Dept. Anatomy, Embryology and Histology, section Histology, University Gent, Louis Pasteurlaan 2, B-9000, Gent, Belgium.
Cytotechnology. 1995 Jan;18(3):227-33. doi: 10.1007/BF00767770.
A two step method to obtain mesenchymal free 3.5 day old chick embryonic notochordsin vitro is presented. 1.) Notochords are isolated by mechanical microdissection from the embryos below the head and above the leg-buds. 2.) The dissected notochords are trypsinized to eliminate contaminating mesenchymal cells, while the perinotochordal sheath (PNS) is retained. After isolation and trypsinization, notochords are cut in standard 8mm lengths, explantedin vitro and incubated at 37°C. Immediately before incubation and after 3 and 6 daysin vitro, notochords are fixed and stained to follow the morphological changes. The total DNA content of notochords is measured before and during maintenancein vitro to evaluate their metabolic activities. Results show that during thein vitro period, the isolated mesenchymal free notochordal fragments can conserve their characteristic architecture. The total DNA content measurements indicate proliferative activity and a high viability of the notochords in ourin vitro system. In the present study, an isolation andin vitro method is offered which might be an effective tool to study the metabolic activities of chick embryonic notochordsin vitro in comparison toin vivo behaviour, in order to study the underlying mechanism of notochord regression.
介绍了一种两步法从 3.5 日龄鸡胚头部以下、腿部以上部位体外分离无中胚层的脊索。1.）通过机械显微解剖法从胚胎中分离脊索。2.）对分离的脊索进行胰蛋白酶消化，以去除污染的间质细胞，同时保留脊索周围鞘（PNS）。分离和胰蛋白酶消化后，将脊索切成标准的 8mm 长度，体外植入并在 37°C 下孵育。在孵育前、孵育 3 天和 6 天后，立即对脊索进行固定和染色，以观察形态变化。在体外维持过程中测量脊索的总 DNA 含量，以评估其代谢活性。结果表明，在体外培养期间，分离的无中胚层脊索片段可以保持其特征结构。总 DNA 含量测量表明，在我们的体外系统中，脊索具有增殖活性和高存活率。本研究提供了一种分离和体外方法，可能是一种有效的工具，用于研究鸡胚脊索的代谢活性，与体内行为进行比较，以研究脊索退化的潜在机制。
