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

Abstract

The application provides a culture medium for long-term culture of primary neurons, application and a long-term culture method of primary neurons, and relates to the technical field of cell biology. The application improves the culture solution components and the operation of the culture method, guarantees that the culture medium provides sufficient nutrients, explores long-time culture technology of in-vitro neurons, and realizes in-vitro survival of neurons for the longest time of 83 days without glial cell co-culture, so as to solve related life science problems.

Description

Technical Field

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

[0002] Primary neuronal in vitro culture refers to the culture of neurons isolated from the brain or spinal cord tissue of embryos or newborn animals in vitro, simulating the in vivo environment. This culture method preserves the basic biological characteristics of neurons, providing an important tool for studying neuronal function, development, and disease mechanisms. Primary neurons are highly similar to in vivo neurons in morphology, synaptic connections, and physiological functions, making them suitable for research on neurodevelopment, disease modeling, and neurofunctional studies. For example, in neurodevelopment research, in vitro culture allows for the study of synapse formation and maturation under in vitro conditions, revealing the molecular mechanisms of synaptic plasticity; it also allows for the observation of neurite extension and branching, studying their regulatory mechanisms, and providing a theoretical basis for the treatment of nervous system diseases. In disease modeling, primary neuronal culture can be used to construct in vitro models of neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease, studying the molecular mechanisms of disease development; and it can also be used to evaluate the protective or toxic effects of drugs on neurons, providing experimental evidence for drug development. For example, in the field of neurofunctional research, electrophysiological recording techniques can be used to study the excitatory and inhibitory synaptic transmission of neurons under in vitro culture conditions, revealing the functional characteristics of neural circuits; it can also be used to study the repair mechanisms of neurons after injury, providing new ideas for the treatment of nerve injuries.

[0003] Currently, primary neurons can typically survive for 2-3 weeks in stereotactic culture (as reported in studies of significantly improving the survival rate of neurons derived from primary cells and stem cells). However, even with optimized culture conditions and current experimental methods, culturing primary neurons in vitro for more than 2 months remains challenging. Nevertheless, long-term viable and stably cultured biological neural networks are a crucial foundation for driving breakthroughs in neuroscience research, brain disease treatment, brain-inspired intelligence development, and biomedical engineering.

[0004] In vitro neuronal culture is an important method for studying neuronal function, and this technique was reported as early as the beginning of the last century. However, after decades of development, the current technology for in vitro neuronal culture still has significant limitations. Firstly, the types of neurons that can be cultured in vitro are limited. Apart from glutamatergic neurons, which are easily cultured in large quantities, other excitatory neurons are not easily cultured on a large scale. Secondly, the survival time of primary cultured neurons is limited, generally not exceeding one month. Thirdly, in conventional primary neuronal culture, to prevent the continuous proliferation of astrocytes from affecting neuronal purity and survival, cytarabine (AraC) is usually added to the culture medium to reduce glial cell contamination by inhibiting DNA synthesis in dividing cells. However, in fact, high concentrations of AraC can induce axonal degeneration and programmed cell death in mature neurons, adversely affecting neuronal growth. Fourthly, in the brain, astrocytes play a crucial supporting role in neuronal survival and information processing by releasing nutrients. However, this nutritional support is lacking in in vitro culture. Furthermore, if direct co-culture is performed, the continuous proliferation of glial cells in the later stages of culture affects neuronal activity and axonal-dendritic extension. Rapid proliferation of glial cells consumes key metabolites such as glucose and glutamine in the culture medium, leading to decreased neuronal activity due to insufficient energy and nutrient supply. Simultaneously, the dense monolayer formed by proliferating glial cells at the culture bottom may hinder the normal extension of neuronal axons and dendrites. Synapse formation requires space, and excessive glial cell proliferation compresses this space, thus inhibiting synaptic network construction. Excessive glial cell proliferation interferes with neuronal activity, and cell purity and coexistence ratios are difficult to control. Because the human brain is protected by the skull and receives nutrients and oxygen through blood and lymphatic circulation while removing waste products, in vitro cultured neurons lack these mechanisms. Therefore, achieving stable culture for more than two months, or even longer, is one of the key issues that urgently needs to be addressed. Summary of the Invention

[0005] This invention aims to at least solve one of the technical problems existing in the prior art. To this end, this invention provides a culture medium, its application, and a method for long-term culture of primary neurons. By improving the composition of the culture medium and refining the operation of the culture method, this invention ensures that the culture medium provides sufficient nutrients, explores a long-term culture technique for isolated neurons, and achieves the longest continuous survival of neurons in vitro for 83 days without glial cell co-culture, thereby solving related life science problems.

[0006] The first aspect of this application discloses a culture medium for long-term culture of primary neurons, the culture medium comprising a basal culture medium and neuronal culture supplementary factors; the neuronal culture supplementary factors comprising: 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;

[0008] Optionally, the content of glucose is 25 mM;

[0009] Optionally, the L-serine content is 0.4 mM;

[0010] Optionally, the KCl content is 10 mM;

[0011] Optionally, the basal culture medium is selected from any of the following: commercial neurobasal culture medium NeurobasalPlus / B27 Plus, commercial brainPhys... TM The preferred medium is the commercially available neurobasal Plus / B27 Plus neuronal culture medium.

[0012] In some embodiments, the neuron culture supplement factor further includes 0.5 μM cytarabine Ara-C and 0.5 μM fluorouracil FUDR, both inhibitors of astrocytes.

[0013] Optionally, the neuron culture supplement factor also includes astrocyte supernatant culture medium, which, after being treated with high-temperature complement inactivation, is mixed with neuron culture medium at a ratio of 1:10.

[0014] Optionally, the oxygen content in each liter of culture medium is in the range of 8-12%, preferably 10%;

[0015] Optionally, the osmotic pressure of the culture medium is in the range of 265-275 mOsm / L; preferably 270 mOsm / L.

[0016] Optionally, a non-porous but gas-permeable semi-permeable membrane is disposed on the culture medium.

[0017] The second aspect of this application discloses the use of the culture medium for long-term culture of primary neurons according to the first aspect of this application in the preparation of primary neurons, the application including preparing neurons by culturing neurons in the culture medium for long-term culture of primary neurons.

[0018] The third aspect of this application discloses a neuron prepared by the application described in the second aspect of this application.

[0019] The fourth aspect of this application discloses a method for long-term culture of primary neurons, the method comprising:

[0020] S101, neurons and astrocytes were extracted and isolated from experimental animals and cultured separately. After processing, neuronal cell suspensions and astrocyte cell suspensions were obtained and cultured separately. The neurons and astrocytes extracted from the animals were separated.

[0021] S102, the inoculation time is recorded as day 0, and the primary neurons are cultured using the culture medium described in the first aspect of this application;

[0022] S103, when the inoculation time is day N, the supernatant culture medium produced during the culture of astrocytes is taken, and after high temperature inactivation of complement, it is mixed with neuronal culture medium at a ratio of 1:10 to form a modified culture medium;

[0023] S104, thereafter the neurons were replaced with half their volume every M days using a modified culture medium and cultured continuously.

[0024] In some embodiments, the method further includes: mounting a non-porous but gas-permeable semi-permeable membrane onto the culture medium;

[0025] Optionally, the semi-permeable membrane is made of FEP material;

[0026] Optionally, the semi-permeable membrane is cut to size according to the size of the culture dish outside the culture medium;

[0027] Optionally, the method further includes: periodically 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.

[0028] This application has the following beneficial effects:

[0029] 1. This 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 goal of high purity, high survival rate, and long culture time can be achieved.

[0030] Specifically, 1) In the brain, the most numerous cells are actually astrocytes. Astrocytes play a crucial role in supporting neuronal survival and information processing by releasing nutrients. However, this nutritional support is lacking in in vitro culture. To supplement the nutrients provided by astrocytes, we culture astrocytes separately, take their supernatant culture medium, and after attenuation treatment, mix it with neuronal culture medium in a certain proportion for long-term culture of primary neurons, which can significantly promote neuronal survival.

[0031] 2) The problem of decreased culture medium stability. Sudden cell death can occur during in vitro culture. This is mainly due to osmotic pressure changes caused by repeated fluid replenishment and replacement. The osmotic pressure of normal culture medium is 270-330 mOsm / L. Long-term culture leads to increased osmotic pressure due to water evaporation and salt concentration, resulting in poor neuronal survival and even sudden death. Our measurement data shows that under normal culture conditions, the osmotic pressure of the culture medium reaches the theoretical tolerance limit of nerve cells after 20 days of culture, and reaches 365 mOsm / L after 35 days of culture. These data indicate that we need to monitor solution evaporation and osmotic pressure changes. To minimize evaporation, a customized FEP semi-permeable membrane was developed. It allows gases such as oxygen to pass through, but has no pores, thereby reducing water evaporation and blocking microorganisms.

[0032] 3) Long-term culture of neural networks resulted in a decrease in firing frequency and a decline in the proportion of firing neurons. Biologically, this is speculated to be due to changes in neural network morphology and a tendency for intrinsic gene expression to minimize energy consumption. Currently, the team believes that the information processing capacity of such networks is significantly reduced. This research found that adjusting the composition of the culture medium can improve neural network activity: the original culture medium contained four components: "activating neurons," "assisting in activating neurons," "pH adjustment," and "energy supply." We adjusted four key substances and controlled the oxygen content, osmotic pressure, and evaporation rate in the culture medium to ensure the optimal culture environment for primary neurons, achieving a significant impact on neural network activity. This is the main component of our newly developed culture medium, enabling the neurons to survive, thrive for longer periods, and fire more neurons.

[0033] 2. This application achieved unexpected results by modifying the composition of the culture medium and the process of long-term culture of primary neurons. Specifically, a batch of neurons cultured for a long period of time were photographed and displayed, with photos taken every 6 days, showing photos taken after 6 to 62 days of culture. It can be seen that the axons and dendrites of neurons were relatively thinner in the first two weeks of culture, and became thicker and the cell bodies more enlarged after long-term culture.

[0034] In summary, by controlling nutrient conditions and culture methods, it is now possible to achieve two months of in vitro culture of mouse neurons, and the improved culture medium enhances neuronal purity and activity. Further extending the in vitro neuronal culture time, a maximum of 83 days of continuous in vitro neuronal survival has been achieved without glial cell co-culture. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying 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 effort.

[0036] Figure 1 This is a schematic diagram of the method flow provided in the fourth aspect of the present invention;

[0037] Figure 2 This is a schematic diagram of a long-term culture system for primary neurons provided in an embodiment of the present invention;

[0038] Figure 3 This is a schematic diagram of a computer device provided in an embodiment of the present invention;

[0039] Figure 4 This is a schematic diagram of the architecture of an exemplary computing device provided in an embodiment of the present invention;

[0040] Figure 5 This is a schematic diagram of the storage medium provided in an embodiment of the present invention;

[0041] Figure 6 This is a schematic diagram illustrating the preparation process of primary mouse neurons provided in this embodiment of the invention; wherein, Figure 6 A is a mouse lying on its back. Figure 6 B involves cutting open the skin of the mouse's abdomen to expose the uterus. Figure 6 C represents the transfer of the mouse embryo's head into a culture dish. Figure 6 D refers to anatomical dissection under a stereoscope. Figure 6 E is the collected centrifuge tube filtrate;

[0042] Figure 7 This is a schematic diagram illustrating the state of neural network activity several days after long-term culture of mouse primary cortical neurons, as provided in this embodiment of the invention; wherein, Figure 7 A is 6 days after cultivation. Figure 7 B represents 12 days after culture. Figure 7 C represents 18 days after culture. Figure 7 D represents 24 days after culture. Figure 7 E represents 30 days after culture. Figure 7 F represents 36 days after culture. Figure 7 G is 42 days after culture. Figure 7 H represents 48 days after culture. Figure 7 I is 54 days after culture. Figure 7 J is 62 days after culture;

[0043] Figure 8 This is an identification diagram of the primary cortical neurons of DIV83 provided in an embodiment of the present invention;

[0044] Figure 9 This is a schematic diagram of a cell culture medium under bright field microscopy provided in an embodiment of the present invention, using the original culture medium, without the addition of astrocyte inhibitors, without the addition of additional nutrients, and without astrocyte supernatant culture medium.

[0045] Figure 10 This is a schematic diagram of the neural network activity after several days of long-term culture of mouse primary cortical neurons, provided by an embodiment of the present invention. The left side shows 470 active neurons detected after 23 days of culture, and the right side shows 240 active neurons detected after 45 days of culture.

[0046] Figure 11 The results show that the firing activity of neurons in the modified culture medium provided in this embodiment of the invention is twice that of neurons in the conventional culture medium. Detailed Implementation

[0047] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0048] In some of the processes described in the specification, claims, and accompanying drawings of this invention, multiple operations appearing in a specific order are included. However, it should be clearly understood that these operations may not be executed in the order they appear herein, or may be executed in parallel. The operation numbers, such as 101, 102, etc., are merely used to distinguish different operations and do not represent any execution order. Furthermore, these processes may include more or fewer operations, and these operations may be executed sequentially or in parallel. It should be noted that the descriptions such as "first," "second," etc., in this document are used to distinguish different messages, devices, modules, etc., and do not represent a sequential order, nor do they limit "first" and "second" to different types.

[0049] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0050] Table 1 shows the main components of the culture medium for long-term culture of primary neurons disclosed in the first aspect of this application and a conventional culture medium. Using the culture medium of this application, the goals of viable, long-term, and high-purity cultures can be achieved. This embodiment discloses a culture medium for long-term culture of primary neurons, comprising a basal culture medium and neuronal culture supplementary factors; the neuronal culture supplementary factors include: 35-45 mM Na+, 20-30 mM glucose, 0.3-0.5 mM L-serine, and 9-11 mM KCl. This culture medium composition is a modified version of the commercially available neuronal culture medium Neurobasal Plus / B27Plus with additional additions. In this embodiment, the corresponding concentration unit "nM" means nmol / L, which indicates that each liter of culture medium contains ×× nmol.

[0051] In some embodiments, the Na+ content is 40 mM; optionally, the glucose content is 25 mM; optionally, the L-serine content is 0.4 mM; optionally, the KCl content is 10 mM.

[0052] In some embodiments, the basal culture medium is selected from any of the following: commercially available neurobasal culture medium Neurobasal Plus / B27 Plus, commercially available BrainPhysic culture medium TM The preferred medium is the commercially available neurobasal Plus / B27 Plus neuronal culture medium.

[0053] In some embodiments, the neuron culture supplement factor further includes 0.5 μM cytarabine Ara-C and 0.5 μM fluorouracil FUDR, both inhibitors of astrocytes.

[0054] Optionally, the neuron culture supplement factor also includes astrocyte supernatant culture medium, which, after being treated with high-temperature complement inactivation, is mixed with neuron culture medium at a ratio of 1:10.

[0055] Optionally, the oxygen content in each liter of culture medium is in the range of 8-12%, preferably 10%;

[0056] Optionally, the osmotic pressure of the culture medium is in the range of 265-275 mOsm / L; preferably 270 mOsm / L.

[0057] Optionally, a non-porous but gas-permeable semi-permeable membrane is disposed on the culture medium.

[0058] Table 1

[0059]

[0060] The second aspect of this application discloses the use of the culture medium for long-term culture of primary neurons according to the first aspect of this application in the preparation of primary neurons, the application including preparing neurons by culturing neurons in the culture medium for long-term culture of primary neurons.

[0061] The third aspect of this application discloses a neuron prepared by the application described in the second aspect of this application.

[0062] Figure 1 This is a schematic flowchart of a long-term culture method for primary neurons provided in the fourth aspect of the present invention. Specifically, the method includes the following steps:

[0063] S101, neurons and astrocytes were extracted and isolated from experimental animals and cultured separately. After processing, neuronal cell suspensions and astrocyte cell suspensions were obtained and cultured separately. The neurons and astrocytes extracted from the animals were separated.

[0064] S102, the inoculation time is recorded as day 0, and the primary neurons are cultured using the culture medium described in the first aspect of this application; here, the method of taking the supernatant and adding it to the culture medium of the primary neurons is adopted, and after the complement is inactivated by high temperature, it is added at a ratio of 1:10.

[0065] S103, when the inoculation time is day N, the supernatant culture medium produced during the culture of astrocytes is taken, and after high temperature inactivation of complement, it is mixed with neuronal culture medium at a ratio of 1:10 to form a modified culture medium;

[0066] S104, thereafter the neurons were replaced with half their volume every M days using a modified culture medium and cultured continuously.

[0067] In some embodiments, N and M are both rational numbers.

[0068] In some embodiments, the method further includes: mounting a non-porous but gas-permeable semi-permeable membrane on the culture medium; in some more specific embodiments, the semi-permeable membrane is made of FEP material;

[0069] In some embodiments, the semi-permeable membrane is cut to size according to the culture dish outside the culture medium; the semi-permeable membrane is designed to minimize evaporation, and a customized FEP semi-permeable membrane is used. It allows gases such as oxygen to pass through, but has no pores, thereby reducing moisture evaporation and blocking microorganisms.

[0070] In some embodiments, the method further includes: periodically monitoring the osmotic pressure in the culture medium and issuing a risk warning when the osmotic pressure is less than 270 or greater than 330 mOsm / L. Periodic sampling and testing are performed using an existing osmometer.

[0071] The innovation motivation for the fourth aspect of this application lies in: 1. Simulating the maturation process of the nervous system and studying its long-term functional characteristics. Neurons require weeks or even months to complete their 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 undergo 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. Supporting the formation and functional research of complex neural networks. The network activities of neurons (such as synchronous firing and signal integration) require sufficient culture time to form stable synaptic connections. The network structure of neurons cultured for a short period 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, facilitating the observation of long-term synaptic remodeling or network degradation under pathological conditions. In addition, when studying the chronic pathological mechanisms of neurodegenerative diseases (such as Alzheimer's disease), long-term culture is needed to simulate disease progression. 3. Some experiments require neurons to survive in vitro for extended periods to achieve specific goals. Examples include: • Drug screening and toxicity testing: Assessing the chronic toxicity or protective effects of drugs on neurons requires stable and long-term culture systems. • Gene editing and phenotypic observation: Viral vector-mediated gene manipulation (such as CRISPR) may take weeks to observe phenotypic changes; long-term culture provides a time window for such experiments. • Disease model construction: Models of hypoxic-ischemic brain injury or chronic neuroinflammation require long-term culture to simulate adaptive changes in neurons within the pathological microenvironment.

[0072] Figure 3 This is a schematic diagram of a computer device provided in 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 code that, when run by the one or more processors, can perform the methods described above.

[0073] The processor in this embodiment can be an integrated circuit chip with signal processing capabilities. The processor can be a general-purpose processor, a digital signal processor (DSP), an application-specific integrated circuit (ASIC), an off-the-shelf programmable gate array (FPGA), or other programmable logic devices, discrete gate or transistor logic devices, or discrete hardware components. It can implement or execute the methods, operations, and logic block diagrams disclosed in this embodiment. The general-purpose processor can be a microprocessor or any conventional processor, and can be based on an x86 or ARM architecture.

[0074] In general, the various exemplary embodiments of this disclosure can be implemented in hardware or dedicated circuitry, software, firmware, logic, or any combination thereof. Some aspects can be implemented in hardware, while others can be implemented in firmware or software that can be executed by a controller, microprocessor, or other computing device. When aspects of embodiments of this disclosure are illustrated or described as block diagrams, flowcharts, or using some other graphical representation, it will be understood that the blocks, apparatuses, systems, techniques, or methods described herein can be implemented as non-limiting examples in hardware, software, firmware, dedicated circuitry or logic, general-purpose hardware or controllers or other computing devices, or some combination thereof.

[0075] For example, the method or apparatus according to embodiments of this disclosure can also be used by means of Figure 4 The architecture of the computing device 3000 shown is used for implementation. For example... 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 devices in the computing device 3000, such as the ROM 3030 or the hard disk 3070, may store various data or files used for processing and / or communication of the methods provided in this disclosure, as well as 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 merely exemplary and can be omitted as needed when implementing different devices. Figure 4 One or more components in the computing device shown.

[0076] This invention also includes a computer-readable storage medium, such as... Figure 5The diagram illustrates a storage medium 4000 provided in an embodiment of the present invention. The computer storage medium 4020 stores computer-readable instructions 4010. When the computer-readable instructions 4010 are executed by a processor, the method described above according to embodiments of the present disclosure can be performed. The computer-readable storage medium in the embodiments of the present disclosure may be volatile memory or non-volatile memory, or may include both volatile and non-volatile memory. Non-volatile memory may be read-only memory (ROM), programmable read-only memory (PROM), erasable programmable read-only memory (EPROM), electrically erasable programmable read-only memory (EEPROM), or flash memory. Volatile memory may be random access memory (RAM), which is used as an external cache. By way of example, but 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 used in the methods described herein is intended to include, but is not limited to, these and any other suitable types of memory.

[0077] This disclosure also provides a computer program product or system, including a computer program that, when executed by a processor, implements the steps of the above-described method.

[0078] In some embodiments, this embodiment also discloses a long-term culture system for primary neurons, such as... Figure 2 As shown, the system includes:

[0079] The cell suspension preparation module 201 is used or configured to extract and isolate neurons and astrocytes from experimental animals and culture them separately, and after processing, obtain neuronal cell suspensions and astrocyte cell suspensions, which are then cultured separately.

[0080] Neuron culture medium module 202 is used or configured to record the inoculation time as day 0, and primary neurons are cultured using the culture medium described in the first aspect of this application;

[0081] The culture medium preparation module 203 is used or configured to take the supernatant culture medium produced during the culture of astrocytes when the inoculation time is day N, and after high-temperature inactivation of complement, mix it with neuronal culture medium at a ratio of 1:10 to form a modified culture medium.

[0082] Neuron continuous culture module 204 is used or configured to replace half the volume of neurons with modified culture medium every M days thereafter, and to continue culturing. Specific implementation examples:

[0084] 1. In vitro culture of mouse cerebral cortex neurons

[0085] Experimental materials: ICR pregnant mice on day 16.5 of gestation (Vitolliwa), medical dissecting instruments (Reward), dissecting 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 tubes (Corning), 50 1 ml centrifuge tubes (Corning), 12-well cell culture plates, 24-well cell culture plates, 100× penicillin-streptomycin solution (Zhongke Maichen, catalog number CC004), 0.25% trypsin solution (Zhongke Maichen, catalog number CC017), L-glutamine (Gibco, catalog number A2916801).

[0086] Experimental steps:

[0087] Preparation of modified culture medium: Based on the commercial neuronal culture medium Neurobasal Plus / B27 Plus with 0.5× penicillin-streptomycin solution and 2 mM L-glutamine, the additional modified components include 40 mM Na+, 25 mM glucose, 0.4 mM L-serine, and 10 mM KCl.

[0088] Preparation of modified culture medium containing astrocyte inhibitors: Add 0.5 μM of astrocyte inhibitors AraC and 0.5 μM of FUDR to the modified culture medium.

[0089] Preparation of mixed culture medium: Astrocyte supernatant culture medium that has undergone high-temperature complement inactivation treatment is added to the modified culture medium and mixed with the modified neuronal culture medium at a ratio of 1:10.

[0090] 1) The day before cell culture, dissolve PDL in PBS buffer 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 2 μg / ml working solution. Add 1 ml of the prepared PDL solution to each well of a 12-well plate and 500 μl to each well of a 24-well plate. Incubate overnight at 37°C. The next day, remove the plate, carefully wash twice with sterile double-distilled water, and air dry before use.

[0091] 2) Use sterilized surgical instruments to cut open the abdominal skin of the pregnant mouse, expose the uterus, remove the intact uterus of the pregnant mouse with the replaced instruments, and transfer it to a 10cm culture dish containing pre-cooled PBS;

[0092] 3) Carefully dissect the mouse embryo using fresh scissors and forceps, and cut off the head of the mouse embryo with the scissors. Transfer the mouse embryo head to a 10cm culture dish containing pre-cooled DMEM medium;

[0093] 4) Dissect the head of the mouse embryo under a stereomicroscope, carefully separate the cerebral cortex, and discard the hippocampus, olfactory bulb, and internal nuclei. Collect sufficient cerebral cortex into a 15 ml centrifuge tube containing pre-cooled DMEM medium with penicillin-streptomycin solution;

[0094] 5) Carefully discard the culture medium in the 15 ml centrifuge tube, gently rinse the cortical tissue twice with pre-cooled phenol red-free DMEM culture medium, add trypsin solution to digest the cortical tissue, place the centrifuge tube in a 37°C water bath for 20 min, and gently shake the centrifuge tube every 5 min to mix the liquid.

[0095] 6) After water bath for 20 minutes, 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 stand for 5 minutes.

[0096] 7) Using a 1ml pipette, aspirate the serum from the centrifuge tube. Carefully pipette the tissue 20 times, then let it stand. Aspirate the supernatant, filter, and add 2ml of pre-cooled culture medium containing fetal bovine serum. Repeat the above steps 2-3 times. Filter through a 40μm filter and collect the filtrate into a new 15ml centrifuge tube. At this point, the neuronal cells will be in the collected filtrate.

[0097] 8) Centrifuge at 80 g for 3 min, carefully discard the supernatant, and retain the cell pellet. Add 1 ml of modified culture medium, gently resuspend the pellet with a 1 ml pipette, mix well, and count using a cell counting chamber. Dilute the cells to 3-4 × 10^5 cells / ml using modified culture medium containing astrocyte inhibitors, and seed them into 24-well plates. Record the time when the isolated primary neurons are seeded into the culture plate as day 0; Figure 6 As shown;

[0098] 9) On the third day of neuron culture, astrocyte supernatant culture medium that has undergone high-temperature complement inactivation treatment was added to the culture medium at a 1:10 ratio to prepare a mixed culture medium. During subsequent culture, half the volume of the mixed culture medium was replaced every 3 days to maintain neuronal viability and basic function. Half-volume replacement means adding fresh culture medium to the culture dish used to culture neurons every 3 days while removing half of the old medium. This is done to maintain an appropriate nutrient concentration in the culture medium and to remove metabolic waste and harmful substances.

[0099] 10) Starting from day 6 of isolation and culture from mice, the morphology of neurons was observed every 6 days under a bright field microscope using a 10× objective lens, and the state of neurons was recorded by taking pictures.

[0100] 2. Long-term culture of neurons in the mouse cerebral cortex

[0101] Under a bright-field microscope, primary cortical neurons on the day of seeding appear as plump, translucent cells, shaped like circles, ovals, or cones, with halos around the cells. Some cells begin to form a small number of processes, about 1-3. After 1 day of culture, the cortical neuron cell bodies enlarge, the number of processes increases (3-5), the processes become longer, and the processes connect with each other. After 4 days of culture, the hippocampal neuron cell bodies further enlarge, the number and length of processes further increase, and the neurons connect to form a network. After 6 days of culture, the neuron cell bodies become larger and plumper, the cytoplasm is abundant, the processes are more numerous, and the network formed by the process connections becomes denser and more complex. After 18 days of culture, the hippocampal neuron processes are relatively large, the neurons are highly mature, and the network formed by the connections is dense and complex. After 30 days of culture, some neuron cells begin to degenerate, and degenerated cell fragments are visible, with metabolic products suspended in the culture medium. After 42 days, the number of degenerated neurons increased, and some neurons showed signs of cell body shrinkage and process dissolution. The amount of degenerated cell debris and metabolic products suspended in the culture medium increased, but most neuronal cell bodies still exhibited strong refractive properties, and the neural networks formed by axonal-dendritic connections became more complex and dense. Figure 7 and Figure 10 As shown, and Figure 9 As a comparative illustration; Figure 11 This study compares the neuronal firing activity in conventional and modified culture media.

[0102] 3. Identification of cortical neurons by immunofluorescence method

[0103] Neurons cultured for 83 days were harvested. Figure 8(Identification image of primary cortical neurons at 83 days old). Cells were washed twice with pre-warmed PBS for 5 min each time. They were then fixed with 4% paraformaldehyde for 15 min, followed by three 5-min washes with PBS. Cells were treated with 0.2% Triton X-100 at room temperature for 15 min, followed by three 5-min washes with PBS. After blocking with 5% BSA bovine serum for 30 min, the cells were discarded, and β-3-Tubulin antibody (1:400) and synapsin-1 antibody (1:200) were added. The cells were incubated overnight at 4°C, followed by three 5-min washes with PBS. 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, and the cells were incubated at room temperature in the dark for 1 h, followed by three 5-min washes with PBS. Then, an appropriate amount of 4,6-diamino-2-phenylamine dihydrochloride (DAPI) was added to stain the cell nucleus, and the cells were observed and photographed using a fluorescence microscope.

[0104] It should be noted that the flowcharts and block diagrams in the accompanying drawings illustrate the architecture, functionality, and operation of possible implementations of systems, methods, and computer program products according to various embodiments of this disclosure. In this regard, each block in a flowchart or block diagram may represent a module, segment, or portion of code containing one or more executable instructions for implementing a specified logical function. It should also be noted that in some alternative implementations, the functions indicated in the blocks may occur in a different order than those indicated in the drawings. For example, two consecutively indicated blocks may actually be executed substantially in parallel, and they may sometimes be executed in reverse order, depending on the functions involved. It should also be noted that each block in the block diagrams and / or flowcharts, and combinations of blocks in the block diagrams and / or flowcharts, can be implemented using a dedicated hardware-based system that performs the specified function or operation, or using a combination of dedicated hardware and computer instructions.

[0105] In general, the various exemplary embodiments of this disclosure can be implemented in hardware or dedicated circuitry, software, firmware, logic, or any combination thereof. Some aspects can be implemented in hardware, while others can be implemented in firmware or software that can be executed by a controller, microprocessor, or other computing device. When aspects of embodiments of this disclosure are illustrated or described as block diagrams, flowcharts, or using some other graphical representation, it will be understood that the blocks, apparatuses, systems, techniques, or methods described herein can be implemented as non-limiting examples in hardware, software, firmware, dedicated circuitry or logic, general-purpose hardware or controllers or other computing devices, or some combination thereof.

[0106] Those skilled in the art will clearly understand that, for the sake of convenience and brevity, the specific working processes of the systems, devices, and units described above can be referred to the corresponding processes in the foregoing method embodiments, and will not be repeated here.

[0107] In the several embodiments provided in this application, it should be understood that the disclosed systems, apparatuses, and methods can be implemented in other ways. For example, the apparatus embodiments described above are merely illustrative; for instance, the division of units is only a logical functional division, and in actual implementation, there may be other division methods. For example, multiple units or components may be combined or integrated into another system, or some features may be ignored or not executed. Furthermore, the coupling or direct coupling or communication connection shown or discussed may be an indirect coupling or communication connection between apparatuses or units through some interfaces, and may be electrical, mechanical, or other forms.

[0108] The units described as separate components may or may not be physically separate. The components shown as units may or may not be physical units; that is, they may be located in one place or distributed across multiple network units. Some or all of the units can be selected to achieve the purpose of this embodiment according to actual needs.

[0109] Furthermore, the functional units in the various embodiments of the present invention can be integrated into one processing unit, or each unit can exist physically separately, or two or more units can be integrated into one unit. The integrated unit can be implemented in hardware or as a software functional unit.

[0110] The exemplary embodiments of this disclosure described in detail above are merely illustrative and not restrictive. Those skilled in the art will understand that various modifications and combinations of these embodiments or their features can be made without departing from the principles and spirit of this disclosure, and such modifications should fall within the scope of this disclosure.

Claims

1. A culture medium for long-term culture of primary neurons, characterized in that, The culture medium includes a basal medium and neuronal culture supplement factors; the basal medium includes: Neurobasal Plus / B27 Plus, 40 mM Na+, 25 mM glucose, 0.4 mM L-serine, 10 mM KCl, 0.5 μM cytarabine Ara-C, and 0.5 μM fluorouracil FUDR; the neuronal culture supplement factors include: astrocyte supernatant culture medium; wherein, the astrocyte supernatant culture medium is treated with high-temperature complement inactivation and then mixed with the basal medium at a ratio of 1:10; in the culture medium for long-term culture of primary neurons, the oxygen content per liter of culture medium ranges from 8-12%; the osmotic pressure of the culture medium for long-term culture of primary neurons ranges from 265-275 mOsm / L.

2. The culture medium for long-term culture of primary neurons according to claim 1, characterized in that, In the culture medium used for long-term culture of primary neurons, the oxygen content per liter of culture medium ranges from 10%.

3. The culture medium for long-term culture of primary neurons according to claim 1, characterized in that, The osmotic pressure of the culture medium used for long-term culture of primary neurons is 270 mOsm / L.

4. The culture medium for long-term culture of primary neurons according to claim 1, characterized in that, A semi-permeable membrane that is non-porous but permeable to gas is set on the culture medium used for long-term culture of primary neurons.

5. The application of the culture medium for long-term culture of primary neurons according to any one of claims 1-4 in the preparation of primary neurons, characterized in that, The application includes preparing neurons by culturing neurons in the medium used for long-term culture of the primary neurons.

6. A method for long-term culture of primary neurons, characterized in that, The method includes culturing neurons in the culture medium for long-term culture of primary neurons as described in any one of claims 1-4.

7. The method for long-term culture of primary neurons according to claim 6, characterized in that, The method further includes: installing a non-porous but gas-permeable semi-permeable membrane on the culture medium used for long-term culture of primary neurons.

8. The method for long-term culture of primary neurons according to claim 7, characterized in that, The semi-permeable membrane is made of FEP material.

9. The method for long-term culture of primary neurons according to claim 7, characterized in that, The semi-permeable membrane is cut to size according to the size of the culture dish outside the culture medium.

10. The method for long-term culture of primary neurons according to claim 6, characterized in that, The method further includes: periodically monitoring the osmotic pressure in the culture medium, and issuing a risk warning when the osmotic pressure is less than 270 mOsm / L or greater than 330 mOsm / L.

11. A computer device, characterized in that, The device includes: 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 6-10.

12. A computer-readable storage medium, characterized in that, It stores a computer program thereon, which, when executed by a processor, implements the steps of the method as described in any one of claims 6-10.

13. A computer program product, comprising a computer program, characterized in that, When executed by a processor, the computer program implements the steps of the method described in any one of claims 6-10.

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