WO2024078647A2 - 一种神经组织单细胞悬液的制备方法 - Google Patents

一种神经组织单细胞悬液的制备方法 Download PDF

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WO2024078647A2
WO2024078647A2 PCT/CN2024/074357 CN2024074357W WO2024078647A2 WO 2024078647 A2 WO2024078647 A2 WO 2024078647A2 CN 2024074357 W CN2024074357 W CN 2024074357W WO 2024078647 A2 WO2024078647 A2 WO 2024078647A2
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solution
preparation
percoll
tissue
working solution
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吴磊
张博
李爽
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华北理工大学
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    • C—CHEMISTRY; METALLURGY
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

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  • the invention belongs to the technical field of biomedicine, and in particular relates to a method for preparing a single-cell suspension of neural tissue.
  • the preparation methods of single cell suspension of solid tissue include mechanical method and enzyme digestion method.
  • Mechanical method includes grinding method, shearing method, homogenization method, etc. Mainly by giving greater pressure to the tissue, cells are released from the tissue.
  • manual mechanical dissociation of tissue causes fluctuations in the yield of live cells due to differences in blowing amplitude and speed, and the repeatability is poor.
  • Enzyme digestion method mainly hydrolyzes collagen fibers and mucopolysaccharide substances between tissues, separates the tight connection between cells, and achieves the purpose of preparing single cell suspension, but the enzyme incubation conditions are difficult to control, and solid tissue cells are easy to adhere to agglomerate, and the yield is low.
  • CNS central nervous system
  • the proportion of lymphocytes is relatively low, the cell purity, activity and yield obtained by these two methods are all low, which is not conducive to technical analysis such as flow cytometry.
  • the purpose of the present invention is to make up for the deficiencies of the prior art and provide a method for preparing a single cell suspension of neural tissue, which can efficiently obtain mononuclear cells from neural tissue, with high cell survival rate, high purity and good cell activity.
  • the present invention provides a method for preparing a single cell suspension of neural tissue, comprising the following steps:
  • the hydrolytic enzyme solution includes a trypsin solution or a papain solution, the size of the fragments is less than 1 mm ⁇ 1 mm ⁇ 1 mm;
  • the nerve tissue includes brain tissue or spinal cord tissue;
  • step (2) filtering the resuspension obtained in step (1), centrifuging, and resuspending the precipitate in 30% Percoll working solution;
  • step (3) adding the resuspension obtained in step (2) to the top of 70% Percoll working solution, centrifuging, and collecting the white blood cell layer;
  • step (3) mixing the white blood cell layer obtained in step (3) with 1 ⁇ Hank's balanced salt solution containing 2% v/v fetal bovine serum, centrifuging, collecting the precipitate and resuspending to obtain the neural tissue single cell suspension;
  • the 30% Percoll working solution is obtained by mixing 100% Percoll working solution and 1 ⁇ Hank's balanced salt solution in a volume ratio of 3:7;
  • the 70% Percoll working solution is obtained by mixing 100% Percoll working solution and 1 ⁇ Hank's balanced salt solution in a volume ratio of 7:3;
  • the 100% Percoll working solution is obtained by mixing the Percoll stock solution and 10 ⁇ PBS in a volume ratio of 9:1; the 10 ⁇ PBS is free of Ca 2+ and Mg 2+ and contains 0.5 mmol/L EDTA.
  • the enzymatic hydrolysis temperature is 55°C
  • the pH is 6.0
  • the time is 30 minutes.
  • the concentration of the enzyme in the hydrolase solution is 20 mg/mL.
  • the centrifugal speed in step (1) is 2000 r/min and the time is 5 min.
  • the filtration in step (2) uses a cell mesh with a pore size of 70 ⁇ m.
  • the centrifugation temperature in step (2) is 4°C
  • the rotation speed is 2000 r/min
  • the time is 15 min.
  • the volume ratio of the resuspension solution in step (3) to the 70% Percoll working solution is 10:3.
  • the centrifugation temperature is 4° C.
  • the rotation speed is 2000 r/min
  • the time is 15 min
  • the acceleration is set to 3
  • the deceleration is set to 2.
  • the centrifugation temperature in step (4) is 4°C, the speed is 2000r/min, and the time is 5min.
  • the resuspension in step (4) uses 1 ⁇ Hank's balanced salt solution containing 2% v/v fetal bovine serum.
  • the present invention uses a combination of mechanical and enzymatic methods to process nerve tissue, and uses Percoll discontinuous density gradient centrifugation to separate mononuclear cells from nerve tissue, providing a high-activity, high-purity cell sample for flow cytometry analysis.
  • the results of evaluating cell activity by trypan blue staining and flow cytometry show that the cell survival rate extracted by this method is high, and the separation process does not affect cell activity and does not change its intracellular and extracellular marker proteins.
  • the present invention determines the optimal centrifugal speed, acceleration, number of cycles and enzyme incubation temperature conditions according to the specific characteristics of neural tissue, and uses a cell mesh with a pore size of 70 ⁇ m to filter the enzymatic tissue, thereby reducing It can effectively avoid cell damage and better avoid cell clumping. It is simple, fast and low-cost to operate and can be completed using common experimental equipment. It is crucial for studying the functions of mononuclear cells in the CNS (including neurons, glial cells, infiltrating lymphocytes, etc.).
  • FIG1 is a comparison of cell survival rates in brain tissue single cell suspensions prepared by different methods
  • Figure 2 is a graph showing the percentage of mononuclear cells in brain tissue prepared by different methods detected by flow cytometry; among them, mechanical is the grinding and homogenization method of comparative example 1, enzymatic is the papain hydrolysis method of comparative example 2, and mechanical+enzymatic is the technical method of the present invention.
  • the present invention provides a method for preparing a single cell suspension of neural tissue, comprising the following steps:
  • the hydrolytic enzyme solution includes a trypsin solution or a papain solution, the size of the fragments is less than 1 mm ⁇ 1 mm ⁇ 1 mm;
  • the nerve tissue includes brain tissue or spinal cord tissue;
  • step (2) filtering the resuspension obtained in step (1), centrifuging, and resuspending the precipitate in 30% Percoll working solution;
  • step (3) adding the resuspension obtained in step (2) to the top of 70% Percoll working solution, centrifuging, and collecting the white blood cell layer;
  • step (3) mixing the white blood cell layer obtained in step (3) with 1 ⁇ Hank's balanced salt solution containing 2% v/v fetal bovine serum, centrifuging, collecting the precipitate and resuspending to obtain the neural tissue single cell suspension;
  • the 30% Percoll working solution is obtained by mixing 100% Percoll working solution and 1 ⁇ Hank's balanced salt solution in a volume ratio of 3:7;
  • the 70% Percoll working solution is obtained by mixing 100% Percoll working solution and 1 ⁇ Hank's balanced salt solution in a volume ratio of 7:3;
  • the 100% Percoll working solution is obtained by mixing the Percoll stock solution and 10 ⁇ PBS in a volume ratio of 9:1; the 10 ⁇ PBS is free of Ca 2+ and Mg 2+ and contains 0.5 mmol/L EDTA.
  • the present invention preferably cuts the nerve tissue into pieces smaller than 1 mm ⁇ 1 mm ⁇ 1 mm to obtain tissue pieces.
  • the nerve tissue preferably includes brain tissue or spinal cord tissue, more preferably brain tissue.
  • the present invention preferably wets the nerve tissue with 1 ⁇ Hank's balanced salt solution before cutting.
  • the present invention After obtaining the tissue fragments, the present invention performs enzymatic hydrolysis on the tissue fragments, collects the precipitate by centrifugation, and resuspends to obtain a first resuspension.
  • the hydrolase solution preferably includes trypsin solution or papain solution, and more preferably papain solution.
  • the enzymatic hydrolysis temperature of the present invention is preferably 55° C.
  • the pH is preferably 6.0
  • the time is preferably 30 min.
  • the concentration of the enzyme in the hydrolase solution is preferably 20 mg/mL.
  • the centrifugal speed is preferably 2000 r/min, and the time is preferably 5 min.
  • the filtration in the present invention preferably uses a cell sieve with a pore size of 70 ⁇ m.
  • the present invention filters the first resuspension, centrifuges, obtains a precipitate, and resuspends it in 30% Percoll working solution to obtain a second resuspension.
  • the centrifugation temperature is preferably 4°C
  • the speed is preferably 2000r/min
  • the time is preferably 15min.
  • the present invention adds the obtained second resuspension to the top of the 70% Percoll working solution, centrifuges, and collects the white blood cell layer.
  • the centrifugation temperature is preferably 4°C
  • the speed is preferably 2000r/min
  • the time is preferably 15min
  • the acceleration is preferably set to 3
  • the deceleration is preferably set to 2.
  • the volume ratio of the resuspension and the 70% Percoll working solution is preferably 10:3.
  • the buffy coat of the present invention is mixed with 1 ⁇ Hank's balanced salt solution containing 2% v/v fetal bovine serum, centrifuged, and the precipitate is collected and resuspended to obtain the single cell suspension of the neural tissue.
  • the centrifugation temperature is preferably 4°C
  • the speed is preferably 2000r/min
  • the time is preferably 5min.
  • the resuspension of the present invention preferably uses 1 ⁇ Hank's balanced salt solution containing 2% v/v fetal bovine serum.
  • the present invention has a high survival rate of mononuclear cells in the neural tissue single cell suspension obtained by the above method, and the separation process does not affect the cell activity and does not change the intracellular and extracellular marker proteins.
  • Cell culture, flow cytometric analysis, and protein and nucleic acid analysis can be further performed. Combining these analysis techniques, quantitative analysis of multiple parameters of single cells can be achieved.
  • rat/mouse brain tissue or spinal cord tissue Place fresh neural tissue (rat/mouse brain tissue or spinal cord tissue) in a 10 cm culture medium and add 1 mL of Hank's balanced salt solution (1 ⁇ HBSS) to keep the brain moist.
  • HBSS Hank's balanced salt solution
  • scalpel blade Use a scalpel blade to cut the brain tissue into pieces smaller than 1 mm ⁇ 1 mm ⁇ 1 mm.
  • the brain tissue single cell suspension was prepared by grinding and homogenizing method, which was different from Example 1 in that papain was not used for enzymatic hydrolysis.
  • the papain enzymatic hydrolysis method is used to prepare a brain tissue single cell suspension.
  • the difference from Example 1 is that the fresh neural tissue (rat/mouse brain tissue or spinal cord tissue) is directly transferred to the tissue dissociation tube in step 2 and neutralized without cutting.
  • the cell survival rates of the brain tissue single cell suspensions obtained in Comparative Example 1, Comparative Example 2 and Example 1 were measured by trypan blue exclusion method, and the results are shown in FIG1 .
  • the present invention adopts a combination of mechanical method and enzymatic method to obtain a high cell survival rate of cells in the brain tissue single cell suspension. Good repeatability.
  • the technical solution provided by the present invention is simple, easy and fast to operate, low cost, and the obtained cells have high activity.
  • Cell culture, flow cytometric analysis, and protein and nucleic acid analysis can be further performed. Combining these analysis techniques, quantitative analysis of multiple parameters of single cells can be achieved.

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Abstract

本发明属于生物医学技术领域，具体涉及一种神经组织单细胞悬液的制备方法。本发明采用机械法和酶法结合的方式对神经组织进行处理，采用Percoll非连续性密度梯度离心法，能够从神经组织中分离出单个核细胞，为流式细胞术技术分析提供了高活性、高纯度的细胞样本。通过台盼蓝染色和流式细胞数对细胞活性进行评价的结果表明，此方法提取的细胞存活率高，且分离过程不影响细胞活性，不改变其胞内和胞外的标记蛋白。

Description

一种神经组织单细胞悬液的制备方法 技术领域
本发明属于生物医学技术领域，具体涉及一种神经组织单细胞悬液的制备方法。
背景技术
组织单细胞悬液制备是细胞生物学中的基础技能，在神经科学的各个研究领域，包括发育研究、疾病病理生理学、药物和毒素筛选，以及神经疾病动物模型的移植等均需要使用分离的单细胞。借助单细胞分析技术研究异质细胞群体间相互作用介导的生物学过程极大推动了神经科学研究的发展，如流式细胞术等，而这些技术都依赖于组织单细胞悬液的制备。然而由于神经细胞体和成千上万的突触紧密结合以及轴突和树突的易断裂阻碍了神经组织单细胞的获得，不合适的组织解离可能降低细胞功能和分子实验中获得的数据质量，产生实验伪象。
目前实体组织单细胞悬液制备方法有机械法和酶消化法。机械法包括研磨法、剪碎法、匀浆法等。主要通过给予组织较大的压力，使细胞从组织中释放出来。但手动机械解离组织由于吹打幅度和速度差异导致活细胞得率的波动，重复性较差。酶消化法主要是水解组织间的胶原纤维和黏多糖物质，分开细胞间的紧密连接，达到制备单细胞悬液的目的，但酶孵育条件较难控制，实体组织细胞易黏连成团，产率较低。此外，由于中枢神经系统(CNS)中细胞种类繁多，且淋巴细胞所占比例又较低，使得这两种方法获得的细胞纯度、活性和产量均较低，不利于进行流式细胞等技术分析。
发明内容
本发明的目的在于弥补现有技术的不足，提供一种神经组织单细胞悬液的制备方法，高效的神经组织中获取单个核细胞，细胞存活率大，纯度高，细胞活性好。
为了实现上述目的，本发明提供了一种神经组织单细胞悬液的制备方法，包括如下步骤：
(1)利用水解酶液对神经组织的碎块进行酶解，离心收集沉淀，重悬；所述水解酶液包括胰蛋白酶液或木瓜蛋白酶液，所述碎块的尺寸小于1mm×1mm×1mm；所述神经组织包括脑组织或脊髓组织；
(2)对步骤(1)得到的重悬液过滤，离心，得到沉淀后利用30％Percoll工作液重悬；
(3)将步骤(2)得到的重悬液加入到70％Percoll工作液上方，离心，收集白细胞层；
(4)将步骤(3)得到白细胞层和含有2％v/v胎牛血清的1×Hank's平衡盐溶液混合，离心，收集沉淀后重悬，得到所述神经组织单细胞悬液；
所述30％Percoll工作液由100％Percoll工作液和1×Hank's平衡盐溶液按照3：7的体积比混合得到；
所述70％Percoll工作液由100％Percoll工作液和1×Hank's平衡盐溶液按照7：3的体积比混合得到；
所述100％Percoll工作液由Percoll原液和10×PBS按照9：1的体积比混合得到；所述10×PBS无Ca2+和Mg2+，含0.5mmol/L EDTA。
优选的，所述酶解的温度为55℃，pH为6.0，时间为30min。
优选的，所述水解酶液中酶的浓度为20mg/mL。
优选的，步骤(1)所述离心的转速为2000r/min，时间为5min。
优选的，步骤(2)过滤使用70μm孔径的细胞筛网。
优选的，步骤(2)所述离心的温度为4℃，转速为2000r/min，时间为15min。
优选的，步骤(3)所述重悬液和70％Percoll工作液的体积比为10：3。
优选的，步骤(3)所述离心的温度为4℃，转速为2000r/min，时间为15min，加速度设置为3，减速度设置为2。
优选的，步骤(4)所述离心的温度为4℃，转速为2000r/min，时间为5min。
优选的，步骤(4)所述重悬使用含有2％v/v胎牛血清的1×Hank's平衡盐溶液。
有益效果：
本发明采用机械法和酶法结合的方式对神经组织进行处理，采用Percoll非连续性密度梯度离心法，能够从神经组织中分离出单个核细胞，为流式细胞术技术分析提供了高活性、高纯度的细胞样本。通过台盼蓝染色和流式细胞数对细胞活性进行评价的结果表明，此方法提取的细胞存活率高，且分离过程不影响细胞活性，不改变其胞内和胞外的标记蛋白。
进一步的，本发明根据神经组织的特定，确定最佳的离心转速、加速度、循环数以及酶孵育温度条件，利用70μm孔径的细胞筛网过滤酶解组织，减少了 细胞的损伤，较好地避免了细胞成团现象，且操作简单、快速，成本低，使用通常的实验设备即可完成，对CNS中单个核细胞(包括神经元、神经胶质细胞、浸润的淋巴细胞等)功能的研究至关重要。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍。
图1为不同方法制备得到的脑组织单细胞悬液中细胞存活率比较图；
图2为流式细胞术检测不同方法制备得到的脑组织单个核细胞百分比图；其中，mechanical为对比例1研磨匀浆法，enzymatic为对比例2木瓜蛋白酶解法，mechanical+enzymatic为本发明技术方法。
具体实施方式
本发明提供了一种神经组织单细胞悬液的制备方法，包括如下步骤：
(1)利用水解酶液对神经组织的碎块进行酶解，离心收集沉淀，重悬；所述水解酶液包括胰蛋白酶液或木瓜蛋白酶液，所述碎块的尺寸小于1mm×1mm×1mm；所述神经组织包括脑组织或脊髓组织；
(2)对步骤(1)得到的重悬液过滤，离心，得到沉淀后利用30％Percoll工作液重悬；
(3)将步骤(2)得到的重悬液加入到70％Percoll工作液上方，离心，收集白细胞层；
(4)将步骤(3)得到白细胞层和含有2％v/v胎牛血清的1×Hank's平衡盐溶液混合，离心，收集沉淀后重悬，得到所述神经组织单细胞悬液；
所述30％Percoll工作液由100％Percoll工作液和1×Hank's平衡盐溶液按照3：7的体积比混合得到；
所述70％Percoll工作液由100％Percoll工作液和1×Hank's平衡盐溶液按照7：3的体积比混合得到；
所述100％Percoll工作液由Percoll原液和10×PBS按照9：1的体积比混合得到；所述10×PBS无Ca2+和Mg2+，含0.5mmol/L EDTA。
本发明优选将神经组织切割成小于1mm×1mm×1mm的碎块，得到组织碎块。在本发明中，所述神经组织优选包括脑组织或脊髓组织，进一步优选为脑组织。本发明优选进行所述切割前，利用1×Hank's平衡盐溶液对神经组织进行湿润处理。
得到组织碎块后，本发明对所述组织碎块进行酶解，离心收集沉淀，重悬，得第一重悬液。在本发明中，所述水解酶液优选包括胰蛋白酶液或木瓜蛋白酶液，进一步优选为木瓜蛋白酶液。本发明所述酶解的温度优选为55℃，pH优选为6.0，时间优选为30min。所述水解酶液中酶的浓度优选为20mg/mL。
在本发明中，所述离心的转速优选为2000r/min，时间优选为5min。本发明所述过滤优选使用70μm孔径的细胞筛网。
得到所述第一重悬液后，本发明对所述第一重悬液过滤，离心，得到沉淀后利用30％Percoll工作液重悬，得到第二重悬液。在本发明中，所述离心的温度优选为4℃，转速优选为2000r/min，时间优选为15min。
得到第二重悬液后，本发明将得到的第二重悬液加入到70％Percoll工作液上方，离心，收集白细胞层。在本发明中，所述离心的温度优选为4℃，转速优选为2000r/min，时间优选为15min，加速度优选设置为3，减速度优选设置为2。所述重悬液和70％Percoll工作液的体积比优选为10：3。
得到白细胞层后，本发明所述白细胞层和含有2％v/v胎牛血清的1×Hank's平衡盐溶液混合，离心，收集沉淀后重悬，得到所述神经组织单细胞悬液。在本发明中，所述离心的温度优选为4℃，转速优选为2000r/min，时间优选为5min。本发明所述重悬优选使用含有2％v/v胎牛血清的1×Hank's平衡盐溶液。
本发明采用上述方式得到的神经组织单细胞悬液中单个核细胞存活率高，且分离过程不影响细胞活性，不改变其胞内和胞外的标记蛋白，可以进一步进行细胞培养、流式细胞分析以及蛋白和核酸的分析，结合这些分析技术，可以实现单个细胞多参数的定量分析。
为了进一步说明本发明，下面结合附图和实施例对本发明提供的一种神经组织单细胞悬液的制备方法进行详细地描述，但不能将它们理解为对本发明保护范围的限定。
实施例1
1.将新鲜神经组织(大鼠/小鼠脑组织或脊髓组织)放入10cm的培养血中，并加入1mL Hank's平衡盐溶液(Hank's balanced salt solution，1×HBSS)，保持大脑湿润。使用手术刀刀片将脑组织切成体积小于1mm×1mm×1mm的碎块。
2.将脑组织碎块转移至组织解离管，选择木瓜蛋白酶(Papain)，用PBS溶解至浓度20mg/mL，向解离管加入2mL酶工作液。
3.将含有酶工作液的解离管混合液pH值调整值6.0，置于水浴锅中，温度 调整值55℃，水浴处理30min。
4.水浴结束后，加速度1500r/min，转速2000r/min离心5min。
5.离心结束后重悬样品，经过70μm孔径的细胞筛网进行过滤，收集细胞滤液至50mL离心管。
6.利用Percoll液梯度分离法分离CNS中单个核细胞，具体步骤如下：
(1)先将Percoll液原液与10×PBS(无Ca2+和Mg2+，含0.5mmol/L EDTA)按9：1的体积比混合，配制为实验用的100％Percoll工作液；
(2)用100％Percoll工作液与1×HBSS按7：3的体积比混合配制成70％Percoll工作液。
(3)将上述50mL离心管中的细胞滤液，4℃，转速2000r/min，离心15min。用7mL 1×HBSS重悬细胞，随即加入3mL 100％Percoll工作液，吹打混匀，即得30％Percoll工作液。
(4)先向15mL离心管中加入3mL的70％Percoll工作液，再用1ml移液枪将30％Percoll工作液缓缓加入到70％Percoll工作液上面。随后应用水平转自离心机，4℃，转速2000r/min，加速度设置为3，减速度设置为2，离心25min。离心后，可在30％～70％Percoll工作液界面之间看见白色细胞层。
(5)用移液枪小心将白色细胞层(约2～3mL)取出至新的离心管中，加入10mL 1×HBSS(含2％v/v胎牛血清)，4℃，转速2000r/min，反复离心重悬，清洗细胞3次，得到脑组织单细胞悬液。
7.重复步骤1～6的操作3次，即进行4个平行试验。
对比例1
采用研磨匀浆法制备脑组织单细胞悬液，与实施例1的区别在于，不利用木瓜蛋白酶进行酶解。
对比例2
采用木瓜蛋白酶解法制备脑组织单细胞悬液，与实施例1的区别在于，将新鲜神经组织(大鼠/小鼠脑组织或脊髓组织)直接转移至步骤2组织解离管中和，不进行切割处理。
测试例1
(1)用台盼蓝拒染法测定对比例1、对比例2和实施例1得到的脑组织单细胞悬液中细胞存活率，结果如图1所示。根据图1可以看出，本发明采用机械法和酶法相结合的方式，得到的脑组织单细胞悬液中细胞的细胞存活率高， 重复性好。
(2)将对比例1、对比例2和实施例1处理好的脑组织单细胞悬液，分别加入FITC标记的Anti-mouse CD4、PE标记的Anti-mouse CD25、APC标记Anti-mouse CD45等表面抗体，冰上避光孵育染色30min，随后加入1×PBS清洗细胞，上流式细胞仪检测脑组织单个核细胞百分比，结果如图2所示。根据图2可以看出，本发明技术方案提取的细胞存活率高，且分离过程不影响细胞活性，不改变其胞内和胞外的标记蛋白。
根据上述内容可以看出，本发明提供的技术方案简单操作简单快速、成本低，获取的细胞活性高，可以进一步进行细胞培养、流式细胞分析以及蛋白和核酸的分析，结合这些分析技术，可以实现单个细胞多参数的定量分析。
尽管上述实施例对本发明做出了详尽的描述，但它仅仅是本发明一部分实施例，而不是全部实施例，人们还可以根据本实施例在不经创造性前提下获得其他实施例，这些实施例都属于本发明保护范围。

Claims (10)

  1. 一种神经组织单细胞悬液的制备方法，其特征在于，包括如下步骤：
    (1)利用水解酶液对神经组织的碎块进行酶解，离心收集沉淀，重悬；所述水解酶液包括胰蛋白酶液或木瓜蛋白酶液，所述碎块的尺寸小于1mm×1mm×1mm；所述神经组织包括脑组织或脊髓组织；
    (2)对步骤(1)得到的重悬液过滤，离心，得到沉淀后利用30％Percoll工作液重悬；
    (3)将步骤(2)得到的重悬液加入到70％Percoll工作液上方，离心，收集白细胞层；
    (4)将步骤(3)得到白细胞层和含有2％v/v胎牛血清的1×Hank's平衡盐溶液混合，离心，收集沉淀后重悬，得到所述神经组织单细胞悬液；
    所述30％Percoll工作液由100％Percoll工作液和1×Hank's平衡盐溶液按照3：7的体积比混合得到；
    所述70％Percoll工作液由100％Percoll工作液和1×Hank's平衡盐溶液按照7：3的体积比混合得到；
    所述100％Percoll工作液由Percoll原液和10×PBS按照9：1的体积比混合得到；所述10×PBS无Ca2+和Mg2+，含0.5mmol/L EDTA。
  2. 根据权利要求1所述的制备方法，其特征在于，所述酶解的温度为55℃，pH为6.0，时间为30min。
  3. 根据权利要求1所述的制备方法，其特征在于，所述水解酶液中酶的浓度为20mg/mL。
  4. 根据权利要求1所述的制备方法，其特征在于，步骤(1)所述离心的转速为2000r/min，时间为5min。
  5. 根据权利要求1所述的制备方法，其特征在于，步骤(2)过滤使用70μm孔径的细胞筛网。
  6. 根据权利要求1所述的制备方法，其特征在于，步骤(2)所述离心的温度为4℃，转速为2000r/min，时间为15min。
  7. 根据权利要求1所述的制备方法，其特征在于，步骤(3)所述重悬液和70％Percoll工作液的体积比为10：3。
  8. 根据权利要求1所述的制备方法，其特征在于，步骤(3)所述离心的温度为4℃，转速为2000r/min，时间为15min，加速度设置为3，减速度设置为2。
  9. 根据权利要求1所述的制备方法，其特征在于，步骤(4)所述离心的温 度为4℃，转速为2000r/min，时间为5min。
  10. 根据权利要求1所述的制备方法，其特征在于，步骤(4)所述重悬使用含有2％v/v胎牛血清的1×Hank's平衡盐溶液。
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