TrueGel3D™ Slow Gelling Hydrogels Preparation Protocol

Introduction

TrueGel3D™ is a chemically-defined hydrogel formed by mixing polymers with crosslinkers. This hydrogel enables the encapsulation of cells to enable their growth in a 3-dimensional environment that better mimics 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 slow gelling polymers, SLO-PVA and SLO-DEXTRAN, permit preparation of hydrogels at a slow gelation rate that facilitates applications like the filling of microchannels or syringes. Incubation times are between 8 and 50 minutes to obtain a solid hydrogel, depending on your choice of polymer and crosslinker (CD cell-degradable crosslinker or PEG non cell-degradable crosslinker).

Reagents preparation

Follow instructions indicated in product datasheets for product preparation.

Make sure that TrueGel3D™ buffers are completely dissolved. Do not cool buffers with ice, as this may cause crystallization of buffer 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 slow kits. Table 1 provides a standard protocol that allows you to prepare a soft hydrogel with or without TrueGel3D™ RGD integrin adhesion peptide OR protein

Table 1. reagent volumes needed to set up a soft SLOW hydrogel with or without TrueGel3D™ RGD integrin adhesion peptide OR protein

Reagents	Reagent volume in µL
	Without TrueGel RGD integrin adhesion peptide	With TrueGel RGD integrin adhesion peptide	With a protein (recommended concentration: 200 µg/ml)
Thiol reactive polymer (SLO-PVA or SLO-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 7.2	2.4	2.4	2.4
water	16.6	15.3	7.1
total	30	30	30

Experimental procedure

1. Pipet water, TrueGel3D™ buffer (pH 7.2) and SLO thiol-reactive polymer (SLO-PVA or SLO-DEXTRAN) in a reaction tube. 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. Incubate sample for 20 min to allow attachment of the RGD peptide to the polymer.
3. Add your cell suspension and protein (if applicable) to the reaction tube containing water, buffer, SLO thiol-reactive polymer and TrueGel3D™ RGD integrin adhesion peptide (if applicable).
   Add the crosslinker of your choice to your mix: depending on the crosslinker you have chosen, please use the appropriate protocol:
   A- When crosslinking with CD cell-degradable crosslinker: within one minute, the mix begins to form a gel and will not be pipettable. Resuspend your cells by gently pipetting before dispensing in the cell culture dish of your choice* to make sure that your cells will be in suspension in the gel. You can test gel formation after 25 min (before adding cell culture medium) by careful probing with a pipet tip.
   B- When crosslinking with PEG non cell-degradable crosslinker: Your mix will remain in the liquid state for up to 10 minutes before forming a gel; once the period of gel formation begins, the mixture will not be pipettable. You can use this period of time to dispense your mix in the sterile culture dish of your choice* but you will need to mix or agitate to resuspend your cells before gelation begins to ensure that they will become suspended in the gel. Gelation will be complete after about 50 minutes. incubation time. You can test gel formation before adding cell culture medium by careful probing with a pipet tip.
   * Culture vessel options include multiwell plates (6-, 24-, 96- well) or any kind of 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 your cell culture medium to the cell culture dish in sufficient volume to cover the gel.
5. Place the culture dish in the tissue culture incubator.
6. Renew the medium after 1 hour.
7. Change the medium as needed during cultivation of cells.

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

You can dissolve TrueGel3D™ Hydrogels containing live or chemically fixed cells 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 volumes of gels are prepared (less than 100 μL), you will need only very small volumes of the TrueGel3D™ RGD integrin adhesion peptide stock solution. We recommend diluting the TrueGel3D™ RGD integrin adhesion peptide stock solution (from 20 mmol/L to 3 mmol/L) with water to enable more convenient pipetting volumes. 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 final mix of water, TrueGel3D™ buffer, thiol-reactive polymer (SLO-PVA or SLO-DEXTRAN), TrueGel3D™ RGD integrin adhesion peptide (if applicable), cell suspension and crosslinker 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 cell suspension volume specified in the protocol 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) is also available for control experiments.