JP5083952B2 - Egg cell culture equipment - Google Patents

Description

  The present invention relates to a cell culture apparatus and a culture method. More specifically, the present invention relates to a culture apparatus particularly suitable for culturing fertilized eggs important in reproductive medicine or regenerative medicine, and a culture method using this apparatus.

  A technique has been developed in which fertilized eggs (zygotes) are produced by in vitro fertilization of sperm and eggs of animals, particularly mammals, and cultured to the stage of cleavage, morula, blastula and even late blastula. A technique has been developed in which fertilized eggs in the stage of blastocysts from this cleavage are transplanted into the uterus to obtain offspring, used for breeding in livestock, and further applied to human infertility treatment.

  However, the success rate of pregnancy due to in vitro fertilization, which is performed as a means of reproductive medicine, particularly infertility treatment, is not high. This success rate is still around 25% since the world's first child-raising in the UK about 30 years ago, and it is hoped that the success rate will be improved. Infertility treatment generally requires the collection of an ovum (in vitro maturation is required for in vitro maturation), in vitro fertilization, and the process of returning a fertilized egg into the uterus. The most important process is a process in which a fertilized egg is developed to a developmental stage (4 to 8 cell stage, blastocyst stage) suitable for transplantation.

Various studies have been made in the past to increase egg cell maturation efficiency and pregnancy efficiency (see Non-Patent Documents 1 to 3).
All of the techniques introduced in these documents attempt to promote culture by applying a physical stimulus (mechanical stress) to an egg cell.

“Gamete Research”, vol. 125, 1984, p4984 “Journal of Experimental Zoology”, vol. 266, 1993, p146 “Biology of Reproduction”, vol. 75, 2006, p45

  However, it has not yet been clearly clarified what physical stimulation is optimal for egg cell culture. As a result, no suitable device has been placed on the market for loading physical stimuli.

As a result of intensive studies on the mode of physical stimulation to be applied when culturing egg cells, the present inventors have found that an incubator filled with egg cells may be rocked, and the present invention has been conceived. It came to do.
That is, the invention of the first aspect of the present invention is defined as follows.
A holding unit for holding the incubator;
A swinging part for swinging the holding part;
A control unit for controlling the swing of the swing unit,
The egg cell culture apparatus, wherein the control unit controls the rocking unit to rock the holding unit so as to apply a physical stimulus to the egg cell in the incubator.

  According to the egg cell culturing apparatus configured as described above, physical stimulation can be given to the egg cells in the incubator with a simple configuration in which the holding unit that holds the incubator is swung. Thereby, an inexpensive culture apparatus can be provided.

As a mode of swinging the holding portion, as a result of experiments by the present inventors, as defined in the second aspect of the present invention, the holding portion is tilted in one direction, and then is kept stationary for a certain period of time, and then opposite It is preferable to incline in the direction and then rest for a certain period of time, and then repeat this (see FIG. 1). Thereby, a physical stimulus of rotation + slip can be given to the egg cell.
Further, as defined in the third aspect of the present invention, the egg cell in the incubator moves at a speed of 2 mm / sec or less, and a shear stress of 1.2 dynes / cm ² or less is applied to the egg cell. As described above, it is preferable to swing the holding portion.

The incubator used in the present invention is not particularly limited as long as egg cells can be cultured. A microdrop of culture broth was formed on the bottom of the petri dish, and eggs were encapsulated in the microdrop, or a plurality of small-diameter holes were formed in the chamber. The thing etc. can be mentioned.
2 and 3 show the configuration of the culture apparatus 1.
The culture apparatus 1 includes a rocking plate 3 and a main body 11.
The rocking plate 3 as a holding portion is made of a resin plate, and a silicon rubber 5 is attached as an anti-slip layer on the upper surface thereof. This silicon rubber 5 is adsorbed to an incubator 20 such as a petri dish. Thereby, even if the rocking | fluctuation plate 3 inclines, the incubator 20 does not move and does not slide down from the rocking | fluctuation plate 3. FIG. In order to more reliably prevent the incubator 20 from sliding down, a convex portion sandwiching the bottom wall of the incubator 20 can be formed on the swing plate 3.

The main body 11 houses a motor 13 as a swinging portion, and the swinging plate 3 is fixed to a rotating shaft 14 of the motor 13. Reference numeral 15 denotes a control unit that controls the rotation of the rotating shaft 14 of the motor 13. An operator can input control parameters (rotation angle, rotation speed, downtime, etc.) of the motor 13 via the input unit 16, and the control unit 15 controls the operation of the motor 13 in accordance with the input control parameters.
In the example shown in FIGS. 2 and 3, the rotating shaft 14 of the motor is fixed almost at the center of the swing plate 3, and the swing plate 3 swings around the center. The rotating shaft 14 of the motor may be connected to the end of the moving plate 3.
Also, a pair of linear motors whose movable elements can move in the vertical direction is prepared, both ends of the rocking plate 3 are supported by the pair of movable elements, and the vertical movement of the movable element is controlled by controlling the vertical movement of the movable element. Oscillation can also be controlled.
Furthermore, the swinging plate can be controlled by supporting the swinging plate with a plurality of columns from below and changing the height of each column. In this case, if the swing plate is supported by at least three columns, the swing plate can be inclined in an arbitrary direction.

According to the culture apparatus having the above-described configuration, a conventional stationary culture system experimental instrument can be used as it is. Specifically, rock cells can be cultured by adding egg cells to a microdrop 21 produced on a petri dish 20 as an incubator or a 4-well culture plate.
This culture method can be applied to in vitro maturation, in vitro fertilization, parthenogenesis, in vitro culture and the like of mammalian egg cells. Furthermore, it can be applied to culture of embryonic stem cells and other cells.
A fertilized egg may be in any stage of zygote, cleavage, morula, and blastula, but an egg in a life cycle other than the fertilized egg (eg, an egg in a follicle, an ovulatory egg, etc.) is placed in a culture container. May be used after being fertilized and fertilized. Further, a fertilized egg after freezing and thawing may be used.
Preferably, a fertilized egg derived from a mammal is used. Examples of mammals include humans, monkeys, cows, horses, sheep, goats, baboons, pigs, dogs, cats, rabbits, guinea pigs, rats, mice and the like.

The culture solution can be arbitrarily selected according to the cells to be cultured. For example, a culture solution such as α-MEM manufactured by GIBCO, IVC manufactured by Medicalt, or HTF manufactured by Irvine can be used. The culture solution may further contain a useful substance for culture. For example, β mercaptoethanol for preventing generation inhibition due to peroxidation by oxygen in the gas phase can be mentioned. As culture conditions, conditions usually used by those skilled in the art for culturing fertilized eggs are employed. For example, the temperature is about 37 to 38 ° C., and the osmotic pressure is about 280 mOsm / kg.
Examples of the present invention will be described below.

A microdrop having a diameter of 10 mm and a volume of 80 μl of M16 medium (Cosmo Bio) was prepared on a 35 mm petri dish (Iwaki). Eight F1 mouse fertilized eggs multiplied by C57BL6 and ICR were placed in the drop. A petri dish containing microdrops was prepared on the rocking culture apparatus shown in FIGS. 2 and 3, and the movement was observed with a stereomicroscope (manufactured by ZEISS). The rocking culture apparatus was operated with a maximum inclination angle of 10 degrees, an inclination change rate of 0.37 rpm, and an inclination holding time of 1 minute. That is, it is tilted to one side and stopped for 1 minute when the tilt angle reaches 10 degrees, and tilted in the opposite direction at an angular velocity of 0.37 rpm every 1 minute, and when the tilt angle reaches 10 degrees, it is 1 minute. Stopped, and so on. When changing the tilt angle, the observed maximum moving speed of the fertilized egg was 0.16 mm / sec, and the observed maximum moving distance was calculated to be 1.4 mm. Their values when maintaining the slope were estimated to be 0.006 mm / sec and 0.3 mm, respectively. Therefore, it was estimated that a shear stress of 0.033 dyne / cm ^{2 at} maximum was applied under this rocking culture condition. This load and the observed maximum migration speed of the embryo are smaller than the values 1.2 dyne / cm ² and 2 mm / sec in Non-Patent Document 3, and the intensity stress described in the literature is continuously Since it is not loaded, it can be said that it exists in the range which does not kill an egg cell.

Table 1 shows the results of examining the above-mentioned shear stress by observing the movement of mouse fertilized eggs while changing several inclination angular velocities. In this table, the estimated value when the inclination angle is changed is described. In any condition, it is considered that the range is such that the egg cell is not killed.

Porcine oocytes-cumulus masses (OCCs) are present on the surface of the ovary 3 brought back from the slaughterhouse by immersion in 0.9% physiological saline supplemented with antibiotics (GIBCO) maintained at 28-37 ° C. In a disposable 50-ml centrifuge tube (Sumitomo Bakelite Co., Ltd.) by aspirating a ~ 6mm middle follicle together with follicular fluid with an 18G needle (Nipro Corp.) attached to a 10-ml disposable syringe (Nipro Corp.) Collected. The collected cell pieces containing OCCs were washed 3 times with TL-HEPES-PVA culture solution in a disposable 50-ml centrifuge tube, and then 3 ml of modified TL- in the culture dish (made by Greiner) with a diameter of 35 mm was obtained from the OCCs alone. Collected in HEPES-PVA culture. What was further washed three times with the same solution was used in the experiment.

As a mature basal medium, a solution obtained by adding β mercaptoethanol (manufactured by SIGMA) to 50 μM to a complete maturation medium for pigs POM (manufactured by Peptide Institute) was used. For maturation culture, a 500-liter modified POM medium was dispensed into a 4-well culture plate (manufactured by NUNC) in a gas incubator maintained at 39 ° C in a gas phase of 5% carbon dioxide and 95% air. (Established by Tabai) and equilibrated overnight.
The collected OCCs were further washed with 3 ml of modified POM in a 35 mm diameter petri dish (manufactured by Greiner), and then the mature OCCs were equilibrated overnight to 45 to 50 per well (500 μl). 5 g after adding 5 μl per well (500 μl) of eCG (1,000 iu / ml concentration), hCG (1,000 iu / ml concentration) and dibutyryl cyclic AMP (100 mM) dissolved in pure water Maturation culture was started by placing it in a gas incubator maintained at 39 ° C under carbon dioxide carbon dioxide, 95% air gas phase conditions.

In the rock culture section of the experimental section, OCCs are placed on the rocking plate of a rocking incubator installed in a gas incubator maintained at 39 ° C with 5% carbon dioxide and 95% air. 4-well culture plates were placed. The rocking condition was carried out under the condition that the rocking plate was maintained at a tilt of 20 degrees for 1 minute, then slowly reversed and conversely maintained at a tilt of 20 degrees for 1 minute. The speed at which the plate tilts was about 0.1 rpm.
The stationary culture group of the control group was placed on the same tray as the rocking incubator in the gas incubator maintained at 39 ° C. under the gas phase conditions of 5% carbon dioxide and 95% air. .
OCCs are cultured for 20 hours under these conditions, eCG (1,000 iu / ml concentration), hCG (1,000 iu / ml concentration) and dibutyryl cyclic, taking care not to destroy the cumulus cell mass structure. After washing 3 times with 3 ml of fresh modified POM without addition of AMP (100 mM) (inside a 35 mm diameter petri dish), eCG (1,000 iu / ml concentration), hCG (1,000 iu / ml concentration) on a 4-well culture plate ) And 500 μl of fresh modified POM without dibutyryl cyclic AMP (100 mM) (5% carbon dioxide, 95% air, gas phase, 39 ° C gas culture) The mixture was equilibrated overnight in a vessel) and further cultured under the same culture conditions for 24 hours.

After the first 20 hours of culture with the addition of eCG (1,000 iu / ml concentration), hCG (1,000 iu / ml concentration) and dibutyryl cyclic AMP (100 mM), a portion of each section of OCCs was observed with a stereomicroscope (Nikon). The diameter of OCCs was measured based on the scale of a micrometer (made by Nikon) taken at the same magnification. The diameter of one OCCs was an average value of numerical values obtained by measuring the diameters in the x-axis and y-axis directions. (Fig. 4)
FIG. 5 shows a graph in which the average cumulus diameter and average oocyte diameter were measured. Compared to the static culture group, the average cumulus diameter increased in the rock culture group, and the average oocyte diameter was almost the same as that in the static culture group. Therefore, it was suggested that the cumulus cell expansion was promoted by rocking culture.

The cumulus cells were removed by pipetting OCCs that had undergone matured culture in a solution containing 0.1% hyaluronidase (manufactured by SIGMA) in modified TL-HEPES-PVA, and the oocytes were made naked. The oocytes are placed in a fusion medium containing 250.3mM sorbitol, 0.3mM HEPES, 0.2% (w / v) bovine serum albumin, 0.1mM Ca-lactate, 0.1mM glutathione in pure water. Place an oocyte on the electrode, apply 5V AC current between these electrodes for 5 seconds, apply 100V DC current for 30μs, and then apply 100V DC current again for 30μs 1 minute later. Oocytes were activated (using BCM, ECM2001M).
Thereafter, these oocytes were maintained in an NCSU 37 culture solution containing 2.2 μg / ml cytochalasin B in a 4-well culture plate at a gas phase of 5% carbon dioxide carbon dioxide and 95% air at 39 ° C. The mixture was transferred to a gas incubator overnight and cultured for 2 hours to suppress the release of the second polar body of the activated egg and doubled.

Thereafter, a solution (5% carbon dioxide carbonic acid carbonate) added to a modified NCSU37 culture solution not containing 3 ml of cytochalasin B in a culture dish (manufactured by Greiner) having a diameter of 35 mm to a concentration of 50 μM. After washing three times in a gas incubator kept at 39 ° C in a gas phase condition of 95% air and 39 ° C overnight, 500 μl was dispensed into 4-well culture plates (NUNC) Transfer to fresh same solution (5% carbon dioxide carbon dioxide, 95% air gas phase condition, equilibrated overnight in a gas incubator maintained at 39 ° C) for 6 days under the same culture conditions as mature culture The culture was continued and the state of initial development was observed under a stereomicroscope (Nikon Corporation).
The results are shown in Table 2. In the rock culture, the cleavage rate and blastocyst arrival rate increased. When the significance probability was calculated by χ 2 test, it was judged that there was a significant difference.

Pab <0.01,
Pcd <0.05 (χ2 test)

The in vitro fertilization method for human unfertilized eggs was examined under the condition of the presence or absence of a rocking incubator. A medium (HTF medium, manufactured by Irvine Scientific) was added to a 4-well culture plate (manufactured by NUNC), and unfertilized eggs were added. Pre-cultured unfertilized eggs for 3 hours under 5% carbon dioxide carbon dioxide, 5% oxygen, 95% air gas phase conditions and culture conditions in a gas incubator (Astech) maintained at 37 ° C. It was. Washed semen was added to a sperm concentration of 100,000 / ml. In the experimental group, the 4-well culture plate was placed on the polycarbonate plate of the rocking culture apparatus shown in FIGS. 2 and 3, and the tilt angle was 10 degrees, the tilt change rate was 0.2 rpm, and the tilt holding time. Drove without. In the control group, the chamber was left in a thermostat. Pronucleus formation was confirmed with a stereomicroscope (Leica) 18 to 20 hours after the maturation. The results are shown in Table 3. Since statistical significance was greater than 0.05, no significant difference was observed, but it was confirmed that the fertilization rate increased.

The fertilization rate was calculated by (2 number of pronucleated embryos) × 100 / ((2 number of prenucleated embryos) + (number of non-pronucleated embryos)). Significance = 0.859

Human in vitro fertilized embryo maturation was compared in the presence or absence of a rocking incubator. Add 0.8 ml of medium (Blast Assistist, manufactured by Medicult) to a 4-well culture plate (manufactured by NUNC), and keep it at 37 ° C in 5% carbon dioxide, 5% oxygen, 95% air gas phase Embryos whose fertilization was confirmed were cultured under the culture conditions in a leaning gas culture vessel (manufactured by Astec). In the rock culture section, the rock culture apparatus shown in FIGS. 2 and 3 was operated at an inclination angle of 10 degrees, an inclination change rate of 3.3 rpm, and an inclination holding time of 1 minute. In the control group, the chamber was left in a thermostat. Four days after the confirmation of fertilization, the maturity of the embryo was confirmed with a microscope (Leica). The results are shown in Table 4. Although there was no significant difference, it was shown that the blastocyst arrival rate increased.

Significance = 0.70

  The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims.

It is a figure which shows the relationship between operation | movement of the culture apparatus of this invention, and the physical stimulation loaded on an egg cell. It is a front view of the culture apparatus of the Example of this invention. It is a side view similarly. Fig. 2 is the porcine oocyte-cumulus cell mass of Example 2. It is the graph which similarly measured the average cumulus diameter and average oocyte diameter in porcine in vitro maturation.

1 Incubator, 3 Oscillating plate, 13 Motor, 15 Control unit, 20 Incubator

Claims (4)

A holding unit for holding the incubator;
A swinging part for swinging the holding part;
A control unit for controlling the operation of the swing unit,
The egg cell culture apparatus, wherein the control unit controls the rocking unit to rock the holding unit so as to apply a physical stimulus to the egg cell in the incubator.   2. The control unit according to claim 1, wherein the control unit tilts the holding unit in one direction, then stops for a certain period of time, then tilts in the opposite direction, then rests for a certain period of time, and repeats the following. Egg cell culture equipment.   The control unit swings the holding unit so that the egg cell in the incubator moves at a speed of 2 mm / sec or less and a shear stress of 1.2 dynes / cm2 or less is loaded on the egg cell. The egg cell culture apparatus according to claim 1 or 2.   The holding part is plate-shaped, and an anti-slip layer is formed on the surface thereof, and the incubator placed on the anti-slip layer is immovable with respect to the holding part even when the holding part is inclined. The egg cell culture apparatus according to any one of claims 1 to 3, wherein the egg cell culture apparatus is provided.