Patent Application
20100056396
The present invention relates to cell array and matrix assembly and in particular to transfer of cells suspended in a fluid from the fluid to a substrate.
An important step in research studies in the field of gene therapy and drugs discovery is the disposition of cells in an array or a matrix on a substrate. This is typically done by preparing a sample containing a certain concentration of cells in a liquid and subsequently dispensing from a needle. To ensure a desired cell size, filtration may be a step in the preparation of the sample. One of the limitations of the technology is the speed with which the array or matrix can be build up. Another limitation is that it is difficult to dispense cells individually from a suspension in a reliable way. In patch clamp technology an array of sub-cellular sized holes in a glass plate can be used for attaching cells by under-pressure; see e.g. Fertig et al., Biophysics Journal, June 2002, 82(6), p. 3056.
Studies within the field of gene therapy and drugs discovery often involve electroporation used for introduction of a substance, such as e.g. DNA, into the cells. This is typically done by deposing a droplet of the substance to each cell and applying an electrical field that temporarily increases the permeability of the cell membrane.
Hence, a more efficient technique for transferring cells from a fluid to a substrate would be advantageous, and in particular a technique that renders preceding filtration of the cells superfluous would be advantageous.
Accordingly, the invention preferably seeks to mitigate, alleviate or eliminate one or more of the above mentioned disadvantages singly or in any combination. In particular, it may be seen as an object of the present invention to improve the efficiency with which cells can be transferred from a fluid in which they are suspended to form an array or a matrix of cells on a substrate. It may be seen as a further object of the present invention to enable transfer of cells within a predefined size range. These objects and several other objects are obtained in a first aspect of the invention by providing an apparatus for transferring cells suspended in a fluid from the fluid to a substrate, the apparatus comprising a container for containing the fluid and a transfer device having an exterior surface, an interior cavity, and a plurality of passages each having a first end at the exterior surface and a second end in fluid communication with the cavity and each having a diameter corresponding to a predefined size of the cells to be transferred, and the apparatus further comprising a pressure regulating device capable of regulating a pressure in the cavity so as to establish a pressure difference between the first and second ends of each passage, and a flow device capable of causing the fluid to flow across the exterior surface when in contact with the fluid.
By appropriate selection of passage diameter, pressure difference and fluid flow velocity, the transfer device can be used to pick up cells within a predefined size range. Cells that are smaller than the diameter of the passages are sucked up through the passages, and very large cells are not retained against the first ends of the passages due to the too large fluid drag on these cells. Therefore, by use of an apparatus according to the present invention, complicated sample preparation steps to select the target cell type from biological samples can be simplified or even omitted. Furthermore, cells are handled individually so that the cell array or matrix on the substrate to which the cells are transferred has only one cell at each separate position. The transfer device may e.g. correspond to an ink jet head as used in a printer, or it may be a flat substrate with an array of nozzles that can be pressurized at one side.
An apparatus according to the present invention may comprise a detector for detecting when passages are blocked by a cell, and a controller for causing the transfer device to move to the second position in which the pressure difference is controllable so that the cells will be released from the passages and attach to the substrate. It may e.g. be required that a predefined fraction of the passages are blocked, before the cells are transferred.
The velocity V of the fluid in the container near the average position of an attached cell is preferably in the range of 0.1 Vchar<V <10 Vchar, where the characteristic velocity is:
V
char
=D
passage
·Δp/12η
Dpassage is the diameter of the passages, Δp is the pressure difference between fluid in the container and in the cavity, and η is the dynamic velocity of the fluid. However, any other relationship between the parameters and any velocity range is covered by the scope of the invention.
The transfer device may also be useable for disposal of substance containing one or more materials that is/are to be brought into one or more cells on transferred cells. Such a substance may e.g. comprise DNA, antisense agents or pharmaceuticals under investigation in the field of gene therapy or drugs discovery.
In an embodiment of the invention, the transfer device further comprises electrodes whereby a potential can be applied to transferred cells. This may e.g. be used to perform electroporation and transfection, but any purpose of applying a potential will be possible within the scope of the invention. The electrodes preferably form a pattern that enables the potential to be applied to perform selective electroporation of one or more cells. Hereby it will e.g. be possible to study the effect of transfection of selected cells only or to perform the electroporation at certain time intervals to study time dependent effects.
Another aspect of the present invention is provided by a method for transferring cells from a fluid to a substrate using an apparatus as described above. The method comprises the steps of establishing contact between the fluid and the exterior surface, establishing the pressure difference between the first and second ends of each passage, establishing fluid flow across the exterior surface so that cells larger than the predefined size are not retained against the first ends of the passages, bringing the first ends of each passage and the substrate in close proximity of each other, and varying the pressure difference so that the cells attach to the substrate.
In embodiments of the invention in which electroporation can be performed by application of a potential, the electrodes may be placed on the substrate, and as described for the transfer device, these electrodes may form a pattern that enables selective electroporation of one or more cells. It is also possible within the scope of the invention to have electrodes both on the transfer device and on the substrate.
The present invention will now be explained, by way of example only, with reference to the accompanying figures, where
The drawings in the figures are not to scale.
An apparatus 1 according to the present invention enables building up an array or a matrix of cells 2 from a biological sample. The cells 2 are initially suspended in a fluid 3 that is filled in a container 4 having an inlet 5 and an outlet 6 as illustrated schematically in
The apparatus 1 comprises a pressure regulating device 14 by use of which a pressure difference can be established between the first and second ends 12, 13 of each passage 11 and thereby between the cavity 10 and the fluid 3 in the container 4. When the exterior surface 9 of the transfer device 8 is in contact with the fluid 3, the pressure difference causes the fluid 3 to flow into the cavity 10 via the passages 11. Hereby cells 2 that are smaller than the diameter of the passages 11 can be sucked up and led away via the cavity 10, whereas larger cells 2 are retained against the first ends 12 of the passages 11. From the cavity 10 the fluid 3 may be led to a second container (not shown). In the situation illustrated in
The transfer device 8 can be use to pick up cells 2 within a predefined size range. This is obtained by an appropriate choice of the diameter of the passages 11 and by application of a fluid flow in the container 4 as described above. The velocity of the fluid 3 and the pressure difference can be adjusted so that cells 2 that are too big cannot be retained against the first ends 12 of the passages 11 but are washed away via the outlet 6 of the container 4. By use of this embodiment of the invention, it therefore becomes unnecessary to filter the sample of cells 2 in a fluid 3 which simplifies the process. Cells 2 may stick together incidentally. If this happens, they will either be washed away due to their total larger size, or they will be separated by the transversal flow.
Theoretical modeling has been used to determine an appropriate relationship for the choice of parameter settings for a given application. It was found that the following equation can be used. The velocity V of the fluid 3 in the container 4 near the average position of an attached cell 2 should preferably be in the range of 0.1 Vchar<V<10 Vchar, where the characteristic velocity is:
V
char
=D
passage
·Δp/12η
Dpassage is the diameter of the passages 11, Δp is the pressure difference between fluid 3 in the container 4 and in the cavity 10, and η is the dynamic velocity of the fluid 3. However, any other relationship between the parameters and any velocity range is covered by the scope of the invention. When a cell 2 is attached to a passage 11, Δp is the pressure difference over the cell 2, i.e. the pressure difference between the fluid 3 at the side of the cell 2 facing the container 4 and the fluid 3 at the other side facing the passage 11.
In the embodiment shown in
An alternative to moving the transfer device 8 is to keep it in a fixed position and to move the container 4 and the substrate 17.
If desired, it may be possible to use different flow velocities for different rows. Hereby it is possible to vary the upper limit of the size range of cells 2 between the rows. This may e.g. be relevant if influence from the cell size, or another parameter directly related thereto, is a variable under investigation in a given research study.
In an (not shown) embodiment of the invention, the transfer device 8 is oriented so that the first end 12 of the passages 11 point upwards before the cells 2 are to be released. Hereby it can be ensured that possible fluid 3 remains are not leaving the passages 11.
In an embodiment of the invention, the transfer device 8 is used not only to build up an array or a matrix of cells 2 as described above but also to perform subsequent electroporation and transfection. After the array or matrix has been built up, the passages 11 and the cavity 10 of the transfer device 8 are emptied, cleaned and filled with DNA, antisense agents, pharmaceuticals or another substance 18 that is to be brought into one or more cells 2. The transfer device 8 is then moved to a position in which the first ends 12 of the passages 11 are in close proximity of the cells 2 on the substrate 17, and the substance 18 is disposed on one or more of the cells 2.
The electroporation can subsequently be performed by applying an electrical potential to the cells 2. The potential can be applied via electrodes 19 on either the transfer device 8 or the substrate 17 or on both.
An alternative to using the same transfer device 8 for transfer of both cells and substance 18 is to use one or more other transfer devices (not shown) for disposing substance 18 than the one used to transfer the cells 2. This alternative may increase the working speed, minimize the waste of fluid 3 and substance 18, and minimize the risk of contamination due to undesired mixing of remains of fluid 3 and substances 18. Such other transfer devices may have a design different from the one used to transfer the cells 2, as they may not comprise e.g. the cavity 10 and the pressure regulating device 14.
In all embodiments described above it is typically important to ensure that the cells 2 are attached to the substrate 17 at predefined positions, and that the substance 18 comprising material to be brought into the cells 2 is disposed precisely on the cells 2. The choice of appropriate alignment technology for this purpose will be obvious for a person skilled in the art.
The passages 11 may be divided into groups each connected to a separate cavity (not shown) so that one or more groups of cells 2 can be released at a time. Hereby it will e.g. be possible to place the cells 2 on the substrate 17 in a pattern different from the one formed by the position of the passages 11 of the transfer device 8. This possibility may alternatively be obtained by use of a piezo-facility known e.g. from an ink-jet head of an ink-jet printer. These options may also be used to apply substance 18 to some of the cells 2 only.
Although the present invention has been described in connection with the specified embodiments, it is not intended to be limited to the specific form set forth herein. Rather, the scope of the present invention is limited only by the accompanying claims. In the claims, the term “comprising” does not exclude the presence of other elements or steps. Additionally, although individual features may be included in different claims, these may possibly be advantageously combined, and the inclusion in different claims does not imply that a combination of features is not feasible and/or advantageous. In addition, singular references do not exclude a plurality. Thus, references to “a”, “an”, “first”, “second” etc. do not preclude a plurality. Furthermore, reference signs in the claims shall not be construed as limiting the scope.
| Number | Date | Country | Kind |
|---|---|---|---|
| 06124973.6 | Nov 2006 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB07/54825 | 11/28/2007 | WO | 00 | 5/22/2009 |
