Claims for Patent: 6,872,569

Summary for Patent: 6,872,569

Title:	In vitro production of haploid germ cells
Abstract:	The present invention relates to a method of in vitro spermatogenesis involving Sertoli cells and diploid germ cells from a testis of a male mammal to yield differentiated haploid spermatids. The present invention also relates to spermatids produced by the method described above, where the spermatids are haploid. The present invention also involves a method of overcoming male infertility in mammals involving the use of the haploid round spermatids produced by the in vitro spermatogenesis method of the present invention. The present invention also relates to isolated haploid spermatids.
Inventor(s):	Lee; Dong Ryul (Seoul, KR), Kaproth; Michael T. (Ithaca, NY), Parks; John E. (Ithaca, NY)
Assignee:	Cornell Research Foundation, Inc. (Ithaca, NY)
Application Number:	10/147,576
Patent Claims:	1. A method of in vitro spermatogenesis comprising: isolating a plurality of Sertoli cells and a plurality of undifferentiated diploid germ cells from a testis of a male mammal; aggregating the Sertoli cells and the germ cells to yield Sertoli-germ cell aggregates; encapsulating the Sertoli-germ cell aggregates to yield a plurality of tubule-like structures containing both Sertoli cells and germ cells; transferring the tubule-like structures to a culture medium; and culturing the tubule-like structures under conditions effective to allow the Sertoli cells and the germ cells to interact, whereby the germ cells progress through spermatocytogenesis and meiosis to produce a plurality of differentiated haploid spermatids. 2. The method according to claim 1, wherein the mammal is selected from the group consisting of a human, a bovine, a porcine, and a rodent. 3. The method according to claim 1, wherein the male mammal is selected from the group consisting of a neonatal male, a juvenile male, a peri-pubertal male, and an adult male. 4. The method according to claim 1, wherein the male mammal is a neonatal bovine ranging in age from 1 to 5 days old. 5. The method according to claim 4, wherein the neonatal bovine is 3 days old. 6. The method according to claim 1, wherein the male mammal is a peri-pubertal bovine. 7. The method according to claim 1, wherein the male mammal is an adult bovine. 8. The method according to claim 1, wherein the undifferentiated diploid germ cell is a gonocyte. 9. The method according to claim 1, wherein said isolating comprises: decapsulating a portion of the testis to yield exposed parenchyma tissue containing a plurality of seminiferous tubules containing Sertoli cells and germ cells; dissociating the Sertoli and germ cells from the seminiferous tubules using a first enzyme solution to yield a mixture comprising peritubular cells, dissociated Sertoli cells, dissociated germ cells, and dissociated seminiferous tubules; separating the dissociated Sertoli cells, germ cells, and seminiferous tubules from the peritubular cells; and incubating the dissociated Sertoli cells, germ cells, and seminiferous tubules in a second enzyme solution which assists in releasing the dissociated Sertoli and germ cells from the seminiferous tubules. 10. The method according to claim 9, wherein said decapsulating comprises: manually decapsulating a portion of tunica albuginea of the testis. 11. The method according to claim 9, wherein said first enzyme solution comprises collagenase, DNase I, soybean trypsin inhibitor, and hyaluronidase in Ca.sup.++, Mg.sup.++ -free PBS. 12. The method according to claim 9, wherein said second enzyme solution comprises collagenase, DNase I, soybean trypsin inhibitor, and hyaluronidase in Ca.sup.++, Mg.sup.++ -free PBS. 13. The method according to claim 1 further comprising: storing the isolated Sertoli cells and germ cells prior to said aggregating. 14. The method according to claim 13 further comprising: storing the isolated Sertoli and germ cells in liquid nitrogen prior to said aggregating and thawing to yield a frozen-thawed suspension of isolated Sertoli cells and germ cells. 15. The method according to claim 1, wherein the said aggregating is carried out using a chemical aggregating agent. 16. The method according to claim 15, wherein the chemical aggregating agent is phytohemagglutinin. 17. The method according to claim 1, wherein said encapsulating is carried out by a chemical encapsulating agent. 18. The method according to claim 17, wherein the chemical encapsulating agent is sodium alginate. 19. The method according to claim 1, wherein the culture medium comprises HEPES-buffered DMEM/F12 medium supplemented with insulin-transferrin-selenium solution, vitamin C, vitamin E, retinoic acid, retinol, pyruvate, bovine FSH, testosterone, antibiotic-antimycotic solution, and bovine calf serum. 20. The method according to claim 19, wherein the culturing of the tubule-like structures is carried out in the culture medium for up to 14 weeks at a temperature in a humidified atmosphere of about 5% carbon dioxide in air. 21. The method according to claim 20, wherein culturing is carried out in the culture medium for a period of 2, 5, 10, or 14 weeks.

Details for Patent 6,872,569

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Bausch & Lomb Incorporated	VITRASE	hyaluronidase	Injection	021640	05/05/2004		2021-05-18
Bausch & Lomb Incorporated	VITRASE	hyaluronidase	Injection	021640	12/02/2004		2021-05-18
Amphastar Pharmaceuticals, Inc.	AMPHADASE	hyaluronidase	Injection	021665	10/26/2004		2021-05-18
Akorn, Inc.	HYDASE	hyaluronidase	Injection	021716	10/25/2005		2021-05-18
Smith & Nephew, Inc.	SANTYL	collagenase	Ointment	101995	06/04/1965		2021-05-18