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

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 a...

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Veröffentlicht in:Tissue engineering. Part C, Methods Methods, 2017-05, Vol.23 (5), p.274-285
Hauptverfasser: Hackethal, Johannes, Mühleder, Severin, Hofer, Alexandra, Schneider, Karl Heinrich, Prüller, Johanna, Hennerbichler, Simone, Redl, Heinz, Teuschl, Andreas
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container_end_page 285
container_issue 5
container_start_page 274
container_title Tissue engineering. Part C, Methods
container_volume 23
creator Hackethal, Johannes
Mühleder, Severin
Hofer, Alexandra
Schneider, Karl Heinrich
Prüller, Johanna
Hennerbichler, Simone
Redl, Heinz
Teuschl, Andreas
description 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.
doi_str_mv 10.1089/ten.tec.2017.0016
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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
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
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