Noninvasive imaging of embryonic stem cell cultures by multiphoton microscopy reveals the significance of collagen hydrogel preparation parameters

摘要

In situ multiphoton microscopy (MPM) imaging method combining two-photon fluorescence (TPF) and second harmonic generation (SHG) optical signals helped to discover localized disaggregation and restructuring of collagen fibers within 3D hydrogels as a result of stem cell culture. We prepared hydrogels at different solid contents (2 g l(-1) or 4 g l(-1)) and self-assembly temperatures (27 degrees C or 37 degrees C). In a separate experiment, we stabilized 2 g l (-1) 37 degrees C hydrogels with the nontoxic cross-linkers carbodiimide (EDC), EDC/N-hydroxysuccinimide (NHS) or genipin. We characterized all collagen hydrogels nondestructively with MPM with and without culturing embryonic stem cells on/within them. We used rheology measurements to assess the flow behavior of the materials we prepared. All crosslinkers reduced the viscous part G '' of the modulus, implying that cross-linked materials are more rigid and non-flowing compared to non-cross-linked hydrogels. We evaluated nine possible scenarios of how embryonic stem cells could be set up to interact with collagen scaffolds when used in tissue engineering or regenerative medicine applications. The cells encapsulated or placed on top of 4 g l(-1) self-assembled hydrogels extended significantly slower than cells interfaced with 2 g l(-1) materials. Cultured cells modified the structure within non-cross-linked materials while the structure within cross-linked hydrogels was less affected. The non-invasive in situ MPM imaging analysis in this study is valuable because it enables longitudinal, 3D analysis of the structural and functional status of thick, opaque scaffolds as they interact with stem cells and are being degraded.