| Method and solutions for cryopreserving oocytes, especially fresh human oocytes -> Monitor Keywords |
|
|
  Method and solutions for cryopreserving oocytes, especially fresh human oocytesUSPTO Application #: 20060105317Title: Method and solutions for cryopreserving oocytes, especially fresh human oocytes Abstract: In a method for cryopreserving fresh human oocytes, freezing and thawing solutions are used, which solutions include 1,2 propanediol (PROH) and sucrose at a concentration of at least 0.3M. The oocytes are exposed for 13-15 minutes to a solution including 1.5M PROH and 0.3M sucrose before starting the freezing process. (end of abstract) Agent: Klein, O'neill & Singh - Irvine, CA, US Inventor: Raffaella Fabbri USPTO Applicaton #: 20060105317 - Class: 435002000 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Maintaining Blood Or Sperm In A Physiologically Active State Or Compositions Thereof Or Therefor Or Methods Of In Vitro Blood Cell Separation Or Treatment The Patent Description & Claims data below is from USPTO Patent Application 20060105317. Brief Patent Description - Full Patent Description - Patent Application Claims
TECHNICAL FIELD [0001] This invention relates to an improved method for cryopreserving oocytes, especially fresh human oocytes. This invention also relates to solutions particularly suitable for cryopreservation of oocytes, especially fresh human oocytes. BACKGROUND ART [0002] It is well known that cryopreservation of unfertilised human ooccytes is a technique which offers several advantages, especially whenever oocytes are to be preserved of patients who are at risk of ovarian hyperstimulation and can not transfer embryos during the in vitro fertilization treatment. However, cryopreservation of oocytes, especially of fresh human oocytes, has run into greater technical difficulties than preservation of male gametes or embryos because of oocytes cytological peculiarity. [0003] The low number of births from human cryopreserved oocytes, reported in literature, shows the technical difficulties of cryopreserving oocytes. Up to now researches carried out on oocytes cryopreservation provide contrasting results regarding the more suitable and less damaging methods for maintaining cellular integrity and for getting a higher rate of viable oocytes. [0004] As yet, a definitive protocol for cryopreserving human oocytes has not been established and the number of oocytes utilised up to now is still too low to determine a definitive methodology to be applied. [0005] Human oocytes survival rate in cryopreservation depends, as well as on oocytes size, also on cryoprotectant used (composition, concentration and exposure time) and on freezing/thawing rate. [0006] In the cryopreservation process, oocytes size is a very important parameter affecting the survival rate because the large quantity of water in ooplasm causes intracellular ice formation during the freezing process: intracellular ice is one of the main responsible factors for intracellular structure damages. [0007] Oocytes cryopreservation protocols usually include the following steps: [0008] a) initially exposing the oocytes to a solution including a permeating cryoprotectant (e.g. 1,2-propanediol (PROH)), which aim is to reduce to a minimum intracellular structure damages caused by water crystallization; [0009] b) subsequently exposing for a time of 2-5 min. the oocytes to a so-called loading solution including a mixture of a permeating cryoprotectant and a non permeating cryoprotectant (e.g. sucrose) to increase oocytes dehydration; [0010] c) slowly cooling to -150.degree. C.; [0011] d) storing in liquid nitrogen (-196.degree. C.); [0012] e) thawing; [0013] f) diluting and removing the cryoprotectants by exposure to so-called thawing solutions and returning to the physiological environment for further manipulations. [0014] Cryoprotectants benefit are related to [0015] I) their concentration, [0016] II) exposure times; [0017] III) the temperature at which they are added to oocytes. [0018] Known methods for cryopreserving oocytes provide for using loading/thawing solutions including sucrose at a concentration of 0.1M or 0.2M. DISCLOSURE OF INVENTION [0019] In a method for cryopreserving oocytes, according to the present invention, it is provided for using loading/thawing solutions including sucrose at a concentration of 0.3M or greater. [0020] In fact, it has been surprisingly noticed that the presence of sucrose at a concentration of 0.3M or greater outside the cell membrane highly increases cell dehydration/rehydration with an improvement of cryopreserved oocytes survival rate. [0021] Furthermore, in a particular embodiment of the invention, the present method provides for exposing oocytes to a loading solution including at least 0.3M sucrose for a time of 15 min before starting the freezing process. [0022] In this latter case, a morphological analysis, effected by an inverted microscope, has shown an oocyte cytoplasmatic volume reduction of about 30% with further dehydration; this also reduces the possibility of intracellular ice formation. BEST MODE FOR CARRYING OUT THE INVENTION [0023] The essential phases of a process, according to the present invention, for cryopreserving fresh human oocytes are described hereinafter. [0024] The following solutions are used in the freezing process: [0025] a PBS solution (Dulbecco's Phosphate Buffered Saline Solution w/o sodium bicarbonate); [0026] an equilibration solution (1.5M PROH); [0027] a loading solution (1.5M PROH+0.3M sucrose); [0028] a SSS solution (Synthetic Serum Substitute) [0029] The composition of the above solutions is as follows: [0030] PBS solution [0031] 8 ml PBS [0032] Equilibration solution (1.5M PROH) [0033] 6.79 ml PBS [0034] 1.21 ml PROH (1,2-propanediol) [0035] Loading solution (1.5M PROH+0.3M sucrose) [0036] 6.79 ml PBS [0037] 1.21 ml PROH [0038] 1.128 gr sucrose [0039] These solutions are used in a slow freezing program. The solutions are prepared, mixed, filtered and conserved at +4.degree. C. It is better to maintain the solutions at room temperature for 15 min before using. [0040] When the above described solutions are ready, a Petri dish is prepared with 2.1 ml of PBS solution+0.9 ml SSS (Synthetic Serum Substitute). In this solution all the oocytes are washed from their culture medium before transferring in the equilibration solution. Then a suitable dish provided with a plurality of wells is prepared: some of the wells contain 0.350 ml of equilibration solution (1.5M PROH)+0.150 ml of SSS (Synthetic Serum Substitute). In this solution 1 or 2 oocytes/well are transferred and maintained for 10 min before transferring in loading solution. [0041] The others wells contain 0.350 ml of loading solution (1.5M PROH+0.3M sucrose)+0.150 ml of SSS (Synthetic Serum Substitute). The oocytes (taken from the equilibration solution) are transferred in the loading solution and rapidly loaded in plastic straws. Afterwards the straws are placed in a programmable freezer, e.g. a Kryo 10 series III (Planer Kryo 10/1.7 GB). [0042] According to a particularly advantageous embodiment of the present method, before starting the freezing program the oocytes are exposed to the loading solution (including sucrose at a concentration of at least 0.3M) for 13-15 min and preferably for about 15 min. Continue reading... Full patent description for Method and solutions for cryopreserving oocytes, especially fresh human oocytes Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Method and solutions for cryopreserving oocytes, especially fresh human oocytes patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. Start now! - Receive info on patent apps like Method and solutions for cryopreserving oocytes, especially fresh human oocytes or other areas of interest. ### Previous Patent Application: Method, apparatus and kit for demonstrating the use of absorbent products Next Patent Application: Compositions and methods for non-targeted activation of endogenous genes Industry Class: Chemistry: molecular biology and microbiology ### FreshPatents.com Support Thank you for viewing the Method and solutions for cryopreserving oocytes, especially fresh human oocytes patent info. IP-related news and info Results in 0.42995 seconds Other interesting Feshpatents.com categories: Tyco , Unilever , Warner-lambert , 3m |
||
