Spheroid-producing device, method for recovering spheroids, and method for producing spheroids

Patent metadata

Field	Value
Publication number	US-11208625-B2
Application number	US-202016864446-A
Country	US
Kind code	B2
Filing date	May 1, 2020
Priority date	Feb 25, 2014
Publication date	Dec 28, 2021
Grant date	Dec 28, 2021

Abstract

The present invention provides a device for producing a large number of uniform spheroids by an easy method. The spheroid-producing device (1) at least includes a first surface (11), a second surface (12), and a plurality of wall surfaces (13). The second surface (12) faces the first surface (11). The respective wall surfaces (13) constitute a plurality of holes penetrating through the first surface and the second surface. In addition, an equivalent diameter of inscribed circles of openings in the first surface (11) is greater than an equivalent diameter of inscribed circles of openings in the second surface (12).

First claim

The invention claimed is:

1. A spheroid-producing hanging drop device comprising:
atop surface that forms an upper surface of the spheroid-producing device:
a bottom surface that is a back side surface of the first surface, and that forms a bottom surface of the spheroid-producing device;
a plurality of wall surfaces extending from the top surface to the bottom surface that define a plurality of wells, each of the plurality of wells having a hole penetrating through the top surface through which cells and media are introduced into each of the plurality of wells, and a hole penetrating through the bottom surface;
wherein a width of the top surface between adjacent wall surfaces defining one of the wells is 2 mm or less;
wherein the top surface between adjacent wells are round or conical or are polygonal cones having vertexes; and
wherein the plurality of wall surfaces are hydrophilic such that a contact angle θc between the plurality of wall surfaces and a culture medium disposed in the plurality of wells is within a range of 0<cos θc<1, as measured by the droplet method.

2. The spheroid-producing hanging drop device according to claim 1 , wherein the holes in the bottom surfaces of the wells are circular, and the diameter of the circular holes in the bottom surfaces of the wells is within a range of 200 micrometers to 1 cm.

3. The spheroid-producing hanging drop device according to claim 1 , wherein at least portions of the wall surfaces are inclined between the top surface and the bottom surface, and make an angle greater than 1 degree and smaller than 90 degrees with respect to the bottom surface.

4. The spheroid-producing hanging drop device according to claim 2 , wherein a radius (cm) of the circular holes in the bottom surfaces of the wells is less than or equal to a value of a variable X that is defined by an equation of
(⅔)α X 2 +pX= 2 γL ,

where p is a water pressure [g/cm 2 ] at the openings in the second surface, a is a specific gravity of the culture medium, and γL [g/cm] is surface tension of the culture medium as measured by the Wilhelmy method.

5. The spheroid-producing hanging drop device according to claim 2 , wherein a diameter of the circular holes in the bottom surfaces of the wells is such that the following equations are satisfied:
γ L cos θ c−γs> 0

and p=γL ×(2/ r )

where p is a water pressure [g/cm 2 ] at the openings in the second surface, γL is surface tension of the liquid medium [g/cm] as measured by the Wilhelmy method, r is a radius of curvature [cm], and γs is surface tension of the plurality of wall surfaces [g/cm].

6. The spheroid-producing device according to claim 3 , wherein a width between one of the wall surfaces constituting one of the holes and another one of the wall surfaces constituting another adjacent one of the holes is 5 mm or less, wherein the width is a width of an end portion that is close to the first surface, in which the two wall surfaces incline at the angle θi with respect to the second surface.

7. The spheroid-producing hanging drop device according to claim 1 , wherein the spheroid-producing device is made of a material selected from the group consisting of acrylic resin, polylactic acid, polyglycolic acid, styrene resin, acrylic styrene copolymer resin, polycarbonate resin, polyester resin, polyvinyl alcohol resin, ethylene-vinyl alcohol copolymer resin, thermoplastic elastomer vinyl chloride resin, silicone resin, silicon resin, and combinations thereof.

8. The spheroid-producing hanging drop device according to claim 7 , wherein the plurality of wall surfaces comprise functional groups formed by a surface modification treatment selected from the group consisting of plasma treatment, corona discharge, UV ozone treatment, and combinations thereof.

9. The spheroid-producing hanging drop device according to claim 7 , wherein the plurality of wall surfaces are coated with a substance selected from the group consisting of inorganic substances, metal, synthetic polymers, dimers, trimers, tetramers, biobased polymers, and combinations thereof.

10. The spheroid-producing hanging drop device according to claim 8 , wherein a portion of the wall surfaces comprise nanometer order microstructures.

11. The spheroid-producing device according to claim 1 , wherein the spheroid-producing device is a molding made of one of or a combination of inorganic substances.

12. The spheroid-producing device according to claim 11 , wherein surfaces of the plurality of wall surfaces are modified by a surface modification treatment selected from the group consisting of plasma treatment, corona discharge, UV ozone treatment, and combinations thereof.

13. The spheroid-producing device according to claim 11 , wherein the plurality of wall surfaces are coated with a substance selected from the group consisting of inorganic substances, metal, polymer, dimers, trimers, tetramers, and combinations thereof.

14. The spheroid-producing device according to claim 1 , wherein front surfaces of the plurality of wall surfaces comprise nanometer order microstructures.

15. The spheroid-producing hanging drop device according to claim 1 , wherein the outer wall surrounding the first surface is a part of a well container.

16. A method for recovering spheroids from the spheroid-producing device according to claim 1 , comprising bringing the second surface of the spheroid-producing device into contact with a solution selected from water, a culture medium, and a buffer solution in order to recover the spheroids.

17. A method for recovering spheroids from the spheroid-producing device according to claim 1 , comprising applying a pressure on the first surface of the spheroid-producing device, whereby the spheroids fly out of the openings in the second surface.

18. A method for producing spheroids using the spheroid producing device according to claim 1 , the method comprising:
pouring a culture medium containing cells into the plurality of holes from the first surface;
forming droplets in the plurality of holes; and
culturing the cells in the droplets in order to produce the spheroids.

19. The method according to claim 18 , further comprising bringing the second surface into contact with a solution selected from one of water, a culture medium, and a buffer solution in order to recover the spheroids.

20. The method according to claim 18 , further comprising applying a pressure on the first surface to thereby destroy the droplets and to cause the spheroids to fly out of the openings in the second surface.

Assignees

• Corning Inc

Inventors

• Sumi Shoichiro

Classifications

• C12N2511/00

  Cells for large scale production · CPC title

• C12N2513/00

  3D culture · CPC title

• C12N5/0062

  General methods for three-dimensional culture · CPC title

• C12N2535/00

  Supports or coatings for cell culture characterised by topography · CPC title

• C12M25/01 Primary

  Drops · CPC title