Separation and culture method of chick embryo forebrain neuron

Abstract

The invention discloses a method for separating and culturing chick embryo forebrain neurons. The method comprises the following steps: (1) carrying out coating treatment on a cell plate by using a poly-D-lysine solution; (2) obtaining chick embryo brain tissue fluid; and (3) culturing neurons. By improving and optimizing a neuron preparation method and a reagent, the chick embryo forebrain neuron with the high purity of 98% or above is obtained.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a method for isolating and culturing neurons in the forebrain of chicken embryos. Background Technology

[0002] The isolation, purification, and culture of primary brain neurons are among the core techniques in neuroscience research. Neurons isolated and cultured from living brain tissue can largely retain their in vivo physiological characteristics, thus more accurately reflecting their biological features. However, the highly differentiated nature of neurons often presents technical challenges when cultured in vitro, such as low survival rates and insufficient purity. In recent years, with the optimization of cell culture techniques, the isolation and culture of primary mammalian neurons has matured, significantly improving cell quality and enabling the efficient isolation and stable culture of neurons from various tissue sources.

[0003] As a classic model of vertebrate development, chicken embryos are widely used in various studies due to their ease of observation and manipulation of embryonic development. Compared to mammalian models, chicken embryonic development is controllable, readily available, inexpensive, and allows for sampling at different embryonic stages. In vitro culture of chicken embryonic forebrain neurons not only helps track neuronal differentiation, synapse formation, and functional maturation but also serves as an in vitro model for viral infection, gene editing, and drug screening. However, standardized and reproducible methods for culturing neurons in chicken embryos are still lacking. Due to the structural and cellular differences between avian and mammalian brains, directly applying optimized culture environments (such as culture medium composition and coating matrix) to mammalian neurons often yields poor results. Therefore, obtaining high-purity chicken embryonic brain neurons remains a pressing technical problem for those skilled in the art. Summary of the Invention

[0004] This invention aims to explore and optimize the isolation and primary culture methods for forebrain neurons in chicken embryos. By systematically comparing the effects of different ages, enzyme digestion conditions, and culture medium configurations on neuron isolation and growth, a suitable method for isolating and culturing forebrain neurons in chicken embryos will be established. This will provide a platform for basic neurobiological research in birds, the elucidation of pathogenic mechanisms of nervous system-related diseases, and the development of potential treatment strategies.

[0005] To solve the above-mentioned technical problems, the technical solution proposed by this invention is as follows: This invention relates to a method for isolating and culturing neurons in the forebrain of a chicken embryo, comprising the following steps: (1) Coating of cell plates with poly-D-lysine solution; (2) Select 7-8 day old chicken embryos, disinfect the eggshell, crack open from the air cell end and peel off the embryo, remove the embryo under sterile conditions and place it in D-Hank's solution pre-cooled at 0-4℃; use sterile ophthalmic forceps to peel off the meninges, remove the brain tissue, and transfer the forebrain to digestion solution pre-cooled at 0-4℃. Use a sterile dropper to blow up and down 15-20 times to disperse the brain tissue, and then place it in a 37℃ CO2 incubator for digestion for 10-20 min; the digestion solution contains trypsin and DNase I; (3) Remove the digested tissue fluid, add FBS for neutralization, and pipette 10-20 times. Filter the tissue fluid through a 40 μm cell filter to remove tissue blocks and large cell clumps. Centrifuge the filtrate, discard the supernatant, add cell culture medium I, count cells, plate them, and incubate at 37℃ in a CO2 incubator for 20-30 h. Discard the supernatant, add cell culture medium II, and incubate at 37℃ in a CO2 incubator for 20-30 h. Change the medium again after 48 h, discard the supernatant, add neuronal culture medium I, and incubate at 37℃ in a CO2 incubator. Then, on day 5, completely change the medium using neuronal culture medium II. Cell culture medium I contains 75-85 mL DMEM + 15-25 mL FBS + 0.5-1.5 mL penicillin / streptomycin per 100 mL; cell culture medium II contains 91-96 mL DMEM + 4-8 mL FBS + 0.5-1.5 mL penicillin / streptomycin per 100 mL. mL of penicillin and streptomycin; the neuronal culture medium I contains chicken cerebrospinal fluid and L-glutamine, and the neuronal culture medium II contains chicken cerebrospinal fluid, chicken serum and L-glutamine.

[0006] In a preferred embodiment of the present invention, the poly-D-lysine solution is formulated to contain 40-60 μg of poly-D-lysine per 1 mL of poly-D-lysine solution.

[0007] In a preferred embodiment of the invention, the digestive fluid contains trypsin and DNase I.

[0008] In a preferred embodiment of the present invention, the digestive solution is formulated as 0.25% trypsin + 0.02% EDTA + 0.01% DNase I + D-Hank's solution.

[0009] In a preferred embodiment of the present invention, the formulation of the neuronal culture medium I is as follows: per 100 mL of neuronal culture medium I, there are 95-97 mL Neurobasal Medium + 2 mL B-27 Serum-Free Supplement (50×) + 80-120 μL chicken cerebrospinal fluid + 0.5-1.5 mL penicillin-streptomycin mixture (100×) + 0.5-1.5 mL L-glutamine solution.

[0010] In a preferred embodiment of the present invention, the formulation of the neuronal culture medium II is as follows: per 100 mL of neuronal culture medium II, there are 85-92 mL of neurobasal medium + 2 mL of B-27 serum-free supplement (B-27 serum-free supplement, 50×) + 80-120 μL of chicken cerebrospinal fluid + 5-10 mL of chicken serum + 0.5-1.5 mL of penicillin-streptomycin mixture (100×) + 0.5-1.5 mL of L-glutamine solution.

[0011] In a preferred embodiment of the present invention, the purity of the neurons is 98% or higher.

[0012] Compared with the prior art, the present invention has the following significant advantages: the present invention obtains chicken embryo forebrain neurons with a purity of over 98% by improving and optimizing the neuron preparation method and reagents. Attached Figure Description

[0013] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0014] Figure 1 Photos of SPF chicken embryos and brain tissue.

[0015] Figure 2 The effect of cell plate treatment on neuronal adhesion.

[0016] Figure 3 Differences in the preparation of neurons from chicken embryos of different ages.

[0017] Figure 4 The effect of DNase enzyme on neuronal growth.

[0018] Figure 5 The effects of digestion with different proteases on neuronal isolation and growth.

[0019] Figure 6 The effects of L-glutamine on neuronal separation and growth.

[0020] Figure 7 The effects of different culture conditions on the later growth state of neurons.

[0021] Figure 8 Morphological changes of primary chicken embryo neurons at different time points.

[0022] Figure 9 Immunofluorescence image of MAP2 in neurons.

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0024] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0025] Before cell preparation, 50 μg / mL of poly-D-lysine solution was added to a 24-well cell plate (500 μL / well), and incubated at room temperature in the dark for 2 h. The cells were then washed three times with sterile deionized water, thoroughly dried, irradiated with UV light for 10 min, and stored at 4℃ in the dark for later use.

[0026] Comparative Example 1 Except for not using PDL wrapping, all other operations are the same as in Example 1.

[0027] Eight-day-old SPF chicken embryos were selected. After sterilizing the eggshells with 70% alcohol, the embryos were cracked open from the air cell end and removed. The embryos were then removed under aseptic conditions and placed in pre-cooled D-Hank's solution at 4°C. The meninges were dissected with sterile ophthalmic forceps, and the brain tissue was removed. The forebrain was transferred to different digestive solutions or tissue separation solutions pre-cooled at 4°C (8 ml digestive solution / 4 brains). The brain tissue was dispersed by blowing up and down 15-20 times with a sterile dropper and then placed in a 37°C CO2 incubator for digestion for 15 min. Figure 1 ).

[0028] Remove the digested tissue fluid, add 4 ml of FBS for neutralization, and pipette 10-20 times. Filter the tissue fluid through a 40 μm cell filter to remove tissue fragments and large cell clumps. Centrifuge the filtrate at 1000 rpm for 5 min. Discard the supernatant, add cell culture medium I, perform cell counting, and plate the cells (2000 cells / mm²). 2), and were placed in a 37℃ CO2 incubator for incubation.

[0029] Culture for 24 h; discard the supernatant, add cell culture medium II, and incubate in a 37℃ CO2 incubator for 48 h. After 48 h, change the medium again, discard the supernatant, add neuronal culture medium I, and incubate in a 37℃ CO2 incubator. Thereafter, change the medium every 2-3 days, discarding the supernatant and adding neuronal culture medium II.

[0030] Cell plates were coated with cells from Example 1 and Comparative Example 1 respectively. Chicken embryo forebrain neurons were isolated and cultured according to the method of Example 2, and the growth status of the neurons after culture was observed.

[0031] Result: As Figure 2 As shown, primary cells adhered well to the cell plate after PDL treatment; in the untreated cell plate, neurons could not adhere effectively and floated in the culture medium as dead cell clusters, indicating that PDL treatment is crucial for the adhesion of primary neurons.

[0032] Brain growth varies among embryos of different ages. To further improve the efficiency of neuron isolation and culture, this study selected SPF chicken embryos at 7, 8, 9, and 10 days of age for primary neuron preparation to obtain the optimal embryo age for brain tissue collection. Neuronal growth was observed 1-4 days after cell preparation.

[0033] Result: As Figure 3 As shown, the cells prepared from the brain tissue of 7-day and 8-day-old chicken embryos showed good growth. From 1 to 4 days post-inoculation, numerous individual spindle-shaped neurons were observed adhering to the wall and gradually forming synapses and neural networks. The cell adhesion of 9-10-day-old chicken embryos significantly weakened. At day 1 (day in vitro, DIV), a large number of dead cells and cell debris appeared in the field of view; at day 2, cells clumped together; and at day 3-4, numerous non-neuronal cells resembling glial cells appeared. Therefore, 7-8 days of age is the optimal time for collecting neurons from chicken embryos.

[0034] To address the issue of cell clustering, 0.01% DNase I was added to a 0.25% trypsin solution as a digestive solution for brain tissue during the preparation of primary cells. The cells were then digested at 37°C for 10-15 minutes, and the growth status of neurons was observed 1-4 days after cell preparation.

[0035] Result: As Figure 4As shown, neurons not digested with DNase I were more prone to clumping and growth after adhesion, and were also more likely to die in the later stages of growth. Neurons digested with DNase I exhibited uniform growth density, mostly showing a single, independent growth state, and their growth status was relatively good at 4 days. These results suggest that neuronal clumping may be related to the release of nucleic acids during cell preparation and digestion. DNase I can effectively degrade these nucleic acid components, preventing neuronal clumping after adhesion.

[0036] To further optimize the selection of proteases and digestion methods during the preparation process, this study employed mechanical digestion, trypsin digestion solution, acutase, and papain digestion solution for brain tissue digestion. Specifically, the brain tissue was transferred to different pre-cooled digestion solutions or tissue separation solutions at 4°C (8 ml digestion solution / 4 brains), and the tissue was dispersed by blowing up and down 15-20 times using a sterile dropper. The tissue was then placed in a 37°C CO2 incubator for 15 min for digestion. Cell morphology was observed 1-4 days after preparation, and the viability and purity of neurons prepared using different methods were compared.

[0037] Result: As Figure 5 As shown, mechanical digestion yielded the largest number of cells and the highest cell density in the field of view, but fewer adherent cells and a cluttered background. Later, numerous cell clusters ruptured and died, indicating significant cell damage. The trypsin digestion group had a relatively lower cell density, but the cells were in good condition; a large number of adherent cells were observed within 1 DIV, with a clean growth background, and neuronal axons and dendrites intertwined to form a stable neural network structure within 2-4 DIVs. The neuronal state in the Accutase digestion group was similar to that in the trypsin digestion group. The papain digestion group showed the least cell damage; a large number of spindle-shaped neurons were observed within 1 DIV, forming long synapses, and the neuronal density reached its maximum in the later stages. In summary, trypsin, accutase, and papain digestion all achieved good digestion effects, with papain digestion causing the least cell damage, but trypsin was the most cost-effective.

[0038] To understand the effect of additives in the culture medium on the growth status of neurons, this study used culture media with and without L-glutamine in the early stage of cell culture to culture isolated nerve cells, and observed and compared the growth status of the cells.

[0039] Result: As Figure 6 As shown, in the group without L-glutamine, some cells in the untreated group showed vacuolation and apoptosis on day 3 of culture; compared with the untreated group, the addition of L-glutamine could inhibit early neuronal apoptosis to some extent.

[0040] Although the neurons showed good growth in the early stages of isolation and culture, by day 5, the cell bodies began to swell, vacuoles appeared in the cytoplasm, and they gradually underwent apoptosis. To further maintain the growth of the neurons, this study explored the conditions for changing the culture medium on day 5 to obtain the optimal added components. The replacement conditions included: partial medium change, complete medium change, or adding chicken serum, B-27, and BDNF to the neuronal culture medium I after a complete medium change.

[0041] Result: As Figure 7 As shown, compared to partial medium replacement, cells with complete medium replacement exhibited a lower degree of vacuolation. Adding 50 μL of chicken serum (i.e., 10% chicken serum) to the culture medium effectively inhibited vacuolation formation, and the neurons maintained good condition. Adding 10 μL of B-27 did not effectively inhibit apoptosis; and adding 10 ng of BDNF also had limited effect, resulting in a large number of neuronal clusters and death on day 8 of growth. Therefore, the addition of chicken serum can effectively maintain the growth status of neurons in the chicken embryo forebrain.

[0042] Eight-day-old SPF chicken embryos were selected. After sterilizing the eggshells with 70% alcohol, the embryos were cracked open from the air cell end and removed under aseptic conditions. The embryos were placed in pre-cooled D-Hank's solution at 4°C. The meninges were dissected using sterile ophthalmic forceps, and the brain tissue was removed. The forebrain tissue was transferred to pre-cooled trypsin digestion solution at 4°C (8 mL digestion solution / 4 brains). The brain tissue was dispersed by pipetting up and down 15-20 times with a sterile pipette and then placed in a 37°C CO2 incubator for 15 min for digestion. The digested tissue fluid was collected, and 4 ml of FBS was added for neutralization. The fluid was then pipetted 10-20 times with a sterile pipette. The tissue fluid was filtered through a 40 μm cell filter to remove tissue fragments and large cell clumps, and centrifuged at 1000 rpm for 5 min. The supernatant was discarded, and cell culture medium I was added for cell counting and plating (2000 cells / mm²). 2 Cells were incubated in a 37°C CO2 incubator for 24 h. The supernatant was discarded, and cell culture medium II was added. The cells were then incubated in a 37°C CO2 incubator for another 24 h. After 48 h, the medium was changed again, the supernatant was discarded, and neuronal culture medium was added. The cells were then incubated in a 37°C CO2 incubator. Subsequently, on day 5, the cell culture medium was discarded, and neuronal culture medium I containing 10% chicken serum was added. The cells were then incubated in a 37°C CO2 incubator. Cell samples were collected 1-5 days after inoculation, and IFA assays were performed using MAP2 polyclonal antibody as the primary antibody to observe neuronal morphology.

[0043] Result: As Figure 8As shown, on day 1 after cell seeding, a large number of neurons adhered to the cell wall, some cells exhibiting a spindle shape and developing synapses. From day 2 to 4 (DIV), the neuronal synapses gradually elongated, with one synapse extending and forming an axon. From day 4 to 5, neurons interacted to form a neural network.

[0044] Chicken embryo neurons were isolated and cultured using a modified and optimized method. Eight-day-old SPF chicken embryos were selected. After sterilizing the eggshells with 70% alcohol, the embryos were cracked open from the air cell end and detached. The embryos were aseptically removed and placed in pre-cooled D-Hank's solution at 4°C. The meninges were dissected using sterile ophthalmic forceps, and the brain tissue was removed. The forebrain tissue was transferred to pre-cooled trypsin digestion solution at 4°C (8 mL digestion solution / 4 brains). The brain tissue was dispersed by blowing up and down 15-20 times with a sterile pipette and then placed in a 37°C CO2 incubator for 15 min for digestion. The digested tissue fluid was collected, and 4 ml of FBS was added for neutralization. The fluid was then blown up and down 10-20 times with a sterile pipette. The tissue fluid was filtered through a 40 μm cell filter to remove tissue blocks and large cell clumps, and centrifuged at 1000 rpm for 5 min. The supernatant was discarded, and cell culture medium I was added for cell counting and plating (2000 cells / mm²). 2 The cells were incubated in a 37°C CO2 incubator for 24 h. The supernatant was discarded, and cell culture medium II was added. The cells were then incubated in a 37°C CO2 incubator for another 24 h. After 48 h, the medium was changed again, the supernatant was discarded, and neuronal culture medium I was added. The cells were then incubated in a 37°C CO2 incubator. The purity of the isolated neurons cultured to day 3 was determined using an IFA assay with MAP2 Polyclonal antibody as the primary antibody.

[0045] Result: As Figure 9 As shown, a large number of red fluorescent cells are present in the field of view. By calculating the ratio of red fluorescent single cells to cell nuclei, fluorescently positive cells account for approximately 98.37 ± 0.21% of all cells.

[0046] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements can be made without departing from the principle of the present invention, and these improvements should also be considered within the scope of protection of the present invention.

Claims

1. A method for isolating and culturing neurons in the forebrain of a chicken embryo, comprising the following steps: (1) Coating of cell plates with poly-D-lysine solution; (2) Select 7-8 day old chicken embryos, disinfect the eggshell, crack open from the air cell end and peel off the embryo, remove the embryo under sterile conditions and place it in D-Hank's solution pre-cooled at 0-4℃; use sterile ophthalmic forceps to peel off the meninges, remove the brain tissue, and transfer the forebrain to digestion solution pre-cooled at 0-4℃. Use a sterile dropper to blow up and down 15-20 times to disperse the brain tissue, and then place it in a 37℃ CO2 incubator for digestion for 10-20 min; the digestion solution contains trypsin and DNase I; (3) Remove the digested tissue fluid, add FBS for neutralization, and pipette 10-20 times. Filter the tissue fluid through a 40μm cell filter to remove tissue blocks and large cell clumps. Centrifuge the filtrate, discard the supernatant, add cell culture medium I, count cells, plate them, and incubate at 37℃ in a CO2 incubator for 20-30 h. Discard the supernatant, add cell culture medium II, and incubate at 37℃ in a CO2 incubator for 20-30 h. Change the medium again after 48 h, discard the supernatant, add neuronal culture medium I, and incubate at 37℃ in a CO2 incubator. Then, on day 5, completely change the medium using neuronal culture medium II. Cell culture medium I contains 75-85 mL DMEM + 15-25 mL FBS + 0.5-1.5 mL penicillin per 100 mL; cell culture medium II contains 91-96 mL DMEM + 4-8 mL FBS + 0.5-1.5 mL penicillin per 100 mL. mL of penicillin and streptomycin; the neuronal culture medium I contains chicken cerebrospinal fluid and L-glutamine, and the neuronal culture medium II contains chicken cerebrospinal fluid, chicken serum and L-glutamine.

2. The method according to claim 1, wherein the poly-D-lysine solution is formulated to contain 40-60 μg of poly-D-lysine per 1 mL of poly-D-lysine solution.

3. The method according to claim 1, wherein the digestive fluid contains trypsin and DNase I.

4. The method according to claim 3, wherein the digestive solution is formulated as 0.25% trypsin + 0.02% EDTA + 0.01% DNase I + D-Hank's solution.

5. According to the method of claim 1, the formulation of the neuronal culture medium I is as follows: per 100 mL of neuronal culture medium I, there are 95-97 mL Neurobasal Medium + 2 mL B-27 Serum-Free Supplement + 80-120 μL chicken cerebrospinal fluid + 0.5-1.5 mL penicillin-streptomycin mixture + 0.5-1.5 mL L-glutamine solution.

6. According to the method of claim 1, the formulation of neuronal culture medium I is as follows: per 100 mL of neuronal culture medium II, there are 85-92 mL Neurobasal Medium + 2 mL B-27 Serum-Free Supplement + 80-120 μL chicken cerebrospinal fluid + 5-10 mL chicken serum + 0.5-1.5 mL penicillin-streptomycin mixture + 0.5-1.5 mL L-glutamine solution.

7. The method according to any one of claims 1-5, wherein the purity of the neuron is 98% or higher.

Images