JP5406481B2 - Embryo culture vessel - Google Patents

Description

  The present invention relates to an embryo culture container for culturing in vitro fertilized embryos or cloned embryos.

  Advances in in vitro culture techniques for mammalian embryos have made it possible to generate in vitro fertilized embryos and somatic cell cloned embryos up to the blastocyst stage. In vitro culture of mammalian embryos is a method of placing a drop of about 50 μl of a culture solution in a plastic petri dish (culture vessel), coating this drop with mineral oil, and then introducing the embryo into the drop to culture the embryo, It is common to do this. However, according to this method, a blastocyst stage embryo cannot be efficiently obtained unless several tens of embryos are assembled and cultured in a drop of the same culture solution (see Non-Patent Document 1).

  On the other hand, embryos that are difficult to produce, such as somatic cell clone embryos, cannot produce and culture many embryos simultaneously. Therefore, it is difficult to efficiently obtain a blastocyst stage embryo from a somatic cell clone embryo or the like by the above method, and development of a culture system capable of efficiently generating even a small number of embryos is required.

In recent years, it has been reported that a small number of embryos can be efficiently generated into blastocysts by a method of culturing embryos in minute wells drilled in the bottom of the culture dish (Well of well; WOW method). . However, since this method manually pierces minute wells, it is difficult to stably pierce wells having the same shape and cannot be easily manufactured (see Non-Patent Document 2).
Nagao Y, Iijima R and Saeki K. Interaction between embryos and culture conditions during in vitro development of bovine early embryos.Zygote. 2008; 16: 127-133. Vajta G, Peura TT, Holm P, Paldi A, Greve T, Trounson AO, Callesen H. New method for culture of zona-included or zona-free embryos: the Well of the Well (WOW) system.Mol Reprod Dev. 2000 ; 55: 256-264.

  Therefore, the present invention is efficient to blastocyst stage embryos by individually culturing embryos that are difficult to cultivate individually because it is difficult to produce a large number of embryos such as somatic cell clone embryos. It is an object to provide an embryo culture vessel that can be generated well and can be easily manufactured.

  As a result of intensive studies by the inventors of the present invention, it is possible to culture even a small number of embryos with high efficiency and high quality by suppressing / preventing material exchange inside and outside the well provided on the surface of the embryo culture apparatus. And the present invention was completed.

That is, embryo culture container according to claim 1 of the present invention, in the embryo culture vessel having wells for culturing accommodating the embryo, Ri synthetic resin name by molding under reduced pressure, after molding there is no inner wall surface diameter 0.2μm or more holes in the wells, and the diameter 200Myuemu～500myuemu, is shall be and wherein the cylindrical well of depth 100μm~300μm are formed.

The embryo culture container according to claim 2 of the present invention is the embryo culture container according to claim 1 , wherein the synthetic resin is a thermosetting resin.

The embryo culture container according to claim 3 of the present invention is the embryo culture container according to claim 2 , wherein the thermosetting resin is of a silicone resin.

The embryo culture container according to claim 4 of the present invention is the embryo culture container according to claim 3 , wherein the silicone resin is polydimethylsiloxane.

  By using the embryo culture container of the present invention, a small number of embryos such as in vitro fertilized embryos and cloned embryos can be cultured with high efficiency, and the quality of the resulting embryos is high. Therefore, in vitro fertilization and somatic cell cloning of mammals can be performed with higher efficiency, which can contribute to the improvement of mammalian varieties. In addition, application to human embryo culture can contribute to the improvement of assisted reproduction technology.

  The embryo culture container of the present invention is an embryo culture container having a well for containing and cultivating an embryo, and has a low porosity on the inner wall surface of the well, and the substance cannot be exchanged substantially inside and outside the well. The details will be described below.

  As shown in FIG. 1, the embryo culture vessel 1 is provided with a well 2 on the surface for accommodating and culturing an embryo. The shape of the embryo culture vessel 1 is not particularly limited, but is generally rectangular. Are preferred. Further, the material of the embryo culture vessel 1 is not particularly limited as long as the material cannot be exchanged substantially inside and outside the well 2, and glass or the like can be used in addition to the synthetic resin. Moreover, although the culture container 1 may be manufactured only with a single material, you may use it combining a some material.

  Examples of the shape of the well 2 include a general cylindrical shape, and a U-shaped, V-shaped, and hemispherical cross section that facilitates cell collection. In addition, the size of the well 2 is such that about 1 to several embryos can enter. When the shape of the well 2 is cylindrical, the diameter is about 200 μm to 500 μm, and the depth is 100 μm to It is about 300 μm.

  The inner wall surface of the well 2 has a low porosity so that various substances such as proteins, hormones and oxygen cannot be exchanged inside and outside the well. Therefore, the substance secreted from the embryo accumulates inside the well, and the secreted substance acts on the embryo itself (autocrine), thereby promoting the growth of the embryo.

Here, the porosity is the number of holes opened in a certain area, and is measured by observing with an atomic force microscope (AFM). Further, the low porosity means that there are only 1,000,000 holes / mm ² or less of pores having a diameter of 0.2 to 0.5 μm.

  Such an embryo culture container 1 is prepared, for example, by preparing a mother mold of an embryo culture container, pouring a thermosetting resin into the mother mold, and then heating and curing the thermosetting resin with a heater or the like under reduced pressure. And can be manufactured by molding. Note that “under reduced pressure” means that the pressure around the culture vessel is lower than atmospheric pressure, and it is preferable to reduce the pressure to a vacuum in order to lower the porosity more efficiently.

  The thermosetting resin can be used without particular limitation as long as the porosity of the inner wall surface of the well is lowered. Among these, a silicone resin, particularly polydimethylsiloxane (hereinafter abbreviated as PDMS) is preferable because it is easy to mold and low cost and has high biocompatibility.

  The embryo culture container of the present invention can also be produced by pouring a photocurable resin into the matrix of the embryo culture container and then irradiating the photocurable resin with light such as ultraviolet rays under reduced pressure. Furthermore, the embryo culture container of the present invention can also be produced by coating the inner wall surface of a well with a resin having low porosity and low material permeability, such as polystyrene resin. In addition, the embryo culture container of the present invention can also be produced by injection molding a thermoplastic resin such as polystyrene under reduced pressure using a matrix having a draft and a release agent.

  Hereinafter, the present invention will be described in more detail based on examples. However, the scope of claims of the present invention is not limited in any way by the following examples.

  An embryo culture container formed by molding PDMS under reduced pressure (hereinafter abbreviated as PDMSW-LP) for the effect of differences in culture containers on the development rate of blastocyst stage embryos using in vitro fertilized embryos, An embryo culture vessel formed by molding PDMS under atmospheric pressure (hereinafter abbreviated as PDMSW-AP), a conventional embryo culture vessel using the WOW method, and a commercially available cell culture plate were used for examination. The details will be described below.

1. Preparation of embryo culture container
PDMSW-AP and PDMSW-LP were fabricated using the matrix. The matrix was produced as follows. First, 1 ml of negative type thick film photoresist (SU-8 3050, manufactured by Kayaku Microchem) is placed on a 3 inch silicon wafer that has been polished on one side, and 2000 rpm using a spin coater (1H-D7, manufactured by MIKASA). For 5 seconds. The solvent was removed from the photoresist by soft baking at 90 ° C. for 40 minutes in a constant temperature drying oven (DO-300FPA, manufactured by ASONE).

  While contacting a photomask (custom product, manufactured by Topic) with the desired pattern on the resist surface, ultraviolet light at 365 nm was irradiated for 18.5 seconds using a simple exposure device. After baking at 90 ° C. for 40 minutes, the substrate was immersed in a developer for about 10 minutes to remove excess resist and washed with isopropanol.

  The completed matrix was fixed to the bottom of the culture dish with an adhesive. On the other hand, PDMS (Slygard184, manufactured by Toray Dow Corning) was mixed and stirred so as to be a base 10: catalyst 1 (weight ratio). The mixed PDMS was transferred to a vacuum desiccator (240 type, manufactured by ASONE) and degassed for about 40 minutes.

  In the case of PDMSW-AP, degassed PDMS was poured into a matrix and heated for about 120 minutes under atmospheric pressure with a film heater (5Ω, manufactured by Kyoritsu Denshi) to solidify PDMS to obtain a PDMS film. In the case of PDMSW-LP, put the master mold in a vacuum desiccator (240 type, manufactured by ASONE), pour the degassed PDMS into the master mold, and deaerate (depressurize to -0.08Mpa) in the vacuum desiccator The PDMS was solidified by heating with a film heater for 120 minutes.

  The obtained PDMS sheet was bonded to the bottom of a glass bottom dish (manufactured by Nippon Genetics) by oxygen plasma treatment. As a result, embryo culture containers, PDMSW-AP and PDMSW-LP in which cylindrical wells having a diameter of 300 μm and a depth of 200 μm were formed were obtained.

  The surfaces of the obtained embryo culture containers, PDMSW-AP and PDMSW-LP were observed with an atomic force microscope (SPI3800, manufactured by SII). The result is shown in FIG. 2A shows the observation result of PDMSW-AP, and FIG. 2B shows the observation result of PDMSW-LP.

From this figure, the surface of PDMSW-AP fabricated under atmospheric pressure has 128 holes (= 1,000,000 holes / mm ² or more) in 10 μm x 10 μm (high porosity) ) Was confirmed. In contrast, it was confirmed that there were no holes (low porosity) on the surface of PDMSW-LP solidified PDMS under reduced pressure.

  The culture vessel by the WOW method was prepared by partially modifying the method described in Non-Patent Document 2. Specifically, it was prepared by providing a microwell (diameter: about 300 μm, depth: about 250 μm) on the bottom of a petri dish (Becton Dickinson) using an aggregation needle (BLS). A Petri dish (manufactured by Becton Dickinson) was used as a control group (aggregate culture method).

2. In vitro fertilization and in vitro fertilized embryo culture In vitro fertilization and in vitro fertilized embryo culture are performed according to the procedures described in the following (1) to (4) according to the literatures 1 and 2 by Saeki et al. And the literature by Brackett et al. went.
Reference 1: Saeki K, Hoshi M, Leibfried-Rutledge ML, First NL. In vitro fertilization and development of bovine oocytes matured in serum-free medium. Biol Reprod. 1991; 44: 256-260.
Reference 2: Saeki K, Nagao Y, Kishi M, Nagai M, Iritani A. Timing of completion of the first meiotic division in bovine oocytes after maintenance of meiotic arrest with cycloheximide and their subsequent development. J Vet Med Sci. 1998 ; 60: 523-526.
Literature by Brackett et al .: Brackett BG, and Oliphant G. Capacitation of rabbit spermatozoa in vitro. Biol Reprod. 1975; 12: 260-274

(1) In vitro maturation culture of bovine ovum The bovine ovary collected at the slaughterhouse was brought back to the laboratory while being kept warm with 25 ° C. physiological saline. Using the 10ml syringe (made by Terumo) equipped with 21G injection needle (made by Terumo), the cumulus egg complex (hereinafter abbreviated as COCs) from the follicle 2-8mm in diameter from the bovine ovary brought home. Aspiration was collected. After selecting only COCs with 3 or more layers of tight cumulus cell layers attached from the collected COCs, 199 medium containing 5% fetal calf serum (Bio West, hereinafter abbreviated as FBS) (Earle salt, In vitro matured culture (5% CO ₂ , 95% air, 39 ° C., saturated humidity) in GIBCO).

(2) Preparation of sperm solution After thawing the frozen semen of Japanese black hair and laminating this frozen semen lysate in a centrifuge tube containing 45% and 90% Percoll solution, centrifuge at 25 ° C, 700xg for 30 minutes, Normal sperm and bad sperm were separated (discontinuous density gradient method). After removing the supernatant, the precipitated normal sperm was suspended in a fertilization culture solution, and the sperm was washed by centrifugation at 25 ° C. and 700 × g for 10 minutes.

  In addition, the fertilization culture solution was prepared by removing glucose from a defined medium (hereinafter abbreviated as DM) and adding 1% (v / v) antibiotic-antifungal solution (GIBCO) instead of penicillin. A culture medium for corrected fertilization (hereinafter abbreviated as corrected DM) was used.

(3) In vitro fertilization
COCs cultured in vitro for 21 hours were introduced into a sperm suspension diluted to 2 × 10 ⁶ sperm / ml with modified DM containing 10 μg / ml parin under liquid paraffin and cultured for 18 hours (39 ° C., 5% Fertilized by CO ₂ , 95% air, saturated humidity). After culturing, sperm and cumulus cells adhering to the ovum were peeled off using a Pasteur pipette that had been processed into an ovum diameter.

(4) Cell culture The obtained embryo is introduced into a modified synthetic oviduct culture medium (hereinafter abbreviated as mSOFM), and up to 168 hours after fertilization by the method shown below depending on the microplate used Cultured (39 ° C., 5% CO ₂ , 5% O ₂ , 90% N ₂ , saturation humidity).

  In the case of the mass culture method (control group), 25 embryos were introduced into each 50 μl mSOFM drop layered with liquid paraffin and cultured. In the case of the WOW method, the PDMSW-AP method, and the PDMSW-LP method, each embryo was introduced and cultured so that there was one embryo per well. Note that air bubbles in the wells formed when PDMSW-AP and PDMSW-LP were covered with mSOFM were removed by sucking air with a vacuum desiccator.

3. Observation and measurement 48 hours after fertilization using a stereomicroscope, observe the embryos, count the number of embryos split, calculate the cleavage rate, and similarly observe the embryos 168 hours after fertilization. The blastocyst stage embryos that developed were counted and the incidence was examined. The results are shown in Table 1. Table 1 summarizes the results of performing the same experiment three times.

  As shown in Table 1, the cleavage rate in each experimental group was almost the same (about 70%). In addition, embryos cultured with PDMSW-AP hardly developed into blastocysts (incidence 4%). In contrast, embryos cultured with PDMSW-LP developed into blastocyst-stage embryos at a rate approximately equal to that of the control group (incidence: 24-25%).

The ontogenic potential of blastocyst-stage embryos is related to the number and percentage of inner cell mass (hereinafter referred to as ICM) and trophoblast cells (hereinafter referred to as TE) contained in the embryo. It is known to do. Therefore, the number of cells in the blastocyst stage embryo was counted according to the following literature by Thouas et al. Specifically, it measured as follows.
Thouas et al .: Thouas GA, Korfiatis NA, French AJ, Jones GM, Trounson AO. Simplified technique for differential staining of inner cell mass and trophectoderm cells of mouse and bovine blastocysts. Reprod Biomed Online. 2001; 3: 25-29.

  First, the blastocyst stage embryo was transferred to a 0.1 mg / ml propidium iodide (PI, SIGMA) solution containing 1% Triton-X100 (manufactured by SIGMA), and left still for 30 seconds to stain TE with red fluorescence. Next, the blastocyst stage embryo is transferred to a 4% (w / v) paraformaldehyde solution containing 10mg / ml hoechst 33342 (manufactured by SIGMA) and left in the dark for 30 minutes. Fluorescently stained. Further, using VECTA SHIELD (manufactured by Vector Laboratory), the stained blastocyst stage embryo was encapsulated on a slide glass. Finally, the slide glass was observed with a fluorescence microscope, and the total number of cells, the number of ICM and TE cells were counted from the obtained fluorescence-stained images, and the ratio of ICM in the whole embryo was calculated. The results are shown in Table 2. In addition, the number of cells in the table is an average value.

  As shown in Table 2, in the control group and PDMSW-LP, the total number of cells (Total) and the ratio of ICM in the whole embryo (ICM / Total) were almost equal. From this, it was found that by culturing bovine in vitro fertilized embryos in an embryo culture vessel (PDMSW-LP) having a low porosity, a blastocyst stage embryo excellent in quality can be obtained.

It is an external appearance perspective view of the embryo culture apparatus of this invention. It is the result of having observed the surface of PDMSW-AP and PDMSW-LP with an atomic force microscope.

Claims (4)

An embryo culture container having a well for containing and culturing an embryo,
Ri synthetic resin name by molding under reduced pressure,
An embryo culture vessel characterized in that a plurality of cylindrical wells having a diameter of 200 μm to 500 μm and a depth of 100 μm to 300 μm are formed on the inner wall surface of the well after molding without a hole having a diameter of 0.2 μm or more . The embryo culture container according to claim 1, wherein the synthetic resin is a thermosetting resin. The embryo culture container according to claim 2 , wherein the thermosetting resin is a silicone resin. The embryo culture container according to claim 3 , wherein the silicone resin is polydimethylsiloxane.