Long-term Video-micrographic Observation on Proliferation and differentiation of Human Eosinphil Precursors in Vitro.

在本研究中，我们使用了一种新的设备来压缩嗜酸性粒细胞前体，使其精细结构变得更加明显，而不会失去其增殖和分化的能力。将脐带血分离的单核细胞置于含有10%牛胎血清、rhIL-3和rhIL-5的Iscove改良Dulbecco培养基中，在含5% CO_2的潮湿环境中培养9天。收集非黏附细胞并置于人纤维蛋白凝块中。在标本上放了一个小的盖玻片。细胞被压缩使用一个新的装置与一个分叉杆施加在覆盖滑动，施加的压力是由一个螺钉调整。细胞在上述相同的培养基中培养。将尼康相衬倒置显微镜置于恒温室中，在培养5-9天的时间内获得延时视频显微图像。在此观察期间，大多数嗜酸性粒细胞前体分裂2-3次，产生自由移动的成熟嗜酸性粒细胞。生成时间从17小时8分到96小时3分不等。在成熟的嗜酸性粒细胞中观察到更长的世代时间的趋势。嗜酸性粒细胞的运动模式随其分化而改变：核为圆形或卵圆形的最不成熟的嗜酸性粒细胞前体，除了偶尔有宽的面纱外，几乎没有运动；中等成熟的嗜酸性粒细胞前体在一个方向上反复刺激和缩回一个相对狭窄的假足；成熟的嗜酸性粒细胞运动较快，呈宽杆状，后端圆形。这项研究表明，在适当的压迫下，人类嗜酸性粒细胞前体可以继续增殖和分化，这使得它们的精细结构变化更容易被观察到。

In the present study, we used a new device to compress eosinophil precursors allowing their fine structures to become more visible without losing their ability to proliferate and differentiate. Mononuclear cells separated from cord blood were cultured in Iscove's modified Dulbecco medium containing 10% bovine fetal serum, rhIL-3 and rhIL-5, for 9 days in a humidified atmosphere containing 5% CO_2. Non-adhering cells were harvested and placed in a human fibrin clot. A small cover slide was placed over the specimen. The cells were compressed using a new device with a forked rod applied on the cover slide, the pressure applied was adjusted by a screw. The cells were cultured in the same medium as described above. A Nikon phase-contrast inverted microscope placed in a thermostatic chamber was used to obtain time-lapsed video-micrography, over 5-9 days of culture. During this observation period, most of the eosinophil precursors divided 2-3 times to yield freely moving mature eosinophils. Generation time varied from 17 hours 8 minutes to 96 hours 3 minutes. A tendency towards a longer generation time in more mature eosinophils was observed. Pattern of motility in the eosinophils changed along with their differentiation : the most immature eosinophil precursors with a round or oval nucleus showed little motility except for occasionally throwing a wide veil ; intermediately mature eosinophil precursors repeatedly prodded and retracted a reatively narrow pseudopod in one direction ; mature eosinophils showed relatively fast motion taking a shape of a broad rod with a round posterior end.This study showed that human eosinophil precursors could continue to proliferate and differentiate under appropriate compression which allow their fine strucutral changes to be more easily visualizad.