JP4665589B2 - Cell culture vessel - Google Patents

Description

  The present invention relates to a cell culture container for accommodating eggs and sperm in the same culture system in in vitro fertilization using the assisted reproduction technique (ART) or culturing fertilized eggs for a certain period.

  In vitro fertilization is performed on infertility cases caused by female factors such as obstruction of the fallopian tube, male factors such as a small number of good sperm, or both factors. Eggs collected from patients And sperm are fertilized outside the body, and after culturing to some extent, return to the body. In infertile cases with a small number of spermatozoa, in order to increase the fertilization probability, fertilization methods in a microculture system called microdrop method, in which the culture solution layer in which sperm before fertilization is floating are made smaller, are often used. Yes.

  Conventionally, in this microdrop method, a bottom surface is a single flat surface and a diameter is used as a container for fertilizing an egg and sperm in the same culture system, or culturing a fertilized egg until the transplantation time to a living body. A petri dish with a thickness of 30 to 60 mm is used. Usually, on the bottom of the petri dish, several culture systems composed of eggs and sperm and a culture solution or culture systems composed of a fertilized egg and a culture solution are produced at intervals and cultured for 3 to 6 days. During the culture, it is necessary to exchange the culture solution of each culture system several times in order to prevent an adverse effect due to an increase in waste products generated by the activity of the culture or a decrease in nutrient components in the culture solution. However, when the petri dish is used, there is a possibility that the culture system produced at intervals moves and merges with another culture solution when exchanging the culture solution.

  On the other hand, a culture vessel provided with a culture compartment enclosed by a peripheral wall is known (for example, see Patent Document 1). This container is for fixing a culture to the bottom face of each accommodation compartment, and filling culture solution so that all the accommodation compartments may be covered, and culturing a culture. In the container, the storage compartment is provided only at the center of the container. Therefore, the liquid level of the culture solution rises due to the surface tension at the edge of the container, so that there is no possibility that the culture cannot be observed from the bottom of the container at the edge. However, this container cannot be used in in vitro fertilization where the culture is not fixed to the bottom. Further, since the container does not fundamentally solve the rising of the liquid level at the edge of the container, it is not possible to form a storage compartment at the edge of the container, and the vicinity of the edge becomes a dead space. End up.

  There has also been proposed a container in which a first recess is formed at the bottom of the container and a second recess is further formed at the bottom of the first recess (see, for example, Patent Document 2). This container is used for the swim up method in which the entire first recess is filled with the culture solution, and the sperm injected into the second recess moves through the culture solution and selectively collects where it reaches the first recess. Is. However, since the container does not take measures against the rise due to the surface tension of the liquid surface at the edge of the storage compartment pointed out in Patent Document 1, the storage compartment has a small capacity for use in the microdrop method or the like. In this case, the microscopic observation from the bottom in the accommodation compartment cannot be performed accurately.

  Furthermore, a culture vessel in which the inside of the vessel is partitioned by a wall surface made of a gel-like substance containing a culture solution is also known (see, for example, Patent Document 3). In this container, since the culture solution stored in the storage compartment is always in contact with the wall surface made of a gel-like substance containing the culture solution, it is not necessary to exchange the culture solution during the culture period. However, since the container is not provided with a measure for the bulge caused by the surface tension of the liquid surface at the edge of the storage compartment, when the storage compartment has a small capacity, the microscopic observation from the bottom in the storage compartment can be accurately performed. I can't do it.

Japanese Utility Model Publication No. 4-12898 JP 2003-289850 A JP 2004-141110 A

  Therefore, the present invention allows the inside of the storage compartment to be observed with a microscope from below the bottom even in a usage method intended for cell culture in a small amount of culture solution such as the microdrop method, and the inside of the container is partitioned without waste. It is an object of the present invention to provide a cell culture container that can be used.

  As a result of intensive studies, the inventor has arranged the boundary portion between the bottom surface and the wall surface of the storage compartment as a curved surface, and arranges the wall surface of the storage compartment in a state inclined with respect to the vertical direction of the bottom surface. The present inventors have found that it is possible to provide a container capable of accurately performing microscopic observation from the bottom of the bottom of the compartment to the edge of the storage compartment, and further capable of designing the storage compartment to have a small capacity.

That is, the present invention
(1) A cell culture container having a bottom portion and a side wall, and having a predetermined capacity of a culture system accommodation compartment therein, the culture system accommodation compartment having a uniform thickness, a wall surface, And a gentle curved surface connecting the bottom surface and the wall surface, and the wall surface of the culture system accommodation compartment is arranged in a state inclined to the outside by 5 to 20 ° with respect to the vertical direction of the bottom surface. A cell culture container,
(2) The cell culture vessel according to (1), wherein a bottom surface and a wall surface of the culture system accommodation section are connected by a curved surface having a curvature radius of 0.1 to 1.0 mm,
(3) The cell culture vessel according to (1) or (2), wherein the culture system accommodating compartment has a volume of 0.01 to 0.10 mL.
(4) The bottom surface of the culture system accommodation compartment uses the bottom of the cell culture container, and the wall surface of the culture system accommodation compartment is connected to the top surface of the bottom of the cell culture container via the curved surface. The cell culture container according to any one of (1) to (3),
(5) The culture system accommodation section is a recess provided at the bottom of the cell culture container, and the upper end of the wall surface of the culture system storage section is flush with the top surface of the bottom of the cell culture container (1 ) To (3) the cell culture container according to any one of
(6) The said culture | cultivation system accommodation division is related with the container for cell cultures in any one of (1)-(5) formed in the said cell culture container inside.

  In the cell culture container of the present invention, the bottom surface and the wall surface of the culture system accommodation compartment are connected with a gentle curved surface, and the wall surface of the culture system accommodation compartment is disposed in an inclined state with respect to the vertical direction of the bottom surface. Thus, it is possible to prevent the liquid level of the culture system from rising due to surface tension at the edge of the storage compartment as in a conventional container. Therefore, microscopic observation from below the bottom surface can be performed accurately also at the edge of the culture system accommodation compartment. In addition, since the culture system accommodation compartment can be designed to have a small capacity and the culture system accommodation compartment can be disposed at the edge of the cell culture container, the cell culture container can be miniaturized. . Furthermore, since the edge of the bottom surface of the culture system accommodation section is a curved surface, the culture after culture can be easily collected.

Hereinafter, the container for cell culture of the present invention will be described in detail based on preferred embodiments shown in the accompanying drawings, but the present invention is not limited to these descriptions.
1 is a top view showing an embodiment of the cell culture container of the present invention, FIG. 2 is a cross-sectional view of the cell culture container shown in FIG. 1, and FIG. 3 is a diagram of the cell culture container shown in FIG. It is a side view. FIG. 4 is a top view showing another embodiment of the cell culture container of the present invention.

As shown in FIG. 2, the cell culture container 1 of the present invention has a bottom 2 and a side wall 3, and a culture system accommodation section 4 having a specified capacity is provided inside. As a material constituting the container 1, a material that can be sterilized by electron beam and has high transparency, such as polystyrene, polyethylene, and polyamide, is preferably used.
The shape of the cell culture vessel 1 is not particularly limited as long as it is easy to hold the cell culture vessel 1 from the outside of the side wall 3 in addition to a cylindrical shape or a polygonal column shape. The size of the cell culture vessel 1 is also not particularly limited. However, when the cell culture vessel 1 is used for a method for cell culture in a small amount of culture solution as in the microdrop method, the necessary number of culture system accommodating compartments 4 are provided. In addition, it is preferably designed to be as small as possible. Specifically, the outer diameter is preferably about 30 to 60 mm.

  The culture system accommodation compartment 4 is a compartmented compartment that can accommodate a culture system comprising a culture such as an egg, sperm, or fertilized egg and a culture solution containing an antibiotic such as cephalotin or Hepes as a buffer solution. It is. The culture system accommodation section 4 is composed of a bottom surface 41 and a wall surface 42, and the bottom surface 41 and the wall surface 42 are connected by a gentle curved surface 43. Moreover, the wall surface 42 which comprises this culture type | system | group accommodation division 4 is inclined and arrange | positioned 5-20 degree outside with respect to the vertical direction of the bottom face 41. As shown in FIG. With such a configuration, the liquid level of the culture system accommodated in the culture system accommodation section 4 can be prevented from rising due to surface tension at the edge. Therefore, the microscopic observation from the bottom of the bottom surface 41 can be accurately performed also at the edge of the culture system accommodation section 4. Moreover, when collecting the culture accommodated in this culture | cultivation accommodation compartment 4, it is easy to insert and operate instruments, such as a pipette. When the inclination of the wall surface 42 is smaller than 5 °, the effect of suppressing the rise of the liquid level due to the surface tension of the culture system is small, and when the inclination exceeds 20 °, the culture rides on the wall surface 42, There is a possibility that accurate microscopic observation from below the bottom surface 41 cannot be performed.

  The shape of the curved surface 43 constituting the culture system accommodation compartment 4 is not particularly limited as long as it is a gently curved surface and does not form a corner at the edge of the bottom surface 41 of the culture system accommodation compartment 4. The curved surface preferably has a curvature radius of 0.1 to 1.0 mm. When the radius of curvature of the curved surface 43 is smaller than 0.1 mm, the culture is sandwiched between the bottom surface 41 and the wall surface 42, and it becomes difficult to collect with a pipette or a catheter. Further, if the curvature radius of the curved surface 43 is larger than 1.0 mm, the culture is likely to climb on the curved surface 43 during the culture, and there is a possibility that the microscopic observation from below the bottom surface 41 cannot be performed accurately in that portion. is there. By having the curved surface 43, the cell culture container 1 of the present invention can more easily collect the cultured product after culturing from the culture system accommodating section 4.

  When the culture system containing compartment 4 is used for the microdrop method, it preferably has a volume of 0.01 to 0.10 mL, and more specifically, the bottom surface 41 is a circle having a diameter of 2 to 10 mm. And the height of the wall surface 42 is preferably 1 to 5 mm. Since the culture system accommodation compartment 4 of the present invention allows microscopic observation from the bottom of the bottom surface 41 to the edge, even if it has a capacity larger than the above capacity, the minimum necessary amount according to the amount of the culture system It is possible to set the capacity.

The culture system storage compartment 4 may be one in which the bottom surface 2 of the cell culture vessel 1 is used as the bottom surface 41 and the wall surface 42 is disposed on the top surface of the bottom portion 2. The recessed part provided in may be sufficient.
In the former case, when the bottom surface 41 of the culture system accommodation compartment 4 uses the bottom 2 of the cell culture container 1, the bottom 2 of the cell culture container 1 also serves as the bottom surface 41 of the culture system accommodation compartment 4. Point to. That is, the culture system accommodation section 4 is formed by dividing the inside of the cell culture container 1 using the wall surface 42. The wall surface 42 is connected to the upper surface of the bottom 2 of the cell culture container 1 through a curved surface 43.
On the other hand, in the latter case, as shown in FIG. 2, the bottom surface 41 of the culture system accommodation section 4 is formed at a position that is one step lower than the bottom 2 of the cell culture container 1. It is preferable that the upper end of the wall surface 42 of the culture system accommodating section 4 is flush with the upper surface of the bottom 2 of the cell culture container 1. The culture system accommodated in the cell culture vessel 1 may be covered with oil such as paraffin in order to prevent the culture solution from evaporating. In the cell culture vessel 1 having the latter configuration, May be used to fill the bottom 2 surface.

In the culture system accommodation section 4, the culture system accommodated therein is observed through the bottom surface 41 from below the bottom surface 41 with a microscope. Therefore, the bottom surface 41 has a uniform thickness, and the average value of the thickness is preferably 0.5 to 3 mm. If the variation in thickness is about ± 0.1 mm, the bottom surface 41 can be used for microscopic observation. There will be no hindrance.
The bottom surface 41 is made of a highly transparent material such as polystyrene, polyethylene, or polyamide in consideration of microscopic observation. As the transparency, microscopic observation is possible if the transmittance of light having a wavelength of 450 nm is 55% or more, but it is preferable that the transmittance is 90% or more because the observation accuracy is higher.

A plurality of the culture system accommodating compartments 4 may be arranged inside the cell culture container 1 as shown in FIG. The arrangement method of the culture system accommodating compartment 4 is not particularly limited, and it may be arranged in a matrix as shown in FIG. 1 or may be arranged in a ring shape as shown in FIG. Furthermore, an identification symbol 44 for recognizing each compartment 4 and a starting symbol 45 may be displayed on the culture system accommodation compartment 4. The symbols 44 and 45 can be appropriately displayed on the bottom surface 41 or the wall surface 42 of the culture system accommodation section 4, the bottom 2 or the side wall 3 of the cell culture vessel 1, and the like.
When a plurality of the culture system accommodation compartments 4 are arranged, the interval between the culture system accommodation compartments 4 is not particularly limited, and the distance is such that the culture systems accommodated in each culture system accommodation compartment 4 are not mixed with each other by vibration or the like. If it is. If the interval is too large, the cell culture container 1 may not be miniaturized. Since the culture system accommodating compartment 4 of the present invention can suppress the liquid level of the culture system from rising due to surface tension at the edge, it can be disposed without any problem even at the edge of the cell culture vessel 1. The inside of the container 1 can be partitioned and used without waste.

In the cell culture vessel 1 of the present invention, a convex portion 31 as shown in FIG. 3 may be formed on the outer peripheral surface of the side wall 3. The convex portion 31 has an effect of preventing slipping when the cell culture container 1 is gripped from the outside of the side wall 3. The convex portion 31 may be provided over the entire outer peripheral surface of the side wall 3, but may be provided intermittently on the outer peripheral surface.
Further, the side wall 3 may be arranged in the vertical direction with respect to the bottom 2 or may be arranged so as to be slightly inclined with respect to the vertical direction of the bottom 2 as shown in FIG. If the side walls 3 are inclined from the vertical direction of the bottom 2, preferably from improved slip effect at the time of gripping the cell culture vessel 1 from the outside of the side wall 3.

It is a top view which shows one Example of the container for cell cultures of this invention. It is sectional drawing of the container for cell cultures shown by FIG. 1 of this invention. It is a side view of the container for cell cultures shown by FIG. 1 of this invention. It is a top view which shows the other Example of the container for cell cultures of this invention of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Cell culture container 2 Bottom part 3 Side wall 31 Convex part 4 Culture system accommodation compartment 41 Bottom face 42 Wall surface 43 Curved surface 44 Identification symbol 45 Symbol

Claims (5)

A cell culture container having a bottom and a side wall, and having a predetermined capacity of a culture system accommodation compartment therein, the culture system accommodation compartment having a uniform thickness, a wall surface, and the bottom surface And a gently curved surface curved outwardly of the culture system accommodation compartment connecting the wall surfaces, and the wall surface of the culture system accommodation compartment is inclined outward by 5 to 20 ° with respect to the vertical direction of the bottom surface A cell culture container , wherein the curvature radius of the curved surface of the culture system accommodation section is 0.1 to 1.0 mm . The container for cell culture according to claim 1, wherein the culture system accommodating compartment has a volume of 0.01 to 0.10 mL. The bottom surface of the culture system accommodation compartment utilizes the bottom of the cell culture container, and the wall surface of the culture system accommodation compartment is connected to the top surface of the bottom of the cell culture container via the curved surface. The container for cell culture according to claim 1 or 2 . The culture system accommodating compartments, the a recess provided in the bottom of the container for cell culture according to claim 1 or 2 the upper end of the wall surface of the culture system housing compartment is a bottom upper surface flush with the container the cell culture The cell culture container according to 1. The cell culture container according to any one of claims 1 to 4 , wherein a plurality of the culture system accommodating compartments are provided inside the cell culture container.

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