Confocal scanning of intervertebral disc cells in 3D: Inside alginate beads and in native microenvironment

The interaction between cells and their extracellular matrix (ECM) is crucial to maintain both tissue and cellular homeostasis. Indeed, cell phenotype is significantly affected by the 3D microenvironment. Although highly convenient, isolating cells from the intervertebral disc (IVD) and growing them in 2D on plastic or glass substrates, causes them to rapidly lose their phenotype and consequently alter their gene and protein expression. While characterization of cells in their native or simulated 3D environment is preferred, such approaches are complexed by limitations in phenotypic readouts. In the current article, we describe a detailed protocol to study nucleus pulposus cells in 3D—embedded in alginate as a permeable cell‐staining reservoir, as well as adaptation for cell staining and imaging in their native ECM. This method allows for detection of phenotypical and cytoskeletal changes in cells within native tissue or 3D alginate beads using confocal microscopy, without the need for histological processing. In this article, we explain easy to adopt protocols that permit visualization of cells in their native matrix or biomimetic 3D system, including (A) how to build an inexpensive and easy to assemble sample holder that allows confocal visualization of cells embedded in alginate beads (B), and how to process rodent nucleus pulposus (C) for better staining and confocal visualization of cells in situ (D). Both without the need of further histological processing.