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Report No. 008 Cell culture utilizing collagen
Report No. Cell culture utilizing collagen
008

Overview

In cell culture, the selection of culture substrates is an important factor that influences the results of the experiments. The cells that comprise the human body are classified into about 200 types [ref. 1], and these cells are in contact with a wide variety of extracellular matrix (ECM*) components. By making the ECM composition of the culture substrate closer to that of the body, we could reproduce "actual in vivo phenomena" in vitro.
*What’s the extracellular matrix (ECM)?
The ECM is an insoluble structure made up of molecules secreted by cells, and it functions as a scaffold for cells. In addition to functioning as a framework that physically and mechanically supports tissues and organs such as skin, heart, kidneys and bones, it also plays a role in controlling the proliferation and differentiation of various cells, including blood vessels and nerves. In mammals, including humans, there are around 270 types of extracellular matrix proteins, including collagens, fibronectins, laminins and proteoglycans [ref. 2, 3]. Furthermore, molecular diversity can be created through mechanisms such as alternative splicing of genes, post-translational modifications including proline hydroxylation and lysine hydroxylation, diverse glycosylation and regulation through enzymatic degradation. Just as collagen fibers vary in thickness and length depending on the magnitude and direction of applied forces on the tissue, ECM components are also influenced by external forces. These wide varieties of changes in ECM components contribute to the elasticity, toughness, and flexibility of tissues.

Extracellular matrix as culture substrate

Of all the ECM components, type I collagen, which exists in large amounts in the body, has been the most extensively studied. Type I collagen molecules, after being produced by fibroblasts and other cells, form fibers and act as a tissue backbone. Type I collagen can be extracted while maintaining its structure in vivo, characterized by a triple-helix conformation (Report #01 Solubilization of insoluble collagen fibers ).The extracted type I collagen can be coated on culture dishes or made into gels by adjusting the temperature, pH, and ionic strength (Fig. 1).



Fig. 1 Treatment of culture dishes with type I collagen.

Cells have cell surface receptors recognizing the ECM components. Cells adhere to collagen via integrins and DDRs (discoidin domain receptors), which regulate various signal transduction pathways involved in cell proliferation and differentiation. In general, cell adhesion and proliferation are enhanced on type I collagen-coated culture dishes. On the other hand, when cultured on type I collagen gels, various phenomena not observed in collagen-coated cultures occur depending on the cell type. For example, epidermal keratinocytes promote adhesion and elongation on collagen-coated culture dishes, whereas cell elongation is inhibited on collagen gels, resulting in apoptosis (Fig. 2, ref. 4). Collagen gels are formed by regularly arranged fibers of collagen molecules, and its physical properties differ from those of collagen molecules alone. It has been suggested that these changes in physical properties also affect cell behavior through receptors such as integrins, which have recently been reported to function as mechanosensors [ref. 5-8].


Fig. 2 Morphological changes in human epidermal keratinocytes cultured on collagen molecules and fibers.

Standard experimental protocols have been established for coating collagen onto culture dishes and preparing gels, making it easy to start experiments. We have various kinds of collagens and reagent kits for laboratory research and three-dimensional culture.

Reference

1. Cooper G, Adams K. The cell: A Molecular Approach, ninth edition. Oxford University Press, (2022)
2. Naba A, Clauser KR, Hoersch S, Liu H, Carr SA, Hynes RO. The matrisome: in silico definition and in vivo characterization by proteomics of normal and tumor extracellular matrices. Mol Cell Proteomics. 11, M111.014647 (2012)
3. Naba A, Clauser KR, Ding H, Whittaker CA, Carr SA, Hynes RO. The extracellular matrix: Tools and insights for the "omics" era. Matrix Biol. 49, 10-24 (2016)
4. Fujisaki H, Hattori S. Keratinocyte apoptosis on type I collagen gel caused by lack of laminin 5/10/11 deposition and Akt signaling. Exp Cell Res. 280, 255-269 (2002)
5. Katsumi A, Orr AW, Tzima E, Schwartz MA. Integrins in mechanotransduction. J Biol Chem. 279, 12001-12004 (2004)
6. Stupack DG. The biology of integrins. Oncology (Williston Park). 21, 6-12 (2007)
7. Sun Z, Guo SS, Fässler R. Integrin-mediated mechanotransduction. J Cell Biol. 215, 445-456 (2016)
8. Jansen KA, Atherton P, Ballestrem C. Mechanotransduction at the cell-matrix interface. Semin Cell Dev Biol. 71, 75-83 (2017)

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