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Cell viability, immunofluorescence analysis and advanced imaging technologies are frequently used to interrogate Matrigel matrix enabled 3D cultures.
Viability can be measured via the detection of DNA synthesis in proliferating cells, based on the incorporation of 5-ethynyl-2′-deoxyuridine (EdU). A protocol can be found below:
The labs of Mina Bissell at Lawrence Berkeley National Laboratory and Joan Brugge at Harvard Medical School have published extensively on 3D models using Matrigel matrix and have included a widely used immunofluorescence analysis preparation method. A few protocols can be found below:
Many labs have studied 3D architecture utilizing advanced imaging technologies. In the publication by Jorgens, et al. many of these methods were employed and are covered in the materials and methods section of the paper. Spheroid/organoid size and morphology, as well as cryogenic techniques, volume electron microscopy, and super-resolution light microscopy have been used to study phenotypic and functional attributes. A protocol can be found below:
Finally, recovery of cells from 3D Matrigel matrix cultures to be used for cell number determination, RNA isolation, and qPCR analysis can be accomplished using Corning cell recovery solution. Using the solution at low temperature (on ice) and applying mechanical disruption such as pipetting or the use of an orbital shaker will help de-polymerize the Matrigel matrix. Cell-cell interactions can be disrupted through the use of chelators and/or proteolytic enzymes such as Trypsin or Dispase.