Yanpaisan W, King N J, Doran P M
Department of Biotechnology, University of New South Wales, Sydney NSW 2052, Australia.
Biotechnol Bioeng. 1998 Jun 5;58(5):515-28. doi: 10.1002/(sici)1097-0290(19980605)58:5<515::aid-bit8>3.0.co;2-g.
Flow cytometry was used to measure cell cycle parameters in Solanum aviculare plant cell suspensions. Methods for bromodeoxyuridine (BrdU) labeling of plant nuclei were developed so that cell cycle times and the proportion of cells participating in growth could be determined as a function of culture time and conditions. The percentage of cells active in the cell cycle at 25 degrees C decreased from 52% to 19% within 7.6 d of culture; presence of a relatively large proportion of non-active cells was reflected in the results for culture growth. While the maximum specific growth rate of the suspensions at 25 degrees C was 0.34 d-1 (doubling time: 2.0 d), the specific growth rate of active cells was significantly greater at 0.67 d-1, corresponding to a cell cycle time of 1.0 d. A simple model of culture growth based on exponential and linear growth kinetics and the assumption of constant cell cycle time was found to predict with reasonable accuracy the proportion of active cells in the population as a function of time. Reducing the temperature to 17 degrees C lowered the culture growth rate but prolonged the exponential growth phase compared with 25 degrees C; the percentage of cells participating in the cell cycle was also higher. Exposure of plant cells to different agitation intensities in shake flasks had a pronounced effect on the distribution of cells within the cell cycle. The proportion of cells in S phase was 1.8 times higher at a shaker speed of 160 rpm than at 100 rpm, while the frequency of G0 + G1 cells decreased by up to 27%. Because of the significant levels of intraculture heterogeneity in suspended plant cell systems, flow cytometry is of particular value in characterizing culture properties and behavior.
采用流式细胞术测定了滨藜属植物细胞悬浮液中的细胞周期参数。开发了用于植物细胞核溴脱氧尿苷（BrdU）标记的方法，以便能够根据培养时间和条件确定细胞周期时间以及参与生长的细胞比例。在25℃下培养7.6天内，处于细胞周期活跃状态的细胞百分比从52%降至19%；培养生长结果反映出存在相对较大比例的非活跃细胞。虽然25℃下悬浮液的最大比生长速率为0.34 d-1（倍增时间：2.0天），但活跃细胞的比生长速率显著更高，为0.67 d-1，对应细胞周期时间为1.0天。发现一个基于指数和线性生长动力学以及细胞周期时间恒定假设的简单培养生长模型能够以合理的准确度预测群体中活跃细胞比例随时间的变化。将温度降至17℃会降低培养生长速率，但与25℃相比会延长指数生长期；参与细胞周期的细胞百分比也更高。在摇瓶中使植物细胞暴露于不同搅拌强度下对细胞在细胞周期内的分布有显著影响。在160 rpm的摇床速度下，处于S期的细胞比例比100 rpm时高1.8倍，而G0 + G1期细胞的频率最多降低27%。由于悬浮植物细胞系统中培养物内部存在显著的异质性水平，流式细胞术在表征培养物特性和行为方面具有特殊价值。
