Claims for Patent: 6,103,523

Summary for Patent: 6,103,523

Title:	Pluripotent rabbit cell lines and method of making
Abstract:	The present invention relates to a rabbit embryonic stem (ES) cell line, comprising at least 70%, preferably 80 to 90% undifferentiated cells and obtainable by isolating the inner cell mass of 5.5 days postcoitus blastocysts and culturing them on feeder cells in rabbit ES medium. The invention further relates to further optimization of derivation and maintainance of the cell line and to the use thereof in inter alia generation of chimeric rabbits.
Inventor(s):	Moreadith; Randall (Chapel Hill, NC), Schoonjans; Luc (Wilsele, BE)
Assignee:	Thromb-X N.V. (Leuven, BE)
Application Number:	08/810,945
Patent Claims:	1. Pluripotent rabbit cell line, comprising at least 70% undifferentiated, pluripotent cells obtainable by isolating an inner cell mass of a 5.5 day postcoitus blastocyst and culturing the inner cell mass cells on feeder cells in rabbit ES medium, wherein the rabbit ES medium comprises high glucose Dulbecco's Modified Eagle Medium, 4 mM L-glutamine, 0.1 mM 2-mercaptoethanol, 148 units/ml penicillin G sodium, 148 microgram/ml streptomycin sulfate, 4 microgram/ml bovine insulin, 10.sup.3 units/ml murine Leukemia Inhibitory Factor, 20% fetal bovine serum and 1.5% MEM non-essential amino acid solution, wherin the fedder cells are 12.5 days old Mouse Embryonic Feeder cells in a density of 3 to 4.times.10.sup.6 cells per 10 cm petri dish, and wherein the inner cell mass cells are cultured over various passages and the inner cell mass cells are trypsinized before each passage with a selective trypsinization medium consisting of 0.1% collagenase, 1% chicken serum and 0.03% trypsin-ETDA in phosphate buffered saline, such that a pluripotent rabbit cell line results. 2. A method for producing a pluripotent rabbit cell line, comprising about 70% undifferentiated pluripotent cells comprising the steps of: a) isolating an inner cell mass of a 5.5 day postcoitus blastocyst, and b) culturing innner cell mass cells on feeder cells in rabbit ES medium, wherein the rabbit ES medium comprises high glucose Dulbecco's Modifed Eagle Medium, 4 mM glutamine, 0.1 mM 2-mercaptoethanol, 148 units/ml penicillin G sodium, 148 microgram/ml streptomycin sulfate, 4 microgram/ml bovine insulin, 10.sup.3 units/ml murine Leukemia Inhibitory Factor, 20% fetal bovine serum and 1.5% MEM non-essential amino acid solution, wherein the feeder cells are 12.5 days old Mouse Embryonic Feeder cells in a density of 3 to 4.times.10.sup.6 cells per 10 cm petri dish, and wherein the inner cell mass cells are cultured over various passages and the inner cell mass are trypsinized before each passage with a selective trypsinization medium consisting of 0.1% collagenase, 1% chicken serum and 0.03% trypsin-EDTA in phosphate buffered saline, such that a pluripotent rabbit cell line results. 3. Selective trypsinization medium, consisting of 0.1% collagenase, 1% chicken serum and 0.03% trypsin-EDTA in phosphate buffered saline.

Details for Patent 6,103,523

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Smith & Nephew, Inc.	SANTYL	collagenase	Ointment	101995	06/04/1965		2016-04-29