Brachmann Isabel, Tucker Kerry L
Interdisciplinary Center for Neurosciences, Institute of Anatomy and Cell Biology, University of Heidelberg, Germany.
J Vis Exp. 2011 Mar 29(49):2309. doi: 10.3791/2309.
For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in which the enhanced version of the green fluorescent protein (EGFP) is exclusively expressed in all neurons of the developing central and peripheral nervous system(1), allowing the possibility to both film the innervation of the forelimb and to manipulate this process with pharmacological and genetic techniques(2). The most critical parameter in the successful cultivation of such slice cultures is the method by which the slices are prepared. After extensive testing of a variety of methods, we have found that a vibratome is the best possible device to slice the embryos such that they routinely result in a culture that demonstrates viability over a period of several days, and most importantly, develops in an age-specific manner. For mid-gestation embryos, this includes the normal outgrowth of spinal nerves from the spinal cord and the dorsal root ganglia to their targets in the periphery and the proper determination of skeletal and muscle tissue. In this work, we present a method for processing whole embryos of embryonic day (E) E10 to E12 into 300 - 400 micrometer slices for cultivation in a standard tissue culture incubator, which can be studied for up to two days after slice preparation. Critical for the success of this approach is the use of a vibratome to slice each agarose-embedded embryo. This is followed by the cultivation of the slices upon Millicell culture membrane inserts placed upon a small volume of medium, resulting in an interface culture technique. One litter with an average of 7 embryos routinely produces at least 14 slices (2-3 slices of the forelimb region per embryo), which varies slightly due to the age of the embryos as well as to the thickness of the slices. About 80% of the cultured slices show nerve outgrowth, which can be measured througout the culturing period(2). Representative results using the tauGFP mouse line are demonstrated.
在许多情况下，将小鼠胚胎作为器官型切片进行体外培养是很有必要的。例如，我们使用一种转基因小鼠品系（tauGFP），其中绿色荧光蛋白（EGFP）的增强版本仅在发育中的中枢和外周神经系统的所有神经元中表达(1)，这使得我们既可以拍摄前肢的神经支配情况，又可以用药理学和遗传学技术来操控这一过程(2)。成功培养此类切片培养物的最关键参数是切片的制备方法。在对多种方法进行广泛测试后，我们发现振动切片机是切取胚胎的最佳设备，这样通常能得到一种在数天内都具有活力的培养物，而且最重要的是，它能以与年龄相关的方式发育。对于妊娠中期的胚胎，这包括脊髓神经从脊髓和背根神经节正常向外生长至外周的靶标，以及骨骼和肌肉组织的正常分化。在这项工作中，我们提出了一种将胚胎期（E）E10至E12的完整胚胎处理成300 - 400微米切片的方法，以便在标准组织培养箱中培养，切片制备后可对其进行长达两天的研究。这种方法成功的关键在于使用振动切片机切取每个包埋在琼脂糖中的胚胎。随后，将切片培养在置于少量培养基上的Millicell培养膜插入物上，从而形成一种界面培养技术。一窝平均有7个胚胎通常能产生至少14个切片（每个胚胎2 - 3个前肢区域的切片），由于胚胎的年龄以及切片的厚度不同，数量会略有差异。大约80%的培养切片显示出神经生长，在整个培养期间都可以对其进行测量(2)。展示了使用tauGFP小鼠品系的代表性结果。
