title: An Effective Method of Atelocollagen Type 1/3 Isolation from Human Placenta and Its In Vitro Characterization in Two-Dimesional and Three-Dimensional Cell Culture Applications.
abstract: Pepsin-solubilized atelocollagen can be used to form highly complex three-dimensional matrices for a broad spectrum of tissue engineering applications. Moreover, it has a long history as a favorable biomaterial in pharmaceutical and medical industries. So far, the main sources for these approaches are collagens from xenogenic sources. Yet, these nonhuman collagens carry a risk of provoking immune reactions in patients. Here we describe an effective method of isolating atelocollagen type 1/3 (COL1/3) from human placenta. By combining a single pepsin digestion step with tangential flow filtration and further precipitation steps, we could purify COL1/3 within only 4 days of processing. The resulting COL1/3 was biochemically characterized by determining residual DNA content, proving the absence of impurities by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) analysis combined with total amino acid quantification, identifying the isolated collagen types by Western blot analysis, and analyzing the spontaneous formation of fibrous structures on freeze-drying via scanning electron microscopy. Finally, the cytocompatibility of the isolated collagen was demonstrated in two dimensional using primary rat hepatocytes and in three dimensional by a sprouting assay of human umbilical vein endothelial cell. The isolation method described not only fulfills demands for cost-efficient bioengineering using a human waste material but also potentially increases overall safety for patients by use of homologous products.
authors.affiliation: 1 Ludwig Boltzmann Institute for Experimental and Clinical Traumatology , Vienna, Austria .|2 Austrian Cluster for Tissue Regeneration , Vienna, Austria .
authors.forename: Johannes
authors.initials: J
authors.lastname: Hackethal
authors.affiliation: 1 Ludwig Boltzmann Institute for Experimental and Clinical Traumatology , Vienna, Austria .|2 Austrian Cluster for Tissue Regeneration , Vienna, Austria .
authors.forename: Severin
authors.initials: S
authors.lastname: Mühleder
authors.affiliation: 3 Research Area Biochemical Engineering, Institute of Chemical Engineering, Vienna University of Technology , Vienna, Austria .
authors.forename: Alexandra
authors.initials: A
authors.lastname: Hofer
authors.affiliation: 4 Center of Biomedical Research, Medical University of Vienna , Vienna, Austria .|5 Ludwig Boltzmann Cluster for Cardiovascular Research , Vienna, Austria .
authors.forename: Karl Heinrich
authors.initials: KH
authors.lastname: Schneider
authors.affiliation: 2 Austrian Cluster for Tissue Regeneration , Vienna, Austria .|6 Department of Biochemical Engineering, University of Applied Sciences Technikum Wien , Vienna, Austria .
authors.forename: Johanna
authors.initials: J
authors.lastname: Prüller
authors.affiliation: 2 Austrian Cluster for Tissue Regeneration , Vienna, Austria .|7 Red Cross Blood Transfusion Service of Upper Austria , Linz, Austria .
authors.forename: Simone
authors.initials: S
authors.lastname: Hennerbichler
authors.affiliation: 1 Ludwig Boltzmann Institute for Experimental and Clinical Traumatology , Vienna, Austria .|2 Austrian Cluster for Tissue Regeneration , Vienna, Austria .
authors.forename: Heinz
authors.initials: H
authors.lastname: Redl
authors.affiliation: 2 Austrian Cluster for Tissue Regeneration , Vienna, Austria .|6 Department of Biochemical Engineering, University of Applied Sciences Technikum Wien , Vienna, Austria .
authors.forename: Andreas
authors.initials: A
authors.lastname: Teuschl
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D003094:Collagen / Q000302:isolation & purification* / Q000378:metabolism; D005260:Female; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D010920:Placenta / Q000378:metabolism*; D011247:Pregnancy; D051381:Rats; D017207:Rats, Sprague-Dawley; D023822:Tissue Engineering / Q000379:methods*
chemical_list: C059821:atelocollagen; D003094:Collagen
keywords: HUVEC sprouting; atelocollagen; pepsin; placenta; rat primary hepatocytes