Hepes 4 2 hydroxyethyl 1 piperazineethanesulfonic acid buffer

The PC-12 pheochromocytoma cell line derived from rat adrenal medulla was obtained from American Type Culture Collection (Manassas, VA). RPMI-1640, trypsin solution, phosphate-buffered saline, fetal bovine serum (FBS), sodium pyruvate (0.1 M), L-glutamine (0.2 M) and penicillin/streptomycin solution (containing 10,000 units·ml⁻¹ penicillin and 10,000 μg·ml⁻¹ streptomycin) were obtained from Quality Biological (Gaithersburg, MD), horse serum (heat inactivated) was purchased from Biosource (Rockville, MD) and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer [1 M, pH 7.4] was obtained from Mediatech, Inc. (Manassas, VA). The PC-12 cells were maintained in RPMI-1640 supplemented with 1 mM HEPES buffer, 10% horse serum, 5% FBS, 1% sodium pyruvate, 1% L-glutamine and 1% penicillin/streptomycin.

Briefly, the ECM powder was aseptically manufactured from reconstituted
decellularized bovine connective tissue (Maverick BioSciences, New Zealand) by
decellularizing, solubilizing and lyophilizing the tissue. The bovine tissues
were shipped to the lab on dry ice to keep the temperature below
−20°C for the duration of the transfer. At the lab the tissues
were decellularized using Triton X-102 (Sigma-Aldrich, St Louis, MO),^{18 (link)} then solubilized in an acidic
pepsin solution, lyophilized and rehydrated with a specified amount of water to
create a solution with a total protein content >5mg/ml.^{15 (link)} The concentrated slurry was
brought to an alkaline pH using sodium hydroxide (Sigma-Aldrich) to inactivate
pepsin, neutralized in a HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid) buffer (Mediatech Inc, Herndon, VA), and brought to iso-osmolarity with
calcium chloride (Sigma-Aldrich) before a final lyophilization step. The
resulting dry ECM material was milled into a powder and loaded into 1 mL Luer
lock syringes (20 mg per syringe). Each syringe was then placed into double
peel-pack package and stored at −20°C protected from light until
use.