There are many ways in which a 3D cell culture model can be characterized and the technique used for a specific assay will depend on the specific research question being asked. For example, understanding how a library of antibody drug conjugates penetrate into a solid tumor model would require the use of high content confocal imaging whereas measuring ATP would only require a plate reading assay. However, for all non-destructive imaging based assays (e.g. plate reading, wide-field microscopy, confocal microscopy) tissue clearing with Visikol® HISTO-M™ allows for a dramatic increase in the number of cells characterized and thus a more accurate readout which is indicative of the entire population of cells and not just those on the periphery.
Traditional characterization techniques
- 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.
Laser vs LED confocal imaging
When choosing a confocal system to image your 3D cell culture model it is important that you choose a system that employ a laser based excitation source instead of an LED based light source. LED light sources are more inexpensive but are non-coherent and thus you will see significant image artifacts and a lack of depth with this type of excitation.
The use of tissue clearing with 3D cell culture models allows for all of the cells within these models to be characterized instead of only characterizing the peripheral 1-3 cell layers. This results in the ability to characterize an entire 3D cell culture model instead of biasing results towards cells on the periphery of these models where they are most exposed to compounds, nutrients and oxygen. The type of imager you use for your research question will be based on the equipment you have access to but also your research question. Below we provide details on the different systems used for image based characterization.
The most powerful application of Visikol HISTO-M tissue clearing is to pair it with confocal microscopy for the complete 3D imaging of 3D cell culture models. Generally, confocal imaging is most ideal with an inverted water dipping objective combined with glass flat bottom plates and a laser based-excitation source. However, with Visikol HISTO-M the imaging depth improvement with water dipping objectives is minimal and for some models a depth of 1 mm can be achieved with an inexpensive air objective. While glass flat bottom plates provide the best imaging quality, many researchers generate their 3D cell culture models using ultra low attachment U-bottom plates which are less ideal for imaging. For these U-bottom plates we highly suggest using a laser based confocal system (e.g. CellInsight CX7 LZR, Perkin Elmer Opera Phenix, GE IN CELL 6000/6500, Yokogawa CV7000) instead of an LED based confocal system (e.g. Perkin Elmer Operetta, Molecular Devices ImageXpress, CellInsight CX7 LED) as the laser based systems will dramatically reduce image artifacts while improving imaging depth and overall image quality. While confocal microscopy allows for every cell within a 3D cell culture model to be depicted in 3D, generating hundreds of images in mutiple channels from a single 3D cell culture model greatly reduces throughput and thus might not be ideal for your research question.
Wide field microscopy
Most researchers do not think that tissue clearing would be amenable to wide field microscopy as Z-sectioning is not possible, However, Visikol HSITO-M tissue clearing will allow for the signal from all of the cells within a 3D cell culture model to be detected which will also reduce the bias imposed by traditional 3D cell culture imaging without tissue clearing. Though Z-sectioning is not possible with wide field microscopy, wide field microscopy allows for much higher throughput and consequently much larger sample sizes.
Tips and tricks for 3D cell culture model imaging
Apparent loss of signal: Occasionally researchers will tell us that Visikol HISTO-M seems to have quenched the fluorescent signal in their sample, since the total signal intensity is reduced compared to non-cleared specimens. The reason for this is not quenching of fluorescence but instead that Visikol HISTO-M will greatly reduce the reflection of light within the 3D cell culture model, reducing the amount of signal reflected back to the camera. A useful analogy is using headlights when it is foggy; the fog (non-cleared samples) reflects most of the light back, increasing the apparent brightness. As such, increased exposure times may be necessary.
DAPI vs. Hoescht: We always suggest that researchers use DAPI instead of Hoescht with tissue clearing.
Centrifuge your plates before imaging: We have noticed that 3D cell culture models can stick to the sides of wells which makes imaging challenging especially if models within a plate are in different Z-planes. Therefore, we suggest waiting for models to settle, centrifuging plates or using magnets and magnetic nano-shuttles to cause models to come to the bottom and center of wells.