Optimizing Collagenase, Fetal Bovine Serum, and Insulin to Isolate, Proliferate, and Differentiate Rat Preadipocyte in vitro

M Rasjad Indra, Satuman Satuman, Edwin Widodo

Abstract


BackgroundThe aim of this study is to develop a method for optimal in vitro proliferation and differentiation of rat adipocyte. MethodsPreadipocyte were isolated from omentum of Rattus norvegicus Wistar using Collagenase type I dan II (Sigma). Cells were cultured in M199 culture containing either 0%, 8% or 10% Fetal Bovine Serum (FBS). Insulin were added into the media once the cells attached to the culture plate Proliferating and differentiated cells were counted and analysed by using Oil Red O dan Hematoxylen (HE) staining. Results This study demonstrated that adipocyte could be isolated using Collagenase type I but not type II. The addition of 10% FBSsignificantly increased the number of preadipocyte and differentiation of adipocyte more than those of 8% FBS and without FBS. The timing of FBS addition was best performed on day 8 using 10% FBS. Specific adipocyte staining using Oil Red O revealed thatthere were core lipids in mature adipocyte. ConclusionsCollagenase tipe I could be used to isolate preadipocyte cells. Supplementation of culture media with 8-10% FBS could enhance the in vitro proliferation of preadipocyte. Standard media M199 containing 10% FBS and insulin may provide an environment to differentiate preadipocyte specifically into adipocyte in vitro.

Keywords


obesity; culture media; implant tissue; adipocyte precursor; proliferation

Full Text:

PDF

References


Ailhaud G1997. Molecular mechanisms of adipocyte differentiation. Journal of Endocrinology. 155, 201-202

Ailhaud G2001. Adipose Tissue Protocol. Humana Press Totowa, New Jersey. 225-228.

Albright and Stern 1998. Methods for growth of cultured cells in serum

free medium. Anal. Biochem 102:255-270.

Anselme K, Broux O, Noel B, Bouxin B, Bascoulergue G, Dudermel AF, Bianchi F, Jean, ls J and Hardouin P 2002In vitro control of human bone marrow stromal cells for bonetissue engineering. Tissue Eng 8:941953.

Bjorntorp P, Karlsson M, Petterson P and Sypniewska G 1980. Differentiation and function of rat adipocyte precursor cells in primary culture. J Lipid Res 21:714723.

DonovanSM and Odle J1994. Growtj factor in milk as mediators of infant development. Annu. Rev. Nutr. 14: 147167

Ethier SP 1986. Serum-free culture conditions for the growth of normal rat mammary epithelial cells in primary culture. In Vitro Cell Dev Biol 22:485490.

Fajas L 2003. Adipogenesis: a cross-talk between cell proliferation and cell differentiation. Ann Med 35:7985.

Freshney RI 1987.Culture of animal cells: a manual of basic technique. 2nd ed. Alan, R. Liss Inc., New York.

Gstraunthaler G2003. Alternatives to the use of fetal bovine serum: serum-free cell culture. Altex 20:275281.

Hauner H, Entenmann G, Wabitsch M, Gaillard D, Ailhaud G, Negrel R and Pfei.er EF 1989. Promoting effect of glucocorticoids on the differentiation of human adipocyte precursor cells cultured in a chemically dened medium. J Clin Invest 84:16631670.

Hemmrich K, Dennis VH, Kathrin C, Sevine H, Norbert P 2005. Optimization of the differentiation of human preadipo-cytesin vitro

Differentiation. 73:28-35.

Intosh A, Bertrand B, Ailhaud G and Grimaldi P 2005. Regulation of adipose cell differentiation. Fatty acids are inducers of the aP2 gene expression. J. Lipid Res. 32:1449-1456.

Kawada T, Aoki N, Kawai R and Sugimoto E1990. Proliferation of 3T3-L1 preadipocytes in a completely de.ned serum free medium. Cell Biol

Int Rep 14:567574.

Kawada Xu, X and P Bjontrorp 2000. Effect dexamethasone on multiplication and differentiation of rat adipose tissue precursor

cells. Exp. Cell Res. 189:247-252.

Krystyna M, Manchel I and Smith AK 2000. Alternative to animal sera for human bone marrow cell expansion: human serum and serum free media. J. Hemat-other 7:413-423.

KollerN, Varzaneh J and Lau DC1998. Extracellular matrix component secreted by microvascular endhotelial cell stimulate preadipocyte

differentiation in vitro. Metabolism 43:906-912.

Mc RobinT,Pham andMiller1976. The mechanism of inhibition of 3T3-L1 preadipocyte differentiation by prostaglan-din. Endocrinology 137: 5641-5650.

PhillippeD,Daniel AK, RoncariandCharles HH1983. Influence of Anatomic Site and Age on the Replication and Differentiation of Rat Adipocyte in Culture. J. Clinical Invest. 1200-1208.

Smahel J 1989 Experimental implantation of adipose tissue fragments. Br J Plast Surg 42:207211.

Tezel TH and Del Priore LV 1998Serum-free media for culturing and serialpassaging of adult human retinal pigment epithelium. Exp Eye Res 66:807815.

Vincent G, IsabelleL, Jacques M and Jean LD2000. Proliferation and differentiation of stromal vascular cells in primary culture differ between neonatal pigs consuming maternal or formula milk. J. Nutr. 130: 1179-1182.

von Heimburg D, Kuberka M, Rendchen R, Hemmrich K, Rau G and Pallua N 2003. Preadipocyte-loaded collagen scaffolds with enlarged pore size for improved soft tissue engineering. Int J Artif Organs 26:10641076.

von Heimburg D, Zachariah S, Heschel I, Kuhling H, Schoof H, Hafemann B and Pallua N 2001 Human preadipocytes seeded on freeze-dried collagen scaffolds investigat-ed in vitro and in vivo. Biomaterials 22:429438.Zulket al.,2002).




DOI: http://dx.doi.org/10.33476/jky.v16i3.244

Refbacks

  • There are currently no refbacks.


Copyright (c) 2016 YARSI Medical Journal

Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

__________________________________________________________________________________________

Copyright of YARSI Medical Journal.

Powered by OJS.

Creative Commons License

This work is licensed under a Creative Commons Attribution- NonCommercial 4.0 International License.