Cells in their physiological setting typically interact with extracellular factors in three dimensions (3D). The extracellular matrix (ECM) is a complex formulation of biological polymers composed of peptides, polysaccharides, proteins, enzymes and signaling molecules that reside outside of cells. Interactions between cells and their surrounding ECM provide cues that can influence cellular polarization, shape, motility, differentiation and many other phenotypes. Traditional cell culture is done on a two-dimensional (2D) substrate, usually polystyrene or glass, and cells grown in this manner often do not fully recapitulate many phenotypes observed by those same cell types in vivo. One approach to making cells cultured in vitro more closely resemble their corresponding physiological tissue type is to embed and culture cells within a 3D hydrogel that mimics the physiological ECM. This strategy is a subset to the larger approach of culturing cells in three-dimensions, typically referred to as 3D cell culture.

Comparison of cellular behavior cultured in 2D vs 3D cell culture

Figure 1. Hydrogels enable more biologically-relevant 3D cell culture.

3D Hydrogel Applications

Cells grown in 3D hydrogels have proven valuable for numerous applications. Traditional immortalized cell lines can be cultured embedded within hydrogels to form multicellular complexes called spheroids. Spheroids have been demonstrated to provide a wealth of information not apparent from the same cells cultured under 2D conditions. Stem cells grown in 3D hydrogels can differentiate into multiple cell types that closely resemble native tissues. They can be programmed to form 3D organoids, differentiated mini-organ structures that have been used as highly predictive, reproducible, and scalable in vitro models of complex physiology. Other applications of culturing cells within hydrogels have focused on interrogating the physical properties of the hydrogel to mechano-sensitive behaviors of cells including the formation of morphological features and cell motility.

Naturally-derived 3D Hydrogels

The most conventional method for mimicking the physiological extracellular matrix in vitro is to use an extract of native ECM from a biological source. Traditionally, the ECM extract from Engelbreth-Holm-Swarm (EHS) murine sarcoma basement membrane has been used for 3D cell culture applications. EHS ECM extract is conveniently liquid at 4 °C and will gelate at room temperature, allowing cells to be easily embedded. This product is sold as ECM Gel hydrogel. For some applications, an ECM extract from human cells is important. MaxGel™ ECM is a human in vitro derived basement membrane extract (BME).

Synthetic 3D Hydrogels

Reconstituting a synthetic hydrogel may be the optimal choice for some applications. A chemically defined hydrogel formulation enhances reproducibility, and the presence and concentration of media components such as growth factors is known. In addition, key physical parameters of a hydrogel may be important for specific applications. These include:

  • elastic modulus
  • pore size
  • hydrogel swelling
  • crosslinking
  • dissociation conditions

We offer several reagents, kits and protocols to be used for developing synthetic hydrogels including HyStem® hydrogel scaffolds, TrueGel3D™ hydrogel kits and Cell-In-a-Box® technology.

Reconstituted hydrogels for 3D cell culture (HYS020)

Figure 2. Synthetic Extracellular Matrix (ECM)


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