Feeder-free method for culture of bovine and porcine spermatogonial stem cells
AssigneeWashington State University Research Foundation
InventorsJon Michael Oatley, Melissa Joan Oatley
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Abstract
The present invention relates to the production and culture of undifferentiated spermatogonial stem cells that can be maintained long term and are feeder free. The resultant feeder-free populations can be used in any of a number of protocols including the generation of progeny bulls. The present invention includes novel methods required for the successful enrichment of bovine spermatogonial stem cells, novel cell lines and other components used for the same, as well as the resultant stem cell compositions.
Claims32
A method of enriching spermatogonial stem cells (SSCs) from a population of testis-derived cells containing at least one SSC, said method comprising:
- a) providing a media that has been preconditioned with a feeder cell line;
- b) contacting said population of testis-derived cells with said preconditioned media under conditions suitable for SSC cell maintenance and enrichment.
A bovine embryonic fibroblast cell line for pre-conditioning media for culture of bovine spermatogonial stem cells BEF1 deposited as ATCC Accession number ______.
A bovine somatic cell line for pre-conditioning media for culture of bovine spermatogonial stem cells BSC1 deposited as ATCC Accession number ______.
A method of isolating spermatogonial stem cells from bovine testicular tissue comprising:
- a. Obtaining testicular tissue from a subject
- b. Incubating said tissue with collagenase to separate spermatogonia and Sertoli cells
- c. Separating tubules from other cell types
- d. Digesting tubules with trypsin to yield a suspension spermatogonial cells and Sertoli cells and
- e. Separating the spermatogonial cells and Sertoli cells from said suspension.
A method for creating a media for maintenance and enrichment of bovine spermatogonial stem cells comprising
- a. Incubating feeder cells selected from the group comprising cell line BEF1 deposited as ATCC Accession number ______ and cell line BSC1 deposited as ATCC Accession number ______ with said media so that said media becomes preconditioned with support for maintenance and enrichment of said spermatogonial stem cell and thereafter
- b. Removing said feeder cells from said media.
A kit for maintaining at least one bovine SSC in a feeder-free culture system, said kit comprising:
- a) a culture system comprising a serum free culture medium wherein said medium has been pre-conditioned with feeder cell lines BEF1 and BSC1;
- b) an applicator, and
- c) instructional material;
- wherein said instructional material comprises instructions for the use of said kit to maintain at least one bovine SSC in said feeder-free culture system.
Dependencies are inferred from the claim text.