Effect of Milk Renewal on Cell Viability In Vitro at Different Time Frames

Abstract The purpose of this study was to evaluate if the renewal of milk as a storage medium, every 12, 24 and 48 h, is able to increase its ability to maintain human periodontal ligament fibroblasts (PDLF) viability over time. PDLF were soaked in Minimum Essential Medium at 37 °C (MEM-37) (positive control), tap water (Water) (negative control) and in skimmed milk (44 wells) at 5 °C and 20 °C. The skimmed milk was renewed every 12 h (Milk-12), 24 h (Milk-24) and 48 h (Milk-48) in 11 wells of each plate, and the milk in the remaining 11 wells of each plate was maintained in situ (not renewed milk) (NRM). After 24, 48, 72, 96 and 120 h, cell viability was determined by the tetrazolium salt-based colorimetric (MTT) assay. Data were statistically analyzed by Kruskal-Wallis, Scheffé and Mann-Whitney tests (a=5%). At 5 °C, only Milk-48 was significantly better than NRM. At 20 °C, NRM was more effective than Milk-12 and Milk-24 in all time periods. In relation to the temperature (5 °C or 20 °C), renewal of milk at 5 °C was better in maintaining cell viability than the renewal at 20 °C. In conclusion, the renewal of milk was able to increase its ability to maintain cell viability only when performed every 48 h in milk maintained at 5 °C.

摘要 本研究旨在评估每隔12、24和48小时更换储存介质脱脂乳，是否能够随培养时间延长提升其维持人牙周膜成纤维细胞（human periodontal ligament fibroblasts, PDLF）活性的能力。将PDLF分别置于37 ℃的最小必需培养基（Minimum Essential Medium, MEM-37，阳性对照）、自来水（Water，阴性对照），以及5 ℃和20 ℃下的脱脂乳（共44孔）中孵育。每个培养板中，11孔脱脂乳分别每隔12 h（Milk-12）、24 h（Milk-24）和48 h（Milk-48）进行更换，剩余11孔脱脂乳则保持原位不更换（non-renewed milk, NRM）。分别于孵育24、48、72、96和120 h时，采用基于四唑盐的比色法（tetrazolium salt-based colorimetric, MTT）检测细胞活性。数据采用Kruskal-Wallis检验、Scheffé检验及Mann-Whitney检验进行统计学分析，检验水准设定为5%。在5 ℃条件下，仅Milk-48组的细胞活性维持效果显著优于NRM组；在20 ℃条件下，各观测时间点NRM组的细胞活性维持效果均优于Milk-12和Milk-24组。对比两种培养温度（5 ℃与20 ℃），5 ℃下更换脱脂乳的细胞活性维持效果优于20 ℃下的更换操作。综上，仅当在5 ℃条件下每隔48 h更换脱脂乳时，方可提升其维持细胞活性的能力。