Placenta Derived Adherent Cell (PDAC) Interaction and Response on Extracellular Matrix Isolated from Human Placenta
- 0 Comments
- 7382 reads
Isolation of pECM. The pECM was isolated from frozen placentas, qualified as described previously.7,8 A frozen human placenta was thawed in 0.5 M sodium chloride, ground in an Anvil meat-grinder (Model MIN0012), and repeatedly washed in 0.5 M sodium chloride and water mixture using a New Brunswick Scientific (Model C25; Edison, NJ) incubator shaker at 23°C. This was followed by a detergent wash with either 1% SDS or deoxycholic acid. The blood-free placental tissue was treated with 0.1–0.5 N sodium hydroxide (3–24 hours), followed by rinsing with PBS to neutralize the pH. The material produced was a stable paste and stored at 4°C.
Biochemical analysis. To determine the biochemical composition of the isolated pECM,a sample of known dry weight was freeze-dried and the dry weight was determined.The pECM was solubilized by either dissolving in 10 mM HCl at 100°C or by treating with pepsin (1 mg/g) in 10 mM HCl at 23°C for 18 hours.The tissue dissolved in 10 mM HCl was used to determine content of fibronectin, laminin, glycosaminoglycans (GAGs), and elastin. The pepsin-solubilized tissue was used to determine collagen content using the SIRCOL dye based assay kits from Accurate Chemical and Scientific (Westbury, NY). Fibronectin and laminin concentrations were determined using a sandwich ELISA obtained from Takara Bio USA (Madison,Wis). Elastin and GAG content were determined using the FASTIN and BLYSCAN dye based assay kits (Accurate Chemical and Scientific).For collagen I, III, and IV, sandwich ELISAs were developed using primary and HRP conjugated secondary antibodies from Rockland Immunochemicals, Inc. (Gilbertsville, Pa).
Preparation of pECM constructs. To prepare sheets of this pECM, an even layer of hydrated pECM paste was sandwiched between 2 medical grade Tyvek sheets from Tolas Health Care (Feasterville, Pa).This construct was loaded into a gel drier (BioRad Model 583) and vacuum was applied overnight at 23°C until the pECM film was dry. Sheets were cut to an appropriate size for cell culture studies.To prepare 3-D structures of the pECM, the pECM paste was filled into various molds and freeze-dried using a benchtop Labconco Freezone 4.5 freeze dryer (Labconco,Kansas City,Mo).To study the stability of the pECM sheets and 3-D molds in cell culture media or water.The constructs were incubated at 37°C up to 1 week in water, saline, or cell culture media.
1. Cabrera-Crespo J, Goncalves VM, Martins EA, Grellet S, Lopes AP, Raw I. Albumin purification from human placenta. Biotechnol Appl Biochem. 2000;31(Pt 2):101–106.
2. Frolik CA, Dart LL, Meyers CA, Smith DM, Sporn MB. Purification and initial characterization of type beta transforming growth factor from human placenta. Proc Natl Acad Sci U S A. 1983;80(12):3676–3680.
3. Li S, Li F,Tan W,Yang N, Jin H, Chen H.An improved, inexpensive method for the large-scale purification of human nerve growth factor. Biotechnol Appl Biochem. 1998;28(Pt 3):215–218.
4. Rhodes RK, Miller EJ. Evidence for the existence of an alpha 1(V) alpha 2(V) alpha 3(V) collagen molecule in human placental tissue. Collagen Relat Res. 1981;1(4):337-343.
5. Edinger J,Ye Q, Paludan C,Wang JL, Hofgartner W, Hariri R. Characterization of placenta derived adherent cells (PDAC): a novel type of stem cells isolated from human placenta. American Society of Hematology, 48th Annual Meeting; December 9–12, 2006; Orlando, Fla.
6. Gharaee-Kermani M, Phan SH. Links role of cytokines and cytokine therapy in wound healing and fibrotic diseases. Curr Pharm Design. 2001;7(11):1083–1103.
7. Hariri RJ, Kaplunovsky AM, Murphy PA. Collagen biofabric and methods of preparing and using the collagen biofabric. US Patent Application Publication No. 20030187515. 2003.
8. Hariri RJ, Kaplunovsky AM, Murphy PA. Collagen biofabric and methods of preparing and using the collagen biofabric. US Patent Application Publication No. 20040048796. 2004.
9. Velling T, Risteli J,Wennerberg K, Mosher DF, Johansson S. Polymerization of type I and III collagens is dependent on fibronectin and enhanced by integrins alpha 11beta1 and alpha 2beta 1. J Biol Chem. 2002;277(40):37377–37381.
10. Dinh T, Pham H,Veves A. Emerging treatments in diabetic wound care.WOUNDS. 2002;14(1):2–10.
11. Bennett NT, Schultz GS. Growth factors and wound healing: Part II. Role in normal and chronic wound healing. Am J Surg. 1993;166(1):74–81.
12. Singer AJ, Clark RA. Cutaneous wound healing. N Engl J Med. 1999;341(10):738–746.
13. Grinnell F. Fibronectin and wound healing. J Cell Biochem. 1984;26(2):107–116.
14. Rollins BJ. Chemokines. Blood. 1997;90(3):909–928.
15. Gerszten RE, Garcia-Zepeda EA, Lim YC, et al. MCP-1 and IL-8 trigger firm adhesion of monocytes to vascular endothelium under flow conditions. Nature. 1999;398(6729):718–723.
16. Fahey TJ 3rd, Tracey KJ, Tekamp-Olson P, et al. Macrophage inflammatory protein 1 modulates macrophage function. J Immunol. 1992;148(9):2764–2769.
17. Low QE, Drugea IA, Duffner LA, et al.Wound healing in MIP-1alpha (-/-) and MCP-1(-/-) mice. Am J Path. 2001;159(2):457–463.
18. Rennekampff HO, Hansbrough JF, Kiessig V, Dore C, Sticherling M, Schroder JM. Bioactive interleukin-8 is expressed in wounds and enhances wound healing. J Surg Res. 2000;93(1):41–54.
19. Lin ZQ,Kondo T, Ishida Y,Takayasu T, Mukaida N. Essential involvement of IL-6 in the skin wound-healing process as evidenced by delayed wound healing in IL-6-deficient mice. J Leukoc Biol. 2003;73(6):713–721.
20. Gallucci RM, Simeonova PP, Matheson JM, et al. Impaired cutaneous wound healing in interleukin-6 deficient and immunosuppressed mice. FASEB J. 2000;14(15):2525–2531.