4D monitoring of stem cell differentiation by dielectric spectroscopic optical coherence tomography

近年来，干细胞的治疗潜力已得到多边认可。尽管如此，监测它们分化成不同谱系的手段是繁琐的和破坏性的，妨碍了3D组织再生的动态研究。在这个项目中，我们建议开发一种没有生物标记剂的成像技术，以在单细胞水平上在时间和空间（4D）上监测分化的干细胞。每个细胞系都具有生物电特征，其将细胞的响应转换为与细胞膜和细胞质的蛋白质含量相关的频率依赖性电磁场。这种生物电特征已经在介电光谱和介电电泳中用于细胞分选。分化中的干细胞根据其各种分化状态具有动态特征。在EMF激发期间涉及的生物机制也通过细胞成分的重组产生细胞的散射性质的变化。这种光学特性的变化将被利用来通过光学相干断层扫描（OCT）在单细胞水平上在3D中监测干细胞分化。OCT是一种无创干涉技术，允许在没有标记剂的情况下进行深度组织成像。OCT的最新进展使相位显微术在样品光路上具有纳米分辨率。通过将介电光谱系统与OCT同步，我们将记录频率相关光学特征（FDOS），并将其与2D和3D中的区分事件相关联。商业脂肪来源的干细胞（ADSC）将根据良好建立的方案在2D培养板和支持3D培养的商业藻酸盐基质中分化成三种不同的谱系（骨、软骨、脂肪组织）。新成像技术（DSOCT）的多模态将用于真实的实时记录差异事件（光学、介电及其组合（FDOS））。DSOCT的灵敏度也将与Hay博士合作，通过将hESC分化为三个不同发育阶段的肝细胞来测试：中内胚层，内胚层和肝内胚层。新的成像技术能够在单细胞水平上连续和非侵入性地跟踪干细胞分化的各个发育阶段，而无需标记剂，这将导致再生医学的许多突破。

In the recent years the therapeutic potential of stem cells has been recognized multilaterally. Still, the means to monitor their differentiation into different lineages are tedious and destructive precluding dynamic studies of 3D tissue regeneration. In this project, we propose to develop an imaging technology without biological labelling agents to monitor in time and space (4D) differentiating stem cell at a single-cell level. Every cell line has a bioelectrical signature that translates the response of a cell to a frequency dependent electro-magnetic field related to the proteins content of the cell membrane and cytoplasm. This bioelectrical signature is already exploited in dielectric spectroscopy and dielectrophoresis for cell sorting. A differentiating stem cell has a dynamic signature according to its various differentiation states. The biological mechanisms involved during EMF excitation generate also a change of the scattering properties of the cell by the reorganization of the cell components. This variation of the optical properties will be exploited to monitor in 3D at the single-cell level stem cell differentiation by Optical Coherence Tomography (OCT). OCT is a non invasive interferometric technique allowing in depth tissue imaging without labelling agent. Recent advances in OCT enables phase microscopy with nanometer resolution on the sample optical path. By synchronising a dielectric spectroscopic system with an OCT we will record a frequency dependent optical signature (FDOS) and will correlate it to differentiating event in 2D and 3D. Commercially adipose derived stem cells (ADSC) will be differentiated into three different lineages (bone, cartilage, adipose tissue) according to well established protocols in 2D culture plate and in commercial alginate matrixes supporting 3D culture. The multimodality of the new imaging technology (DSOCT) will be used to record differentiating events (optical, dielectric, and their combination (FDOS)) in real time. The sensitivity of the DSOCT will also be tested in collaboration with Dr. Hay by differentiating hESC into liver cells at three different developmental stages: mesendoderm, endoderm and hepatic endoderm. The new imaging technology ability to track various developmental stages of stem cell differentiation continuously and non invasively at the single-cell level without labelling agents will lead to many breakthroughs in regenerative medicine.