TrueGel3D™ is a biochemically defined hydrogel formed by mixing polymers with crosslinkers. Compared to other hydrogels based on a biological extract from animal cells (e.g. mouse EHS tumor cells), TrueGel3D™ does not contain any products of animal origin that could interfere with or contaminate experiments. Moreover, components that are included in TrueGel3D™ preserve viability and allow critical features of the natural extracellular matrix (ECM) to be mimicked. They replicate native cellular environments similar to those of tissues by supporting cell adhesion and migration. TrueGel3D™ technology provides mechanical and biochemical cues to investigate both morphological and physiological properties of cells in the 3D environment. Finally, these hydrogels allow encapsulation of cells for growth in a 3D environment that more closely mimics native tissue environments when compared with traditional 2D cell culture conditions.
Most importantly, TrueGel3D™ has a defined composition, unlike basement membranes extracts derived from animal cells where a mix of ECM components and growth factors can interfere with the cells because it creates a non-native environment.
The TrueGel3D™ technology is a four component system consisting of:
Figure 1.TrueGel3D™ technology
Figure 2.TrueGel3D™ protocol flowchart
If you need to optimize your gel environment (e.g. change in stiffness, adhesion molecules, ECM proteins, etc.), other TrueGel3D™ kits are available and are listed in Table 1.
*During this time, the mix with all components remains liquid; after that, it begins to form a gel and can no longer be pipetted.
**For a more detailed view of recommended gels by application, please refer to Table 2.
The moderate gelation time is only available for pre-configured TrueGel3D™ hydrogel, CD cell-degradable crosslinker and RGD peptide (Cat. No.TRUE1-1KT). Fast and slow gelation kits are listed in Table 1.
*TrueGel3D™ RGD adhesion peptide (provided as separate component) needs to be added
Polymers and crosslinkers with differential properties provide options for gel requirements
TrueGel3D™ hydrogel systems employ one of two types of polymers: a synthetic, non-degradable polyvinyl alcohol (PVA) or an enzymatically-degradable dextran. Both polymers are functionalized either with fast or slow thiol-reactive groups.
Polymers (PVA and dextran) are inert and do not adhere to cells. However, they can be conjugated with bioactive components such as adhesion peptides or combined with extracellular matrix proteins to allow cell adhesion and mimic relevant physiological conditions.
Crosslinkers in TrueGel3D™ are also of two types. The PEG non-cell-degradable crosslinker is made up of polyethylene glycol with thiol (SH) groups at each end. The CD cell-degradable crosslinker is similar to PEG non-cell-degradable crosslinker, but includes peptidic sequences, creating cleavage sites for matrix metalloproteases (MMPs). Gels with CD cell-degradable crosslinker allow cells to spread and migrate by cleaving conjugated peptide if cell adhesion molecules are also a component of the gel.
Variables affecting the gelation rate:
Gel formation occurs best at equimolar concentration (starting from 1.8 mmol/L) of thiol (crosslinker) and thiol-reactive groups (polymer). However, a stiffer gel can be formed with a higher concentration of thiol and thiol-reactive groups.
In addition, the reaction rate is also controlled by hydrogen ion concentration (pH). At a lower pH (acidic environment) the deprotonated thiolate anions react slowly with thiol-reactive groups and slow down the network formation between polymer and crosslinker. Thus, buffers in the kits (pH 5.5 and 7.2) can be mixed to generate an intermediate pH to aid in optimization of gelation time. Both buffers include phenol red to facilitate pH monitoring.
A suspension of freshly prepared cells from embryonic chick heart tissue was embedded in a Dextran-based hydrogel modified with RGD Peptide and crosslinked with matrix metalloprotease-cleavable crosslinker (TRUE1). Aggregated heart muscle cells are surrounded by fibroblasts. Heart muscle cells courtesy of Dr. Udo Kraushaar (NMI Reutlingen, Electrophysiology Group, Germany).