Our laboratory has developed DNA-crosslinked polyacrylamide hydrogels, a dynamic hydrogel system, to better understand the effects of modulating tissue stiffness on cell function. Here, we provide schematics, descriptions, and protocols to prepare these hydrogels.
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Method Article
Preparation of DNA-crosslinked Polyacrylamide Hydrogels
In This Article
Summary
Abstract
Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, the effect of tissue elasticity on cell function is a major area of mechanobiology research because tissue stiffness modulates with disease, development, and injury. Static tissue-mimicking materials, or materials that cannot alter stiffness once cells are plated, are predominately used to investigate the effects of tissue stiffness on cell functions. While information gathered from static studies is valuable, these studies are not indicative of the dynamic nature of the cellular microenvironment in vivo. To better address the effects of dynamic stiffness on cell function, we developed a DNA-crosslinked polyacrylamide hydrogel system (DNA gels). Unlike other dynamic substrates, DNA gels have the ability to decrease or increase in stiffness after fabrication without stimuli. DNA gels consist of DNA crosslinks that are polymerized into a polyacrylamide backbone. Adding and removing crosslinks via delivery of single-stranded DNA allows temporal, spatial, and reversible control of gel elasticity. We have shown in previous reports that dynamic modulation of DNA gel elasticity influences fibroblast and neuron behavior. In this report and video, we provide a schematic that describes the DNA gel crosslinking mechanisms and step-by-step instructions on the preparation DNA gels.
Introduction
Static and dynamic substrates are two categories of biomaterials that were developed to study the effects of tissue elasticity or stiffness on cell function. Static substrates are unable to change their physical properties after they are fabricated and/or once cells are plated. Polyacrylamide (PA) gels were the first two-dimensional, static substrates that were synthesized for mechanobiology investigations 5,17. PA gels are easy to prepare, inexpensive, versatile, and can be fabricated with a broad range of elastic moduli. Although these technical advantages make PA gels a commonly applied substrate, static substrates are not indicative of the dynamic natur....
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Protocol
NOTE: The entire protocol from gel preparation to cell processing takes a minimum of six days. Estimated time for gel preparation is 8 hr plus an O/N incubation. Estimated time for gel immobilization and DNA annealing is 8 hr plus an O/N rinsing step. Estimated time for gel functionalization is 2 hr. Time for cell plating and growth is dependent on culture type and application, but a minimum of four days is required.
1. Preparation of DNA Gels
NOTE: Prepare DNA gels in three distinct steps. First, individually polymerize SA1 and SA2 ssDNA into a PA backbone. These solutions are called SA1 polymerized solution and S....
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Results
Prior to our studies, cell-ECM interactions were observed on static compliant biomaterials or on irreversible and unidirectional dynamic substrates. These substrates do not accurately reflect the dynamic nature of the cellular microenvironment. Our work shifts the existing technical paradigms by providing a more physiologic model for studying cell-ECM interactions on softening and stiffening dynamic biomaterials. In previous studies, we analyzed the effects of dynamic substrates on cells by examining various cell morphol.......
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Discussion
The ability of DNA gels to soften or stiffen before and after cell adhesion makes them an ideal model to study the role of dynamic tissue stiffness on cell function. All three designs have been used in mechanical and biological studies. However, all three designs have similar elasticities at various crosslink percentages, indicating crosslink length does not influence DNA gel elasticity (Table 2). In contrast, acrylamide concentration affects elasticity. These designs may differ in crosslinking kinetics .......
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Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors would like to thank: Dr. Frank Jiang, Dr. David Lin, Dr. Bernard Yurke and Dr. Uday Chippada for their contributions on developing the DNA gel technology; Dr. Norell Hadzimichalis, Smit Shah, Kimberly Peterman, Robert Arter for their comments and edits of this manuscript; funding sources including the New Jersey Commission on Spinal Cord Research (Grant #07A-019-SCR1, N.A.L.) and New Jersey Neuroscience Institute (M.L.P.); and publishers of Tissue Engineering, Part A for permission to reprint Figures 2 and 4 and Biomaterials for permission to reprint Figure 3.
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| ssDNA | Integrated DNA Technologies (Coralville, Iowa) idtdna.com | Do not vortex ssDNA. Gentle invert the vial and/or pipette solution to mix. | |
| PBS with calcium and magnesium | Any brand. | ||
| 100x Tris-EDTA buffer (TE buffer) | Sigma-Aldrich (St. Loius, MO) sigmaldrich.com | T9285 | |
| 10x Tris-Borate-EDTA buffer (TBE buffer) | Sigma-Aldrich (St. Loius, MO) | 93290 | TBE is a reproductive toxin. |
| 40% Acrylamide solution | Fisher Scientific (Pittsburg, PA) | BP14021 | Acrylamide is a toxin. |
| Ammonium persulfate (APS) | Sigma-Aldrich (St. Loius, MO) | A3678 | Prepare a 2% solution in TE buffer. APS is a toxin and irratant. |
| Tetramethylethylenediamine (TEMED) | Sigma-Aldrich (St. Loius, MO) | T9281 | Prepare a 20% solution in TE buffer. TEMED is flammable, a corrosive, and a toxin. |
| 12 mm diameter round coverglass | Fisher Scientific (Pittsburg, PA) fishersci.com | 12-545-82 | |
| Norland optical adhesive 72 | Norland Products (Cranbury, NJ) norlandprod.com | NOA72 | |
| 24-well tissue culture plate | Any brand. | ||
| Microcentrifuge tubes | Any brand. | ||
| Sulfo-SANPAH | ProteoChem or Thermo Fisher, (Rockland, IL) proteochem.com or thermofisher.com | C111 or 22589 | Prepare a 0.315 mg/ml solution in water immediately before use. Dissolve at 37 °C and filter sterilze. It is normal to observe undisolved sulfo-SANPAH in the filter. Sulfo-SANPAH is light sensitive and, therefore, the solution should be protect from light until UV exposure. |
| Poly-D-Lysine (PDL) | Sigma-Aldrich (St. Loius, MO) | P6407 | Prepare a 0.2 mg/ml solution in water and filter sterilize. |
| Collagen Type I | Affymetrix (Santa Clara, CA) affymetrix.com | 13813 | Prepare a 0.2 mg/ml solution in 0.2 N acetic acid. Solution needs to remain cold at all times to avoid polymerization. Acetic acid is a flammable, toxic, and corrosive. |
| 22 x 60 cover glass | Fisher Scientific (Pittsburg, PA) | 12-544-G | |
| Positive-displacement pipette | Gilson, Inc (Middletown, WI) gilson.com | F148504 | |
| Heat block | Fisher Scientific (Pittsburg, PA) | 11-718 | |
| UV light source | Place gels as close as possible to the UV light. UV light can cause skin or eye injury. | ||
| Thermometer | Any brand. | ||
| Nitrogen gas | GTS-Welco (Flemington, NJ) www.praxairmidatlantic.com/ | NI 5.0UH-R |
References
- Balaban, N. Q., et al. Force and focal adhesion assembly A close relationship studied using elastic micropatterned substrates. Nat Cell Biol. 3 (5), 466-472 (2001).
- Peppas Brannon-Peppas, L., Peppas, N. A.
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