Polymers Division, Material Measurement Laboratory, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA.
Biomaterials. 2012 Mar;33(7):2119-26. doi: 10.1016/j.biomaterials.2011.11.058. Epub 2011 Dec 20.
The focus on creating tissue engineered constructs of clinically relevant sizes requires new approaches for monitoring construct health during tissue development. A few key requirements are that the technology be in situ, non-invasive, and provide temporal and spatial information. In this work, we demonstrate that optical coherence microscopy (OCM) can be used to assess cell viability without the addition of exogenous probes in three-dimensional (3D) tissue scaffolds maintained under standard culture conditions. This is done by collecting time-lapse images of speckle generated by sub-cellular features. Image cross-correlation is used to calculate the number of features the final image has in common with the initial image. If the cells are live, the number of common features is low. The number of common features approaches 100% if the cells are dead. In control experiments, cell viability is verified by the addition of a two-photon fluorescence channel to the OCM. Green fluorescent protein transfected human bone marrow stromal cells cultured in a transparent poly(ethylene glycol) tetramethacrylate hydrogel scaffold is used as the control system. Then, the utility of this approach is demonstrated by determining L929 fibroblast cell viability in a more challenging matrix, collagen, an optical scatterer. These results demonstrate a new technique for in situ mapping of single cell viability without any exogenous probes that is capable of providing continuous monitoring of construct health.
关注创建具有临床相关尺寸的组织工程构建体需要新的方法来监测组织发育过程中的构建体健康。一些关键要求是该技术为原位、非侵入式，并提供时间和空间信息。在这项工作中，我们证明了光学相干显微镜（OCM）可以在不添加外源性探针的情况下用于评估三维（3D）组织支架中细胞活力，这些支架在标准培养条件下维持。这是通过采集由亚细胞特征产生的散斑的时移图像来完成的。图像互相关用于计算最终图像与初始图像共有的特征数量。如果细胞存活，则共同特征的数量较低。如果细胞死亡，则共同特征的数量接近 100％。在对照实验中，通过向 OCM 添加双光子荧光通道来验证细胞活力。转染绿色荧光蛋白的人骨髓基质细胞在透明聚（乙二醇）四甲基丙烯酸酯水凝胶支架中培养用作对照系统。然后，通过确定在更具挑战性的基质胶原（一种光散射体）中 L929 成纤维细胞活力来证明该方法的实用性。这些结果证明了一种新的无需任何外源性探针即可原位映射单细胞活力的技术，该技术能够提供构建体健康的连续监测。
