JP5753148B2 - Method for culturing spheroids using cell culture carrier and cell culture carrier - Google Patents

Method for culturing spheroids using cell culture carrier and cell culture carrier Download PDF

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JP5753148B2
JP5753148B2 JP2012244030A JP2012244030A JP5753148B2 JP 5753148 B2 JP5753148 B2 JP 5753148B2 JP 2012244030 A JP2012244030 A JP 2012244030A JP 2012244030 A JP2012244030 A JP 2012244030A JP 5753148 B2 JP5753148 B2 JP 5753148B2
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culture
recess
cell culture
channel
carrier
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JP2014090703A (en
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松本 卓也
卓也 松本
奈緒子 小尾
奈緒子 小尾
英樹 村上
英樹 村上
正雄 河合
正雄 河合
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日本写真印刷株式会社
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  The present invention relates to a cell culture carrier that is a container for culturing spheroids (cell mass) such as embryonic stem cells (ES cells).

  Conventionally, synthetic resin, glass petri dishes, well plates, etc. (hereinafter referred to as conventional containers) have been used as cell culture carriers for three-dimensionally culturing animal cells including humans to form spheroids.

  Also, conventionally, the lower member and the upper member have a two-stage structure, and the lower member is a plate-like body made of a ceramic porous body, and a plurality of concave portions that are cell culture sites are arranged. A cell culture carrier (see, for example, Patent Document 1), which is a plate-like body placed on the upper surface of the lower member and having a through hole at a position corresponding to the recess, has been proposed. This cell culture carrier is hereinafter referred to as an improved carrier.

Such conventional cell culture carriers have the following problems.
(1) Since the spheroid adheres to the wall surface in both the conventional container and the improved carrier, a peeling process is required for taking out the spheroid. The improved carrier has a two-stage structure, and there is a device for facilitating spheroid removal by removing the upper member, but a peeling treatment is still necessary.
(2) The improved carrier uses a porous material for the lower member so that nutrients and waste products can enter and exit the spheroid. On the other hand, the porous material is opaque to visible light, and the progress of spheroid growth in the cell culture carrier cannot be observed with a microscope.
(3) When spheroids are hypoxic, they are effective for long-term storage of spheroids. Large spheroids are required to make the spheroids hypoxic. However, when spheroids are cultured in a conventional container or an improved carrier up to large spheroids, the spheroids cannot maintain a spherical shape due to their own weight.

JP 2010-263868 A

  Therefore, the present invention is a cell culture carrier that, when spheroids are cultured, does not adhere to the wall surface, ensures that metabolites enter and exit the spheroids, can observe spheroids using an optical microscope, and can support spherical retention of spheroids It is a problem to obtain. Another object of the present invention is to obtain a method for producing such a cell culture carrier.

  Means for solving the problems will be described below. For ease of understanding, description will be made with reference numerals corresponding to the embodiments of the present invention, but the present invention is not limited to the embodiments. A number as a symbol may indicate a part or the like collectively. When an individual part or the like is indicated in an embodiment described later, an alphabetic suffix may be added after the number.

The cell culture carrier according to one aspect of the present invention is a cell culture carrier having a culture recess,
The cell culture support (6) is provided with a plurality of culture recesses (7),
An individual inflow channel (21) directed downward from the bottom surface of the culture recess, a main channel inflow channel (22) communicating with the plurality of individual inflow channels, and an overflow channel (24) directed laterally from the upper surface of the culture recess. The plurality of culture recesses are conducted through the overflow channel,
The individual inflow channel has a cross-sectional area of 19.5 μm 2 or more and 1970 μm 2 or less,
The cell culture carrier is transparent to visible light and has an impermeable property.

In a preferred embodiment of the present invention, the shape of the upper end surface of the culture recess may be a circle, the diameter (Dop) thereof may be 50 μm or more and 2000 μm or less, and the shape of the upper end surface of the culture recess is It is a circle, and when the diameter is Dop and the height of the culture recess is H, the following equation (1) may be satisfied.
0.4Dop ≦ H ≦ 0.6Dop Equation (1)

In another preferred embodiment of the present invention,
The cell culture carrier is obtained by laminating a middle layer plate on a lower layer plate, and laminating an upper layer plate on the middle layer plate,
The main line inflow passage is formed in the lower plate,
The individual inflow channel is formed in the middle layer plate,
The culture recess and the overflow channel are formed in the upper plate,
The lower layer plate and the upper layer plate are synthetic resin plates created by transferring the original shape,
The intermediate layer plate may be a photoresist film having a desired shape formed by an exposure operation and a development operation.

A cell culture device according to another aspect of the present invention comprises a cell culture carrier and a pump according to the present invention,
A liquid supply port that is a liquid supply outlet of the pump and a main line inflow channel inlet that is an inlet of the main line inflow channel are connected, and a part of the overflow channel is a waste liquid port,
The culture solution is sent from the pump to the culture recess and discarded from the waste solution port.

A method for producing a cell culture carrier according to another aspect of the present invention includes:
A method for producing a cell culture carrier according to the present invention, comprising:
The intermediate layer plate is laminated on the lower layer plate, and the upper layer plate is laminated on the intermediate layer plate,
The main line inflow passage is formed in the lower plate,
The individual inflow channel is formed in the middle layer plate,
The culture recess and the overflow channel are formed in the upper plate,
The lower layer plate and the upper layer plate are synthetic resin plates created by transferring the original shape,
The intermediate layer plate is formed into a photoresist film to form a photoresist film, the photoresist film is exposed to form an exposed photoresist film, and the exposed photoresist film is developed to form the intermediate layer plate having a desired shape. It is what.

  The present invention described above, preferred embodiments of the present invention, and components included in these can be implemented in combination as much as possible.

  Since the cell culture carrier according to the present invention is a visible light transparent cell culture carrier having a culture recess, a specific individual inflow channel, and a specific overflow channel, along with other matters specifying the invention, The culture solution which passes through the individual inflow channel and reaches the culture recess and is drained from the overflow channel can be flowed. Under such a culture fluid flow, spheroids always exist in a floating state in the culture recess and do not adhere to the wall surface. Therefore, spheroids can be easily taken out from the cell culture carrier. Also, the floating helps the spheroids retain their spherical shape. Furthermore, since the culture solution can be always flowed into and out of the culture recess, and the culture solution flow flows in a certain direction, exchange of spheroid metabolites is easy.

  Since the cell culture device according to the present invention includes the cell culture carrier according to the present invention and a specific pump, there is an advantage that the feeding of the culture solution is further facilitated and the exchange of spheroid metabolites is further facilitated.

  The method for producing a cell culture carrier according to the present invention includes a step of producing a middle layer plate including individual inflow channels by molding, exposing and developing a photoresist together with the steps of specifying other inventions. There is an advantage that the process of forming the flow path can be easily and accurately performed.

FIG. 1 is a perspective view of the first cell culture carrier. FIG. 2 is a cross-sectional view of the culture recess row, and is a cross-sectional view in a plane indicated by a cut surface 9 in FIG. FIG. 3 is a perspective view of the second cell culture carrier. FIG. 4 is a cross-sectional view of the cell recess row, and is a cross-sectional view in a plane indicated by a cut surface 19 in FIG. FIG. 5 is a cross-sectional view showing the laminated structure of the first cell culture carrier. FIG. 6 is an explanatory view showing a processing step of the intermediate layer plate. FIG. 7 is an explanatory view showing the processing steps of the lower layer plate.

  Hereinafter, the cell culture carrier according to the embodiment of the present invention and the production method thereof will be further described with reference to the drawings. In the drawings referred to in this specification, in order to facilitate the understanding of the present invention, some of the components are schematically illustrated in an exaggerated manner. For this reason, the dimension, ratio, etc. between components may differ from a real thing. Further, the dimensions, materials, shapes, relative positions, etc. of the members and parts described in the embodiments of the present invention are not intended to limit the scope of the present invention to those unless otherwise specified. It is merely an illustrative example.

  Referring to FIG. 1, the first cell culture carrier 6 has a rectangular parallelepiped shape and has a plurality of first culture recesses 7. The shape of the 1st cell recessed part 7 is a column shape, and the column-shaped upper surface is opening on the upper surface of the rectangular parallelepiped mentioned above. Three first culture recesses 7 form a culture recess array 8 in series.

  The culture recess row 8f includes three first culture recesses 7fl, 7fm, and 7fn. Similarly, the culture recess row 8g includes three first culture recess portions 7gl, 7gm, and 7gn, and the culture recess row 8h includes three first culture recess portions 7hl, 7hm, and 7hn.

  Each culture recess 7 has an individual inflow passage 21 that extends downward from the inner bottom surface thereof. The lower end of the individual inflow channel 21 communicates with the main inflow channel 22. A side wall opening of the first cell culture carrier 6 at one end of the main line inflow path 22 is a main line inflow path inlet 23.

  A groove is carved on the upper surface of the first cell culture carrier 6, and the groove communicates with the inner surface of the first culture recess 7 at the upper end of the first culture recess 7. Such a groove is the overflow channel 24.

  With reference to FIG. 1 and FIG. 2, in this embodiment, three individual inflow channels 21 communicate with a single main inflow channel 22, and the upper ends of the three individual inflow channels 21 are the individual first cultures. It leads to the recesses 7fl, 7fm, and 7fn. The upper end of the first culture recess 7fn and the upper end of the first culture recess 7fm communicate with each other via an overflow channel 24 that is a groove formed in the horizontal direction. The upper end of the first culture recess 7fm and the upper end of the first culture recess 7fl communicate with each other via an overflow channel 24. The upper end of the first culture recess 7fl is electrically connected to the waste liquid port 25 through the overflow channel 24.

  The culture recess array is a unit of a plurality of culture recesses that share the main line inlet passage 23 and the waste liquid outlet 25. The number of culture recess rows included in a single cell culture carrier is not particularly limited, and may be single or plural. If the number of the culture recess rows is plural, there is an advantage that the flow of the culture solution becomes uniform in each culture recess.

  Usually, the liquid feeding port of the pump 3 is connected to the main line inflow passage inlet 23. The culture solution is sent from the pump 3. For example, a syringe pump or a peristaltic pump can be suitably used as the pump 3.

  The liquid fed from the pump 3 passes through the individual inflow path 21 and enters the first culture recess 7. Since the individual inflow path 21 is open to the bottom surface of the first culture recess 7, the liquid feed from the pump 3 flows into the first culture recess 7 upward from the bottom surface. This upward flow becomes a force that causes the spheroid in the first culture recess 7 to float. When spheroids are cultured in a floating state, the spheroids do not adhere to the bottom surface or inner surface of the culture recess 7. Moreover, the force which maintains a spheroid in a spherical shape works, and the spherical shape of a spheroid does not collapse by its own weight.

  In order to float the spheroid efficiently, it is desirable that the individual inflow channel is opened at the center of the bottom surface. Even in the second cell culture carrier described in detail later, it is preferable that the individual inflow channel is opened at the bottom end of the second culture recess having a hemispherical shape.

  In the first cell culture carrier 6, the first culture recess 7 has a cylindrical shape, but in the present invention, the shape of the culture recess is not limited to a cylindrical shape. The shape of the culture recess is, for example, a hemispherical shape, a semi-elliptical sphere shape, or a polygonal pyramid shape. Among these, a cylindrical shape and a hemispherical shape are preferable. If it is a cylindrical shape or a hemispherical shape, spheroids are more likely to float. Furthermore, if it is hemispherical, the flow of the culture solution is improved.

  FIG. 3 is a perspective view of a second cell culture carrier having a hemispherical second culture recess, FIG. 4 is a cross-sectional view of the culture recess array, and a cross-sectional view in a plane indicated by a cut surface 19 in FIG. It is. The second cell culture carrier 16 and the first cell culture carrier 6 are the same except for the shape of the culture recess. Hereinafter, the second cell culture carrier 16 will be briefly described.

  Referring to FIG. 3, the second cell culture carrier 16 has a rectangular parallelepiped shape and has a plurality of second culture recesses 17. The shape of the cell recess 17 is a hemispherical shape, and the upper surface of the hemispherical shape is opened on the upper surface of the rectangular parallelepiped described above. Three second culture recesses 17 form a culture recess array 18 in series.

  The culture recess row 18f includes three second culture recesses 17fl, 17fm, and 17fn. Similarly, the culture recess row 18g includes three second culture recess portions 17gl, 17gm, and 17gn, and the culture recess row 18h includes three second culture recess portions 17hl, 17hm, and 17hn.

  Each second culture recess 17 has an individual inflow passage 21 that extends downward from the inner bottom surface thereof. The lower end of the individual inflow channel 21 communicates with the main inflow channel 22. A side wall opening of the second cell culture carrier 16 at one end of the main line inflow path 22 is a main line inflow path inlet 23.

  A groove is carved on the upper surface of the second cell culture carrier 16, and the groove communicates with the inner surface of the culture recess 17 at the upper end of the culture recess 17. Such a groove is the overflow channel 24.

  3 and 4, in this embodiment, three individual inflow channels 21 are connected to a single main inflow channel 22, and the upper ends of the three individual inflow channels 21 are the individual second cultures. It leads to the recesses 17fl, 17fm, and 17fn. The upper end of the second culture recess 17fn and the upper end of the second culture recess 17fm communicate with each other via an overflow channel 24 that is a groove formed in the horizontal direction. The upper end portion of the second culture recess portion 17fm and the upper end portion of the second culture recess portion 17fl are in communication with each other via an overflow channel 24. The upper end of the second culture recess 17fl is electrically connected to the waste liquid port 25 through the overflow channel 24.

  Usually, the liquid feeding port of the pump 3 is connected to the main line inflow passage inlet 23. The liquid fed from the pump 3 passes through the individual inflow path 21 and enters the second culture recess 17. Since the individual inflow path 21 is opened at the bottom surface of the second culture recess 17, the liquid feed from the pump 3 flows into the second culture recess 17 upward from the bottom surface.

  Below, the common matter of the 1st cell culture carrier 6 and the 2nd cell culture carrier 16 is demonstrated.

The area of the cross section of the individual inflow passage 21 is preferably 19.5 μm 2 or more and 1970 μm 2 or less. When the individual inflow channel is formed in a perfect circle, the cross-sectional area corresponds to a true circle having a diameter of 5 μm to 50 μm. This cross-sectional area is equivalent to the cross-sectional area of cells that form spheroids (especially undifferentiated embryonic stem cells). If the cross-sectional area of the individual inflow channel is within this range, the cultured cells pass through the individual inflow channel and flow into the main line. It is because it does not enter the road. Although the cross-sectional shape is preferably a perfect circle, it is not easy to process a cylindrical hole with a perfect cross-section whose cross-sectional area is in the above range. Therefore, the cross-sectional shape may be a shape that approximates a perfect circle, and the cross-sectional shape may be an ellipse, a quadrangle, a polygon, or the like.

  The first culture recess 7 in the first cell culture carrier has a cylindrical shape, and the upper end surface is circular. The second culture recess 17 in the second cell culture carrier has a hemispherical shape, and the upper end surface is circular. The diameter of the upper end surface of the circular culture recess is defined as Dop. Let H be the height of the culture recesses 7 and 17. In the first culture recess 7, the height H is the distance between the upper end surface and the bottom surface. In the second culture recess 17, the height H is the distance between the upper end surface and the apex located on the lower side.

  There is no particular limitation on the length of the Dop. The preferred Dop length is 50 μm or more and 2000 μm or less, and particularly preferably 50 μm or more and 100 μm or less. This range is equivalent to the diameter of a general spheroid and is a size suitable for culturing a single spheroid in a single culture recess.

There is no particular limitation on the ratio of Dop and H. A ratio satisfying the formula (1) is preferable.
0.4Dop ≦ H ≦ 0.6Dop Equation (1)
This is because, if the ratio is within this range, the processing of the culture recess, which is a recess, is easy.

  The cell culture carriers 6 and 16 are transparent to visible light. This is to contribute to the observation of cultured cells using an optical microscope. The cell culture carriers 6 and 16 are usually made of a synthetic resin such as PDMS (polydimethylsiloxane) or a photoresist. The synthetic resin which is such a material has a water-impermeable property.

  Subsequently, one preferable production method of the cell culture carrier will be described. The cell culture carrier is made by stacking the lower layer plate 51, the middle layer plate 52 and the upper layer plate 53 in this order. In FIG. 5, the boundary surface between the lower layer plate 51 and the middle layer plate 52 is indicated by a broken line. Similarly, the boundary surface between the middle layer plate 52 and the upper layer plate 53 is indicated by a broken line. The lower plate 51 has a groove on the upper surface. The lower surface of the middle layer plate 52 covers the groove and forms the main line inflow passage 22. That is, the main line inflow passage 22 is formed in the lower layer plate 51.

  The middle layer plate 52 has a through hole. The through hole is electrically connected to the main line inflow path 22 in the lower layer plate 51. Further, the through hole is electrically connected to the culture recess 7 in the upper layer plate. The through hole is an individual inflow passage 21. That is, the individual inflow passage 21 is formed in the middle layer plate 52.

  The upper layer plate 53 has a through hole. The upper surface of the middle layer plate 52 covers the bottom open surface of the through hole to form the culture recess 7. That is, the culture recess 7 is formed in the upper layer plate 53. An overflow channel 24 is also formed in the upper layer plate 53.

  FIG. 6 shows a method for manufacturing the middle layer plate 52. As shown in (1), a thin layer 62 of photoresist is formed on a base 61 such as siliconware. The thin layer 62 may be formed by various coating methods. A preferred coating method is a spin coating method. This is because the smoothness of the surface of the thin layer 62 is excellent. As shown in (2), a thin layer 62 of photoresist is exposed through a photomask 63 and partially cured. Develop as shown in (3). Development is the removal of unnecessary portions of the thin layer 62 of photoresist. The base 61 and the thin photoresist layer 62 are separated to obtain the middle layer plate 52 as shown in (4).

  The thickness of the intermediate layer 52, that is, the thickness of the thin layer 62 of photoresist is preferably in the range of 20 μm to 200 μm. This is because within this range, the strength as the intermediate layer plate 52 can be obtained, and a thin layer can be formed with a uniform thickness and a smooth surface.

  If the intermediate layer plate is manufactured by photoetching, a through hole having a diameter in the range of 5 μm to 50 μm can be easily formed.

  FIG. 7 shows a manufacturing method of the lower layer plate 51 formed by transferring a photoresist mold. As shown in (1), a thin layer 62 of photoresist is formed on a base 61 such as siliconware. As shown in (2), a thin layer 62 of photoresist is exposed through a photomask 63 and partially cured. Development is performed as shown in (3) to form a photoresist mold 64.

  As shown in (4), PDMS 65 in a fluidized state is poured into the photoresist mold 64 and cured by heating. Thereby, the shape of the photoresist mold 64 is transferred to the PDMS 65. The PDMS 65 is separated from the photoresist mold 64 to obtain the lower layer plate 51 as shown in (5).

  The thickness of the lower layer plate 51 is not particularly limited. The depth of the groove forming the main line inflow channel is about 10 μm to 50 μm, and may be any thickness as long as the groove can be dug.

  The upper layer plate 53 in the first cell culture carrier can be formed by creating a photoresist mold in the same manner as the lower layer plate 51 and transferring it. Since the upper layer plate 53 in the second cell culture carrier includes a curved surface, it may be prepared by creating a mold and transferring it.

  Further, the lower layer plate of the first cell culture carrier and the lower layer plate of the second cell culture carrier may be prepared by preparing a mold and transferring it.

  Although the embodiment according to the present invention has been described in detail with reference to the drawings, a specific configuration example is not limited to this embodiment, and design changes and the like within a scope not departing from the gist of the present invention are possible. Even if it exists, it is included in this invention.

3 pump 6 first cell culture carrier 7 first culture recess 8 culture recess array 9 cut surface 16 second cell culture support 17 second culture recess 18 culture recess array 19 cut surface 21 individual inflow path 22 trunk inflow path 23 Main line inlet passage 24 Overflow passage 25 Waste liquid port 51 Lower layer plate 52 Middle layer 53 Upper layer plate 61 unit 62 Thin layered photoresist 63 Photomask 64 Photoresist type 65 PDMS
H Height of the culture recess Dop Diameter of the top surface of the culture recess that is circular

Claims (2)

  1. In the method for culturing spheroids (cell mass) performed using a cell culture carrier,
    The cell culture carrier is
    The cell culture carrier is provided with a plurality of culture recesses,
    An individual inflow channel that extends downward from the bottom surface of the culture recess, a main channel inflow channel that communicates with the plurality of individual inflow channels, and an overflow channel that extends in a lateral direction from the top surface of the culture recess, Conducting through an overflow channel,
    The individual inflow channel has a cross-sectional area of 19.5 μm 2 or more and 1970 μm 2 or less,
    The cell culture carrier is transparent to visible light and has water-impermeable properties ,
    Put the spheroid in the culture recess,
    Using a cell culture carrier characterized in that, during a spheroid culture period, a culture solution is passed through the trunk inflow channel, the individual inflow channel, and the culture recess in one direction in order to flow to the overflow channel A spheroid culture method to be performed.
  2. In a cell culture carrier having a culture recess,
    The cell culture carrier is provided with a plurality of culture recesses,
    An individual inflow channel that extends downward from the bottom surface of the culture recess, a main channel inflow channel that communicates with the plurality of individual inflow channels, and an overflow channel that extends in a lateral direction from the top surface of the culture recess, Conducting through an overflow channel,
    The individual inflow channel has a cross-sectional area of 19.5 μm 2 or more and 1970 μm 2 or less,
    The cell culture carrier is transparent to visible light and has water-impermeable properties,
    During the spheroid culture period in which the spheroid (cell mass) is put in the culture recess, the culture solution passes through the trunk inflow channel, the individual inflow channel, and the culture recess in order in one direction to reach the overflow channel. A cell culture carrier that flows .
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