WO2023029909A1 - 一种透射电镜观察用原位体外培养细胞的样品处理方法 - Google Patents

一种透射电镜观察用原位体外培养细胞的样品处理方法 Download PDF

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WO2023029909A1
WO2023029909A1 PCT/CN2022/111315 CN2022111315W WO2023029909A1 WO 2023029909 A1 WO2023029909 A1 WO 2023029909A1 CN 2022111315 W CN2022111315 W CN 2022111315W WO 2023029909 A1 WO2023029909 A1 WO 2023029909A1
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solution
cell
agar gel
agar
embedding
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PCT/CN2022/111315
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French (fr)
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朱昌来
贺小琴
刘小曼
刘芳
王莹洁
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南通大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/2806—Means for preparing replicas of specimens, e.g. for microscopal analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/36—Embedding or analogous mounting of samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/405—Concentrating samples by adsorption or absorption
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
    • G01N2001/2873—Cutting or cleaving
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/36—Embedding or analogous mounting of samples
    • G01N2001/364—Embedding or analogous mounting of samples using resins, epoxy

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  • the invention belongs to the field of biotechnology, and in particular relates to a sample processing method for in situ in vitro cultured cells for transmission electron microscope observation.
  • Transmission electron microscope observation is currently the only method that can observe and study the ultrastructure inside cells from the field of morphology.
  • the sample preparation needs to be processed by enzymatic digestion or mechanical scraping, and the cells are aggregated into clusters by centrifugation, and then processed according to conventional methods.
  • enzymatic digestion or mechanical scraping and centrifugation to collect cells have great disadvantages: First, the observed cells may have great changes in shape, surface structure, and internal ultrastructure, which seriously affects the authenticity of cell ultrastructure. Second, enzymatic digestion or mechanical scraping and centrifugation destroy the original mutual positional relationship between adjacent cells in the state of culture and growth, which has great significance for many specific scientific researches. Adverse effects, such as in vitro observation of myelination under the co-culture of Schwann cells and DRG neuron axons.
  • the present invention provides a sample processing method for in situ in vitro cultured cells for transmission electron microscope observation, which can effectively avoid enzyme digestion or When the cells are collected by mechanical scraping, the original growth position and ultrastructural changes of the cells are caused, and the original ultrastructural characteristics of the cultured cells in different growth stages or under the intervention of other experimental conditions are restored to the greatest extent.
  • an embodiment of the present invention provides a sample processing method for in situ in vitro cultured cells for transmission electron microscope observation, including the following process:
  • step S3 Subsequent processing is performed on the sample prepared in step S2, and after being fixed with osmium acid and rinsed, the agar gel block containing cell growth is cut off with a scalpel and micro tweezers, and transferred to another culture plate or culture dish ;
  • step S4 Perform subsequent dehydration and infiltration on the sample prepared in step S3, and perform sample embedding in a capsule-type embedding plate;
  • the step S1 specifically includes the following process:
  • the step S2 specifically includes the following process:
  • step S2-2 Take the cell suspension of the above cell density, add it to the cell culture plate or cell culture dish containing the agar gel prepared in step S1, and culture the cells with the cell suspension prepared in step S2-1
  • the plates or cell culture dishes were placed in a cell culture incubator at 37°C, saturated humidity, and 5% CO 2 for static culture for 12-24 hours.
  • the step S3 specifically includes the following process:
  • step S3-1 Take out the sample culture plate or culture dish in step S2, discard the cell culture medium, rinse with PBS or other seepage liquid for 10 minutes, and rinse 3 times in total;
  • S3-3 Use a scalpel or microscopic tweezers to gently cut and pick out the agar from the culture plate to protect the integrity of the agar gel. Put the cell-containing side up and place it on another cell culture plate or culture dish The bottom of the well or the bottom of the dish.
  • the step S4 specifically includes the following process:
  • step S4-1 dehydrating the samples prepared in step S3 with 50% ethanol, 70% ethanol, 80% ethanol, 95% ethanol, and absolute ethanol, respectively, for 15 minutes in each step;
  • the step S4-4 specifically includes the following process: prepare a commercially available capsule-type tissue embedding plate, add half the amount of embedding agent into the embedding hole, and quickly cut the agar gel containing cell components into About 1mm2 in size, carefully placed at the bottom of the embedding hole of the embedding plate, with the cell side facing the bottom, add enough embedding agent (to fill the embedding hole), gently arrange the gel blocks neatly, and place them in the oven. Embed at 37°C, 45°C, and 60°C for 24 hours respectively.
  • the epoxy resin embedding agent includes liquid A and liquid B; wherein, liquid A is configured from 6.2ml Epon812 and 10ml DDSA (dodecyl succinic anhydride); liquid B is composed of 10ml Epon812 and 80ml MNA (methyl Inner methine tetrahydrophthalic anhydride) is configured; liquid A and liquid B are mixed in a ratio of 2:8 or 1:9, fully stirred evenly, and a total of 1.5% of DMP-30 ( 2,4,6-tris(dimethylaminomethyl)phenol).
  • liquid A is configured from 6.2ml Epon812 and 10ml DDSA (dodecyl succinic anhydride)
  • liquid B is composed of 10ml Epon812 and 80ml MNA (methyl Inner methine tetrahydrophthalic anhydride) is configured
  • liquid A and liquid B are mixed in a ratio of 2:8 or 1:9, fully stirred evenly, and a total of 1.5%
  • step S5 specifically includes the following process: taking out the solidified sample embedding block in S4, performing conventional trimming, semi-thin sectioning and positioning after staining with 1% toluidine blue, ultrathin sectioning, 1% uranyl acetate and lemon Stained with lead acid and observed under a transmission electron microscope.
  • the present invention utilizes agar to have mechanical characteristics similar to biological tissues and cells by inoculating cultured cells on the surface of the agar gel, both components can be sliced smoothly under an ultra-wave microtome, and the agar gel has an electron microscope It does not show the structure under high magnification, does not combine with electron stains (lead citrate, uranyl acetate), and is resistant to the high-energy electron beam bombardment of transmission electron microscopy.
  • the components as a whole are subjected to routine processing for observation of biological samples by transmission electron microscope: fixation, dehydration, infiltration, embedding, microtome sectioning and staining. The process of this method is simple and convenient, and the cost is low, so it is suitable for popularization.
  • the method of the present invention can avoid the observation of the cell culture sample prepared by the conventional method, the cell enzyme digestion or scraping method in the sample processing process, etc. in the steps of collecting and centrifuging cells to cause changes in the original growth position of the cultured cells And ultrastructural changes, which makes the judgment of the experimental results fallacious. It is suitable for observing the ultrastructure of cultured cells in vitro, which has strict requirements on the structural information such as the original position of cell growth and mutual relationship in the observation of cultured cell samples by transmission electron microscopy. It is of great significance for the in situ electron microscope observation of cultured cells and the study of the ultrastructural relationship between adjacent cells.
  • Fig. 1 is the transmission electron microscope observation figure of in vitro in situ cell culture Schwann cells and dorsal root ganglion explants by the method of the present invention; obvious myelin sheath structure (the position indicated by the arrow) can be seen in the figure.
  • Fig. 2 is a transmission electron microscope observation picture of co-cultured Schwann cells and DRG explants treated with conventional enzyme digestion method, and no myelin sheath structure is seen in the picture.
  • Reagents required mainly include: agar, phosphate buffer, sodium cacodylate solution, 25% glutaraldehyde for electron microscopy, osmic acid, ethanol, propylene oxide, Epon 812 epoxy resin embedding agent (including Epon812, MNA, DDSA, DMP-20), lead citrate and uranyl acetate, etc.
  • Block co-cultivation is taken as an example to illustrate the operation and implementation process of the present invention by strictly studying the relevant mechanism of myelin sheath; another conventional enzymatic digestion method is set up as a contrast, and the specific implementation steps are not described in detail here), fume hood, embedding plate, oven , tissue slicer, stereoscope, ultra-bo slicer and 120KV transmission electron microscope, etc.
  • Preparation of the required agar gel Weigh 2 grams of agar, add 100ml of double distilled water, heat in a microwave oven to fully dissolve, absorb the above-mentioned unsolidified 2% agar hot solution (45-60°C), add it to the sterile 12-cell For the culture well of the well culture plate, absorb the amount of agar solution so that the solution covers the bottom of the well or the bottom of the dish to a depth of about 2mm. Place the cell culture plate on a flat table and wait for the solution to cool and solidify at room temperature.
  • Liquid A Epon812 6.2ml; DDSA (dodecyl succinic anhydride) 10ml;
  • Liquid B Epon812 10ml; MNA (methyl endomethylene tetrahydrophthalic anhydride) 80ml;
  • Aspirate the unsolidified 2% agar hot solution (45-60°C), add it to the culture wells of a sterile 12-well cell culture plate, absorb the amount of agar solution so that the solution covers the bottom of the well to a depth of about 2mm.
  • the above-mentioned dried agar gel was prepared to be sterilized by ultraviolet radiation for 2 hours in an ultra-clean bench.
  • the cells to be inoculated are adjusted to the required cell density with the complete medium used to cultivate the cells, and the cell suspension of the cells to be inoculated is prepared (the present invention uses rat-derived Schwann cells and rat dorsal root ganglia for co-culture in vitro, Take the study of myelin-related mechanisms as an example).
  • the DRG explants and Schwann cells were planted in the above-mentioned agar gel and placed in a cell culture incubator at 37° C., saturated humidity, and 5% CO 2 for static culture for 21 days. Take out the cell culture plate in step 9, discard the cell culture medium, rinse with PBS for 10 minutes, rinse 3 times in total, and discard the PBS.
  • each step is 15 minutes. Dehydrate with absolute ethanol for 15 minutes. After repeating twice in total, add the mixture of absolute ethanol: propylene oxide (v/v) (v/v) 1:1, 1:2 to replace the dehydrating agent. Each step is 30 minutes; A mixture of water ethanol and propylene oxide; add pure propylene oxide for 30 minutes, then discard the propylene oxide. Replace them with the mixture of epoxy resin embedding agent and propylene oxide in a volume ratio of 1:2, 1:1, and 2:1, respectively, for 3 hours per step. Subsequent replacement with pure epoxy resin embedding agent, a total of 3 hours.
  • Example 2 After carrying out the experiment according to the experimental steps of Example 2, the results were observed by transmission electron microscope: the in situ cell culture group processed by the transmission electron microscope observation of the present invention using the sample processing method of in situ cultured cells in vitro, as shown in Figure 1, Obvious myelin sheath structure can be observed in the in situ cell culture group; however, the conventional enzyme digestion method does not observe the myelin sheath structure because the position of the original cell is changed after digestion and has a corresponding impact on the ultrastructure of the cell, such as Figure 2 shows.
  • the in situ transmission electron microscope observation of the in situ cell culture group can restore the original ultrastructural characteristics of the cells in different growth stages or under the intervention of other experimental conditions to the greatest extent.

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Abstract

本发明公开了一种透射电镜观察用原位体外培养细胞的样品处理方法，通过制备琼脂凝胶置于细胞培养板的孔底，并对琼脂凝胶进行酸化、漂洗及表面的阳离子活性处理，将待观察细胞接种于所得琼脂凝胶表面，在后续样品处理制备中，经过锇酸固定、漂洗后，用手术刀和显微镊切去含有细胞生长的琼脂凝胶块，转移至另一培养板中，进行后续脱水、渗透，并在胶囊型包埋板中进行样品包埋，随后进行常规的切片、染色及上机观察。该方法可以避免常规样品处理方式所造成的培养细胞原始生长位置的改变和超微结构变化，而使得实验结果判断的谬误；同时，对于培养细胞的原位电镜观察及研究相邻细胞间超微结构关系具有重要意义。

Description

一种透射电镜观察用原位体外培养细胞的样品处理方法
本申请要求于2021年08月30日提交中国专利局、申请号为CN202111003683.2、发明名称为“一种透射电镜观察用原位体外培养细胞的样品处理方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于生物技术领域，具体涉及一种透射电镜观察用原位体外培养细胞的样品处理方法。
背景技术
透射电子显微镜观察是目前唯一能够从形态学领域对细胞内部的超微结构进行观察、研究的手段。通常地，培养的贴壁细胞的透射电镜观察，样品制备时需要先采用酶消化法或机械刮取的方式对贴壁细胞进行处理，并通过离心将细胞聚集成团，然后按照常规方法处理。但酶消化或机械刮取并离心收集细胞法存在很大的缺点：一是所观察细胞在外形、表面结构、内部超微结构形态均可能会发生极大变化，严重影响细胞超微结构的真实性和后期对观察结果分析判断的科学性；二是酶消化或机械刮取并离心法破坏了细胞在培养生长状态下相邻细胞间的原始相互位置关系，对很多特定的科学研究具有极大的不利影响，如体外观察施万细胞与背根节神经元轴突共培养下的成髓鞘。
发明内容
发明目的：针对现有技术中存在问题或不足，本发明提供一种透射电镜观察用原位体外培养细胞的样品处理方法，能有效避免培养细胞样品的透射电镜观察样品制备过程中酶消化法或机械刮取法收集细胞时，造成细胞原始生长位置的改变及超微结构变化，最大程度地还原了培养细胞在不同生长期或其它实验条件干预下的原位生长细胞的原始超微结构特征。
为实现上述发明目的，本发明的实施例提供了一种透射电镜观察用原位体外培养细胞的样品处理方法，包括以下过程：
S1、通过制备1～4％的琼脂凝胶置于细胞培养板或培养皿的孔底，并对所得琼脂凝胶进行酸化、漂洗及表面的阳离子活性处理；
S2、将待观察细胞接种于所得琼脂凝胶表面；
S3、对步骤S2所制备的样品进行后续处理，再经过锇酸固定、漂洗后，用 手术刀和显微镊切去含有细胞生长的琼脂凝胶块，转移至另一培养板或培养皿中；
S4、对步骤S3所制备的样品进行后续脱水、渗透，并在胶囊型包埋板中进行样品包埋；
S5、随后进行常规的切片、染色及上机观察。
优选的，所述步骤S1具体包括以下过程：
S1-1、称取1～4克琼脂粉盛放于洁净玻璃锥形瓶中，加入100ml双蒸水，经微波炉加热充分溶解后配制1～4％琼脂溶液；
S1-2、吸取上述尚未凝固的1～4％琼脂热溶液，琼脂热溶液的温度为45～60℃，添加至无菌细胞培养板的培养孔或细胞培养皿，吸取琼脂溶液量以溶液覆盖孔底或皿底深度2mm；
S1-3、将上述盛有琼脂溶液的细胞培养板或细胞培养皿静置在平整的台面，室温下待溶液冷却凝固；
S1-4、吸取2％冰醋酸至上述细胞培板或细胞培养皿中凝固的琼脂凝胶表面，室温下酸化处理3小时；
S1-5、弃去冰醋酸溶液，加入0.1mmol/L磷酸盐缓冲液(PBS)静置漂洗10分钟，弃去洗液；
S1-6、重复上述漂洗环节3次，弃去漂洗液，自然晾干；
S1-7、在上述晾干的琼脂凝胶表面，加入0.25％胰蛋白酶溶液进行琼脂凝胶表面的阳离子活性处理，室温静置2小时，或4℃过夜；
S1-8、弃去胰蛋白酶溶液，加入PBS漂洗10分钟；
S1-9、弃去上述漂洗液，重复漂洗2次，弃去漂洗液，室温自然晾干琼脂凝胶备用；
S1-10、将上述晾干的琼脂凝胶备在超净台内进行紫外辐照灭菌。
优选的，所述步骤S2具体包括以下过程：
S2-1、将待接种细胞，用培养该细胞所用的完全培养基调整成所需的细胞密度，制备待接种细胞的细胞悬液；
S2-2、吸取上述细胞密度的细胞悬液，添加至含有步骤S1所制备的琼脂凝胶的细胞培养板或细胞培养皿中，将步骤S2-1中所制备的具有细胞悬液的细胞培养板或细胞培养皿放置在37℃、饱和湿度、含5％CO 2的细胞培养箱中静置 培养12～24h。
优选的，所述步骤S3具体包括以下过程：
S3-1、取出步骤S2的样品培养板或培养皿，弃去细胞培养液，PBS或其它渗液漂洗10分钟，共漂洗3次；
S3-2、加入2.5％～4％戊二醛固定液，充分覆盖细胞表面，4℃固定过夜或室温固定2小时，弃去戊二醛溶液，PBS或其它渗液漂洗10～15分钟，共3～4次；弃去漂洗液，加入1％锇酸，室温避光固定2小时后，PBS或其它渗缓冲液漂洗10分钟，共计2次；
S3-3、利用手术刀或显微镊工具，轻轻将琼脂从培养板中切割挑出，保护琼脂凝胶的完整性，将含有细胞面朝上，置于另一细胞培养板或培养皿的孔底或皿底。
优选的，所述步骤S4具体包括以下过程：
S4-1、将步骤S3所制备的样品分别以50％乙醇、70％乙醇、80％乙醇、95％乙醇、无水乙醇进行梯度脱水，每步15分钟；
S4-2、使用无水乙醇再次脱水15分钟后，依次加入混合液A及混合液B置换脱水剂，其中，混合液A为无水乙醇：环氧丙烷(v/v)＝1:1的混合液、混合液B为无水乙醇：环氧丙烷(v/v)＝1:2的混合液，每次30min，弃去无水乙醇与环氧丙烷的混合液；加入纯环氧丙烷，30分钟，弃去环氧丙烷；
S4-3、依次置换成环氧树脂包埋剂与环氧丙烷按体积比分别为1:2、1:1、2:1的混合液，每步3小时；
S4-4、随后置换为纯环氧树脂包埋剂包埋3小时。
优选的，所述步骤S4-4具体包括以下过程：准备市售胶囊型组织包埋板，在包埋孔里加入一半量的包埋剂后，将含有细胞成分的琼脂凝胶快，切成约1mm 2大小，小心放置于包埋板包埋孔底部，细胞面朝向底部，添加至足量包埋剂(填满包埋孔)，轻轻将凝胶块摆放整齐，放置烘箱内，分别于37℃、45℃、60℃各包埋24小时。
优选的，环氧树脂包埋剂包括A液与B液；其中，A液由6.2ml Epon812和10ml DDSA(十二烷基琥珀酸酐)配置而成；B液由10ml Epon812和80ml MNA(甲基内次甲基四氢邻苯三甲酸酐)配置而成；A液与B液按2:8或1:9的比例混合，充分搅拌均匀，并缓慢滴加总量为1.5％的DMP-30(2,4,6-三(二 甲胺基甲基)苯酚)。
进一步的，所述步骤S5具体包括以下过程：取出S4固化后的样品包埋块，进行常规修块、半薄切片1％甲苯胺蓝染色后进行定位，超薄切片，1％醋酸铀及柠檬酸铅染色，透射电镜下观察。
本发明的上述技术方案的有益效果如下：
(1)本发明通过将培养细胞接种在琼脂凝胶表面，利用琼脂具有与生物组织及细胞相似的机械特征，两者成分均可在超波切片机下顺利切片，且琼脂凝胶在具有电镜高放大倍数下不显现结构、不与电子染色剂(柠檬酸铅、醋酸铀)结合、耐受透射电镜的高能电子束轰击的特征，将接种在琼脂凝胶表面的单层生长细胞与凝胶成分作为一个整体，进行透射电镜生物样品观察的常规处理：固定、脱水、渗透、包埋、切片机切片及染色。此法过程简单方便、成本低廉，适宜推广。
(2)本发明的方法可以避免常规方法制备的细胞培养样品在透射电镜观察，在样品处理过程中的细胞酶消化或刮擦法等在收集、离心细胞等步骤造成培养细胞原始生长位置的改变和超微结构变化，而使得对实验结果判断的谬误。适用于透射电镜观察培养细胞样品中，对细胞生长原始位置、相互关系等结构信息有严格要求的体外培养细胞超微结构观察的样品。对于培养细胞的原位电镜观察及研究相邻细胞间超微结构关系具有重要意义。
附图说明
图1为本发明的方法对体外原位细胞培养施万细胞与背根节植块的透射电镜观察图；图中可见明显的髓鞘结构(箭头所指位置)。
图2为常规酶消化法处理施万细胞与背根节植块共培养的透射电镜观察图，图中未见髓鞘结构。
具体实施方式
为使本发明要解决的技术问题、技术方案和优点更加清楚，下面将结合具体实施例进行详细描述。
实施例一、实验准备
1、所需试剂：主要包括：琼脂、磷酸盐缓冲液，二甲胂酸钠溶液、25％电镜专用戊二醛，锇酸、乙醇、环氧丙烷、Epon 812环氧树脂包埋剂(包括Epon812，MNA，DDSA，DMP-20)，柠檬酸铅和醋酸铀等。
2、所需实验材料及设备：三角烧瓶、微波炉、手术刀、显微镊、细胞培养板(皿)、培养的目的细胞(本发明以体外大鼠来源原代施万细胞及背根节植块共培养以严究成髓鞘相关机制为例来说明本发明的操作实施过程；另设立常规的酶消化法为对照，具体实施步骤未在此详述)，通风橱、包埋板、烘箱、组织切片机、体视镜、超博切片机和120KV透射电镜等。
3、所需琼脂凝胶配制：称取琼脂2克，加入100ml双蒸水，微波炉加热充分溶解吸取上述尚未未凝固的2％琼脂热溶液(45～60℃)，添加至无菌的12细胞孔培养板的培养孔，吸取琼脂溶液量以溶液覆盖孔底或皿底约2mm深度。将细胞培养板静置在平整的台面，室温下待溶液冷却凝固。
4、0.2M二甲胂酸钠溶液配制：二甲砷酸钠4.28g溶于去离子水80ml，调整pH至7.4，定容至100ml。
5、4％戊二醛固定液配制：25％电镜专用戊二醛原液16ml，0.2M二甲砷酸钠钠缓冲液50ml，去离子水34ml。
6、0.1M磷酸盐缓冲液配制：Na 2HPO 4·12H 2O 29.01g，NaH 2PO 4·2H 2O 2.96g，去离子水定容至1000ml，调pH值至7.4。
7、1％锇酸配制：将商品化的锇酸安剖瓶表面清洁后，放置于洁净的200ml棕色试剂瓶内，用血管钳夹碎安剖瓶，小心加入100ml双蒸水，震荡溶解后置于4℃冰箱里，隔天摇晃震荡助溶，配制完成3～4天后可用。
8、梯度乙醇溶液配制：分别将30ml、50ml、70ml、80ml、95ml无水乙醇加双蒸水至100ml。
9、Epon 812包埋剂配制：
A液：Epon812 6.2ml；DDSA(十二烷基琥珀酸酐)10ml；
B液：Epon812 10ml；MNA(甲基内次甲基四氢邻苯三甲酸酐)80ml；
将A液与B液按冬季1:9、夏季2:8的比例混合，充分搅拌均匀，并缓慢滴加总量约为1.5％的DMP-30(2,4,6-三(二甲胺基甲基)苯酚)。
10、1％甲苯胺蓝溶液配制：将1克甲苯胺蓝加入99ml 0.1M磷酸盐缓冲液里溶解。
11、1％柠檬酸铅配制：称取4克NaOH，溶于100ml双蒸水中，得到0.1M NaOH溶液；称取柠檬酸钠3.52克，硝酸铅2.66克于100ml容量瓶中，加蒸镏水至80ml，震荡摇晃，1小时后缓慢加入0.1M NaOH溶液10ml，待溶液澄清 后加双蒸水定容至100ml。
12、1％醋酸铀溶液配制：称取1克醋酸铀，溶于99ml 50％乙醇中。
实施例二、实验步骤
称取2克琼脂粉盛放于洁净玻璃锥形瓶中，加入100ml双蒸水，经微波炉加热充分溶解后配制2％琼脂溶液。
吸取上述尚未凝固的2％琼脂热溶液(45～60℃)，添加至无菌的12孔细胞培养板培养孔，吸取琼脂溶液量以溶液覆盖孔底约2mm深度。
将上述盛有琼脂溶液的细胞培养板静置在平整的台面，室温下待溶液冷却凝固。
吸取1ml 2％冰醋酸至上述12孔细胞细胞培板中凝固的琼脂凝胶表面，室温下静置3小时。
弃去冰醋酸溶液，加入PBS静置漂洗10分钟，弃去洗液。重复上述漂洗环节3次，弃去PBS，自然晾干备用。
在上述晾干的琼脂凝胶表面，加入0.25％胰蛋白酶溶液进行琼脂凝胶表面的阳离子活性处理，室温静置2小时。
弃去0.25％胰蛋白酶溶液，加入PBS漂洗10分钟，重复漂洗2次，弃去漂洗液，室温自然晾干琼脂凝胶备用。
将上述晾干的琼脂凝胶备在超净台内进行紫外辐照灭菌2小时。
将待接种细胞，用培养该细胞所用的完全培养基调整成所需的细胞密度，制备待接种细胞的细胞悬液(本发明以大鼠来源施万细胞与大鼠背根节体外共培养，以研究成髓鞘相关机制为例)。
将背根节植块与施万细胞种植在上述琼脂凝胶中并放置在37℃、饱和湿度、含5％CO 2的细胞培养箱中静置培养21天。取出步骤9中的细胞培养板，弃去细胞培养液，PBS漂洗10分钟，共漂洗3次，弃去PBS。
每孔加入2ml 4％戊二醛固定液，4℃固定过夜，弃去戊二醛溶液，PBS漂洗10分钟，共4次；弃去漂洗液，每孔加入1％锇酸，室温避光固定2小时后，PBS漂洗10分钟，共计2次，弃去PBS漂洗液。
利用手术刀、显微镊，轻轻将琼脂从培养板中切割挑出，特别注意，将含有细胞面朝上，置于另一12孔细胞培养板的培养孔的孔底。
50％、70％、80％、95％、无水乙醇梯度脱水，每步15分钟。无水乙醇脱 水15分钟，共计重复二次后，依次加入无水乙醇：环氧丙烷(v/v)为1:1、1:2的混合液，置换脱水剂，每步30min；弃去无水乙醇与环氧丙烷的混合液；加入纯环氧丙烷，30分钟，弃去环氧丙烷。依次置换成环氧树脂包埋剂与环氧丙烷按体积比分别为1:2、1:1、2:1的混合液，每步3h。随后置换为纯环氧树脂包埋剂，共计3小时。
准备市售胶囊型组织包埋板，在包埋孔里加入约一半量的包埋剂后，将含有细胞成分的琼脂凝胶快，切成约1mm 2大小，小心放置于包埋板包埋孔底部，细胞面朝向底部，添加至足量包埋剂(盛满包埋孔)，轻轻将凝胶块摆放整齐，放置烘箱内，分别于37℃、45℃、60℃各包埋24小时。取出固化后的样品包埋块，进行常规修块、半薄切片1％甲苯胺蓝染色后进行定位，超薄切片，1％醋酸铀及柠檬酸铅染色，透射电镜下观察。
实施例三 实验结果
按照实施例二的实验步骤进行实验后，通过投射电镜观察，结果可见：本发明的透射电镜观察用原位体外培养细胞的样品处理方法所处理的原位细胞培养组，如图1所示，原位细胞培养组可以观察到明显的髓鞘结构；而采用常规的酶消化法，因消化后，改变原始细胞位置，并对细胞超微结构有相应影响，没有观察到成髓鞘结构，如图2所示。
通过本发明的透射电镜观察用原位体外培养细胞的样品处理方法所处理的原位细胞培养组的投射电镜图(图1)，与常规的酶消化法消化后的投射电镜图(图2)进行对比，原位细胞培养组的原位透射电镜观察，可最大程度地还原细胞在不同生长期或其它实验条件干预下的原位细胞原始的超微结构特征。
以上所述是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明所述原理的前提下，还可以作出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (8)

  1. 一种透射电镜观察用原位体外培养细胞的样品处理方法，其特征在于，包括以下过程：
    S1、制备1～4％的琼脂凝胶置于细胞培养板或培养皿的孔底，并对所得琼脂凝胶进行酸化、漂洗及表面的阳离子活性处理；
    S2、将待观察细胞接种于所得琼脂凝胶表面；
    S3、对步骤S2所制备的样品进行后续处理，再经过锇酸固定、漂洗后，用手术刀和显微镊切去含有细胞生长的琼脂凝胶块，转移至另一培养板或培养皿中；
    S4、对步骤S3所制备的样品进行后续脱水、渗透，并在胶囊型包埋板中进行样品包埋；
    S5、随后进行常规的切片、染色及上机观察。
  2. 根据权利要求1所述的样品处理方法，其特征在于，所述步骤S1具体包括以下过程：
    S1-1、称取1～4克琼脂粉盛放于洁净玻璃锥形瓶中，加入100ml双蒸水，经微波炉加热充分溶解后配制1～4％琼脂溶液；
    S1-2、吸取上述尚未凝固的1～4％琼脂热溶液，琼脂热溶液的温度为45～60℃，添加至无菌细胞培养板的培养孔或细胞培养皿，吸取琼脂溶液量以溶液覆盖孔底或皿底深度2mm；
    S1-3、将上述盛有琼脂溶液的细胞培养板或细胞培养皿静置在平整的台面，室温下待溶液冷却凝固；
    S1-4、吸取2％冰醋酸至步骤S1-3细胞培板或细胞培养皿中凝固的琼脂凝胶表面，室温下酸化处理3小时；
    S1-5、弃去冰醋酸溶液，加入0.1mmol/L磷酸盐缓冲液静置漂洗10分钟，弃去洗液；
    S1-6、重复上述漂洗环节3次，弃去漂洗液，自然晾干；
    S1-7、在上述晾干的琼脂凝胶表面，加入0.25％胰蛋白酶溶液进行琼脂凝胶表面的阳离子活性处理，室温静置2小时，或4℃过夜；
    S1-8、弃去胰蛋白酶溶液，加入PBS漂洗10分钟；
    S1-9、弃去上述漂洗液，重复漂洗2次，弃去漂洗液，室温自然晾干琼脂 凝胶备用；
    S1-10、将上述晾干的琼脂凝胶备在超净台内进行紫外辐照灭菌。
  3. 根据权利要求1所述的样品处理方法，其特征在于，所述步骤S2具体包括以下过程：
    S2-1、将待接种细胞，用培养该细胞所用的完全培养基调整成所需的细胞密度，制备待接种细胞的细胞悬液；
    S2-2、吸取上述细胞密度的细胞悬液，添加至含有步骤S1所制备的琼脂凝胶的细胞培养板或细胞培养皿中，将步骤S2-1接种有细胞悬液的细胞培养板或细胞培养皿放置在37℃、饱和湿度、含5％CO 2的细胞培养箱中静置培养12～24h。
  4. 根据权利要求1所述的样品处理方法，其特征在于，所述步骤S3具体包括以下过程：
    S3-1、取出步骤S2的样品培养板或培养皿，弃去细胞培养液，PBS或其它渗液漂洗10分钟，共漂洗3次；
    S3-2、加入2.5％～4％戊二醛固定液，充分覆盖细胞表面，4℃固定过夜或室温固定2小时，弃去戊二醛溶液，PBS或其它渗液漂洗10～15分钟，共3～4次；弃去漂洗液，加入1％锇酸，室温避光固定2小时后，PBS或其它渗缓冲液漂洗10分钟，共计2次；
    S3-3、利用手术刀或显微镊工具，轻轻将琼脂从培养板或培养皿中切割挑出，保护琼脂凝胶的完整性，将含有细胞面朝上，置于另一细胞培养板或培养皿的孔底或皿底。
  5. 根据权利要求1所述的样品处理方法，其特征在于，所述步骤S4具体包括以下过程：
    S4-1、将步骤S3所制备的样品分别以50％乙醇、70％乙醇、80％乙醇、95％乙醇、无水乙醇进行梯度脱水，每步15分钟；
    S4-2、使用无水乙醇再次脱水15分钟后，依次加入混合液A及混合液B置换脱水剂，其中，混合液A为无水乙醇：环氧丙烷(v/v)＝1:1的混合液、混合液B为无水乙醇：环氧丙烷(v/v)＝1:2的混合液，每次30min，弃去无水乙醇与环氧丙烷的混合液；加入纯环氧丙烷，30分钟，弃去环氧丙烷；
    S4-3、依次置换成环氧树脂包埋剂与环氧丙烷按体积比分别为1:2、1:1、2:1的混合液，每步3小时；
    S4-4、随后置换为纯环氧树脂包埋剂包埋3小时。
  6. 根据权利要求5所述的样品处理方法，其特征在于，所述步骤S4-4具体包括以下过程：准备市售胶囊型组织包埋板，在包埋孔里加入一半量的纯环氧树脂包埋剂后，将含有细胞成分的琼脂凝胶快，切成约1mm 2大小，小心放置于包埋板包埋孔底部，细胞面朝向底部，添加足量包埋剂以填满包埋孔，轻轻将凝胶块摆放整齐，放置烘箱内，分别于37℃、45℃、60℃各包埋24小时。
  7. 根据权利要求5所述的样品处理方法，其特征在于，环氧树脂包埋剂包括A液与B液；其中，A液由6.2ml Epon812和10ml DDSA十二烷基琥珀酸酐配置而成；B液由10ml Epon812和80ml MNA甲基内次甲基四氢邻苯三甲酸酐配置而成；A液与B液按2:8或1:9的比例混合，充分搅拌均匀，并缓慢滴加总量为1.5％的DMP-302,4,6-三(二甲胺基甲基)苯酚。
  8. 根据权利要求1所述的样品处理方法，其特征在于，所述步骤S5具体包括以下过程：取出S4固化后的样品包埋块，进行常规修块、半薄切片1％甲苯胺蓝染色后进行定位，超薄切片，1％醋酸铀及柠檬酸铅染色，透射电镜下观察。
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