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

Tissue Eng Part C Methods. 2017 May;23(5):274-285. 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.

Keywords: HUVEC sprouting; atelocollagen; pepsin; placenta; rat primary hepatocytes.

MeSH terms

  • 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*


  • atelocollagen
  • Collagen