279508 Click-Based, MMP-Degradable and Photodegradable Hydrogels for 3D Cell Culture and Cell Recovery

Thursday, November 1, 2012: 1:06 PM
Cambria West (Westin )
Mark W. Tibbitt, Department of Chemical and Biological Engineering, University of Colorado, Boulder, CO and Kristi S. Anseth, Department of Chemical and Biological Engineering, University of Colorado, Howard Hughes Medical Institute, Boulder, CO

Enzymatically responsive hydrogels have shown promise as 3D culture platforms that facilitate mammalian cell proliferation, differentiation, migration, and organogensis.1 Recent work has focused on the development of matrix-metalloproteinase (MMP) susceptible hydrogels, in which the local degradation rates are dictated by the level of MMP expression of the cells. While these systems enable the observation of cell behavior in well defined and realistic 3D culture platforms, less discussed are the difficulties in performing standard biological analyses in 3D.2  For example, it is often difficult to quantify cell function as the recovery of RNA and protein becomes increasingly difficult in 3D cultures.

To overcome these challenges, a photodegradable and MMP-degradable PEG-based hydrogel was synthesized via copper-free click chemistry for the 3D culture and recovery of mammalian cells. Gels were formed and degraded with light.  3T3 fibroblasts, hMSCs, and neural precursor cells were cultured within the hydrogels and recovered by photoerosion of the hydrogel.  Photorecovered 3T3s were plated and grown to confluency to demonstrate that photodegradation does not damage cells.  Recovered hMSCs were used to demonstrate the ease of purification of protein and RNA.

Photodegradable, PEG-based hydrogels were synthesized by reacting tetra–yne functionalized four-armed poly(ethylene glycol) with a di-azide functionalized, matrix metalloproteinase susceptible and photodegradable peptide at room temperature for 5 min via copper-free click chemistry.3  The gels were formed with a shear modulus of 1 ± 0.2 kPa and were degraded with 365nm or 400-500nm irradiation.  To render the gels more amenable to cell culture, azide functionalized adhesive peptides, RGDS and YIGSR, were included in the gel formulation. 3T3s and hMSCs were encapsulated in RGDS-functionalized gels and spread or migrated through the gel during culture as MMPs cleaved the peptide linkers within the gel.  Neural progenitor cells were encapsulated in YIGSR and RGDS-functionalized gels and extended extensive axonal processes by day 7.  Cell spreading and process extension was not observed in control gels.

This peptide functionalized gel system affords the unique ability to use light to recover entrapped cells at any point in space or time during 3D culture as the gel is photodegradable.  3T3 cells were released from the gel by light and grown to confluency on TCPS, demonstrating that cells recovered by photodegradation remain viable.  hMSCS were photoreleased from the gel and recovered to isolate protein and RNA for western blots and qRT-PCR analyses.  Ongoing work is comparing the quality of the isolated protein and RNA to traditional recovery methods.  In all, photodegradable and MMP-degradable hydrogels comprise a unique class of materials that affords 3D cell culture and recovery for the efficient and facile analysis of cell function during 3D culture.

References

  1. Fairbanks BD, Schwartz MP, Halevi AE, Nuttelman CR, Bowman CN, Anseth KS, Adv Matls 21 (2009) 5005-5010
  2. Tibbitt MW, Anseth KS, Biotech Bioeng 103 (2009) 655-663
  3. DeForest CA, Anseth KS Nat Chem 3 (2011) 925-931

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See more of this Session: Hydrogel Biomaterials
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