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

• Published in: Tissue engineering. Part C, Methods Vol. 23; no. 5; pp. 274 - 285
• Main Authors: Hackethal, Johannes, Mühleder, Severin, Hofer, Alexandra, Schneider, Karl Heinrich, Prüller, Johanna, Hennerbichler, Simone, Redl, Heinz, Teuschl, Andreas
• Format: Journal Article
• Language: English
• Published: United States 01.05.2017
• Subjects: Animals; Cell Culture Techniques - methods; Cells, Cultured; Collagen - isolation & purification; Collagen - metabolism; Female; Hepatocytes - cytology; Hepatocytes - metabolism; Human Umbilical Vein Endothelial Cells - metabolism; Humans; In Vitro Techniques; Male; Placenta - metabolism; Pregnancy; Rats; Rats, Sprague-Dawley; Tissue Engineering - methods; HUVEC sprouting; atelocollagen; placenta; rat primary hepatocytes; pepsin
• ISSN: 1937-3392
• DOI: 10.1089/ten.tec.2017.0016

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.