Minuth Will W, Strehl Raimund
Department of Molecular and Cellular Anatomy, University of Regensburg, D-93053 Regensburg, University Street 31, Germany.
Biomed Mater. 2007 Jun;2(2):R1-R11. doi: 10.1088/1748-6041/2/2/R01. Epub 2007 Mar 7.
Epithelia act as biological barriers, which are exposed to different environments at the luminal and basal sides. To simulate this situation and to improve functional features an in vitro gradient perfusion culture technique was developed in our laboratory. This innovative technique appears to be simple at first sight, but the performance needs practical and theoretical knowledge. To harvest intact epithelia after a long-term gradient culture period of many days, leakage, edge damage and pressure differences in the system have to be avoided so that the epithelial barrier function is maintained continuously. Unexpectedly, one of the major obstacles are micro-injuries in the epithelia caused by gas bubbles, which arise during transportation of the medium or due to respiration of the cultured tissue. Gas bubbles randomly accumulate either at the luminal or basal fluid flow of the gradient perfusion culture container. This phenomenon results in fluid pressure differences between the luminal and basal perfusion compartments of the gradient container, which in turn leads to damage of the barrier function. Consequently, the content of gas bubbles in the transported culture medium has to be minimized. Thus, our technical concept is the reduction of gas bubbles while keeping the content of oxygen constant. To follow this strategy we developed a new type of screw cap for media bottles specifically designed to allow fluid contact only with tube and not with cap material. Furthermore, a gas expander module separates gas bubbles from the liquid phase during transportation of the medium. Finally, a new type of gradient culture container allows a permanent elimination of transported gas bubbles. Application of this innovative equipment optimizes the parallel transportation of fluid in the luminal and basal compartments of a gradient culture container.
上皮组织作为生物屏障，在管腔侧和基底侧暴露于不同环境。为模拟这种情况并改善功能特性，我们实验室开发了一种体外梯度灌注培养技术。这项创新技术乍一看似乎很简单，但实际操作需要实践和理论知识。为在多天的长期梯度培养期后收获完整的上皮组织，必须避免系统中的渗漏、边缘损伤和压力差，以便上皮屏障功能得以持续维持。出乎意料的是，主要障碍之一是由气泡引起的上皮组织微损伤，这些气泡在培养基运输过程中或由于培养组织的呼吸而产生。气泡随机积聚在梯度灌注培养容器的管腔或基底液流处。这种现象导致梯度容器的管腔和基底灌注隔室之间出现流体压力差，进而导致屏障功能受损。因此，必须将运输的培养基中的气泡含量降至最低。所以，我们的技术理念是在保持氧气含量恒定的同时减少气泡。为遵循这一策略，我们开发了一种新型的培养基瓶螺帽，专门设计成仅允许流体与管子接触，而不与螺帽材料接触。此外，一个气体膨胀模块在培养基运输过程中将气泡与液相分离。最后，一种新型的梯度培养容器可永久消除运输过程中的气泡。应用这种创新设备可优化梯度培养容器管腔和基底隔室中流体的平行运输。
