Methods of in vitro oocyte development
Methods of preparing ovarian tissue for primordial follicle growth are presented comprising the steps: providing an ovarian tissue sample comprising cortical tissue and stromal tissue; removing damaged tissue from the ovarian tissue sample where present; removing excess stromal tissue from the ovarian tissue sample where present; and then mechanically stretching the ovarian tissue sample along at least one dimension of the ovarian tissue sample, such that the size of the ovarian tissue sample along the at least one dimension is increased by at least 10%. Methods of growing viable oocyte in vitro, and methods of preparing individual ovarian follicles for growth are also presented.
1. A method of preparing ovarian tissue for primordial follicle growth comprising the steps:
providing an ovarian tissue sample comprising cortical tissue and stromal tissue;
removing damaged tissue from the ovarian tissue, sample where present;
removing excess stromal tissue from the ovarian tissue sample where present; and then
mechanically stretching the ovarian tissue sample along at least one dimension of the ovarian tissue sample, such that the size of the ovarian tissue sample along the at least one dimension is increased by at least 10%.
2. The method according to claim 1 , wherein the ovarian tissue sample is mechanically stretched along multiple dimensions of the ovarian tissue sample.
3. The method according to claim 1 , wherein the thickness of the layer of stromal tissue is reduced to between 1 and 5 mm after the step of removal of excess stromal tissue from the ovarian tissue sample.
4. The method according to claim 3 , wherein the thickness of the layer of stromal tissue is reduced to 3 mm after the step of removal of excess stromal tissue, from the ovarian tissue, sample.
5. The method according to claim 1 , wherein the ovarian tissue sample is cut into a plurality of smaller ovarian tissue fragments after the step of mechanically stretching the ovarian tissue sample.
6. The method according to claim 1 , wherein the cortical tissue within the ovarian tissue sample forms a cortical surface on a first side of the ovarian tissue sample and the stromal tissue forms a stromal surface on a second side of the ovarian tissue, opposed to the first side.
7. The method according to claim 6 , wherein the surface area of the cortical surface is at least 1 to 2 times the surface area of the stromal surface after the step of mechanically stretching the ovarian tissue sample.
8. The method according to claim 7 , wherein the surface area of the cortical surface is about 1.5 times the surface area of the stromal surface after the step of mechanically stretching the ovarian tissue sample.
9. The method according to claim 1 , wherein the cortical tissue comprises a population of endogenous ovarian follicles, and wherein further endogenous ovarian follicles within the population of endogenous ovarian follicles with a diameter greater than 80 μm are mechanically removed from the ovarian tissue.
10. The method according to claim 1 , wherein the ovarian tissue sample is cultured in a serum-free media comprising follicle stimulating hormone (FSH) for at least twenty four hours.
11. The method according to claim 10 , wherein the serum-free media may comprise between 0.5 ng/ml to 2.5 ng/ml FSH.
12. The method according to claim 10 , wherein a population of isolated ovarian somatic support cells are introduced into the cortical tissue of the ovarian tissue sample after culturing for at least twenty four hours.
13. The method according to claim 12 , wherein the isolated somatic support cells are DDX4 negative.
14. The method according to claim 12 , wherein the ovarian tissue sample are then cultured for a further period of at least four days after the step of introducing the population of isolated ovarian somatic support cells.