REGULATION OF SPONTANEOUS ACTIVITY AND GROWTH OF EMBRYONIC CHICK HEART CELLS IN TISSUE CULTURE
REGULATION OF SPONTANEOUS ACTIVITY AND GROWTH OF EMBRYONIC CHICK HEART CELLS IN TISSUE CULTURE
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DOI：
10.1016/0012-1606(67)90025-5
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发表时间：
1967-01-01
影响因子：
2.7
通讯作者：
DEHAAN, RL
中科院分区：
文献类型：
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作者：
DEHAAN, RL
1.1.在组织培养中表现出自发活动的胚胎心脏细胞的百分比（% BC）取决于各种各样的环境参数，包括组织解聚的模式、培养基的组成、细胞接种物的密度和其他培养条件，以及从其中取出细胞的胚胎的年龄。在0.05%胰蛋白酶中孵育三个8分钟的周期，解离7天的心脏，释放出最大数量的活细胞;当使用足够的组织进行解离以使所得细胞悬液达到2至3 × 106个细胞/ml或更多时，获得最佳结果。为了能够测定% BC，必须以足够低的密度接种细胞以防止广泛的细胞间接触。当密度超过100个细胞/mm 2时，许多细胞会与相邻细胞接触，从而无法区分细胞是由于自身的生电活动而搏动，还是对来自起搏相邻细胞的脉冲的反应。培养基中使用的血清在很大程度上影响自发搏动的细胞的百分比。总共测试了56个血清样本，产生了一系列的效果：从明显有毒的血清到给出“最佳”结果的血清。如果用血清制成的标准低钾培养基中的细胞外观健康，并且始终产生40- 50%BC，则认为血清样品是最佳的。在成人血清K+水平（4-5 meq/l）下，培养7天的心脏中约有20%的细胞自发跳动。增加细胞外K+会导致一些细胞停止活动。将K+降低至1 meq/l允许最大数量的潜伏起搏器变得活跃。用4、7、12和18天的胚胎心脏细胞测定了K+浓度与% BC的剂量反应曲线。从7天的心脏中获得最大数量的自发活性细胞。在最佳条件下，用1 meq/l K+培养基，活性范围在40和50%BC之间，平均值为44%BC。在单个平板中记录的最大活性细胞数量为64%BC。年龄较大的心脏产生的起搏器逐渐减少，在18天时最低平均值约为10% BC。早于7天的心脏也产生较少的自发活性细胞。4天的心脏培养物对培养基中升高的K+相对不敏感。由此可见，早期胚胎心脏的细胞内高Na+含量和快速Na+通量特征代表了早期起搏细胞可以维持足够大的钠钾电导率比率的装置，以允许在羊膜形成之前由卵子提供的高钾环境中自发活动的开始。两种类型的心脏细胞可以在培养物中进行形态学区分：F细胞，其具有典型的成纤维细胞的外观;和M细胞，其类似于骨骼肌的成肌细胞。F细胞占40-50%; M细胞占50- 60%。大多数显性起搏器是M细胞。然而，这些符合F细胞所有形态学标准的细胞中的一小部分（小于1%）也是自发活性的。在培养的心脏细胞中，有丝分裂活动可以仅仅通过鸡胚提取物的存在或不存在而被诱导或阻止。通过透析CEE以去除K+，可以获得表现出快速有丝分裂和高% BC的培养物。它的结论是，没有必要的拮抗作用之间存在的增长和维持分化的功能状态，在胚胎心脏细胞培养的条件下描述。
1.1. The percentage of embryonic heart cells that exhibit spontaneous activity (% BC) in tissue culture is dependent upon a wide variety of environmental parameters, including mode of tissue disaggregation, composition of the culture medium, density of cell inoculum, and other culture conditions, as well as the age of the embryo from which the cells are taken.2.2. Dissociating 7-day hearts with three 8-minute incubation cycles in 0.05% trypsin liberates a maximal number of viable cells; best results are obtained when enough tissue is used for dissociation to bring the resultant cell suspension to 2 to 3 × 106cells/ml or more.3.3. To be able to determine % BC, clls must be plated at densities low enough to prevent extensive intercellular contacts. At densities above about 100 cells/mm2many cells make contact with neighbors, making it impossible to distinguish whether a cell is beating because of its own electrogenic activity or as a response to an impulse conducted from a pacemaking neighbor.4.4. The serum used in a culture medium influences, to a marked degree, the percentage of cells which beat spontaneously. A total of 56 serum samples were tested, yielding a range of effects: from those sera which were clearly toxic to those which gave “optimal” results. A sample of serum was considered optimal if cells in standard low-potassium medium, made with that serum, were healthy in appearance, and consistently yielded 40–50% BC.5.5. At adult serum levels of K+(4–5 meq/l) approximately 20% of the cells in a culture from 7-day heart beat spontaneously. Increasing extracellular K+causes some cells to cease activity. Decreasing the K+to 1 meq/l permits a maximal number of latent pacemakers to become active. Dose-response curves relating K+concentration to % BC have been determined with cells from embryonic hearts aged 4, 7, 12, and 18 days.6.6. The greatest number of spontaneously active cells is obtained from 7-day hearts. Under optimal conditions, with 1 meq/l K+medium, activity ranged between 40 and 50% BC, with a mean of 44% BC. The greatest number of active cells ever recorded in a single plate was 64% BC. Older hearts yielded progressively fewer pacemakers, to a minimum mean of about 10% BC at 18 days. Hearts earlier than 7 days also yielded fewer spontaneously active cells.7.7. Four-day heart cultures are relatively insensitive to elevated K+in the culture medium. From this it is suggested that the high intracellular Na+-content and rapid Na+-flux characteristic of early embryonic hearts represent devices whereby early pacemaker cells can maintain a ratio of sodium: potassium conductance sufficiently large to permit the onset of spontaneous activity in the high-potassium environment provided by the egg before amnion formation.8.8. Two types of heart cells may be distinguished morphologically in cultures: F cells, which have the appearance of typical fibroblasts; and M cells, which resemble myoblasts of skeletal muscle. F cells comprise 40–50% of the cells; M cells 50–60%. Most overt pacemakers are M cells. However, a small proportion (less than 1%) of these which meet all the morphological criteria of F cells, are also spontaneously active.9.9. Mitotic activity in heart cell cultures can be induced or prevented merely by the presence or absence of chick-embryo extract. Cultures can be obtained which exhibit both rapid mitosis and high % BC, by dialyzing CEE to remove K+. It is concluded that no necessary antagonism exists between growth and the maintenance of a differentiated functional state in embryonic heart cells cultured under the conditions described.