96 Prolonged Duration Of Oxygen Equilibration Is Required For Hypoxic Culture Conditions
96 Prolonged Duration Of Oxygen Equilibration Is Required For Hypoxic Culture Conditions
复制标题
96 缺氧培养条件需要延长氧平衡时间
DOI：
10.1136/bjsports-2014-094114.95
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发表时间：
2014
影响因子：
18.4
通讯作者：
Smith R
中科院分区：
文献类型：
--
作者：
Smith R
前言模拟生理条件进行低氧素体外研究需要特别考虑培养基的溶解氧水平（DO）预处理。增殖和表型的高度分化的细胞从组织，如肌腱，韧带和骨髓，可以改变当暴露于不同的氧气水平比在他们的正常nich.1建立缺氧培养条件内的媒体需要特定的时间范围取决于最终的缺氧水平所需的。2在这项研究中，DO水平达到设定点所需的平衡时间进行了研究。我们表明，DO平衡是没有达到几个小时，并可能有影响的读数在早期取样的实验组织，如asex vivotendon explores.MethodsFour液体介质进行了测试：蒸馏去离子水，Dulbecco的磷酸盐缓冲盐水，含有10%胎牛血清（FBS）的Dulbecco改良Eagle培养基（D10）和含有15%FBS的DMEM（D15）（所有初始均在环境大气气体组成下平衡）在37 ℃的加湿培养箱中暴露于8%O2（平衡5%CO2，87%N2），并使用Jenway 970便携式DO 2 meter（Jenway，UK）测量DO水平长达8小时。还使用磁棒搅拌（S）培养基以研究混合对氧溶解速率的影响。实时监测的温度measured.ResultsThere是一个逐渐减少的DO对设定点（8%），虽然设定点，直到8小时，在所有媒体（图1）。两种培养基的DO降低率无显著差异。搅拌促进在前2小时内更快的氧气去除，然而在随后的时间点没有观察到这种初始优势。在D10中培养的细胞的存在并不影响DO减少模式相比，S和非搅拌（NS）conditions.DiscussionIn S和NS组的最低8小时预处理需要达到指定的氧气设定点。这些结果表明，预处理的培养基在缺氧实验和优化这一时期是必不可少的。这个预处理期至关重要，因为DO逐渐降低至设定点会不断改变实验的细胞和分子特征，并可能影响早期采样。参考资料Chung DJ等人Res Vet Sci. 2012;92（1）：66- 75 Newby D.et al. Placenta. 2005;26（4）：353-357
IntroductionMimicking physiological conditions for hypoxicin vitrostudies requires special consideration regarding the preconditioning of culture media with respect to dissolved oxygen level (DO). The proliferation and phenotype of highly differentiated cells from tissues such as tendons, ligaments and bone marrow, can alter when exposed to different oxygen levels than in their normal niche.1Establishment of hypoxic culture conditions within media requires specific time frames depending on final level of hypoxia desired.2In this study the equilibration time required for DO level reaching the set point was investigated. We show that DO equilibrium is not attained for several hours and may have implications on readouts in early sampling of experimental tissues such asex vivotendon explants.MethodsFour liquid media were tested: distilled de-ionised water; Dulbecco’s phosphate buffer saline; Dulbecco’s modified eagle medium containing 10% foetal bovine serum (FBS) (D10) and DMEM containing 15% FBS (D15) (all initially equilibrated at ambient atmospheric gas composition) were exposed to 8% O2(balance 5% CO2, 87% N2) in a humidified incubator at 37oC and DO level measured up to 8 h with a Jenway 970 portable DO2metre (Jenway, UK). Media was also stirred (S) using a magnetic bar to study the effect of mixing on oxygen dissolving rate. Real-time monitoring of the temperature was measured.ResultsThere was a gradual reduction of DO toward the set point (8%) although the set point was not reached until 8 h in all media (Figure 1). There was no significant different between the media in the rate of DO reduction. Stirring facilitated faster oxygen removal in the first 2 h period however this initial advantage was not observed at later time points. The presence of cells cultured in D10 did not influence the DO reduction pattern compared to S and non-stirring (NS) conditions.DiscussionIn both S and NS group a minimum of 8 h pre-conditioning was required to reach the designated oxygen set point. These results show that preconditioning of culture media in hypoxic experiments and optimisation of this period is essential. This pre-conditioning period is critical as the gradual decrease of DO towards the set point can constantly alter the cellular and molecular profile of the experiment and may impact early sampling.ReferencesChung DJ,et al. Res Vet Sci. 2012;92(1):66–75Newby D.et al. Placenta. 2005;26(4):353–357