ESI BIO
Production Manager
Cell-culture standards have been evolving steadily since the practice emerged in the early twentieth century. With the scientific community becoming increasingly aware of the importance of accurately reproducing the in vivo niche during in vitro experiments, techniques, reagents and substrates have changed to provide increasingly repeatable and physiologically relevant results. That's especially true in the fields of cancer and stem cell biology: The mechanisms that contribute to many of the most common end points in these fields, from proliferation and differentiation to gene expression and drug resistance, can all be impacted by culture conditions. In particular, researchers are more commonly factoring into their experiments that cells in vivo do not generally grow in two-dimensional sheets but rather in complex three-dimensional (3D) structures.
Researchers have devised several strategies to 3D cell culture, including microgravity suspension systems, hanging drops, microcarriers and scaffolds, and all have strengths and weaknesses. Weaknesses can include unnatural and unyielding plastic components, the need for specialized equipment, harsh chemical manipulations and toxic or undefined reagents. The biocompatible HyStem® hydrogels from ESI BIO avoid many of these undesirable elements. Being both easily customizable in physical and chemical properties and also applicable to a range of culture formats and cell types, the HyStem hydrogel platform provides a flexible, customizable base for experimentation.
HyStem defined
ESI BIO’s HyStem technology is based on the reaction between thiol-modified biopolymers and a thiol-reactive crosslinker to produce a hydrogel that gels in situ and can be used as both a cell-culture scaffold and cell-delivery vehicle. The common thread in all HyStem hydrogels is hyaluronic acid (HA)—an evolutionarily conserved glycosaminoglycan with excellent water-retention capabilities that is found in many tissue types and species. HyStem also includes a polyethylene glycol-based crosslinker to tune gelation, and can be formulated to include cellular attachment sites, growth factors and small molecules, the most common of which are gelatin as a basic attachment protein and heparin for slow growth-factor release.
The standard HyStem-C formula for adherent cell types contains 0.4% Glycosil (thiol-modified HA), 0.4% Gelin-S (thiol-modified porcine gelatin) and 0.2% Extralink (poly (ethylene glycol) diacrylate). These components are provided as dry, lyophilized solids containing phosphate-buffered saline salts and are reconstituted in water. When combined, the resulting clear liquid can be manipulated easily and gels within 45 minutes. This format makes it easy to alter the final gel composition to influence such parameters as gelation time, stiffness and porosity. It is therefore a simple matter for researchers to screen their particular cell types against an array of hydrogel conditions to find the one that best supports physiologic growth and behavior.
Optimizing conditions
Because of the widely different nature of cells used in research, HyStem hydrogel kits have been formulated to be readily customizable.
The simplest approach an experimenter can take to modify cell-culture conditions is to change the reconstitution volume. Although the standard use instructions indicate reconstituting the components to 10 mg/mL each, the biopolymers in Glycosil and Gelin-S can be reconstituted in half the volume, and Extralink is soluble up to about 150 mg/mL. By starting with a more concentrated solution and diluting with PBS, gel stiffness and gelation time can be controlled across two to three orders of magnitude. For example, a neural cell line that is accustomed to the soft conditions in the brain may benefit from a more dilute, looser hydrogel than chondrogenic cells that require a more rigid environment to induce natural behavior.
For adherent cell types, the hydrogel should have relevant attachment sites covalently linked to the matrix. The HA and collagens in Gelin-S provide a good foundation for many cell types, including mesenchymal stem cells, endothelial cells, adipocytes and hepatic cells, but it may be necessary to supplement or replace the collagen for specific experiments. To that end, any proteins that will react with thiol or acryl groups can be added to the liquid components and incorporated into the matrix. For example, if a researcher aims to build an animal-free tumor model, replacing the Gelin-S with recombinant laminin, and a peptide containing an RGD attachment sequence and a terminal cysteine or maleimide, will allow for cancer-cell attachment and aid in vascularization. Similarly, growth factors (GF) and cytokines may be added to the liquid components, as desired.
Typically, a protein that is not covalently linked to the hydrogel and is smaller than 75 kDa will elute out of the gel via osmosis. This rate can be controlled by formulation changes, with more crosslinking due to higher concentrations of Glycosil and HA reducing the rate of release. For a more controlled approach, Glycosil is available with thiolated heparin (HyStem-HP Hydrogel Kit). The immobilized heparin mimics the heparin sulfate proteoglycans normally present in the extracellular matrix; it slows the release of GF and prevents proteolysis. With this approach, far less GF is required to achieve cell signaling than when it is added directly to the media. Again, users must experiment with the protein concentration to determine what is optimal for a given experiment, but optimization is as easy as adding proteins or peptides of interest to the liquid gel components and screening encapsulated cells for the desired phenotype, behavior or expression profile.
The goal, of course, is to achieve the most physiologically relevant culture system possible, and HyStem hydrogels have been used successfully to culture, characterize and deliver numerous cell types in many different systems. For specific recommendations and examples relevant to a particular field of research, please visit our web site or contact us directly.
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