- Hydrogels are suitable for culturing primary cells, stem cells and cell lines.
- Cells can be encapsulated or grown on the hydrogel surface in 6- to 384-well plates or tissue culture inserts.
- Hydrogels can be varied by user to possess the desired compliance.
- Tested for bacteria, endotoxins, and lactate dehydrogenase-elevating virus (LDEV)
In the past, large amounts of proteases and enzymes were required to digest Extracel® and HyStem® hydrogels for several hours to recover encapsulated cells. These harsh conditions can potentially damage sensitive cells. In addition, introduction of animal-based enzymes may hinder clinical applications of the cells where growth in an animal-free milieu is important. PEGSSDA® now allows a researcher to quickly and gently dissolve Extracel or HyStem non-enzymatically, thereby easily recovering cells either grown on top or within the hydrogel. PEGSSDA contains two internal disulfide bonds which are rapidly reduced with small amounts of reducing agent (such as N-acetyl cysteine or glutathione), causing gel liquefaction.
PEGSSDA is a pentablock PEGDA molecule composed of four PEG molecules (MW 2000 Da) and two interdigitated disulfide bonds. Only the external PEG molecules are acrylated (see below). The final molecular weight is approximately 8500 Da1. Like PEGDA, PEGSSDA is a thiol-reactive crosslinker that covalently reacts with the thiol groups in Glycosil®, HyStem®, Heprasil®, and Gelin-S® to form viscoelastic hydrogels1. PEGSSDA is lyophilized in water.
PEGSSDA is used to chemically crosslink Glycosil, HyStem, Heprasil, and Gelin-S. The gelation time ranges from as short as 5 minutes1 to as long as a couple of hours, depending upon the amount of PEGSSDA used and the concentration/dilution of the Glycosil, HyStem, Gelin-S and /or Heprasil. While we use thiol chemistry to crosslink our hydrogels, other researchers have used photoinitiators such as Darocur 1173. This process adds an extra level of complexity, as many photoinitiators are toxic to cells and the UV light used to crosslink the hydrogel can damage cells.
Uses of PEGSSDA
PEGSSDA is easy to use, non-toxic, and cytocompatible1. It not only allows gentle and and quick recovery of cells grown in or on Extracel or HyStem in the presence of N-acetyl cysteine or glutathione, but also serves as an alternative to PEGDA hydrogels when cell recovery is crucial.
Structure of PEGSSDA
For research use only
PEGSSDA® (PEGSSDA, disulfide-containing polyethylene glycol diacrylate) is packaged in 0.25mL aliquots. Vials are blanketed by argon and under a slight vacuum.
- Store PEGSSDA in the original vial unopened at -20 °C for up to one year.
- Reconstituted solutions can be stored at -20˚C for ~ one month.
INSTRUCTIONS FOR USE
PEGSSDA is prepared by dissolving the lyophilized solid in the DG Water. When reconstituted, it will be in 1x phosphate buffered saline (PBS) buffer pH ~7.4.
PEGSSDA should be prepared in the following manner:
- Allow the PEGSSDA vial to come to room temperature.
- Under aseptic conditions using a syringe and needle add to the vial the 0.25 mL of DG Water as indicated on the label.
- Invert several times to dissolve.
- PEGSSDA is used to chemically crosslink hydrogels made from Glycosil, Heprasil, HyStem, and Gelin-S. PEGSSDA does not form a hydrogel on its own.
- Typically PEGSSDA is used in a 4:1 volume ratio with Glycosil, Heprasil, Gelin-S, and HyStem as follows:
- 0.25 mL PEGSSDA is crosslinked with 1.0 mL Glycosil
- 0.25 mL PEGSSDA is crosslinked with 0.5 mL Glycosil + 0.5 mL Gelin-S
- 0.25 mL PEGSSDA is crosslinked with 1.0 mL Heprasil
- 0.25 mL PEGSSDA is crosslinked with 0.5 mL Heprasil + 0.5 mL Gelin-S
- 0.25 mL PEGSSDA is crosslinked with 1.0 mL HyStem
- 0.25 mL PEGSSDA is crosslinked with 0.5 mL HyStem + 0.5 mL Gelin-S
- Note the gelation time varies depending upon the amount of PEGSSDA, the amount of Glycosil, Heprasil. or HyStem, and the amount of Gelin-S used. Hydrogels that include Gelin-S will typically have longer gelation times than those made with only Glycosil, Heprasil, or HyStem.
- Note: Gelin-S will not form a hydrogel when mixed with PEGSSDA.
- Note: Hydrogels made using only PEGSSDA and Glycosil, Heprasil, or HyStem will not support cell attachment.
Dissolution of gels with cells on top and encapsulated (gel volume of 0.6 mL) in a 24 well plate.
Note: The following procedure was optimized particularly for the aforementioned gel geometry. Dissolution of gels with alternate geometry and/or volumes may require adjustments to the protocol.
- Make up the appropriate amount of 40mM N-Acetyl-L-Cysteine in 1X PBS or media and pH to 7.4.
- Add 2 mL of 40mM N-Acetyl-Cysteine to the top of each gel and let sit at 37°C for 0.5 hours. Agitation by orbital shaking will help decrease dissolution time.
- After 0.5 hours mix the gel and N-Acetyl-L-Cysteine together by pipetting up and down with a 1 mL pipet until there is no longer any resistance upon filling the pipet tip.
- Let incubate at 37°C for another 0.5 hours.
- Mix again as in step 3.
- Repeat this process for a total time of 2h from the first addition a N-Acetyl-L-Cysteine.
- Remove liquid from well and place in conical centrifuge tube. Add PBS to a total of 5mL of liquid.
- Centrifuge at 1000 RPM for 5 minutes.
- Aspirate off PBS and process cells as desired.
Note: Product has been manufactured under aseptic conditions and tested for bacteria and fungus.
- J. Zhang, A. Skardal, and G.D. Prestwich, “Engineered extracellular matrices with cleavable crosslinkers for cell expansion and easy cell recovery,” Biomaterials, 29(34), 4521-31 (2008).