Over the last five years 3D cell cultures have begun to be readily adopted into the drug discovery pipeline due to their improved in vivo relevancy compared to traditional 2D monolayer in vitro models. However, though these models are becoming more commonly used in drug discovery studies, there is currently a major limitation in how they are analyzed as researchers are currently using methods originally developed for 2D analysis. The current paradigm for analyzing these models consists of one of the following techniques which have their own individual limitations:
- Traditional Pathology: Cell culture models can be analyzed using traditional pathology where they are removed from well plates and processed. Though this method is capable of acquiring pathological information from tissues, the size of these models makes this process super low-throughput, tedious and expensive.
- Dissolution Based Assays: Many researchers are currently using dissolution based assays where cell culture models are homogenized and analyzed using a fluorescence probe such as Promega’s CellTiter-Glo Luminescent kit. While these assays are high-throughput, they do not capture the 3D data held within these models which makes them intrinsically more valuable than traditional 2D cell culture models.
- Wide Wield Imaging: Well plates can be characterized using wide field imaging where fluorescent markers such as GFP, immunofluorescence and chemical dyes are imaged. Though these assays are high-throughput, they suffer from a fundamental limitation which is that light attenuation results in an imaging depth of only a few cell layers. Therefore, only data from the periphery of the model is depicted and there is a significant bias to only characterize the periphery of these models which differs substantially from the interior.
- Confocal Imaging: Confocal imaging of 3D cell culture models can be high-throughput through use of a high content system such as the Thermo Fisher CX7 LZR, Molecular Devices ImageXpress, Yokogawa CV7000, Perkin Elmer Opera Phenix/CLS Operata or General Electic IN CELL 6000/6500. While these devices allow for a reduction in out of plane light, improved image quality and Z-stacks, they are still limited by light attenuation and thus only the periphery of these models is characterized.
Therefore, the major limitation with the current 3D cell culture imaging paradigm is that none of the current techniques are capable of characterizing these models in their entirety and acquring truly 3D data. To address this problem, we have developed our Visikol HISTO-M reagent which rapidly renders tissues transparent. We have shown that through the application of Visikol HISTO-M reagent to 3D cell culture models that we can dramatically improve both wide field imaging and confocal microscopy.