The present disclosure provides methods, device, assemblies, and systems for dispensing and visualizing single cells. For example, provided herein are systems and methods for dispensing a dispense volume into a plurality of wells of a multi-well device, where, on average, a pre-determined number of cells (e.g., 1-20) are present in the dispense volume, and determining, via a cellular label, the number of cells present in each of the plurality of wells. Such dispensing and cell detection may be repeated a number of times with respect to wells identified as having less than the pre-determined number of cells in order increase the number wells in the multi-well device containing the desired number (e.g., a single cell).
Geneticists are striving to characterize complex diseases like cancer, autoimmune and neurological disorders, but finding the underlying mechanisms driving these diseases has been elusive. Somatic mutations, spontaneous variants that accumulate in cells over a lifetime, are a major factor that drives disease onset and reoccurrence. As cells accumulate new mutations, they form polyclonal cell populations that co-exist with normal cells. Sequencing bulk cell populations can mask the underlying heterogeneity of these unique rare cell types, making it difficult to distinguish them from normal germline mutations. The best way to reveal these differences and visualize the clonal architecture is to sequence individual cells in the population. While single-cell sequencing can help uncover mechanisms of complex disease, traditional approaches are expensive, labor intensive, and require large sample input. What is needed are methods to isolate single cells that, for example, are amenable for use with multi-well devices.
Reference "Arraycount, an algorithm for automatic cell counting in microwell arrays" discloses an algorithm to analyze cell microarray images by automatically detecting a microwell array and then counting visible cells in each microwell. Reference
The invention is defined in the claims.
Hereinafter, the terminology "embodiment" relates to the disclosure and only forms part of the invention if it falls within the scope of the claims. present disclosure provides methods, device, assemblies, and systems for dispensing and visualizing single cells. For example, provided herein are systems and methods for dispensing a dispense volume into a plurality of wells of a multi-well device, where, on average, a pre-determined number of cells (e.g., 1-20) are present in the dispense volume, and determining, via a cellular label, the number of cells present in each of the plurality of wells. Such dispensing and cell detection may be repeated a number of times with respect to wells identified as having less than the pre-determined number of cells in order increase the number wells in the multi-well device containing the desired number (e.g., a single cell). In certain embodiments, single-cell analysis (e.g., sequencing) is performed in the wells with a single cell.
Embodiments of the present disclosure provide a method to rapidly select, dispense and visualize and analyze single cells, comprising: a) using a Poisson distribution to dilute and dispense cells at a single cell per well concentration into a plurality of wells of a microfluidic device; and b) visualizing said wells using a microscope and rapid microscope image analysis software to detect, visualize, and select those wells bearing single cells. In some embodiments, the method further comprises the step of performing additional dispense cycles of either cell-containing solutions into individual wells specifically identified as having a cell count of zero (e.g., repeated one or more times) or more than 1 cell. In other embodiments, image analysis and chip mapped positions (e.g., via a dispense map file) direct selection of wells for addition of reagents and materials permitting further biochemical or biophysical and / or cell viability based-investigation.
Further embodiments provide a system, comprising: a) a microfluidic device; and b) a microscope component comprising a microscope and computer software and a computer processor configured to perform rapid microscope image analysis software to detect, visualize, and select wells bearing single cells. In certain embodiments, the system further comprises a dispense map file.
In certain embodiments, the methods further comprise c) dispensing a first reagent into at least one (e.g., 1 ... 5 ... 10 ... 500 ... 5000 ... 10,000; all or most wells in multi-well chip) of said selected wells bearing the defined number of cells (e.g., in wells with one and only one cell in a particular well). In some embodiments, the first reagent is selected from the group consisting of: cell culture components, cell health measuring reagents, cell differentiation reagents, drugs, WTA, WGA, nucleic acid, proximity ligation assay (PLA) reagents, cell surface markers, labeled antibodies, unlabeled antibodies, detection reagents associated with antibody detection, nanomaterials, reagents for lysing cells, reagents for detection and/or resolution of nucleic acids or lipids or carbohydrates or protein cell components. In particular embodiments, the defined number of cells per well is 1, or 2, or any number more than 1, or zero.
In certain embodiments, provided herein are methods comprising: a) dispensing a dispense volume of a cell suspension into each of a plurality of wells in a multi-well device, wherein the cell suspension comprises cells present in the cell suspension at a concentration such that, on average, X cell(s) (e.g., 0.1 to 100 cells) is/are present in the dispense volume, and wherein the dispensing is performed with a liquid dispensing system; b) labeling at least a portion of the cells with a first detectable label before and/or after the dispensing; and c) determining the number of cell(s) present in each of at least some of the plurality of wells (e.g.., by visualizing stained cells, or sequencing methods).
In some embodiments, X is between 1 and 100 cells (e.g., 1 ... 5 ... 10 ... 15 ... 20 ... 30 ... 40 ... 50 ... 60 ... 70 ... 80 ... 90 ... or 100 cells). In certain embodiments, X is one. In further embodiments, the number of cell(s) in at least one of the plurality of wells is determined to be zero (e.g., at least 20% of the wells result in liquid dispensed, but no cell). In other embodiments, the number of cells in at least one of the plurality of wells is between zero and forty. In further embodiments, the methods further comprise: d) dispensing a dispense volume of the cell suspension into at least some of the wells determined to have less than X cells.
In other embodiments, the methods further comprise: e) dispensing a first additional volume which is approximately equal to the dispense volume, but is free of cells, into at least some of the wells determined to have X cells or more than X cells (e.g., such that the volume of the liquid dispensed into all the wells on a chip remains the same). In other embodiments, the methods further comprise: e) determining the number of cell(s) present in each of the wells previously determined to have less than X cells. In further embodiments, the methods further comprise: f) dispensing a dispense volume of the cell suspension into wells determined to have less than X cells. In other embodiments, the methods further comprise: g) dispensing a second additional volume which is equal to (or about equal to) the dispense volume, but is free of cells, into at least some of the wells determined to have X cells or more than X cells.
In some embodiments, the methods further comprise: labeling at least a portion of the cells with a second detectable label before and/or after the dispensing and/or determining steps. In additional embodiments, the methods further comprise: determining which, if any, of the plurality of wells contain a cell with the second label. In additional embodiments, the determining comprises visualizing the first detectable label in each of the at least some of the plurality of wells. In some embodiments, the determining comprising capturing a first image of at least some of the plurality of wells using an image capturing system, wherein the first image indicates the number of cells present in each of the wells in the first image. In additional embodiments, the image capturing system comprises a camera linked to a magnifying lens. In other embodiments, the image capturing system further comprises a computer, wherein the computer comprises a computer processor, computer memory, and image analysis software. In further embodiments, the image analysis software is configured to analyze the first image and generate: i) a first list of which wells contain less than X cells, ii) a second list of which wells contain X cells; and iii) third list of wells than contain more than X cells. In additional embodiments, the image analysis software generates instructions, for the liquid dispensing system, to dispense a dispense volume into each of the first list of wells. In further embodiments, the methods further comprise: d) dispensing, based on the first image, a dispense volume into at least some of the wells with less than X cells. In other embodiments, the methods further comprise: e) capturing a second image of at least some of the wells with less than X cells prior to the dispensing, wherein the second image indicates, via the first detectable label, the number of cell(s) present in each of the wells with less than X cells prior to the dispensing.
In other embodiments, the liquid dispensing system comprises: i) a plurality of fluidic dispensing channels, ii) a source container containing the cells suspension; and iii) a robotic movement component attached to the fluidic dispensing channels, wherein the robotic movement component is moveable between the source container and the multi-well device. In other embodiments, the liquid dispensing system is automated and is configured to receive instructions from the image analysis software. In some embodiments, the first detectable label comprises a cell stain. In certain embodiments, the first detectable label is selected from the group consisting of: cell culture components, cell health measuring reagents, cell differentiation reagents, drugs, WTA, WGA, nucleic acid, proximity ligation assay (PLA) reagents, cell surface markers, labeled antibodies, unlabeled antibodies, detection reagents associated with antibody detection, nanomaterials, reagents for lysing cells, reagents for detection and/or resolution of nucleic acids or lipids or carbohydrates or protein cell components. In particular embodiments, the cells are epithelial cells, organ cells, skin cells, bacterial cells, human cells, circulating cancer cells, stem cells, hematopoietic stem cells, or any other type of cells.
In certain embodiments, the methods further comprise: d) conducting a biological reaction in at least one of the wells determined to have X cell(s). In other embodiments, the methods further comprise: d) conducting a biological reaction in at least 10% of the wells determined to have X cell(s). In further embodiments, the biological reaction comprises a sequencing reaction. In additional embodiments, the sequencing reaction employs nucleic acid barcode sequences. In further embodiments, the multi-well device comprises at least 30 wells (e.g., 30 ... 75 ... 150 ... 400 ... 1000 ... 4,000 ... 10,000 ... 20,000 ... or 30, wells). In other embodiments, the multi-well device comprises at least 1000 wells. In particular embodiments, the multi-well device comprises a multi-well chip (e.g., with nano or micro wells). In other embodiments, the second detectable label is specific for circulating cancer cells and/or cancer stem cells and/or for any other type of target cell desired to be detected. In additional embodiments, the second detectable label comprises an antibody, its conjugate or an antigen binding portion of an antibody.
In certain embodiments, the cells in the cell suspension are purified from tumor or normal tissue. In other embodiments, dispensing volume is between 5 and 9000 nl (e.g., 5 ... 25 ... 100 ... 500 .. 1000 ... 4000 ... 9000 nl). In further embodiments, the labeling the cells is before the dispensing. In additional embodiments, the labeling the cells is after the dispensing.
In certain embodiments, provided herein are systems and kits comprising: a) a multi-well device comprising a plurality of wells; b) a liquid dispensing system configured to dispense a dispense volume into each of the plurality of wells; and c) a cell suspension comprising cells present in the cell suspension at a concentration such that, on average, X cell(s) (e.g., 0.1 to 100 cells) is/are present in the dispense volume.
In other embodiments, X is between 1 and 20 cells (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ... 15 ... or 20 cells). In further embodiments, X is one. In particular embodiments, the liquid dispensing system is configured to dispense the dispense volume into each of the plurality of wells in an automated or semi-automated fashion. In other embodiments, the systems further comprise: d) an image capturing system that captures a first image of at least some of the plurality of wells, and determines the number of cells present in each of the wells in the first image. In other embodiments, the image capturing system comprises a camera linked to a magnifying lens. In further embodiments, the image capturing system further comprises a computer, wherein the computer comprises a computer processor, computer memory, and image analysis software. In further embodiments, the image analysis software is configured to analyze the first image and generate: i) a first list of which wells contain less than X cells; and/or ii) a second list of which wells contain X cells; and/or iii) a third list of which wells contain more than X cells. In some embodiments, the image analysis software if configured to generate instructions for the liquid dispensing system to dispense a dispense volume into each of the first list of wells. In other embodiments, the liquid dispensing system comprises: i) a plurality of fluidic dispensing channels, ii) a source container containing the cells suspension; and iii) a robotic movement component attached to the fluidic dispensing channels, wherein the robotic movement component is moveable between the source container and the multi-well device. In additional embodiments, the systems further comprise a first and/or second detectable labels capable of labeling the cells in the cell suspension (e.g., one label to label all of the cells, and another to label cells of interest, such as stem cells or circulating tumor cells). In other embodiments, the cells in the cell suspension are labeled with a first and/or second detectable label. In certain embodiments, the first detectable label comprises a cell stain. In some embodiments, the first detectable label is selected from the group consisting of: cell culture components, cell health measuring reagents, cell differentiation reagents, drugs, WTA, WGA, nucleic acid, proximity ligation assay (PLA) reagents, cell surface markers, labeled antibodies, unlabeled antibodies, detection reagents associated with antibody detection, nanomaterials, reagents for lysing cells, reagents for detection and/or resolution of nucleic acids or lipids or carbohydrates or protein cell components.
In some embodiments, provided herein are methods comprising: a) dispensing a dispense volume of a cell suspension into each of a plurality of wells in a multi-well device, wherein the cell suspension comprises cells present in the cell suspension at a concentration such that, on average, one cell is present in the dispense volume, and wherein the dispensing is performed with a liquid dispensing system; b) labeling the cells with a first detectable label before and/or after the dispensing; and c) determining if zero, one, or multiple (e.g., two, three, four, or more) cells are present in each of at least some of the plurality of wells (e.g., visualizing with a microscope; generating a digital image; or generating computer data; that indicates the number of cells in each well). In certain embodiments, the methods further comprise d) generating a report (e.g., computer code, computer file, written report, or electronic report) that indicates if there is zero, one, or multiple cells in each of the at least some of the plurality of wells.
In particular embodiments, the methods further comprise: d) dispensing a dispense volume of the cell suspension into at least one of the wells determined to have zero cells. In other embodiments, the methods further comprise: d) dispensing a dispense volume of the cell suspension into at least 50% (e.g., 50% ... 67% ... 75% ... 85% ... 95% ... or 100%) of the wells determined to have zero cells. In other embodiments, the methods further comprise: e) determining if zero, one, or multiple cells are present in each of the wells that were previously determined to have zero cells. In additional embodiments, the methods further comprise: f) dispensing a dispense volume of the cell suspension into wells twice determined to have zero cells. In other embodiments, the methods further comprising: g) determining that at least 50% of all of said plurality of wells present in said multi-well device have a single cell (e.g., at least 50% ... 55% ... 60% ... 65% ... 75% ... 80% ... 85% ... or 90%).
In some embodiments, the determining comprises visualizing the detectable label in each of the at least some of the plurality of wells. In other embodiments, the determining comprising capturing a first image of at least some of the plurality of wells using an image capturing system, wherein the first image indicates if zero, one, or multiple cells are present in each of the wells in the first image. In other embodiments, the image capturing system comprises a camera linked to a magnifying lens. In further embodiments, the image capturing system further comprises a computer, wherein the computer comprises a computer processor, computer memory, and image analysis software. In additional embodiments, the image analysis software is configured to analyze the first image and generate: i) a first list of which wells contain zero cells and/or ii) a second list of which wells contain one cell. In further embodiments, the image analysis software generates instructions, for the liquid dispensing system, to dispense a dispense volume into each of the first list of wells. In some embodiments, the methods further comprise d) dispensing, based on the first image, a dispense volume into at least some of the wells with zero cells. In additional embodiments, the methods further comprise: e) capturing a second image of at least some of the wells with zero cells prior to the dispensing, wherein the second image indicates, via the first detectable label, if zero, one, or multiple cells are present in each of the wells with zero cells prior to the dispensing. In certain embodiments, methods further comprise centrifuging the multi-well device (e.g., to collect cells in the bottom of each well) prior to the determining step.
In certain embodiments, the liquid dispensing system comprises: i) a plurality of fluidic dispensing channels, ii) a source container containing the cells suspension; and iii) a robotic movement component attached to the fluidic dispensing channels, wherein the robotic movement component is moveable between the source container and the multi-well device. In other embodiments, the liquid dispensing system is automated and is configured to receive instructions from the computer (e.g., from image analysis software). In other embodiments, the first detectable label comprises a cell stain (e.g., Hoechst stain). In other embodiments, the first detectable label is selected from the group consisting of: cell culture components, cell health measuring reagents, cell differentiation reagents, drugs, WTA, WGA, nucleic acid, proximity ligation assay (PLA) reagents, cell surface markers, labeled antibodies, unlabeled antibodies, detection reagents associated with antibody detection, nanomaterials, reagents for lysing cells, reagents for detection and/or resolution of nucleic acids or lipids or carbohydrates or protein cell components. In certain embodiments, the first detectable label is specific for circulating cancer cells and/or stem cells and/or cancer stem cells (e.g., specific for CD44, CD133, ALDH1, etc.).
In some embodiments, the cells are circulating cancer cells. In other embodiments, the cells are stem cells. In further embodiments, the cells are cancer stem cells (e.g., breast CSCs, ovarian CSCs, colon CSCs, prostate CSCs, pancreatic CSCs, etc.). In further embodiments, the methods further comprise: d) conducting a biological reaction in at least one of the wells determined to have a single cell. In other embodiments, the methods further comprise: d) conducting a biological reaction in at least 50% (e.g., at least 50% ... 70% ... 90% ... or 100%) of the wells determined to have one cell. In some embodiments, the biological reaction comprises a sequencing reaction, and/or a PCR reaction, and/or a cell lysis reaction). In particular embodiments, the sequencing reaction employs nucleic acid barcode sequences.
In certain embodiments, the multi-well device comprises at least 50 wells (e.g., 50 ... 100 ... 150 ... 400 ... 689 ... 900 ... or more). In additional embodiments, the multi-well device comprises at least 1000 wells (e.g., 1000 ... 1500 ... 2500 ... 5000 ... 5184 ... 10,000 .. 20,000 ... or more). In other embodiments, the multi-well device comprises a multi-well chip.
In particular embodiments, the methods further comprise labeling at least some of the cells with a second detectable label before and/or after the dispensing in step a). In certain embodiments, the second detectable label is specific for circulating cancer cells and/or cancer stem cells. In other embodiments, the second detectable label comprises an antibody or an antigen binding portion of an antibody. In some embodiments, the cells in the cell suspension are purified from tumor tissue. In other embodiments, the dispensing volume is between 25 and 500 nl or between 500 nl and 1 ul. In further embodiments, the labeling the cells is before the dispensing. In further embodiments, the labeling the cells is after the dispending.
In some embodiments, provided herein are systems comprising at least two of the following: a) a multi-well device comprising a plurality of wells; b) a liquid dispensing system configured to dispense a dispense volume into each of the plurality of wells; and c) at least one component selected from: i) a cell suspension comprising cells present in said cell suspension at a concentration such that, on average, X cell(s) is/are present in said dispense volume, ii) a dispense map file that provides instructions to said liquid dispensing system for dispensing liquid into cells in said multi-well device that contain X cells.
In certain embodiments, the liquid dispensing system is configured to dispense the dispense volume into each of the plurality of wells in an automated or semi-automated fashion (see, e.g.,
In certain embodiments, the systems further comprise: d) an image capturing system configured to capture a first image of at least some of the plurality of wells, and determine if zero, one, or multiple cells are present in each of the wells in the first image. In other embodiments, the image capturing system comprises a camera linked to a magnifying lens (e.g., a CCD camera linked to microscope optics). In other embodiments, the image capturing system further comprises a computer, wherein the computer comprises a computer processor, computer memory, and image analysis software. In some embodiments, the image analysis software is configured to analyze the first image (and second, third, fourth, etc. images) and generate: i) a first list of which wells contain zero cells and/or ii) a second list of which wells contain one cell. In certain embodiments, the image analysis software if configured to generate instructions for the liquid dispensing system to dispense a dispense volume into each of the first list of wells.
In particular embodiments, the liquid dispensing system comprises: i) a plurality of fluidic dispensing channels, ii) a source container containing the cells suspension; and iii) a robotic movement (e.g., arm) component attached to the fluidic dispensing channels, wherein the robotic movement component is moveable between the source container and the multi-well device.
In some embodiments, the systems further comprise a first and/or second detectable label capable of labeling the cells in the cell suspension. In other embodiments, the cells in the cell suspension are labeled with a first and/or second detectable label. In particular embodiments, the detectable label comprises a cell stain. In some embodiments, the first detectable label is selected from the group consisting of: cell culture components, cell health measuring reagents, cell differentiation reagents, drugs, WTA, WGA, nucleic acid, proximity ligation assay (PLA) reagents, cell surface markers, labeled antibodies, unlabeled antibodies, detection reagents associated with antibody detection, nanomaterials, reagents for lysing cells, reagents for detection and/or resolution of nucleic acids or lipids or carbohydrates or protein cell components.
In certain embodiments, the multi-well device comprises at least 50 wells. In other embodiments, the multi-well device comprises at least 1000 wells. In further embodiments, the multi-well device comprises a multi-well chip. In other embodiments, the systems further comprise a second detectable label that is specific for circulating cancer cells and/or cancer stem cells. In other embodiments, the second detectable label comprises an antibody or an antigen binding portion of an antibody. In certain embodiments, the cells in the cell suspension are purified from tumor tissue. In other embodiments, the dispensing volume is between 25 and 500 nl, or between 50 nl and 1 µl.
The present disclosure provides methods, device, assemblies, and systems for dispensing and visualizing single cells. For example, provided herein are systems and methods for dispensing a dispense volume into a plurality of wells of a multi-well device, where, on average, a pre-determined number of cells (e.g., 1-20) are present in the dispense volume, and determining, via a cellular label, the number of cells present in each of the plurality of wells. Such dispensing and cell detection may be repeated a number of times with respect to wells identified as having less than the pre-determined number of cells in order increase the number wells in the multi-well device containing the desired number (e.g., a single cell). In certain embodiments, single-cell analysis (e.g., sequencing) is performed in the wells with a single cell.
Cell heterogeneity is a general feature of biological tissues and cells in general. Geneticists are striving to characterize complex diseases including cancer, autoimmune and neurological disorders. However, determining the underlying mechanisms driving these diseases remains elusive. As cells accumulate new mutations, they may form polyclonal cell populations that co-exist with normal cells. As a consequence, sequencing bulk cell populations can mask the underlying heterogeneity of these unique rare cell types, rendering it difficult to "find needles in the haystack." An alternate approach to reveal intra-population/inter-cell differences is to assess the nucleic acid sequences in selected individual cells from a population. Single-cell analyses have been used to define subpopulations with distinct DNA and RNA expression profiles. In summary, it is widely believed that single-cell analysis may uncover previously "hidden" mechanisms of complex disease.
A core requirement in the single-cell field is to clearly and unambiguously detect that the sample being assessed only contains a single cell. Traditional single cell isolation approaches including: FACS instrumentation (Becton Dickinson) microfluidic capture (Fluidigm) limited or widely dispersed cell dilution methods are too expensive, labor intensive, require large sample input methods, and do not readily scale into the need for more cells within standard molecular biology workflows. On the other hand, random deposition of cells may be unpredictable /stochastically distributed, making predictions of cell distributions unwieldy.
An alternate approach is to dispense cells into reaction chambers such that the average over many such dispenses results in a single cell being dispensed. A statistical description of this phenomenon is known as the Poisson distribution. In theory, dispensing a single cell per well (n= exactly 1 cell, but not 0, 2, 3, 4, 5, 6 etc cells) is constrained by theta theoretical maxima = of 36.8% of wells will contain exactly 1 cell. However, the Poisson distribution however can be leveraged to alter the input cell concentration to a very wide range of occupancy rates. A tradeoff in optimizing for a desired number of cells per well (i.e., 1 cell / well) exists. More specifically, optimizing to achieve a desired ratio (10:1 ratio where lambda approaches 0.185) of wells containing a single cell may result in an unsatisfactory percentage of wells without any cells (> 82%). A similar approach attempting to specifically target 1 cell per well alongside a size separation approach has recently been reported. However, in that case, possibly due to the physical constraints in the cell capture device employed, only 10% of wells contained single cells. However, that methodology is complex and requires specialized reagents. In that case, possibly due to physical constraints of that system, only 10% of wells contained single cells. Critically, the size constriction component of such devices cannot exclude the possibility that each well contains only a single cell.
Emulsion-based methods, for selecting single cells include placing cells in water-in-oil emulsions. Such systems offer the advantage of insulating against cross contamination. However, these oil-separated compartments are difficult to manipulate. Moreover, such emulsions often require vortexing that depend on standard unselected Poisson statistics to achieve clonality. However, these approaches lead to only a small fraction of occupied and a large number of unoccupied compartments. As a consequence, emulsions are generated in microfluidic systems which increase cost and bear the significant disadvantage that once an emulsion is formed, it is difficult to exchange additional material in wells in a controlled fashion. Moreover, emulsion PCR is optionally performed using conditions that are not easily generalizable.
It is difficult to isolate single cells without expensive and complicated equipment. Moreover, such system cannot typically capture more than 384 single cells. As a result, provided herein are statistical methods combined with microscopy to visualize the cells in microfluidic chips (e.g., those sold by Wafergen, Freemont, CA). Work conducted during the development of embodiments of the present disclosure employed the Poisson distribution to dispense cells, and solves a problem associated with a Poisson distribution. In brief, the Poisson distribution is statistically limiting (Table 1) for dispensing single cells because the microchip will either have far too many wells with zero cells (vast underutilization of chip capacity) or too many wells with two or more cells (undermining and confounding the "single-cell" analysis).
This disclosure overcomes the statistical limitations of technologies that have, for example, (a) only a single dispense opportunity at their disposal, and/or, (b) no visual confirmation of which wells actually contain single cells. In some embodiments, it is established with 99% confidence (or greater) that the samples submitted for downstream genomic or other analysis contain one and only one cell. The present disclosure provides, in certain embodiments, a simple, robust commercially applicable method that employs Poisson statistics, robotic liquid handling and microscopy based image analysis (e.g., in combination with software) in an iterative manner to rapidly and accurately identify single cells in wells of a multi-well device (e.g., >2,000 or more single cells per chip in a 5,184 well format). Such methods are scalable to larger numbers as desired. Solving this problem has considerable commercial, scientific and ultimately medical value.
This disclosure describes the use of microfluidic (e.g., WaferGen SmartChip) technology to isolate and process single cells for either DNA, RNA, and/or other applications. Cells are diluted using Poisson statistics such that on average 1 cell per dispense volume is dispensed. In certain embodiments, microscopy (e.g., magnifying optics) is used to visualize each well and directly know if that well contains a single cell. In certain embodiments, multisample dispensers (e.g., as shown in
In certain embodiments, when wells are identified as having received zero cells, a a second (and third) optional Recursive Poisson Distribution (RPD) step may be employed to circumvent the statistical limitations of the Poisson distribution, thereby raising single cell occupancy rates on-chip from a theoretical maxima of 37% to > 50%. The RPD in this disclosure refers to the iterative cycle of, (a) dispensing cell-containing solutions into reaction vessels (wells, chambers, etc.) in a chip, (b) visualization of cells on-chip in individual wells, (c) identifying the on-chip cell counts (equal to zero, equal to one, and greater than one) in individual wells by software-aided microscopy, and, (d) performing additional dispense cycles of cell-containing solutions into individual wells specifically identified in the previous round as having a cell count of zero. The objective of RPD is to maximize the number of occupied reaction vessels (wells, chambers, etc.) containing a single-cell (or some other desired number of cells) above the theoretical limitations Poisson distribution for a single dispense. This disclosure does not place a limit on the number of iterative cycles.
In summary, the utility, straight forward nature and robustness of this approach commend it for use, for example, in a variety of situations where commercially relevant R&D, screening, compound analysis, and/or diagnostics on single cell nucleic acids are to be performed.
In some embodiments, this disclosure describes methods of isolating individual cells and transferring them into individual wells of microfluidic (e.g., Wafergen's SmartChip wells). For example, cells are first stained with the commonly available supravital dye Hoechst 33342 that emits a strong blue fluorescence when bound to DNA. The cells are counted, diluted to contain 1 cell per dispense volume, added to a source container (e.g. 384 well plate) and dispensed directly into a deep-well chip using a robotic micro-liquid dispenser (e.g., Wafergen Multiple Sample Nano Dispenser (MSND)). The multi-well chip is centrifuged to collect cells in the bottom of each well. Each well is then visualized by automated microscopy and image analysis to categorically confirm if either 0, 1, 2, 3 or 4 cells are dispensed in each well. This quality control step is both important and unique as it rapidly and definitively identifies the contents of wells in each of the wells in the chip. This exemplary process is illustrated in
In word conducted during development of embodiments of the present disclosure, it was routinely observed that between 14% and 29% of a 5184 well chip (n=5 chips seeded with different concentrations of cells) had single cells. These single cell occupancy rates convert to between 725 to 1451 single cells in a single 5184 well chip. Those occupancy rates are between 7 and 15 times higher than the single cell market leader (Fluidigm) single cell 96 cell recovery rates. An experiment was performed in which the input cells were titrated across a four-fold relative concentration range in order to assess the "on-chip" cell count distribution profile following a single dispense using the MSND (
In this study, the single cell count was 29.8% for the cell concentration at the apparent maxima (e.g., for the Relative Concentration of Input Cells = 3.0). Overall, the relative distributions of cell counts (e.g., equal to zero, equal to one, and greater than one) over the input cell concentration range examined adequately mirror a Poisson distribution (see Table 1).
In certain embodiments, the methods and systems of the present disclosure have the benefit of being relatively straight forward to perform, and highly robust with minimal training. The disclosure offers numerous advantages relative to current single cell isolation approaches, removing the need for complex and expensive low throughput microfluidics system (e.g., Fluidigm C1 chip) and elimination of excessive physical manipulation of the cells (FACS). It has the benefit of being an open system such that a variety of Biochemical and Molecular Biology protocols can be performed in these wells. Emulsion PCR is not necessary. Moreover, it isolates significantly more single cells than other systems with minimal training or equipment required. In certain embodiments, performing one or more recursive Poisson distribution step(s) is contemplated to circumvent the statistical limitations (Table 1) of the Poisson distribution raising single cell occupancy rates to, for example, greater than 50% (e.g., greater than 50% ... 60% ... 75% ... 85% ... or more).
The theoretical basis for increasing the single cell occupancy rate above the statistical limitations of the Poisson distribution for a single dispense step (
In certain embodiments, automated systems and methods are employed to determine which wells of a multi-well testing device contain the desired number of dispensed cells (e.g., 1 cell per well). In particular embodiments, software for automated imaging and automated well selection is employed to determine which cells contain the desired number of cells. In this regard, a dispense map (e.g., as shown in
One exemplary embodiment, with reference to
Confirmation of orientation and assessment of well location is achieved by analysis of a dispensed dye (e.g., fluorescent dye) located in known well positions (fiducial wells) of each chip (see
Once well locations are identified image analysis is performed to identify the number of objects within each well which meet automated or user defined image analysis thresholds that define the presence or absence of a cell(s). These parameters may include, but are not limited to, object and background fluorescence intensity, size and roundness. In addition, results from multiple wavelengths may be combined for each object to assess parameters such as cell viability. Image analysis results are subsequently summarized by array position (row and column position) and associated with barcodes and image analysis values of any given array position permitting well-level user review.
Importantly, the well selection software (e.g., Wafergen's CellSelect software) will generate a report indicating if a well is a candidate single cell or other user defined well, to receive a dispense of a subsequent reagent. These wells are then used to construct the dispense map or file or filter file used in follow-on biochemistry steps.
Example output from CellSelect software is presented in
Upon completion of analysis of all imaged wells, a reagent dispense map (aka a "dispense or filter file") or is created. Well selection and subsequent dispense file mapping can be completely automated or user-edited in CellSelect software (or other well selection software) to either include or exclude wells per user requirements. This dispense map is used to program a dispensing component, such as Wafergen's MSND, to selectively deliver reagents to appropriate wells for further processing such as reverse transcription.
The present disclosure is not limited by the type of cells that are employed. In certain embodiments, cancer cells, circulating cancer cells, stem cells, and cancer stem cells are employed. Most cancer deaths appear to be caused by metastatic spread and growth by circulating tumor cells at distant organs. Circulating tumor cells (CTCs), CTC clusters (two or more individual CTCs bound together), and cancer stem cells (CSCs) may be initially localized, latent systemic, or post-adjuvant treatment depleted. Consequently, CTCs and the relevant stem cells are frequently present at low numbers within a large background of normal non-cancerous cells. The low frequency of these cells generates a complex "needle in a haystack" analysis problem for detecting the required cancer cell signal within the large 'noise" background. Detection of cancer cell specific cell surface markers and analysis of these cells is deeply relevant to understanding the biology of metastatic spread. The methods and systems provided herein allow isolation and analysis of such important cancer cells.
Single-cell, multiple-cell and cell clusters may initially be either enriched or depleted from a cell or tissue milieu or population, based on the presence of antigenic / phenotypic cell-surface or intra-cellular markers including but not restricted to: protein, lipid, carbohydrate (i.e. glycosylation) post-translational modifications of those moieties, nucleic acids and their modifications, or varying combinations of these moieties. Detection of cell surface markers in single cells -including cancer cells- and transferring those cells into discrete individual wells of a microfluidic device (e.g., Wafergen's SmartChip wells) is performed with the methods and systems described herein. In other embodiments, labelled cells may be dispensed directly into wells and antigenic moieties detected directly in chip via standard or automated microscopy using a variety of widely available fluorescence filters.
Methods of circulating tumor cell (CTC) enrichment and visualization are known in the art and may be employed for generating (and later visualizing) the initial cell suspension employed in the methods and systems described herein. For example, Table 1 of
The present disclosure is not limited by the type of multi-well testing devices (e.g., plates or chips) employed. In general, such devices have a plurality of wells that contain, or are dimensioned to contain, liquid (e.g., liquid that is trapped in the wells such that gravity alone cannot make the liquid flow out of the wells). One exemplary chip is WAFERGEN's 5184-well SMARTCHIP. Other exemplary chips are provided in
The overall size of the multi-well devices may vary and it can range, for example, from a few microns to a few centimeters in thickness, and from a few millimeters to 50 centimeters in width or length. Typically, the size of the entire device ranges from about 10 mm to about 200 mm in width and/or length, and about 1 mm to about 10 mm in thickness. In some embodiments, the chip is about 40 mm in width by 40 mm in length by 3 mm in thickness.
The total number of wells (e.g., nanowells) on the multi-well device may vary depending on the particular application in which the subject chips are to be employed. The density of the wells on the chip surface may vary depending on the particular application. The density of wells, and the size and volume of wells, may vary depending on the desired application and such factors as, for example, the species of the organism for which the methods of this disclosure are to be employed.
The present disclosure is not limited by the number of wells in the multi-well device or the number of wells in the multi-well source device. A large number of wells may be incorporated into a device. In various embodiments, the total number of wells on the device is from about 100 to about 200,000, or from about 5000 to about 10,000. In other embodiments the device comprises smaller chips, each of which comprises about 5,000 to about 20,000 wells. For example, a square chip may comprise 125 by 125 nanowells, with a diameter of 0.1 mm
The wells (e.g., nanowells) in the multi-well devices may be fabricated in any convenient size, shape or volume. The well may be about 100 µm to about 1 mm in length, about 100 µm to about 1 mm in width, and about 100 µm to about 1 mm in depth. In various embodiments, each nanowell has an aspect ratio (ratio of depth to width) of from about 1 to about 4. In one embodiment, each nanowell has an aspect ratio of about 2. The transverse sectional area may be circular, elliptical, oval, conical, rectangular, triangular, polyhedral, or in any other shape. The transverse area at any given depth of the well may also vary in size and shape.
In certain embodiments, the wells have a volume of from about 0.1 nl to about 1 ul. The nanowell typically has a volume of less than 1 ul, preferably less than 500 nl. The volume may be less than 200 nl, or less than 100 nl. In an embodiment, the volume of the nanowell is about 100 nl. Where desired, the nanowell can be fabricated to increase the surface area to volume ratio, thereby facilitating heat transfer through the unit, which can reduce the ramp time of a thermal cycle. The cavity of each well (e.g., nanowell) may take a variety of configurations. For instance, the cavity within a well may be divided by linear or curved walls to form separate but adjacent compartments, or by circular walls to form inner and outer annular compartments.
A well of high inner surface to volume ratio may be coated with materials to reduce the possibility that the reactants contained therein may interact with the inner surfaces of the well if this is desired. Coating is particularly useful if the reagents are prone to interact or adhere to the inner surfaces undesirably. Depending on the properties of the reactants, hydrophobic or hydrophilic coatings may be selected. A variety of appropriate coating materials are available in the art. Some of the materials may covalently adhere to the surface, others may attach to the surface via non-covalent interactions. Non-limiting examples of coating materials include silanization reagent such as dimethychlorosilane, dimethydichlorosilane, hexamethyldisilazane or trimethylchlorosilane, polymaleimide, and siliconizing reagents such as silicon oxide, AQUASIL, and SURFASIL. Additional suitable coating materials are blocking agents such as amino acids, or polymers including but not limited to polyvinylpyrrolidone, polyadenylic acid and polymaleimide. Certain coating materials can be cross-linked to the surface via heating, radiation, and by chemical reactions. Those skilled in the art will know of other suitable means for coating a nanowell of a multi-well device, or will be able to ascertain such, without undue experimentation.
An exemplary multi-well device (e.g., chip) may have a thickness of about 0.625 mm, with a well have having dimensions of about 0.25 mm (250 um) in length and width. The nanowell depth can be about 0.525 mm (525 um), leaving about 0.1 mm of the chip beneath a given well. A nanowell opening can include any shape, such as round, square, rectangle or any other desired geometric shape. By way of example, a nanowell can include a diameter or width of between about 100 µm and about 1 mm, a pitch or length of between about 150 µm and about 1 mm and a depth of between about 10 µm to about 1 mm. The cavity of each well may take a variety of configurations. For instance, the cavity within a nanowell may be divided by linear or curved walls to form separate but adjacent compartments.
The wells (e.g., nanowells) of the multi-well device may be formed using, for example, commonly known photolithography techniques. The nanowells may be formed using a wet KOH etching technique, an anisotropic dry etching technique, mechanical drilling, injection molding and or thermo forming (e.g., hot embossing).
Reagents contained within the liquid in the multi-well device depend on the reaction that is to be run with the single cell that is deposited into each well. In an embodiment, the wells contain a reagent for conducting the nucleic acid amplification reaction. Reagents can be reagents for immunoassays, nucleic acid detection assays including but not limited to nucleic acid amplification. Reagents can be in a dry state or a liquid state in a unit of the chip. In an embodiment, the wells contain at least one of the following reagents: a probe, a polymerase, and dNTPs. In another embodiment, the wells contain a solution comprising a probe, a primer and a polymerase. In various embodiments, each well comprises (1) a primer for a polynucleotide target within said standard genome, and (2) a probe associated with said primer which emits a concentration dependent signal if the primer binds with said target. In various embodiments, each well comprises a primer for a polynucleotide target within a genome, and a probe associated with the primer which emits a concentration dependent signal if the primer binds with the target. In another embodiment, at least one well of the chip contains a solution that comprises a forward PCR primer, a reverse PCR primer, and at least one FAM labeled MGB quenched PCR probe. In an embodiment, primer pairs are dispensed into a well and then dried, such as by freezing. The user can then selectively dispense, such as nano-dispense, the sample, probe and/or polymerase.
In other embodiments of the disclosure, the wells may contain any of the above solutions in a dried form. In this embodiment, this dried form may be coated to the wells or be directed to the bottom of the well. The user can add a mixture of water and the captured cells to each of the wells before analysis. In this embodiment, the chip comprising the dried down reaction mixture may be sealed with a liner, stored or shipped to another location.
The multi-well devices, with a single cell in each well, may be used for genotyping, gene expression, or other DNA assays preformed by PCR. Assays performed in the plate are not limited to DNA assays such as TAQMAN, TAQMAN Gold, SYBR gold, and SYBR green but also include other assays such as receptor binding, enzyme, and other high throughput screening assays.
In some embodiments cells are subjected (e.g., after lysis and/or other processing steps) to amplification and/or sequencing analysis. Conducting one or more amplification reactions may comprise one or more PCR-based amplifications, non-PCR based amplifications, or a combination thereof. Illustrative non-limiting examples of nucleic acid amplification techniques include, but are not limited to, polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), nested PCR, linear amplification, multiple displacement amplification (MDA), real-time SDA, rolling circle amplification, circle-to-circle amplification transcription-mediated amplification (TMA), ligase chain reaction (LCR), strand displacement amplification (SDA), and nucleic acid sequence based amplification (NASBA). Those of ordinary skill in the art will recognize that certain amplification techniques (e.g., PCR) require that RNA be reversed transcribed to DNA prior to amplification (e.g., RT-PCR), whereas other amplification techniques directly amplify RNA (e.g., TMA and NASBA).
The polymerase chain reaction (
Transcription mediated amplification (
The ligase chain reaction (
Strand displacement amplification (
Other amplification methods include, for example: nucleic acid sequence based amplification (
In some embodiments, nucleic acid sequencing methods are utilized (e.g., for detection of amplified nucleic acids). In some embodiments, the technology provided herein finds use in a Second Generation (a.k.a. Next Generation or Next-Gen), Third Generation (a.k.a. Next-Next-Gen), or Fourth Generation (a.k.a. N3-Gen) sequencing technology including, but not limited to, pyrosequencing, sequencing-by-ligation, single molecule sequencing, sequence-by-synthesis (SBS), semiconductor sequencing, massive parallel clonal, massive parallel single molecule SBS, massive parallel single molecule real-time, massive parallel single molecule real-time nanopore technology, etc.
A number of DNA sequencing techniques are suitable, including fluorescence-based sequencing methodologies (See, e.g.,
Next-generation sequencing (NGS) methods share the common feature of massively parallel, high-throughput strategies, with the goal of lower costs in comparison to older sequencing methods (see, e.g.,
In pyrosequencing (
In the Solexa/Illumina platform (
Sequencing nucleic acid molecules using SOLiD technology (
In certain embodiments, the technology finds use in nanopore sequencing (see, e.g.,
In certain embodiments, the technology finds use in HeliScope by Helicos BioSciences (
The Ion Torrent technology is a method of DNA sequencing based on the detection of hydrogen ions that are released during the polymerization of DNA (see, e.g.,
The technology finds use in another nucleic acid sequencing approach developed by Stratos Genomics, Inc. and involves the use of Xpandomers. This sequencing process typically includes providing a daughter strand produced by a template-directed synthesis. The daughter strand generally includes a plurality of subunits coupled in a sequence corresponding to a contiguous nucleotide sequence of all or a portion of a target nucleic acid in which the individual subunits comprise a tether, at least one probe or nucleobase residue, and at least one selectively cleavable bond. The selectively cleavable bond(s) is/are cleaved to yield an Xpandomer of a length longer than the plurality of the subunits of the daughter strand. The Xpandomer typically includes the tethers and reporter elements for parsing genetic information in a sequence corresponding to the contiguous nucleotide sequence of all or a portion of the target nucleic acid. Reporter elements of the Xpandomer are then detected. Additional details relating to Xpandomer-based approaches are described in, for example,
Other single molecule sequencing methods include real-time sequencing by synthesis using a VisiGen platform (
Reagents for any suitable type of assay may be added to the wells of the multi-well chip (e.g., using a multi-well dispenser, such as the one from WAFERGEN BIOSYSTEMS). Such reagents may be added to the wells before or after a cell (e.g., a single cell) is added to a well. In certain embodiments, protein detection assay components (e.g., anti-body based assays) are added to the wells. In other embodiments, SNP detection assay components are added to the wells. In other embodiments, nucleic acid sequencing assay components are added to the wells. In certain embodiments, nucleic acid sequence assay components that employ barcoding for labelling individual mRNA molecules, and/or for labeling for cell/well source (e.g., if wells pooled before sequencing analysis), and/or for labeling particular multi-well chips (e.g., if wells from two or more multi-well chips are pooled prior to sequencing) are employed. Examples of such barcoding methodologies and reagents are found in Pat. Pub.
In certain embodiments, the barcode tagging and sequencing methods of
In further embodiments image and chip mapped wells within the chip are dynamically and/or statically selected for further analysis by a combination of single or multiple addition of reagents for detection and/or resolution of nucleic acids or lipids or carbohydrates or protein cell components reagents.
An example of the use of cell surface markers including positive and negative control antibody (Ab) staining, dispense and imaging of single and multiple cells in a Wafergen chip is shown in
SK-BR-3 cells were washed and collected into 1 mL of 1x PBS (no Mg++, no Ca++, pH 7.4; 37 °C). One-half of the volume of the above suspension was incubated with 10 uL of an appropriate, conjugated monoclonal antibody (mouse anti-human ERBB2 (HER2)-APC; R&D Systems; FAB1129A) against the cell surface antigen ERBB2 (HER2/neu). The remaining half-volume of the suspension was incubated in parallel with an appropriate negative control antibody for immunostaining, namely, a conjugated, isotype-matched control antibody (mouse IgG2B-APC; R&D Systems; IC0041A).
In brief, conjugated antibody incubations were performed at 37 °C for one hour. Subsequently, a volume of 4.5 mL of pre-warmed 1x PBS (37 °C) was added, and the cells were centrifuged at 200 x g for 5 minutes at room temperature. Pelleted cells were resuspended in 200 µL of pre-warmed 1x PBS (37 °C) then stained for - 20 minutes in 1x Hoechst 33342 supravital stain at 37 °C. Eighty microliter aliquots of each suspension were dispensed into the appropriate receiving wells of a 384-well plate. A Wafergen MSND (multi-sample nanodispenser; see
A field of view (FOV), displaying 36 individual wells on a chip, indicates a batch of SK-BR-3 cells that were dual-stained with Hoechst 33342 dye and the APC-conjugated monoclonal antibody targeting the HER2/neu antigen. All of the SK-BR-3 cells are expected to stain using Hoechst 33342 supravital dye (
A second field of view displaying 36 individual wells from a different region of the same 5184 well chip shows a batch of SK-BR-3 cells that were dual-stained with Hoechst 33342 dye and the APC-conjugated negative control monoclonal antibody mouse IgG2B-APC (
The image data in
In this example, three samples were examined. Two samples were composed of a single species: human U87-MG-(Red Fluorescent protein positive) cells or mouse NIH/3T3 cells. The third sample was composed of a mixture of the individual single species samples. Cells were stained and dispensed using an MSND (multi-sample nano-dispenser; Wafergen Inc.) permitting a Poisson distribution of cell counts in the wells of a WaferGen SMARTCHIP array. That array was subsequently imaged, and single-cell containing wells selected and other analyte containing wells were selected to be processed into first and second strand cDNA. The cDNA was extracted from the chip using the methods described earlier. The cDNA was PCR amplified off-chip to yield DNA amplicon "libraries" suitable for NGS amplification and readout. Sequence reads from the sample libraries can then be mapped to the specific sample (i.e species genome) to confirm firstly that the cell and RT buffer selection process were capable of selecting specific wells, and secondly, that selected wells from the mixed cell sample give rise to sequencing reads mapping to only one species. In effect, the latter can be used to calculate the single cell occupancy and partitioning ability of the system.
In brief, human U87-MG-RFP and mouse NIH 3T3 cells were separately washed in 1x PBS, counted and stained with Hoechst 33342 and propidium iodide. For cell mixtures, an aliquot of each cell line was mixed in 1:1 ratio. RNA derived from K562 cells (12 pg) and 1x PBS were employed as positive and negative process controls respectively.
Samples and controls were loaded in a 384-well source plate for dispense onto a SmartChip array using the Multisample Nanodispenser (MSND). Each well of the SmartChip array contains a pre-printed sample identifying "barcode" primer where the 3' oligodT sequence on those primers is used to anneal to polyadenylated stretches of nucleic acid sequences. The barcodes serve as molecular addresses for the mRNA molecules captured from each cell after lysis. Post control and sample dispense, the array was blotted, sealed and centrifuged at 300 x g for 5 minutes at 22°C. The array was then imaged. Post-imaging, the array was placed in a freezer (-80°C) to await cell and sample selection and subsequent reagent addition. Single cells and other analyte containing wells were selected based on their Hoechst 33342 signal using CellSelect (™) image analysis software.
A total of 1,268 "single-cell" containing wells were identified. Of these 1,268 wells, 90 wells of each single species cells, 382 wells from the mixed cell sample alongside 4 each of the positive and negative control wells, were also selected for processing. Once well selection was complete, those wells were mapped to receive RT reagents. In brief, the array was removed from the freezer, thawed at room temperature for 10 minutes and centrifuged at 3,220 x g for 3 minutes at 4°C. The array was placed in a thermal cycler and barcoded oligodT primers in each well were permitted to anneal to polyadenylated stretches in nucleic acids (primarily mRNA) after nucleic acid temperature denaturation (3 minutes at 72°C) and annealing during a combined centrifugation / temperature step (3,220 x g for 3 minutes at 4°C). The array was transferred to the MSND for RT dispense. Using the dispense map created by the analysis software, 50nL of an RT mix containing MMLV RT and a second strand synthesis "template switching oligo" were added to selected wells. The array was centrifuged at 3,220 x g for 3 minutes at 4°C to localize the array contents in each well and incubated for 90 minutes at 42°C. Pooled (barcoded) sample cDNA products were collected via second centrifugation (as above) after inverting the chip and placing into an extraction device (as described in
After sequencing, reads from single-species samples and the mixture of human and mouse cells were separately aligned to the human and mouse genomes. Reads that aligned to both genomes were identified as alignments to homologous genomic regions and excluded from analysis. Reads that uniquely aligned to a single genome for each barcode were identified. Barcodes that primarily align to one genome were identified to be from that genome. Of 382 wells processed from the mixed cell sample, reads from 138 and 234 single-cell containing wells unambiguously aligned to human and mouse genomes respectively. Five well barcodes registered fewer than 25,000 alignments to either genome (Low Aligned Reads) and were excluded from further analysis. 5 of 382 wells from the mixed cell sample that gave rise to sequencing reads mapping to both species was used to calculate the multiple cell occupancy rate and partitioning ability of the system. In brief, 1.3% of barcodes show almost equal number of alignments to both genomes. In conclusion, the single cell occupancy and partitioning ability of the system is 98.7%, ie -99% of wells were single-cell containing wells as shown in
Although the disclosure has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the disclosure that are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.
This list of references cited by the applicant is for the reader's convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.