NL1040089C2

NL1040089C2 - Micro well plate to distribute single cells in single wells, and methods to use such plate.

Info

Publication number: NL1040089C2
Application number: NL1040089A
Authority: NL
Inventors: Cornelis Johannes Maria Rijn; Arjan Gerhardus Johannes Tibbe; Leonardus Wendelinus Mathias Marie Terstappen
Original assignee: Vycap B V
Priority date: 2013-03-12
Filing date: 2013-03-12
Publication date: 2014-09-15

Description

MICRO WELL PLATE TO DISTRIBUTE SINGLE CELLS IN SINGLE WELLS, AND METHODS TO USE SUCH PLATE

The present invention is related to a method for seeding single objects into individual wells, with the possibility to add reagents to individual wells, and in combination with the possibility to retrieve and transfer these cells from these wells to a different platform for further interrogation of the specific cell by applying PCR, RT-PCR, FISH or comparable DNA and RNA analysis methods. The presented invention is in particular suited for obtaining single cells and/or microorganisms suspended in fluid samples. The invention is well suited for use in healthcare, life science and medical treatment applications as well as food safety and food technology. Though the method is especially designed for these fields it can have use in other fields as well.

Single cell technologies are of extreme importance in cases where only very few events are present in a sample. Examples of these are bacteria in bodily fluids and circulation tumor cells (CTC) in blood. By collecting these single events and subsequent perform downstream analysis on the collected events, such as analyzing DNA mutation and RNA/protein expression at single cell level, the signature of these events can be established which can lead to more specific treatment, development of new treatments and understanding of the underlying biological processes.

A common method to isolate cells is to mechanically separate the cells by wells. Depending on the intended application a microwell device can be designed in numerous ways and with numerous different materials. Well-shaped structures of 10 and 20 pm in diameter have been fabricated using PDMS stamping of PEG polyethylene glycol) onto silicon substrates (Suh et al., 2004), and polystyrene substrates (Dusseiller et al., 2005). Mid-sized wells have been fabricated by surface engineered PEG on glass, creating arrays for improved optical cell imaging with wells capable of harboring more than one cell, such as 30x30 pm (Revzin, 2003) or 15x15 pm (Revzin et al.,2005) wells.

Suspensions of single cells are normally seeded manually into these microwells, and the cells are randomly positioned in the wells by gravitation/sedimentation. To minimize the chance of having multiple cells in a single well, diluted cell suspensions are used which results in a low percentage of wells that are filled. Other methods for seeding single cells in individual wells use wells with a dimension that has a volume that can only hold a single cell with the result that no additional reagent volume can be added to the individual well, which limits their use. Remaining cells outside a well are flushed away, sometimes followed by another round of cell loading to increase the final number of captured cells. In cases where only a limited amount of cells are present, as for example in the analysis of CTC, it is not allowed to have cells outside the wells and to flush these away.

When working with larger wells micromanipulation is the mostly used technology, to retrieve the cells from the wells. An example of cell retrieval from smaller cell-sized wells using micromanipulation was demonstrated by Tokimitsu et al., 2007. Cell retrieval and/or removal are critical parameters for microwell chips in general, i.e. can the cells be retrieved by post analysis or are they lost? Many single-cell micro-chips are designed for continuous analysis on chip without the possibility for the investigator to retrieve interesting cells or clones. However, techniques for retrieval and manipulation of cells are very important, since screening attempts often end up in particularly interesting findings regarding only a few cells worthwhile to analyze further.

The issues identified above have been resolved in the present invention. We have designed a micro well plate for capturing and distributing single cells in individual wells, comprising a micro well plate having micro wells with a bottom plate, a sample supply side and a sample discharge side, wherein at least one individual well is provided with a bottom plate having at least one pore to pass sample liquid from the supply side to the discharge side, and according to the invention characterized in that if one object or cell of interest is collected on the bottom plate of the well, the sample flow rate through that particular well strongly reduces herewith minimizing the possibility that multiple cells or objects of interest can enter the same well. At best a single cell or object of interest should be able to close at least one pore of the well bottom plate and will promote single cell capture. For this the base of every well is provided with a single pore or a set of pores. When a fluidic sample with the objects of interest is applied to the microwells, the fluid will enter the wells at the supply side and will leave the wells through the pores at the bottom of the well at the sample discharge side. Hydrodynamic forces take the objects of interest along with the flow and objects are collected at the bottom of the well on the pores (with a dimension smaller than the objects) herewith strongly reducing or stopping the sample flow rate through that particular well herewith minimizing the possibility that multiple cells or objects of interest can enter the same well in a later stage. With preference a single cell or object of interest is able to close most of the pores present in the well bottom plate. Very good results have been obtained when the bottom plate is provided with only a single pore and having a size smaller or comparable than the cell or object of interest. The advantage of a single pore is that it is immediately totally closed after the capture of a single cell herewith preventing that other cells in the sample fluid can enter the well. Also the flux through one pore with size d is higher than the flux through N pores with size d/N, and this enables a relatively fast flow of the sample fluid.

Thin bottom plates with pores have a preference to be used and can according to the invention best be made in the same way as micromachined microsieves are being made. Micromachined microsieves have been introduced comprising a supporting silicon substrate and a thin ceramic membrane layer with precisely etched pores. In this way mechanically stable and thin membranes with high pressure strength can be made, even when the membrane has a thickness of only a few hundred nanometer. In the present invention the design and dimension of the support structure of the microsieve has been modified such that the open support structure itself will form the microwell plate. Microsieves have a number of specific advantages such as a very low flow resistance, regular and precise pore geometry and an optically flat surface. According to the invention the bottom plate of the wells near the pores has a thickness less than ten times and preferably less than three times the diameter of the pores, herewith enabling a high sample fluid flow through the pores. Furthermore the micro well plate is chemically inert and has no disadvantageous fluorescence back light scattering, herewith avoiding unwanted chemical reactions and facilitating the staining and detection of the micro-objects with a fluorescence microscope. With preference the pores or single pore in a bottom plate are centered in the middle of the well, in order to promote microscopic observation. After the capture of a single cell in a well the pores can be further sealed with various methods to allow a chemical/biological reaction between the collected object and an added reagent without cross interference between different wells. The pores in the bottom of the wells can be closed by using many different methods, such as by using a plastic foil or plate, a thin fixating material or the deposition of a hydrophobic agent. Examples of reagents that can be added are, e.g. fluorescence labels, PCR reagents, DNA amplification reagents or reagent that can lyse the cells. After completion of the reaction the fluid can be removed from each individual well by using micro-pipetting or by opening the pores at the bottom of the well.

Another part of the invention focusses on the retrieval of collected objects of interest. Micro-pipetting the single cells from the individual wells are a possibility, but require a skilled operator with a large possibility on loss cells. Alternatively according to the invention a novel method has been developed in removing or punching out the bottom plus captured object from a preselected well. After collection of the punched bottom of an individual well with an individual object, it can be easily transferred to a microscope slide, a tube, a sample cup, or a specific well of a (standardized) PCR well plate, allowing the use of standard commercially available reagents and platforms to further interrogate the collected single events. To enable the removal of the bottom plate of an individual well, a bottom plate from a ceramic material such as silicon nitride with a thickness between 200 nm and 2 micrometer has been found advantageous.

DETAILED DESCRIPTION OF THE INVENTION WITH SPECIFIC EMBODIMENTS EXAMPLES

Figure 1 A) Impression of a microsieve with wells. Wells are arranged in a square in the center of the sieve. Dimensions are not on scale. B) Enlargement of A, each well is closed by a membrane that contains a single pore such that the object of interest is able to strongly reduce the flow into that particular well. In this particular impression each well contains one pore. C) Drawing of the cross-section of a well with typical dimensions and a single cell that is closing one of the pores.

Figure 2 Principles of seeding single cells in individual wells. Hydrodynamic forces drag the cells into the wells, where these close the pore in the bottom plate of the well. The flow through that pore is strongly reduced and as a result the chance that a second cell enter the well is minimized.

Figure 3 Single cells in individual wells. Each well contained exactly one pore with a diameter of 5 microns. Cells are SKBR-3 cells fluorescently labeled with Cytotracker orange.

Figure 4 Chance of having multiple cells in a single well when cells are randomly distributed using gravity versus the forced seeding using the microsieves with wells.

Figure 5 Principles of adding reagents to the wells. A) Submerging the membrane side of the wells into reagent. The reagent will by capillary action or by pressure forced upward through the pore into the well. B) Micro pipetting reagents into the well. C) Photograph shows microsieve with wells that are partially filled with reagents by submerging the microsieve in reagents. D) Photograph of microsieve with wells where one well is filled by printer technologies.

Figure 6 Schematic illustration of the process steps for the analysis of the DNA of cells captured in individual cavities.

Figure 7 Principles of retrieving a cell by punching and micropipetting. A) The collected cells are transferred to a reagent tube by punching the bottom of a well together the collected cell into an analysis tube for further analysis. B) The collected cell is removed from the well by using a micropipette to take out the collected cell and transfer the cell.

Figure 8 Examples of punched bottoms from the well with cells.

Example 1: Design and manufacturing of a microwell plate

In a silicon wafer 1 with a thickness (h) of 380 micrometer large cavities in the form of wells 5 are provided with a dry or wet etching method known in the art. On the bottom of the wells 5 a silicon nitride membrane 2 is provided with a number of pores 3, e.g. with a diameter between 0.2 and 20 micrometer, typically with a size smaller that the objects of interest (Fig. 1). The silicon nitride layer 2 is low stress silicon nitride with a thickness (t) between of 0.2 and 2 micrometer. For other materials or substrate materials other micromachining methods can be used to form the cavities in the substrate, such as molding, electroplating, lasering, etc.

The large cavities or wells 5 are facing towards the sample fluid and can be used to capture e.g. cells or microorganisms and can be used as a bio reagent chamber. Figure 1A presents a microwell plate with round cavities 5 having a diameter (d) of 100 microns in a 3 x 3 mm2 area. A zoom in on the wells with single pores is presented in Figure IB. The thickness (t) of the bottom plate is smaller than the diameter of the pores 3 to achieve a low flow resistance. Here each cavity 5 has only a single pore. In case a cell 4 enters the cavity it will land onto the pore, hereby enhancing the flow resistance and forcing other cells to enter a different cavity. In this way the chance that multiple cells are present in a single cavity is severely decreased. This microwell plate is very well suited used for the analysis of single cells 4 that are present at very low densities (typically a few per milliliter). Examples are tumor cells that are present in bodily fluids, such as pleural, spinal and urine fluid. Amongst the tumor cells other, non-malignant cells are present in these fluids. To be able to analyze the DNA of the individual tumor cells it is important that their DNA is not mixed with DNA of other cells. As such the cell content from each of the collected cells needs to be kept isolated to be able to analyze the DNA constituents of individual cells. Additionally the top of cavity walls can be supplied with an additional hydrophobic layer 6 as an extra measure to prevent mixing of the contents of individual wells. The hydrophobic layer 6 can be applied by applying a silane with a hydrophobic endgroup such as an alkane to a pretreated silicon nitride layer or any other method known in the art.

Example 2 Seeding of single cells in individual wells

Fig 2 presents a schematic illustration of seeding single cells in individual wells. A sample fluid containing objects of interest, in this case a sample fluid with cells 4 is added to the sample supply side, the side with the large cavities of the microsieve. The fluid flows in the wells and flows out of the well through the pore in the bottom plate of the membrane. In this case each well is provided with a single pore that has a dimension smaller than the objects of interest. The objects of interest are dragged by the flow and hydrodynamic forces into the well, Fig 2A. As a result the objects of interest will land on the pore of a well herewith strongly reducing or stopping the flow rate through the pores hereby minimizing the chance that a second object enters the same well (Fig 2B). This process continues, Fig 2C, until all sample has passed through the wells. The end result is that all cells contain one single cell, Fig 2D.

Figure 3 presents a photograph of single cells seeded in single wells. In this particular example SKBR-3 cells are fluorescently labeled with cytotracker orange and are distributed in a fluid. The number of available wells versus the number of cells is 1 : 0.95. As becomes clear from the photograph 96% of the wells that do contain cells contain a single cell.

Figure 4 presents a graph that compares the percentage of cell containing wells that do contain one as a function of the ratio between the number of cells and the number of available wells, for the situation where the cells sink into wells under gravity or using the seeding method according to the invention. The dotted line presents the situation in case the number of cells sinks into the wells by gravity only. The distribution of the number of cells per well is given by the Poisson distribution in the case of gravity, whereas the seeding method (solid line) as presented in this invention gives a much higher percentage. At a ratio of cell/wells of e.g.0.8 the seeding method that uses gravity only results in 65% of the wells that have a single cell whereas the seeding method described results in 96% of the wells that have a single cell, an increase of 31%.

Example 3 Methods for filling wells with reagent.

Figure 5A presents a schematic illustration for filling the wells by submerging the bottom plate with the pore into reagents 9. The reagents are forced to move into the wells through the pores. Fig 5C shows a photograph of the wells that have been filled with reagents. At the time the image was acquired approximately 80% of the wells were filled with the reagents already. This method can especially be used when the same reagents need to be added to all wells without losing cells.

Alternatives to submerging the perforated micro well plate is pipetting or printing reagents in individual wells, well by well. Fig 5B presents a schematic image of pipetting reagents 14 using a micropipette 15. To prevent that the reagents will not leak from the pores the pores are closed with a sealing sheet 13 before the reagents are added.

Instead of using a micropipette reagents can be printed in the wells using inkjet technology. The photograph of image 5D presents a part the well plate and one of the wells is filled with reagents using inkjet printing technology.

Example 4 Procedure for DNA analysis of individual cells

In Fig.6 a procedure for analysis is depicted in 5 steps with a micro well plate 6 showing three wells.

Step 1:

The seeding of the cells 5 is depicted and the perforated micro well plate is brought into contact with an absorbing body 7. The sample fluid passes through the micro wellplate towards the absorbing body 7 while leaving the cells 5 behind on the pores. Next the cells are labeled with fluorescence labels and fluorescence microscopy identifies the wells that contain the cells of interest. The locations of these cavities are stored to be able to revisit these wells after amplification of the DNA has completed. Step2:

The micro wellplate is moved towards the compartment that contains the reagents for DNA amplification. In this example the microsieve with wells containing pores at the bottom is dipped into the fluid 8. The fluid 8 will move through the pores towards the cells and fills all the wells.

Step 3:

After the fluids have been picked up the wellplate is pressed onto a seal 9. This will prevent fluids to escape through the pores while incubating. If needed a series of reagents can be picked-up with or without drying / washing / fixation of the sample between each step.

Step 4:

While the pores are closed the PCR reaction and / or DNA amplification reaction cycle is started to amplify DNA or part of the DNA. The amplified DNA 10 stays inside the well of the collected cell. If needed the temperature can be cycled between a minimum and a maximum temperature.

Step 5:

Two options are possible: 1: During amplification a fluorescence label against a specific DNA sequence was incorporated. In this case the presence of a specific sequence can be detected using for example the fluorescence intensity where the fluorescence light is collected by an objective 11 as is used in Real Time PCR reactions.

2: The amplified DNA is transferred to another platform to further analyze it, e.g. sequencing, using for example a pipette tip 12 that has a dimension smaller than the diameter of a well.

Example 5, retrieving objects from the wells

Figure 7, shows two different approaches to retrieve individual cells form the wells. Fig 7A illustrates a method that is based on removing the whole bottom, including the collected cells, from the well by punching the bottom out. The bottom with cell is punched, by a puncher 16 into a reaction tube 17 that it suitable for the next analysis step, e.g. in wells of the PCR well plate. Depending on the requirements different materials for the puncher can be used, for example stainless steel or glass pipettes.

Removal of the whole bottom will only work for brittle, non-elastic materials and the silicon nitride bottom as used in this in this invention is very well suited for this.

Alternatively micropipettes can be used to remove the cell from the microsieve well as is illustrated in Fig 7B; A small suction force is applied to the micropipette 18 to hold the cells and next it can be removed from the well.

Examples of bottoms + cells are present in Fig 8. The photograph shows 6 cells and fragments of 6 bottoms. In this case the bottoms have been punched on a slide instead of a reaction tube.

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Claims

A method for capturing and distributing individual cells in a microwell plate, wherein the microwell plate comprises at least one well with a bottom, a sample inflow and a sample outflow side, wherein the bottom contains at least one pore for fluid from the sample inflow side to pass sample outflow, characterized in that if one object or cell of interest is collected at the bottom of an individual well, sample inflow is greatly reduced, minimizing the chance of multiple objects ending up in the same well .

A microwell plate for a method according to claim 1, wherein a single cell or object of interest can seal at least one single pore from the bottom of the well.

The microwell plate of claim 1, wherein a single cell or object of interest can close off substantially all pores of the bottom of a well.

Microwell plate according to claim 1, 2 or 3, wherein the bottom in the vicinity of the pore has a thickness of less than ten, preferably less than three times the diameter of the pore.

A microwell plate according to claim 1, 2, 3 or 4, wherein the bottom of each well is provided with a single pore.

A microwell plate according to any one of the preceding claims, wherein the pore in the bottom is centered in the center of the well.

Microwell plate according to one of the preceding claims, wherein the bottom consists of a ceramic material.

A microwell plate according to claim 7, wherein the soil together with the object of interest is expressed in a (reaction) well of a standard microtiter plate to further investigate the object of interest by PCR, RT-PCR, FISH or similar DNA and RNA analysis methods

A microwell plate according to any one of the preceding claims, wherein the top of the microwell plate is provided with a hydrophobic layer on the sample inflow side to prevent the contents of the individual wells from mixing.

Microwell plate according to any one of the preceding claims, wherein the pores of the microwell plate are closed with the aid of a foil or plate, before adding reagents to the microwell plate.

A microwell plate according to any of the preceding claims, wherein the microwell plate is used directly as a microtiter plate.