TrueGel3D™ Fast Gelling Hydrogels Preparation Protocol

Introduction

TrueGel3D™ is a chemically-defined hydrogel formed by mixing polymers with crosslinkers. This hydrogel permits the encapsulation of cells to put them in a three-dimensional environment to better mimic the native tissue environment.

TrueGel3D ™ consists of two types of polymers: polyvinyl alcohol (PVA), a synthetic non-degradable polymer, and dextran polymer, which can be degraded by enzymatic reaction with TrueGel3D™ enzymatic cell recovery solution. Both polymers are functionalized with either fast or slow thiol-reactive groups.

The fast thiol-reactive groups include maleimide groups along the polymer backbone that react rapidly with crosslinkers to from hydrogels, from within seconds to a few minutes, depending on the gel composition and pH applied.

Reagents preparation

Follow instructions indicated in product datasheets for reagent preparation.

Make sure that TrueGel3D™ buffers are completely dissolved.  Do not cool buffers with ice, as this may cause crystallization of salts.

To avoid oxidation of the thiol groups, do not expose RGD integrin adhesion peptide, PEG non-cell-degradable crosslinker, or CD cell-degradable crosslinker to air and room temperature longer than necessary. Make sure to close reagent caps immediately after each use.

Use culture medium, PBS, or physiological solution to prepare your stock cell suspension or other biological sample.

Experimental procedure

IMPORTANT: please read the complete protocol before mixing your reagents.

Hydrogel mix preparation considerations:

Different parameters can be modified and optimized when using TrueGel3D™ hydrogel fast kits. Table 1 shows reagent volume modifications that allow you to prepare a soft hydrogel with or without TrueGel3D™ RGD adhesion peptide OR protein.

Table 1. reagents volume to set up a soft FAST hydrogel with or without TrueGel3D™ RGD integrin adhesion peptide OR protein

Reagents	Reagent volume in µL
	Without TrueGel3D™ RGD integrin adhesion peptide	With TrueGel3D™ RGD integrin adhesion peptide	With a protein (recommended concentration: 200 µg/ml)
Thiol reactive polymer (FAST-PVA or FAST-DEXTRAN)	2	2.5	4
Crosslinker (PEG non cell-degradable crosslinker or CD cell-degradable crosslinker)	3	3	6
Cell suspension	6	6	6
TrueGel3D™ RGD integrin adhesion peptide	-	0.8	-
Protein (200 µg/ml)	-	-	4.5
TrueGel3D™ buffer pH 5.5	2.4	2.4	2.4
water	16.6	15.3	7.1
total volume	30	30	30

Experimental procedure

1. Mix water, TrueGel3D™ buffer (pH 5.5) and FAST thiol-reactive polymer (FAST-PVA or FAST-DEXTRAN) in a reaction tube and mix well.
2. (If TrueGel3D™ RGD integrin adhesion peptide is not needed, skip to step 3), Add the TrueGel3D™ RGD integrin adhesion peptide and mix immediately to ensure homogenous modification of FAST thiol-reactive polymer with the peptide. Incubate sample for 5 minutes to allow attachment of the RGD peptide to the maleimide groups of the polymer.
3. Distribute the crosslinker (PEG non-cell-degradable crosslinker or CD cell-degradable crosslinker) on the surface of a sterile culture dish*. Do not spread out the crosslinker solution that needs to be kept as a drop.
   * You can use multiwell plates (6-, 24-, or 96-well) or any appropriate sterile culture plate or flask. Non-tissue culture treated polystyrene is recommended. For imaging, we recommend glass slides with cell culture chambers, such as Millicell^® EZ SLIDE 8-well glass, sterile (product number PEZGS0816)
4. Add cell suspension and your protein (if applicable) to the reaction tube containing water, buffer, FAST thiol-reactive polymer and TrueGel3D™ RGD integrin adhesion peptide (if applicable).
5. Pipet 27 μL of the mix (if no protein added) or 24 μL of the mix (if protein added) and add it to the crosslinker-primed cell culture plate; mix quickly by pipetting up and down three times, avoiding the formation of air bubbles. Leave the mix on the surface of the culture dish. Wait approximately 3 minutes to allow the gel to form.
6. Add cell culture medium to the cell culture dish in sufficient volume to cover the gel.
7. Incubate culture dish in the tissue culture incubator.
8. After one hour, replace the culture medium.
9. Change the medium as required during cultivation of cells.
   Note: With the TrueGel3D™ hydrogel chemistry, gel will begin to form within a few seconds of mixing. For this reason, it is important to complete the mixing step as quickly as possible to avoid gel formation in the pipet tip. You can test gel formation before adding cell culture medium by careful inspection with a pipet tip.

Dissolving TrueGel3D™ FAST-DEXTRAN Hydrogels with TrueGel3D™ enzymatic cell recovery solution:

TrueGel3D™ Hydrogels containing live or chemically fixed cells can be dissolved by adding TrueGel3D™ enzymatic cell recovery solution to the culture medium. For example, a 30 μL gel can be dissolved with 300 μL of a 1:20 dilution of TrueGel3D™ enzymatic cell recovery solution in medium (30-60 minutes incubation, 37 °C). After dissolution of the gel, centrifuge the cell suspension and suspend the pelleted cells in fresh medium or physiological buffer. Repeat this washing procedure once or twice to remove remaining TrueGel3D™ enzymatic cell recovery solution. The removal of enzyme is important if cells are being embedded again in dextran hydrogels to continue culture. If TrueGel3D™ enzymatic cell recovery solution is not removed completely, it can destabilize the newly set hydrogel.

Gel preparation variations

Preparation of small gel volumes:

If small-volume gels are prepared (less than 100 μL), correspondingly very small volumes of the TrueGel3D™ RGD integrin adhesion peptide stock solution are required. To avoid the pipetting of such small volumes, we recommend reducing the concentration of the TrueGel3D™ RGD integrin adhesion peptide stock solution from 20 mmol/L to 3 mmol/L by dilution with water to increase the volume to be pipetted. Should this dilution of the adhesion peptide stock solution be performed, the volume of water in the mix must be reduced accordingly to fill to the final volume.

Preparation of multiple gels of same composition:

For the preparation of multiple gels of the same composition, the mix of water, TrueGel3D™ buffer (pH 5.5) and FAST thiol-reactive polymer (FAST-PVA or FAST-DEXTRAN), TrueGel3D™ RGD integrin adhesion peptide and cells suspension may be scaled up in a single master mix. Mix thoroughly before dispensing to ensure an equal number of cells in each gel.

Preparation of plain gels (without cells) or embedding other specimens:

If no cells are included in the gel, e.g. for encapsulation of tissues or preparation of plain gels, replace the volume of cell suspension with cell culture medium, PBS or any other physiologically compatible solution of your choice.

TrueGel3D™ RGD integrin adhesion peptide replacements for control experiments:

TrueGel3D™ Thioglycerol can be added to the gel instead of the TrueGel3D™ RGD integrin adhesion peptide. In this case, the gel does not provide cell attachment sites and can be used as a control for RGD Peptide-modified gels. TrueGel3D™ scramble RGD integrin adhesion peptide (catalog numbers TRUESRGD-1EA and TRUESRGD-3EA) are available for control experiments.