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Publication numberCN102391984 A
Publication typeApplication
Application numberCN 201110352089
Publication date28 Mar 2012
Filing date9 Nov 2011
Priority date9 Nov 2011
Also published asCN102391984B
Publication number201110352089.4, CN 102391984 A, CN 102391984A, CN 201110352089, CN-A-102391984, CN102391984 A, CN102391984A, CN201110352089, CN201110352089.4
Inventors吕小岩, 周钦, 孙环, 石运莹, 陈铁林, 魏于全
Applicant四川大学华西医院
Export CitationBiBTeX, EndNote, RefMan
External Links: SIPO, Espacenet
In-vitro culture method for embryo lungs
CN 102391984 A
Abstract
The invention belongs to the biotechnology field, relates to an in-vitro organ culture method for embryo lungs of mammals, and particularly relates to the isolated organ culture for the lungs in embryonic periods of mice. The in-vitro organ culture method solves the main technical problems that a conventional culture method has the disadvantages of needing special materials and occupying large space. The in-vitro organ culture method for the embryo lungs adopts the technical scheme that the method comprises the steps as follows: carrying tablets are put in cultivation holes formed on a porous culture plate and are matched with the cultivation holes in size, and a cultivation space is formed at the bottom part of each cultivation hole; secondly, the obtained mammalian embryo lungs are put on the carrying tablets, culture mediums are added in, and the lungs are led to be positioned at the junctions of the culture mediums and air; and thirdly, the lungs are cultured in an incubator under the temperature of 37 DEG C, and then solution changing of the culture mediums is carried out every one day or every two days. By adopting the in-vitro organ culture method, large number of in vitro cultured lung organs that are grown stably and are convenient for subsequent operation can be obtained at low cost, and the requirements of large-scale drug screening can be satisfied.
Claims(9)  translated from Chinese
1.哺乳动物胚胎肺的体外器官培养方法,其特征在于包括以下步骤:a、在多孔培养板的培养孔中放入尺寸与之适配的承载片,在培养孔底部构建出培养空间;b、将取得的哺乳动物胚胎肺放置于承载片上,加入培养基,使胚胎肺处于培养基和空气的液-气交界面;c、36C〜38C培养箱中培养,然后每1到2天进行培养基换液。 1. mammalian embryonic lung organ culture in vitro method, comprising the steps of: a, the wells multiwell plate was placed with the size of the fitted sheet bearing, constructed in the bottom of the hole cultured culture space; b , mammalian embryonic lung placed on the acquired carrier sheet, added to the medium, the embryo lung in liquid medium and air - air interface; c, 36 C~38 C incubator, then every 1 to two days carried the medium was changed.
2.根据权利要求1所述的哺乳动物胚胎肺的体外器官培养方法,其特征在于:步骤a 所述的多孔培养板为标准的M孔培养板。 Organ culture in vitro method according to claim 1, wherein the mammalian embryonic lung, characterized in that: said step a perforated plate is a standard M-well culture plate.
3.根据权利要求1所述的哺乳动物胚胎肺的体外器官培养方法,其特征在于:在使用多孔培养板的各孔间隙加入湿度平衡液,以减缓培养基挥发。 Organ culture in vitro method according to claim 1, wherein the mammalian embryonic lung, characterized in that: in each well of multi-well plate using a gap moisture equilibrium solution is added, in order to reduce the volatile medium.
4.根据权利要求1所述的哺乳动物胚胎肺的体外器官培养方法,其特征在于:步骤b 中加入培养基后调整承载片于培养孔的底部的中心,哺乳动物胚胎肺位于承载片的中部。 Organ culture in vitro method according to claim 1, wherein the mammalian embryonic lung, characterized in that: the step b is added after the medium was adjusted to the carrier sheet in the center of the bottom of the wells, mammalian embryonic lung central carrier sheet .
5.根据权利要求1所述的哺乳动物胚胎肺的体外器官培养方法,其特征在于:步骤c 中所述的培养基采用半量递增的方式换液,即每次换液时保留上次培养基体积的一半在原培养孔中,然后将比上次培养基体积的一半按体积分数还多增加4% -16%的新鲜培养基加入培养孔。 Organ culture in vitro method according to claim 1, wherein the mammalian embryonic lung, characterized in that: step c using half the amount of the medium was changed incrementally, i.e., retains the last time the medium was changed every half the volume of the original culture wells, then half of the medium volume than the last by the volume fraction of more than 4% -16% of the wells of fresh medium was added.
6.根据权利要求1所述的哺乳动物胚胎肺的体外器官培养方法,其特征在于:步骤a 所述的承载片为透明玻璃圆片。 Organ culture in vitro method according to claim 1, wherein the mammalian embryonic lung, characterized in that: said step a carrier sheet is transparent glass wafer.
7.根据权利要求1所述的哺乳动物胚胎肺的体外器官培养方法,其特征在于:所述湿度平衡液为无菌的磷酸盐缓冲液或蒸馏水。 Organ culture in vitro method according to claim 1, wherein the mammalian embryonic lung, characterized in that: said moisture equilibrium solution sterile phosphate buffer or distilled water.
8.根据权利要求1所述的哺乳动物胚胎肺的体外器官培养方法,其特征在于:所述胚胎肺为分离的胚胎期10〜13天的小鼠胚胎肺。 In vitro organ culture method according to claim 1, wherein the mammalian embryonic lung, characterized in that: the embryonic lung is isolated mouse embryonic embryonic lung 10~13 days.
9.根据权利要求1所述的哺乳动物胚胎肺的体外器官培养方法,其特征在于:将胚胎肺平放。 Organ culture in vitro method according to claim 1, wherein the mammalian embryonic lung, characterized in that: the embryonic lung flat.
Description  translated from Chinese

胚胎肺体外培养方法 Embryonic lung in vitro method

技术领域 Technical Field

[0001] 本发明属于生物技术领域,具体涉及哺乳动物的胚胎肺的体外器官培养方法,特别是小鼠胚胎期肺的离体器官培养。 [0001] The present invention belongs to the field of biotechnology, in particular to a method of in vitro organ culture mammalian embryonic lung, in particular mouse embryonic lung organ culture in vitro.

背景技术 Background

[0002] 哺乳动物的器官发育研究能够在完整器官的背景下了解多个基本的细胞生物学过程。 [0002] The mammalian organ development research to understand more of the basic processes of cell biology in the context of the complete organ. 这在遗传学的时代特别重要,因为哺乳动物如小鼠等中的基因缺失或突变可直接与人类先天畸形相关联。 This is particularly important in the era of genetics, because mice and other mammals such as deletions or mutations in the gene can be directly associated with human congenital malformations.

[0003] 体外器官培养属于组织培养的一种高级形式,它能再现器官的发育过程,模拟器官在不同状态及条件下的功能状态,是是了解器官发育过程,研究疾病致病机制及药物筛选的重要技术手段。 [0003] In vitro organ culture belongs to an advanced form of tissue culture, it can reproduce the process of organ development, simulation of organ functional status in different states and conditions is to understand the process of organ development, the study of disease pathogenesis and drug screening The important technical means. 这对于通过分支形态发生机制培养的器官肺特别有用。 This is particularly useful mechanism for the occurrence of lung organ culture by branching morphogenesis. 小鼠胚胎肺的发育始于胚胎9. 5天的前肠内胚层喉气管沟的外凸,然后形成芽基逐渐形成支气管分支。 Mouse embryonic lung development begins with 9.5 days of embryonic foregut endoderm laryngotracheal groove convex, then gradually formed blastema bronchial branches. 这时候每个芽基包含着三层,表皮,包绕的间充质和间皮层,最后形成呼吸系统的肺。 This time each bud group contains three layers, the epidermis, wrapped between mesenchymal and between the cortex, and finally the formation of pulmonary respiratory system. 而这些分支,分层和肺的器官形成需要例如表皮生长因子(EGF)成纤维生长因子(FGF)等细胞因子和信号通路的调控。 These branches, stratification and lung organogenesis needs such as epidermal growth factor (EGF) fibroblast growth factor (FGF) and other cytokines and signal transduction pathway. 为了确定哪些因子对这些过程有影响,早期胚胎肺的体外培养实验体系就应运而生。 In order to determine which factors affect these processes, in vitro experimental system came into being early embryonic lung.

[0004] 目前比较通用的胚胎肺的体外培养体系是基于Trowell 1959年Hie culture of mature organs in a synthetic medium. Exptl. CeZZ Res. 16 :118-147,1959.其后许多人对其做了改进,但其核心为利用大口径培养皿(35mm,60mm),加入适量培养基,将孔径5um 左右的聚碳酸脂滤膜漂在培养基上。 [0004] The present system is relatively common in vitro embryonic lung is based Trowell 1959 年 Hie culture of mature organs in a synthetic medium Exptl CeZZ Res 16:.... 118-147,1959 followed many of its improvements have been made , but its core is the use of large diameter dish (35mm, 60mm), adding an appropriate amount medium around the aperture 5um polycarbonate membrane floating in the media. 然后将小鼠胚胎肺放置于滤膜上,置于气液交界处培养(图1、2、3、4)。 The mice were then placed on the embryo lung membrane, is placed at the junction of the gas-liquid culture (Figure 1, 2). 但是上述方法的使用需要(1)使用聚碳酸脂滤膜等特殊材料,(2)浪费大量的培养皿空间,(胚胎肺与培养基的接触面积有限不利于进一步有效的实验干预,(4) 并且不利于大规模胚胎肺培养的应用。 But the need to use the above method (1) using polycarbonate membrane and other special materials, (2) waste a lot of dish space, the contact area ( embryonic lung and medium limited against further effective experimental intervention (4 ) and is not conducive to the application of large-scale cultivation of embryonic lung.

发明内容 DISCLOSURE

[0005] 本发明的目的是解决常规培养方法需要使用聚碳酸脂滤膜等特殊材料,浪费大量的培养皿空间等问题。 Objective [0005] The present invention is to solve the conventional culture method requires the use of special materials such as polycarbonate membrane, wasting a lot of dish space and other issues.

[0006] 本方案解决方案是提供一种哺乳动物胚胎肺的体外器官培养方法,其特征在于包括以下步骤: [0006] The program solution is to provide in vitro mammalian embryonic lung organ culture method comprising the steps of:

[0007] a、在多孔培养板的培养孔中放入尺寸与之适配的承载片,在培养孔底部构建出培养空间; [0007] a, in culture wells multiwell plate was placed with the size of the fitted sheet bearing, constructed in the bottom of the hole cultured culture space;

[0008] b、将取得的哺乳动物胚胎肺放置于承载片上,加入培养基,使胚胎肺处于培养基和空气的液-气交界面; [0008] b, mammalian embryonic lung will get placed in on-chip carrier, added to the medium, the embryo lung in a liquid medium and the air - air interface;

[0009] c、36C〜38C培养箱中培养,然后每1到2天进行培养基换液。 [0009] c, 36 C~38 C incubator, and then the medium was changed every 1-2 days.

[0010] 其中,上述方法步骤a所述的多孔培养板为标准的M孔培养板GX 6孔排列,孔直径15. 6mm,孔底面积1. 9m2)。 [0010] wherein the method steps a porous plate according to a standard M-well culture plate GX 6-hole arrangement, the hole diameter 15. 6mm, hole bottom area 1. 9m2). [0011] 其中,上述方法步骤a所述的多孔培养板仅为液体培养基的承载容器,并不与胚胎肺直接接触,可以使用国产的灭菌的标准M孔培养板。 [0011] wherein the method steps of the perforated plates is only carrying a container of the liquid medium, not in direct contact with the embryonic lung, can be used domestically sterilized standard M-well culture plate.

[0012] 其中,上述方法步骤a中所述的承载片为透明玻璃圆片。 [0012] wherein the carrier sheet in the above-described process steps is a transparent glass wafer. 与培养的胚胎肺直接接触,应进行相应的细胞生物学处理。 Direct contact with cultured human embryonic lung, should make the appropriate biological treatment cells. 承载片的大小略小于培养孔孔底面积,为培养孔的50-80%大小即可。 Carrier sheet size slightly smaller than the area of the hole bottom wells for 50-80% of the size of the wells can be.

[0013] 其中,上述方法步骤a中在在使用的多孔培养板的各孔间隙加入湿度平衡液,以减缓培养基挥发。 [0013] wherein, in the above-described process steps each well of a perforated plate used in the liquid moisture balance added gap to slow volatile medium.

[0014] 其中,上述方法中所述湿度平衡液为无菌的磷酸盐缓冲液或蒸馏水。 [0014] wherein, in the above-described method for the sterile liquid equilibrium humidity phosphate buffer or distilled water.

[0015] 其中,上述方法步骤b中所述胚胎肺为分离的胚胎期10〜13天的小鼠肺原基。 [0015] wherein the method steps b in the embryonic lung is isolated embryonic mouse lung primordia 10~13 days.

[0016] 其中,上述方法步骤b中所述的肺放置方式为:将胚胎肺平放。 [0016] wherein, lung placement in the above-described method steps b is: the embryonic lung flat. 使得胚胎肺处于培养基的液-气交界处。 So embryonic lung in liquid medium - air junction.

[0017] 其中,上述方法步骤b中加入培养基后调整承载片于培养孔的底部的中心,哺乳动物胚胎肺位于承载片的中部。 [0017] wherein the method step b Adjusted added to the medium carrier sheet on the bottom of the wells in the center of the mammalian embryonic lung in the middle of carrying tablets.

[0018] 其中,上述方法c步骤中所述的培养基采用半量递增的方式换液,即每次换液时保留上次培养基体积的一半在原培养孔中,然后将比上次培养基体积的一半按体积分数还多增加4% -16%的新鲜培养基加入培养孔。 [0018] wherein, in the above-described method step c using half the amount of the medium was changed incrementally, i.e., the last half of the retention medium volume was changed every time when the original culture wells, and the volume of the medium than the last Press half of the volume fraction of more than 4% -16% of fresh culture medium was added to the hole. 优选多增加培养基体积一半的8% -13%,最优为10% -12%。 Preferably a multi-medium volume increase of 8% -13% half, and most of 10% -12%.

[0019] 本发明方法的有益效果在于:克服了目前所有方法的占用空间大缺点,大大提高了空间利用率,同时也降低了成本,便于大规模培养。 [0019] The method has the advantages that: Overcoming the space currently occupied by major drawback of all methods, greatly improving the utilization of space, but also reduces the cost, ease of mass culture. 由于肺与培养基的接触较多,提高了实验干预的效率。 Lung due to contact with the media more, improve the efficiency of the experimental intervention. 小体积培养减少了培养基的用量,只需ISOul左右的培养基培养即可。 Reducing the amount of the small volume of culture medium, just about the culture medium can ISOul. 克服了现有技术需要无需昂贵的进口材料,进口滤膜,占用大的培养空间和资源才能取得好效果的偏见,成本大大降低。 Overcome the prior art requires no expensive imported materials, import filters, culture occupy a large space and resources in order to achieve good results in prejudice, greatly reduce the cost. 做实验干预时,也可以减少干预试剂的量就可达到预期的浓度或效果。 Experiment intervention can also reduce the amount of intervention agents can achieve the desired concentration or effect. 组装和操作简单,只需将处理好的盖玻片放入培养孔,放置胚胎肺,加入培养基培养即可,简单易行。 Assembly and operation is simple, just deal with good cover slip into the wells, place the embryonic lung, adding culture medium can be, easy. 有更好的肺的培养形态,由于没有支撑膜的遮挡,便于观察肺的气管分支,如可以使用通用的细胞倒置显微镜观察肺在体外培养情况。 There are better lung morphogenesis, because there is no support film occlusion, easy to observe the lung airway branching, can be used as a common inverted microscope lung cells in vitro culture conditions. 而加入的盖玻片也方便了后续操作,如可进行原位杂交,免疫荧光等操作。 Join coverslips also facilitates subsequent operations can be carried out as in situ hybridization, immunofluorescence and other operations. 本发明方法体外培养胚胎肺超过10天,且肺部支气管平滑肌可有节律的收缩运动,具有好的应用前景。 The method of the present invention is cultured embryonic lung more than 10 days, and the lungs of bronchial smooth muscle contraction may have rhythm, with good prospects.

附图说明 Brief Description

[0020] 图1、现有的胚胎肺培养方法的体系示意图。 [0020] FIG. 1, the system existing embryonic lung culture method. Fig. l、60mm培养皿;2、胚胎肺;3聚碳酸 l, 60mm dish; 2, embryonic lung; 3 Polycarbonate

酯滤膜;4、培养基。 Ester membrane; 4, medium.

[0021] 图2、现有的胚胎肺培养方法的体系示意图。 [0021] FIG. 2, the system existing embryonic lung culture method of FIG. l、60mm培养皿;2、胚胎肺;3聚碳酸 l, 60mm dish; 2, embryonic lung; 3 Polycarbonate

酯滤膜;4、培养基。 Ester membrane; 4, medium.

[0022] 图3、本发明胚胎肺培养方法的体系示意图。 [0022] FIG. 3, the system embryonic lung culture method of the present invention. Fig. 1、M孔培养板上的培养孔;2胚胎肺; 3、培养基;4、承载片。 1, M-well culture plate wells; (2) embryonic lung; 3 medium; 4, the carrier sheet.

[0023] 图4、本发明胚胎肺培养方法的体系示意图。 [0023] FIG 4, system embryonic lung culture method of the present invention. Fig. 1、M孔培养板上的培养孔;2胚胎肺; 3、培养基;4、承载片。 1, M-well culture plate wells; (2) embryonic lung; 3 medium; 4, the carrier sheet.

[0024] 图5、显示的是小鼠胚胎期12. 5天的胚胎进行培养十天的生长情况。 [0024] Figure 5 shows the embryonic mouse 12.5 days embryos were cultured growth of ten days.

[0025] 图6为视频截图显示在胚胎肺培养的第五天,此后肺部支气管平滑肌出现有节律的收缩运动,其中箭头所指示的部位为运动幅度相当明显的部位,分别位于肺部大支气管和两个肺叶小的气管分支。 [0025] FIG. 6 is displayed in the video capture cultured embryonic lung fifth day, after lung bronchial smooth muscle rhythmic contraction occurs, wherein the portion indicated by the arrow portion of the amplitude of motion is quite obvious, it is located the pulmonary bronchi and two lobes small airway branches.

[0026] 图7、更换培养基体积方案。 [0026] FIG. 7, the replacement medium volume programs.

[0027] 图8、标准M孔细胞培养板的图片,以示其各孔间存在的用于加湿度平衡液的空隙。 [0027] 8, the standard M-well cell culture plate image view to show the presence of apertures between their respective gaps for adding moisture equilibrium solution. 每相邻4培养孔间围成一个间隙,标准M孔细胞培养板有15个可添加湿度平衡液的间隙。 4 adjacent wells each surrounded by a gap, standard M-well cell culture plate 15 can be added to liquid moisture balance gap. 图中1为培养孔间的间隙,2为培养孔。 FIG. 1 is a culture gap between the holes, 2 wells.

[0028] 图9、标准M孔细胞培养板的照片,以示其各孔间存在的用于加湿度平衡液的空隙。 [0028] FIG. 9, the standard M-well cell culture plate photograph to show the presence of apertures between their respective gaps for adding moisture equilibrium solution. 每相邻4培养孔间围成一个间隙,标准M孔细胞培养板有15个可添加湿度平衡液的间隙。 4 adjacent wells each surrounded by a gap, standard M-well cell culture plate 15 can be added to liquid moisture balance gap. 图中1为培养孔间的间隙,2为培养孔。 FIG. 1 is a culture gap between the holes, 2 wells.

具体实施方式: DETAILED DESCRIPTION:

[0029] 以下结合附图通过具体实施方式,对本发明进行详细说明。 [0029] DETAILED DESCRIPTION OF THE DRAWINGS By way of the present invention will be described in detail.

[0030] 本发明提供了一种哺乳动物胚胎肺的体外器官培养方法,包括以下步骤: [0030] The present invention provides a method of in vitro organ culture of mammalian embryonic lung, comprising the steps of:

[0031] a、在多孔培养板的培养孔中放入尺寸与之适配的承载片,在培养孔底部构建出培养空间; [0031] a, in culture wells multiwell plate was placed with the size of the fitted sheet bearing, constructed in the bottom of the hole cultured culture space;

[0032] b、将取得的哺乳动物胚胎肺放置于承载片上,加入培养基,使胚胎肺处于培养基和空气的液-气交界面; [0032] b, mammalian embryonic lung will get placed in on-chip carrier, added to the medium, the embryo lung in a liquid medium and the air - air interface;

[0033] c、36C〜38C培养箱中培养,然后每1到2天进行培养基换液。 [0033] c, 36 C~38 C incubator, and then the medium was changed every 1-2 days.

[0034] 其中,上述方法步骤a所述的多孔培养板为标准的M孔培养板GX 6孔排列,孔直径15. 6mm,孔底面积1. 9m2)。 [0034] wherein the above-described method steps of the perforated plate is a standard M-well plates GX 6 holes aligned hole diameter 15. 6mm, the hole bottom area 1. 9m2).

[0035] 其中,上述方法步骤a所述的多孔培养板仅为液体培养基的承载容器,并不与胚胎肺直接接触,可以使用国产的灭菌的标准M孔培养板。 [0035] wherein the method steps of the perforated plates is only carrying a container of the liquid medium, not in direct contact with the embryonic lung, can be used domestically sterilized standard M-well culture plate.

[0036] 其中,上述方法步骤a中所述的承载片为透明玻璃圆片,与培养的胚胎肺直接接触,必需进行相应的通用细胞生物学处理。 [0036] wherein the carrier sheet in the above-described process steps is a transparent glass disc, in direct contact with the cultured embryonic lung, accordingly necessary common biological treatment cells. 承载片尺寸应小于孔径大小,大小为培养孔的50-80%大小,形状匹配。 Carrier sheet size should be smaller than the pore size, the size of the wells of 50-80% of the size, shape matching. 最好与培养孔一样为圆形的透明玻璃圆片。 Preferably the wells as circular transparent glass wafer. 承载片一般应贴在底部。 Carrier sheet should generally be attached to the bottom.

[0037] 其中,上述方法中可以在使用的多孔培养板的各孔间隙加入湿度平衡液,以减缓 [0037] wherein the method can be added humidity balanced solution in the pores of the porous plate gap used to slow

培养基挥发。 Medium volatile.

[0038] 其中,上述方法中所述湿度平衡液为无菌的磷酸盐缓冲液或蒸馏水。 [0038] wherein, in the above-described method for the sterile liquid equilibrium humidity phosphate buffer or distilled water.

[0039] 其中,上述方法步骤b中所述胚胎肺为分离的胚胎期10〜13天的小鼠胚胎肺。 [0039] wherein the method steps b in the embryonic lung is isolated embryonic mouse embryonic lung 10~13 days.

[0040] 其中,上述方法步骤b中所述的肺放置方式为:将胚胎肺平放。 [0040] wherein, lung placement in the above-described method steps b is: the embryonic lung flat. 使得胚胎肺处于培养基的液-气交界面。 So embryonic lung in liquid medium - air interface.

[0041] 其中,上述方法步骤b中加入培养基后调整承载片于培养孔的底部的中心,哺乳动物胚胎肺位于承载片的中部。 [0041] wherein the method step b Adjusted added to the medium carrier sheet on the bottom of the wells in the center of the mammalian embryonic lung in the middle of carrying tablets.

[0042] 其中,上述方法c步骤中所述的培养基采用半量递增的方式换液,即每次换液时保留上次培养基体积的一半在原培养孔中,然后将比上次培养基体积的一半按体积分数还多增加4% -16%的新鲜培养基加入培养孔。 [0042] wherein, in the above-described method step c using half the amount of the medium was changed incrementally, i.e., the last half of the retention medium volume was changed every time when the original culture wells, and the volume of the medium than the last Press half of the volume fraction of more than 4% -16% of fresh culture medium was added to the hole.

[0043] 本发明方法中使用的各种试剂和材料,按本领域常识都应为无菌的,操作也应在无菌条件下进行。 [0043] the various reagents and materials for use in the method of the present invention, according to general knowledge in the art should be sterile, the operation should be carried out under sterile conditions. [0044] 本发明方法使用的承载片可以是各种材质的适于组织细胞培养的薄片,最常用和最优的是透明玻璃圆片。 The method of the invention using the carrier sheet [0044] This may be a sheet of various materials suitable for tissue culture, the most common and the best is a transparent glass wafer. 承载片使用前应进行充分的清洗和灭菌处理,在需要的情况下,可以用合适的基质预处理,如多聚赖氨酸,明胶或者胶原等。 It should be carried out before the carrier sheet using adequate cleaning and sterilization process, in case of need, you can use a suitable substrate pre-treatment, such as polylysine, gelatin or collagen.

[0045] 本发明方法中使用的多孔培养板可以是常规的能应用于细胞组织培养的普通M 孔板,其孔最好是底面积在1〜3平方厘米的平底圆孔。 Perforated plates used in the method of the invention [0045] This may be a conventional tissue culture can be applied to ordinary M plate, which hole is preferably 1 ~ 3 cm in the bottom area of the flat bottom hole. 最常见的是标准的M孔平底细胞培养板,材质是玻璃或者塑料均可。 The most common is a standard M-well flat bottom cell culture plate, the material may be glass or plastic. 标准规格为4X 6孔排列布,孔直径15. 6mm,孔底面积1. 9m2。 Standard specifications for 4X 6-hole arrangement cloth, hole diameter 15. 6mm, the hole bottom area 1. 9m2.

[0046] 本发明方法中使用的湿度平衡液必须无菌,无毒。 Moisture equilibrium method was used in the invention [0046] This must be sterile, non-toxic. 最好用无菌的磷酸盐缓冲液或蒸馏水,根据使用多孔培养板确定用量。 Preferably sterile phosphate buffer solution or distilled water, the amount determined in accordance with the use of porous plates. 多孔培养板孔间具有间隙(参见图8,图9),在本发明中可用于添加湿度平衡液,一般的标准M孔平底细胞培养板每个孔间间隙使用的湿度平衡液体积应大于200ul,最好是500ul。 Between perforated plates hole with a gap (see Fig. 8 and 9), in the present invention can be used to add liquid moisture balance, moisture balance fluid volume between the general standard M-well flat bottom cell culture plates used for each hole clearance should be larger than 200ul , preferably 500ul.

[0047] 本发明方法中使用的胚胎肺来源为一般实验用的小鼠的胚胎。 Embryonic lung source for use in the inventive method [0047] The general experimental use of embryonic mice. 实验小鼠胚胎肺可以为胚胎期10〜13天的肺原基(肺原基是本领域发育早期的胚胎肺的一种叫法,一般包括从食管分支出的气管和肺叶)。 Mice embryonic lung 10~13 days for embryonic lung primordia (lung primordia early embryo development in the art of one lung is called, generally ranging from trachea and esophagus branching lobes). 而每个承载片上最好仅培养1个小鼠胚胎肺。 The best piece on each train carrying only a mouse embryonic lung. 胚胎肺最好平放,即按横切面水平放置于承载片中央以便肺原基能发育成形态良好形态。 Embryonic lung best flat, ie horizontal cross-section in the center of the carrier sheet so that the lungs can develop into good shape primordial form. 而培养基加入后是使胚胎肺处于培养基和空气的液-气交界面,本领域的这种描述是指培养基不能完全淹没胚胎肺,但也不能使其直接暴露在空气中,而是要使其在上表面形成一层培养基的液膜,这就称之为处于培养基和空气的液-气交界面。 And after the medium was added to make embryonic lung in a liquid medium and the air - air interface, this description refers to the art medium is not completely submerged in fetal lung, but can not make it directly exposed to the air, but To make it on the surface layer of the medium of film, which is referred to in the media and air liquid - gas interface.

[0048] 在获取胚胎肺时,一定要保证手术中胚胎肺的完整性(不要破坏胚胎肺气管分支,以免出现肺发育形态异常)。 [0048] in obtaining embryonic lung, we must ensure the integrity of embryonic lung surgery (do not destroy the embryo lung tube branch, in order to avoid abnormal lung development form).

[0049] 本发明方法中使用培养基可为本领域通用的各种细胞培养基,比如dulbecco' s modifi ed eagle,s medium(DMEM,通用的培养基,如商家Invitrogen 货号11965 的产品, 或也可参考网上公开的:http://baike. baidu. com/view/3501452. htm 记载的DMEM(H)细胞培养基(粉末型)成分配制)或者fegle,s minimal essential medium(EMEM,通用的培养基,配方公知。然后在选择的培养基中加入体积分数为5-20%的FBS(胎牛血清)或者F12营养因子anvitrogen公司产品,品名:营养因子混合物,货号:21700),一般还可添加按终浓度0. lmg/ml添加抗生素penicillin和str印tomycin (青霉素和链霉素)防止污 [0049] The method of the present invention, the medium used in the art may be common to a variety of cell culture media, such as dulbecco 's modifi ed eagle, s medium (DMEM, generic media, such as a business product Invitrogen Num 11965, or also Refer to the online disclosure: http:... // baike baidu com / view / 3501452 htm described DMEM (H) cell culture medium (powder type) component formulation) or fegle, s minimal essential medium (EMEM, common culture The base, the recipe is known and then select the media at the concentrations of 5-20% FBS (fetal bovine serum) or F12 nutritional factors anvitrogen company's products, product name: a mixture of trophic factors, Item: 21700), the general may be added according to a final concentration of 0. lmg / ml penicillin and antibiotics str India tomycin (penicillin and streptomycin) to prevent contamination

^fe ο ^ Fe ο

[0050] 本发明方法中的培养条件为通用细胞培养条件。 [0050] The method of the present invention, the culture conditions for general cell culture conditions. 一般温度为36C〜38C;最好在37C下,体积分数为5%二氧化碳的环境中培养。 Usually temperature 36 C~38 C; preferably at 37 C, the volume fraction of 5% carbon dioxide environment culture.

[0051] 本发明方法中使用的培养基可每隔M〜48小时,去掉旧培养基并加入等体积新培养基继续培养。 Culture method [0051] The present invention can be used in every M~48 hour, remove the old medium and add an equal volume of fresh medium and cultured. 还可以采用的优选的换液方式是采用半量递增的方式换液,即每次换液时保留上次培养基体积的一半在原培养孔中,然后将比上次培养基体积的一半还按体积分数多增加4% -16%的新鲜培养基加入培养孔。 Preferred methods may also be used to change liquid is to use half the amount was changed incrementally, i.e., the last half of retention medium volume was changed every time when the original culture wells, then half of the medium volume further than the last by volume Score more than 4% -16% of fresh culture medium was added to the hole. 优选多增加培养基体积一半的8% -13%, 最优为11%。 Preferably more than half of the medium volume increase of 8% -13%, and most 11%. 若用标准M孔平底细胞培养板时,培养基体积可用每孔150-200ul,最佳的体积应为每孔180ul。 If the standard M-well flat bottom cell culture plate, medium volume available per hole 150-200ul, the best volume per well should 180ul. 换培养基时将比上次培养基体积的一半多5〜15ul的新鲜培养基加入培养孔。 The volume of the medium than the last time more than half of medium exchange 5~15ul fresh medium was added wells. 优选8〜12ul,最佳为IOul。 Preferably 8~12ul, best of IOul.

[0052] 实施例一:使用本发明方法培养Balb/c小鼠胚胎期11. 5天肺。 [0052] Example I: Culture Balb / c mouse embryonic lung 11.5 days using the method of the invention.

[0053] 1、准备承载片:用直径12mm的玻璃圆盖片作为承载片,购买于海门市华凯实验玻璃仪器有限公司。 [0053] 1, to prepare the carrier sheet: 12mm diameter circular glass cover sheet as a carrier sheet, purchased in Haimen Experimental Glass Instrument Co., Ltd. Huakai. 首先,用蒸馏水配制体积分数为的盐酸溶液,在室温摇动清洗圆盖片一个小时。 First, using distilled water as the volume fraction of the hydrochloric acid solution, shaken at room temperature for one hour to clean the dome sheet. 用蒸馏水清洗后,放入体积分数为70%的酒精溶液中,摇动清洗过夜。 After washing with distilled water, placed in a volume fraction of 70% alcohol solution, shaken clean overnight. 用IOcm 玻璃平皿盛放清洗后的圆盖片,浙干液体后放入高温蒸汽灭菌锅内,121C灭菌40分钟。 IOcm glass plates with round bloom after cleaning cover sheet, Zhejiang dry the liquid into the high-temperature steam sterilization pan, 121 C sterilized 40 minutes. 烘干后置于超净工作台备用。 Dried and placed in a clean bench spare.

[0054] 2、组装培养装置:用无菌的镊子夹取处理好的圆盖片放置于无菌的M孔培养板的培养孔中。 [0054] 2, the assembly culture device: with sterile forceps Deal circular cover plate is placed in a sterile culture wells M-well culture plate. 无菌的M孔培养板为购置于Costar (康宁公司,美国)的35M型组织培养板。 M-well culture plate for purchase in sterile Costar (Corning, USA) 35M, tissue culture plates.

[0055] 3、获得胚胎期11. 5天小鼠肺:以受孕Balb/c母鼠,来源于四川大学华西医院基因工程小鼠中心SPF级(无特殊病原菌)动物房,检测到阴道栓当天记为E0. 5天开始计算胚胎发育时间,在第Ell. 5天从动物饲养房取出孕鼠。 [0055] 3, obtained 11.5 days mouse embryonic lung: A pregnant Balb / c female mice, from West China Hospital Center genetically engineered mice in SPF (specific pathogen free) animal housing, detected pessaries day recorded as E0. 5 day computing embryonic development time, at Ell. 5 天 removed from the animal husbandry room in pregnant rats. 引颈法处死孕鼠后,用酒精消毒孕鼠及解剖器械。 Pregnant rats were killed after eagerly, with alcohol pregnant rats and anatomical instruments. 取出小鼠胚胎后于冰上预冷的解剖液(IX PBS,HBSS或者DMEM)中解剖,体视镜下取出胚胎肺,用ImL吸头转移到装有培养基(DMEM+10% FBS) 1. 5ml的EP管中。 Remove the mouse embryos were dissected after ice-cold (IX PBS, HBSS or DMEM) anatomy, embryonic lung removed stereoscopic microscope, transferred to the tip with ImL containing medium (DMEM + 10% FBS) 1 . 5ml of EP tube.

[0056] 4、在超净工作台内,用ImL的移液器转移胚胎肺到培养孔,每孔一个。 [0056] 4, in a clean bench, with pipettes ImL transferred to culture embryonic lung holes, each hole. 用移液器吸走培养孔中残余培养基并加入ISOul培养基。 Wells with pipette away the residual medium and add ISOul media. 用酒精灯高温灭菌尖镊子,待冷后调整胚胎肺至园盖片中部,并调整园盖片位置使得胚胎肺位于培养孔正中。 High temperature sterilization with alcohol lamp tipped forceps and cool embryonic lung adjusted to Central Park, the cover sheet, and adjust the position of the cover sheet garden makes embryonic lung wells located in the middle. 培养体系的构建参见图3和图4的示意图。 Construction schematic see Figure 3 and Figure 4 system culture.

[0057] 5、小心将种好的胚胎肺放置于细胞培养箱于37C,5% CO2中培养M小时。 [0057] 5. Carefully kind of good embryo lung cells placed in an incubator at 37 C, 5% CO2 cultured M hours.

[0058] 6、用移液器将培养孔中保留原培养基90ul,并丢弃剩余培养基。 [0058] 6 culture wells with a pipette to retain the original medium 90ul, and discard the remaining media.

[0059] 7、加入90+10ul的新鲜培养基到培养孔,混勻后于细胞培养箱中培养。 [0059] 7, 90 + 10ul fresh medium was added to the wells, and mix after cell incubator.

[0060] 8、培养M小时后,然后同步骤6和7进行半量递增换液:留95ul原培养基+105ul [0060] 8. After incubation M h, then steps 6 and 7 with half the amount of incremental change solution: stay 95ul original medium + 105ul

新培养基。 New media.

[0061] 9、每隔M小时换液。 [0061] 9. The medium was changed every M-hour. 更换培养基体积方案如图7所示。 Medium volume replacement scheme shown in Figure 7.

[0062] 10、培养10天效果(见附图5)。 [0062] 10, training 10 days effect (see Figure 5).

[0063] 图5显示的是小鼠胚胎期12. 5天的胚胎进行培养十天的生长情况。 [0063] FIG. 5 shows a mouse embryo 12.5 days embryos were cultured growth of ten days. 培养胚胎肺的时间可超过10天,且肺气管分支充分的分支并展开,获得了很好的形态。 The time to develop embryonic lung may be more than 10 days, and lung tube branch full of branches and expand, get a good shape.

[0064] 图6为视频截图显示在胚胎肺培养的第五天,此后肺部支气管平滑肌出现有节律的收缩运动,其中箭头所指示的部位为运动幅度相当明显的部位,分别位于肺部大支气管和两个肺叶小的气管分支。 [0064] FIG. 6 is displayed in the video capture cultured embryonic lung fifth day, after lung bronchial smooth muscle rhythmic contraction occurs, wherein the portion indicated by the arrow portion of the amplitude of motion is quite obvious, it is located the pulmonary bronchi and two lobes small airway branches. 说明培养肾脏接近体内情况,有一定的高级生理功能。 Description cultured kidney approaching the in vivo situation, there are some advanced physiological functions.

[0065] 经多次试验,利用上述方法使用E12. 5天的胚胎肺进行体外培养几乎是100%成功率,采用1个孔1个胚胎肺的方式,24孔平板所有孔可培养M个,最佳是使用8个内圈的孔进行培养。 [0065] After numerous experiments, using the above method using E12. 5 days were cultured embryonic lung is almost 100% success rate, using a hole an embryonic lung way, 24-well culture plate all holes of M, Best is to use the inner ring of holes 8 culture. 24孔板(13cmX8. 5cmX2. 5cm)培养24个胚胎肺。 24-well plates (13cmX8. 5cmX2. 5cm) cultured 24 embryonic lung. 即约11立方厘米空间1个肺,而现有技术最小是一般3. 5cmX3. 5cmX2. 5cm养一个,即约31立方厘米空间1个肺,另外本方案M个肺的培养在一起可联合起来作为相同环境的严格控制的操作,生长一致性更好,也更便于实现大规模药物筛选。 That is about 11 cubic centimeters of space a lung, whereas the prior art are generally the smallest 3. 5cmX3. 5cmX2. 5cm raise a, or about 31 cubic centimeters of space a lung, in addition to the M train the program to unite together the lung As of the same strictly controlled operating environment, growth consistency better and easier to implement large-scale drug screening.

7 7

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Classifications
International ClassificationC12N5/073
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