Humabiologics HumaDerm- Human Skin Collagen Type I, Lyophilized

HumaDerm- Human Skin Collagen Type I, Lyophilized (SCL)

HumaDerm is ideal for many applications including coating tissue culture surfaces to support rapid cell attachment and growth or for making hydrogel. The optimal concentration may vary depending on cell type and application and must be titrated for best results. It is soluble in dilute acid at pH ~2.0.

Product Description

Type I Collagen is the most abundant type of collagen in the human body, as a major structural matrix protein in skin, and many other tissues (bone, tendon, and fibrous connective tissues) [1]. There are a number of types of collagen identified to date, and all are composed of molecules containing three polypeptide chains arranged in a triple helical conformation [2]. The types of collagen differ slightly in the primary amino acid sequence of their polypeptide chains. Type I collagen is a heterotrimer composed of one α2(I) chain and two α1(I) chains [3].

Various types of cell surface receptors present on diverse types of mammalian cells recognize the collagen triple helix structure and facilitate cell attachment to collagen materials, including films and scaffolds. The most characterized cell receptors are the integrins α1β1 and α2β1 [4]. Many cell types express both forms of integrin, including mesenchymal stem cells (MSCs), fibroblasts, endothelial cells, chondrocytes, osteoblasts, and lymphocytes[3, 5-7]. Smooth muscle cells interact with collagen via α1β1, and epithelial cells attach via α2β1 [8, 9]. Collagen type I is characterized by the presence of three regions on SDS-PAGE corresponding to molecular weights. The alpha region has a molecular weight of 100 KDa and consists of two α1 chains and one α2 chain. The beta region has a molecular weight of 200 KDa and consists of two α1 chains fused together and α1 α2 chains fused together. The gamma region has a molecular weight of 300 KDa and consists of overlapping two α1 and one α2 chains. HumaDerm has all three regions and chains with very minimal fragmented proteins in between. HumaDerm is ideal for many applications including coating tissue culture surfaces to support cell attachment and growth or for making hydrogel.

Tissue Source

Type I collagen is isolated from human skin sourced strictly from American Association of Tissue Banks (AATB) accredited and FDA registered tissue banks and organ procurement organizations (OPOs). Humabiologics strives to meet research needs by providing high quality biomaterials obtained from tissue partners who comply with requirements for transplantable human tissues under 21 CFR 1271 of the Food and Drug Administration (FDA).

Precautions and Disclaimer

HumaDerm is obtained from human tissue that has been tested and found negative for a minimum of HIV-1 and -2, hepatitis B, and hepatitis C, as well as other infectious agents. Please review the Safety Data Sheet (SDS) for information regarding hazards and safe handling practices. HumaDerm is for research use only and is not intended for human use, diagnosis, screening, household, food or other uses.

Storage/Reconstitution

HumaDerm should be stored at -20 °C upon receipt. Collagen is a stable protein if it is stored frozen while minimizing freeze-thaw cycles. Research grade products have a shelf life of 6 months after opening.

HumaDerm can be reconstituted in HCl or acetic acid. HumaDerm is soluble in 10-20 mM HCl with a final pH of 1.9-2.1. Reconstitution may take 3 hours at ambient temperature with vigorous shaking/vortexing or overnight at 4 °C. Solution can be briefly centrifuged to remove bubbles. Do not let the temperature exceed 30°C during reconstitution. After reconstitution, solution should be used immediately or stored at 4 °C for no more than 3 months.

Collagen-Coated Tissue Cultureware

Note: The following are general recommendations. Researcher should optimize parameters based on their specific applications. Optimal collagen concentrations for tissue culture surface coatings may depend on application and cell type and must be determined for each application. Typically, 10 - 100 µg/ml of collagen is used.

1. Reconstitute HumaDerm in 10-20 mM HCl with a pH of 1.9-2.1 to a concentration of 0.1 mg/ml. It is recommended to use ultrapure sterile water to make the 10-20 mM HCl.

2. Mix by shaking or vortexing until the solution is homogeneous.

3. Aseptically add collagen solution to tissue culture surface to evenly cover entire surface.

4. Cover and incubate collagen coated surfaces at 37 °C for at least 2 hours. The optimal incubation time may vary depending on the experiment and cell type and should be determined for each application.

5. Transfer collagen coated surfaces to biosafety cabinet and aspirate excess solution from coated surface careful not to scratch the surface. Remove lid to allow for air drying overnight.

6. Rinse the coated culture surface with sterile PBS or culture medium to remove residual acid.

7. Use coated cultureware immediately or keep sterile and store at 2-10 °C.

Making 3D Collagen Gels

Note: All procedures should be performed in a cold room or on ice. All reagents should be cooled prior to making the hydrogel. To prevent premature gelation, keep solutions between 2-10 °C. Concentrations will need to be tested to determine the optimal gel concentration for each application. HumaDerm has been tested and found to form a gel at ≥ 0.5 mg/ml final concentration.

1. Reconstitute HumaDerm in 10-20 mM HCl with a pH of 1.9-2.1 to desired concentration. It is recommended to use ultrapure sterile water to make the 10-20 mM HCl.

2. Prepare 10x culture medium or 10x PBS.

3. Combine 9 volumes of chilled collagen solution with 1 volume of chilled 10x concentrated medium or 10x PBS.

4. Gently swirl or pipette the mixture repeatedly.

5. Adjust the pH to 7.0-8.0 using NaOH, preferably around 7.4. Concentration and pH will affect the speed and strength of the gel. Once pH has been adjusted, cells can be added to the solution if desired.

6. Incubate at 37 °C for 30-120 minutes for gel formation depending on final concentration.

References

1. Bornstein, P. and H. Sage, Structurally distinct collagen types. Annu Rev Biochem, 1980. 49: p. 957-1003.

2. Tanzer, M.L., Cross-linking of collagen. Science, 1973. 180(4086): p. 561-6.

3. Heino, J., The collagen family members as cell adhesion proteins. Bioessays, 2007. 29(10): p. 1001-10.

4. Barczyk, M., S. Carracedo, and D. Gullberg, Integrins. Cell Tissue Res, 2010. 339(1): p. 269-80.

5. Heino, J., The collagen receptor integrins have distinct ligand recognition and signaling functions. Matrix Biol, 2000. 19(4): p. 319-23.

6. Ivaska, J., et al., Cell adhesion to collagen. Is one collagen receptor different from another? Vol. 30. 1998. 273-283.

7. Goessler, U.R., et al., In vitro analysis of integrin expression in stem cells from bone marrow and cord blood during chondrogenic differentiation. J Cell Mol Med, 2009. 13(6): p. 1175-84.

8. Ebert, E.C. and A.I. Roberts, Human intestinal intraepithelial lymphocytes bind to mucosal mesenchymal cells through VLA4 and CD11A. Cell Immunol, 1996. 167(1): p. 108-14.

9. Gilcrease, M.Z., Integrin signaling in epithelial cells. Cancer Lett, 2007. 247(1): p. 1-25.