True Gel3D™ Hydrogel Troubleshooting Guide

The TrueGel3D™ hydrogel pre-configured kit (TRUE1) is used to prepare a chemically defined hydrogel with stiffness of ~400 Pa (shear modulus) that can be customized to match that of the native cell environment. The TrueGel3D™ hydrogel is formed by crosslinking of RGD (arginylglycylaspartic acid) degradable polymer with CD cell-degradable crosslinker. The cells are encapsulated during crosslinking and can adhere to the polymer through the RGD peptide and grow within the hydrogel. The CD cell-degradable crosslinker is composed of matrix metalloprotease (MMP)-cleavable peptide (Pro-Leu-Gly-Leu-Trp-Ala) that enables cells to spread and migrate by secreting matrix metalloproteases (MMP1, MMP3, MMP7, and MMP9).

The hydrogel can be further dissolved by TrueGel3D™ Enzymatic Cell Recovery Solution (TRUEENZ) for post-culture analysis including RT-PCR, Western blots and other molecular biological studies.

Issues	Reasons	Solutions
Weak/low gel formation	Thiol-reactive groups (polymer) and thiol groups (crosslinker) are not near equimolar concentration	Balance the thiol-reactive groups of polymer with thiol groups of crosslinkers
	Excess of peptide with thioglycerol results in few thiol-reactive groups	Balance the thiol groups of peptide and crosslinker with thiol-reactive groups of polymer
	Low pH caused by acid in peptide sample	Optimize the buffer pH: For a slow gelling system use a buffer with pH 7.2. For a fast gelling system use a buffer with pH 5.5
	Weak network formed because of deteriorated reactive groups	Use fresh aliquots
Fast gelation	pH is too high	Use buffer with pH 5.5 to slow down the gelation
Too slow gelation process	pH is too slow	Use buffer with pH 7.2 to accelerate the gelation
Decrease in cell viability	Lack of adhesion factor in polymer	Add appropriate adhesion peptide or proteins
	Acidic environment	Use neutral pH during gel setup
Incomplete dissolution of dextran polymer even after 1 hour incubation with TrueGel3DTM enzymatic cell recovery solution	High gel strength	Increase the incubation period for 2 hours and increase concentration of TrueGel3D™ enzymatic cell recovery solution
	Reduced TrueGel3D™ enzymatic cell recovery solution activity	Use fresh aliquot of enzyme