Rabinowitz Claudette, Moiseeva Elisabeth, Rinkevich Baruch
Israel Oceanography and Limnological Research, National Institute of Oceanography, Tel Shikmona, PO Box 8030, Haifa, 31080, Israel.
Cell Tissue Res. 2016 Dec;366(3):693-705. doi: 10.1007/s00441-016-2495-6. Epub 2016 Sep 13.
We report here a novel approach for the extraction, isolation and culturing of intact ectodermal tissue layers from a model marine invertebrate, the sea anemone Nematostella vectensis. A methodology is described in which a brief exposure of the animal to the mucolytic agent N-acetyl-L-cysteine (NAC) solution triggers the dislodging of the ectodermis from its underlying basement membrane and mesoglea. These extracted fragments of cell sheets adherent to culture-dish substrates, initially form 2D monolayers that are transformed within 24 h post-isolation into 3D structures. These ectodermal tissues were sustained in vitro for several months, retaining their 3D structure while continuously releasing cells into the surrounding media. Cultures were then used for cell type characterizations and, additionally, the underlying organization of actin filaments in the 3D structures are demonstrated. Incorporation of BrdU and immunohistochemical labeling using p-histone H3 primary antibody were performed to compare mitotic activities of ectodermal cells originating from intact and from in vivo regenerating animals. Results revealed no change in mitotic activities at 2 h after bisection and a 1.67-, 1.71- and 3.74-fold increase over 24, 48 and 72 h of regeneration, respectively, depicting a significant correlation coefficient (p < 0.05; R = 0.74). A significant difference was found only between the control and 3-day regenerations (p = 0.016). Cell proliferation was demonstrated in the 3D ectodermis after 6 culturing days. Moreover, monolayers that were subjected to Ca++/Mg++ free medium for the first 2 h after isolation and then replaced by standard medium, showed, at 6 days of culturing, profuse appearance of positive p-histone H3-labeled nuclei in the 3D tissues. Cytochalasin administered throughout the culturing period abolished all p-histone H3 labeling. This study thus depicts novel in vitro tissue culturing of ectodermal layers from a model marine invertebrate, demonstrating the ease with which experiments can be performed and cellular and molecular pathways can be revealed, thus opening studies on 2D tissue organizations and morphogenesis as well as the roles of cellular components in the formation of tissues in this organism.
我们在此报告一种从海洋无脊椎动物模型海葵星状海葵（Nematostella vectensis）中提取、分离和培养完整外胚层组织层的新方法。本文描述了一种方法，即让动物短暂暴露于溶粘蛋白剂N - 乙酰 - L - 半胱氨酸（NAC）溶液中，会促使外胚层从其下方的基底膜和中胶层上脱落。这些附着在培养皿底物上的细胞片片段最初形成二维单层，在分离后24小时内转变为三维结构。这些外胚层组织在体外维持了几个月，保持其三维结构，同时不断向周围培养基中释放细胞。然后将培养物用于细胞类型表征，此外，还展示了三维结构中肌动蛋白丝的基础组织。进行了BrdU掺入和使用磷酸化组蛋白H3一抗的免疫组织化学标记，以比较来自完整动物和体内再生动物的外胚层细胞的有丝分裂活性。结果显示，二等分后2小时有丝分裂活性无变化，在再生24、48和72小时后分别增加了1.67倍、1.71倍和3.74倍，呈现出显著的相关系数（p < 0.05；R = 0.74）。仅在对照组和3天再生组之间发现显著差异（p = 0.016）。培养6天后，在三维外胚层中证明了细胞增殖。此外，分离后最初2小时置于无Ca++/Mg++培养基中，然后换成标准培养基的单层细胞，在培养6天时，在三维组织中显示出大量磷酸化组蛋白H3标记阳性的细胞核。在整个培养期间施用细胞松弛素消除了所有磷酸化组蛋白H3标记。因此，本研究描述了从海洋无脊椎动物模型中进行外胚层层的新型体外组织培养，证明了进行实验的简便性以及揭示细胞和分子途径的可能性，从而开启了关于二维组织组织和形态发生以及该生物体中细胞成分在组织形成中的作用的研究。
