WO2015162920A1

WO2015162920A1 - Plates for Culture of Biological Samples

Info

Publication number: WO2015162920A1
Application number: PCT/JP2015/002190
Authority: WO
Inventors: Tomoko Jomura; Takashi Miyazawa
Original assignee: Toyo Gosei Co., Ltd.
Priority date: 2014-04-23
Filing date: 2015-04-22
Publication date: 2015-10-29

Abstract

A method for cultivating cells and composite body including spheroids and a plate by which spheroids have been cultivated is disclosed.

Description

Plates for Culture of Biological Samples

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit under 35 U.S.C. section 119(e) of U.S. Provisional Patent Application Serial No. 61/983,267 filed on April 23, 2014, the disclosure of which is hereby incorporated herein in its entirety by this reference.

An aspect of the present invention relates to the fields of plates suitable for biological samples such as cell, bacterium, virus and protein. Another aspect of the present invention relates to the fields of screening methods of which typical applications are drug development and biopsy.

Background

A plate for culturing cells which has a lattice structure disclosed in US patent 5792653 (date of patent: August 11, 1998).

A plate relating to an aspect of the present invention includes a member. The member has a first plane, a second plane and a plurality of structural objects each of which has at least one of a bottom in a recess and a top in a protrusion; a first distance from the second plane to a boundary between each of the plurality of structural objects and the first plane is different from a second distance between the second plane and the at least one of the bottom and the top; and at least five structural objects of the plurality of structural objects are arranged at a third distance from one structural object of the plurality of structural objects.

With regard to the plate, it is preferred that the plurality of structural objects are configured such that at least a part of the plurality of structural objects can contact a biological sample.

With regard to the plate, it is preferred that six structural objects of the plurality of structural objects including the at least five structural objects are arranged at the third distance from the one structural object.

With regard to the plate, it is preferred that the one structural object and two structural objects of the six structural objects constitute an equilateral triangle substantially.

In this specification and claim of this patent application, the term “substantially” for equilateral triangle means that the ratio of the difference between the shortest side among three sides constituting the equilateral triangle and the longest side of the three sides to the longest side is equal to or smaller than 0.15.

With regard to the plate, it is preferred that the two structural objects are arranged at positions proximate to each other.

With regard to the plate, it is preferred that the six structural objects constitute a regular hexagon substantially. In this specification and claim of this patent application, the term “substantially” for regular hexagon means that the ratio of the difference between the shortest side among six sides constituting the regular hexagon and the longest side of the six sides to the longest side is equal to or smaller than 0.15.

With regard to the plate, it is preferred that the one structural object and two structural objects of the six structural objects are included in a regular hexagon.

A plate relating to an aspect of the present invention includes a member. The member has a first plane and a second plane; a plurality of first portions and a second portion are formed in the first plane; an affinity of the plurality of first portions to a sample is different from an affinity of the second portion to the sample; and at least five first portions of the plurality of first portions are arranged at a third distance from one first portion of the plurality of first portions.

Such difference in affinity to such sample between the plurality of first portions and the second portion can be formed by surface treatment or patterning.

Alternatively, such difference in affinity can be formed by formation structures such as recess and protrusion.

Examples of such sample are chemical compound and biological samples such as normal cell and cancer cell, bacterium, virus and protein.

With regard to the plate, it is preferred that six first portions of the plurality of first portions including the at least five structural portions are arranged at the third distance from the one first portions.

With regard to the plate, it is preferred that the one first portion and two first portions of the six first portions constitute an equilateral triangle substantially.

With regard to the plate, it is preferred that the two first portions are arranged at positions proximate to each other.

With regard to the plate, it is preferred that the six first portions constitute a regular hexagon substantially.

With regard to the plate, it is preferred that the one first portion and two first portions of the six first portions are included in a regular hexagon.

A plate relating to an aspect of the present invention includes a member. The member has a first plane and a second plane opposite to the first plane; a plurality of first portions, a second portion, a plurality of third portions and a fourth portion are formed in the first plane; a first interval between two first portions of the plurality of first portions arranged at positions proximate to each other is different from a second interval between two third positions of the plurality of third positions arranged at positions proximate to each other.

With regard to the plate, it is preferred that the plurality of first portions, the second portion, the plurality of third portions and the fourth portion are configured such that at least a part of the plurality of first portions, the second portion, the plurality of third portions or the fourth portions can contact a biological sample.

With regard to the plate, it is preferred that each of the two first portions is surrounded by the second portion; and each of the two third portions is surrounded by the fourth portion.

With regard to the plate, it is preferred that at least four first portions of the plurality of first portions are arranged at the first interval from one first portion of the plurality of first portions; and at least four third portions of the plurality of third portions are arranged at the second interval from one third portion of the plurality of first portions.

With regard to the plate, it is preferred that six first portions of the plurality of first portions are arranged at the first interval from one first portion of the plurality of first portions; and at least six third portions of the plurality of third portions are arranged at the second interval from one third portion of the plurality of first portions.

With regard to the plate, it is preferred that the six third portions constitute a regular hexagon.

With regard to the plate, it is preferred that the six first portions constitute a regular hexagon; and the six third portions constitute a regular hexagon.

With regard to the plate, it is preferred that a first affinity of the plurality of first portions to a sample is different from a second affinity of the second portion to the sample; and a third affinity of the plurality of third portions to the sample is different from a fourth affinity of the fourth portion to the sample.

Examples of such sample are chemical compound and biological samples such as normal cell, cancer cell, bacterium, virus and protein.

With regard to the plate, it is preferred that the first affinity is greater than the second affinity; and the third affinity is greater than the fourth affinity.

With regard to the plate, it is preferred that each of the plurality of first portions has a structural object of which shape is at least one of a recess and a protrusion.

With regard to the plate, it is preferred that each of the plurality of third portions has a structural object of which shape is at least one of a recess and a protrusion.

With regard to the plate, it is preferred that the plurality of first portions and the second portion are formed in a first area; and the plurality of third portions and the fourth portion are formed in a second area.

With regard to the plate, it is preferred that the first area is surrounded by a first fence; and the second area is surrounded by a second fence.

With regard to the plate, it is preferred that the plate further includes a frame and each of the first fence and the second fence is a part of the frame.

With regard to the plate, it is preferred that a plurality of fifth portions and a sixth portion are formed in the first plane; and a third interval between two fifth portions of the plurality of fifth portions arranged at positions proximate to each other is different from the first interval and the second interval.

With regard to the plate, it is preferred that the plurality of fifth portions and the sixth portion are surrounded by a third fence.

A method for culturing cells relating to an aspect of the present invention is carried out using any one of the plates above.

With regard to the method, it is preferred that an interval suitable for forming a spheroid effectively between the first interval and the second interval is examined by the method.

A screening method relating to an aspect of the present invention is carried out using spheroids formed by culture of cells by any one of the above methods. The screening method can be applied to examination of pharmacological effects of compounds or drugs.

A method for manufacturing a drug relating to an aspect of the present invention is carried out based on the results of pharmacological effects examined by the screening method.

A composite body relating to an aspect of the present invention includes: a plate; and cells which are positioned on bottoms of well of the plate.

With regard to the composite body, it is preferred that the composite body further includes: a sheet which covers at least the wells. Typical examples of such sheet are formed of polymer such as ethyl ethylcellulose, polypropylene and polybutadiene.

With regard to the composite body, it is preferred that the sheet contacts edges surrounding the wells.

With regard to the composite body, it is preferred that the sheet adheres to edges surrounding the wells.

With regard to the composite body, it is preferred that the sheet is permeable to oxygen. The sheet may be permeable to moisture.

With regard to the composite body, it is preferred that the composite body is carried at a temperature equal to or lower than 4 degrees Celsius.

With regard to the composite body, it is preferred that the cells have been cultivated. It is more preferred that the cells have been cultivated three-dimensionally.

With regard to the composite body, it is preferred that the cells constitute spheroids.

A manufacturing method of a composite body relating to an aspect of the present invention includes: a step of cultivating biological sample such as cell, bacterium and virus on any one of the plates explained above.

With regard to the manufacturing method of a composite body, it is preferred that the method further includes a step of covering the cultivated sample by a sheet.

In the drawings, which illustrate what is currently considered to be the best mode for carrying out the invention:

FIG. 1 shows an overview of a plate relating to an aspect of the present invention and arrangements of affinity regions formed in bottoms of wells of the plate.

FIG. 2 shows arrangements of affinity regions formed in bottoms of wells of a plate relating to an aspect of the present invention.

FIG. 3 shows a cross-sectional view of a well bottom in which affinity regions are formed.

FIG. 4 shows a cross-sectional view of a well bottom in which recesses are formed.

FIG. 5 shows a cross-sectional view of a well bottom on which protrusions are formed.

FIG. 6 shows spheroids formed by cultivation of DLD-1 using affinity regions of which pitches are 400 micrometers, 600 micrometers and 800 micrometers.

FIG. 7 shows spheroids formed by cultivation of SKBR3 using affinity regions of which pitches are 400 micrometers, 600 micrometers and 800 micrometers.

FIG. 8 shows time-dependent changes of numbers of cells by cultivation of DLD-1.

FIG. 9 shows time-dependent changes of numbers of cells by cultivation of SKBR3.

FIG. 10 shows outlines of spheroids formed form DLD-1 and SKBR3.

FIG. 11 shows coefficient of variations (CV) of widths of spheroids formed form DLD-1 and SKBR3.

FIG. 12 shows process from cultivation of cells and packing of cultivated cells.

FIG. 13 shows packing of spheroids with a resin with oxygen permeability.

DETAILED DESCRIPTION

Experimental Procedures

An exemplified plate relating to an aspect of the present invention has 96 wells in total each of which is surrounded by a black frame as shown in FIG.1. A portion of the black frame corresponds to an example of the first fence or the second fence explained above. The constituent material of the bottoms of the wells is a resin such as cyclolefin resin. Cycloolefin resin is useful because of its high transparency if the plate is used for imaging. Typically, the thickness of the resin is around 190 micrometers if the constituent material is cycloolefin resin. Plural circular regions which are 100 micrometers in diameter and have high affinity to biological samples such as cells compared to the periphery of the regions are formed in the bottom of each of the wells. This enables to anchor and cultivate biological samples. The density of the regions differs depending on parts of the plate. The circular affinity regions are arranged in three different pitches, i.e. 400, 600 and 800 micrometers pitches. The numbers of the circular regions per well are 220, 100 and 50 for 400, 600 and 800 micrometers pitches, respectively.

Three of the circular affinity regions arranged at positions proximate to each other constitute an equilateral triangle. Six of the circular affinity regions arranged at positions proximate to one of the circular affinity regions constitute a regular hexagon substantially.

Alternatively, affinity regions on two straight lines intersecting at an acute angle are arranged at the same distance. Typically, the angle formed by the two straight lines is 60 degrees.

Another plate relating to an aspect of the present invention has a square arrangement of affinity regions substantially as shown in FIG. 2. Four of the circular affinity regions arranged at positions proximate to each other constitute a square substantially. Alternatively, affinity regions on two straight lines intersecting at right angle are arranged at the substantially same distance.

In this specification and claim of this patent application, the term “substantially” for square means that the ratio of the difference between the shortest side among four sides constituting the square and the longest side of the four sides to the longest side is equal to or smaller than 0.15.

FIG. 3 shows a cross-sectional view of the circular affinity regions formed the well bottom. The affinity regions can be formed by a surface treatment or photolithography technique. The affinity regions are surrounded by a fence contacting plane A. The fence prevents liquid containing a sample dispensed into the well from leaking. The fence may be a portion of frame.

Instead of formation of affinity regions, structural objects such as recess or protrusion are formed to fix and cultivate biological samples such as cell, bacterium, virus and protein. FIG.4 shows recesses formed in the bottom of the well explained above while FIG. 5 shows protrusions formed on the bottom of the well.

Each of the recesses is constituted by a bottom and a wall as shown in FIG. 4. The wall extends from a boundary between plane A (the surface of the well bottom) and the recess toward the bottom of the recess. The recesses are surrounded by a fence contacting plane A. The fence prevents liquid containing a sample dispensed into the well from leaking. The fence may be a portion of frame.

Each of the protrusions is constituted by a top and a wall as shown in FIG. 5. The wall extends from a boundary between plane A and the protrusion toward the top of the protrusion. The protrusions are surrounded by a fence contacting plane A. The fence prevents liquid containing a sample dispensed into the well from leaking. The fence may be a portion of frame.

The surface conditions of at least one of the wall and the bottom or the top can be adjusted by a surface treatment or selection of material used for the wall and the bottom or the top.

Cultivations of human colorectal adenocarcinoma cells DLD-1 and human breast adenocarcinoma cells SKBR3 were carried out using the plate as shown in FIG. 1.

Each of two suspensions which contains human colorectal adenocarcinoma cells DLD-1 and human breast adenocarcinoma cells SKBR3, respectively, is dispensed into wells of the plate in which the circular affinity regions are arranged in the three different pitches, i.e. 400, 600 and 800 micrometers pitches.

Typical numbers of cells to be seeded for one well are 2,800-5,600, 1,200-2,400 and 650-1,300 for 400, 600 and 800 micrometers pitches, respectively. On this occasion, the numbers of cells seeded in each of the wells were 2,800, 1,200 and 650 for 400, 600 and 800 micrometers pitches, respectively. The seeded cells were incubated in the presence of 5 % humidified CO₂ at 37 degrees Celsius. The incubation medium is replaced with fresh one every 1-3 days.

Spheroids formed from DLD-1 on the well bottoms having affinity regions with 400, 600 and 800 micrometers pitches after 14 days of culture are shown in FIG 6. Apparent formations of spheroids were observed for all of 400, 600 and 800 micrometers pitches. This indicates that culture of biological samples on a surface with affinity regions constituting substantially regular hexagons or equilateral triangles is effective for formation of spheroids.

The spheroids formed on the well bottom having affinity regions of which pitch is 800 µm was the largest in size among the spheroids formed on the well bottoms having affinity regions with the three pitches.

FIG. 8 shows time-dependent changes of numbers of cells by cultivation of DLD-1 on the well bottoms having affinity regions with the three pitches. The changes of numbers of cells were observed by detecting changes of amounts of luminescence emitted from the spheroids. Remarkable growth of spheroids was observed for 800 µm pitch even after 10 days of culture.

Spheroids formed from SKBR3 on the well bottoms having affinity regions with 400-, 600- and 800-µm pitches after 14 days of culture are shown in FIG 7. Apparent formation of spheroids was observed for 400-µm pitch while apparent formations of spheroids were not observed for 600- and 800-µm pitches under this condition.

FIG. 9 shows time-dependent changes of numbers of cells by cultivation of SKBR3 on the well bottoms having affinity regions with the three pitches. The changes of numbers of cells were observed by detecting changes of amounts of luminescence emitted from the spheroids. The growth of spheroids on the well bottom having affinity regions of which pitch is 400 micrometers stagnates after 10 days of culture.

FIG. 10 shows outlines of spheroids formed from DLD-1 and SKBR3 and axes of length measurements of spheroids. The spheroid formed from DLD-1 is subglobular while the spheroid formed from SKBR3 has an irregular shape as seen from FIG. 10. Axis L indicates a line including the maximum length of the spheroid. Axis L indicates a line which passes through the midpoint (M) of the maximum length and is perpendicular to the Axis L. Axes RD and LD are lines which pass through midpoint M and intersect with Axis L at 45 and 135 degrees, respectively. The distance between points at the intersections of each of the four axes with the outlines is gauged.

FIG. 11 shows coefficients of variations (CV) of widths of spheroids formed form DLD-1 and SKBR3 measured by the foregoing method. All of the spheroids were formed on the well bottom having affinity regions of which pitch is 400 µm.

Coefficients of variation of the distances gauged using the four axes is estimated for each of 15 spheroids formed from DLD-1 on the well bottom having affinity regions constituting regular hexagons substantially.

Coefficients of variation of the distances gauged using the four axes is estimated for each of 15 spheroids formed from DLD-1 on the well bottom having affinity regions constituting squares substantially.

Coefficients of variation of the distances gauged using the four axes is estimated for each of 15 spheroids formed from SKBR3 on the well bottom having affinity regions constituting regular hexagons substantially.

Coefficients of variation of the distances gauged using the four axes is estimated for each of 15 spheroids formed from SKBR on the well bottom having affinity regions constituting squares substantially.

The coefficients of variation regarding the spheroids formed from DLD-1 on the well bottom with regular hexagonal pitch range from 1.86 to 7.89 while the coefficients of variation regarding the spheroids formed from DLD-1 on the well bottom with square pitch range from 2.71 to 17.05.

In other words, the spheroids formed for DLD-1 on the well bottom with regular hexagonal pitch have tendency to be more subglobular compared to those formed from DLD-1 on the well bottom with square pitch.

The coefficients of variation regarding the spheroids formed form SKBR3 on the well bottom with regular hexagonal pitch range from 7.80 to 24.95 while the coefficients of variation regarding the spheroids formed form SKBR3 on the well bottom with square pitch range from 4.87 to 28,81.

The spheroids formed from SKBR3 essentially have tendency to have irregular shapes. In other words, the plates relating to an aspect of the present invention enable to form spheroids from cells having irregular shapes such as SKBR3.

Especially, a plate having affinity regions or structural objects of which pitches differ from each other as shown in FIG 1 or FIG 2 is of great utility even for cells having irregular shapes because an optimum pitch of the affinity regions or structural objects for can be determined by minimum trials.

In response to cell type, an optimum plate or pitch of affinity regions or structural objects is determined. Cell type can be determined by parameters regarding size or shape explained above.

In one instance, a cell of which coefficient of variation of distances obtained based on the four axes explained above is equal to or greater than 10 can be judged to have an irregular shape.

FIG. 12 shows process from cultivation of cells and packing of cultivated cells. Feeder cells (3T3 Swiss-albino) are seeded on the bottoms of wells of the plate. The feeder cells are cultivated for one day.

Hepatocytes are seeded above the bottoms and cultivated for two days. Medium is replaced by fresh one. Spheroids and the plate are covered with a resin sheet with oxygen permeability. Spheroids accompanied with the plate are shipped to customers with keeping them at a predetermined temperature. Typically, such predetermined temperature is equal to or lower than at 10 degrees Celsius. The customers can use the spheroids after opening the package.

FIG. 13 shows packing of spheroids with a resin with oxygen permeability.
It is preferred that the resin adheres to portion including edges surrounding the wells. This enables to prevent spheroids or medium in one well from transferred to other wells.

It is preferred that the resin adheres to portion including edges surrounding the wells. This enables to prevent spheroids or medium in one well from transferred to other wells.

Claims

A plate, comprising
a member,
wherein:
the member has a first plane, a second plane and a plurality of structural objects each of which has at least one of a bottom in a recess and a top in a protrusion;
a first distance from the second plane to a boundary between each of the plurality of structural objects and the first plane is different from a second distance between the second plane and the at least one of the bottom and the top; and
at least five structural objects of the plurality of structural objects are arranged at a third distance from one structural object of the plurality of structural objects.
The plate of claim 1,
wherein the plurality of structural objects are configured such that at least a part of the plurality of structural objects can contact a biological sample.
The plate of claim 1 or 2,
wherein six structural objects of the plurality of structural objects including the at least five structural objects are arranged at the third distance from the one structural object.
The plate of any one of claims 1-3,
wherein the one structural object and two structural objects of the six structural objects constitute an equilateral triangle substantially.
The plate of claim 4,
wherein the two structural objects are arranged at positions proximate to each other.
The plate of any one of claims 1-5,
wherein the six structural objects constitute a regular hexagon substantially.
The plate of claim 6,
wherein the one structural object and two structural objects of the six structural objects are included in a regular hexagon substantially.
A plate, comprising
a member,
wherein:
the member has a first plane and a second plane;
a plurality of first portions and a second portion are formed in the first plane;
an affinity of the plurality of first portions to a sample is different from an affinity of the second portion to the sample; and
at least five first portions of the plurality of first portions are arranged at a third distance from one first portion of the plurality of first portions.
The plate of claim 8,
wherein six first portions of the plurality of first portions including the at least five structural portions are arranged at the third distance from the one first portions.
The plate of claim 8 or 9,
wherein the one first portion and two first portions of the six first portions constitute an equilateral triangle substantially.
The plate of claim 10,
wherein the two first portions are arranged at positions proximate to each other.
The plate of any one of claims 8-11,
wherein the six first portions constitute a regular hexagon substantially.
The plate of any one of claims 8-12,
wherein the one first portion and two first portions of the six first portions are included in a regular hexagon substantially.
The plate of any one of claims 8-13,
wherein the sample is any one of chemical compound and biological sample
A plate, comprising
a member,
wherein:
the member has a first plane and a second plane opposite to the first plane;
a plurality of first portions, a second portion, a plurality of third portions and a fourth portion are formed in the first plane;
a first interval between two first portions of the plurality of first portions arranged at positions proximate to each other is different from a second interval between two third positions of the plurality of third positions arranged at positions proximate to each other.
A screening method,
wherein the screening method is carried out using spheroids formed by culture of cells by the method of any one of claims 1-15.
A composite body, comprising:
a plate of any one of claims 1-15; and
cells which are positioned on bottoms of well of the plate.
The composite body of claim 17, further comprising:
a sheet which covers the wells.
The composite body of claim 17 or 18,
wherein the sheet contacts edges surrounding the wells.
The composite body of any one of claims 18-19,
wherein the sheet adheres to edges surrounding the wells.