Every cell counts!

While 3D cell culture models are being adopted in the drug discovery space for their improved in vivo relevancy, the imaging techniques used to characterize these models have serious room for improvement. The problem is due to the thickness and opacity of the 3D cell culture models. Since they are too thick, light cannot penetrate to the center of the tissues, and so only the outer 2-3 layers of cells can be detected.  This causes the dark centers often seen in images of 3D cell culture models. Unfortunately, this effect introduces bias into results, since only the outermost cells can be detected, and those cells are the most exposed to oxygen, nutrients, and drug compound. Treatment with Visikol HISTO-M solves this problem.

Confocal Imaging

Clearing with Visikol HISTO-M renders the 3D model transparent, allowing for the detection of every single cell using confocal imaging with a High Content instrument.

 NCI-H2170 spheroids approx 250 um in diameter labeled with nuclear stain. Left is in PBS and right is the same spheroid after clearing with Visikol HISTO-M.

NCI-H2170 spheroids approx 250 um in diameter labeled with nuclear stain. Left is in PBS and right is the same spheroid after clearing with Visikol HISTO-M.

  3D cell culture in PBS, not cleared. A large majority of cells are not observable.

3D cell culture in PBS, not cleared. A large majority of cells are not observable.

  3D cell culture after clearing with Visikol HISTO-M - every cell in the model can be detected

3D cell culture after clearing with Visikol HISTO-M - every cell in the model can be detected

  Number of cells characterized in before and after clearing NCI-H2170 3D cell culture model

Number of cells characterized in before and after clearing NCI-H2170 3D cell culture model

Results are clear with Visikol HISTO-M

 
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   VISIKOL & INSPHERO APP NOTE    Application of Visikol HISTO-M clearing with InSphero 3D InSight™ microtissues and high-throughput confocal imaging.  ↓ Download Note

VISIKOL & INSPHERO APP NOTE

Application of Visikol HISTO-M clearing with InSphero 3D InSight™ microtissues and high-throughput confocal imaging.

↓ Download Note


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Improved sensitivity and reduction of bias

Significant differences between cleared and non-cleared tumor spheroids have been observed in the dose response curves of common antiproliferatives. The application of tissue clearing to 3D cell culture characterization has been shown to increase sensitivity to measure dose response by an order of magnitude due to the increased number of cells detected in cleared spheroids. Furthermore, highly significant differences in dose response are measured between cleared and non-cleared tumor spheroids due to the increased level of proliferation in the outermost cells.

 NCI-H2170 spheroids dosed with cisplatin and evaluated for cell proliferation  (Ki67 positive cells); A) Dose response relative to vehicle control; B) Absolute cell proliferation score

NCI-H2170 spheroids dosed with cisplatin and evaluated for cell proliferation  (Ki67 positive cells); A) Dose response relative to vehicle control; B) Absolute cell proliferation score