Production of an acellular matrix from amniotic membrane for the synthesis of a human skin equivalent.

Human amniotic membrane (HAM) has useful properties as a dermal matrix substitute. The objective of our work was to obtain, using different enzymatic or chemical treatments to eliminate cells, a scaffold of acellular HAM for later use as a support for the development of a skin equivalent. The HAM was separated from the chorion, incubated and cryopreserved. The membrane underwent different enzymatic and chemical treatments to eliminate the cells. Fibroblasts and keratinocytes were separately obtained from skin biopsies of patients following a sequential double digestion with first collagenase and then trypsin-EDTA (T/E). A skin equivalent was then constructed by seeding keratinocytes on the epithelial side and fibroblasts on the chorionic side of the decellularizated HAM. Histological, immunohistochemical, inmunofluorescent and molecular biology studies were performed. Treatment with 1% T/E at 37 °C for 30 min totally removed epithelial and mesenchymal cells. The HAM thus treated proved to be a good matrix to support adherence of cells and allowed the achievement of an integral and intact scaffold for development of a skin equivalent, which could be useful as a skin substitute for clinical use.

Cryoconservation of peptide extracts from trypsin digestion of proteins for proteomic analysis in a hospital biobank facility.

We tested a semiautomated protocol for the proper storage and conservation in a hospital biobank of tryptic peptide extracts coming from samples with low and high protein complexity for subsequent mass spectrometry analysis. Low-complexity samples (serum albumin, serotransferrin. and alpha-S1-casein) were loaded in replicates in SDS-PAGE and subjected to standard in-gel trypsin digestion. For LC-MALDI-TOF/TOF analysis, purified ß-galactosidase and human serum samples were in-solution digested following standard procedures and desalted with C18 stage-tips. In both cases, peptides extracts were aliquoted in individually 2D coded tubes, vacuum-dried, barcode-read, and stored in an automated -20 °C freezer in the Biobank facility. Samples were kept dried at -20 °C until the corresponding time-point of analysis, then reconstituted in the proper buffer and analyzed by either MALDI-TOF/TOF (peptide fingerprinting and MS/MS) or LC-MALDI-TOF/TOF following a highly reproducible pattern to ensure the reproducibility of the results. Protein identification was done with either Mascot or Protein Pilot as search engines using constant parameters. Over a period of 1 year we checked six different time points at days 0, 7, 30, 90, 180, and 365. We compared MS and MS/MS protein score, number of identified peptides, and coverage of the identified proteins. In the low complexity samples, the number of peptides detected gradually decreased over time, especially affecting the MS score. However, two of the three proteins - serum albumin and serotransferrin - were identified by both PMF and MS/MS at day 90. By day 180, only MS/MS identification in some replicates was possible. By LC-MS/MS, ß-galactosidase and the most abundant serum proteins were identified with good scores at all time points even by day 365, with no detectable peptide loss or decrease in the fragmentation efficiency, although a progressive decrease in peptide intensity indicates that detection of low abundant proteins could not be optimal after very long periods of time. Our results encourage us to use the biobank facility in the future for long-term storage - up to 3 months - of dried peptide extracts.