CARS and SHG microscopy to follow the collagen production in living human corneal fibroblasts and mesenchymal stem cells in fibrin gel 3D cultures

TL;DR

This study demonstrates the use of combined CARS and SHG microscopy as a non-invasive, label-free method to monitor collagen production and stem cell differentiation in 3D cultures over time, with high sensitivity and depth.

Contribution

It introduces a multimodal imaging technique for real-time, long-term observation of collagen synthesis and cell differentiation in thick 3D scaffolds, advancing regenerative medicine tools.

Findings

Enabled detection of small collagen amounts within hours

Allowed imaging of thick tissue sections up to 4 mm

Confirmed scaffold-induced stem cell differentiation

Abstract

Coherent anti-Stokes Raman scattering (CARS) microscopy is combined with second harmonic generation (SHG) technique in order to follow the early stage of stem cell differentiation within a 3D scaffold. CARS microscopy can detect lipid membranes and droplet compartments in living cells and SHG microscopy enables a strong imaging contrast for molecules with a non-centrosymmetric ordered structure like collagen. One of the first evidence of hMSCs differentiation is the formation of an extracellular matrix (ECM) where the collagen protein is its main component. This work demonstrated the multimodal CARS and SHG microscopy as a powerful non-invasive label free technique to investigate the collagen production dynamic in living cell 3D cultures. Its ability to image the cell morphology and the produced collagen distribution on a long term (4 weeks) experiment allowed to obtain important information about the cell-scaffold interaction and the ECM production. The very low limit reached in detecting collagen has permitted to map even the small amount of collagen produced by the cells in few hours of culture. This demonstrates multimodal CARS and SHG microscopy as a novel method to follow cells collagen production and cells differentiation process. In addition the experiment shows that the technique is a powerful tool for imaging of very thick sections (about 4 mm). The study conducted on mesenchymal stem cell in fibrin gel cultures confirmed that differentiation stimulus is induced by the scaffold. The monitoring of stem cell differentiation within a scaffold in a non-destructive way will be an important advantage in regenerative medicine and tissue engineering field.