Product Number 3448-020-K
3D Cultures exhibit cellular behaviors and morphologies similar to those seen in vivo, however, the adaptation of these models for studying biochemical processes has been impeded by the challenge of separating intact cells from extra-cellular proteins comprising the hydrogel. Commonly, proteases are employed to degrade these extracellular proteins, however, proteases also degrade proteins on the cell surface and protease activity may carry over into lysate preparations. Non-enzymatic methodologies have also been described for depolymerizing extracellular matrix proteins, although the implementation of these protocols remains problematic for some researchers. Cultrex® 3D Culture Cell Harvesting Kit provides an optimized and standardized solution for the isolation and normalization of cell lysates from 3-D Culture Matrix™ BME or Laminin I for subsequent biochemical analysis.
These procedures should be performed in a biological hood utilizing aseptic technique to prevent contamination.
Optimal depolymerization of BME or Laminin I requires at least a five-fold excess of 1X Cell Harvesting Buffer and is more efficient with larger ratios. The reaction should be performed in 15 ml conical tubes for optimal cell pelleting. Larger volumes and high density hydrogels may require dividing samples into multiple tubes for optimal depolymerization.
Figure 1. Morphology of MCF-10A (A,B) and MCF-7 cells (C,D) in traditional 2D Culture and 3D BME Culture, Scale = 250 µm
Figure 2.Harvesting Cells From 3D Culture.
Figure 3. Evaluation of RNA expression via RT-PCR (A) and protein expression via Western blotting (B) for GAPDH from MCF-10A and MCF-7 mammary epethelial cells isolated from either traditional 2D culture or 3D BME culture.