ECM Suspension Preparation: A Technique to Obtain Homogenous Suspension from Tissue-derived Extracellular Matrix

Extracellular matrix or ECM, a complex support network in tissues, consists of proteoglycans and proteins.

To obtain homogenized ECM, begin by taking a measured amount of decellularized ECM powder in a tube. Now, add an acidified buffer to the tube to form an ECM suspension.

Simultaneously, in another tube, dissolve alpha-amylase - a glycolytic enzyme - in the same acidified buffer. The acidic pH of this buffer facilitates the optimal activity of the alpha-amylase enzyme.

Next, supplement the tube containing ECM suspension with alpha-amylase enzyme solution. Incubate the tube with constant agitation. Alpha-amylase hydrolyses the glycans - the carbohydrate moieties - present in the proteoglycans.

Centrifuge to pellet the protein-rich ECM fraction, leaving the digested glycans and spent enzymes in the supernatant. Discard the supernatant.

Resuspend the pellet in sodium chloride solution. The chloride ions extract the proteins from the ECM pellet into the suspension. Centrifuge and discard the sodium chloride-containing supernatant.

Next, add acetic acid to the protein-containing ECM pellet. Acetic acid further solubilizes ECM protein aggregates and brings them in suspension.

Use a homogenizer to obtain a uniform ECM suspension. Store the homogeneous ECM suspension at 4 °C for further downstream analysis.

Weigh out 250 milligrams of either minced or cryomilled ECM and transfer it to a 15-milliliter centrifuge tube. Add 5 milliliters of 0.22 molar NaH₂PO₄ buffer to the centrifuge tube and mark the liquid level on the tube. Then, prepare an alpha-amylase stock solution by adding 7.5 milligrams of alpha-amylase to 1 milliliter of 0.22 molar NaH₂PO₄ buffer.

Next, add 100 microliters of the alpha-amylase stock solution to the sample. Then, add 0.22 molar NaH₂PO₄ to obtain a final volume of 10 milliliters. Agitate the suspension continuously at 300 rpm and room temperature for 72 hours.

Centrifuge the suspension at 1,500 x g for 10 minutes. Carefully collect and discard the supernatant without disturbing the digested ECM pellet. Then, use 10 milliliters of 5% NaCl diluted in double distilled water to resuspend the pelleted material and agitate continuously for 10 minutes at 300 rpm.

Repeat the wash two more times before using 10 milliliters of double distilled water to resuspend the pellet. Then, agitate the suspension continuously at 300 rpm at room temperature for 10 minutes. After centrifuging the sample again, carefully collect and discard the supernatant without disturbing the digested ECM pellet.

Add 0.2 molar acetic acid to the 5-milliliter mark and vortex. Then, continuously agitate the suspension at 120 rpm and 37 degrees Celsius overnight. The following day, using a hand-held homogenizer equipped with a 10-millimeter wide saw tooth probe, homogenize the ECM suspension at room temperature in 10-second intervals until no visible fragments remain.