rational numbers.

[0033] In some embodiments, the method further comprises: installing a non-porous but gas-permeable semipermeable membrane on the culture medium; in some more specific embodiments, the semipermeable membrane is made of FEP; In some embodiments, the semipermeable membrane is cut according to the size of the culture dish outside the culture medium; the setting of the semipermeable membrane minimizes evaporation, and the FEP semipermeable membrane is customized. It can pass gases such as oxygen, but has no holes, thereby reducing water evaporation and blocking microorganisms.

[0034] In some embodiments, the method further comprises: regularly monitoring the osmotic pressure in the culture medium, and providing a risk warning when the osmotic pressure is less than 270 or greater than 330 mOsm / L. Regular sampling and regular testing are performed using an existing osmotic pressure meter.

[0035] The innovative motivation of the fourth aspect of this application is: 1. Simulate the maturation process of the nervous system and study long-term functional characteristics. Neurons take weeks or even months to complete the maturation process in vivo, including the extension of axons and dendrites, the establishment of synaptic connections, and the stabilization of electrophysiological properties. In vitro culture, neurons need to go through a similar time window to reach functional maturity. Long-term cultured neurons are more suitable for studying dynamic processes such as synaptic plasticity and neurotransmitter release. 2. Support the formation and functional research of complex neural networks. The network activities of neurons (such as synchronous discharge and signal integration) require sufficient culture time to form stable synaptic connections. The network structure of neurons cultured in the short term may not be fully developed and cannot reflect the complexity of the nervous system in vivo. For example, neurons cultured for more than 21 days can form more mature neural networks, which is convenient for observing long-term synaptic remodeling or network degeneration under pathological conditions. In addition, when studying the chronic pathological mechanisms of neurodegenerative diseases (such as Alzheimer's disease), it is necessary to simulate the progression of the disease through long-term culture. 3. Some experiments require neurons to survive in vitro for a long time to achieve specific goals, such as: • Drug screening and toxicity testing: A stable and long-term survival culture system is required to evaluate the chronic toxicity or protective effects of drugs on neurons. • Gene editing and phenotypic observation: Viral vector-mediated gene manipulation (such as CRISPR) may take several weeks to observe phenotypic changes, and long-term culture provides a time window for such experiments. Construction of disease models: For models such as hypoxic-ischemic brain injury or chronic neuroinflammation, long-term culture is required to simulate the adaptive changes of neurons in the pathological microenvironment.

[0036] Figure 3 is a schematic diagram of a computer device provided by an embodiment of the present invention, such as Figure 3 As shown, the device 2000 may include: one or more processors 2010, and one or more memories 2020; wherein the memories store computer-readable codes, and when the computer-readable codes are run by the one or more processors, the method described above may be executed.

[0037] The processor in this embodiment can be an integrated circuit chip with signal processing capabilities. The above processor can be a general-purpose processor, a digital signal processor (DSP), an application-specific integrated circuit (ASIC), a field-programmable gate array (FPGA) or other programmable logic devices, discrete gates or transistor logic devices, discrete hardware components. The disclosed methods, operations and logic block diagrams in the embodiments of the present disclosure can be implemented or executed. The general-purpose processor can be a microprocessor or the processor can also be any conventional processor, etc., which can be an X86 architecture or an ARM architecture.

[0038] In general, various example embodiments of the present disclosure may be implemented in hardware or dedicated circuits, software, firmware, logic, or any combination thereof. Certain aspects may be implemented in hardware, while other aspects may be implemented in firmware or software that may be executed by a controller, microprocessor, or other computing device. When various aspects of the disclosed embodiments are illustrated or described as block diagrams, flow charts, or using some other graphical representation, it will be understood that the blocks, devices, systems, techniques, or methods described herein may be implemented in hardware, software, firmware, dedicated circuits or logic, general purpose hardware or controllers or other computing devices, or some combination thereof as non-limiting examples.

[0039] For example, the method or device according to the embodiment of the present disclosure may also be implemented by Figure 4 The architecture of the computing device 3000 shown in FIG. Figure 4 As shown, the computing device 3000 may include a bus 3010, one or more CPUs 3020, a read-only memory (ROM) 3030, a random access memory (RAM) 3040, a communication port 3050 connected to a network, an input / output component 3060, a hard disk 3070, etc. The storage device in the computing device 3000, such as ROM 3030 or hard disk 3070, may store various data or files used for processing and / or communication of the method provided by the present disclosure and program instructions executed by the CPU. The computing device 3000 may also include a user interface 3080. Of course, Figure 4 The architecture shown is only exemplary and can be omitted according to actual needs when implementing different devices. Figure 4 One or more components of a computing device are shown.

[0040] The embodiment of the present invention also provides a computer-readable storage medium, such as Figure 5As shown, it is a schematic diagram of a storage medium 4000 provided in an embodiment of the present invention, and a computer readable instruction 4010 is stored on the computer storage medium 4020. When the computer readable instruction 4010 is executed by a processor, the method according to the embodiment of the present disclosure described with reference to the above figures can be executed. The computer readable storage medium in the embodiment of the present disclosure can be a volatile memory or a non-volatile memory, or can include both volatile and non-volatile memories. The non-volatile memory can be a read-only memory (ROM), a programmable read-only memory (PROM), an erasable programmable read-only memory (EPROM), an electrically erasable programmable read-only memory (EEPROM) or a flash memory. The volatile memory can be a random access memory (RAM), which is used as an external cache. By way of example and not limitation, many forms of RAM are available, such as static random access memory (SRAM), dynamic random access memory (DRAM), synchronous dynamic random access memory (SDRAM), double data rate synchronous dynamic random access memory (DDRSDRAM), enhanced synchronous dynamic random access memory (ESDRAM), synchronous link dynamic random access memory (SLDRAM), and direct memory bus random access memory (DR RAM). It should be noted that the memory of the methods described herein is intended to include, but is not limited to, these and any other suitable types of memory. It should be noted that the memory of the methods described herein is intended to include, but is not limited to, these and any other suitable types of memory.

[0041] The embodiments of the present disclosure further provide a computer program product or system, including a computer program, which implements the steps of the above method when executed by a processor.

[0042] In some embodiments, this embodiment also discloses a long-term culture system of primary neurons, such as Figure 2 As shown, the system comprises: The cell suspension preparation module 201 is used or configured to extract and separate neurons and astrocytes from the experimental animals and culture them separately, and obtain neuron cell suspension and astrocyte cell suspension after treatment, and culture them separately; The neuron culture medium module 202 is used or configured to record the inoculation time as day 0, and the primary neurons are cultured using the culture medium described in the first aspect of the present application; The culture medium preparation module 203 is used or configured to take the supernatant culture fluid produced during the astrocyte culture process when the inoculation time is the Nth day, treat it with high temperature inactivated complement, and then mix it with the neuron culture medium at a ratio of 1:10 to form a modified culture medium; A neuron continuous culture module 204 is added, which is used or configured to replace half of the volume of neurons with the improved culture medium every M days thereafter, and continue to culture; Specific embodiment:

[0043] 1. In vitro culture of mouse cerebral cortical neurons Experimental materials: ICR pregnant mice on day 16.5 of pregnancy (Weitong Lihua), medical dissection instruments (Rayward), dissection microscope (Zeiss, catalog number 37081), -20℃ ice box, sterile 1× PBS buffer (Zhongke Maichen, catalog number CC008), sterile DMEM medium containing phenol red (Zhongke Maichen, catalog number CM15019), Neurobasal Plus Medium (Gibco, catalog number A3582901), PDL (sigma, catalog number P6407), fetal bovine serum (sigma, catalog number 12003C), double distilled water, cell counting chamber, constant temperature water bath, centrifuge (eppendorf), 40 μm filter membrane (BD Falcon, catalog number 352350), 15 ml centrifuge tube (Corning), 50 ml centrifuge tube (Corning), 12-well cell culture plate, 24-well cell culture plate, 100× penicillin-streptomycin solution (Zhongke Maichen, product number CC004), 0.25% trypsin solution (Zhongke Maichen, product number CC017), L-glutamine (Gibco, product number A2916801).

[0044] Experimental steps: Preparation of improved culture medium: Based on the commercial neuronal culture medium Neurobasal Plus / B27 Plus supplemented with 0.5× penicillin-streptomycin solution and 2 mM L-glutamine, additional improved ingredients include 40 mM Na+, 25 mM Glucose, 0.4 mM L-serine, and 10 mM KCl.

[0045] Preparation of modified culture medium containing astrocyte inhibitors: Add astrocyte inhibitors AraC 0.5 μM and FUDR 0.5 μM to the modified culture medium Preparation of mixed culture medium: Add astrocyte supernatant culture fluid treated with high temperature inactivated complement to the improved culture medium and mix with the improved neuron culture medium at a ratio of 1:10.

[0046] 1) One day before cell culture, dissolve PDL in PBS buffer solution to prepare a solution with a final concentration of 0.1 mg / ml. Then dilute the 0.1 mg / ml PDL stock solution 50 times to prepare a 2μg / ml working solution. Add 1 ml of the prepared PDL solution to each well of the 12-well plate and 500 μl to each well of the 24-well plate. Place in a 37°C cell culture incubator for overnight coating. Take out the next day, carefully wash twice with sterile double distilled water, and dry for later use; 2) Use sterilized surgical instruments to cut open the abdominal skin of the pregnant mouse to expose the uterus, use replaced instruments to remove the intact uterus of the pregnant mouse, and transfer it to a 10 cm culture dish containing pre-cooled PBS; 3) Use new scissors and forceps to carefully peel off the mouse embryo, and use scissors to cut off the head of the mouse embryo. Transfer the head of the mouse embryo to a 10 cm culture dish containing pre-cooled DMEM medium; 4) Dissect the head of the mouse embryo under a stereoscope, carefully separate the cerebral cortex, discard the hippocampus, olfactory bulb and internal nuclei. Collect enough cerebral cortex into a 15 ml centrifuge tube containing pre-cooled DMEM medium containing penicillin-streptomycin solution; 5) Carefully discard the culture medium in the 15 ml centrifuge tube, gently rinse the cortical tissue twice with pre-cooled DMEM medium without phenol red, add trypsin solution to digest the cortical tissue, place the centrifuge tube in a 37°C water bath for 20 minutes, and gently shake the centrifuge tube every 5 minutes to mix the liquid; 6) After 20 minutes in the water bath, remove the centrifuge tube, wait for the tissue to settle, carefully discard the supernatant, add 3 ml of pre-cooled culture medium containing fetal bovine serum, and let it stand for 5 minutes; 7) Use a 1ml pipette to absorb the serum in the centrifuge tube, carefully blow and aspirate the tissue, blow and aspirate 20 times, let it stand, aspirate the supernatant, filter, add 2 ml of pre-cooled fetal bovine serum medium, and repeat the above steps 2 to 3 times. Filter with a 40μm filter and collect the filtrate into a new 15ml centrifuge tube. At this time, the neuronal cells are in the collected filtrate; 8) Centrifuge at 80 g for 3 min, discard the supernatant carefully, and keep the cell pellet. Add 1 ml of modified culture medium, gently resuspend the pellet with a 1 ml pipette, blow evenly, count using a cell counting plate, dilute the cells with modified culture medium containing astrocyte inhibitors according to 3~4×10^5 cells / ml, and inoculate them into 24-well plates. The time when the isolated primary neurons are planted in the culture plate is recorded as day 0; if Figure 6 As shown; 9) On the third day of neuron culture, add the supernatant culture fluid of astrocytes treated with high temperature inactivated complement to the culture medium, and add it to the modified neuron culture medium at a ratio of 1:10 to prepare a mixed culture medium. In the subsequent culture, replace half of the volume of neurons with mixed culture medium every 3 days to maintain the survival state and basic functions of neurons; half volume replacement means: replace half of the volume of neurons with mixed culture medium every 3 days, which means adding fresh culture medium to the culture dish used to culture neurons every 3 days, and removing half of the old culture medium. This is done to maintain the appropriate nutrient concentration of the culture medium and remove metabolic waste and harmful substances.

[0047] 10) Starting from the sixth day of isolation and culture from mice, the morphology of neurons was observed under a microscope with a 10× objective lens every six days, and the status of neurons was recorded by taking photos; 2. Long-term culture of mouse cerebral cortical neurons Under the bright field of the microscope, it can be seen that on the day of primary cortical neuron planting, the cell bodies are plump and translucent, with round, oval, and conical shapes, and there are halos around the cells. Some cells begin to form a small number of protrusions, about 1 to 3; after 1 day of culture, the cell bodies of cortical neurons are enlarged, the protrusions increase (3 to 5), the protrusions become longer, and the cell protrusions are interconnected. After 4 days of culture, the cell bodies of hippocampal neurons further enlarge, the protrusions increase and become longer, and the neurons are connected to form a network; after 6 days of culture, the cell bodies of neuronal cells become larger and plump, the cell cytoplasm is rich, the protrusions are more abundant, and the network formed by the protrusion connection is more dense and complex. After 18 days of culture, the protrusions of hippocampal neurons are relatively thick, the neurons are highly mature, and the network formed by the connection is dense and complex; after 30 days of culture, some neuronal cells begin to degenerate, and degenerated cell fragments can be seen, and metabolites are suspended in the culture medium. After 42 days, the number of degenerating neurons increased, and a small number of neuronal cell bodies were found to shrink, and their processes dissolved. The number of degenerated cell fragments and metabolites suspended in the culture medium increased, but most neuronal cell bodies still had strong refractive index, and the neural network formed by the axon-dendrite connection was more complex and dense. Figure 7 and Fig.10 As shown, Fig. 9 As a control schematic diagram; Fig.11 Comparison of neuronal firing activity in conventional culture medium and improved culture medium.

[0048] 3. Identification of cortical neurons by immunofluorescence Neurons were taken after 83 days of culture ( Figure 8The figure shows the identification of primary cortical neurons at 83 days old. The cells were washed twice with preheated PBS for 5 min, fixed with 4% paraformaldehyde for 15 min, and then washed three times with PBS for 5 min each time. The cells were treated with 0.2% TritonX-100 at room temperature for 15 min, and washed three times with PBS for 5 min each time. The cells were blocked with 5% BSA bovine serum for 30 min, and then aspirated and discarded, and β-3-Tubulin antibody (1:400) and synapsin-1 antibody (1:200) were added dropwise, incubated at 4°C overnight, and then washed three times with PBS for 5 min each time. Alexa Fluor 488-labeled donkey anti-mouse IgG (1:500) and Alexa Fluor 594-labeled donkey anti-rabbit IgG (1:500) were added to the cells, incubated at room temperature and protected from light for 1 hour, and washed three times with PBS for 5 min each time. Then, an appropriate amount of 4,6-diamino-2-phenylindole dihydrochloride (DAPI) was added to stain the cell nucleus, and the cell nucleus was observed and photographed using a fluorescence microscope.

[0049] It should be noted that the flowcharts and block diagrams in the accompanying drawings illustrate the possible architecture, functions and operations of the systems, methods and computer program products according to various embodiments of the present disclosure. In this regard, each box in the flowchart or block diagram can represent a module, a program segment, or a part of a code, and the module, program segment, or a part of the code contains one or more executable instructions for implementing the specified logical function. It should also be noted that in some alternative implementations, the functions marked in the box can also occur in a different order from the order marked in the accompanying drawings. For example, two boxes represented in succession can actually be executed substantially in parallel, and they can sometimes be executed in the opposite order, depending on the functions involved. It should also be noted that each box in the block diagram and / or flowchart, and the combination of boxes in the block diagram and / or flowchart can be implemented with a dedicated hardware-based system that performs a specified function or operation, or can be implemented with a combination of dedicated hardware and computer instructions.

[0050] In general, various example embodiments of the present disclosure may be implemented in hardware or dedicated circuits, software, firmware, logic, or any combination thereof. Certain aspects may be implemented in hardware, while other aspects may be implemented in firmware or software that may be executed by a controller, microprocessor, or other computing device. When various aspects of the disclosed embodiments are illustrated or described as block diagrams, flow charts, or using some other graphical representation, it will be understood that the blocks, devices, systems, techniques, or methods described herein may be implemented in hardware, software, firmware, dedicated circuits or logic, general purpose hardware or controllers or other computing devices, or some combination thereof as non-limiting examples.

[0051] Those skilled in the art can clearly understand that, for the convenience and brevity of description, the specific working processes of the systems, devices and units described above can refer to the corresponding processes in the aforementioned method embodiments and will not be repeated here.

[0052] In the several embodiments provided in the present application, it should be understood that the disclosed systems, devices and methods can be implemented in other ways. For example, the device embodiments described above are only schematic. For example, the division of the units is only a logical function division. There may be other division methods in actual implementation, such as multiple units or components can be combined or integrated into another system, or some features can be ignored or not executed. Another point is that the mutual coupling or direct coupling or communication connection shown or discussed can be an indirect coupling or communication connection through some interfaces, devices or units, which can be electrical, mechanical or other forms.

[0053] The units described as separate components may or may not be physically separated, and the components shown as units may or may not be physical units, that is, they may be located in one place or distributed on multiple network units. Some or all of the units may be selected according to actual needs to achieve the purpose of the solution of this embodiment.

[0054] In addition, each functional unit in each embodiment of the present invention may be integrated into one processing unit, or each unit may exist physically separately, or two or more units may be integrated into one unit. The above-mentioned integrated unit may be implemented in the form of hardware or in the form of software functional units.

[0055] The exemplary embodiments of the present disclosure described in detail above are merely illustrative and not restrictive. It should be understood by those skilled in the art that various modifications and combinations may be made to these embodiments or their features without departing from the principles and spirit of the present disclosure, and such modifications should fall within the scope of the present disclosure.

Claims

1. A culture medium for long-term culture of primary neurons, characterized in that: The culture medium comprises a basic culture medium and a neuron culture supplement factor; the neuron culture supplement factor comprises: 35-45 mM Na+, 20-30 mM Glucose, 0.3-0.5 mM L-serine, and 9-11 mM KCl.

2. The medium for long-term culture of primary neurons according to claim 1, characterized in that The Na+ content is 40 mM; Optionally, the content of Glucose is 25 mM; Optionally, the content of L-serine is 0.4 mM; Optionally, the content of KCl is 10 mM; Optionally, the basal culture medium is selected from any one of the following: commercial neuronal culture medium Neurobasal Plus / B27Plus, commercial culture medium BrainPhys TM .

3. The medium for long-term culture of primary neurons according to claim 1, characterized in that The neuron culture supplement factor also includes astrocyte inhibitors 0.5 μM cytosine arabinoside Ara-C and 0.5 μM fluorouridine FUDR; Optionally, the neuron culture supplement factor also includes astrocyte supernatant culture fluid, which is treated with high temperature inactivated complement and then mixed with neuron culture medium at a ratio of 1:10; Optionally, the oxygen content in each liter of the culture medium composition ranges from 8 to 12%, preferably 10%; Optionally, the osmotic pressure of the culture medium is in the range of 265-275 mOsm / L; preferably 270 mOsm / L; Optionally, a semipermeable membrane without pores but permeable to gas is arranged on the culture medium.

4. Use of the medium for long-term culture of primary neurons according to any one of claims 1 to 3 in preparing primary neurons, characterized in that: The application includes preparing neurons after culturing neurons in the medium for long-term culture of primary neurons.

5. A neuron, characterized in that: Prepared by the use as claimed in claim 4.

6. A method for long-term culture of primary neurons, characterized in that: The method comprises: S101, extracting and separating neurons and astrocytes from experimental animals and culturing them separately, and obtaining neuronal cell suspensions and astrocyte cell suspensions after treatment, and culturing them separately; S102, recording the inoculation time as day 0, and culturing primary neurons using the culture medium according to any one of claims 1 to 3; S103, when the inoculation time is day N, the supernatant culture fluid produced during the astrocyte culture process is taken, treated with high temperature to inactivate complement, and then mixed with neuron culture medium at a ratio of 1:10 to form a modified culture medium; S104: Thereafter, half the volume of neurons was replaced with the modified culture medium every M days and culture was continued.

7. The long-term culture method of primary neurons according to claim 6, characterized in that: The method further comprises: installing a semipermeable membrane without pores but permeable to gas on the culture medium; Optionally, the semipermeable membrane is made of FEP material; Optionally, the semipermeable membrane is cut according to the size of the culture dish outside the culture medium; Optionally, the method further comprises: regularly monitoring the osmotic pressure in the culture medium, and giving a risk warning when the osmotic pressure is less than 270 or greater than 330 mOsm / L.

8. A computer device, characterized in that: The device comprises: a memory and a processor; the memory is used to store a computer program; the processor executes the computer program to implement the steps of the method according to any one of claims 6 or 7.

9. A computer-readable storage medium, characterized in that: A computer program is stored thereon, and when the computer program is executed by a processor, the steps of the method according to any one of claims 6 or 7 are implemented.

10. A computer program product, comprising a computer program, characterized in that When the computer program is executed by a processor, the steps of the method according to any one of claims 6 or 7 are implemented.

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