Extracellular matrix proteins and tools for cell culture optimization
Animal cells and tissue culture techniques are constantly being improved to optimize in vitro cell culture conditions. Protein coating of the extracellular matrix (ECM), chemical or physical modification of the cell culture vessel, have been shown to be efficient methods to better mimic cell behaviour in vivo. We describe the different coatings available below, with some highlights of new technologies. Background In the 1900s, animal tissues were grown on glass surfaces, but since they require careful cleaning procedures, researchers began experimenting with disposable plastic culture containers made of polystyrene. However, plastic grows containers have certain limitations:
- Difficulty in cell growth and cell attachment in serum-free media
- Change in the shape, polarity and morphology of cells.
- Increases cell proliferation and decreases differentiation.
- Less reactive to hormones and growth factors.
The researchers then began coating the container with biological materials (biological coating) and synthetic polymers (chemical coating) that can enhance cell attachment, growth, and differentiation. Cell growth on coated surfaces is a more relevant representation of the natural environment compared to cell growth on flat 2D plastic surfaces. This technique can be qualified as a 2D physiological environment or 2.5D cell culture condition.
COMPONENTS OF THE EXTRACELLULAR MATRIX
Tissues are not only packed with cells; most of the volume contains extracellular space and is filled with a complex network of proteins called the extracellular matrix (ECM). The components of the ECM in most tissues are secreted by fibroblasts and are classified into proteoglycans and fibrous proteins (collagen, elastin, fibronectin, and laminin). These components provide structural support and facilitate cell communication. Integrins, the cell surface transmembrane proteins that link the cytoskeleton of cells to ECM, activate signalling pathways that regulate cell proliferation, morphology, adhesion, and death.
Collagen is the most abundant protein in mammals and constitutes 25% of the total protein mass. It is composed of three polypeptide chains (called alpha chains) arranged in a helical conformation, rich in glycine and proline residues. There are more than 20 different types of collagens, of which collagen I, II, III, V, and XI are fibrillar collagens that are commonly found in connective tissue.
Type IX and XII collagens are fibril-associated collagens, which bind fibrils to each other and to extracellular matrix components. While type IV and VII collagen is network-forming collagen that makes up most of the basal lamina. In tissues, collagen provides structural support, strength, and endurance, and in cell culture, it is used to study cell growth, differentiation, and migration.
Elastin is a hydrophobic protein of 750 amino acids, rich in proline and glycine. However, unlike collagen, these amino acid residues are not glycosylated. Tropoelastin, a soluble precursor secreted in the extracellular space, assembles into insoluble elastic fibres and sheets. The elastic fibres provide the necessary resistance so that the fabrics can recoil after a transitory stretch.
Fibronectin is a large (220 kDa) glycoprotein composed of two polypeptide chains (dimer) linked by disulfide bonds at one end. Each polypeptide further folds into functionally and structurally distinct domains that bind to various ECM components (glycosaminoglycans, proteoglycans, and collagen) and cell surface proteins.
Fibronectin is secreted by a wide variety of connective tissue cells, including fibroblasts, chondrocytes, Schwann cells, macrophages, intestinal epithelial cells, and hepatocytes. Fibronectin is a multifunctional protein involved in cell spreading and adhesion. It also regulates cell morphology, cell migration, cytoskeletal organization, hemostasis, and wound repair.
Laminin is a major component of the basal lamina. It is made up of three long polypeptide chains (called α, β and γ) that are held together by disulfide bonds and are arranged in an asymmetric cross shape. Laminin acts like glue, holding cells and the ECM together. It has active domains for collagen binding, cell adhesion, heparin-binding, and neurite outgrowth fragment. Laminin modulates cell growth, motility, and signalling pathways.
Vitronectin is a 459 amino acid glycoprotein found in the ECM and blood. It circulates in the blood as a single chain residue of 75 kDa or as a two-chain residue of 65 kDa and 10 kDa. Vitronectin interacts with polysaccharides (glycosaminoglycans) and proteoglycans, acting as a cell adhesion molecule.
Although vitronectin and fibronection have similar functions and have an Arg-Gly-Asp cell recognition sequence, they are structurally and immunologically distinct. Vitronectin acts as an inhibitor of the complement cytolytic pathway and has a physiological role in the coagulation pathway. In addition, it promotes cell migration, proliferation, differentiation and dissemination of endothelial and neoplastic cells.
CHEMICAL / SYNTHETIC COATINGS
- Polylysine and polyornithine
The coating of synthetic polymers (polyamino acids) facilitates the binding of both cells and proteins. Poly amino acids such as polylysine and polyornithine create a positive charge on polystyrene and increase the positively charged sites available for cell attachment. They are also used in combination with binding factors that can promote electrostatic interaction between negatively charged ions in the membrane. cell and positively charged ions of binding factors on the surface of the culture.
- Cytosoft coating
The stiffness of the substrate influences cell functions. CytoSoft® plates are coated with a thin layer of biocompatible silicone, with various stiffnesses that cover a wide physiological range. The surface of the gels forms stable covalent bonds with proteins, facilitating the coating of the gel with binding factors (ECM components) and plaque cells. The following are the advantages of CytoSoft® boards:
- Optically clear and with low autofluorescence.
- Silicone gels are not susceptible to hydrolysis.
- Silicone gels are stable, they do not dry out or swell.
- Resistant to tearing or cracking
- Rigidity hardly changes during long storage periods
- Trypsin and collagenase can be used to harvest cells.
- Resistant to biochemical degradation after enzyme treatment.
Coating the culture surface with ECM proteins and synthetic polymers greatly influences cell behaviour. The observed response depends on both the cell type and the coating used as a substrate. Cells in contact with binding factors survive longer and can also be cultured in the absence of serum factors. Binding factors can sequester and store growth factors, controlling the Spatio-temporal regulation of the factors and facilitate cross-communication between growth factor receptors and ECM receptors.
It also defines mechanical properties and instructs cells to differentiate under permissive conditions. ECM proteins also induce intracellular signalling through the cell surface receptor in synergy with growth factor signalling. As cell culture evolves, more components and combinations are needed to better mimic the in vivo conditions of cells. tissues and deciphering the language of the extracellular matrix between cells.