Extracellular matrix hydrogel derived from decellularized tissues enables endodermal organoid culture

ABSTRACT: Porcine (Sus scrofa domesticus) small intestinal mucosal layers from the ‘Pietrain’ breed were used. To harvest the intestine, one end was cut in situ and the internal tube was pulled out leaving behind the external layer and mesentery. The retrieved mucosal tissue is then extensively cleaned with deionized water (dH2O), opened longitudinally, cut into 1 cm pieces and placed in dH2O to begin the first step of decellularization. The 1cm pieces of intestine were placed in dH2O overnight at 4°C and then in 4% sodium deoxycholate for 4hr at room temperature (RT). This was followed by a washing step in PBS (phosphate buffer saline) for 30min at RT and then 2000kU DNase-I in 1M NaCl for 3hr at RT. The tissue was then placed in milliQ water and washed for 3 days at 4C, changing the water daily. A magnetic stirrer was used throughout the decellularization process. The decellularized porcine intestine was freeze dried overnight or until completely dry, milled into a thin powder using a mini mill, gamma irradiated (17 kGy for 10h) and stored at -20C until further use. The lyophilized ECM powder was split into 3 biological replicates, each processed independently and analyzed in triplicate by LC-MS/MS. The powder was resuspended in lysis buffer and two spike-in proteins (each at 0.5 µg/100 µg of protein powder) were added: carnitine monooxygenase oxygenase subunit (cntA, D0C9N6) from Acinetobacter baumannii, and CTP synthase (CTPsyn, Q9VUL1) from Drosophila melanogaster. Protein extraction was performed by heating at 90°C for 10 min, and centrifuging at maximum velocity for 10 min at 4°C. ECM-derived proteins were reduced in 0.1 M dithiothreitol (DTT) at 95°C for 5 min, dissolved in 8 M urea solution after cooling down to room temperature, alkylated with 55 mM iodoacetamide for 30 min at 25°C in the dark. Alkylated proteins were purified using Microcon YM-10 filter unit for 8 times at 14000g for 40min followed by trypsin digestion for 16 h at 37°C. pH was adjusted to 3 by addition of formic acid. Peptides were desalted by C-18 column and dried into powder and were then re-suspended in 30 µl 0.1% acetic acid for the following mass spectrometry analysis. Protein identification by liquid chromatography–tandem mass spectrometry (LC-MS/MS) was performed using Thermo Fusion Mass Spectrometer with Thermo Easy-nLC1000 Liquid Chromatography. 130 min of LC-MS gradients were performed by increasing organic proportion. The first level of MS was detected by Orbitrap with parameter of Resolution at 120K, Scan Rang at 300-1800 m/z, Mass Tolerance at 10 ppm. The second level of MS was isolated by Quadrupole, activated by HCD and detected by Orbitrap. The Orbitrap Resolution for the second level of MS was 30K. Peak files .mzXML were obtained by RawConverter starting from .raw files. Starting from peak files, the mass spectrometry–derived data were searched against a human protein database (Uniprot Homo sapiens reference proteome, UP000005640) by MaxQuant v. 1.6.7.0. Oxidation of methionine residues and acetyl of protein N-term were set as variable modifications. Carbamidomethyl on cysteine was set as fixed modification. Peptide-spectrum matches (PSMs) were adjusted to a 1% and then assembled further to a final protein-level false discovery rate (FDR) of 1%.

INSTRUMENT(S): Orbitrap Fusion ETD

ORGANISM(S): Sus Scrofa (ncbitaxon:9823)

SUBMITTER: Nicola Elvassore  

PROVIDER: MSV000084136 | MassIVE | Fri Jul 26 04:14:00 BST 2019

SECONDARY ACCESSION(S): PXD014775

REPOSITORIES: MassIVE