3D Cell Culture

Introduction

Cellular encapsulation within hydrogels is becoming increasingly important and popular for cell culture and tissue engineering applications since it is more life-like compared to culture in 2-D.  Hydrogels are water-swollen networks which provide tissue-like properties and a 3-D microenvironment similar to physiological conditions.  In addition, the polymers which compose hydrogels can be designed to be stable or degrade as new extracellular matrix is deposited by the cells growing within it.   In the past decade, a great deal of progress has been made in engineering these microenvironments by incorporating specific biochemical cues to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells1.

Polyethylene glycol diacrylate (PEGDA) and polyethylene glycol tetraacrylate (PEGTA) are linear or cruciate PEGs, respectively, which form a hydrogel in the presence of a photoinitiator (such as Irgacure 2959).  These acrylated PEGs benefit not only from PEGs inherent hydrophilicity and biocompatibility but also from their flexibility in functionalization and use.  In one instance, since PEG-based hydrogels are non-biodegradable and resist cell attachment, a PEG-based hydrogel was engineered to mimic the degradability and cellular attachment qualities of the natural extracellular matrix2.  Briefly, acrylated ABA block co-polymers of PEG (A) and degradable peptide sequences (B) were synthesized (a collagenase-sensitive sequence, GGLGPAGGK; a plasmin-sensitive sequence, GGVRNGGK; and an elastase-sensitive sequence,AAAAAAAAAK, were used)2.  In addition, small amounts of acryloyl-PEG functionalized with the RGDS sequence for cellular attachment was also included.  In another instance, photodegradable (nitrobenzyl ether-derived) moieties were inserted into PEG-based co-polymers allowing user-controlled degradation for further understanding material regulation of live cell function3. 

Assay Discription

Acrylated PEG (10% wt/vol) is dissolved in HEPES-buffered saline (HBS), pH 7.4, and sterilized by filtration through a syringe filter (0.2 micron). 0.05% of 4-(2-hydroxyethoxy)phenyl-(2-hydroxy-2-propyl)  ketone (Irgacure 2959) photoinitiator is then added to the polymer solution. Cells are then mixed into PEGDA-Irgacure 2959 solution at a concentration of 2x106 cells/ml and exposed to UV light (365 nm, 10 mW/cm2, 1 min exposure or until gelled). 

Key Variables

  1. Concentration of PEGDA or PEGTA
  2. Molecular weight of PEGDA
  3. Concentration of Photoinitiator
  4. Time of Irradiation

References

  1. Khetan S and Burdick J, Cellular encapsulation in 3D hydrogels for tissue engineering. J. Vis. Exp. (2009).
  2. Gobin AS and West JL, Cell migration through defined, synthetic extracellular matrix analogues. FASEB J. (2002).
  3. Kloxin AM et al, Photodegradable hydrogels for dynamic tuning of physical and chemical properties. Science (2009) 324: 59-63.