Hepatocyte cell line derived from the epiblast of pig blastocysts

• US 5,532,156 A
• Filed: 10/08/1993
• Issued: 07/02/1996
• Est. Priority Date: 10/08/1993
• Status: Expired due to Term

• Current Assignee: The United States of America As Represented By The Secretary of Agriculture
• Original Assignee: United States Department Of Agriculture
• Inventors: Talbot, Neil, Pursel, Vernon G., Powell, Anne M., Rexroad, Caird E. Jr.
• Primary Examiner(s): Knode, Marian C.
• Assistant Examiner(s): DADIO, SUSAN
• Application Number:
  US08/133,589
• Time in Patent Office: 998 Days
• Field of Search: 435/240.2, 435/240.21, 435/240.23, 435/240.243
• US Class Current: 435/325
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• CPC Class Codes: C12N 5/0672 Stem cells; Progenitor cells; Precursor cells; Oval cells

Abstract

Continuous cultures of pluripotent parenchymal hepatocytes were derived from the epiblasts of pig blastocysts. The cultures are feeder-dependent and grow slowly with doubling times of 3 to 4 days. They differentiate into large secretory duct-like structures or form small canaliculi. Alternatively, the cells accumulate droplets that stain intensely with oil red O, a lipid-specific stain. α-Fetoprotein and albumin mRNA expression increases as the cells differentiate in culture.

Claims

1. An isolated cell line of normal pluripotent parenchymal hepatocytes derived from a primary culture of pig epiblast cells.

2. The cell line of claim 1, where said cell line has all the identifying characteristics of PICM-19, ATCC No. CRL 11519.

3. A method of preparing a cell line from the epiblast of the pig blastocyst, wherein said cell line comprises normal pluripotent parenchymal hepatocytes, said method comprising,

a) removing a blastocyst from the uterus of a pig,

b) immunodissecting the inner cell mass from said blastocyst,

c) dissecting the epiblast from the inner cell mass and culturing the epiblast for a time sufficient for colony formation, wherein a primary culture is obtained,

d) subculturing the epiblast colonies, from step c) to obtain a secondary culture, and

e) isolating said hepatocytes from the secondary culture of step d).

4. The method of claim 3, wherein said primary and said secondary cultures are grown on feeder layers.

5. The method of claim 4, wherein said feeder layers comprise STO feeder cells.