CN114410582B - Glial cell and neuron co-culture method - Google Patents

The invention discloses a glial cell and neuron co-culture method, relates to the technical field of cell culture, and solves the technical problems of few sources, high culture cost, complex culture process and low survival rate of the existing contact co-culture materials; mainly comprises the following steps: collecting glial cells and neurons from brain tissues of adult mice or aged mice, combining and inoculating, sucking half of the suspension cells and the culture medium into another hole when clear suspension cell spheres are observed, supplementing the suspension cells and the culture medium with the complete culture medium, continuously culturing, continuously distributing the suspension cells and the culture medium until the cell density reaches the contact co-culture requirement, and culturing for 25-30 days to form contact co-culture of the glial cells and the neurons and form a neuron network and a glial background more stably; the invention takes adult mice or aged mice as sources, has easy sources, low cost and good repeatability, can obtain cells with certain yield, has long cell survival time, and is suitable for long-time observation.

一种胶质细胞和神经元共培养方法A method for co-culture of glial cells and neurons

技术领域Technical field

本发明涉及细胞生物学技术领域，更具体的是涉及细胞培养技术领域。The present invention relates to the technical field of cell biology, and more specifically to the technical field of cell culture.

背景技术Background technique

现有的接触式共培养常常需要将两种细胞分别抽提，分别培养后再合并共培养，过程复杂且混合后存活率低，且需要以乳鼠或胎鼠为材料，材料来源少，细胞培养成本较高，产量相对较低。Existing contact co-culture often requires the two types of cells to be extracted separately, cultured separately, and then combined for co-culture. The process is complicated and the survival rate after mixing is low. In addition, suckling mice or fetal mice are required as materials, and there are few sources of materials and cells. The cost of cultivation is high and the yield is relatively low.

发明内容Contents of the invention

本发明的目的在于：为了解决上述技术问题，本发明提供一种胶质细胞和神经元共培养方法。The purpose of the present invention is: in order to solve the above technical problems, the present invention provides a co-culture method of glial cells and neurons.

本发明为了实现上述目的具体采用以下技术方案：In order to achieve the above object, the present invention specifically adopts the following technical solutions:

一种胶质细胞和神经元共培养方法，包括以下步骤：A method for co-culture of glial cells and neurons, including the following steps:

S1：从成年鼠或老年鼠的脑组织中提取神经元及胶质细胞，通过梯度密度离心法分离获得第二层细胞和第三层细胞，将细胞沉淀用完全培养基重悬后接种到孔板中；S1: Extract neurons and glial cells from the brain tissue of adult mice or aged mice, separate the second layer of cells and the third layer of cells by gradient density centrifugation, resuspend the cell pellet in complete culture medium and inoculate it into the wells in the board;

S2：接种第1-3天不换液，也不做任何处理；S2: Do not change the medium or perform any treatment on the 1st to 3rd day of inoculation;

S3：3天后可观察到清晰的悬浮细胞球，开始吸出一半培养基及其中细胞到另一孔中，用完全培养基对两孔培养液进行补足，再继续培养2天。S3: After 3 days, clear suspended cell spheres can be observed. Start to aspirate half of the culture medium and the cells in it to another well. Use complete culture medium to supplement the culture medium in both holes and continue culturing for another 2 days.

S4：第5天观察细胞数量，如果神经球细胞较多悬浮，再次吸出一半液体及悬浮细胞到一个空孔中，用S3的方法补足培养液；S4: Observe the number of cells on the 5th day. If there are more neurosphere cells suspended, suck out half of the liquid and suspended cells into an empty hole again, and use the method of S3 to replenish the culture medium;

S5：连续分配悬浮细胞及培养基2-3次后，细胞密度达到接触式共培养要求，观察细胞生长状态，经过25-30天的培养，胶质细胞和神经元形成接触式共培养，较为稳定的形成神经元网络和胶质背景。S5: After continuously distributing suspended cells and culture medium 2-3 times, the cell density reaches the requirements of contact co-culture. Observe the cell growth status. After 25-30 days of culture, glial cells and neurons form contact co-culture, which is relatively Stable formation of neuronal networks and glial background.

作为优选，培养在37℃二氧化碳培养箱内进行。Preferably, the culture is performed in a 37°C carbon dioxide incubator.

作为优选，接种时，2ml细胞悬液平均接种到6孔中。Preferably, when inoculating, 2 ml of cell suspension is inoculated into 6 wells on average.

作为优选，所述完全培养基包括：25ml NeurobasalA、0.5ml B27、Glutamax和庆大霉素，其中Glutamax终浓度为200mM，庆大霉素终浓度为10mg/ml)。Preferably, the complete culture medium includes: 25 ml NeurobasalA, 0.5 ml B27, Glutamax and gentamicin, where the final concentration of Glutamax is 200mM and the final concentration of gentamicin is 10mg/ml).

作为优选，S1包括以下步骤：Preferably, S1 includes the following steps:

S11：将成年鼠或老年鼠麻醉消毒，解剖，将其大脑取出，大脑中的海马体放入冰上带有HABG的盘子中进行运输并称重，剥离海马后两侧大脑半球暴露的皮质取出称重，放入4℃含有HABG的试管中；S11: Anesthetize and disinfect adult or elderly mice, dissect them, and remove their brains. The hippocampus in the brain is placed in a plate with HABG on ice for transportation and weighing. After peeling off the hippocampus, the exposed cortex of the cerebral hemispheres on both sides is taken out. Weigh and put into a test tube containing HABG at 4°C;

S12：将装有组织的试管转移到30℃的摇动水浴中，同时将装有木瓜蛋白酶的试管与装有HABG的试管一起放入浴槽中，摇动8分钟以平衡温度；S12: Transfer the test tube containing the tissue to a shaking water bath at 30°C. At the same time, place the test tube containing papain and the test tube containing HABG into the bath and shake for 8 minutes to balance the temperature;

S13：用大口径移液器将组织转移到含有木瓜蛋白酶的试管中，以刚好足以悬浮组织的速度摇晃30分钟；S13: Use a large-bore pipette to transfer the tissue into a test tube containing papain, and shake for 30 minutes at a speed just enough to suspend the tissue;

S14：用大口径移液管和少量木瓜蛋白酶将切片转移到装有HABG的试管中，在室温下静置5分钟；S14: Use a large-bore pipette and a small amount of papain to transfer the slices to a test tube containing HABG, and let it sit at room temperature for 5 minutes;

S15：反复吹吸组织悬液，在45秒内研磨十次，形成碎片；S15: Repeatedly blow and aspirate the tissue suspension, and grind it ten times within 45 seconds to form fragments;

S16：让碎片静置1分钟，然后将上清液转移到空管中，将沉淀物重新悬浮在HABG中，重复操作两次，合并每次获得的上清液，制成6-12ml的悬浮细胞；S16: Let the fragments stand for 1 minute, then transfer the supernatant to an empty tube, resuspend the pellet in HABG, repeat the operation twice, and combine the supernatants obtained each time to make a 6-12ml suspension cell;

S17：通过梯度密度离心法分离细胞，取第二层细胞及第三层细胞，吸出的细胞用HABG重悬再离心2次，清洗其中碎片及对神经元生长有害的液体。S17: Separate cells by gradient density centrifugation. Take the second and third layers of cells. Resuspend the aspirated cells with HABG and centrifuge twice to clean the debris and liquid harmful to neuron growth.

作为优选，S15中研磨包括将组织吸进移液管中，没有气泡，然后立即将内容物排回管中，没有气泡。Preferably, grinding in S15 involves sucking the tissue into the pipette without air bubbles and then immediately expelling the contents back into the tube without air bubbles.

作为优选，S17中的分离具体为：将细胞悬浮液涂抹在准备好的OptiPrep密度梯度的顶部，细胞悬液应该漂浮在梯度的顶部，在22℃，1,900转/分下，离心梯度15分钟。As a preference, the separation in S17 is as follows: apply the cell suspension on the top of the prepared OptiPrep density gradient. The cell suspension should float on the top of the gradient. Centrifuge the gradient for 15 minutes at 22°C and 1,900 rpm.

作为优选，在细胞分离前3-24小时将蒸压过的玻璃盖玻片放在孔板当中，用poly-Lyscm–2底物包被玻片，超过1小时或过夜后，吸出并用PBS清洗三次，在通风橱中晾干1小时以上。Preferably, place autoclaved glass coverslips in the well plate 3-24 hours before cell isolation, coat the slides with poly-Lyscm-2 substrate, and after more than 1 hour or overnight, aspirate and wash with PBS Three times, dry in a fume hood for more than 1 hour.

作为优选，所有操作均在室温的层流无菌罩中进行。Preferably, all operations are performed in a laminar flow sterile hood at room temperature.

本发明的有益效果如下：The beneficial effects of the present invention are as follows:

1.本发明相对于现有的技术步骤更为简化，培养过程中处理较为容易；本技术去除了以往培养过程中，常规在分析细胞后5小时内全量换液的步骤，能够充分的保留早期短时间内未能贴壁的细胞。本发明能用简化的步骤来完成较为复杂的接触式共培养，得到形态功能完整的神经细胞。1. Compared with the existing technical steps, the present invention is more simplified and easier to handle during the culture process; this technology eliminates the routine step of changing the entire medium within 5 hours after analyzing the cells in the previous culture process, and can fully retain early stage cells. Cells that fail to adhere to the wall within a short period of time. The present invention can use simplified steps to complete relatively complex contact co-culture and obtain nerve cells with complete morphology and function.

2.本发明方法分离的神经元和胶质细胞来源于成年鼠或者老年鼠，来源较容易寻找，需要的成本较低，一只大鼠的培养细胞产量亦可完成部分特定的研究，避免大量乳鼠甚至胎鼠的使用，更加符合动物实验的伦理要求，成年鼠脑组织细胞提取后能够稳定的获得接触式共培养的两种细胞，细胞形态美观，结构完整，功能成熟，是研究神经元和胶质细胞的理想对象。2. The neurons and glial cells isolated by the method of the present invention are from adult mice or old mice. The sources are easier to find and the cost is lower. The cultured cell output of one rat can also complete some specific research, avoiding a large number of The use of suckling mice and even fetal mice is more in line with the ethical requirements of animal experiments. After extracting cells from adult mouse brain tissue, two types of cells can be stably obtained by contact co-culture. The cells have beautiful morphology, complete structure and mature functions. They are ideal for studying neurons. ideal target for glial cells.

3.本发明在细胞分离过程中将胶质细胞和神经元一起采集，不分开采集，合并两层细胞进行接种，依据两种细胞的天然特性，将二者混合培养，自然选择出共生的两种细胞，解决了以往单独获得神经元和胶质细胞后需要用阿糖胞苷纯化神经元，之后再合并胶质细胞共培养的复杂过程。3. In the present invention, glial cells and neurons are collected together during the cell separation process, rather than collected separately. The two layers of cells are combined for inoculation. Based on the natural characteristics of the two cells, they are mixed and cultured to naturally select the two symbiotic cells. Seed cells solve the complicated process of obtaining neurons and glial cells separately in the past, which required purifying the neurons with cytarabine and then combining the glial cells for co-culture.

4.常规技术需要在5小时后进行半量换液，本发明方法与常规培养方法不同，由于在培养细胞初期换液其实会损失细胞生长过程中产生的生长因子，经观察初期不换液，细胞并未死亡，避免了生长因子的损失。4. Conventional technology requires a half-amount of medium change after 5 hours. The method of the present invention is different from the conventional culture method. Since changing the medium in the early stage of culturing cells will actually lose the growth factors produced during the cell growth process, it has been observed that if the medium is not changed in the early stage, the cells will did not die, avoiding the loss of growth factors.

5.本发明在培养过程中，将培养基中的一半悬浮细胞及培养液转移到另一个准备好的培养板孔中；由于采集的细胞当中，含有一定数量的神经元前体细胞，是形成神经球的重要细胞，且多数悬浮于细胞液中，如果此时换液，大量已经成球的细胞可能被丢弃，而本发明保留换液的培养基，继续培养可最大限度利用已经提取的细胞，提高细胞产量和存活率。5. During the culture process of the present invention, half of the suspended cells and culture fluid in the culture medium are transferred to another prepared culture plate well; since the collected cells contain a certain number of neuron precursor cells, they are the Important cells of neurospheres, and most of them are suspended in the cell fluid. If the medium is changed at this time, a large number of cells that have formed into balls may be discarded. However, the present invention retains the medium for changing the medium and continues to culture to maximize the use of the extracted cells. , improve cell yield and survival rate.

附图说明Description of drawings

图1是本发明细胞转移培养过程示意图；Figure 1 is a schematic diagram of the cell transfer and culture process of the present invention;

图2是本发明胶质神经元接触式共培养的显微图，其中，图A和a为X10、相差；图B和b为X40、相差。Figure 2 is a micrograph of contact co-culture of glial neurons of the present invention, in which Figures A and a are X10, phase difference; Figures B and b are X40, phase difference.

具体实施方式Detailed ways

为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合实施例，对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, not All examples.

因此，以下提供的实施例的详细描述并非旨在限制要求保护的本发明的范围，而是仅仅表示本发明的选定实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。Therefore, the detailed description of the embodiments provided below is not intended to limit the scope of the claimed invention, but rather to represent selected embodiments of the invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present invention.

实施例1Example 1

本实施例提供一种胶质细胞和神经元共培养方法，包括以下内容：This embodiment provides a method for co-culturing glial cells and neurons, including the following:

(一)试剂准备：(1) Reagent preparation:

1.B27(Invitrogen，货号17504)用于神经元培养1.B27 (Invitrogen, Cat. No. 17504) is used for neuronal culture

2.层粘连蛋白(10mg ml–1；例如，Sigma，货号L2020)，可加入到多聚赖氨酸当中一起包被盖玻片2. Laminin (10mg ml–1; for example, Sigma, Cat. No. L2020) can be added to polylysine to coat the coverslip.

3.庆大霉素(10mg ml–1；Invitrogen，货号15710)3. Gentamicin (10 mg ml–1; Invitrogen, Cat. No. 15710)

4.Glutamax(200mM；Ala–Gln的稳定二肽；Invitrogen，目录号35050-061)4.Glutamax (200mM; stable dipeptide of Ala–Gln; Invitrogen, catalog number 35050-061)

5.NeurobasalA(Gibco目录号10888022)，接触式共培养中完全培养基的主要成分5.Neurobasal A (Gibco catalog number 10888022), the main component of complete media in contact co-culture

6.Poly-D-Lys用于包被经过无菌处理的盖玻片6.Poly-D-Lys is used to coat sterile-processed coverslips

7.OptiPrep密度1.32(Sigma，目录号D1556)用于密度梯度离心试剂配制7. OptiPrep density 1.32 (Sigma, catalog number D1556) is used for density gradient centrifugation reagent preparation

8.HABG制备：含有60ml HA、1.2ml B27、0.176ml GIn(最终0.5mM)的培养基8. HABG preparation: medium containing 60ml HA, 1.2ml B27, 0.176ml GIn (final 0.5mM)

9.NeurobasalA完全培养基：其制备需要25ml NeurobasalA、0.5ml B27，另加入Glutamax和庆大霉素(Glutamax终浓度200mM，庆大霉素终浓度10mg/ml)，该培养基用于分离细胞后种板前重悬细胞沉淀以及接种后细胞培养的整个过程。9.NeurobasalA complete medium: Its preparation requires 25ml NeurobasalA, 0.5ml B27, and adding Glutamax and gentamicin (Glutamax final concentration 200mM, gentamicin final concentration 10mg/ml). This medium is used after cell isolation. The entire process of resuspending cell pellets before seeding and culturing cells after seeding.

(二)神经元及胶质细胞分离步骤(2) Steps for isolating neurons and glial cells

所有操作均在室温(20–23℃)的层流无菌罩中进行。All operations were performed in a laminar flow sterile hood at room temperature (20–23°C).

1.培养皿准备1. Petri dish preparation

在准备分离神经元时，在细胞分离前3-24小时将蒸压过的玻璃盖玻片放在24孔板当中，用100ml poly-Lyscm–2底物包被玻片。超过1小时或过夜后，吸出并用PBS清洗三次，在通风橱中晾干1小时以上。When preparing to isolate neurons, place autoclaved glass coverslips in a 24-well plate 3-24 hours before cell isolation, and coat the slides with 100 ml poly-Lyscm–2 substrate. After more than 1 hour or overnight, aspirate and wash three times with PBS and dry in a fume hood for more than 1 hour.

2.组织分离的准备2. Preparation for Tissue Isolation

(1)选取6个-9个月龄(也可9月龄以上)SD大鼠，按照动物伦理操作要求处理大鼠(放入装有2ml异氟醚的容器中)(有些实验室使用戊巴对成年大鼠进行麻醉，运动停止后大约15秒，取出大鼠并通过脚趾捏合缺乏退出反射来确认麻醉。(1) Select 6 to 9-month-old SD rats (can also be over 9 months old) and handle the rats according to the requirements for animal ethical operation (put them in a container with 2 ml of isoflurane) (some laboratories use amylamine Anesthetize an adult rat and approximately 15 seconds after cessation of movement, remove the rat and confirm anesthesia by lack of exit reflex by toe pinch.

(2)在断头台上斩首，用70％乙醇消毒头部。解剖头骨顶部的皮肤以暴露头骨；将剪刀插入椎管，小心地将一侧的颅骨切近前部，避免损伤大脑，用镊子抓住头骨的底部，向上和向前抬起以暴露大脑；切断嗅球和视神经。轻轻地将大脑从颅骨中取出，转移到带有4℃Hibernate的解剖盘中。小心避免接触毛皮。(2) Behead on the guillotine and disinfect the head with 70% ethanol. Dissect the skin on top of the skull to expose the skull; insert scissors into the spinal canal and carefully cut one side of the skull closer to the front to avoid damaging the brain, grab the base of the skull with forceps and lift upward and forward to expose the brain; cut off the olfactory bulb and optic nerve. Gently remove the brain from the skull and transfer to a dissecting dish with 4 °C Hibernate. Be careful to avoid contact with fur.

3.组织分离3. Tissue separation

(1)在35毫米直径的培养皿中，在4℃下将大鼠大脑中的海马或其他大脑区域或脊髓快速解剖到2ml HABG中。要解剖海马体，将大脑的背侧朝上定向，以便可以看到两个半球的清晰中线。将镊子展开约2毫米，食指放在弯曲镊子的两个边缘的顶部，向下插入背中线约一半深度；挤压以切断脑连合。使用两个尖端轻轻地将一个半球剥离到一边，并切断海马/隔膜交界处。用镊子将海马体放入冰上带有HABG的盘子中进行运输并进行称重。(1) In a 35 mm diameter Petri dish, quickly dissect the hippocampus or other brain regions or spinal cord from the rat brain into 2 ml HABG at 4°C. To dissect the hippocampus, orient the dorsal side of the brain upward so that the clear midline of both hemispheres can be seen. Spread the forceps approximately 2 mm, place the index finger on top of both edges of the curved forceps, and insert down into the dorsal midline approximately halfway to the depth; squeeze to sever the cerebral commissures. Use both tips to gently peel one hemisphere to the side and sever the hippocampal/septal junction. Use forceps to place hippocampi into a dish with HABG on ice for transport and weigh.

(2)剥离海马后两侧大脑半球暴露的皮质取出称重，放入4℃的含有HABG的50毫升管中，其中HABG有5毫升。(2) Remove the exposed cortex of both cerebral hemispheres after peeling off the hippocampus, weigh it, and put it into a 50-ml tube containing HABG at 4°C, including 5 ml of HABG.

(3)将装有组织的50毫升试管转移到30℃的摇动水浴中，同时将装有木瓜蛋白酶的50毫升管与装有HABG的15毫升管一起放入浴槽中，摇动8分钟以平衡温度。(3) Transfer the 50 ml test tube containing the tissue to a shaking water bath at 30°C. At the same time, place the 50 ml tube containing papain and the 15 ml tube containing HABG into the bath and shake for 8 minutes to balance the temperature. .

(4)用大口径移液器将组织转移到含有木瓜蛋白酶的30℃管中，以刚好足以悬浮组织的速度摇晃30分钟(170转/分)。(4) Use a large-bore pipette to transfer the tissue to a 30°C tube containing papain, and shake for 30 minutes at a speed just enough to suspend the tissue (170 rpm).

(5)用大口径移液管和尽可能少的木瓜蛋白酶将切片转移到装有HABG的15毫升管中，在30℃下含有2毫升HABG，并在室温下静置5分钟。(5) Use a large-bore pipette and as little papain as possible to transfer the slices to a 15 ml tube containing 2 ml of HABG at 30°C and let stand at room temperature for 5 minutes.

(6)用抛光尖端的玻璃巴斯德吸管反复吹吸组织悬液，在45秒内研磨十次。研磨包括将组织吸进移液管中，没有气泡，然后立即将内容物排回管中，没有气泡。研磨太轻不会破坏组织，注意吹吸过程中避免气泡的产生。(6) Use a glass Pasteur pipette with a polished tip to repeatedly blow and aspirate the tissue suspension, and grind it ten times within 45 seconds. Trituration involves drawing the tissue into the pipette without air bubbles and then immediately expelling the contents back into the tube without air bubbles. Grinding too lightly will not damage the tissue. Be careful to avoid the generation of bubbles during the blowing and suction process.

(7)让碎片静置1分钟，然后将上清液转移到15毫升的空管中。将管中的沉淀物重新悬浮在2ml HABG中，再重复操作两次，合并每次获得的上清液，制成6-12ml的悬浮细胞。(7) Let the fragments sit for 1 minute, then transfer the supernatant to a 15 ml empty tube. Resuspend the sediment in the tube in 2 ml of HABG, repeat the operation twice more, and combine the supernatants obtained each time to make 6-12 ml of suspended cells.

(8)通过密度梯度离心分离细胞(8) Cell separation by density gradient centrifugation

小心地将细胞悬浮液涂抹在准备好的OptiPrep密度梯度的顶部，细胞悬液应该漂浮在梯度的顶部，对于皮质，使用了两个梯度。Carefully spread the cell suspension on top of the prepared OptiPrep density gradient. The cell suspension should float on top of the gradient. For the cortex, two gradients were used.

22℃下以800g(在摆动桶离心机中为1,900转/分)离心梯度15分钟，还将装有Neurobasal培养基的管子在4℃下转移到室温。Centrifuge the gradient at 800g (1,900 rpm in a swing-barrel centrifuge) for 15 minutes at 22°C. Also transfer the tube containing Neurobasal medium to room temperature at 4°C.

离心后细胞将被分成4层，取第2、3层用于共培养。After centrifugation, the cells will be divided into 4 layers, and the 2nd and 3rd layers will be used for co-culture.

(9)将第2、3层吸出，吸出的细胞需用HABG重悬再离心2次，清洗其中碎片及对神经元生长有害的液体。(9) Aspirate the 2nd and 3rd layers. The aspirated cells need to be resuspended in HABG and centrifuged twice to clean the debris and liquid harmful to neuronal growth.

4.种板4. Seed board

将细胞沉淀用完全培养基重悬后，平均接种到24孔板的6孔中，一般2ml悬液接种6孔留出后两排孔，用于后续的转移培养。补全培养基约0.5ml每孔，放于37℃二氧化碳培养箱培养。After the cell pellet is resuspended in complete culture medium, it is evenly inoculated into 6 wells of a 24-well plate. Generally, 2 ml of suspension is inoculated into 6 wells, leaving the last two rows of holes for subsequent transfer culture. Add about 0.5ml of culture medium to each well and place it in a 37°C carbon dioxide incubator for culture.

5.培养5. Cultivate

(1)接种第1-3天不换液也不做任何处理。(1) No medium change or any treatment is performed on the 1st to 3rd day of inoculation.

(2)3天后可观察到清晰的悬浮细胞球，开始吸出一半培养基及其中细胞到另一孔中，如图1所示，用完全培养基对两孔培养液进行补足，一般平均达到500ul，再继续培养两天。(2) After 3 days, clear suspended cell balls can be observed. Start sucking out half of the culture medium and the cells in it into another well. As shown in Figure 1, use complete culture medium to supplement the culture medium in both holes, generally reaching an average of 500ul. , continue culturing for two more days.

(3)第5天观察细胞数量，如果神经球细胞较多悬浮，再次吸出一半液体及悬浮细胞到一个空孔中，用同样方法补足培养液。(3) Observe the number of cells on the 5th day. If there are more suspended neurosphere cells, suck out half of the liquid and suspended cells into an empty hole again, and use the same method to replenish the culture medium.

(4)连续分配悬浮细胞及培养基2次后，细胞密度达到接触式共培养要求。(4) After continuously distributing suspended cells and culture medium twice, the cell density reaches the requirements for contact co-culture.

(5)观察细胞生长状态。(5) Observe the cell growth status.

(6)经过25-30天的培养，胶质细胞和神经元形成接触式共培养，较为稳定的形成神经元网络和胶质背景。(6) After 25-30 days of culture, glial cells and neurons form a contact co-culture, forming a relatively stable neuronal network and glial background.

6.细胞生长状态观察6. Observation of cell growth status

培养成功的状态表现为：神经元细胞贴附在胶质细胞表面，胶质细胞紧贴培养皿底部，形成一宽广平坦的背景，表面的神经元与背景里的细胞不在一个层面，细胞立体，有清晰轮廓，突起丰富可互相连接成网。The state of successful culture is as follows: neuronal cells are attached to the surface of glial cells, and glial cells are close to the bottom of the culture dish, forming a broad and flat background. The neurons on the surface are not on the same level as the cells in the background, and the cells are three-dimensional. It has a clear outline and abundant protrusions that can be connected to form a network.

如图2所示，是在第27天时细胞共培养的显微图，从图中可以看到，神经元细胞贴附在胶质细胞表面；A、B图中为在底部贴附的胶质细胞，a、b图中主要聚焦在表面神经元，背景中胶质细胞仍可见。As shown in Figure 2, it is a micrograph of cell co-culture on day 27. It can be seen from the picture that neuronal cells are attached to the surface of glial cells; pictures A and B show the glial cells attached at the bottom. Cells, in images a and b, the main focus is on surface neurons, while glial cells are still visible in the background.