CN106591119B

CN106591119B - Cell culture device

Info

Publication number: CN106591119B
Application number: CN201510665395.1A
Authority: CN
Inventors: O·艾格勒; S·伍德赛德
Original assignee: StemCell Technologies Inc
Current assignee: StemCell Technologies Inc
Priority date: 2015-10-15
Filing date: 2015-10-15
Publication date: 2021-10-15
Anticipated expiration: 2035-10-15
Also published as: CN106591119A

Abstract

The cell culture device comprises a well. There is a plurality of pores within the pore and a first common fluid volume within the pore above the pores. Within each microwell is a set of sub-microwells, and within each microwell above the set of sub-microwells is a second common fluid volume.

Description

Cell culture device

Technical Field

The present disclosure relates to cell culture devices. In particular, the present disclosure relates to cell culture devices, such as multi-well plates, that can be used in colony formation assays.

Background

The following is not an admission that any of the following is part of the prior art or the common general knowledge of a person skilled in the art.

Colony Forming Cell (CFC) assays of non-adherent cells are typically performed in a semi-solid or gel-like medium that prevents convective fluid flow from moving the cells, and thus limits the distance that daughter cells move from the location of the parent cell. This results in the formation of multi-cellular colonies derived from a single cell as the daughter cells continue to divide. Colony formation assays can provide quantitative information about the number of individual viable progenitor cells in a sample and allow the isolation and sampling of individual colonies for subcloning or further analysis. In the case of stem or progenitor cells, CFC assays may also allow the classification of colonies into different lineages based on morphology. Thus, CFC assays may allow for the quantification and lineage identification of progenitor cells in a sample.

Microporous devices have also been used for CFC assays. Such devices are intended to capture individual cells at defined locations to allow their manipulation and study.

Disclosure of Invention

The following summary is provided to introduce the reader to the more detailed discussion that follows. This summary is not intended to limit or define the claims.

According to one aspect, the cell culture device comprises a well. There is a plurality of pores within the pore and a first common fluid volume within the pore above the pores. There may be a set of sub-microwells within each microwell, wherein within each microwell above the set of sub-microwells is a second common fluid volume.

The well can be at least partially defined by at least one well sidewall, and a well bottom wall. Each microwell can be at least partially defined by at least one microwell sidewall extending upward from the well bottom wall. Each sub-microwell can be at least partially defined by at least one sub-microwell sidewall extending upward from the well bottom wall. Each sub-pore may be further defined by a portion of one of the pore sidewalls.

The well bottom wall can be transparent or translucent.

Each set of sub-microwells may contain 4 sub-microwells arranged in a 2x2 array. In alternative examples, the sub-wells are arranged in an array of another configuration such as 2x1, 3x1, 3x2, 3x3, or larger.

Each of the sub-microholes may comprise a sub-microhole top and a sub-microhole bottom, and each of the sub-microholes may gradually decrease in cross-sectional area from the sub-microhole top to the sub-microhole bottom. For example, each sub-microhole can be frustoconical or frustoconical.

Each microwell may comprise a microwell top and a microwell bottom, and each microwell may taper in cross-sectional area from the microwell top to the microwell bottom. For example, each microwell may be frustoconical or frustoconically shaped.

The sub-pores, micropores, and pores may be integrally formed.

The cell culture device may comprise a magnetic or magnetizable element positioned below the sub-microwells. The magnetizable elements may be wire grids. The well can be at least partially defined by a well bottom wall, and the wire grid can be embedded within the well bottom wall.

Each microwell may have a microwell top and an opposing microwell bottom, and the top of each microwell may have a microwell width of at least 100 microns. The microwell depth of each microwell between the top and the bottom can be at least 75 microns.

Each microwell may have a microwell top and a microwell bottom. Each microwell may comprise a maximum dimension at the top of the microwell and a microwell depth between the top of the microwell and the bottom of the microwell. The ratio of the maximum dimension to the depth of the micropores may be from 1.1:1 to 1.9: 1.

According to another aspect, the cell culture device comprises a well. A plurality of micropores are formed in the hole. Each comprising a microwell top and a microwell bottom. Each microwell comprises a maximum dimension at a top of the microwell, and a microwell depth between the top of the microwell and a bottom of the microwell. The ratio of the maximum dimension to the depth of the micropores is from about 1.1:1 to 1.9: 1.

The maximum dimension may be at least 140 microns. The microwell depth may be at least 75 microns.

Each of the micropores may taper in cross-sectional area from the top of the micropore to the bottom of the micropore. For example, each microwell may be frustoconical or frustoconically shaped.

The well can be at least partially defined by at least one well sidewall, and a well bottom wall. The well bottom wall can be transparent or translucent. Each microwell may be at least partially defined by at least one microwell sidewall extending upward from a well bottom wall.

The cell culture device may further comprise a magnetic or magnetizable grid positioned below the microwells. The well may be at least partially defined by a well bottom wall, and the grid may be embedded within the well bottom wall.

The micro-holes and the pores may be integrally formed.

The cell culture apparatus may further comprise a first common fluid volume above the microwells within the wells.

The cell culture apparatus may further comprise a set of sub-microwells within each microwell. Each set of sub-microwells may contain 4 sub-microwells arranged in a 2x2 array.

The well can be at least partially defined by at least one well sidewall, and a well bottom wall. Each microwell may be at least partially defined by at least one microwell sidewall extending upward from a well bottom wall. Each of the sub-microwells may be at least partially defined by at least one sub-microwell sidewall extending upward from a well bottom wall. Each of the sub-microwells may be further defined by a portion of one of the microwell sidewalls.

The sub-micro-holes may be formed integrally with the micro-holes and the pores.

The cell culture device can further comprise a second common fluid volume within each microwell above the set of sub-microwells.

In one embodiment, a cell culture device is provided comprising:

a well defined by at least one well sidewall, and a well bottom wall;

a plurality of microwells within the well, each microwell defined by at least one microwell sidewall extending upward from the well bottom wall and separating a microwell fluid volume from a microwell fluid volume of an adjacent microwell, and a first common fluid volume within the well above the microwell, each microwell comprising a microwell top and a microwell bottom, and wherein the microwell sidewalls between adjacent microwells meet to form a vertex; and

a set of sub-microwells within each microwell for containing one or more cells within each microwell above the set of sub-microwells and a second common fluid volume within each microwell above the set of sub-microwells, each sub-microwell comprising a sub-microwell top, a sub-microwell bottom, and at least one sub-microwell sidewall extending upward from the well bottom wall separating a sub-microwell fluid volume from a sub-microwell fluid volume of an adjacent sub-microwell.

In one embodiment, each sub-microwell is further defined by a portion of one of the microwell sidewalls. In one embodiment, the well bottom wall is transparent or translucent.

In one embodiment, each set of sub-microwells comprises 4 sub-microwells arranged in a 2x2 array. In one embodiment, each set of sub-microwells comprises sub-microwells arranged in an array of 2x1, 3x1, 3x2, 3x3, or larger.

In one embodiment, each sub-pore gradually decreases in cross-sectional area from the top of the sub-pore to the bottom of the sub-pore, optionally wherein each sub-pore is frustoconical or frustoconically shaped. In one embodiment, each microwell tapers in cross-sectional area from the top of the microwell to the bottom of the microwell, optionally wherein each microwell is frustoconical or frustoconically shaped.

In one embodiment, one or more of the sub-wells, micro-wells, and wells are integrally formed.

In one embodiment, the cell culture device comprises a magnetic or magnetizable element positioned below the sub-microwells. Optionally, the magnetizable element is a wire grid embedded in the wall of the hole bottom.

In one embodiment, the top of the microwells have a width of at least 100 microns. In one embodiment, the width of the top of the microwells is at least 250 microns, at least 400 microns, at least 500 microns, or about 50 microns to 1000 microns. In one embodiment, each microwell has a depth between the top and the bottom of at least 25 microns. In one embodiment, each microwell has a depth between the top and the bottom of at least 75 microns, at least 100 microns, at least 200 microns, at least 300 microns, or about 25 microns to 900 microns.

Optionally, and/or additionally, in one embodiment, each microwell comprises a maximum dimension at the top of the microwell and a microwell depth between the top of the microwell and the bottom of the microwell, and the ratio of the maximum dimension to the microwell depth is between 1.1:1 and 1.9: 1. In one embodiment, the largest dimension is at least 140 microns. In one embodiment, the microwell depth is at least 75 microns.

In one embodiment, the angle of the microwell sidewalls with respect to the vertical is less than 30 degrees. In one embodiment, the angle of the microporous sidewall with respect to the vertical direction is less than 20 degrees.

Any feature or combination of features described herein is included so long as the features included in any such combination are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary skill in the art.

Drawings

In the detailed description, reference is made to the accompanying drawings in which:

FIG. 1A is an image of colonies typically observed in a hematopoietic colony assay, showing many individuals with colonies with large degrees of overlap;

FIG. 1B is an image of colonies typically observed in a hematopoietic colony assay, showing one colony with multiple centers derived from a single ancestor;

FIG. 2 is a perspective view of an exemplary cell culture device of the present disclosure;

FIG. 3 is a top view of the cell culture apparatus of FIG. 2;

FIG. 4 is an enlarged view of the area shown in box 4 of FIG. 3;

FIG. 5 is a cross-section taken along line 5-5 of FIG. 4;

FIG. 6 is a perspective cut-away view of the area shown in box 4 in FIG. 3;

FIG. 7 is an enlarged view of the area indicated in circle 7 in FIG. 5, showing the magnetizable grid embedded in the bottom of the hole;

FIG. 8 shows an image of a plurality of microwell configurations taken by a bright field microscope;

FIGS. 9A to 9C are images showing immobilization of fluorescent beads in microwells;

FIGS. 10A to 10E are images showing examples of colony formation in different microwell configurations;

FIG. 11A is an image taken by a bright field microscope of a cell culture device comprising microwells and sub-microwells within microwells;

FIG. 11B is an image taken using a color CCD scanner of a cell culture device comprising microwells and sub-microwells within microwells;

FIGS. 12A to 12C show a CCD line sensor (with

An Epson V500 scanner with a 12-line color sensor) images of stained colonies in a cell culture device comprising microwells;

FIG. 13 is a graph showing the correlation between the count of stem cell progenitors in a cell culture device comprising microwells and a standard CFC assay;

FIG. 14 is a graph showing the increased linear range of a CFC assay based on microwells when using a Poisson distribution to correct a plurality of colony forming cells seeded per microwell;

FIG. 15A is an image taken by a bright field microscope of microwells containing magnetic microcarrier beads;

FIG. 15B is an image taken by a brightfield microscope of the microwell of FIG. 15A after 7 days of incubation;

figure 16 is a graph showing that magnetic assisted sedimentation does not affect colony formation in microwell-based CFC assays.

Detailed Description

A number of apparatuses or methods will be described below to provide examples of embodiments of each claimed invention. The embodiments described below do not limit any claimed invention, and any claimed invention may encompass methods or apparatus not described below. The claimed invention is not limited to devices or methods having all of the features of any one device or method described below or features common to multiple devices or all of the devices described below. It is possible that the apparatus or method described below is not any exclusive embodiment granted by the assignee of the present patent application. Any invention disclosed in the apparatus or method described below and any invention not entitled to antedate such patent by grant of this patent application may be the subject of another protective document such as a continuation of the patent application and the applicant, inventor or owner does not intend to disclaim, exclude, or contribute to the public any such invention by the disclosure in this document thereof.

Semi-solid media may present some limitations for CFC assays for quantification or subcloning of individual cells. Handling of the treatment vessel may interfere with the colonies as the cells are not firmly immobilized in the medium. For example, perturbations such as frequently moving the culture dish or adding liquid reagents to the culture may interfere with the colonies. This may limit the types and number of manipulations that can be performed on these cultures.

In addition, because there is no physical boundary within the culture, some colonies may overlap, making it difficult to determine whether a contiguous population of cells is a colony derived from a single progenitor cell, or represents a single colony with multiple centers that are produced by daughter cells that have migrated a short distance from the original progenitor cell. This may result in an erroneous count of the total number of colonies.

Furthermore, the morphological characteristics of the colonies of different mature cell lineages are often not sufficiently unique, so that the classification of these colonies can be a subjective process that tends to be highly variable. In the case of CFC assays on hematopoietic progenitor cells, for example, it can be difficult to distinguish colonies derived from granulocytic progenitors, monocytic progenitors and megakaryocytic progenitors, only allowing reliable differentiation of the major lineage types (erythroid and myeloid). In order to reliably classify colonies according to the type of progenitor cell from which they are derived, specific labeling and staining methods may be required. Such methods often rely on the introduction of probe molecules that recognize specific cell surface markers or intracellular components. Staining methods typically involve the fixation of cells and a series of subsequent washing, staining and destaining procedures. These methods are incompatible with colony assays for non-adherent cells in semi-solid media, as the addition of fixing, staining and washing solutions can interfere with the colonies.

Another limitation of standard CFC assays in semi-solid media is that multiple colonies appear near another colony or multiple colonies with overlapping boundaries, as shown in figure 1A. Such colonies may be difficult to distinguish from multicenter colonies derived from single cells, as shown in fig. 1B. This not only results in a highly subjective analysis of colony counts, but also complicates the extraction of individual colonies from the culture for further expansion or subcloning due to the presence of foreign cells from neighboring colonies.

Although some of the above mentioned disadvantages are overcome using known microwell devices, in known microwell devices, colonies often spread out of the volume of the microwells. This results in spreading into adjacent microwells and/or washing away of cells in the colonies during conventional processing. Furthermore, in known microwell devices it is common for more than one progenitor cell to be seeded into each microwell, thereby resulting in the growth of more than one colony in each microwell. This may lead to errors in counting colonies.

The present disclosure relates to cell culture devices comprising microwells. The cell culture apparatus may be used in a colony formation assay and may overcome some or all of the disadvantages mentioned above. In particular, as described in further detail below, cell culture devices as described herein can better compartmentalize individual colonies, can better contain the volumes that house the colonies as they grow so that they do not exceed the microwells, and can result in the secure capture of multicellular colonies in a multi-day culture to prevent or reduce their chance of spreading or diffusing between microwells during operations such as adding or removing solution. This may allow the cell culture apparatus to be used for simple quantitative colony formation assays lasting about 4-20 days. Furthermore, this can separate colonies from their nearest neighbors and reduce the occurrence of cell overlap, enabling objective counting of colonies and extraction of individual colonies without contamination of unrelated cells.

In addition, a cell culture device as described herein can effectively immobilize cells, thereby eliminating the need to immobilize the sample prior to staining. This may enable sensitive live cell staining methods (e.g., staining of colonies for reliable classification of colony types) that do not alter the metabolic and physical characteristics of the cells. In conjunction with specific methods of cell staining, the colonies in the microwells can be classified into subtypes (e.g., for hematopoietic colonies: erythroid, myeloid, granulocytic, megakaryocytic, monocytic, etc.). Furthermore, the cell culture apparatus described herein allows for imaging of cells.

Referring to fig. 2 and 3, an exemplary cell culture apparatus 100 is shown. Cell culture device 100 comprises at least one well 102. As used herein, the term "well" generally refers to any fluid reservoir in which cells in a liquid culture medium may be placed for the culture of cells. In the example shown, cell culture apparatus 100 is in the form of a multi-well plate and contains 6 wells 102. In alternative examples, a cell culture device in the form of a multi-well plate may comprise an alternative number of wells, such as 24 wells or 96 wells. The wells of a multi-well plate are typically circular in cross-section rather than rectangular as shown in fig. 2. However, any container having a generally flat bottom is acceptable. In a further alternative, the cell culture apparatus may be in another suitable form, such as a cell culture dish (as used in the examples section below). In such an example, the cell culture device may comprise only one well (i.e., a single fluid reservoir of the cell culture dish).

Referring to fig. 2 and 5, each well 102 includes a well top 104 and a well bottom 106. Further, each well 102 is defined by at least one well sidewall 108, and a well bottom wall 110.

The apertures may have any suitable shape. In the example shown, the well 102 is generally square and is defined by 4 well sidewalls 108, and a well bottom wall 110. Further, the holes may have any suitable size. For example, the well may have a volume of about 30 μ L to about 10L. For example, the lower limit of the range for one well of a 384-well plate is about 30 μ L, and the upper limit of the range for a typical 6-well plate is about 100 mL. For Qtray, the volume is about 1.1L, and for plates filled with the footprint (footprint) of a standard incubator rack, the volume can be as high as 10L. Thus, in one embodiment, the well has a volume of about 30 μ L to about 10L. In another embodiment, the well has a volume of about 30 μ L to 100 mL. In yet another embodiment, the well has a volume of about 30 μ L to about 6 mL.

Referring now to fig. 4-6, within each well 102 are a plurality of micropores 112, and a first common fluid volume 114 (shown in fig. 5) above the micropores 112. Specifically, the micropores 112 are at the well bottom 106 of each well and are spaced apart from the well top 104. The space at the well top 104 of each well 102 forms a first common fluid volume 114 that is in communication with each micro-well 112.

Microwell 112 provides a volume into which individual progenitor cells can be seeded and grown into colonies. That is, a liquid medium containing progenitor cells can be placed in each well 102. The cell culture apparatus 100 can then be centrifuged, forcing the progenitor cells to the well bottom 106 of each well 102 and into the microwells 112, such that the individual cells are isolated into the microwells 112 and can grow to form colonies. Alternatively, the cells may be allowed to settle into microwells 112 under the influence of gravity. The first common fluid volume 114 allows each microwell 112 within a given well 102 to share a common medium such that cells within the microwells 112 are cultured under substantially identical conditions.

Referring to fig. 5 and 6, each microwell 112 comprises a microwell top 116 and a microwell bottom 118. Each microwell 112 is defined by at least one microwell sidewall 120 extending between a microwell top 116 and a microwell bottom 118, and a microwell floor 122 at the microwell bottom.

Referring to fig. 5, in the example shown, the microwell floor 122 of each microwell 112 within a given well 102 is formed by the well floor wall 110 of the given well 102. Further, the microwell sidewalls 120 extend integrally upward from the well bottom wall 110, and the microwell sidewalls 120 of the microwells 112 adjacent to the well sidewalls 108 are integrally formed with the well sidewalls 108.

In an alternative example, the well bottom wall and the well side wall may be formed separately from the well bottom surface and the well side wall. For example, the microwells may be formed as inserts that are placed on and optionally secured to the bottom wall of the wells (as described in the examples section below).

The micropores may have any suitable shape. Referring to fig. 4, in the example shown, each microwell 112 is generally square at a microwell top 116 and is defined by 4 microwell sidewalls 120. Further, referring to fig. 5 and 6, each microwell 112 tapers in cross-sectional area from a microwell top 116 to a microwell bottom 118. More specifically, in the example shown, each micro-hole 112 is generally frustoconical. This shape generally facilitates seeding of progenitor cells within microwells 112, rather than between adjacent microwells 112. More specifically, in the example shown, because the microwells 112 are generally frustoconical, the microwell sidewalls 120 of adjacent microwells 112 meet at an apex 124 such that cells generally cannot be seeded between the microwells 112.

In examples where the micropores 112 taper in cross-section from the micropore top 116 to the micropore bottom 118, the angle of the micropore sidewalls 120 relative to the vertical (also referred to herein as the "wall angle") may be any suitable angle. In some examples, the angle may be less than about 30 degrees, such as 10 to 20 degrees. In other examples, the angle may be as small as 2 degrees. As shown in the examples section below, as the wall angle decreases, the volume of the microwells increases, resulting in an increase in the number of accommodated cell colonies.

In the example shown, the microwell sidewalls 120 extend at the same angle from the microwell top 116 to the microwell bottom 118. In an alternative example (not shown), the microwell sidewalls may comprise a first portion extending downward from the top of the microwell at a first angle and a second portion extending downward from the first portion at a second angle. The second angle may be less than the first angle (e.g., the second angle may be 0 degrees). This may allow the microwell sidewalls of adjacent microwells to meet at the apex, while still allowing the microwell floor to be relatively large and the volume of the microwell to be relatively large.

In an alternative example (not shown), each microwell may be generally circular at the top of the microwell, and may be generally frustoconical. In yet another alternative example, each microwell may be another suitable shape, such as triangular, rectangular, trapezoidal, or hexagonal, at the top of the microwell.

In the example shown, the micro-pores 112 within a given pore 102 are generally of the same shape and size. In alternative examples (not shown), the micropores within a given pore may have different shapes and sizes.

In the example shown, each micropore 112 has a generally central axis of symmetry. In the example shown (not shown), one or more of the microwells may not have a central axis of symmetry.

Generally, the colonies formed in the microwells can have an average size of about 10 to 100,000 cells; however, some colonies can grow to have over 100 million cells. It is expected that a cell colony of 100 ten thousand cells will have a volume of about 1.0. mu.L. Since the microwells in known microwell devices are not generally intended for cell culture, for the most part they are not sized to accommodate occasional large colonies. However, in one known microwell device, the volume of the microwells is about 0.1 μ L or greater (Ungrin WO 2008/106,771), which can accommodate these large colonies. As mentioned above, in these microporous devices, cells tend to still spread out of the volume of the micropores. Surprisingly, it has now been determined that by tailoring the dimensions of a microwell such that the ratio of its maximum dimension at the top to its depth (hereinafter also referred to as the "maximum dimension to depth ratio") is less than 1.9:1, and more particularly, 1.9:1 to 1.1:1, the spreading of cells out of the microwell can be reduced and immobilization of larger colonies can be achieved.

For example, referring to fig. 6, each microwell 112 is generally square at microwell top 116, and has a microwell width 101 and a microwell length 103 at top 116 and a microwell depth 105 between microwell top 116 and microwell bottom 118. Since micro-apertures 112 are generally square at top 116, the largest dimension across the top is diagonal 107. Thus, if the micropore depth 105 is about 1mm, the length of the line 107 will be less than 1.9mm in order to have a maximum dimension to depth ratio of less than 1.9: 1. For example, the microwell width 101 may be 1mm and the microwell length 103 may be 1mm, such that the length of the line 113 is about 1.4 mm.

In an alternative example, the microwell width, microwell length, and microwell depth may be another dimension. For example, the microwell width and microwell length can be generally 100 microns or greater, and more specifically 500 microns or greater, and the microwell depth can be generally 75 microns or greater. In an example where the microwells are square at the top and the width and length of the microwells at the top is 500 microns, the maximum dimension at the top would be about 707 microns. In such an example, to have a maximum dimension to depth ratio of less than 1.9:1, the microwell depth would be greater than about 372 microns.

In an alternative example (not shown) where the micro-holes have different shapes, the maximum dimension across the top may be another dimension. For example, if the microwells are circular at the top, the largest dimension would be the diameter at the top.

The density and total number of micropores within each pore may vary depending on the size and shape of the micropores and the size and shape of the pores. In some examples, the density of micropores within each pore may be 0.5-4.0 micropores per square millimeter. In a specific example, each well may contain about 960 micropores.

Referring still to fig. 4-6, in the example shown, there is a set of sub-micropores 126 within each micropore 112 and a second common fluid volume 128 within each micropore 112 above the set of sub-micropores 126.

As noted above, in known microwell devices, it is common for more than one progenitor cell to be seeded into each microwell, thereby resulting in the growth of more than one colony in each microwell. By providing a set of sub-microwells 126 in each microwell 112, progenitor cells will generally be separated into adjacent sub-microwells 126 and grow into individual colonies even if more than one progenitor cell is seeded into each microwell 112.

Furthermore, as will be described in more detail below, the sub-microwells 126 can be sized to accommodate an average colony (e.g., a colony of up to 100,000 cells) rather than a large colony. Thus, the sub-microwells 126 will be of sufficient size to accommodate the majority of cell colonies that grow; however, if large colonies do grow in the sub-micropores 126, they may grow into the second common fluid volume 128 and will be contained within the micropores 112 that accommodate the sub-micropores 126.

In addition, by providing a sub-microwell 126 within each well 112, cells from the microcolony can be concentrated at the microwell floor 122. This may enhance the ability to detect microcolonies by bright field microscopy. This may allow for a faster colony assay by detecting colonies at an earlier time point or may allow for the detection of progenitor cells with lower proliferative potential, for example.

Referring to fig. 5 and 6, each sub-well 126 comprises a sub-well top 130 and a sub-well bottom 132. In addition, each sub-microvia 126 is defined by at least one sub-microvia sidewall 134 extending between a sub-microvia top 130 and a sub-microvia bottom 132, and a sub-microvia bottom 136 at the sub-microvia bottom 132.

The micropores 126 may have any suitable shape. In the example shown, each sub-microwell 126 is generally square at a sub-microwell top 130 and is defined by 4 sub-microwell sidewalls 134.

Referring to fig. 5, in the example shown, the sub-nanopore floor 136 of each sub-nanopore 126 within a given pore 102 is formed by the pore floor wall 110 of the given pore 102. Further, of the 4 sub-well sidewalls 134 of each sub-well 126, two are formed by a portion of the well sidewall 120 and the other two integrally extend upward from the well bottom wall 110.

In an alternative example (not shown), any of the microwell bottom wall, the microwell sidewalls, the sub-microwell bottom surface, and the sub-microwell sidewalls may be formed from a separate material. For example, a given set of sub-microwells may be formed as an insert that is positioned on the floor of a microwell.

Referring also to fig. 5 and 6, each sub-pore 126 tapers in cross-section from a sub-pore top 130 to a sub-pore bottom 132. More specifically, in the example shown, each sub-pore 126 is generally frustoconical. Similar to microwells 112, this configuration facilitates progenitor cells to be seeded within a sub-microwell 126, rather than between adjacent sub-microwells 126. More specifically, in the example shown, where the sub-microholes 126 are generally frustoconical, the sub-microhole sidewalls 134 of adjacent sub-microholes 126 meet at a vertex 138 such that cells are not generally seeded between the sub-microholes 126.

In an alternative example (not shown), each sub-microwell may be generally circular at the top of the sub-microwell, and may be generally frustoconical. In yet another alternative example, each sub-microwell may be another suitable shape, such as triangular, rectangular, trapezoidal, or hexagonal, at the top of the sub-microwell.

In examples where the submicron pores are tapered in cross-section from the top of the submicron pores to the bottom of the submicron pores, the angle of the submicron pore sidewalls relative to the vertical direction may be any suitable angle. In some examples, the angle may be less than about 30 degrees, such as 10-20 degrees. In other embodiments, the angle may be as low as 2 degrees.

As described above, the sub-microwells 126 can be sized to accommodate an average colony. For example, the sub-microholes can have a size of about 3x 10^-6μ L to a volume of about 1.0 μ L. For example, referring to FIG. 5, the sub-microholes 126 may have a width 109 at the top of about 30 μm to 1mm and a depth 113 between the top and bottom of about 30 μm to 1 mm. In addition, the sub-microporous sidewall 134 may extend at an angle of about 1 to 37 degrees from vertical.

Each microwell 112 may contain any suitable number and arrangement of sub-microwells 126. In the example shown, each microwell 112 contains 4 sub-microwells 126, the 4 sub-microwells 126 arranged in a 2x2 array. In alternative examples, the sub-microholes may be arranged in other configurations such as in an array of 2x1, 3x1, 3x2, 3x3, or larger.

Referring also to fig. 5 and 6, the well bottom wall 110 also forms a micro-well bottom surface 122 and a sub-well bottom surface 136, which may be translucent or transparent. This may allow observation of the cell colonies within the cell culture device 100, for example by microscopy or other visual imaging methods. In the example shown, the sub-microporous bottom surfaces 136 are generally flat, and each sub-microporous bottom surface 136 is generally coplanar. This can assist in observing the cell colonies under a microscope. However, in an alternative example (not shown), the bottom surface of the sub-micro-holes may have another shape, for example circular. In another alternative (not shown), the sub-micropores may not include a sub-micropore floor. For example, the sub-nanopore sidewalls may meet at an apex.

In some examples (not shown), the bottom surface of a microwell and/or the bottom surface of a sub-microwell may contain a demarcation line as a marker to determine the location of a microwell or a sub-microwell within a cell culture device.

In other examples (not shown), the interior surface of the cell culture device can be coated with a hydrophobic coating. The hydrophobic coating can minimize or reduce meniscus formation when the liquid is placed in the cell culture device, which can facilitate uniform distribution of a sample placed in the cell culture device.

In other examples (not shown), the interior surface of the cell culture device can be treated to promote wetting so that the micro-and sub-micro-wells are more easily filled with liquid.

As described above, in some examples, the cells may be centrifuged or passed throughGravity seeding into microwells 112 and sub-microwells 126. In an alternative example, magnetic forces may be used to seed cells into microwells 112 and sub-microwells 126. For example, in use, cells may be treated with magnetic particles such as

Particles or other magnetic particle labels. The particular cell type of interest can be coupled to the magnetic particles using a mixture of antibodies specific for cell surface markers on the target cells and an active moiety (such as dextran) on the carrier particles. Using this mixture, antibody complexes are formed that cross-link the target cells with the magnetic particles to form a mixture of suspended magnetic particles, magnetic particles and target cell complexes, and unbound non-target cells. The cell suspension may then be deposited in the well 102 and may be subjected to a magnetic field gradient in the direction of the well bottom wall 110. The particles will move in the direction of the gradient and collect at the bottom 132 of the sub-microwells. Unwanted cells that remain in suspension can be washed out of well 102, leaving only the target cells to form colonies during subsequent incubations.

Referring now to FIG. 7, in some examples, a cell culture device can include a magnetic or magnetizable element located below the sub-microwells to increase the magnetic field gradient and increase the speed at which particles collect at the bottom of the sub-microwells. In the example shown, the magnetizable elements comprise magnetizable wire grids 140, which are embedded in the bottom wall 110 of the hole. A magnetic field gradient may magnetize the wire grid 140 and increase the magnetic field gradient.

In an alternative example (not shown), the wire grid may be configured to attract the magnetically labeled elements to specific locations within each well.

Examples

EXAMPLE 1 production of cell culture device

The cell culture apparatus as described above is prepared by making the microwells as inserts and inserting them into culture dishes. Some micropores contain sub-micropores and some do not. The micro-holes are made with a variety of maximum dimension to depth ratios, as described above.

Several female molds of micro-porous construction were produced by CNC machining of solid aluminum discs. These circular dies are 35mm in diameter and 20mm thick and exhibit surfaces of opposite topology with micropores. The microporous insert is produced by casting a Polydimethylsiloxane (PDMS) based elastomer in a mold, followed by curing the elastomer to form a flexible disc containing micropores. Specifically, the elastomer was prepared from a 10: 1(w/w) homogeneous mixture of SylGard 184 (Dow Corning) elastomer and curing agent. This mixture of silicone components was exposed to vacuum (< 10mTorr, 1 hour) to remove any volatile components prior to casting into an aluminum mold. 1.5-1.7 g of elastomer was slowly poured onto the mold surface and allowed to spread to form a layer of uniform thickness on the mold. The base of the mold was then placed on a hot plate heated to 180 ℃. Due to the minimum thickness of the mold and the high conductivity of the aluminum material, rapid heat transfer to the mold surface results in rapid curing of the silicone elastomer. After a period of heating of 5min, the mold was removed from the hot plate and cooled to ambient temperature by briefly placing on an aluminum plate cooled to 0 ℃. The hardened PDMS gel was demolded by gently pulling one edge of the cast product to remove the tray containing the micro-pores.

The microporous inserts were sterilized by dry heat (135 ℃ for 1 hour) prior to insertion into 35mm petri dishes (Becton-Dickinson, 35-1008). A drop of 125 μ L of the above mixture of SylGard elastomer and curative was placed in the center of the dish and the microwell insert was then adhered to the dish by inserting the microwell insert aseptically into the dish with the array surface facing up. The inserts were sealed into the wells by incubating the petri dishes with the array inserts in an oven at 80-85 deg.C (2-4 hours) to heat cure the adhesive layer of SylGard.

An example of some microwell arrays produced in this manner is shown in fig. 8, which is a top view image of the microwells of an insert taken by a bright field microscope. The dimensions of the micropores are shown in the following table, with the maximum dimension to depth ratio being shown as the ratio of the maximum horizontal dimension to the depth:

structure of the device	Wall angle (degree)	Micropore width (mm)	Depth of micro-hole (mm)	Maximum dimension to depth ratio
					A	37	0.8	0.38	3.0∶1
B	20	0.8	0.5	2.3∶1
					C	15	0.8	0.5	2.3∶1
D	15	1.0	1.0	1.4∶1

Example 2 Effect of pore architecture on immobilization of particles

The ability of cell culture devices with different sized microwells to compartmentalize particles was evaluated. The cell culture devices tested in this example did not contain sub-microwells. The evaluation was done using fluorescent polystyrene microparticles (Bangs FS06F, 7.3 μm diameter). A suspension of microparticles is placed in several individual microwells of several cell culture devices. All microwells are square on top of the microwells. The micropores have the following dimensions:

structure of the device	Wall angle (degree)	Micropore width (mm)	Depth of micro-hole (mm)	Maximum dimension to depth ratio
					A	15	1.0	1.0	1.4∶1
B	15	0.8	0.5	2.3∶1
					C	37	0.8	0.38	3.0∶1

Allowing the particles to settle by gravity into the pores; the remaining wells were left empty and contained Phosphate Buffered Saline (PBS) only. The cell culture apparatus is then subjected to physical perturbation methods representative of the procedures typically used for cell culture applications. Specifically, the washing step was performed by removing the above PBS and replacing with fresh PBS in a volume of 2.0 mL. The cell culture apparatus is then subjected to rapid lateral (side-to-side) movement. Whether or not the particles appear to spread from well to well in the microwell around the particle-containing well was observed and imaged by a fluorescence microscope (Leica DMIL inverted microscope, 4x objective). An image of the pores around the particle-containing pores is shown in fig. 9.

In fig. 9, the first image in each column is a bright field view of the microwell after placing a fluorescent bead near the center of the cell culture device. The next image in each column shows the fluorescence observed in the microwells prior to any manipulation of the cell culture apparatus. The last image in each column shows the fluorescence observed in the microwells after washing and physically moving the cell culture device.

No particle transfer was seen in the images obtained using the fluorescence microscope after the operation. This shows that microwells with a maximum dimension to depth ratio of 1.4: 1 to 3.0: 1 effectively limit the movement of small particles during conventional cell culture operations.

Example 3 Effect of well configuration on cell-containing colonies

The artificial blood progenitor cells in liquid medium are seeded into cell culture dishes containing microwells with various configurations. The cell culture devices tested in this example did not contain sub-microwells. All microwells are square on top of the microwells. The dimensions of the micropores are summarized in the table below:

structure of the device	Wall angle (degree)	Micropore width (mm)	Depth of micro-hole (mm)	Maximum dimension to depth ratio
					A	37	0.8	0.38	3.0∶1
B	30	1	0.75	1.9∶1
					C	20	1	0.75	1.9∶1
D	15	1	0.75	1.9∶1
					E	15	1	1	1.4∶1

At about 7 colonies/cm²The cell culture apparatus was seeded at colony density and cells were sedimented into the microwells by slow centrifugation. The seeded cell culture device was then incubated at 37 ℃ in a permissive culture environment and containing 5% CO₂Is incubated in a humidified atmosphere. Colony formation was monitored at two-day intervals and any evidence of microwell-to-microwell spread of cells was recorded.

Figure 10 shows the characteristics of colony formation in 5 microwell configurations.

With respect to configuration A, early in the culture, it can be seen that colonies remain confined to the bottom of the microwells. After the culture continued to grow for a total of 14 days, the colonies had grown beyond the microwells and cells appeared in most of the wells surrounding the wells containing the colonies. It is clear that the microwells with this configuration were insufficient to accommodate colonies after 14 days of culture. Therefore, it is not possible to count the number of progenitor cells at the time of seeding using this configuration.

Still referring to fig. 10, with respect to constructs B-E, in all cases, cells were observed to be confined to individual wells at time points early in the culture, when colonies were still small. At later time points of culture, a decrease in the micropore-to-micropore spread of cells was seen with increasing micropore depth or decreasing wall angle. The configuration with the largest pore volume (configuration E) showed that the colonies were completely confined to individual wells and no microwell-to-microwell spread occurred after 14 days of culture.

Additional experiments were performed using microwell configuration B-E, in which hematopoietic progenitor cells from human cord blood in liquid medium were collected at 21-26 colonies/cm²Is seeded into pre-wetted microwells and allowed to settle by gravity into the microwells. Evaluation ofColony formation number and colony acceptance of microwells 7 or 14 days after inoculation. For both 7 and 14 day cultures, as shown in the table below, the number of colonies observed increased as the wall angle increased.

The exception to this trend in the 7 day CFC assay was configuration B. In this assay, there is a high cellular background in all microwells, which makes it difficult to accurately count microcolonies. This cellular background is most likely due to the high rate of overflow and spreading to adjacent microwells due to the shallow wall angle. These data show that for micropores having a maximum dimension to depth ratio in the range of 1.9:1 to 1.1:1, wall angles of less than 30 degrees, more specifically less than 20 degrees, may also be desirable.

Example 4 evaluation of cell culture devices comprising sub-microwells

The artificial blood progenitor cells in the liquid medium are seeded into a cell culture dish containing a set of sub-wells within each well. The microwells within the cell culture device are square at the top of the microwells and have a microwell width of 1.0mm, a microwell depth of 1.0mm 105, and a wall angle of 15 degrees. The sub-microwells are square at the top of the sub-microwells, with a microwell width of 0.37mm, a microwell depth of 0.5mm and a wall angle of 15 degrees.

At about 7 colonies/cm²The cell culture apparatus was seeded at colony density and cells were sedimented into sub-microwells by slow centrifugation. The seeded cell culture device was then incubated at 37 ℃ in a permissive culture environment and containing 5% CO₂Is incubated in a humidified atmosphere. After 7 days of incubation the cultures were observed for colony formation by bright field microscopy and imaged using a CCD digital camera (fig. 11A). The wells indicated by the arrows contain colonies derived from hematopoietic stem cell progenitors after incubation at 37 ℃ for 7 days. Subsequent use of the live cell marker MTTCells were stained (3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide) to visualize colonies by macroscopic analysis using a CCD color scanner (fig. 11B). Briefly, the staining method involves incubating the culture in a medium containing MTT until sufficient color formation is observed. The stained cells are then washed by removing the culture medium (by pipetting) and adding and removing the appropriate wash buffer to the submicron wells. This washing step was repeated until no background coloration was evident.

Colonies were found to be firmly immobilized within the submicron wells and there were no free floating cells before or after the staining and washing steps. Dense and discrete colonies that can be easily counted microscopically are observed and comparable colony counts are obtained using both microscopic and macroscopic methods.

FIG. 11A shows colonies growing in the sub-microwells and FIG. 11B shows the staining results. At least 6 positive sub-wells and a cluster of 4 sub-wells appear in this image, the 4 sub-wells being clustered together in the upper right corner of the image. Based on the total colony frequency in this image, this set of 4 wells was most likely generated by a single ancestor with a high proliferative potential and grown beyond sub-microwells. This example clearly shows the advantage of using sub-micropores.

Example 5 staining of colonies

Three staining methods commonly used for staining of biological and cell culture samples were evaluated in cell culture dishes containing microwells with various configurations. The cell culture devices tested in this example did not contain sub-microwells. The method comprises the following steps: (a) labeling cells with an antibody against a cell surface marker coupled to the enzyme Alkaline Phosphatase (AP), followed by addition of a naphthol phosphate substrate and Fast-Red chromogen resulting in the production of a Red precipitate, (b) staining of live cells using MTT (3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide) as a metabolic substrate, MTT being converted by the live cells into a visible dye, and (c) non-specific staining using the histological lining dye Evans Blue (Evans Blue). For each staining method, the cell culture apparatus containing the colonies was washed by removing the medium (by pipetting) and adding a suitable wash buffer to the microwells, followed by removal of the wash buffer after a short incubation period. This washing step was repeated as required for each staining protocol. The results of the staining are shown in fig. 12.

Figure 12A is an example of colonies stained with alkaline phosphatase-conjugated antibodies. These colonies were contained in a cell culture device with microwells that were square at the top of the microwells, with microwell width of 0.8mm, wall angle of 15 degrees, and microwell depth of 0.5 mm. Colonies were visibly stained red with minimal background staining. In addition, no interference with colonies was observed when subjected to the staining procedure. FIGS. 12B and 12C show colonies contained within a cell culture device having microwells that are square at the top of the microwells, with microwells having 1.0mm openings in width, wall angles of 15 degrees, and microwell depths of 1.0 mm. Colonies were stained with evans and MTT, respectively. As with antibody labeling, no interfering colonies were observed and the colonies were stained at a high level of contrast to background.

Example 6 quantification of colony-forming progenitor cells

Colony forming cultures of hematopoietic progenitor cells are performed in a liquid culture in a cell culture apparatus, while standard colony forming cell assays are performed on the same cell sample in semi-solid medium. The cell culture devices tested in this example did not contain sub-microwells. A cell suspension in a liquid medium is seeded into a cell culture device having microwells that are square at the top of the microwells, with a microwell width of 1.0mm, a microwell depth of 1.0mm, and a wall angle of 15 degrees. Cells were sedimented into the microwells by slow centrifugation. Will be in a semi-solid medium (Methocult)^TMStemcell Technologies) were plated onto standard cell culture dishes. In either case, the dishes were inoculated with approximately 5-15 colonies/cm²Followed by a permissive culture environment at 37 ℃ and containing 5% CO₂Is incubated in a humidified atmosphere. Colony formation was assessed after 14 days of culture and the total number of colonies was compared.

FIG. 13 is a graph showing the correlation between the number of colonies observed in the cell culture apparatus comprising microwells and standard cell culture dishes (normalized to the number of colonies per 1000 cells seeded into the dish). The significant correlation with a slope of about 1 demonstrates the good agreement of the colony assay in the cell culture apparatus comprising microwells with the current standard CFC assay. The cell culture apparatus described in the present application thus provides a suitable method for quantification of colony forming progenitor cells.

Example 7 Linear Range of CFC assay

The cell culture apparatus containing the microwells was pre-wetted with liquid medium by slow centrifugation. The cell culture devices tested in this example did not contain sub-microwells. The microwells tested in this example were square at the top of the microwells, with a microwell width of 1mm, a microwell depth of 1mm and a wall angle of 15 degrees. Seeding a liquid suspension of hematopoietic progenitor cells from frozen phenanthrene-separable human cord blood into a cell culture device and allowing it to settle by gravity at about 7-57 colonies/cm²The expected density of cells settled into the microwells at a cell concentration of from 1x10⁴The number of cells/microwell was increased stepwise to 1x10⁵Individual cells/microwell. The cell culture device is in a permissive culture environment at 37 deg.C and contains 5% CO₂Is incubated in a humidified atmosphere. The total number of colonies from the cell culture apparatus was evaluated 7 days after inoculation.

In fig. 14, the number of positive wells (data points shown as diamonds) did not increase linearly with cell seeding concentration. A positive well may be derived from a single ancestor, or from more than one ancestor. The more progenitors added to a given microwell culture vessel (varying with progenitor frequency, cell concentration, and volume added), the higher the likelihood that each microwell will inoculate more than one progenitor. In this example, the culture vessel has approximately 1000 microwells and is therefore about 50% positive when each dish plate has the highest cell number. Based on the observed frequency of positive wells, the output of the assay can be linearized by determining the expected number of progenitors required to generate the number of positive wells using a poisson distribution. Figure 14 shows that once this correction is applied, the relationship between the number of plated cells and progenitors is linear (data points are shown as squares). The corrected number represents an estimate of the total number of progenitors in the initial sample.

Example 8 magnetic isolation of colony-forming progenitor cells

The cell culture apparatus of example 7 having the same configuration of microwells was humidified with an aqueous buffer (PBS containing 2% FBS) to remove any air remaining in the microwells. Mononuclear cells in human cord blood samples were enriched by ficoll (Stemcell Technologies, 07907) density gradient centrifugation and the cells were resuspended in the above buffer. This cell suspension was mixed with dextran-coated magnetic microparticles (Stemcell Technologies, D-microparticles) and an anti-dextran/anti-CD 34 antibody mixture (Stemcell Technologies, CD34+ selection mixture). After incubation to allow specific binding of the hematopoietic progenitor cells in suspension to the magnetic microcarriers, the mixture is added to a cell culture device. The cell culture apparatus was then placed on a flat magnet (LifeSep 384F). Microcarriers were seen to migrate down the magnetic field gradient and settled into the microwells. The suspension was observed to brighten in less than 2 minutes, while a dark precipitate was seen to form on the bottom surface of the microwells. Hematopoietic progenitor cells are expanded by pipetting off excess supernatant and replacing with liquid medium containing cytokines. Additional cultures in the same configuration of cell culture devices and in semi-solid media were inoculated as controls, as described in example 6 above. Cell culture apparatus and controls were in a permissive environment (37 ℃, 5% CO)₂Wet incubator) for a period of 7 days and observing the formation of cell colonies within the wells.

Brightfield microscopy revealed a uniform distribution of microcarrier beads in the microwells of the cell culture device. However, within each microwell, the beads were found to aggregate toward the edge of the microwell corresponding to the direction of the magnetic field gradient (fig. 15A). Although the magnetic beads obscured individual cells after seeding, it was observed that colonies formed in each well throughout the cell culture apparatus after 7 days of culture (FIG. 15B). The total colony count in the cell culture device in which the hematopoietic progenitor cells were selectively sedimented using magnetic microcarriers was comparable to that observed in the control cell culture device and in cultures performed in semi-solid medium in Petri dishes (Petri dishes) (fig. 16). This demonstrates that in a cell culture device comprising microwells, quantitative colony assays for specific cell types can be performed by selectively depositing the desired cells into the microwells using magnetic microcarriers and antibody mixtures specific for unique markers on the cell surface.

Claims

1. A cell culture apparatus, comprising:

a well defined by at least one well sidewall, and a well bottom wall;

2. The cell culture assembly of claim 1, wherein each sub-microwell is further defined by a portion of one of the microwell sidewalls.

3. The cell culture assembly of claim 1, wherein the well bottom wall is transparent or translucent.

4. The cell culture assembly of claim 1, wherein each set of sub-microwells comprises 4 sub-microwells arranged in a 2x2 array.

5. The cell culture apparatus of claim 1, wherein each set of sub-microwells comprises a plurality of sub-microwells arranged in an array of 2x1, 3x1, 3x2, 3x3, or greater.

6. The cell culture apparatus of claim 1, wherein each sub-microwell gradually decreases in cross-sectional area from the top of the sub-microwell to the bottom of the sub-microwell.

7. The cell culture assembly of claim 6, wherein each sub-microwell is frustoconical or frustro-conical.

8. The cell culture apparatus of claim 1, wherein each microwell tapers in cross-sectional area from the top of the microwell to the bottom of the microwell.

9. The cell culture assembly of claim 8, wherein each microwell is frustoconical or frustoconically shaped.

10. The cell culture apparatus of claim 1, wherein the sub-microwells, and wells are integrally formed.

11. The cell culture apparatus of claim 1, further comprising a magnetic or magnetizable element positioned below the sub-microwells.

12. The cell culture apparatus of claim 11, wherein the magnetizable element is a wire grid embedded in the bottom wall of the well.

13. The cell culture apparatus of claim 1, wherein the microwell tops have a width of at least 100 microns and/or each microwell has a depth between the top and the bottom of at least 75 microns.

14. The cell culture apparatus of claim 1, wherein each microwell comprises a maximum dimension at a top of the microwell and a microwell depth between the top of the microwell and the bottom of the microwell, and a ratio of the maximum dimension to the microwell depth is 1.1:1 to 1.9: 1.

15. The cell culture assembly of claim 14, wherein the largest dimension is at least 140 microns.

16. The cell culture assembly of claim 14, wherein the microwell depth is at least 75 microns.

17. The cell culture assembly of claim 1, wherein the angle of the microwell sidewalls relative to vertical is less than 30 degrees.

18. The cell culture assembly of claim 17, wherein the angle of the microporous sidewall with respect to vertical is less than 20 degrees.