Technical protocols used for GrowDex cultures

Cells can be fluorescently dyed either prior to embedding in GrowDex hydrogels, or dyed whilst embedded in situ, growing in GrowDex hydrogels. This has been shown in many publications. Some example protocols are summarized below.

Fixing cells with PFA

Fixing cells in GrowDex hydrogels with paraformaldehyde prior to immune-fluorescence staining are detailed in Sheard et. al., (2019) [8] and Auvinen et. al., (2019) [9]. Cells cultured in GrowDex hydrogels can be fixed using paraformaldehyde (PFA) whilst embedded in the GrowDex hydrogel, or the cells can be liberated from the hydrogel using GrowDase, and the fixed.

Whilst cells are embedded in GrowDex hydrogels, remove the supernatant, and add 4% PFA for 15 – 20 mins. Incubate the cells at 4 °C or RT as per standard practice. After incubation, remove the PFA and wash the cells twice with PBS for 5 mins. After washing with PBS, the fixed cells embedded in GrowDex can be prepared for immuno-fluorescent staining including permeabilization and anti-body staining.

Cells can be fluorescently stained with fluorescent antibodies (primary or primary/secondary conjugated) whilst embedded in situ in GrowDex hydrogels or following GrowDase digestion of GrowDex. This has been shown in many publications [6, 8-11]. Some example protocols are summarized below.

It is possible to mix different ECM molecules with the GrowDex hydrogels. These include Collagen, Laminin and Fibronectin.

GrowDex hydrogels have been combined with transwell inserts and used in many applications. For example, cells can be embedded inside GrowDex hydrogels which can be placed inside the inner chamber of the transwell insert [8, 10, 15-17], where GrowDex hydrogels have been used to assist with cell seeding on the underside of a transwell insert [18], or where GrowDex hydrogels have been used as a barrier matrix for a chemo-attractant assay [19]. Below are a few example protocols extracted from the publications mentioned above.

Several different cell viability assays can be successfully used for the 3D cell cultures in GrowDex. GrowDex allows the diffusion of the reagent compounds to reach the cells in the hydrogel. Please find the protocols for different viability assays below.