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Isolation and Culture of Parenchymal and Nonparenchymal Cells from Neonatal Swine Liver^1,2,

Virginia Polytechnic Institute and State University, Blacksburg 24061 and U.S. Department of Agriculture, Beltsville, MD 20705

Abstract

Procedures for the isolation and monolayer culture of hepatocytes and nonparenchymal (Kupffer and endothelial) cells from livers of neonatal pigs (1 to 15 d of age) are described. Cell suspensions were obtained by a modification of the two-step collagenase perfusion technique. Hepatocytes were collected by low-speed centrifugation and nonparenchymal cell populations were purified by centrifugal elutriation. Hepatocytes were readily maintained in arginine-free medium fortified with either fetal calf serum or bovine serum albumin and oleate for periods as long as 6 d. The ability of cultured hepatocytes to incorporate ³ H-leucine and ³ H-thymidine into protein and DNA, respectively, demonstrated that cells were metabolically active for at least 3 d in culture. The ³ H-leucine incorporation into total cell protein was constant regardless of animal age at the time of cell isolation, while incorporation of ³ H-thymidine was influenced by animal age. Incorporation of both precursors was dependent upon duration of culture period in vitro and the type of medium (serum-free vs serum-containing) in which the cells were maintained. Morphological observation and analysis of the DNA and protein levels of hepatocyte monolayers suggest that cells did not replicate during the 3-d incubation period. The ability to isolate and culture metabolically active, nonreplicating hepatocytes from neonatal pigs in a serum-free medium affords opportunities for investigation of the influence of specific hormones and specific growth factors on the uptake and metabolism of nutrients by the liver. Similarly, the neonatal pig will serve as a useful model for the characterization of hepatic nonparenchymal cell metabolism during the neonatal period.

¹ This project was supported in part by grants from the National Institutes of Health (AM 36394) and the Virginia Pork Industry Commission to M. L. Failla and a Sigma Xi Research Grant-in-Aid to T. J. Caperna.

² We thank Dr. T. O. Sitz for isolation and chromatographic analysis of hepatocyte RNA and Ms. Sheila Early for assistance with preparation of the manuscript.

³ Dept. of Biochem. and Nutr., Virginia Polytechnic Institute and State Univ., Blacksburg, VA 24061.

⁴ To whom reprint requests should be sent.

⁵ Dept. of Anim. Sci., Virginia Polytechnic Institute and State Univ., Blacksburg, VA 24061.

⁶ USDA, Agricultural Research Service, Beltsville Agricultural Research Center, Animals Science Institute, Nonruminant Anim. Nutr. Lab., Beltsville, MD 20705.

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