Preparation of Decellularized Biological Scaffolds for 3D Cell Culture

Methods Mol Biol. 2017;1612:15-27. doi: 10.1007/978-1-4939-7021-6_2.


The biggest challenge of designing and implementing an in vitro study is developing a microenvironment that most closely represents the interactions observed in vivo. Decellularization of tissues and organs has been shown to be an effective method for the removal of potentially immunogenic constituents while preserving essential growth factors and extracellular matrix (ECM ) proteins necessary for proper cell function. Enzymatic digestion of decellularized tissues allows these tissue-specific components to be reconstituted into bioactive hydrogels through a physical crosslinking of collagen. In the following protocol, we describe unique decellularization methods for both dermis and urinary bladder matrix (UBM) derived from porcine tissues. We then provide details for hydrogel formation and subsequent three-dimensional (3D) culture of two cell types: NIH 3T3 fibroblasts and C2C12 myoblasts .

Keywords: Cell culture; Decellularization; Extracellular matrix; Hydrogel; Scaffolds.

MeSH terms

  • Animals
  • Cell Culture Techniques / methods*
  • Cell Line
  • Collagen / metabolism
  • Cross-Linking Reagents / metabolism
  • Extracellular Matrix / metabolism
  • Fibroblasts / cytology*
  • Fibroblasts / metabolism
  • Hydrogels / metabolism
  • Mice
  • Myoblasts / cytology*
  • Myoblasts / metabolism
  • NIH 3T3 Cells
  • Swine
  • Tissue Engineering / methods
  • Tissue Scaffolds*


  • Cross-Linking Reagents
  • Hydrogels
  • Collagen