# US 2017/0283771 A1 · Methods of in vitro Oocyte Development > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2017/0283771 A1 - Document ID: US20170283771A1 - Status: Published application - Kind: A1 - Application number: 15/513,628 - Filed: 2015-09-24 - Published: 2017-10-05 - Earliest claimed: 2014-09-24 - Assignees: The University Court of the University of Edinburgh - Applicants: The University Court of the University of Edinburgh - Inventors: Evelyn Elizabeth TELFER; Marie MCLAUGHLIN - Web page: https://patentgrep.com/patent/US20170283771A1 ## CPC - C12N 5/0611: {Primordial germ cells, e.g. embryonic germ cells [EG]} - C12N 5/0037: {Serum-free medium, which may still contain naturally-sourced components} - C12N 5/0081: {Purging biological preparations of unwanted cells} - C12N 5/0609: {Oocytes, oogonia (fertilised oocytes C12N5/0604)} - C12N 5/0682: {Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells (oocytes C12N5/0609)} - A01N 1/147: Carriers for immersion in cryogenic fluid for slow freezing or vitrification - C12N 5/0018: {Culture media for cell or tissue culture (media for specific animal cell type C12N5/06)} - C12N 2500/25: Insulin-transferrin; Insulin-transferrin-selenium - C12N 2500/38: Vitamins - C12N 2500/90: Serum-free medium, which may still contain naturally-sourced components - C12N 2501/105: Insulin-like growth factors [IGF] - C12N 2501/11: Epidermal growth factor [EGF] - C12N 2501/125: Stem cell factor [SCF], c-kit ligand [KL] - C12N 2501/16: Activin; Inhibin; Mullerian inhibiting substance - C12N 2501/31: Pituitary sex hormones, e.g. follicle-stimulating hormone [FSH], luteinising hormone [LH]; Chorionic gonadotropins - C12N 2501/392: Sexual steroids - C12N 2502/243: Cells of the female genital tract, non-germinal ovarian cells - C12N 2527/00: Culture process characterised by the use of mechanical forces, e.g. strain, vibration - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - A01N: PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF (preservation of food or foodstuff A23); BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES (preparations for medical, dental or toiletry purposes which kill or prevent the growth or proliferation of unwanted organisms A61K); PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS ## Abstract 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. ## Claims ### Claim 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%. ### Claim 2 (depends on 1) The method according to claim 1, wherein the ovarian tissue sample is mechanically stretched along multiple dimensions of the ovarian tissue sample. ### Claim 3 (depends on 1) The method according to claim 1, wherein the greatest increase in the size of the ovarian tissue sample is achieved when the ovarian tissue sample is mechanically stretched along the dimension of the ovarian tissue sample that is parallel to that of the longest connecting fibres of the ovarian tissue sample. ### Claim 4 (depends on 1) 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. ### Claim 5 (depends on 4) The method according to claim 4, wherein the thickness of the layer of stromal tissue is reduced to about 3 mm after the step of removal of excess stromal tissue from the ovarian tissue sample. ### Claim 6 (depends on 1) 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. ### Claim 7 (depends on 1) 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. ### Claim 8 (depends on 7) The method according to claim 7, wherein the surface area of the cortical surface may be at least 1 to 2 times the surface area of the stromal surface after the step of mechanically stretching the ovarian tissue sample. ### Claim 9 (depends on 8) The method according to claim 8, 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. ### Claim 10 (depends on 1) The method according to claim 1, wherein the cortical tissue comprises a population of endogenous ovarian follicles, and growth of the endogenous ovarian follicles may be facilitated during the step of mechanically stretching the ovarian tissue sample. ### Claim 11 (depends on 10) The method according to claim 10, wherein endogenous ovarian follicles within the population of endogenous ovarian follicles with a diameter greater than 80 μm are mechanically removed from the ovarian tissue. ### Claim 12 (depends on 1) The method according to claim 1, wherein a population of ovarian stem cells is introduced into the ovarian tissue sample after the step of mechanically stretching the ovarian tissue sample. ### Claim 13 (depends on 12) The method according to claim 12, wherein a subset of the population of ovarian stem cells differentiate into exogenous ovarian follicles within the cortical tissue. ### Claim 14 (depends on 13) The method according to claim 13, wherein the exogenous ovarian follicles are labelled with a marker to allow the exogenous ovarian follicles to be differentiated from the endogenous ovarian follicles. ### Claim 15 (depends on 14) The method according to claim 14, wherein the marker is a fluorescent marker. ### Claim 16 (depends on 1) 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. ### Claim 17 (depends on 16) The method according to claim 16, wherein the serum-free media may comprise between 0.5 ng/ml to 2.5 ng/ml FSH. ### Claim 18 (depends on 16) The method according to claim 16, 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. ### Claim 19 (depends on 18) The method according to claim 18, wherein the isolated somatic support cells are DDX4 negative. ### Claim 20 (depends on 18) The method according to claim 18, 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. ### Claim 21 (canceled) ### Claim 22 (canceled) ### Claim 23 (canceled) ### Claim 24 (canceled) ### Claim 25 (canceled) ### Claim 26 (canceled) ### Claim 27 (canceled) ### Claim 28 (canceled) ### Claim 29 (canceled) ### Claim 30 (canceled) ### Claim 31 (canceled) ### Claim 32 (canceled) ### Claim 33 (canceled) ### Claim 34 (canceled) ### Claim 35 (canceled) ### Claim 36 (canceled) ### Claim 37 (canceled) ### Claim 38 (canceled) ### Claim 39 (canceled) ### Claim 40 (canceled) ### Claim 41 (canceled) ### Claim 42 (canceled) ### Claim 43 (canceled) ### Claim 44 (canceled) ### Claim 45 (canceled) ### Claim 46 (canceled) ### Claim 47 (canceled) ### Claim 48 (canceled) ### Claim 49 (canceled) ### Claim 50 (canceled) ### Claim 51 (canceled) ### Claim 52 (canceled)