Patent Application: US-200913061222-A

Abstract:
the present invention provides a practical , non - invasive , and efficient method for cryopreservation of an in - vitro - produced porcine embryo . the inventive method treats the np embryo at the one - cell or cleavage stage prior to compaction with high osmolality followed by high speed centrifugation . the high osmolality treatment enlarges the periviteline space , and with centrifugation , enables the separation of the lipids from the cytoplasm . the lipid - separated embryos after high osmolality treatment have been successfully cryopreserved and later recovered and transferred to produce live offspring .

Description:
unless otherwise defined , all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs . all publications , patent applications , patents , and other references mentioned herein are incorporated by reference in their entirety . the present invention , building on the prior studies , teaches that the lipid removal , or separation , at an early embryo developmental stage is critical for cryopreservation of an ivp ( ivf - or nt - derived ) porcine embryo , and that besides swelling the zona pellucid through partial enzymatic digestion , the perivitelline space of an ivp porcine embryo may be enlarged by condensing the volume of the embryo to enable easy lipid removal / separation . the invention also discloses that exposing an ivp embryo to a high osmolality treatment may condense the embryo but preserve the vitality of the embryo . furthermore , the inventive lipid - separation methods may be employed to treat multiple embryos at once , in contrast to the current delipation procedures that require micromanipulation of each individual oocyte or embryo . referring to fig1 , which is a flow diagram of the inventive cryopreservation and recovery process via the inventive embryo - condensing method . step 1 in fig1 is to provide ivp embryos at a pre - selected early developmental stage , specifically the one - cell or cleavage stage prior to compaction . any standard ivp procedure , such as ivf or nt , may be adapted . according to one embodiment of the inventive method , the in - vitro - fertilization process may start with the oocytes aspirated from the antral follicles of one or multiple pig ovaries . the oocytes may be cultured to maturity in a maturation medium for a period of time and denuded . an exemplary maturation medium may contain tcm 199 ( gibco , 31100035 , grand island , n . y .) with 0 . 1 % pva , 3 . 05 mmol / l glucose , 0 . 91 mmol / l sodium pyruvate , 0 . 57 mmol / l cysteine , 0 . 5 μg / ml lh , 0 . 5 μg / ml fsh , 10 ng / ml epidermal growth factor , 75 μg / ml penicillin and 50 μg / ml streptomycin . the oocytes may be cultured in the maturation medium for about 40 - 44 h at 38 . 5 ° c ., 5 % co 2 in humidified air . after the maturation , the oocytes may be denuded by removing the cumulus cells via vortexing for a short period of time , such as about 4 minutes , in tl - hepes [ 20 ] supplemented with 0 . 1 % pva and 0 . 1 % hyaluronidase . the denuded oocytes may be stored in many different media before insemination . an excellent medium may contain tcm199 with 0 . 6 mmol / l nahco 3 , 2 . 9 mmol / l hepes , 50 μg / ml penicillin , 60 μg / ml streptomycin , 30 mmol / l nacl and 3 mg / ml bsa [ 26 ]. any standard insemination procedure may be followed to produce the ivp embryos . according to one embodiment , the denuded oocytes with a polar body may be first transferred to a suitable ivf medium to be combined with a sperm suspension . an exemplary ivf medium may contain a modified tris - buffered medium with 113 . 1 mmol / l nacl , 3 mmol / l kcl , 7 . 5 mmol / l cacl 2 , 5 mmol / l sodium pyruvate , 11 mmol / l glucose , 20 mmol / l tris , 2 mmol / l caffeine , and 2 mg / ml bsa . according to another embodiment of the invention , nt - derived embryos may start with nuclear transfer donor cells and commercial oocytes . the nuclear transfer donor cells may be collected from a transgenic piglet or produced through genetic modification of wild type cells . after the oocytes are allowed to mature , the cumulus cells are removed from the oocytes by vortexing for about 4 min in tl - hepes supplemented with 0 . 1 % pva and 0 . 1 % hyaluronidase . the first polar body and the adjacent cytoplasm from these oocytes are then aspirated while in manipulation medium with 7 . 0 μg / ml cytochalasin b . a donor cell is then transferred into the pervitelline space . fusion and activation can be accomplished simultaneously with two 30 is pulses of 1 . 2 kv / cm in fusion / sctivation medium ( such as 0 . 3 m mannitol , 1 . 0 mm cacl 2 , 0 . 1 mm mgcl 2 , and 0 . 5 mm hepes ). alternatively fusion and activation may be accomplished stepwise , first exposing in a fusion only medium with a lower concentration of calcium ( such as 0 . 3 m mannitol , 0 . 1 mm cacl 2 , 0 . 1 mm mgcl 2 and 0 . 5 mm hepes ), then exposing to 200 μm thimerosal for about 10 min in the dark and then 8 mm dtt for 30 min to activation [ 21 ] or any other suitable method . after insemination or nt , the ivp embryos are cultured to the one - cell or cleavage stage ( zygote , 2 - cell or 4 - cell stage , prior to compaction ). any suitable culture procedure may be adapted . according to one embodiment , the ivp embryos ( derived from in - vitro - fertilization or nt ) may be cultured in a variety of different culture media at 38 . 5 ° c ., 5 % co 2 in air for 28 to 30 hours to select 2 - cell stage embryos . an exemplary culture medium , pzm3 [ 27 ], may contain nacl 108 . 0 mmol / l , kcl 10 . 0 mmol / l , kh 2 po 4 0 . 35 mmol / l , mgso 4 . 7h 2 o 0 . 4 mmol / l , nahco 3 25 . 07 mmol / l , na - pyruvate 0 . 2 mmol / l , ca ( lactate ) 2 . 5h2o 2 . 0 mmol / l , glutamine 1 . 0 mmol / l , hypotaurine 5 . 0 mmol / l , bme amino acid solution 20 ml / l , mem amino acid solution 10 ml / l , gentamicin 0 . 05 mg / ml , bsa 3 mg / ml , with osmolarity at 288 ± 2 , and ph at 7 . 3 ± 2 . step 2 in fig1 is to increase the osmolality of the embryos to condense the embryo . several different media formulations may be employed to increase the osmolality in order to condense the volume of an oocyte or embryo . the invention provides examples using nacl or sucrose at different concentrations ( resulting different osmolalities ), but other formulations that result in a higher osmolality and subsequent shrinkage of the volume of the cell ( s ) should work . according to one embodiment of the inventive method , the osmolality may be increased by adjusting the osmolality of a medium where the embryos are submerged . for example , nacl or sucrose may be added to a stock medium with about 300 mosmo ( such as a stock solution with 300 - 310 mosmo with 7 . 0 μg / ml cytochalasin b and 0 . 1 mg / ml bsa ) to result a medium with various osmolalities , such as 350 , 400 , 500 , 600 and 800 - 850 mosmo . the ivp - derived embryos may be exposed to a pre - selected high osmolality medium for a short period of time , about 5 to 10 min , before centrifugation . step 3 in fig1 is to separate the lipid from the condensed embryos , normally through centrifugation . for example , the condensed embryos in the high osmolality medium can be centrifuged at 13 , 400 × g for about 6 to 20 min . the centrifugation condition ( force or duration ) may likely be varied to a large range of centrifugation force and time as long as it is sufficient to achieve full lipid separation while preserve the vitality of the embryos . a shorter duration may work especially if the force is increased . likewise a longer duration may be necessary if a lower force is used . pro - longed exposure to high osmolality may affect the vitality of an embryo . the lipid separation may be checked after about 12 hours of culturing ; in some cases ( especially for nt - derived embryos , since they are relatively more valuable ) a second round of high osmolality treatment and subsequent centrifugation may be applied to achieve a relatively complete lipid separation . the second high osmolality treatment may be an optional as long as the embryos remain at the cleavage stage prior to compaction . the lipid - separated embryos are then allowed to develop further to the blastocyst stage step 4 , in fig1 . specifically , the lipid - separated embryos may be cultured in pzm3 for 3 to 6 days for the embryo to attain the blastocyst stage . step 5 in fig1 is the vitrification of the further - developed embryos . the embryos may be vitrified via any standard method / procedure . according to one embodiment , the further - developed embryos may be vitrified at the blastocyst stage by using a modified open pulled straw (&# 39 ; ops &# 39 ;) method . specifically , the further - developed embryos may be placed in an equilibration solution for a short period of time ( such as about 2 min ) followed by exposure to a vitrification solution , then loaded into an ops straw and immediately plunged into liquid nitrogen . an exemplary equilibration solution may contain 10 % ethylene glycol , 10 % dimethyl sulfoxide (&# 39 ; dmso &# 39 ;), while an exemplary vitrification solution may contain 20 % ethylene glycol , and 20 % dmso . the process before plunging into nitrogen may be conducted on a 38 . 5 ° c . warm stage . the duration from exposure to the vitrification solution to plunging into nitrogen is generally short ranging between about 25 to 30 seconds . steps 6 to 8 in fig1 are steps to recover the preserved embryos and transfer such into a recipient . specifically , the vitrified embryos may be thawed by immersing into a buffer solution ( such as sucrose ) for a period of time at a slightly elevated temperature ( such as about 38 . 5 ° c .). the thawed embryos may be treated with 0 . 5 % pronase to soften and remove the zona pellucida . the lipid - separated and zona - removed embryos may then be transferred to the oviduct or uterus of a recipient or surrogate . fig2 ( a )-( f ) are the photos of ivf - derived embryos after high osmolality treatment ( at 400 mosm with nacl ). fig2 ( a ) shows the embryos cultured for several hours after high osmolality treatment and centrification . fig2 ( b ) and 2 ( c ) show the embryos at blastocyst stage . fig2 ( d ) shows the embryos after vitrification and warming . fig2 ( e ) shows the embryos after removal of their zona pellucida . fig2 ( f ) shows the re - expanded embryos after in vitro culture in brl cell conditioned medium . fig3 ( a )-( f ) are the photos of the development of the nt - derived embryos after high osmolality treatment with nacl or sucrose . fig3 ( a ) and 3 ( b ) show the nt - derived embryos cultured for several hours after high osmolality treatment and centrifugation ; fig3 ( c ) and 3 ( d ) show the embryos cultured further to the blastocyst stage ; fig3 ( e ) shows the embryo warmed after vitrification ; and fig3 ( f ) shows the re - expanded embryos after in vitro culture in brl cell conditional medium . the invention further studied the effects of different reagents ( adjusting osmolality ), different osmolality and centrifugation time on the rate of lipid separation . the invention finds that different reagents , such as nacl or sucrose , have similar effects on lipid separation ; the ideal osmolality for lipid separation ranges from about 350 to about 500 mosm ; and centrifugation duration ranging from about 6 minutes to about 20 minutes at a suitable centrifugation force / speed also has a positive effort on the lipid separation rate . fig4 shows the photos of the in - vitro - fertilized embryos treated with different osmolalities ( adjusted with nacl or sucrose ) before and after centrifugation . row 1 shows the photos of embryos exposed at different osmolalities , 300 mosm ( the control ), 400 mosm ( adjusted with nacl ), 600 mosm ( adjusted with sucrose ), and 800 mosm ( adjusted with sucrose ), with condensation clearly shown at the elevated osmolalities ( compared to the control ). row 2 lists the corresponding photos of embryos after centrifugation . the bridge - like structure after centrifugation ( indicating incomplete lipid separation ) can be seen in the control ; complete lipid separation can be observed in the embryo exposed at 400 mosm , while large bridge - like structures are present in the embryos treated with 600 or 800 mosmo . fig2 indicates that osmolality at about 400 mosm provides the most complete lipid separation , when osmolatlity increased to 600 and above , the lipid separation is hindered . the invention further quantitatively evaluated the impacts of the different osmolalities and centrifugation conditions on the lipid separation rate , the embryos &# 39 ; development , and the hatching ability . based on the data included in tables 1 ( ivf - derived embryos , osmolality adjusted with nacl ), 2 ( ivf - derived embryos , osmalility adjusted with sucrose ), and 3 ( nt - derived embryos , osmalility adjusted with both nacl and sucrose , all three tables attached ), the preferred condition for lipid separation is to expose the ivp embryos to osmolality ranging from above 300 to about 500 mosm , preferably from about 350 to about 450 mosm , followed by centrifugation for about 6 to about 20 minutes at 13 , 400 × g speed . the centrifugation time may be shorten or extended depending upon the centrifugation speed . however , extending centrifugation time may subject the embryos to prolonged exposure to high osmolality , which may have adverse impact on the vitality of the embryos . furthermore , in table 3 , the second high osmolality treatment is elected for the nt - derived embryos that failed to condense upon the first round of treatment , which increases the total lipid separation rate . the second high osmolality treatment may be elected as long as the embryos are still at the cleavage stage prior to compaction . table 4 evaluates the pregnancy and offspring data on the ivf - derived embryos preserved by high osmolality treatment , centrifugation and vitrification . among the data included in table 4 , three surrogates out of nine established pregnancies and produced normal offspring . one surrogate received the embryos treated with osmolality at 350 mosm and produced five piglets , three males and two females ; one surrogate received embryos treated with 400 mosm and produced four piglets , two males and two females ; and one surrogate received the embryos treated with 450 mosm and produced three piglets , one male and two females . table 5 lists the transfer , pregnancy , and offspring data of the nt - derived embryos after high osmolality treatment , centrifugation , and vitrification . three embryo transfers were performed and recorded . for each transfer , 80 to 90 embryos with the zona pellucida softened or removed by pronase treatment were transferred into the surrogates . two of the three surrogates receiving the embryos treated with 400 mosmo with 6 min centrifugation and the one receiving embryos treated with 350 mosmo with 20 min centrifugation resulted in pregnancy , with the former a single male piglet was produced . the data in tables 4 and 5 demonstrates that the inventive method , especially when applying the preferred range of osmolality ( from about 350 to about 450 mosm ), is a successfully cryopreservation method for the ivp embryos . while the invention has been described in connection with specific embodiments thereof , it will be understood that the inventive methodology is capable of further modifications . this patent application is intended to cover any variations , uses , or adaptations of the invention following , in general , the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features herein before set forth and as follows in scope of the appended claims . 1 . polge c , wilmut i , rowson lea . the low temperature preservation of cow , sheep , and pig embryos . cryobiology 1977 ; 11 : 560 . 2 . wilmut i . the low temperature preservation of mammalina embryos . journal of reproduction and fertility 1972 ; 31 : 513 - 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