WO2011162826A2 - Group culture system and method with helper embryos - Google Patents
Group culture system and method with helper embryos Download PDFInfo
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- WO2011162826A2 WO2011162826A2 PCT/US2011/001135 US2011001135W WO2011162826A2 WO 2011162826 A2 WO2011162826 A2 WO 2011162826A2 US 2011001135 W US2011001135 W US 2011001135W WO 2011162826 A2 WO2011162826 A2 WO 2011162826A2
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M21/00—Bioreactors or fermenters specially adapted for specific uses
- C12M21/06—Bioreactors or fermenters specially adapted for specific uses for in vitro fertilization
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12M23/00—Constructional details, e.g. recesses, hinges
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0012—Cell encapsulation
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0604—Whole embryos; Culture medium therefor
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- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/02—Coculture with; Conditioned medium produced by embryonic cells
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- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/70—Polysaccharides
- C12N2533/76—Agarose, agar-agar
Definitions
- the present embodiments generally relate to the development of embryos and, more particularly, to the development of embryos in group cultures.
- oocytes or eggs for fertilization employ a number of techniques for retrieving oocytes or eggs for fertilization, such as in vitro fertilization, and each technique possess different drawbacks. Particular problems with different collection techniques are evident in group culturing methods.
- large numbers of oocytes can be collected from slaughterhouse ovaries, which involves the collection and grading of cumulus- oocyte-complexes (COCs) from slaughterhouses.
- COCs cumulus- oocyte-complexes
- the anonymous collected ovaries can then be sliced and flushed for the collection of immature oocytes.
- bovine are often bred specifically for milk production or as beef cattle and reproductive cells are preferred from animals with desirable genetic pedigrees.
- the pedigree and genetic characteristics associated with oocytes collected from a slaughterhouse are completely unknown, presenting an unattractive option for breeding because there is no assurance an embryo will possess good genetics or desirable genetic traits.
- Ovum pick up is an ultrasound guided technique for collecting COCs from the ovaries living animals, such as bovine, and is described by Pieterse et al. in Transvaginal ultrasound guided follicular aspiration of bovine oocytes. Theriogenology 1991 ; 35:19-24, incorporated herein by reference.
- the live donors provide known genetics and pedigree, which can easily be tracked with their reproductive cells. In this way, oocytes can be procured from elite donors with superior genetics.
- OPU sessions produce a limited number of COCs.
- Bos taurus or Bos indicus, or derivative hybrids Some bovine produce an average of eight or more (Bos taurus or Bos indicus, or derivative hybrids) oocytes per OPU session and other species and/or breeds have been shown to produce fewer COCs per session.
- mice Canseco et al., Embryo density and medium volume effects on early murine embryo development. J Assis Reprod Genet 1992; 9:454-57), sheep (Gardner et al. Enhanced rates of cleavage and development for sheep zygotes cultured to the blastocyst stage in the absence of serum and somatic cells: amino acids, vitamins, and culturing embryos in groups stimulate development. Biol Reprod 1994; 50:390-400), and cattle (Khurana et al. Effects of oocyte quality, oxygen tension, embryo density, cumulus cells and energy substrates on cleavage and morula/blastocyst formation of bovine embryos.
- Certain embodiments described herein meet the needs set forth above and relate to a method and system of group culturing desired embryos in the proximity of helper embryos in order to improve the development of the desired embryos.
- Certain embodiments relate to methods and systems for physically separating helper embryos from desired embryos in order to maintain the pedigree information of the desired embryos and to provide the developmental benefits of group culturing for the desired embryos.
- the method can include the steps of obtaining at least one embryo; obtaining at least one helper embryo; and culturing the at least one embryo with the at least one helper embryo. Separation can be maintained between the at least one helper embryo and the at least one embryo during the step of culturing by embedding the at least one helper embryo in a gel or solid suspension.
- the suspension can then be cultured in close proximity to the at least one embryo.
- the suspension can comprise an agarose chip permeable to supporting and promoting chemical factors.
- the helper embryos can be embedded in the agarose chip by: providing a solution containing agarose; melting the solution; adding at least one helper embryo to the melted solution; and aspirating the at least one helper embryo and melted agarose solution to form a chip.
- Another embodiment relates to a method of culturing embryos by collecting a first group of oocytes; fertilizing the first group of oocytes to form a group of helper embryos; collecting a second group of oocytes; fertilizing the second group of oocytes with sex sorted sperm to form a group of sorted embryos; and culturing the helper embryos with the sorted embryos wherein the groups of embryos are physically separated.
- a method for embedding embryos in an agarose chip can include the steps of producing a solution with agarose and NaCl; autoclaving the solution; heating the solution to melt the agarose; cooling the solution to about a normal temperature for embryos; adding embryos into the solution; aspirating the embryos and agarose solution into an instrument with a channel; and releasing the embryos and agarose solution on a cooler surface for solidifying the agarose.
- Additional embodiments relate to containers or systems such as Petri dishes with wells or other incubation spaces for maintained embryos in close proximity to helper embryos.
- the wells or other incubation spaces can contain helper embryos, which can be separated from additional embryos by membranes, or other porous materials such as mesh.
- the system can include at least one well and a plurality of helper embryos within the well or wells.
- the helper embryos can be embedded within each respective well or physically separated in a portion of the well by a membrane, a mesh or another permeable divider.
- FIG. 1A-1B illustrates a container in accordance with certain embodiments of the present invention.
- FIG. 2A-2F illustrate various embodiments of wells in accordance with certain embodiments of the present invention.
- FIG. 3 illustrates a flow diagram of a method in accordance with certain embodiments of the present invention.
- FIG. 4A-4D illustrates group culturing embryos in accordance with certain embodiments of the present invention.
- the embryos can be from any mammalian species, including, but not limited to, bovine, equine, porcine, cervine, human, sheep, goat and other species.
- the method can include obtaining at least one embryo.
- the at least one embryo can interchangeably be referred to as a desired embryo, a free floating embryo, or a targeted embryo in various embodiments and should be understood to be the target embryo or group of target embryos for development.
- the at least one embryo can be produced from oocytes fertilized in vitro, by an ICSI (intracytoplasmic sperm injection) technique, or by other know fertilization methods.
- the at least one embryo can also be obtained thought nuclear transfer procedure, such as a cloned embryo.
- the embryo can also be obtained thought chemical activation, physical activation or a combination of both of an oocyte, resulting in a parthenogenetic embryo.
- the embryo can be produced by fertilizing an oocyte with sex sorted sperm. Specific examples are given regarding embryos, but zygotes or fertilized eggs may be used prior to cleavage in some embodiments.;
- the oocyte or oocytes used to produce the desired or targeted embryo can originate from a pedigreed mammal with desirable genetic characteristics or from an anonymous donor.
- the oocytes can be obtained through an OPU procedure, through flushing, through collection from slaughterhouse ovaries, or by other means known in the art for collecting oocytes, or COCs, or derivative of stem cells, or derivative of induced pluripotent stem cells (iPS).
- OPU can be performed as described by Pieterse et al. in Transvaginal ultrasound guided follicular aspiration of bovine oocytes. (Theriogenology 1991 ; 35:19-24), the entire text of which is incorporated herein by reference.
- helper embryos can be produced from oocytes acquired in any of the same ways described above.
- Helper embryos can be embryos from the same species or from different species compared to the at least one embryo, or the targeted embryo.
- rabbit helper embryos can be used to promote the development of targeted bovine embryos.
- the helper embryos can be obtained by fertilizing oocytes from anonymous donors or from a pedigreed animal such as a bovine with desirable genetic characteristics.
- the helper embryos can be obtained by fertilizing oocytes with sorted sperm, such as sex sorted sperm ) or with conventional un-sorted sperm.
- the helper embryo or embryos can also be obtained through nuclear transfer procedure.
- the helper embryos can also be obtained thought chemical activation of an oocyte, physical activation or an oocyte or a combination of both resulting in parthenogenetic embryos.
- the oocytes used to produce helper embryos can be obtained through an OPU procedure, through flushing, through collecting from slaughterhouse ovaries, or by other means known in the art for collecting oocytes or COC, or derivative of stem cells, or derivative of induced pluripotent stem cells (iPS).
- the helper embryos can be lacking in pedigree or particular genetic characteristics of interest and can be obtained from gametes of anonymous donors. However, helper embryos are not limited to embryos produced from one or more anonymous donors.
- the helper embryos can then be cultured with the at least one embryo, or the targeted embryos. All the embryos can be cultured in a space, such as a well, and can be collected in a fluid volume of between about ⁇ , and 1 ⁇ ,.
- the helper embryos can physically be separated or segregated from the at least one embryo by a membrane, other porous structure, or can be embedded in a permeable suspension. Other methods of separating the targeted or desired embryos from the helper embryos are contemplated beyond these specific examples. Such a separation only needs to be a physical separation so the desired embryos can be identified and collected to maintain the integrity of their pedigree and genetic characteristics.
- the separation can allow biochemical and physiological exchanges to and from the helper embryos so that promoting factors, embryotrophic factors, and other chemicals benefiting embryo development are exchanged between the two groups.
- the helper embryos can be embedded in a gel or solid suspension wherein the suspension is cultured with the at least one embryo.
- the gel or solid would generally have permeability to embryotrophic factors produced by the both groups of embryos.
- the suspension can comprise an agarose chip.
- the agarose chip can be formed by melting a solution containing agarose, adding helper embryos to the melted solution; and aspirating the helper embryos and melted agarose solution to form a chip.
- the helper embryos can be separated from the remaining embryos by a membrane, a mesh material or a porous layer.
- the present invention contemplates any suitable material can be used which sufficiently physically separates the helper cells, while remaining permeable for the various promoting factors and embryotrophic factors to provide group culturing benefits.
- the combined total embryos from the helper embryos and the at least one embryo in a group culture can be at least, but not limited to, ten embryos.
- the total number of embryos in a group culture can be about 20 to about 40 embryos.
- the group culture occurs in a smaller volume of medium, 1-1 ⁇ , for example, as few as a single embryo could be cultured with a single helper embryo.
- the referenced embryo totals can be formed with any combination of helper embryos.
- one to nine or more helper embryos can be used for a group culture of ten total embryos.
- one to nine or more of the desired embryos can be used for a group culture of ten total embryos.
- the helper embryos are kept between about 2° Celsius and about 45°Celsius after being embedded in the solid or gel suspension.
- a culturing container with incubation spaces, such as wells, for culturing groups of embryos can be a Petri dish or another container with a generally flat bottom surface.
- the culturing container can include at least one well on the bottom surface.
- Helper embryos can be placed in the well and either embedded in an agarose chip, or contained within the well by a membrane.
- the well is divided with a divider for separating helper embryos.
- the well can be divided into two concentric areas.
- the helper cells can be placed in either of the inner or outer area, and the remaining embryos, the embryos targeted for development, can be placed in the remaining area.
- each well can further comprise a raised portion.
- the raised portions can further comprise inner wells.
- the helper embryos can then be stored in the inner wells and embedded in a material, or separated with a membrane.
- An advantage in embedding the helper embryos in contrast to the remaining targeted embryos resides in the extra stresses and process steps, including temperature changes, embedded embryos endure.
- the helper embryos and the remaining targeted embryos can interchangeably be placed on either side as long as each group is physically separated.
- helper embryos can be embedded in a gel or solid suspension and wherein the suspension is cultured with the at least one embryo.
- a low melting point agarose can be used to create an agarose chip in which helper embryos can be embedded.
- an agarose chip can be formed by producing a solution with agarose and NaCl.
- the agarose can have a gelling point and a low melting point and make up roughly 1-20% of the solution.
- the solution can be sterilized by autoclaving and then heated to 65° Celsius or higher in order to melt the agarose.
- the melted agarose can then be cooled to a suitable temperature for embryos.
- the solution can be cooled to between about 35° Celsius and 45° Celsius, and more particularly can be cooled to 39° Celsius.
- this method is contemplated for use with bovine, equine, porcine, cervine, human, sheep, goat and other species.
- the solution would be cooled to the appropriate temperatures for any given species.
- embryos can be added to the solution.
- the embryos can be in the two to eight cell stage of development.
- embryos can be used with more than eight cells.
- oocytes, zygotes, or fertilized eggs can be used before cleavage.
- the solution, including the embryos can be aspirated into an instrument with a channel, such as a pipette.
- the desired number of embryos can be aspirated with the surrounding agarose solution, and then released into a cooler medium between about 2° Celsius and 35° Celsius. This cooler temperature will solidify the agarose solution around the embryos embedding the embryos in a sausage shaped agarose chip.
- This agarose chip provides a sufficient means to separate the embedded helper embryos, with any embryos outside the chip in order to prevent mixing. This chip also allows chemicals to exchange between the separated groups of embryos, including embryotrophic factors.
- a container 100 is illustrated with a plurality of wells 102.
- the container 100 can serve as a culturing system for culturing helper embryos alongside targeted embryos.
- Cross section AA is taken through the center of the container 100 and provides a view of the sidewall 106 and the bottom 108. Additionally, a bottom surface 1 10 can be seen in the well 102.
- Such a container 100 can be a Petri dish or another container with a sufficient bottom for the inclusion of wells 102.
- the plurality of wells 102 can each accommodate helper embryos, which can be embedded within the wells 102, or can be embedded in an agarose chip or other gel, which is placed into the wells 102.
- FIG. 2A-2F illustrate several configurations within the well 102 for placing helper embryos 1 14 in the vicinity of at least one targeted embryo 1 12, while maintaining a physical separation between the two groups of embryos.
- FIG. 2A illustrates a cross section of a well 102 with helper embryos 1 14 embedded in a gel or solid 104 on the bottom surface of the well 102 formed in the bottom 108 of a container.
- the targeted embryos 1 12 are placed above the solid or gel 104 and can be in a free floating relationship.
- the targeted embryos 1 12 can include at least one embryo having desirable genetic characteristics and/or a know pedigree, and may interchangeable be referred to as the desirable embryos.
- the solid or gel 104 can be an agarose material within which the helper embryos 1 14 are embedded.
- FIG. 2B illustrates another embodiment of a well 102 having a raised portion 1 10.
- the raised portion 1 16 can have an inner well 1 16 capable of holding cells, such as helper embryos 1 14.
- the helper embryos 1 14 can be physically separated from the targeted embryos 112 with a membrane 1 18 covering the inner well 1 16.
- the membrane 1 18 should maintain separation between the two groups of embryos while permitting promoting factors to exchange between the groups.
- FIG. 2C illustrates another embodiment of the well 102, where a membrane or porous barrier 122 divides the well 102 horizontally.
- the membrane 122 is illustrated roughly in the center of the well 102, but the well 102 can be divided asymmetrically for the helper embryos 1 14 and the targeted embryos 1 12.
- the membrane or porous barrier 122 functions to separate the groups of cells while chemicals, including promoting factors, are still exchanged between the groups of embryos.
- FIG. 2D illustrates a close up view of a well 102 having a membrane 124 across the surface of the well 102.
- Helper embryos 1 14 can be placed in the well 102, and then the well can be isolated with a membrane 124. Subsequently, at least one targeted embryo 1 12 can be place on the membrane in the proximity of the helper embryos 1 14.
- the at least one targeted embryo 1 12, or desirable embryo can be placed into the well 102 and sealed with a membrane 124 with helper embryos 1 14 subsequently placed over the membrane 124, as long as each group of cells in maintained separated, but in close physical proximity.
- FIG. 2E and 2F illustrate an embodiment where each of the helper embryos 1 14 and the targeted embryos 1 12 are placed in the bottom of a well 102.
- the helper embryos 1 14 can be confined in a first region 130 adjacent to the targeted embryos 1 12 confined in the second region 126.
- the first region 130 can be defined by a barrier 132.
- the barrier 132 can be walls covered by a membrane, a porous structure, or even a solid or a gel.
- the barrier can be an agarose chip within which the helper embryos 1 14 are embedded.
- FIG. 2E and 2F illustrate an embodiment where each of the helper embryos 1 14 and the targeted embryos 1 12 are placed in the bottom of a well 102.
- the helper embryos 1 14 can be confined in a first region 130 adjacent to the targeted embryos 1 12 confined in the second region 126.
- the first region 130 can be defined by a barrier 132.
- the barrier 132 can be walls covered by a membrane,
- FIG. 3 illustrates a block diagram in accordance with an embodiment of one method in accordance with the present invention.
- at least one embryo is obtained, such as a targeted embryo or group of targeted embryos. As previously discussed, this embryo, or group of embryos can be selected for the desirable genetic traits of the donor.
- an oocyte for producing the embryo can be obtained through a session of OPU on a donor. Collected oocytes can then be fertilized in vitro and monitored for cleavage.
- other methods of obtaining oocytes are envisioned within the scope of the present invention, and other techniques, such as flushing or collection from slaughter hour ovaries, can be used.
- helper embryos are obtained.
- Helper embryos can be obtained in any of the same ways as the embryos from step 210. Additionally, there is no particular need to maintain the identity of the donors for the oocytes used to produce helper embryos thereby allowing more cost efficient methods of collecting oocytes and producing helper embryos.
- oocytes can be obtained from slaughterhouse ovaries, in the case of bovine. The oocytes can then be fertilized in vitro, or artificially activated, or nuclear transferred, and monitored for cleavage.
- Helper embryos can be the same species or different species as compared to the at least one embryo. Steps 210 and 220 can be performed in any order, or even at the same time.
- the helper embryos can then be cultured with the at least one embryo, or with the targeted embryos.
- the groups of embryos can be cultured in a close proximity.
- culturing can occur in a droplet with a volume of medium of about 1 -50 ⁇ .
- the step of culturing the groups of embryos together can further include the step of separating the groups of embryos, and maintaining a physical separation throughout the culturing process. The physical separation of groups helps ensure the integrity of embryos created from oocytes with known donors.
- Physical separation can be maintained by embedding the helper embryos in a gel or solid suspension which is permeable to various embryotrophic factors.
- the helper embryos can be embedded in an agarose chip.
- Physical separation can also be maintained by a membrane, a mesh, a porous structure or another permeable barrier.
- ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇
- FIG. 4A-4D in vitro development of fertilized bovine embryos cultured in groups with agarose embedded helper embryos can be seen.
- FIG. 4A illustrates embryos (2-8 celled stage) embedded in 1 % agarose chips after IVF, indicated by black arrow.
- FIG. 4A illustrates a single 1 (indicated by white arrow) embryo in the vicinity of the agarose chip, while FIG. 4B illustrates three (indicated by white arrow).
- Each of these embryos can be cultured, freely and separately, in a 50 ⁇ culture droplet together with either 9 or 7 embryos embedded in an agarose chip.
- the total number of embryos per droplet for the embodiment illustrated is 9 and 7 respectively.
- Animals were restrained in a squeeze chute and prepared for follicular aspiration as described by Pieterse et al., Theriogenology 1991 ; 35: 19-24, incorporated herein by reference.
- Aspiration medium consisted of phosphate-buffered saline (PBS) with the addition of 10 IU/mL heparin and 0.1% polyvinyl alcohol. OPU was scheduled twice weekly, a total of 4 replicates were performed. COCs of Grade A to C were selected for subsequent IVM and IVF.
- PBS phosphate-buffered saline
- Embryos were produced as described by Xu et al. Developmental potential of vitrified Holstein cattle embryos fertilized in vitro with sex-sorted sperm. Journal of Dairy Science 89:2510-18 (2006), the text of which is incorporated herein by reference.
- COCs were matured for 22 h in 75 ⁇ L ⁇ droplets of Medium 199 (Invitrogen) containing Earle's salts, L-glutamine, 2.2 g/L sodium bicarbonate and 25 mM Hepes, supplemented with 10 % (vol/vol) fetal bovine serum (FBS; Hyclone, Logan, UT), 0.5 g/mL ovine FSH (National Institute of Diabetes and Digestive and Kidney Disease, NIDDK, Los Angeles), 5.0 ⁇ /mL ovine LH (NIDDK), and 1 .0 ⁇ g/mL estradiol 17- ⁇ . Droplets were covered with mineral oil and contained 15 to 20 oocytes.
- FBS fetal bovine serum
- NIDDK National Institute of Diabetes and Digestive and Kidney Disease
- NIDDK National Institute of Diabetes and Digestive and Kidney Disease
- NIDDK 5.0 ⁇ /mL ovine LH
- estradiol 17- ⁇
- Fertilization was accomplished by use of frozen/thawed semen from bulls of known fertility and previously tested for IVF efficiency in our laboratory.
- sperm was subjected to a swim-up procedure for 1 h. Following centrifugation, the sperm pellet was re-suspended to achieve a concentration of 2 x 106/mL. The final concentration of sperm was 1 x 106/mL in 50 ⁇ droplets of TALP fertilization medium supplemented with 10 ⁇ g/mL heparin after adding both sperm and COCs.
- IVF droplets were covered with mineral oil, and sperm/COCs co-incubation was allowed for 20 to 22 h.
- SOF synthetic oviduct fluid
- presumptive zygotes were moved into 50 ⁇ L ⁇ culture droplets in serum free medium described above, and cultured for 40 hours (total 46 h post IVF) prior to adding helper embryos embedded in agarose chips to the culture.
- a 1 % solution of agarose, with low gelling and melting points (A-9414) was prepared in saline and then sterilized by autoclaving. Solidified agarose was stored at 2-8 °Celsius prior to use.
- the sterile agarose was melted by warming it in a 65 °Celsius water bath, and maintaining it on a 39 "Celsius warming plate until inserting helper embryos. Cleaved helper embryos at 2-8 celled stage were selected and transferred into a Petri dish containing the melted agarose at a temperature between 35 and 39 °Celsius.
- cleaved helper embryos Five, seven or nine cleaved helper embryos were aspirated into a hand-made capillary along with agarose to form a sausage-like gel (chip) (FIG. 4), and then released into culture medium at 25-30 °Celsius.
- the agarose/embryo chips were transferred into 50 ⁇ droplets of SOF medium containing the free embryos or targets embryos, and culture continued for an additional 5 days.
- the embedded helper embryos could easily be observed within the agarose chip, and although diffusion could take place between them and the shared medium, they remained physically separated from the free or targeted embryos (FIG. 4).
- Example 1 Determining the minimum number of free embryos to culture with agarose embedded helpers for effective in vitro development: IVF with conventional semen
- This experiment was designed to establish the minimum number of cleaved embryos that could be cultured in a single droplet without compromising their development to blastocysts.
- IVF was preformed with oocytes from slaughterhouse ovaries, and conventional semen, then after 40-46 hours cleaved embryos at either 1 , 3, 5, 10 or 20 per group were cultured in a 50 ⁇ ⁇ medium droplet for an additional 5 days under the environmental conditions described above.
- the optimal number to maximize blastocyst yield was determined to be > 10. Therefore, cleaved embryos (2-8 celled) in groups of either 1 , 3 or 5, to approximate the numbers likely available from OPU/IVF, were cultured together with either 9, 7 or 5 helpers embedded in agarose chips (Fig. 1), thus, bringing the total number cultured in each droplet to 10.
- CE cleaved embryos (2-8 celled).
- the total number of oocytes used for IVM and IVF in Experiment 1 was 3460, and 40-46 hours post IVF, 72.1% oocytes cleaved to 2-8 celled stage.
- Blastocyst development rate increased from 6.6 %, to 1 1.1%, to 24.4 % in the groups of 1 , 3, or 5 embryos per droplet, respectively. However, even the best development achieved was still significantly lower than that of the group with 10 embryos per droplet (24.4% vs. 39.2%). When the number of embryos was increased to 20 per droplet, the blastocyst rate reached a plateau (43.3%) not significantly greater than achieved with 10 embryos per droplet.
- the blastocyst development of the free floating embryos was 49.5, 41.2 and 39.2%, respectively, which was not significantly different from that of the controls when 10 (FIG. 4D) or 20 embryos were cultured per droplet (Table 1 ).
- the agarose embedded embryos, in these groups of 10 developed to blastocysts (Fig. 4C) at a similar rate of 38.5, 42.3 and 43.5%, respectively. There was no difference in the development between the free-floating and embedded groups or the targeted group and the helper group.
- Example 2 Determining the effect of agarose on the development of embryos in vitro This experiment was performed to determine whether the addition of agarose has any effect on blastocyst development.
- the 2 x 2 experimental design was arranged to test both the number of embryos per group (3 vs.10), and effect of agarose (with vs. without).
- Cleaved embryos were derived from IVF with conventional semen and matured oocytes collected from slaughterhouse ovaries. The culture was same as in Experiment 1. The results are shown in Table 3. Table 3. In vitro development of small number of IVF embryos cultured in groups with the addition of agarose chips
- a total of 71 1 oocytes were fertilized with conventional semen, and the cleavage rate at 40-46h after IVF reached 68.5 ⁇ 5.3%.
- the cleaved embryos were then utilized to test the effect of agarose.
- the development to morula and blastocyst stage in the group of 3 per droplet was 30.0-37.8% and 23.4-27.8%, respectively, which was significantly lower than their counterparts with 10 per droplet (morula 46.0-48.9%, blastocyst 41.5-46.7%).
- the addition of an agarose chip did not promote embryo development to the blastocyst stage, nor did it have any detrimental effect on development.
- Example 3 In vitro developmental potential of OPU/IVF derived embryos when group cultured with agarose embedded helper embryos: IVF with X-sorted sperm
- Oocytes were collected by OPU, and fertilized in vitro, by standard procedures described above, using X-sorted sperm.
- cleaved embryos derived from OPU/IVF in groups of 3 were cultured with either 0 or 7 helper embryos embedded in agarose chips, in droplets containing 50 ⁇ culture medium.
- the number of cultured embryos per droplet was either 3 (OPU group) or 10 (OPU+helper).
- OPU group 3
- OPU+helper 10
- the cleavage to 2-8 celled stage, and development to morulae and blastocysts, except for the values of BLs/2-8 celled were calculated based on the total number oocytes collected by OPU and used for IVF.
- the p values with different letters (a, b) within the same column differ (p ⁇ 0.05).
- the basic concepts of the present invention may be embodied in a variety of ways.
- the invention involves numerous and varied embodiments of shipping container and methods of making and using the shipping container including, but not limited to, the best mode of the invention.
- each element of an apparatus or each step of a method may be described by an apparatus term or method term. Such terms can be substituted where desired to make explicit the implicitly broad coverage to which this invention is entitled. As but one example, it should be understood that all steps of a method may be disclosed as an action, a means for taking that action, or as an element which causes that action. Similarly, each element of an apparatus may be disclosed as the physical element or the action which that physical element facilitates.
- a or “an” entity refers to one or more of that entity; for example, “an embryo” refers to one or more of the embryos.
- the terms “a” or “an”, “one or more” and “at least one” can be used interchangeably herein.
- the applicant(s) should be understood to claim at least: i) the methods disclosed and described for culturing embryos, ii) systems for separating embryos and for group culturing of embryos, iii) similar, equivalent, and even implicit variations of each of these systems and methods, iv) those alternative embodiments which accomplish each of the functions shown, disclosed, or described, v) those alternative designs and methods which accomplish each of the functions shown as are implicit to accomplish that which is disclosed and described, vi) each feature, component, and step shown as separate and independent inventions, vii) the applications enhanced by the various systems or components disclosed, viii) the resulting products produced by such systems or components, ix) methods and apparatuses substantially as described hereinbefore and with reference to any of the accompanying examples, x) the various combinations and permutations of each of the previous elements disclosed.
Abstract
Description
Claims
Priority Applications (2)
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CA2803814A CA2803814A1 (en) | 2010-06-23 | 2011-06-24 | Group culture system and method with helper embryos |
BR112012032921A BR112012032921A2 (en) | 2010-06-23 | 2011-06-24 | group cultivation system and method with helper embryos |
Applications Claiming Priority (2)
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US35786910P | 2010-06-23 | 2010-06-23 | |
US61/357,869 | 2010-06-23 |
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WO2011162826A2 true WO2011162826A2 (en) | 2011-12-29 |
WO2011162826A3 WO2011162826A3 (en) | 2014-04-10 |
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PCT/US2011/001135 WO2011162826A2 (en) | 2010-06-23 | 2011-06-24 | Group culture system and method with helper embryos |
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BR (1) | BR112012032921A2 (en) |
CA (1) | CA2803814A1 (en) |
WO (1) | WO2011162826A2 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102559585A (en) * | 2012-02-03 | 2012-07-11 | 兰诺生物技术无锡有限公司 | Culture medium for in vitro culture of bovine embryo |
Citations (2)
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US20080145860A1 (en) * | 2001-04-13 | 2008-06-19 | Lee Ike W | Encapsulated cell indicator system |
US20100036492A1 (en) * | 2008-07-06 | 2010-02-11 | The Curators Of The University Of Missouri | Osteochondral implants, arthroplasty methods, devices, and systems |
-
2011
- 2011-06-24 CA CA2803814A patent/CA2803814A1/en not_active Abandoned
- 2011-06-24 WO PCT/US2011/001135 patent/WO2011162826A2/en active Application Filing
- 2011-06-24 BR BR112012032921A patent/BR112012032921A2/en not_active IP Right Cessation
Patent Citations (2)
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US20080145860A1 (en) * | 2001-04-13 | 2008-06-19 | Lee Ike W | Encapsulated cell indicator system |
US20100036492A1 (en) * | 2008-07-06 | 2010-02-11 | The Curators Of The University Of Missouri | Osteochondral implants, arthroplasty methods, devices, and systems |
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GOPICHANDRAN ET AL.: 'The effect of paracrine/autocrine interactions on the in vitro culture of bovine preimplantation embryos.' REPRODUCTION vol. 131, 2006, pages 269 - 277 * |
SHEN ET AL.: 'Endothelial cells stimulate self-renewal and expand neurogenesis of neural stem cells.' SCIENCE vol. 304, 2004, pages 1338 - 1340 * |
WILLADSEN ET AL.: 'The viability of early cleavage stages containing half the normal number of blastomeres in the sheep.' J REPROD FERT vol. 59, 1980, pages 357 - 362 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102559585A (en) * | 2012-02-03 | 2012-07-11 | 兰诺生物技术无锡有限公司 | Culture medium for in vitro culture of bovine embryo |
Also Published As
Publication number | Publication date |
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BR112012032921A2 (en) | 2015-10-06 |
CA2803814A1 (en) | 2011-12-29 |
WO2011162826A3 (en) | 2014-04-10 |
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