Potential biological explanation of stimulation of colony growth in semi-solid agar by cytotoxic agents.
Potential biological explanation of stimulation of colony growth in semi-solid agar by cytotoxic agents.
复制标题
细胞毒性剂刺激半固体琼脂中集落生长的潜在生物学解释。
DOI：
10.1038/bjc.1983.279
复制
发表时间：
1983
影响因子：
8.8
通讯作者：
Sipes,NJ
中科院分区：
文献类型：
--
作者：
MeyskensJr,FL;Thomson,SP;Hickie,RA;Sipes,NJ
单细胞在半固体培养基中生长成集落已被广泛用作正常细胞和转化细胞的克隆形成潜力的量度（Park等人，1971; Thomson & Rauth，1974;库尔特奈，1976; Metcalf，1977; Buick等人，1977; Hamburger等人，1977; Salmon，1980）和作为细胞转化的体外标志物（Macpherson & Montagnier，1964; MacAllister & Reed，1968）。细胞在半固体培养基中的生长也已用于测量各种细胞毒性和非细胞毒性剂对克隆原性肿瘤细胞的作用（Salmon等人，1978;特维特等人，1980，1982; Von霍夫等人，1981; Meyskens等人，1981年）。接种的细胞数量与细胞团和集落形成（此处定义为生长单位）之间的关系应明确定义，以确保对克隆形成细胞扰动的有效解释。形成生长单位的增殖频率和程度被认为是肿瘤样品的克隆形成能力以及培养条件的函数。然而，文化条件的许多影响仍然不明确。因此，由于克隆形成试验通常是封闭的非补料系统，我们检查了接种的细胞数量、形成的生长单位数量、含有不同数量细胞的生长单位的相对频率和生长单位内形成的细胞总数之间的关系。我们发现，克隆形成细胞在琼脂中的克隆效率和增殖特性明显取决于接种的细胞数量。小鼠黑色素瘤细胞（CCL 53.1）在Falcon塑料烧瓶中的培养基（FIO加10%马血清和2%热激活胎牛血清）中生长为单层。用Tryodes溶液处理除去细胞并产生单细胞悬浮液。将不同数量的细胞接种在直径为35 mm的皮氏培养皿中的1.0 ml含0.3%琼脂的培养基（Bacto）中，超过1.0 ml含0.5%琼脂的培养基中。将板在37 ℃下在潮湿的5%CO2空气气氛中孵育18天。在随机选择的1、2.5或6.25 mm 2区域中对每组细胞（包含一个以上细胞）进行计数。将每单位面积的生长单位的平均数+ se乘以908 mm 2/35 mm培养皿，以获得每个培养皿的生长单位总数。不同直径的生长单位的数量使用生长单位的相对频率（通过用千分尺直接测量每个平板200个生长单位并乘以每个平板的生长单位总数获得）计算。构建列线图以确定每个生长单位的细胞数。这通过在含有< 8个细胞的生长单位中直接目视观察来完成。对于较大的生长单位，细胞的堆积和拥挤阻碍了准确计数，因此用显微操作器以每10 μ m间隔直径摘取5-10个生长单位，并在染色的制备物中计数细胞。通过将每种尺寸的生长单位的频率乘以每个生长单位的细胞数量并求和，计算生长单位内的细胞总数。群体倍增数的估计值是最小值，因为假设所有子代细胞都能够倍增，并且没有细胞损失发生。我们检查了与接种的鼠黑素瘤细胞数量相关的生长单位的大小和频率。平板接种的细胞数量与生长单位大小分布之间的关系见图1A、B。生长单位的大小随着接种细胞数量的增加而减小，即使在低数量的细胞中也是如此。
Growth of single cells into colonies in semisolid medium has been widely used both as a measure of the clonogenic potential of normal and transformed cells (Park et al., 1971; Thomson & Rauth, 1974; Courtenay, 1976; Metcalf, 1977; Buick et al., 1977; Hamburger et al., 1977; Salmon, 1980) and as an in vitro marker for cellular transformation (Macpherson & Montagnier, 1964; MacAllister & Reed, 1968). Growth of cells in semisolid medium also has been used to measure the effect of various cytotoxic and non-cytotoxic agents on clonogenic tumour cells (Salmon et al., 1978; Tveit et al., 1980, 1982; Von Hoff et al., 1981; Meyskens et al., 1981). The relation between the number of cells plated and the formation of clusters and colonies, defined here as growth units, should be clearly defined to assure validinterpretation of perturbations of clonogenic cells. The frequency and extent of proliferation to form growth units has been regarded as a function of the clonogeniccapacity of the tumour sample together with the conditions of culture. However, many of the effects of the conditions of culture remain undefined. Therefore, because clonogenic assaysare generally closed non re-fed systems, we have examined the relationship between the number of cells plated, the number of growth units formed, the relative frequency of growth units containing different numbers of cells, and the total number of cells formed within the growth units. We found that the cloning efficiency and proliferative characteristics of clonogenic cells in agar is significantly determined by the number of cells plated. Murine melanoma cells (CCL 53.1) were grown in medium (FIO plus 10% horse and 2% heatinactivated foetalcalf-serum) as monolayers in plastic Falcon flasks. Treatment with Tryodes solution removed the cells and produced suspensions of single cells. Different numbers of cells were plated in 1.0 ml of medium containing0.3% agar (Bacto) over 1.0 ml of 0.5% agar in medium in 35mm diameter Petri dishes. The plates were incubated in a humidified, 5% Co2, air atmosphere at 37 C for 18 days. Every group of cells (containing greater than one cell) was counted in randomly selected 1, 2.5, or 6.25 mm2 areas. The mean+ se number of growth units per area was multiplied by 908 mm2 per 35mm culture dish to obtain the total number of growth units per culture dish. The number of growth units of different diameters was calculated using the relative frequency of growth units by size, obtained by direct measurement of 200 growth units per plate with a micrometer, and multiplying by the total number of growth units per plate. A nomogram was constructed to determine the number of cells per growth unit. This was done by direct visual observation in growth units containing< 8 cells. For larger growth units stacking and crowding of cells prevented accurate counting so 5-10 growth units at each 10pm interval diameter were plucked with a micromanipulator and the cells counted in stained preparations. The total numbers of cells within the growth units were calculated by multiplying the frequency of growth units of each size by the number of cells per growth unit and summing. The estimate of the number of population doublings is a minimum number as it was assumed that all progeny cells were capable of doubling and no cell loss occurred.We examined both the size and frequency of the growth units in relation to the number of murine melanoma cells plated. The relationship between the number of cells plated and the distribution of growth units by size is shown in Figure 1A, B. The size of the growth units decreased as the number of cells plated increased, which was true even at low numbers of …