The cellular microenvironment plays a crucial role in cell morphology, growth, proliferation, and migration. Extracellular matrix (ECM) is an intricate network composed of extracellular macromolecules and minerals, providing essential physical scaffolding for the cellular constituents and initiating crucial biochemical and biomechanical cues that are necessary for tissue morphogenesis, differentiation and homeostasis.

In traditional two-dimensional (2D) cell culture systems, cells are cultured in flat dishes, flasks, or tubes with nutrient media. Cells mostly grow as monolayers and these flattened cells can only receive cellular signals on their ventral surfaces. In addition, the lack of cell-cell and cell-extracellular matrix interactions may alter cell morphology, develop abnormal polarization, and deviate phenotypic expression and/or genotypic characteristics. The physiology of cells cultured in vitro in three-dimensional (3D) systems resembles that of their counterparts in vivo much better than the physiology of cells cultured in conventional 2D culture systems. Over the past few decades, a wide variety of materials have been developed to culture cells within 3D ECM mimics to circumvent the limitations of traditional 2D cell culture.

Hydrogels are considered as a highly attractive material for developing 3D analogs of native ECM, due to their ability to simulate the nature of most soft tissues. Hydrogels are hydrophilic 3D cross-linked polymeric networks consisting of interconnected microscopic pores. These networks possess high water contents, which facilitates the transport of oxygen, nutrients, and soluble factors. Furthermore, hydrogels can be formed under mild and cytocompatible conditions and can be easily modified to possess cell adhesion ligands, as well as the desired viscoelasticity and degradability.

3-D Life Biomimetic Hydrogels

Amerigo Scientific offers 3-D Life hydrogel products for generating biomimetic hydrogels for 3D cell culture. The 3-D Life biomimetic hydrogels consist of a polymer functionalized with thiol-reactive groups and a thiol-containing crosslinker. The chemical cross-linking reaction is non-toxic to cells and occurs rapidly at ambient temperature to prevent cell settlement before the gel is formed.

Setup of 3-D Life Hydrogels

The procedure for setting up 3-D Life hydrogels containing embedded cells is very simple and does not require specific equipment or chemical expertise.

Three simple steps to embed cells in peptide-modified hydrogelsFigure 1. Three simple steps to embed cells in peptide-modified hydrogels

  1. Polymers, buffers, water, and peptides are mixed and incubated to allow the peptide to covalently attach to a portion of the thiol-reactive groups of the polymer.
  2. A cell suspension is added to the peptide-polymer conjugate solution.
  3. Cells and the peptide-conjugated polymers are mixed with crosslinker for gel formation. After forming, the gel is immersed in culture medium and the culture dish can be placed in an incubator for cell cultivation.

Features of 3-D Life Hydrogels

  • Defined composition of biologically inert synthetic polymers and biopolymers
  • User-controlled biomimetic modifications (e.g., peptides, proteins)
  • Robust gel formation and handling
  • Wide range of ligand density (up to 5 mmol/l)
  • Tunable gel stiffness
  • Protease-free dissolution of gels for easy cell recovery

Thiol-reactive Polymers and Thiol-functionalized Crosslinkers

Polyvinyl alcohol (PVA) or dextran can be used as thiol-reactive polymers. PVA polymer-based hydrogels are stable and cannot be degraded. In contrast, dextran-based hydrogels can be degraded by glucanases, so that cells can be isolated from such hydrogels for further use. Both polymers can be cross-linked with polyethylene glycol (PEG-Link, which cannot be degraded by cells), a polyethylene glycol peptide conjugate (CD-Link, which contains a matrix metalloproteinase-cleavable peptide that allows cells to cleat the cross-link and diffuse and migrate in gels), or HyLink (a hyaluronic acid crosslinker). The thiol-reactive polymers and the thiol-containing crosslinkers may be freely matched and mixed to produce a gel of defined properties.

PVA, dextran and PEG are water-soluble polymers that do not bind to cells and do not interfere with cellular functions. Thus, our 3-D Life hydrogel components support cells in an inert gel, which provides an ideal basis for the addition of bioactive factors such as adhesion peptides, extracellular matrix proteins, or their fragments to analyze cell phenotypes.

Thiol-reactive polymers and thiol-containing crosslinkers

Thiol-reactive polymers and thiol-containing crosslinkers

Figure 2. Thiol-reactive polymers and thiol-containing crosslinkers (PEG-Link, CD-Link, and HyLink)

Product Range

Amerigo Scientific offers 3-D Life Hydrogel Kits containing a thiol-reactive polymer, a crosslinker, and a 10x concentrated salt and buffer solution. Thiol-reactive polymers are PVA or dextran with functionalized thiol-reactive groups along their back bone. Covalent bonds can be formed between these thiol-reactive groups and thiol groups under cell-compatible reaction conditions. Depending on the type of the thiol-reactive group, the covalent bond formation proceeds at a fast or a slow rate.

• Fast Gelling

For fast gelling hydrogels, PVA or dextran is functionalized with sufficient thiol-reactive maleimide groups along their backbone for modification with bioactive factors and for crosslinking. Maleimides react specifically and very rapidly with thiol groups, so hydrogels can be formed within a few seconds.

Categories Product Name Major Components
Fast Gelling Polymers 3-D Life FG-Dextran Maleimide-functionalized dextran (Mal-Dextran)
3-D Life FG-PVA Maleimide-functionalized polyvinyl alcohol (Mal-PVA)
Fast Gelling Hydrogel Kits 3-D Life Dextran-PEG Hydrogel FG Mal-Dextran and PEG-Link
3-D Life Dextran-CD Hydrogel FG Mal-Dextran and CD-Link
3-D Life PVA-PEG Hydrogel FG Mal-PVA and PEG-Link
3-D Life PVA-CD Hydrogel FG Mal-PVA and CD-Link

• Slow Gelling

For slow gelling hydrogels, PVA or dextran is functionalized with thiol-reactive groups which react at a slower rate with thiol groups than maleimides, but with a similar reaction mechanism.

Categories Product Name Major Components
Slow Gelling Polymers 3-D Life SG-Dextran Dextran functionalized with thiol-reactive groups (SG-Dextran)
3-D Life SG-PVA Polyvinyl alcohol functionalized with thiol-reactive groups (SG-PVA)
Slow Gelling Hydrogel Kits 3-D Life Dextran-PEG Hydrogel SG SG-Dextran and PEG-Link
3-D Life Dextran-CD Hydrogel SG SG-Dextran and CD-Link
3-D Life PVA-PEG Hydrogel SG SG-PVA and PEG-Link
3-D Life PVA-CD Hydrogel SG SG-PVA and CD-Link

• Hydrogel Components

Amerigo Scientific also offers hydrogel components that involved in the formation of hydrogels, including crosslinkers, cell adhesion peptides, dextranases, and buffers. 3-D Life adhesion peptides are highly purified peptides that contain thiol groups for attachment to a hydrogel matrix, and motifs of the adhesion peptides can be recognized by cellular surface receptors. Thus, matrix-bound adhesion peptides enable cells to adhere to the hydrogel matrix, which is a prerequisite for the growth, differentiation and survival of many cell types.

Categories Product Name Size
PEG Crosslinkers 3-D Life PEG-Link 200 µl
3-D Life PEG-Link 3x 200 µl
Cell-degradable Crosslinkers 3-D Life CD-Link 200 µl
3-D Life CD-Link 3x 200 µl
Hyaluronic Acid Crosslinkers 3-D Life HyLink 200 µl
3-D Life HyLink 3x 200 µl
Cell Adhesion Peptides 3-D Life RGD Peptide 1 µmol
3-D Life RGD Peptide 3x 1 µmol
3-D Life Scrambled RGD Peptide 1 µmol
3-D Life Scrambled RGD Peptide 3x 1 µmol
Dextranase 3-D Life Dextranase 500 µl
Buffers 3-D Life 10x CB-Buffer (pH 5.5) 600 µl
3-D Life 10x CB Buffer (pH 7.2) 600 µl

Application Examples

• Epithelial Cyst Formation in 3-D Life Hydrogels

Confocal laser scanning microscopy of MDCK cells cultured 9 days in 3-D Life PVA Hydrogel modified with 5 mmol/l thioglycerol (left) or 5 mmol/l RGD peptide (right). Red: actin cytoskeleton; green: nuclei. Scale bar: 50 µm.Figure 3. Confocal laser scanning microscopy of MDCK cells cultured 9 days in 3-D Life PVA Hydrogel modified with 5 mmol/l thioglycerol (left) or 5 mmol/l RGD peptide (right). Red: actin cytoskeleton; green: nuclei. Scale bar: 50 µm.


• Fibroblast Spreading in Cell-degradable 3-D Life Hydrogels

Confocal laser scanning microscopy of 3T3 fibroblasts cultured 14 days in 3-D Life PVA-Hydrogel modified with RGD peptide and crosslinked with either 3-D Life PEG-Link (left) or 3-D Life CD-Link (right). Pictures show collapsed stacks of confocal frames representing a height of 300 µm of the gel. Red: nuclei; green: actin cytoskeleton. Scale bar: 200 µmFigure 4. Confocal laser scanning microscopy of 3T3 fibroblasts cultured 14 days in 3-D Life PVA-Hydrogel modified with RGD peptide and crosslinked with either 3-D Life PEG-Link (left) or 3-D Life CD-Link (right). Pictures show collapsed stacks of confocal frames representing a height of 300 µm of the gel. Red: nuclei; green: actin cytoskeleton. Scale bar: 200 µm.

Application Note: Fibroblast Spreading in Cell-Degradable 3-D Life Hydrogels


• Co-culture of Tumor and Stroma Cells in 3-D Life Hydrogels

Epifluorescence microscopy of mono- and co-culture of MCF-7 cancer cells and human primary dermal fibroblasts in 3-D Life Hydrogels. A: MCF-7 cells cultured alone, B: fibroblasts cultured alone, C: co-culture of MCF-7 and fibroblasts. Red: actin cytoskeleton; green: nuclei. Scale bar: 100 µm.Figure 5. Epifluorescence microscopy of mono- and co-culture of MCF-7 cancer cells and human primary dermal fibroblasts in 3-D Life Hydrogels. A: MCF-7 cells cultured alone, B: fibroblasts cultured alone, C: co-culture of MCF-7 and fibroblasts. Red: actin cytoskeleton; green: nuclei. Scale bar: 100 µm.


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