Connect public, paid and private patent data with Google Patents Public Datasets

In-vitro culture method for embryo lungs

Info

Publication number
CN102391984A
CN102391984A CN 201110352089 CN201110352089A CN102391984A CN 102391984 A CN102391984 A CN 102391984A CN 201110352089 CN201110352089 CN 201110352089 CN 201110352089 A CN201110352089 A CN 201110352089A CN 102391984 A CN102391984 A CN 102391984A
Authority
CN
Grant status
Application
Patent type
Prior art keywords
culture
lungs
method
vitro
organ
Prior art date
Application number
CN 201110352089
Other languages
Chinese (zh)
Other versions
CN102391984B (en )
Inventor
吕小岩
周钦
孙环
石运莹
陈铁林
魏于全
Original Assignee
四川大学华西医院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date

Links

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.

Description

胚胎肺体外培养方法 The method of in vitro embryonic lung

技术领域 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, particularly relates to a method in organ culture embryonic lung mammal, especially ex vivo organ culture embryonic mouse lung.

背景技术 Background technique

[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] studied organ development in mammals can understand more basic cellular processes 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 age of genetics, such as in a mammal such as a mouse gene deletion or mutation may 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, can reproduce an organ during development, simulation state organ function in different states and conditions are 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 for organ culture occur through the mechanism of lung 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 began 9.5 days embryonic foregut endoderm laryngotracheal convex groove, and then gradually formed blastema bronchial branch. 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。 Each time this blastema contains three layers, the epidermis, between and surrounding mesenchymal mesothelial layer, 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, and stratification formation requires pulmonary organs such as epidermal growth factor (EGF) fibroblast growth factor (FGF) and the like cytokine signaling pathways regulated. 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。 To determine which of these factors affect the process of early embryonic lung vitro experimental system is made.

[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] In vitro culture system is currently more common embryonic lung is based on Trowell 1959 Nian Hie culture of mature organs in a synthetic medium Exptl CeZZ Res 16:.... 118-147,1959 followed by many of its improvements have been made , but its core is a large-diameter culture dish using (35mm, 60mm), was added an appropriate amount of the medium, the pore size of about 5um polycarbonate filter floating on the medium. 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 embryonic lung membrane, is placed at the junction of the culture liquid (Fig. 1,2,3,4). 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,(¾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。 However, the above method requires the use of (1) the use of special materials like polycarbonate filter, (2) waste a lot of space dish, a contact area (¾ embryonic lung culture medium is not conducive to further limited effective experimental intervention, (4 ) and is not conducive to large-scale application of embryonic lung cultures.

发明内容 SUMMARY

[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 problems of the conventional culture method requires the use of special materials like polycarbonate membrane, waste a lot of space dish.

[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 present embodiment is to provide a solution mammalian embryonic lung organ culture in vitro 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, into the carrier sheet with adapted dimensions of the wells of a multiwell culture plate in culture, the culture space in the bottom of the culture construct aperture;

[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, the acquired mammalian embryonic lung placed on the carrier sheet, added to the medium, so that in the embryonic lung liquid medium and the air - air interface;

[0009] c、36°C〜38°C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 every 1-2 days the medium was changed.

[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 above process steps the porous plate is a standard M-well culture plate hole arrangement GX 6, 15. 6mm diameter hole, the 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 above-described method steps according to a multiwell plate containing a liquid carrier medium only, not in direct contact with the embryonic lung, can be made using standard sterilization 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, in the above-described process steps according to a carrier sheet is a transparent glass disc. 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 the cultured embryonic lung, cell biology should be treated accordingly. 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。 The carrier sheet is slightly smaller than the size of the hole bottom area of ​​the culture wells, 50-80% of the size of the wells can.

[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 said method step a humidity Ping Heng buffer was added to each well in a multiwell plate using a gap to slow media evaporation.

[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 the above process liquid Ping Heng moisture phosphate buffer or sterile 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, in the above method step b is isolated embryonic lung 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 disposed above method step b according to: embryonic lung flat. 使得胚胎肺处于培养基的液-气交界处。 Such that the liquid medium in the embryonic lung - gas 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 above process added to the medium after step b adjust the center of the carrier sheet to the bottom of the wells, the mammalian embryonic lung central carrier sheet.

[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 the above process steps c half the amount of the culture medium was changed incrementally, i.e., the last half of each retention volume of the medium in the original culture medium was changed when the hole, and then the last time the volume of medium half by increasing the volume fraction of more than 4% -16% fresh medium was added wells. 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 increased half 8% -13%, optimally 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] Advantageous effects of the present invention is the method wherein: space overcomes the major disadvantage of all methods, greatly improves the space utilization, but also reduces the cost, ease of large scale culture. 由于肺与培养基的接触较多,提高了实验干预的效率。 As more contacts with the media lung, 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。 Small volume of culture medium reduces the amount of, only the culture medium to about 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。 To overcome the existing technology needs without expensive imported materials, import filters, occupies a large space of culture and resources in order to achieve good results 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。 When doing experiments intervention, intervention can also reduce the amount of reagent concentration or can achieve the desired 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 as simple as well coverslips placed in wells treated, placed embryonic lung, was added to the culture medium, 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。 Culture has better lung morphology, because there is no shielding of the supporting film, easy to observe the tracheal bifurcation lung, may be used as a common cell lung inverted microscope 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。 Coverslips and also to facilitate the addition of a subsequent operation, such as may be performed 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 in vitro embryonic lung than 10 days, and the lungs may have bronchial smooth muscle contraction rhythm, has good application prospect.

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, a schematic diagram of a conventional system embryonic lung culture method. 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, the medium.

[0021] 图2、现有的胚胎肺培养方法的体系示意图。 [0021] FIG. 2, a schematic diagram of a conventional system embryonic lung culture method. 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, the medium.

[0022] 图3、本发明胚胎肺培养方法的体系示意图。 [0022] FIG. 3, a schematic diagram of the method of culturing embryonic lung system of the present invention. 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 hole; embryonic lung 2; 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, a schematic diagram of the method of culturing embryonic lung system of the present invention. 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 hole; embryonic lung 2; 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] FIG. 5 shows embryonic 12.5 days mouse embryos were cultured for ten days growth.

[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 a screenshot of a video display in cultured embryonic lung fifth day after rhythmic contraction of bronchial smooth muscle lungs occurs, wherein the portion indicated by an arrow a motion of considerable magnitude obvious parts, which are located the pulmonary bronchi and small airway branches two lobes.

[0026] 图7、更换培养基体积方案。 [0026] FIG. 7, the volume of the medium was changed scheme.

[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, M standard well cell culture plate in FIG picture, to show that the space between the holes for the presence of added moisture Ping Heng liquid. 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 between each adjacent wells surrounded by a gap, M standard well cell culture plate 15 may be added with a solution of the moisture Ping Heng gap. 图中1为培养孔间的间隙,2为培养孔。 Figure 1 is a gap between the hole culture, 2 to culture 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 photograph M-well cell culture plate, to show that the space between the holes for the presence of added moisture Ping Heng liquid. 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 between each adjacent wells surrounded by a gap, M standard well cell culture plate 15 may be added with a solution of the moisture Ping Heng gap. 图中1为培养孔间的间隙,2为培养孔。 Figure 1 is a gap between the hole culture, 2 to culture 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] The following specific embodiments in conjunction with the accompanying drawings, 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 organ culture in vitro 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, into the carrier sheet with adapted dimensions of the wells of a multiwell culture plate in culture, the culture space in the bottom of the culture construct aperture;

[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, the acquired mammalian embryonic lung placed on the carrier sheet, added to the medium, so that in the embryonic lung liquid medium and the air - air interface;

[0033] c、36°C〜38°C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 every 1-2 days the medium was changed.

[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 process steps the porous plate is a standard M-well culture plate hole arrangement GX 6, 15. 6mm diameter hole, 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 above-described method steps according to a multiwell plate containing a liquid carrier medium only, not in direct contact with the embryonic lung, can be made using standard sterilization 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, in the above-described process steps according to a carrier sheet is a transparent glass disc, in direct contact with cultured embryonic lung, necessary to perform the processing corresponding generic cell biology. 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 less than the pore size, pore size of 50-80% of the culture size, shape matching. 最好与培养孔一样为圆形的透明玻璃圆片。 Like wells preferably circular transparent glass wafer. 承载片一般应贴在底部。 Carrier sheet typically 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 above process may be added to each well of the humidity balance liquid multiwell plates used in the gap 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 the above process liquid Ping Heng moisture phosphate buffer or sterile 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, in the above method step b is isolated embryonic lung 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 disposed above method step b according to: embryonic lung flat. 使得胚胎肺处于培养基的液-气交界面。 Such that the liquid medium in the embryonic lung - 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 above process added to the medium after step b adjust the center of the carrier sheet to the bottom of the wells, the mammalian embryonic lung central carrier sheet.

[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 the above process steps c half the amount of the culture medium was changed incrementally, i.e., the last half of each retention volume of the medium in the original culture medium was changed when the hole, and then the last time the volume of medium half by increasing the volume fraction of more than 4% -16% fresh medium was added wells.

[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 used in the process of the 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。 [0044] The method of the present invention the carrier sheet may be a sheet material suitable for a variety of tissue culture, the most common and the most 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。 Before using the carrier sheet should be fully cleaned and sterilized, in case of need, a suitable matrix may be pretreated, such as polylysine, gelatin, collagen, or the like.

[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。 [0045] The method of the present invention, perforated plates can be used in common can be applied to a conventional M-well plate tissue culture cells, preferably the bottom area of ​​the pores is 1 ~ 3 cm flat circular 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 plates, glass or plastic material can be. 标准规格为4X 6孔排列布,孔直径15. 6mm,孔底面积1. 9m2。 4X 6 standard hole arrangement for the cloth, pore diameter 15. 6mm, well 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。 [0046] The method of the present invention, the moisture balance liquid used 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。 Buffer or distilled water is preferably sterile phosphate, an amount determined in accordance with the use of multiwell 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 having a clearance hole (see FIG. 8, FIG. 9), in the present invention may be used to add liquid moisture Ping Heng, the moisture balance between the volume of each well was general standard M-well flat bottom cell culture plate using a gap should be greater 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 origin used in the method of the invention [0047] This is a general experimental mouse embryos used. 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)。 Experimental Mouse embryonic lung may embryonic primordia 10~13 days lung (pulmonary primordium is a name for early embryonic lung development in the art, generally comprises from trachea and esophagus lung branching). 而每个承载片上最好仅培养1个小鼠胚胎肺。 And each of the carrier sheet is preferably a culture of mouse embryonic lung only. 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。 Preferably embryonic lung flat, i.e., horizontally in the transverse plane according to the carrier sheet so that a central lung primordia can develop into a good morphological 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。 After the medium was added to make the medium and the embryonic lung in liquid air - air interface, this description refers to a medium of the present art is not completely submerged embryonic lung, but it can not be directly exposed to the air, but so as to form a liquid film on the surface layer of the medium, which is called a liquid medium and the air - air 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] When obtaining embryonic lung, we must ensure the integrity of the embryonic lung surgery (not embryonic lung damage branch pipe, in order to avoid lung development morphological abnormality).

[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 may be used in the media art various general-purpose cell culture medium, such dulbecco 's modifi ed eagle, s medium (DMEM, common medium, such as merchant Invitrogen 11965 Num products, or else reference may be publicly available online: http:... // baike baidu com / view / 3501452 DMEM htm described in (H) cell culture medium (powder type) component is formulated) or fegle, culture s minimal essential medium (EMEM, general the base, formulations known in the selection medium and then added to a volume fraction of 5-20% FBS (fetal bovine serum) or F12 nutritional factors anvitrogen products, Name: nutritional factor cocktail, catalog: 21700), in general may be added with a final concentration of 0. lmg / ml penicillin and antibiotic str printing tomycin (penicillin and streptomycin) to prevent fouling

^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 culture conditions of the process of the present invention is a general cell culture conditions. 一般温度为36°C〜38°C;最好在37°C下,体积分数为5%二氧化碳的环境中培养。 Usually a temperature of 36 ° C~38 ° C; preferably at 37 ° C, the volume fraction of 5% carbon dioxide in the culture environment.

[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。 Method medium [0051] The present invention may be used in every M~48 hours, remove the old medium and adding 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。 The preferred embodiment may also be employed was changed is the use of half the amount was changed incrementally, i.e., the last half of each retention volume of the medium in the original culture medium was changed when the hole, then the volume of the medium further than the last half by volume score more than the increase of 4% to 16% fresh medium was added wells. 优选多增加培养基体积一半的8% -13%, 最优为11%。 Preferably a multi-medium volume increased half 8% -13%, optimally 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 plates, medium volume per well available 150-200ul, optimal 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。 Than the previous volume of the medium during the medium exchange with fresh medium was added 5~15ul than half the culture wells. 优选8〜12ul,最佳为IOul。 Preferably 8~12ul, most preferably 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 a: cultured using the methods of the invention Balb / c mice 11.5 days of embryonic lungs.

[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, the carrier sheet prepared: 12mm diameter glass cover sheet as a carrier sheet, for later experiments in Haimen Hua Kai Glass Instruments. 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, washed dome sheet shaken at room temperature for one hour. 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, washing shaken 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,121°C灭菌40分钟。 IOcm glass plate with a circular accommodating the cleaning cover sheet into Zhejiang autoclavble liquid dry pot, 121 ° C sterilization for 40 minutes. 烘干后置于超净工作台备用。 After drying clean bench placed in standby.

[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 culture device is assembled: a gripping Handled dome sheet is placed in wells M-well plate sterile with sterile forceps. 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。 Sterile M-well culture plate was purchased from Costar (Corning, United States) tissue culture plate 35M type.

[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, to obtain embryonic mouse lung 11.5 days: to become pregnant Balb / c female mice, from Sichuan University, West China Hospital Center genetically engineered mice in SPF (specific pathogen-free) animal house, detected vaginal suppository day referred to as a start to E0. 5 developmental embryonic day period, removed from the pregnant rats at the animal houses Ell. 5 days. 引颈法处死孕鼠后,用酒精消毒孕鼠及解剖器械。 After eagerly pregnant rats were killed, pregnant mice and dissecting instrument with alcohol. 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。 Anatomical solution (IX PBS, HBSS or DMEM) mice were removed after embryo dissected on ice-cold, stereo microscope embryonic lung removed, transferred to a tip containing medium (DMEM + 10% FBS) 1 with ImL . 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, using the transfer pipette to culture embryonic lung ImL of 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。 Pipetted away and the hole remaining in the culture medium was added ISOul medium. 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。 Alcohol lamp autoclaved tipped forceps and cool embryonic lung adjusted to the middle of the cover sheet garden, park and adjust the position of the flap is located such that the wells in the middle embryonic lung. 培养体系的构建参见图3和图4的示意图。 Construction Referring to schematic FIGS. 3 and 4 culture system.

[0057] 5、小心将种好的胚胎肺放置于细胞培养箱于37°C,5% CO2中培养M小时。 [0057] 5, the kind of good embryonic lung carefully placed in a cell culture incubator at 37 ° C, 5% CO2 M h in culture.

[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, with a pipette hole to retain the original culture medium was 90 uL, and the remaining medium is discarded.

[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, fresh medium was added to the 90 + 10ul culture wells, 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, M h after culturing, and then with half-steps 6 and 7 increment amount was changed: the original medium leaving 95ul + 105ul

新培养基。 New media.

[0061] 9、每隔M小时换液。 [0061] 9, the medium was changed every M hours. 更换培养基体积方案如图7所示。 Replace the medium volume embodiment shown in Fig.

[0062] 10、培养10天效果(见附图5)。 [0062] 10, the effect for 10 days (see FIG. 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 the embryonic mouse embryos were 12.5 days for ten days culture growth. 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。 Time may be cultured embryonic lung than 10 days, and lung branch pipe branched and expand sufficiently to give a good morphology.

[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 a screenshot of a video display in cultured embryonic lung fifth day after rhythmic contraction of bronchial smooth muscle lungs occurs, wherein the portion indicated by an arrow a motion of considerable magnitude obvious parts, which are located the pulmonary bronchi and small airway branches two lobes. 说明培养肾脏接近体内情况,有一定的高级生理功能。 Description approaching train kidneys in vivo situation, there are certain 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, the use of E12. 5-day embryonic lung cultured in vitro by the above method is almost 100% success rate, using an aperture of an embryonic lung way, all holes may be 24 well plate culture of M, preferred is the use of an inner ring 8 wells cultured. 24孔板(13cmX8. 5cmX2. 5cm)培养24个胚胎肺。 24-well plates (13cmX8. 5cmX2. 5cm) cultured embryonic lung 24. 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。 I.e., a space of about 11 cm3 lung, whereas the prior art is generally the smallest 3. 5cmX3. 5cmX2. 5cm a support, i.e., a space of about 31 cm3 lungs, additional lung culture of the present embodiment M may combine together the same as the operation strictly controlled environment, growth consistency is better, and easier to implement large-scale drug screening.

7 7

Claims (9)

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、36°C〜38°C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, into the carrier sheet in a size adapted thereto wells multiwell plate, constructed in the bottom of the culture the culture space hole; B , mammalian embryonic lung placed on the acquired carrier sheet, added to the medium, so that in the embryonic lung liquid medium and the air - air interface; c, 36 ° C~38 ° C incubator, then every 1 to for 2 days 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 mammalian embryonic lung claim 1, wherein: said step of perforated plates 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 mammalian embryonic lung claim 1, wherein: the humidity balance was added to each well in a multiwell plate using a gap to slow media evaporation.
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 mammalian embryonic lung claim 1, wherein: step (b) After the medium was added to adjust the carrier sheet to the bottom of the wells of 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 mammalian embryonic lung claim 1, wherein: step c using half the amount of the medium was changed incrementally, i.e., each time when the medium was changed last reserved half the volume of the original culture wells, and the volume of the medium than the last half by increasing the volume fraction of more than 4% -16% fresh medium was added wells.
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 mammalian embryonic lung claim 1, wherein: the step of said carrier sheet is a transparent glass disc.
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 mammalian embryonic lung claim 1, wherein: said liquid Ping Heng humidity phosphate buffer or sterile 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。 Organ culture in vitro method according to claim mammalian embryonic lung claim 1, wherein: the embryonic lung as a separate embryonic mouse 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 mammalian embryonic lung claim 1, wherein: the flat embryonic lung.
CN 201110352089 2011-11-09 2011-11-09 In-vitro culture method for embryo lungs CN102391984B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN 201110352089 CN102391984B (en) 2011-11-09 2011-11-09 In-vitro culture method for embryo lungs

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN 201110352089 CN102391984B (en) 2011-11-09 2011-11-09 In-vitro culture method for embryo lungs

Publications (2)

Publication Number Publication Date
CN102391984A true true CN102391984A (en) 2012-03-28
CN102391984B CN102391984B (en) 2014-06-18

Family

ID=45859368

Family Applications (1)

Application Number Title Priority Date Filing Date
CN 201110352089 CN102391984B (en) 2011-11-09 2011-11-09 In-vitro culture method for embryo lungs

Country Status (1)

Country Link
CN (1) CN102391984B (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5344454A (en) * 1991-07-24 1994-09-06 Baxter International Inc. Closed porous chambers for implanting tissue in a host
US5453278A (en) * 1991-07-24 1995-09-26 Baxter International Inc. Laminated barriers for tissue implants

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5344454A (en) * 1991-07-24 1994-09-06 Baxter International Inc. Closed porous chambers for implanting tissue in a host
US5453278A (en) * 1991-07-24 1995-09-26 Baxter International Inc. Laminated barriers for tissue implants

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
O.A.TROWELL: "The culture of mature organs in a synthetic medium", 《EXPERIMENTAL CELL RESEARCH》, vol. 16, 31 December 1959 (1959-12-31), XP024790337, DOI: doi:10.1016/0014-4827(59)90201-0 *
叶露等: "PFOS/PFOA对斑马鱼(Brachydanio rerio)胚胎致毒效应研究", 《环境科学》, vol. 30, no. 6, 30 June 2009 (2009-06-30) *

Also Published As

Publication number Publication date Type
CN102391984B (en) 2014-06-18 grant

Similar Documents

Publication Publication Date Title
Guo et al. Creation of engineered cardiac tissue in vitro from mouse embryonic stem cells
Ling et al. Identification of pulmonary Oct-4+ stem/progenitor cells and demonstration of their susceptibility to SARS coronavirus (SARS-CoV) infection in vitro
US5496722A (en) Method for producing non-neoplastic, three dimensional, mammalian tissue and cell aggregates under microgravity culture conditions and the products produced therefrom
US5858783A (en) Production of normal mammalian organ culture using a medium containing mem-alpha, leibovitz L-15, glucose galactose fructose
Takezawa A strategy for the development of tissue engineering scaffolds that regulate cell behavior
Carrier et al. Cardiac tissue engineering: cell seeding, cultivation parameters, and tissue construct characterization
Takezawa et al. Collagen vitrigel: a novel scaffold that can facilitate a three-dimensional culture for reconstructing organoids
Kim et al. Nanopatterned cardiac cell patches promote stem cell niche formation and myocardial regeneration
Shachar et al. The effect of immobilized RGD peptide in alginate scaffolds on cardiac tissue engineering
Ferreira et al. Cord blood-hematopoietic stem cell expansion in 3D fibrin scaffolds with stromal support
US20080057578A1 (en) Process and substrate for culturing cartilage cell, material for reproducing biological tissue containing cartilage cell, and cartilage cell
Marlovits et al. Chondrogenesis of aged human articular cartilage in a scaffold-free bioreactor
Galindo et al. Expression of ΔNp63 in response to phorbol ester in human limbal epithelial cells expanded on intact human amniotic membrane
Kaeffer Mammalian intestinal epithelial cells in primary culture: a mini-review
JP2007312736A (en) Substrate for cell culture
Desroches et al. Functional scaffold-free 3-D cardiac microtissues: a novel model for the investigation of heart cells
Leung et al. A modular approach to cardiac tissue engineering
Gerlach et al. Dynamic 3D culture promotes spontaneous embryonic stem cell differentiation in vitro
Danes et al. Epithelial line from normal human colon mucosa
CN101352586A (en) Method for preparing full-thickness skin for toxicity test by stem cell raft type cultivation
Kapur et al. Human adipose stem cells maintain proliferative, synthetic and multipotential properties when suspension cultured as self-assembling spheroids
Davidson et al. Topographically induced self-deformation of the nuclei of cells: dependence on cell type and proposed mechanisms
Wang et al. Reconstruction of renal glomerular tissue using collagen vitrigel scaffold
Palmiero et al. Engineered dermal equivalent tissue in vitro by assembly of microtissue precursors
Parsons et al. Efficient derivation of human cardiac precursors and cardiomyocytes from pluripotent human embryonic stem cells with small molecule induction

Legal Events

Date Code Title Description
C06 Publication
C10 Entry into substantive examination
C14 Grant of patent or utility model
CF01