WO2011083478A2

WO2011083478A2 - Methods of loading cells

Info

Publication number: WO2011083478A2
Application number: PCT/IL2011/000024
Authority: WO
Inventors: Mordechai Deutsch; Elena Afrimzon
Original assignee: Seng Enterprises Ltd.
Priority date: 2010-01-11
Filing date: 2011-01-10
Publication date: 2011-07-14
Also published as: WO2011083478A3

Abstract

A method of improving wettability a miniwell array. The method comprises providing a working surface having a miniwell array of plurality of miniwells, applying a pre-loading treatment with an alcoholic solution on top of the miniwell array, applying a buffer solution on top of the miniwell array, and loading a cell suspension on the miniwell array.

Description

METHODS OF LOADING CELLS

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to method of improving the wettability of microstructures cell substrates and system of analyzing individual cells and, more particularly, but not exclusively, to method and system of analyzing individual cells using a multi miniwell plate, such as, a nanowell plate, a picowell plate, a micropore plate, a nanopore plate, and/or a picopore plate.

In the art, various different methods for studying living cells are known. Multiwell plates having 6, 12, 24, 48, 64, 96, 384 or even 1536 wells on a standard ca. 8.5 cm by ca. 12.5 cm footprint are well known in the art. Such multiwell plates are provided with a 2" by 3" array of rectangular packed wells, n being an integer. The diameter of the wells of a plate depends on the number of wells and is generally greater than about 250 microns (for a 1536 well plate). The volume of the wells depends on the number of wells and the depth thereof but generally is greater than about 5xl0^"6 liter (for a 1536 well plate). The standardization of the multiwell plate format is a great advantage for researchers, allowing the use of standardized products including robotic handling devices, automated sample handlers, sample dispensers, plate readers, observation components, plate washers, software and such accessories as multifilters.^" Multiwell plates are commercially available from many different suppliers. Multiwell plates made from many different materials are available, including but not limited to u glass, plastics, quartz and silicon. Multiwell plates having wells where the inside surface is coated with various materials, such as active entities, are known.

Although exceptionally useful for the study of large groups of cells, common multiwell plates are not suitable for the study of individual cells or even small groups of cells due to the large, relative to the cellular scale, size of the wells. Cells held in such wells either float about a solution or adhere to a well surface. When cells float about in a well, specific individual cells are not easily found for observation. When cells adhere to a well surface, the cells adhere to any location in the well, including anywhere on the bottom and walls of the well. Such variability in location makes high-throughput imaging (for example, for morphological studies) challenging as acquiring an individual cell and focusing thereon is extremely difficult. Such variability in location also makes high-throughput signal processing (for example, detection of light emitted by a single cell through fluorescent processes) challenging as light must be gathered from the entire area of the well, decreasing the signal to noise ratio. Further, a cell held in a well of a multiwell plate can be physically or chemically manipulated (for example, isolation or movement of a single selected cell or single type of cell, changing medium or introducing active entities) only with difficulty. Further, the loading of multiwell plates when loaded with one cell per well, expressed in terms of cells held singly in the wells per unit area is very low (about 1536 cells in 65 cm², or 24 cells cm²).

A number of methods and devices have been developed for the study of individual cells or a small number of cells as a group. Many such methods are based on using a miniwell-bearing device, such as a picowell-bearing device. A picowell-bearing device is a device for the study of cells that has at least one picowell bearing component for study of cells. A picowell-bearing component is a component having at least one, but generally a plurality of picowells, each picowell configured to hold at least one cell. The term picowell is general term that describes 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 is general and includes such features as dimples, depressions, tubes and enclosures. As used herein a picowell may also be any small well that is physical feature that localizes a cell or group of cells to a specific area on a planar surface of the picowell-bearing component using by physical confinement. The picowell-bearing component may be a carrier, for example, a substantially planar component such as a chip, plate, sheet or slide. Since cells range in size from about 1 microns to about 100 (or even more) microns 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 known in the art have dimensions of between about 1 micron up to about 200 microns, depending on the exact implementation. For example, a device designed for the study of single isolated 20 micron cells typically has picowells of dimensions of about 20 microns. In other cases, larger picowells are used to study the interactions of a few cells held together in one picowell. For example, a 200 micron picowell is recognized as being useful for the study of the interactions of two or three cells, see PCT Patent Application No. ILOl/100992 published as WO 03/1035824. One feature that increases the utility of a picowell bearing device is that each individual picowell is individually addressable. By individual addressability is meant that each picowell can be registered, found, observed or studied without continuous observation. For example, while cells are held in picowells of a picowell-bearing component, each cell is characterized and the respective picowell wherein that cell is held is noted. When desired, the observation component of the picowell-bearing device is directed to the location of the picowell where a specific cell is held. One method of implementing individual addressability is by the use of fiducial points or other features (such as signs or labels), generally on the picowell-bearing component. Another method of implementing individual addressability is by arranging the picowells in a picowell-array and finding a specific desired picowell by counting. Another method of implementing individual addressability is by providing a dedicated observation component for each picowell.

Mrksich and Whitesides, Ann. Rev. Biophys. Biomol. Struct. 1996, 25, 55-78; Craighead et al., J. Vac. Sci. Echnol. 1982, 20, 316; Singhvi et al., Science 1994, 264, 696-698; Aplin and Hughes, Analyt. Biochem. 1981, 113, 144-148 and U.S. Pat. No. 5,324,591 all teach devices including arrays of spots of cell-attracting or cell-binding entities on a plate. In such devices, the spots serve as picowells, binding cells through a variety of chemical bonds. In U.S. Patent No. 6,103,479, the picowell-bearing component is a transparent carrier provided with a non-uniform array of picowells, each well functionalized with chemical entities that bind to cells specifically or non- specifically. Each picowell is of approximately 200 to 1000 micron diameter and is configured to hold a plurality of cells. The inter picowell areas are hydrophobic so as not to attract cells. In addition to the carrier, a device of U.S. Pat. No. 6,103,479 is provided with a glass, plastic or silicon chamber-bearing plate in which individually addressable microfluidic channels are etched, the chamber-bearing plate configured to mate with the carrier. When mated, the carrier and chamber-bearing plate constitute a cassette in which each cell is bound to the carrier and isolated in a chamber provided with an individual fluid delivery system. Reagents are provided through the fluid delivery system and observed by the detection of fluorescence.

In PCT Patent Application No. US99/04473 published as WO 99145357 is taught a picowell-bearing component produced by etching the ends of a bundle of optical fibers (apparently of glass) while leaving the cladding intact to form a picowell- bearing component that is a bundle of tubes. The size of the hexagonal picowells is demonstrated to be as small as 7 micron wide, 5 micron deep and having a volume of about 1 .45xl0^"13 liter.

SUMMARY OF THE INVENTION

According to some embodiments of the present invention there is provided a method of improving wettability of a miniwell array. The method comprises providing a working surface having a miniwell array of plurality of miniwells, applying an alcoholic solution on top of the miniwell array, applying a buffer solution on top of the miniwell array, and loading a cell suspension on the miniwell array.

Optionally, the alcoholic solution comprises at least 70% ethanol.

Optionally, the applying comprises applying between 70 μΐ and 80 μΐ of the alcoholic solution for each 96 of the plurality of miniwells.

Optionally, the temperature of the alcoholic solution is below 15 Celsius degrees.

Optionally, the method further comprises cooling the miniwell array to a temperature of less than 0 Celsius degrees before the loading.

Optionally, the method further comprises waiting at least 3 minutes after the buffer solution is applied.

Optionally, the miniwell array is not dried after the buffer solution being applied.

Optionally, the method further comprises processing the miniwell array with plasma wetting before the loading.

Optionally, the plurality of miniwells comprises a member of a group consisting of picowells, nanowells, microwells, picopores, nanopores, and micropores.

Optionally, the providing comprises providing a Petri dish, laboratory slide, or microplate having the working surface thereon.

According to some embodiments of the present invention there is provided a method of improving wettability of a miniwell array. The method comprises providing a working surface having a miniwell array of plurality of miniwells, applying a buffer solution having a temperature of at least about 60 Celsius degrees on top of the miniwell array, applying a desired medium on top of the miniwell array, and loading a cell suspension on the miniwell array. Optionally, the applying comprises applying between 70 μΐ and 80 μΐ of the buffer solution for each 96 of the plurality of miniwells.

Optionally, the temperature of the buffer solution is above 75 Celsius degrees.

Optionally, the method further comprises waiting at least 3 minutes after the desired medium is applied.

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 DRAWINGS

Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. 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 embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

In the drawings:

FIG. 1 is a flowchart of a method of loading cells onto a miniwell array for facilitating analysis of individual cells, according to some embodiments of the present invention; FIG. 2 is a schematic illustration of a miniwell-bearing device having a working surface of a plurality of miniwells, according to some embodiments of the present invention;

FIG. 3 is another flowchart of another method of loading cells onto a miniwell array for facilitating analysis of individual cells, according to some embodiments of the present invention; and

FIGs. 4A-4E are images of a segment of a φ250 μιη 175 μηι deep embossing miniwell array in different wetting stages.

DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to method and system of analyzing individual cells and, more particularly, but not exclusively, to method and system of analyzing individual cells using a multi miniwell plate, such as, a nanowell plate, a picowell plate, a micropore plate, a nanopore plate, and/or a picopore plate.

According to some embodiments of the present invention there is provided a method of loading cells onto a miniwell array. The method is based on a working surface having a miniwell array of plurality of miniwells. An alcoholic solution, optionally, cold, is applied on top of the miniwell array. The alcoholic solution optionally comprises ethanol in a concentration of more than 60%, for example, between about 75% and about 95%, for example 90%. Optionally, the alcoholic solution is cooled to a below zero. Cycles degrees, for example -20°. Optionally, the process is now halt for more than 3 minutes, for example about 5 minutes. Now, a buffer solution, such as phosphate buffered saline (PBS), is applied on top of the miniwell array. Optionally, the buffer solution is not dried or washed before the cell suspension is loaded. This process increases the occupancy percentage of the miniwell array when the cell suspension is loaded.

According to some embodiments of the present invention there is provided another method of loading cells onto a miniwell array. This method is also based on a working surface that has a miniwell array of plurality of miniwells. First, a buffer solution, such as PBS, is applied on the miniwell array. The buffer solution is optionally heated to a temperature of about 60 Celsius degrees before being applied on top of the miniwell array. Optionally, the process is now halt for more than 3 minutes, for example about 5 minutes. Now, a desired medium is applied on top of the miniwell array. Optionally, the buffer- solution and the desired medium are not dried or washed before the cell suspension is loaded. Similarly to the method outlined above, this process also increases the occupancy percentage of the miniwell array when the cell suspension is loaded.

Optionally, the miniwell array underwent a plasma wetting to enhance its occupancy percentage when the cell suspension is loaded. Optionally, the plasma wetting is adjusted to the type of loaded cells.

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 drawings and/or the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

Reference is now made to FIG. 1, which is a flowchart of a method of loading cells onto a working surface of a miniwell-bearing device having a plurality of miniwells with relatively high occupancy percentage for facilitating analysis of individual cells, according to some embodiments of the present invention. The method enhances the wetting of the working surface by cell suspensions. As used herein, a miniwell means a picowell, a nanowell, a microwell a picopore, a nanopore, a micropore and the like.

When a miniwell-bearing device is used, cells are loaded onto a miniwell array in a cell suspension before being analyzed. The loading may be performed through a pipette. One of the common cells loading procedure is the administration of a cell suspension over the miniwell array. During loading, cells sediment from the top of the miniwell array, forced to settle down on the bottom of the wells due to the packing configuration of the cells, for example as described in U.S Patent Application No. 10/492,531 filed on June, 29, 2009. However, the occupancy percentage of the loaded miniwell-bearing device averagely does not exceed 90%. As the cells are provided in a cell suspension, the water repellency of the working surface of the miniwell-bearing device, which may be referred to herein as hydrophobicity, prevents from the cells to sediment into all the miniwells. In addition, the surface tension of some cell suspension droplets tends to minimize their surface trying to achieve a spherical shape. On contact with the miniwells array, either complete or incomplete wetting may occur depending on the structure of the miniwell array and the fluid tension of the droplets. The hydrophobicity may be defined according to the angle at which a cell suspension droplet meets the top plane of the miniwell array, an angle which may be referred to herein as a contact angle. The higher the contact angle is, the higher the hydrophobicity of a surface. By reducing the hydrophobicity of the miniwell array, the wetting thereof increases and the occupancy percentage averagely surpasses 90%.

First, as shown at 101, a mini well-bearing device, such as a plate, having a plurality of miniwells is provided. FIG. 2 depicts an exemplary miniwell-bearing device, for example as described in International Patent Application No. WO/2009/081409 filed on December 25, 2008, which is incorporated herein by reference. Optionally, the miniwell-bearing device 12 includes a working space 11 such as a transparent glass or polymer microscope slide, for example between 0.17 mm and 1 mm thick, 2.54 cm wide, and 7.62 cm long, though other standard sizes on which a circular 1 cm diameter miniwell array 20 for the study of cells has been embossed, for example from a UV- curable adhesive such as NOA-63 and/or NOA-81 (Norland Products Inc., Cranbury, NJ, USA, USA) which the specification thereof is incorporated herein by reference or etched. Optionally, each miniwell has a 20 micrometer (μηι) diameter and 12, 12.5, 15 and/or 20 μπι depth. The miniwell-bearing device may be any Petri dish, laboratory slide, and/or microplate having a working space with a miniwell array as described above, optionally embossed. The miniwell-bearing device may be any of the laboratory slides described in U.S Patent Application No. 2009/0111141, filed on April 20, 2006. U.S Patent Application No. 2009/0105095 filed on July 26,^' 2005, and U.S Patent Application No. 2008/0241874 filed on June 6, 2008 and/or U.S Patent Application No. 2008/0009051 filed on August 24, 2005, which are incorporated herein by reference.

Optionally, the embossing is performed, for example, by applying a drop of the fluid precursor of the adhesive and curing the fluid precursor while in contact with a die (made, for example, from a metal such as Teflon-coated metal, glass, PDMS or silicone rubber) having a negative of the well array. The drop of adhesive disperses between the slide and the die and forms a thin layer, for example, between 10 and 100 micrometers thick. After the adhesive has set, the die is peeled away or otherwise removed. Alternatively to embossing, the glass is etched. In an exemplary embodiment of the invention, a layer is provided over the glass, but not over the miniwells, or not over the active miniwells, to match the height of the wells to the conduits. Optionally or alternatively, the thickness of the conduit layers is selected to have desired properties (e.g., of flow rate and/or cell dislocation), taking into account the thickness of the miniwell area 20. In an exemplary embodiment of the invention, the entire glass slide is etched or embossed with miniwells, and these are covered over by adhesive tape or adhesive or another layer, except in area of the active miniwells.

It should be noted that any other element having a miniwell array may be provided.

Now, as shown at 102, a layer of a solution that comprises alcohol is applied on the miniwell array 20 of the miniwell-bearing device 10, referred to herein as alcoholic solution. Optionally, the alcoholic solution includes ethanol, isopropanol, propylene glycol, methanol, butanol, and/or propanol. Optionally, the alcoholic solution comprises at least 60% alcohol, for example a concentration of between about 70% and about 95%.

When loading is performed, the alcoholic solution increases the surface tension of the cell suspension about to be loaded on the miniwell array 20. The surface tension is increased as the attraction between molecules of the cell suspension and molecules of the alcoholic solution is stronger than the attraction between molecules of the cell suspension and molecules of an unlayered version of the miniwell array 20. For example, see data published in the Dormund data bank software and separation technology DDBST, www.ddbonline.ddbst.de/EE/ll%20SFT%20(Surface%

20Tension).shtml, which is incorporated herein by reference.

Additionally or alternatively, as shown at 103, the temperature of the miniwell array 20 of the miniwell-bearing device and optionally the alcoholic solution is reduced to below zero. For example, the temperature of the miniwell array 20 is reduced, for example by one or more cooling elements, such as cooling ribs and/or refrigerating ribs. In another example, the solution of alcohol applied on the miniwell array 20 of the miniwell-bearing device, for example ethanol, is an ice cooled solution, optionally in a temperature of less than -15°C, for example, about -20°C. For example, a solution of 70- 80 microliters (μΐ) is applied on a Petri dish device or on each 96 wells of a miniwell plate and 10-15 μΐ for product life cycle assessment (pLCA). The inventor surprisingly discovered that by cooling the temperature of the alcoholic solution and/or the miniwell array 20 with the alcoholic solution increases, when loading is performed, the occupancy percentage of the cell suspension in the loaded miniwell-bearing device is increased. It should be noted that this phenomena occur even though the surface tension of alcohol decreases with temperature. For example, see data published in the Dormund data bank software and separation technology (DDBST), www.ddbonline.ddbst.de/EE/ll %20SFT%20(Surface%20 Tension). shtml, which is incorporated herein by reference.

Optionally, as shown at 104, the process is halt for a waiting period, for example for about 5 minutes, so as to allow the alcoholic solution to spread over and into the miniwells of the miniwell array 20. The wetting of the miniwell array 20, which is defined as the contact between the alcoholic solution and the miniwell array 20, increases during the waiting period. In such a manner, the effect of the alcoholic solution on the loaded cell suspension is increased.

Now, as shown at 105, the miniwell array 20 of the miniwell-bearing device is washed, for example, with a buffer solution or solvent, such as water, a solution of PBS and/or another medium. Optionally, the miniwell array 20 is not dried before the loading.

Now, as shown at 106, a cell suspension is loaded onto the miniwell array 20 of the miniwell-bearing device, for example, as known in the art. The process depicted in FIG. 1 reduces the proportion of the loaded cells which settle down between holes rather than inside them and increases the loading efficiency, causes a reduction in the waste of cells, which might be curtail when the sample size is limited.

According to some embodiment of the present invention, the miniwell array 20 underwent a plasma wetting process. The plasma wetting process decreases the contact angle of the cell suspension on the miniwell array 20. This improves cell specific attachment by controlling surface chemical structures, surface energies and surface charge states; see P. I. John, Plasma Sciences and the Creation of Wealth, Tata- McGraw-Hill, New Delhi, 2005, which is incorporated herein by reference. In such an embodiment, the alcoholic solution may be applied in room temperature. For clarify, this process may be performed, simultaneously or sequentially, on a plurality of miniwell arrays which are embossed and/or etched on one or more working surfaces, such as shown at 11.

Reference is now also made to FIG. 3, which is a flowchart 200 of another method of loading cells onto a miniwell array 20, according to some embodiments of the present invention. Similarly to method depicted in FIG. 1, the method 200 enhances the wetting of the miniwell array 20 by cell suspensions. Block 101 is as described above.

Now, as shown at 202, a buffer solution or a solvent, such as a PBS, is applied on the miniwell array 20 and, as shown at 203, the provided miniwell-bearing device 10 is heated. Optionally, the solvent is heated so as to heat the provided miniwell-bearing device 10.

For example, the heated buffer solution is PBS solution warmed to between about 75 °C and about 80°C. Optionally, the volume of the applied heated solvent is between about 70 μΐ and about 80 μΐ for Petri dish device or 96 miniwells and between about 10 μΐ and 15 μΐ for a pLCA. The surface tension of the buffer solution decreases with the increase of temperature.

Now, as shown at 204, the process is halt, for example, for a waiting period, for example of about 5 minutes. The waiting period enhances the wetting of the miniwell array 20, which is the contact between the buffer solution and the miniwell array 20. In such a manner, the effect of the buffer solution on the cell suspension is increased, see L Courbin, Dynamics of wetting: from inertial spreading to viscous imbibitions. J. Phys.: Condens. Matter 21 (2009) 464127 (13pp), which is incorporated herein by reference.

Now, as shown at 205, the heated solvent is replaced with a desired medium, such as BPS, Roswell park memorial institute (RPMI) medium and/or any other form of medium used in cell culture.

Now, as shown at 204, a cell suspension is loaded onto the miniwell array 20 of the miniwell-bearing device, for example as known in the art. Optionally, the miniwell array 20 is not dried before the loading, which is optionally performed as described above, as shown at 106. For clarify, this process may be performed, simultaneously or sequentially, on a plurality of miniwell arrays which are embossed and/or etched on one or more working surfaces, such as shown at 12.

According to some embodiment of the present invention the miniwell array 20 underwent a plasma wetting process. The plasma wetting process decreases the contact angle of the cell suspension on the miniwell array 20. This improves cell specific attachment by controlling surface chemical structures, surface energies and surface charge states; see P. I. John, Plasma Sciences and the Creation of Wealth, Tata- McGraw-Hill, New Delhi, 2005, which is incorporated herein by reference. In such an embodiment, the buffer solution may be applied in temperature of about 60°C.

It is expected that during the life of a patent maturing from this application many relevant systems and methods will be developed and the scope of the term a miniwell and a miniwell array is intended to include all such new technologies a priori.

As used herein the term "about" refers to ± 10 %.

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

The phrase "consisting essentially of" means that the composition or method may include additional ingredients and/or steps, but only if the additional ingredients and/or steps do not materially alter the basic and novel characteristics of the claimed composition or method.

As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

The word "exemplary" is used herein to mean "serving as an example, instance or illustration". Any embodiment described as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments and/or to exclude the incorporation of features from other embodiments.

The word "optionally" is used herein to mean "is provided in some embodiments and not provided in other embodiments". Any particular embodiment of the invention may include a plurality of "optional" features unless such features conflict. 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 there between.

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.

Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

Reference is now made to an example, which together with the above description, illustrates some embodiments of the invention in a non limiting fashion. FIGS. 4A-4E is a set of images of a segment of a φ250 μηι 175 μηι deep embossing miniwell array. FIG. 4A depicts the deep embossing miniwell array before the loading of cells. FIG. 4B depicts the deep embossed miniwell array after being loaded and perfectly filled (wetted) with cell media in the loading method depicted in FIG. 1 and described above. FIGS. 4C-4E depict the same deep embossing miniwell array after being loaded without any preparation, for example without the preparation process which is part of the method depicted in FIG. 1 and described above. As can be shown from these images, loading cell media according to the method depicted in FIG. 1 achieve much better occupancy percentage at the miniwell array.

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.

All 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 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 of improving wettability of a miniwell array, comprising: providing a working surface having a miniwell array of plurality of miniwells; applying an alcoholic solution on top of said miniwell array;

applying a buffer solution on top of said miniwell array; and

loading a cell suspension on said miniwell array.

2. The method of claim 1, wherein said alcoholic solution comprises at least 70% ethanol.

3. The method of claim 1, wherein said applying comprises applying between 70 μΐ and 80 μΐ of said alcoholic solution for each 96 of said plurality of miniwells.

4. The method of claim 1, wherein the temperature of said alcoholic solution is below 15 Celsius degrees.

5. The method of claim 1, further comprising cooling said miniwell array to a temperature of less than 0 Celsius degrees before said loading.

6. The method of claim 1, further comprising waiting at least 3 minutes after said buffer solution is applied.

7. The method of claim 1, wherein said miniwell array is not dried after said buffer solution being applied.

8. The method of claim 1, further comprising processing said miniwell array with plasma wetting before said loading.

9. The method of claim 1, wherein said plurality of miniwells comprises a member of a group consisting of picowells, nanowells, microwells, picopores, nanopores, and micropores.

10. The method of claim 1, wherein said providing comprises providing a Petri dish, laboratory slide, or microplate having said working surface thereon.

11. A method of improving wettabilit of a miniwell array, comprising: providing a working surface having a miniwell array of plurality of miniwells; applying a buffer solution having a temperature of at least about 60 Celsius degrees on top of said miniwell array;

applying a desired medium on top of said miniwell array; and

loading a cell suspension on said miniwell array.

12. The method of claim 11, wherein said applying comprises applying between 70 μΐ and 80 μΐ of said buffer solution for each 96 of said plurality of miniwells.

13. The method of claim 11, wherein the temperature of said buffer solution is above 75 Celsius degrees.

14. The method of claim 11, further comprising waiting at least 3 minutes after said desired medium is applied.

15. The method of claim 11, wherein said miniwell array is not dried after said buffer solution being applied.

16. The method of claim 11, further comprising processing said miniwell array with plasma wetting before said loading.

17. The method of claim 11, wherein said plurality of miniwells comprises a member of a group consisting of picowells, nanowells, microwells, picopores, nanopores, and micropores.

18. The method of claim 11, wherein said providing comprises providing a Petri dish, laboratory slide, or microplate having said working surface thereon.