Frequently used strategies to isolate ECM proteins from human placenta and adipose tissue

Background: The natural extracellular matrix (ECM) provides the optimal environment for cells. Many enzymatic or non-enzymatic based strategies to extract ECM proteins from tissues were published over the last years. However, every single isolation strategy reported so far is associated with specific bottlenecks. Experiment: In this study, frequently used strategies to isolate extracellular matrix (ECM) from human placenta or adipose tissue using Tris-, serum, or pepsin-based buffers were compared. The resulting ECM proteins were biochemically characterized by analysis of cellular remnants using HOECHST DNA staining, glycosaminoglycan (GAG) content by dimethylemethylene blue (DMMB), visualization of protein bands using SDS PAGE analysis combined with amino acid quantification and assessment of the pro-angiogenic profile using an angiogenesis array. Results: Tris-NaCl extracted ECM proteins showed a high heterogenic degree of extracted proteins, bioactive growth factors and GAGS, but no collagen-I. Active serum extracted ECM showed significant lower DNA remnants when compared to the Tris-NaCl isolation strategy. Pepsin-extracted ECM was rich in collagen-I and low amounts of remaining bioactive growth factors. This strategy was most effective to reduce DNA amounts when compared to the other isolation strategies. Pepsin-extracted ECM from both tissues easily gelled at 37°C, whereas the other extracted ECM strategies did not gel at 37°C (Tris-NaCl: liquid; serum: sponge). Conclusions: All relevant characteristics (DNA residues, ECM diversity and bioactivity, shape) of the extracted ECM proteins highly depend on its isolation strategy and could still be optimized.