WO2008078326A2

WO2008078326A2 - Device for studying cells of thixotropic material and method of manufacure thereof

Info

Publication number: WO2008078326A2
Application number: PCT/IL2007/001607
Authority: WO
Inventors: Mordechai Deutsch; Assaf Deutsch
Original assignee: Seng Enterprises Ltd.
Priority date: 2006-12-26
Filing date: 2007-12-25
Publication date: 2008-07-03
Also published as: WO2008078326A3

Abstract

Provided in some embodiments are novel planar well-bearing components made of a thixotropic material, the components having a relatively homogeneous thickness, and on which a surface is located an array of wells, where the bottoms of the individual wells of a component being relatively coplanar, each well of a size and shape suitable for retaining a cell therein. Provided in some embodiments are novel methods for producing a planar well-bearing component useful for holding and studying living cells including impressing a well-array into a surface of a thixotropic material.

Description

DEVICE FOR STUDYING CELLS OF THIXOTROPIC MATERIAL AND METHOD OF MANUFACTURE THEREOF

The present application gains benefit of the filing date of US patent application No. 60/876,998 filed 26 December 2006 which is incorporated by reference as if fully set forth herein.

FIELD AND BACKGROUND OF THE INVENTION The present invention, in some embodiments thereof, relates to methods and devices useful for studying living cells, especially a large number of living cells as individuals, as well as methods of making the device.

The study of cell behavior is important in many fields including biology, medicine and pharmacology. Since cell-functions include many interrelated pathways, cycles and chemical reactions and since there is a large variation of cell biochemistry amongst similar cells, the study of a bulk of cells, whether the bulk is homogenous or heterogeneous, does not provide sufficiently detailed or interpretable results: rather a comprehensive study of cell biological activity is advantageously performed by examining single isolated living cells as individuals. Thus, the use of single-cell assays is one of the most important tools for understanding biological systems and the influence thereupon of various stimuli such as exposure to active entities.

In order to understand cell behavior, for example, such as the response to stimuli such as various biological modulators, two fundamental research capabilities are required (i) the ability to track temporal behavior of large groups of cells as individuals for periods of minutes, hours and even days and (ii) the ability to identify and study cell heterogenity, a phenomenon existing even in synchronized cell lines. For this to be achieved, it is necessary to use of high-throughput single live cell assays which allow the study of real-time responses to stimuli in large and heterogeneous cell populations at an individual cell level in order to: study each cell from a plurality of cells as an individual, observing the realtime response of the cells to intervention using probes to monitor morphological responses, intracellular and extra-cellular parameters, as well as cell-cell interaction; perform long-term, non-intrusive, repeated measurements on intact, living, adherent or non-adherent cells, including bone marrow cells;; perform multiple functional assays on living cells, followed by immuno- staining and chromatic staining on the same cells following fixation; perform kinetic measurements of non-synchronous activities in individual cells; analyze and compare actual quantitative measurements of sub-populations and individual cells as opposed to recording the mean values of entire population; and perform quantitative measurements on a cellular-to-molecular level.

In the art, a number of method and devices have been developed for the study of individual cells or a small number of cells as a group.

Exceptionally useful are the devices and methods taught in the PCT Patent Applications published as WO2003/035824, WO2004/113492, WO2005/007796, WO2006/003664, WO2006/080000 and WO2007/052245 of the Inventor, all of which are included by reference as if fully set forth herein. In some embodiments, the above art teaches devices for the study of cells including a picowell-bearing component. In some embodiments, a picowell-bearing component is a component having at least one, but generally a plurality of pico wells, each picowell configured to hold at least one cell. The term "picowell" is general and refers to a "small well", that is physical feature that localizes a cell to a specific area on a planar surface of the picowell-bearing component primarily by physical confinement. In some embodiments, a picowell-bearing component is a "carrier", a substantially planar component such as a chip, plate, sheet or slide. The term "picowell" is generally a discrete cavity of a size and shape suitable for retaining cells therein where the size and shape are defined by some physical features such as walls. The term "picowell" includes physical features such as wells, dimples, depressions, pits, tubes and enclosures.

Since cells range in size from about 1 micrometers to about 100 (or even more) micrometers diameter there is no single picowell size that is appropriate for holding a single cell of any type. That said, the dimensions of the typical individual picowell in the picowell-bearing components in the above references have dimensions suitable for accommodating cells having diameters of between about 1 micrometer up to about 200 micrometers, depending on the exact implementation, for example round or hexagonal picowells having a diameter of from about 1 micrometer up to about 200 micrometers. For example, a picowell-bearing component of a device designed for the study of single isolated 8 to 20 micrometer diameter cells typically has picowells of dimensions of about 20 micrometers. In some embodiments, larger picowells are used to study the interactions of a few cells held together in one picowell. For example, a 200 micrometer picowell is recognized as being useful for the study of cell spheroids or the interactions of two, three or more cells as discussed in the PCT Patent Application published as WO 2003/035824 of the Inventor

A feature that increases the utility of some embodiments of the picowell- bearing devices taught in the above references is that the picowells are juxtaposed, that is to say the walls separating the individual picowells are thin (relative to the size of the picowells) so that the area occupied by a picowell-array is substantially entirely made up of picowells with little or no inter picowell area, as depicted in Figure 1.

Figure 1 is a reproduction of a photograph of part of a picowell-array 2 on a carrier described hi the PCT patent application published as WO2003/035824. In Figure 1 is seen a plurality of hexagonal picowells 4, some holding living cells 6. It is seen that walls 8 separating picowells 4 are less than 1 micrometer wide so that the interpicowell area of picowell array 2 makes up only a minor percentage of the total area of picowell-array 2. This feature allows near tissue-density packing of cells, especially in embodiments configured to hold a single cell in each picowell. For example, a typical device of the PCT patent application published as WO2003/035824 having a 2 mm by 2 mm picowell-array of hexagonally-packed juxtaposed picowells 4 having a 10 micrometer diameter includes about 61600 picowells. This feature also allows simple loading of the picowells with cells: a liquid containing suspended cells is introduced in the volume above picowells of the appropriate size. Since there is little inter-picowell area, cells necessarily settle in the picowells. After the cells settle, a flow of liquid applied in parallel to the surface of the picowell array either washes away cells accidentally resting on a wall separating picowells, or pushes such a cell into an unoccupied picowell. In such a way, the only cells in the proximity of the picowell array are cells held in a picowell, where each picowell holds only the number of cells for which it is configured. For example, one cell per picowell, two cells per picowell or three cells per picowell.

The use of a device for studying cells including a pico well-bearing component generally involves loading picowells with cells in a physiological medium and then observing the cells as individuals while exposing the cells to various stimuli to study the effect of the stimuli on the individual cells. In some of the above-referenced PCT Patent Applications, the picowell-bearing component is a thin, transparent carrier and the cells are observed from below with an inverted microscope or similar device, for example by detection of light emitted by fluorescence or direct optical observation of the cells. In such embodiments, it is important that the bottoms of the picowells on which the cells rest be as coplanar as possible: coplanarity allows for optical observation of many cells (whether by scanning or simultaneously using a wide-angle observation component) without the need for time consuming and difficult-to- implement refocusing. In such embodiments it is also important that the carrier be as flat and planar as possible, be of a uniform thickness and be as homogenous as possible so that the carrier be as optically neutral as possible, to allow observation of the cells with as little distortion as possible.

In the above cited PCT patent applications, a number of methods of producing a carrier are discussed. For example, using standard etching techniques, a picowell array and other desired features are etched on a positive master, e.g., on a borosilicate glass plate or other etchable material. From the positive master a negative die (also, mold, stamp or template) is made, for example by deposition of a metal (such as nickel) or application of a fluid precursor material that sets in contact with the positive master. The die is separated from the positive master. When it is desired to make the carrier, the die is contacted with a layer of fluid precursor material that is cured while in contact with the die. Once the fluid precursor material has cured, the die and the carrier are separated, leaving a picowell array on the surface of the carrier.

It has been found that although highly effective, in some embodiments the above-described method has disadvantages. Often, the produced die is thin and relatively fragile. In some instances, separation of the die from the positive master distorts or tears the master and/or the die. Even when separation is performed carefully, internal stress or local inconsistencies produced during the step of applying the material making up the die or during a curing step may cause bending or warping of the die. When the die is contacted with the layer of fluid precursor material to make the picowell array, the produced die may not be sufficiently planarity or homogenous. As the picowells are micrometer-sized features and as it is often desired to observe cell organelles, even slight inconsistencies of planarity or homogeneity reduce the quality of the produced carrier. The multiple contacting of the die with a precursor material and the subsequent separation of the die and carrier means that an individual die has a relatively short lifetime, so that each die can be used only a limited number of times. In some embodiments, it is difficult, although not impossible, to avoid any inclusions or air bubbles in a produced carrier. Many produced carriers must be discarded as a result of post-production inspection.

The problems discussed above are exceptionally significant when practicing the teachings of the PCT patent application published as WO 2005/007796 where in some embodiments it is desired to produce a device that substantially constitutes a 96- well plate, where on the bottom of each one of the 96 micro wells of the 96- well plate is a picowell array. It would be preferable not only that the bottoms of the picowells of an individual microwell be coplanar, but also that that bottoms of the picowells of different microwells be coplanar.

Further, it is desired that devices according to the teachings of the above- referenced PCT patent applications be mass-producible at a low cost rather than individually crafted.

It would be highly advantageous to have a cheap method to produce a picowell-bearing carrier suitable for the study of cells where the picowells have a high degree of coplanarity, where the method is suitable for mass production of such carriers.

SUMMARY OF THE INVENTION

Some embodiments of the present invention provide a method of producing a well-bearing carrier for the study of cells that is devoid of at least some of the shortcomings of the prior art. Some embodiments of the present invention provide a novel well-bearing carrier. According to an aspect of some embodiments of the present invention there is provided a method for making a planar well-bearing component suitable for the study of cells comprising: a) providing a thixotropic material; b) forming the thixotropic material into a layer having a surface; and c) pressing a surface of a die including a negative of an array of wells against the layer surface with sufficient pressure so as to impress an array of wells on the layer surface wherein the array of wells comprises wells having a dimension on the layer surface of no more than about 500 micrometers, the wells of a size and shape suitable for retaining at least one cell of a certain type therein. According to some embodiments of the invention, the layer of thixotropic material is formed on a sheet of support material. According to some embodiments of the invention the method further comprises: cutting the layer of thixotropic material and the sheet of support material.

^"According to some embodiments of the invention, the die surface is substantially planar.

According to some embodiments of the invention, the die surface is curved. According to some embodiments of the invention, the die surface is cylindrical. According to some embodiments of the invention, the die surface is cylindrical and the pressing of the die surface against the sheet surface comprises rolling the die surface over the layer surface.

According to an aspect of some embodiments of the present invention there is also provided a well-bearing component useful for the study of living cells, comprising: a layer having an array of wells on a surface thereof, wherein the array of wells comprises wells having a dimension on the layer surface of no more than about 500 micrometers, the wells of a size and shape suitable for holding at least one cell of a certain type (and in some embodiments, not more than one) therein, wherein the layer is made of a thixotropic material.

According to some embodiments of the invention, the layer of thixotropic material rests on a sheet of support material. According to some embodiments of the invention, the thixotropic material is substantially transparent. According to some embodiments of the invention, the thixotropic material is a thixotropic Sol-Gel. According to some embodiments of the invention, the thixotropic material is a thixotropic Sol-Gel including nanoparticles.

According to some embodiments of the invention, the thixotropic material is biologically compatible, that is to say does not have toxic or injurious effect on cells held in wells of the well array when immersed in a medium.

The sheet of support material is of any suitable material, for example polyester, polycarbonate, polyethylene terephthalate, polystyrene, and glass.

According to some embodiments of the invention, the sheet of support material is of an optically transparent material.

The wells may be of any suitable shape, for example round, hexagonal, pentagonal, square or triangular.

According to some embodiments of the invention, two adjacent wells of the array of wells are juxtaposed and separated by a well-wall. According to some embodiments of the invention, the distance between two adjacent the wells is not more than 150%, not more than 130%, not more than 120% and even not more than 110% of the well-dimension.

According to some embodiments of the invention, the wells are configured to hold no more than one living cell of a certain type. According to some embodiments of the invention, the wells are configured to hold no more than a predetermined number of living cells of a certain type, for example not more than four, not more than three and even not more than 2 cells of a certain type.

According to some embodiments of the invention, the wells have dimensions on the surface of no more than about 100 micrometers, not more than about 50 micrometers and even not more than about 25 micrometers.

According to some embodiments, the wells have a depth that is in the order of the size of the cell which the well is configured to hold. Thus, in some embodiments, the wells have a depth no more than about 150%, no more than about 120% and even no more than about 100% of the dimension of the well on the surface. According to some embodiments, the depth of the wells is less than the dimension of the well on the surface. According to some embodiments of the invention, the wells have a depth of no more than about 30 micrometers, no more than about 20 micrometers, and even no more than about 10 micrometers.

According to an aspect of some embodiments of the present invention there is provided a cylindrical die for use with a rotating die press, comprising: a) a cylindrical surface; and b) on the surface, the negative of an array of wells, wherein the array of wells comprises wells having a dimension of no more than about 200 micrometers, the wells of a size and shape suitable for holding at least one cell of a certain type. Such a cylindrical die is useful for preparing a well-bearing component as described above.

Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE FIGURES

Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying figures. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of some embodiments of the invention. In this regard, the description taken with the figures makes apparent to those skilled in the art how some embodiments of the invention may be practiced. In the figures:

FIG. 1 (prior art) is a reproduction of a photograph of a cell-populated picowell array of a carrier, the picowell-bearing component of a device of the PCT patent application published as WO2003/035824 of the Inventor;

FIG. 2 A is a schematic depiction of a roller press useful in implementing some embodiments of the method of the present invention, where a rotating cylindrical roller die impresses an array of wells on the surface of a layer of a thixotropic material;

FIG. 2B is a reproduction of a photograph of the surface of the cylindrical roller die showing the negative of an array of wells; FIG. 3 is a reproduction of a photomicrograph of the surface of a thixotropic layer Including an array of wells, each well being 8 micrometer deep and 20 micrometer wide on the surface of the layer;

FIGS. 4A-4E depict a device similar to a 96- well plate where the bottom of each of the 96 (micro)wells is an array of (pico)wells impressed in a layer of thixotropic material; and

FIGS. 5A-5F are reproductions of SEM images of carriers of the present invention.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION The present invention, in some embodiments thereof, relates to methods and devices useful for studying living cells, especially a large number of living cells as individuals, as well as methods of making the device.

Some embodiments of the present invention are of methods for the production of carriers, devices that are substantially planar well-bearing components made of a thixotropic material as described above having a surface provided with physical features including wells having a size and shape suitable for retaining at least one cell of a certain type therein.

Some embodiments of a carrier of the present invention are a device useful for the study of living cells, comprising a planar layer having an array of wells on a surface thereof, wherein the array of wells comprises wells having a dimension on the surface of the layer of no more than about 500 micrometers, the wells of a size and shape suitable for retaining at least one cell of a certain type therein, wherein the layer is made of a thixotropic material.

In some embodiments, carriers of the present invention or carriers made using the method of the present invention have advantageous properties such as homogeneous thickness, high planarity, relatively few surface imperfections, very sharp and well-defined surface features, the bottoms of the wells of the well array are coplanar and/or the material from which the carriers are made is highly homogeneous so that the carriers are optically neutral.

In some embodiments, the methods of the present invention allow for the exceptionally cheap and/or efficient production of carriers, including large sheets of carriers (e.g., greater than 1 cm², in embodiments having some or all of the above advantageous properties.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details of construction and the arrangement of the components and/or methods set forth in the following description and/or illustrated in the figures and/or the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to". The term "consisting of means "including and limited to".

The term "consisting essentially of means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure. As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a well" or "at least one well" may include a plurality of compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from

3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging/ranges between" a first indicate number and a second indicate number and "ranging/ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

Herein, by "array of wells" is meant a group of two or more picowells, preferably a plurality of picowells, preferably a plurality of picowells arranged in an orderly fashion. Typical arrangements include hexagonal arrays (picowells arrayed in staggered rows so that each picowell has six equidistant neighboring picowells) or rectangular arrays (picowells arrayed in rows so that each picowell has four equidistant neighboring picowells in a cross shape).

Some embodiments of the present invention include components that are transparent or are made of a transparent material. By "transparent" is meant that the component or material is substantially transparent to at least one wavelength of light (preferably a range of wavelengths) in at least part of the visible light spectrum, the ultraviolet light spectrum and/or of infrared radiation, preferably the visible light spectrum.

It is important to note that some aspects of embodiments of the present invention are related to embodiments of devices for the study of cells including a well-bearing component, especially picowell-bearing devices described by the Inventor in the PCT patent applications published as WO2003/035824, WO2004/113492, WO2004/077009, WO2005/007796, and WO2006/080000 which are all included by reference as if fully set forth herein.

Method of making a well-bearing component suitable for the study of cells

In some embodiments, the present invention provides a method for making a planar well-bearing component suitable for the study of cells by impressing a layer of thixotropic material with an array of wells of an appropriate size and arrangement. Thus, in some embodiments, the present invention is of a method for making a planar well-bearing component suitable for the study of cells comprising: a) providing a thixotropic material; b) forming said thixotropic material into a layer having a surface; and c) pressing a surface of a die including a negative of an array of wells against said layer surface with sufficient pressure so as to impress an array of wells on said layer surface wherein said array of wells comprises wells having a dimension on said layer surface of no more than about 200 micrometers, said wells of a size and shape suitable for retaining cells therein.

In some embodiments, the layer of thixotropic material is formed on a sheet of support material. In some embodiments, the method further comprises cutting said layer of thixotropic material and said sheet of support material.

In some embodiments, the die surface is substantially planar, and in some such embodiments impressing is performed by stamping said surface of said layer with said die surface.

In some embodiments, the die surface is curved. In some embodiments, the die surface is cylindrical. In some embodiments, the die surface is cylindrical and said pressing of said die surface against said sheet surface comprises rolling said die surface over said layer surface.

An advantage of some embodiments using a cylindrical die surface that rolls over the surface of the layer of thixotropic material is that such a method is continuous and allows production of broad and effectively unlimited lengths of well arrays, enabling well arrays to be manufactured relatively cheaply. Further, in some embodiments, excess thixotropic material is "squeezed-out" so that the thickness of the impressed layer of thixotropic material as well as the depth and height of impressed features such as the well array is very consistent, so that the quality of the produced well arrays is very high. In some such embodiments, a die is supported on a rigid support so that the die is maintained in a flat state and has relatively wear, further reducing the price of manufacture of well arrays.

It has been found that in embodiments it is possible to provide a very accurate reproduction of the micrometer scale features of a carrier, with a sufficient degree of coplanarity and flatness of the picowell bottoms (within the focal depth of a microscope objective, and in embodiments less than about 1 micrometer, less than about 0.5 micrometer and even less than about 0.2 micrometer).

It has been found that in embodiments it is possible to provide a carrier having a very consistent layer thickness. It has been found that in embodiments, the method produces very few if any inclusions, for example of gas bubbles.

It has been found that the method assists in providing a produced well-beairng component that is exceptionally optical neutrality and is naturally warp resistant. As the thixotropic material becomes fluid during the impressing of the carrier features by contact with the rotating die and solidifies upon relief of the pressure, the produced feature-bearing layer is stress free and homogenous, contributing to exceptional optical neutrality.

A well-bearing component suitable for the study of cells

In some embodiments, the present invention provides devices that are planar well-bearing components suitable as components of devices for the study of cells, such as described, for example, in the cited PCT patent applications of the Inventor.

In some embodiments, such devices are termed "carriers" or "cell-chip" carriers as in the cited PCT applications.

In some embodiments, a device of the present invention useful for the study of living cells, comprises: a planar layer having an array of wells on a surface thereof, wherein the array of wells comprises wells having a dimension on the layer surface of no more than about 500 micrometers, the wells of a size and shape suitable for retaining a cell therein, wherein the layer is made of a thixotropic material, where the thixotropic material is, in an non-disturbed state viscous to a degree sufficient to maintain micronic features at room temperatures.

In some embodiments, the layer of thixotropic material rests on a sheet of support material. In some embodiments, the sheet of support material is an optically transparent material. Suitable materials include glasses (e.g., sodalime glass and borosilicate glass, especially optical grade glass) and plastic or polymer support materials (e.g., polyethylene terephthalate, polycarbonate, polyester, polystyrene).

In some embodiments, the thixotropic material is biologically compatible (that is not substantially cytotoxic, is substantially devoid of biologial activity) or coated with a biologically compatible layer, does not have a toxic or injurious effect on cells held in wells of the well array when immersed in a medium] In embodiments, the thixotropic material from which the carrier is substantially impervious to water, or coated with a layer that is substantially impervious to water.

In embodiments, the thixotropic material from which the carrier is non- fluorescent.

In embodiments of the present invention, the thixotropic material from which the carrier is made is substantially transparent so as to allow observation of cells held in the wells. By transparent is especially meant transparent to one or more frequencies of electromagnetic radiation in the visible, ultraviolet or infrared spectra. In embodiments of the present invention, the thixotropic material from which the carrier is made is optically neutral. It is important to note that surprisingly it has been found that the method of the present invention assists in providing a carrier of the present invention with optical neutrality. As the thixotropic material becomes fluid during the impressing of the carrier features and solidifies upon relief of the pressure, the produced feature-bearing layer is stress free and homogenous, contributing to exceptional optical neutrality.

A thixotropic material suitable for implementing some embodiments of the present invention is a Sol-Gel material, especially nanoparticle Sol-Gel material similar to those described in U.S Patent 6,855,371 (all included by reference as if fully set forth herein). Such material are easily spread in even layers of the appropriate thickness between about 5 and 500 microns, generally between about 10 and 60 micrometers.

Characteristics of some Sol-Gel materials renders the materials suitable for use in making a well-bearing component, including optical inertness, biological comaptibility and chemical stability. Further, it has been found that amongst thoxotropic materials, certain Sol-Gel materials, especially certain nanoparticle Sol- Gel materials allow the accurate and repetitive production of sheets of picowell arrays with sufficient coplanarity (within the focal depth of a microscope objective, and in embodiments less than about 1 micrometer, less than about 0.5 micrometer and even less than about 0.2 micrometer) of the picowell bottoms over large areas, for example areas corresponding to the size of standard 96-well plates. In some embodiments, to improve the optical properties of a produced well- bearing component the surface of the layer of the thixotropic material, such as the Sol-Gel is coated with a film of material as an ami reflecting coating (ARC). When, such embodiments are used for the study of cells, the ARC coating is an intermediate layer between the thixotropic material (e.g., for Sol-Gels n ~ 1.5) and a biological solution (n -1.33). Such an anti reflective coating is preferably transparent, optically inert, and biological compatible.

In some embodiments, a layer of thixotropic material, such as a Sol-Gel, is hardened and/or toughened, in the usual way. In some embodiments, the wells of a well-bearing component are round or hexagonal, although in some embodiments, wells are triangular, square, pentagonal or any other suitable shape. Although wells of a well array of a well-bearing component may be arranged in any suitable arrangement, in some embodiments wells are hexagonally packed, allowing a high loading of cells per unit area^" In some embodiments, two adjacent wells of a well-bearing component are juxtaposed and separated by a well-wall, as disclosed in the cited PCT patent applications. In some embodiments, the distance between two adjacent wells is not more than 150%, not more than 130%, not more than 120%, not more than 110% and even not more than 105% of the well-dimension. The wells of a well-bearing component are generally of any size so as to hold at least one cell of a certain type. In some embodiments that are directed to the study of cells as individuals, it is generally preferred that the wells be small so as to avoid having a large number of cells held in any one well.

For example, in some embodiments, a cell well-bearing component is configured for study of lymphocytes having a typically diameter of about 6 micrometers. In some such embodiments a well-bearing component has wells of about 6 to 10 micrometer dimension on the surface of the well-bearing component so that the lymphocytes enter and are held in the wells, one in each well.

For example, in some embodiments, a cell well-bearing component is configured for study of oocytes or single-cell spheroids having a typically diameter of about 400 micrometers. In some such embodiments a well-bearing component has wells of about 400 to 500 micrometers dimension on the surface of the well-bearing component so that the oocytes or spheroids enter and are held in the wells, one in each well.

Thus, generally, the dimensions of the wells are generally less than about 500, 400, 200, 100, 50, 25 or even less than about 10 micrometers. By dimensions is meant the usual meaning of the word and is dependent on the shape of the wells. For example, for hexagonal or circular wells, the term dimension refers to diameter. For square or triangular wells is meant the longest dimension of the square or triangle, respectively. The exact size of wells of any given well-bearing component is determined by the type of cells or alternately or additionally by the amount of cells to be studied using the carrier. Since different types of cells have different sizes, generally a carrier of the present invention will have wells of a size to accommodate one or more cells of the type to be studied. Most preferred is that a well be of a size so as to hold no more than one cell of the type to be studied at any one time. In other embodiments, a well size is determined by the size of a predetermined number of a certain type of cells, so as to allow holding of that predetermined number of that type of cell, for example two cells, three cells, or even four cells.

To ensure that no more than a limited number of cells are held in a given well, and that a held cell is able to efficiently absorb nutrients and release waste, the depth of a well is generally not more than the order of the size of the cell (or spheroid) which the well is configured to hold. Thus, in some embodiments, the wells have a depth no more than about 150%, no more than about 120% and even no more than about 100% of the dimension of the well on the surface. According to some embodiments, the depth of the wells is less than the dimension of the well on the surface. Thus in some embodiments, the depth of a well is no more than about 500, not more than about 200, not more than about 100, not more than about 50, not more than about 30, not more than about 20 and even not more than about 10 micrometers deep.

In some embodiments of the present invention, wells are dimples, depressions, or pits on the. surface of well-bearing component. In other embodiments, the wells are substantially enclosures of dimensions such that substantially an entire cell of a certain type is containable within the enclosure, each enclosure having an opening at the surface, the opening defined by a first cross section of a size allowing passage of a cell of the certain type.

In some embodiments of the present invention, a surface of a well-bearing component is provided with fluid transport channels and other such features as discussed in detail in the PCT patent applications published as WO2003/035824 and WO 2004/113492.

An embodiment of a method of the present invention for making a planar well-bearing component is described with reference to Figures 2 A and 2B. In Figure 2A, a roller press 10 is schematically depicted (components not to scale) including a rotating cylindrical roller die 12 with a cylindrical die surface 14 made of silicone rubber rotatably suspended on an axle 16 over counter table 18. The height of axis 16 and consequently die surface 14 above the surface of counter table 16 is adjustable with an accuracy of greater than 1 micrometer with the help of a height adjusting mechanism (not depicted). Roller press 10 is also provided with a rotating support feeder 20 configured to hold a rolled up precursor sheet 22 of support material 24 onto which was applied a layer 26 of thixotropic material. In the embodiment depicted in Figure 2A, support material 24 is 1 mm thick polyester while layer 26 is a 20 micrometer thick of a thixotropic nanoparticle-containing Sol-Gel material.

Figure 2B is a reproduction of a photomicrograph of surface 14 of cylindrical roller die 12 showing an array of domes 28 which constitute the negative of an array of hexagonally-packed circular wells, each well 8 micrometers deep and 20 micrometers in diameter. For use, the height of axle 16 is adjusted so that die surface 14 is a desired height above counter table 18, e.g., 1020 micrometers. Precursor 22 is fed from rotating support feeder 20 at a rate that matches the rate of rotation of cylindrical roller die 12 so that support material 24 rests on counter table 18 and the surface of layer 24 of thixotropic material faces upwards. Before approaching cylindrical roller die 12, the thixotropic material making up layer 26 is in a non-disturbed state that is viscous enough to retain micrometer scale features at room temperature. As cylindrical roller die 12 rolls over the surface of layer 26 of thixotropic material, die surface 14 applies sufficient pressure on the surface of layer 26 of thixotropic material, reducing the viscosity of the thixotropic material to the point where the thixotropic material flows around and adopts the shape of die surface 14 and specifically domes 28 which constitute a well array.

When surface 14 of cylindrical roller die 12 moves away from the surface of layer 26 of thixotropic material, the viscosity of the thixotropic material increases so that the thixotropic material soldifies while retaining the shape of the well array impressed on the surface of layer 26 of thixotropic material. Figure 3 is a reproduction of a photomicrograph of the surface of layer 26 showing an array 30 of wells 32, each well being 8 micrometer deep and 20 micrometer wide on the surface of layer 26 where two adjacent wells 32 are separated by a wall 34 that is less than 1 micrometer wide so that the distance between the two adjacent wells 32- is not more than 110% of the well-dimensions (less than 21 micrometer interwell distance / 20 micrometer well width).

In the embodiments discussed above, a die surface was only provided with negative of a well array so as to impression of only a well array on a surface of a layer of thixotropic material. In some embodiments, negative of other features are also provided on a die surface so that other features, e.g., fluid conduits, fiducial features (see, for example, the PCT patent applications of the Inventor cited herein) are impressed on the surface of the layer of thixotropic material.

In the embodiments discussed above, a continuous well array 30 is made on layer 26 of thixotropic material formed on a sheet of support material 24 and is potentially cut to an appropriate size in a subsequent step. In some embodiments, a die is provided with a cutting component, such as a cutting blade, to automatically produce a planar well-bearing component of a desired size concurrently with the imprinting of the well array in the layer of thixotropic material.

Device for the study of cells Generally, a well-bearing component as described above is integrated in a device suitable for the study of cells. Integration of a well-bearing component such as described above into a device may be done in any of a number of ways with which one skilled in the art is familiar, including methods analogous to methods described in the cited PCT patent applications of the Inventor.

In some embodiments, such devices are substantially similar to a device such as disclosed in the PCT patent application published as WO2005/007796, which are substantially multi-well plates such as prior art 96-well plates, where there bottom of each well (microwell) includes the smaller wells (picowells) of a well-bearing component (a carrier) such as described above. In embodiments, such a device comprises a picowell-bearing component and a microwell- wall component. In Figures 4A-4E is depicted a device 36 resembling a prior art 96-well plate where the bottom surface of each one of the 96 microwells 38 is an array 30 of picowells 4. In Figure 4A, the components making up device 36 are depicted separately.

In Figure 4A, three components of 96-well plate device 36 for the study of cells are depicted: a (micro)well-wall component 40 defining the walls of the 96 miorowells 38; a (pico)well-bearing component 42 (also termed a "carrier") is a flexible sheet of thin, substantially optically neutral material (1 mm thick polyester) which entire upper surface is a picowell array 30 of picowells 4 in a layer of thixotropic material (a Sol-gel as described above); and a base plate 42 defining 96 observation ports 46.

When the three components are assembled to constitute device 36, 96 microwells 38 are defined by microwell-wall component 40, each microwell 38 including a bottom surface covered substantially entirely by a picowell array 30 that is a part of the microwell array of picowell-bearing component 42. In Figure 4B, device 36 is depicted fully assembled from above. In Figure 4C, a close up view of device 36 shows picowell arrays 30 on the bottom surface of microwells 38. In Figure 4D, a hottom surface of a microwell 38 is shown magnified so as to show 20 micrometer picowells 4 making up picowell array 30.

Base plate 44 provides rigidity, strength, and prevents bending and flexing of picowell arrays 30. Additionally, base plate 44 defines observation ports 46 that allow a microscope lens 48 to brought very close to picowell arrays 30 from below to observe cells 6 held in picowells 4 with little distortion, loss of resolution or light intensity as depicted in Figure 4E. In some embodiments (such as device 36 depicted in Figures 4), a well- bearing component (such as 42) is made as described above, where substantially the entire upper surface of the well-bearing component comprises a picowell array. In some embodiments a well-bearing components is made as described above, where substantially the upper surface of the well-bearing component comprises a plurality of picowell arrays separated by gaps designated as adhesion surfaces for a microwell- wall component. The dimensions of the well-bearing component are of any suitable size. For example, when a device is of the dimensions of a standard 96-well plate (such as device 36), a well-bearing component is generally between about 7 cm and 8 cm wide and between 10.6 and 12.2 cm long.

A microwell-wall component is substantially a grid-like component that, when attached to the well-bearing component, constitutes the walls of the micro wells. For example, when a device is of the dimensions of a standard 96-well plate, a microwell- wall component is generally between about 7 cm and 8 cm wide and between 10.6 and 12.2 cm long and includes 96 circular (6.6 mm) walls arranged in an 8 x 12 array. For assembly, the microwell-wall component is properly positioned relative to the well-bearing component and attached thereto. Suitable methods include, but are not limited to, hot welding, ultrasonic welding and the use of adhesives, especially light-curable adhesives. In some embodiments, insert molding of the microwell-walls to an already fabricated well-bearing component is preferred as there is no need to produce the microwell-wall component separately eliminating the need for a separate assembly step, there is no cell toxicity resulting from the use of adhesives and there is no need for rinsing of the produced device. In general, an appropriately sized well-bearing component is placed inside a mold, and the precursor of the material (e.g., polycarbonate, polystyrene) from which the microwell-walls are made is injected into the mold, allowing high adhesion between the well-bearing component that serves as the base of the device and the microwell-walls, and reducing the chance of leakage between adjacent microwells. If the injection molding is performed in a clean facility (e.g., a medical equipment production facility) the devices thus produced are immediately ready for packaging and sterilization without any need for further cleaning or rinsing. In some embodiments, reactive molding of the microwell-walls to an already fabricated well-bearing component is preferred. In such embodiments generally a resin and a hardener are mixed as a precursor, an appropriately sized well-bearing component is placed inside a mold, and the precursor of the material from which the microwell-walls are made is injected into the mold. Although generally considered slower than insert molding, reactive molding shares many of the characteristics of insert molding.

In some embodiments, a well-bearing component is sufficiently robust to serve as a base plate for such a device. In some embodiments, a well-bearing component is a relatively thin material

(e.g., polyester film or polycarbonate film, e.g., 1 mm thick or even 0.5 mm thick) which is preferably attached to a rigid base plate as a structural support. An advantage of such embodiments is that it is simple to provide equipment (such as a press) to impress a well array on a layer of thixotropic material on a thin sheet of support material without fear of damaging the support material during impression of the well array on the surface of the layer of the thixotropic material. Further, in some embodiments (such as device 36), a base plate includes observation windows corresponding to the microwells defined by the microwell-wall component.

In some embodiments, such a device is a multiwell plate comprising a plurality of (micro)wells wherein at the bottom surface of at least one (micro)well of the plurality of (micro)wells is a plurality of (pico)wells. Preferably, the plurality of (micro) wells of the device comprises 6n (micro) wells arranged in a 2n by 3n array, where n is an integer greater than 0, the (micro)wells preferably being arranged in rectangular packing. Preferred pluralities of (micro)wells are the commonly known pluralities of (micro)well such as 6, 24, 96, 384 and 1536 (micro)wells. Most preferred are devices of 96 (micro)wells and 384 (micro)wells as these formats are most popular and have many available accessories including fluid-handling accessories such as fluid-handling robots.

In some embodiments, such a device is in a physical format different from that of a multiwall plate, for example, that of a microscope slide, a petri dish or any other vessel or device useful for the study of cells. Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find support in the following examples. Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following example, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following example.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

A planar well-bearing component for the study of cells was manufactured at the Leibniz-Institut fuer Neue Materialien GmbH (Leibniz, Germany) in accordance with the teachings of the present invention and the cytotoxicity thereof was evaluated.

On the cylindrical surface of a cylindrical roller die (similar to roller die 12) of a roller press having a width of 9 cm and a diameter of 1 cm was secured a 9 cm wide sheet of silicone rubber on which surface was a negative of a hexagonally-packed array of circular 20 micrometer wells separated by walls that were less than 1 micrometer thick so that the distance between two adjacent wells less than 21 micrometers, 105% of a well diameter (resembling the surface depicted in Figure 2B), the sheet made by curing a fluid silicone rubber precursor on an etched glass postive of the array. The thus-fashioned cylindrical roller die was mounted on an appropriate roller press so that the surface of the cylindrical roller die was 1020 micrometers above the planar stainless steel counter table.

A 20 micrometer thick layer of a transparent thixotropic nanoparticle- containing Sol-Gel material (labeled "POS") was prepared on a polyester sheet (1 mm thick, 9 cm wide and 14 cm long) which served as a support material. The polyester sheet were laid on the stainless steel counter table so that the polyester sheet contacted the counter table. The polyester sheets were passed between the counter table and the cylindrical roller die so that the cylindrical roller die rolled over the upper surface of the layer of the thixotropic material.

As the cylindrical roller die rolled over the layer of thixotropic material, the die surface applied pressure on the surface of the thixotropic material, reducing the viscosity of the thixotropic material to the point where the thixotropic material flowed around and adopted the shape of the well array on the die surface.

When the surface of the cylindrical roller die moved away from the thixotropic material, the viscosity of the thixotropic material increased so that the thixotropic material soldified while retaining the well array impressed on the surface of the thixotropic material.

In such a way, a 9 cm wide and 14 cm long planar well-bearing component inncluding a well-array comprising hexagonally packed circular wells 20 micrometers in diameter and 8 micrometers deep with a distance between two adjacent wells of less than 21 micrometers was made, where the polyester sheet serve as a support material. The picowell array on the surface of the thixotropic material labeled of the well-bearing component is depicted in Figures 5A-5F. The depth of the thixotropic material on the polyester sheets was 20 micrometers with a variance of less than ±0.5 micrometers. The bottoms of all the wells were within better than ±0.5 micrometers of coplanar. The sheet showed no signs of warping or curving. The index of the refraction of the thixotropic material making up the well-bearing component (carrier) was approximately 1.5.

The cytotoxicity of the carrier was compared to a plain polyester sheet equivalent to the support material and to that of a standard 96-well polystyrene plate (F96 Micro Well™ Plate from Nunc™ of Thermo Fisher Scientific, Waltham, MA, USA) using U937 cells with PI staining. The results shown in Table 1 demonstrate that the carrier is non-cytotoxic.

As a standard 96- well multiwell plate has dimensions of about 8 by 12 cm, the well-bearing component is suitable for fashioning a device similar to device 36.

It is expected that during the life of a patent maturing from this application many suitable thixotropic materials will be developed and the scope of the claims is intended to include all such new materials a priori.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements. AU publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In case of conflict, the patent specification, including definitions, will control. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

Claims

WHAT IS CLAIMED IS:

1. A method for making a planar well-bearing component suitable for the study of cells comprising: a) providing a thixotropic material; b) forming said thixotropic material into a layer having a surface; and c) pressing a surface of a die including a negative of an array of wells against said layer surface with sufficient pressure so as to impress an array of wells on said layer surface wherein said array of wells comprises wells having a dimension on said layer surface of no more than about 500 micrometers, said wells of a size and shape suitable for retaining at least one cell of a certain type therein.

2. The method of claim 1, wherein said layer of thixotropic material is formed on a sheet of support material.

3. The method of claim 2, further comprising: cutting said layer of thixotropic material and said sheet of support material.

4. The method of any of claims 1 to 3, wherein said die surface is substantially planar.

5. The method of any of claims 1 to 4, wherein said die surface is curved.

6. The method of claim 5, wherein said die surface is cylindrical.

7. The method of claim 6, wherein said die surface is cylindrical and said pressing of said die surface against said sheet surface comprises rolling said die surface over said layer surface.

8. A well-bearing component useful for the study of living cells, comprising: a layer having an array of wells on a surface thereof, wherein said array of wells comprises wells having a dimension on said layer surface of no more than about 500 micrometers, said wells of a size and shape suitable for holding at least one cell of a certain type therein, wherein said layer is made of a thixotropic material.

9. The well-bearing component of claim 8, wherein said layer of thixotropic material rests on a sheet of support material.

10. The method of claim 1 or the well-bearing component of claim 8 wherein said thixotropic material is substantially transparent.

11. The method of claim 1 or the well-bearing component of claim 8 wherein said thixotropic material is a thixotropic Sol-Gel.

12. The method or well-bearing component of claim 11, wherein said thixotropic material is a thixotropic Sol-Gel including nanoparticles.

13. The method of claim 1 or the well-bearing component of claim 8, wherein said thixotropic material is biologically compatible.

14. The method of claim 3 or the well-bearing component of claim 9, wherein said sheet of support material is of an optically transparent material.

15. The method of claim 1 or the well-bearing component of claim 8, wherein two adjacent said wells of said array of wells are juxtaposed and separated by a well- wall.

16. The method or well-bearing component of claim 15, wherein the distance between two adjacent said wells is not more than 150% of said well- dimension.

17. The method of claim 1 or the well-bearing component of claim 8, wherein said wells are configured to hold no more than one living cell of a certain type.

18. The method of claim 1 or the well-bearing component of claim 8, wherein said wells are configured to hold no more than a predetermined number of living cells of a certain type.

19. The method of claim 1 or the well-bearing component of claim 8, wherein said wells have dimensions on said surface of no more than about 200 micrometers.

20. A cylindrical die for use with a rotating die press, comprising: a) a cylindrical surface; and b) on said surface, the negative of an array of wells, wherein said array of wells comprises wells having a dimension of no more than about 500 micrometers, said wells of a size and shape suitable for holding at least one cell of a certain type.