Method for transferring and repositioning biological samples - Patents.com

Abstract

A method for repositioning and transferring N distinct biological samples from N distinct first locations arranged in an irregular pattern to M distinct second locations arranged in a regular pattern on a transfer device, the transfer device comprising at least M adhesive transfer regions, the at least M adhesive transfer regions being formed from at least one flexible sheet material, and N≧2 and N≧M.

Description

[Technical field]

[0001] The present invention relates to a method for the transfer and relocation of a plurality of distinct biological samples from a plurality of distinct first locations, for example on a microscope slide, to a plurality of distinct second locations, such as wells on a receiving plate. [Background technology]

[0002] In medical diagnostics, as well as in research and development, there is a need to be able to isolate rare cells occurring in low numbers within larger solid tissue samples or small tissue samples.

[0003] Rare cells or tissue samples are often surrounded by tissue, and although there are techniques that allow cutting these cells from the sample, such as laser microdissection (LMD), the transfer of these rare cells is performed in an individual manner, i.e. by lifting a specific target rare cell or cluster of rare cells from the substrate and transferring it to a sample well. The rare cells are generally placed on a microscope slide, where they must be identified by optical, especially fluorescent, microscopic examination and transferred to a dedicated receptacle for further analysis, such as genetic analysis.

[0004] For example, it is known to use the inner surface of a microcentrifuge tube cap or Eppendorf as a transfer surface to transfer LMD samples. Using a robotic tube holder, the transfer surface on the inner surface of the cap of an open tube is brought into contact with the LMD sample by gently and perpendicularly pushing the transfer surface from above against the sample, causing the sample to adhere to the inner surface of the microcentrifuge cap. The cap is then pulled away with the sample attached. Once this is complete, the microcentrifuge tube is closed and centrifuged. The applied centrifugal force causes the sample to detach from the inner surface of the cap and fall into the well portion of the microcentrifuge tube.

[0005] This method of transferring just one cell is time and labor intensive, and therefore requires the use of a lot of material and equipment space. In addition to the above drawbacks, the cells are stored in individual receptacles from which they may need to be transferred again individually for further analysis into a container capable of holding multiple samples in multiple wells, such as a 96-well plate. That additional transfer step further increases the risk of losing at least a portion of the analytical material.

[0006] It is therefore an object of the present invention to provide a method, together with an instrument and an apparatus, in which the above-mentioned complications of known transfer methods are reduced. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention provides methods, devices and apparatus that facilitate the transfer and relocation of multiple spatially distinct biological samples from multiple spatially distinct locations on the tissue sample to multiple spatially distinct locations on a transfer device, from the tissue sample's inherent "unique" pattern to a regular pattern, such as a rectangular or circular array. [Means for solving the problem]

[0008] A first object of the present invention is to provide a method for repositioning and transferring N separate biological samples from N separate first locations arranged in an irregular pattern to M separate second locations arranged in a regular pattern on a transfer device comprising at least M adhesive transfer areas, the at least M adhesive transfer areas being formed of at least a flexible sheet material, N≧2 and N≧M, the method comprising the following steps in the following order: a. positioning one adhesive transfer region of the at least M adhesive transfer regions to overlap one distinct biological sample of the N distinct biological samples in its first position in a vertical direction, and optionally decreasing the distance between the first position of the distinct biological sample and the one adhesive transfer region in a vertical direction before or after the positioning step; b. stretching the flexible sheet material of the one adhesive transfer region of the at least M adhesive transfer regions into contact with the one distinct biological sample of the N distinct biological samples at the first location and to adhere the one distinct biological sample of the N distinct biological samples to the flexible sheet material of the one adhesive transfer region of the M adhesive transfer regions; c. shrinking the flexible sheet material of the one adhesive transfer region of the at least M adhesive transfer regions to remove the one distinct biological sample of the N distinct biological samples from its first location and transfer the one distinct biological sample of the N distinct biological samples to a second location on the transfer device, optionally increasing a vertical distance between the first location of the one distinct biological sample and the second location of the one distinct biological sample on the one adhesive transfer region after said shrinking step; and d. Repeating steps a-c individually for each of the remaining ones of the N separate biological samples, such as removing each of the remaining ones of the N separate biological samples from its first location and transferring each of the remaining ones of the N separate biological samples to its second location.

[0009] One advantage of the method according to the first object of the present invention is that the biological samples can be removed and transferred directly to the transfer device without any intermediate steps, and a single transfer device can thus load multiple biological samples in an orderly array, which facilitates downstream handling of multiple biological samples.

[0010] It is understood that the method according to the first object of the present invention can be carried out in a situation where the sample support plate is oriented so that the separate biological samples face vertically downwards, or in a situation where the sample support plate is oriented so that the separate biological samples face vertically upwards. The sample support plate may be inverted to the preferred orientation of the sample support plate. However, regardless of the orientation of the sample support plate, the transfer device is placed on the side of the sample support plate that contains the separate biological samples. Thus, when the sample support plate is oriented so that the separate biological samples face vertically downwards, the transfer device is placed below the sample support plate so that at least M adhesive transfer areas face the separate biological samples, and when the sample support plate is oriented so that the separate biological samples face vertically upwards, the transfer device is placed above the sample support plate so that at least M adhesive transfer areas face the separate biological samples. It is noted that the adhesion of the biological samples to the adhesive transfer areas is such that it allows the method to be carried out in either orientation.

[0011] In a preferred embodiment of the method according to the first object of the invention, N may be equal to M.

[0012] In a preferred embodiment of the method according to the first object of the present invention, said method further comprises the following steps: e. Attaching an expansion plate having at least M holes to the transfer device such that the cross-sectional areas of the at least M holes overlap in the vertical direction with at least M adhesive transfer regions of the transfer device to form at least M wells containing biological samples, preferably containing biological samples at their bottoms, wherein the side walls of each of the at least M wells are defined at least in part, preferably entirely, by the inner walls of the at least M holes of the expansion plate, and the at least M adhesive transfer regions at least in part define the bottoms of the at least M wells.

[0013] The advantage of the above-mentioned preferred embodiment of the method according to the first object of the present invention is that by attaching the extension plate, further transfer of the biological samples to a separate multi-well plate for downstream analysis can be avoided, since the attachment of the extension plate results in a "sample-loaded" state of the side walls of the wells surrounding each biological sample. Each newly formed well can then receive a liquid, such as a lysis buffer, that allows further analysis of the biological sample. In a more preferred embodiment, the cross-sectional area and cross-sectional shape of the at least M holes are essentially the same as the area and shape of the at least M adhesive transfer regions of the transfer device in the vertical direction. In another more preferred embodiment, the cross-sectional area and cross-sectional shape of the at least M holes are smaller than the area and shape of the at least M adhesive transfer regions of the transfer device in the vertical direction, in which case the part of the adhesive transfer region surrounding the cross-sectional area and cross-sectional shape of the at least M holes forms a seal gasket between the transfer device and the extension plate. It is understood that the extension plate with at least M holes to the transfer device forms a seal at the contact surface between the at least M adhesive transfer regions of the transfer device and the extension plate with at least M holes when attached to the transfer device. It is further understood that the expansion plate having at least M holes can be attached to the transfer device using any suitable means, such as mechanical fastening means such as clamps, screws, interlocking pins and recesses, latches, etc., or other means such as adhesives.

[0014] In a preferred embodiment of the method according to the first object of the invention, the extension plate may be made of metal or of a synthetic thermoplastic polymer, for example a polyolefin, or a polymeric resin, such as a silicone resin.

[0015] In a preferred embodiment of the method according to the first object of the invention, the extension plate may include integrated caps for closing the formed wells.

[0016] In a preferred embodiment of the method according to the first object of the present invention, said method further comprises, after step e, the following step: f. Closing the wells formed in step e, preferably using cap strips or cap mats, or integral caps of the extension plate.

[0017] In a preferred embodiment of the method according to the first object of the present invention, the expansion plate having at least M holes may be equipped with a gasket to enhance the seal between the expansion plate and the transfer device. This may be particularly advantageous if the cross-sectional area and cross-sectional shape of the at least M holes are larger in the vertical direction than the area and shape of the at least M adhesive transfer regions of the transfer device.

[0018] In another preferred embodiment of the method according to the first object of the present invention, said method further comprises the following steps: e. Positioning the transfer device so that the at least M adhesive transfer areas vertically overlap at least M wells on a receiver plate having at least M wells and so that the at least N separate biological samples face at least M wells of the receiver plate, optionally reducing the vertical distance between the at least M adhesive transfer areas of the transfer device and the bottoms of the at least M wells of the receiver plate, and simultaneously or sequentially releasing each of the separate biological samples from the at least M adhesive transfer areas of the transfer device into the at least M wells of the receiver plate.

[0019] When including step e, the method according to the first object of the invention allows potentially simplifying the task of transferring multiple samples from multiple locations by performing roughly N contacting and N lifting steps and one transfer step for the transfer of N samples (2N+1) instead of N contacting and N lifting steps and N transfer steps (3N) when transferring N samples one by one.

[0020] In a preferred embodiment of the method according to the first object of the invention, the receiver plate may include an integrated cap for each well of the receiver plate to close the well.

[0021] In a preferred embodiment of the method according to the first object of the present invention, said method further comprises, after step e, the following step: f. Closing the wells of the receiver plate, preferably using cap strips or cap mats, or integral caps of the receiver plate.

[0022] In a preferred embodiment of the method according to the first object of the present invention, said method further comprises a step after step d, preferably between steps d and e, of treating the N separate biological samples on the at least M adhesive transfer areas of the transfer device with a releasing solution comprising a releasing agent, preferably with an aqueous releasing solution comprising a releasing agent selected among proteolytic enzymes such as trypsin. In particular, this can facilitate the simultaneous or sequential release of each of the separate biological samples in step e from the at least M adhesive transfer areas of the transfer device into the at least M wells of a receiver plate. For example, the use of a releasing solution allows for shorter centrifugation times or less acceleration when releasing the samples from the transfer device in a centrifuge device.

[0023] In a preferred embodiment of the method according to the first object of the present invention, in step a, the distance between the first location of the distinct biological sample and the adhesive transfer area may be decreased in the vertical direction, and in step c, the distance between the first location of one distinct biological sample and one adhesive transfer area is increased in the vertical direction. When the transfer device approaches the biological sample, it can more easily capture the biological sample by stretching / contracting the sheet material of the adhesive transfer area, since less stretching / contraction is required. Similarly, after the biological sample is captured, the transfer device may be retracted to facilitate repositioning in the horizontal plane for the next iteration of capturing the next biological sample.

[0024] However, the step of approaching / retreating the transfer device is optional because the transfer device can approach and remain in vertical proximity during repetition of the method if the distance between the first location of one distinct biological sample and one adhesive transfer region in the vertical direction is small enough to allow successful uptake of the biological sample by stretching / contracting the sheet material of the adhesive transfer region alone. This can be the case, for example, when the distance between the adhesive transfer region and the first location of the biological sample is smaller than the diameter of the adhesive transfer region in the case of the preferred circular adhesive transfer region, or when the distance between the adhesive transfer region and the first location of the biological sample is smaller than one side of the adhesive transfer region in the case of a square adhesive transfer region.

[0025] In general, when decreasing the vertical distance between the first location of the distinct biological sample and the adhesive transfer area, or when increasing the vertical distance between the first location of the distinct biological sample and the second location of the distinct biological sample on the adhesive transfer area, the threshold is less than 3, 4 or 5 mm, which means that when decreasing the distance, the distance decreases until it is less than 3, 4 or 5 mm, and when increasing the distance, the distance increases until it is more than 3, 4 or 5 mm. However, the threshold may also be 6, 7, 8, 9, 10 or 15 mm.

[0026] In a preferred embodiment of the method according to the first object of the invention, the flexible sheet material is further optically transparent, preferably transparent at least in the visible spectrum (VIS). When the flexible sheet material is optically transparent, different advantages are realized depending on the configuration of the device used to carry out the method. In one configuration, the device comprises an optical unit capable of optically identifying the biological sample from the opposite side of the flexible sheet to the sample, i.e. from behind the flexible sheet material, through the flexible sheet. In another configuration, the device comprises an optical subunit capable of illuminating the sample by a light source from the opposite side of the flexible sheet to the sample, i.e. from behind the flexible sheet material. In one configuration, the device comprises an optical unit capable of optically identifying the biological sample by an optical sensor, such as a camera, from the opposite side of the flexible sheet to the sample, i.e. from behind the flexible sheet material, through the flexible sheet, and illuminating the sample by a light source from the opposite side of the flexible sheet to the sample, i.e. from behind the flexible sheet material.

[0027] In a preferred embodiment of the method according to the first object of the invention, the stretching of the sheet material of one of the M adhesive transfer regions is carried out by applying pneumatic, hydraulic or mechanical pressure. By applying pneumatic, hydraulic or mechanical pressure, the stretching of the sheet material of one of the M adhesive transfer regions may be individually controlled, preferably with respect to both the degree of stretching and the duration of the stretching. It is noted that when the applied pressure is removed, the flexible sheet material springs back to its original shape.

[0028] In a preferred embodiment of the method according to the first object of the invention, the stretching of the sheet material of one of the M adhesive transfer regions is carried out by applying mechanical pressure by means of a plunger, which has the advantage that it is not subject to pneumatic or hydraulic leaks that may hinder the correct operation of the method.

[0029] In a preferred embodiment of the method according to the first object of the invention, the plunger preferably comprises or consists of an optically transparent material, for example a light guide. The light guide may be configured to emit light towards the sample through the optically transparent flexible sheet material forming the adhesive transfer region. The plunger can thus advantageously serve both to stretch the sheet material and to illuminate the biological sample for better identification by optical microscopy. Alternatively, the light guide may be configured to receive light from the sample through the sheet material in the adhesive transfer region. The plunger can then advantageously serve both to stretch the sheet material and to provide an image of the sample to the imaging device.

[0030] In a preferred embodiment of the method according to the first object of the invention, the N separate biological samples are transferred simultaneously. The simultaneous transfer of samples is simpler than transferring each of the samples individually. The simultaneous transfer can be performed by applying centrifugal force or vibration to the N separate biological samples. To achieve this, the transfer device is attached to a receiver plate, for example a multi-well plate, and the unit consisting of the receiver plate and the transfer device can be centrifuged.

[0031] In a preferred embodiment of the method according to the first object of the present invention, at least N distinct biological samples in a first position are placed on a sample support plate, such as a microscope slide. The sample support plate, such as a microscope slide, may have a tissue section sample, such as a cryotome or microtome section, on which the biological sample is separated. Typically, the tissue section sample is covered with a synthetic polymer layer, such as a laser-sensitive layer, so that the target biological sample can be cut out by laser microdissection. Thus, in most cases, the spatially distinct biological sample comprises a cell or a cluster of cells and one or more synthetic polymer layers attached to the cell or cluster of cells.

[0032] In a preferred embodiment of the method according to the first object of the present invention, the entire area of ​​the flexible sheet material or at least the adhesive transfer area of ​​the flexible sheet material is coated with an adhesive, for example a silicone resin, which can increase the adhesion of the biological sample to the flexible sheet material and facilitate the uptake of the biological sample in the adhesive transfer area.

[0033] In a preferred embodiment of the method according to the first object of the invention, the flexible sheet material comprises or consists of a thermoplastic polymeric material selected from among polyolefins, polyesters, polycarbonates or polyamides. Alternatively, the flexible sheet material comprises or consists of an elastomeric polymeric material such as a silicone resin, for example polydimethylsiloxane (PDMS). These polymeric materials have been found to have good mechanical properties such as elasticity.

[0034] In a preferred embodiment of the method according to the first object of the invention, the flexible sheet material, in particular the thermoplastic polymeric material, has a thickness of 40 to about 400 micrometers, preferably 40 to about 200 micrometers. This thickness range has been found to provide sufficient optical transparency and sufficient mechanical properties such as elasticity. Alternatively, the flexible sheet material, in particular the elastomeric polymeric material, has a thickness of up to 5 mm, preferably up to 2 mm.

[0035] In a preferred embodiment of the method according to the first object of the present invention, the flexible sheet material is a cast film or a blown film.

[0036] In a preferred embodiment of the method according to the first object of the invention, the flexible sheet material comprises or consists of a cyclic olefin copolymer or a cyclic olefin polymer. Cyclic olefin copolymers and cyclic olefin polymers have good optical properties in VIS and UV, which is advantageous when the method uses a light source and an optical microscope to identify biological samples, and they show strong resistance to tearing and ripping. At the same time, these polymers have also been found to show good adhesion to biological samples as is, i.e. without the need to apply an adhesive.

[0037] In a preferred embodiment of the method according to the first object of the invention, N is at least 6, 12, 24, 48, 96, 384.

[0038] In a preferred embodiment of the method according to the first object of the present invention, the at least M wells on the receiver plate are M wells on a multiwell plate, preferably M wells on a 96-well plate, with the condition that M is less than or equal to 96.

[0039] In a preferred embodiment of the method according to the first object of the present invention, the at least M wells on the receiver plate are M wells on a multiwell plate, preferably M wells on a 384-well plate, with the condition that M is less than or equal to 384.

[0040] In a preferred embodiment of the method according to the first object of the present invention, the at least N distinct biological samples are at least N distinct laser microdissected samples, in particular laser microdissected samples of microtome sections, such as cryotome sections or paraffin microtome sections.

[0041] In a preferred embodiment of the method according to the first object of the present invention, the transfer device comprises a support plate having at least M holes and supporting the sheet material, the M adhesive transfer areas being defined by the overlap of the cross-sectional areas of said holes and the sheet material, the holes being preferably circular or polygonal and the plate being made of metal or polymer.

[0042] The transfer device may be formed by a support plate having at least M holes, the sheet material being attached to a surface of the support plate, in particular the surface facing the biological sample. In this case, the sheet material is essentially flush with the surface of the plate of the transfer device. Alternatively, the transfer device formed by a support plate having at least M holes may include two plates with vertically overlapping holes, the two plates having a flexible sheet material sandwiched between them. In this case, the sheet material is essentially embedded in the holes to an extent that depends on the thickness of the plate relative to the surface of the plate of the transfer device.

[0043] In a preferred embodiment of the method according to the first object of the present invention, the at least M holes have the same size and / or shape.

[0044] In a preferred embodiment of the method according to the first object of the present invention, the adhesive surface areas are arranged on the transfer device in a regular pattern that corresponds to the pattern of the second locations, i.e., for example, in an array, more preferably in an array that at least partially corresponds to or overlaps with the array of wells of a 6, 12, 24, 48, 96, 384 well plate. For example, the adhesive surface areas may be arranged on the transfer device in an 8x12 array or a 16x24 array. The regular pattern on the transfer device may correspond to an array, preferably an 8x12 array or a 16x24 array.

[0045] In a preferred embodiment of the method according to the first object of the present invention, the at least M adhesive transfer regions are formed by a plurality, in particular at least M separate sheets, of flexible sheet material. For example, the at least M adhesive transfer regions may be formed by a plurality of tabs of flexible sheet material, each of which overlaps one or more holes in the plate of the transfer device. The tabs may be circular with a diameter larger than the diameter of the holes in the plate of the transfer device, or may be essentially rectangular pieces with a length and width each larger than the diameter of the holes in the plate of the transfer device.

[0046] In a preferred embodiment of the method according to the first object of the present invention, the at least M adhesive transfer regions are formed by a single continuous sheet of flexible sheet material, which covers and overlaps all holes in the support plate of the transfer device. The use of a single continuous sheet of flexible sheet material allows for easier assembly of the transfer device compared to the use of multiple tabs of flexible sheet material.

[0047] In a preferred embodiment of the method according to the first object of the invention, the transfer device comprises a frame and a panel, the frame preferably being made of metal or polymer and the panel being formed by a single continuous sheet of flexible sheet material, the single sheet of flexible sheet material spanning an area that includes the adhesive surface area.

[0048] A further object of the present invention is to provide a transfer device for use in a method for relocation and transfer of N distinct biological samples from N distinct first locations arranged in an irregular pattern to M distinct second locations arranged in a regular pattern on said transfer device, preferably for use in a method according to the first object above.

[0049] In a preferred embodiment of the transfer device according to a further object of the present invention, the transfer device comprises at least M adhesive transfer regions formed from at least one sheet of flexible sheet material, said transfer device comprising a support plate with at least M holes, the at least M adhesive transfer regions being defined by the overlap of the cross-sectional areas of the holes and the sheet material, the holes being preferably circular or polygonal, the plate being made from a metal or a polymer, or said transfer device comprising a frame and a panel, the frame being made from a metal or a polymer, the panel being formed by one continuous sheet of flexible sheet material. By way of example, in the transfer device, each of the at least M adhesive transfer regions may take up one biological sample (N is equal to M) or may take up more than one sample (N is greater than M).

[0050] In a preferred embodiment of the transfer device according to a further object of the present invention, the entire area of ​​the flexible sheet material or at least the adhesive transfer area of ​​the flexible sheet material is coated with an adhesive, for example a silicone resin, which can increase the adhesion of the biological sample to the flexible sheet material and facilitate the uptake of the biological sample in the adhesive transfer area.

[0051] In a preferred embodiment of the transfer device according to a further object of the present invention, the flexible sheet material comprises or consists of a thermoplastic polymer material selected from among polyolefins, polyesters, polycarbonates or polyamides, which have been found to have good mechanical properties such as elasticity.

[0052] In a preferred embodiment of the transfer device according to a further object of the present invention, the flexible sheet material comprises or consists of an elastomeric polymeric material, for example selected from silicone resins such as PDMS, which have been found to have good mechanical properties such as elasticity.

[0053] In a preferred embodiment of the transfer device according to a further object of the present invention, the flexible sheet material has a thickness of from 40 to about 400 micrometers, preferably from 40 to about 200 micrometers, which thickness range has been found to provide sufficient optical transparency and sufficient mechanical properties such as elasticity.

[0054] This thickness range has been found to provide sufficient optical transparency and sufficient mechanical properties such as elasticity. Alternatively, the flexible sheet material, particularly the elastomeric polymeric material, has a thickness of up to 5 mm, preferably up to 2 mm. This thickness range has been found to provide sufficient optical transparency and sufficient mechanical properties such as elasticity.

[0055] In a preferred embodiment of the transfer device according to a further object of the present invention, the flexible sheet material is a cast film or a blown film.

[0056] In a preferred embodiment of the transfer device according to a further object of the present invention, the flexible sheet material comprises or consists of a cyclic olefin copolymer or a cyclic olefin polymer. Cyclic olefin copolymers and cyclic olefin polymers have good optical properties in VIS and UV, which is advantageous when the method uses a light source and an optical microscope to identify biological samples, and are highly resistant to tearing and ripping. At the same time, these polymers have also been found to show good adhesion to biological samples as is, i.e. without the need to apply adhesives.

[0057] In a preferred embodiment of the transfer device according to a further object of the present invention, the transfer device comprises a support plate having M holes, the M adhesive transfer regions being essentially flush with the surface of the plate, said surface of the plate preferably facing at least N distinct biological samples. When the M adhesive transfer regions are essentially flush with the surface of the support plate facing the N distinct biological samples, the degree of stretching / contraction required to lift the biological samples can be reduced compared to when the flexible sheet material is embedded in holes against the outer surface of the support plate, thereby providing a more robust transfer method and / or robust operation of the transfer device. When the M adhesive transfer regions are essentially flush with the surface of the support plate, the manufacture of the transfer device is simplified, since in at least some embodiments only one support plate is required, as opposed to at least two support plates being required when the M adhesive transfer regions are embedded in holes.

[0058] A further third object of the present invention is to provide an apparatus for carrying out the method for repositioning and transferring according to the first object of the present invention, preferably using a transfer device according to the further object of the present invention.

[0059] For example, a third object of the present invention is to provide an apparatus comprising: a first detection unit, preferably a microscope or a fluorescence microscope unit, configured to optically detect the N distinct biological samples at their respective distinct first locations; ii. a second positioning unit, e.g. a single-axis robot or a robotic arm, configured to perform steps a-d according to the method of the invention on the N distinct biological samples detected in step i, said second positioning unit comprising a pneumatic, hydraulic or mechanical subunit capable of stretching and contracting the sheet material of the adhesive transfer region, said subunit preferably being connected to an optical subunit capable of illuminating the samples by a light source optically coupled to a transparent plunger of the mechanical subunit; iii. a third unit, comprising: A. Attach the extension plate to the transfer device, or B. Position the transfer device against the multi-well plate.

[0060] In a preferred embodiment of the device according to the third object of the present invention, the device further comprises a fourth unit configured to apply a releasing solution comprising a releasing agent, preferably an aqueous releasing solution comprising a releasing agent selected from among proteolytic enzymes such as trypsin, to the M adhesive transfer regions of the transfer device.

[0061] Further embodiments of the invention are defined in the dependent claims.

[0062] Preferred embodiments of the present invention will be described below with reference to the drawings, which are intended to illustrate, but not to limit, the preferred embodiments of the present invention. The description of the drawings is as follows: [Brief description of the drawings]

[0063] [Figure 1] The figure shows a transfer device (1) formed by a plate (P) having four holes (2, 2', 2'', 2'''), each covered with a flexible sheet material, a first adhesive transfer region (3) arranged such that the first adhesive transfer region (3) defined by the first hole (2) and a first biological sample (4) are at least partially overlapping vertically, the biological sample being located on a sample support plate (5). The biological samples (4, 4', 4'', 4''') are randomly distributed within a tissue sample (6) on the sample support plate (5). [Diagram 2]The transfer instrument (1) is shown being moved downward after positioning to vertically decrease the distance between the adhesive transfer region (3) and the first biological sample (4). Once the adhesive transfer region (3) is within proximity of the first biological sample (4), the sheet material of the adhesive transfer region (3) is stretched towards the first biological sample (4) at its first position to contact and adhere to the first biological sample (4), and then contracted to lift the first biological sample (4*) from its first position. [Diagram 3] The transfer device (1) is shown being moved upwards to increase the distance vertically between the adhesive transfer area (3) and the first position of the first biological sample (4*). [Figure 4] The transfer device (1) is shown being moved in a horizontal plane such that a further adhesive transfer area (3') defined by the hole (2') and a further biological sample (4') at least partially overlap vertically, the biological sample (4') being located on a sample support plate (5). [Diagram 5] The transfer device (1) is shown being moved downwards in order to vertically decrease the distance between the adhesive transfer area (3') and the further biological sample (4'). [Figure 6] When the adhesive transfer region (3') is within proximity of a further biological sample (4'), the sheet material of the adhesive transfer region (3') is shown to be stretched towards the biological sample (4') at its first position so as to contact and adhere to the biological sample (4'), and then retracted to lift the biological sample (4'*) from its first position, as shown in more detail in FIG. 2 for the adhesive transfer region (3) and biological sample (4). [Figure 7] The transfer device (1) is shown to be moved vertically upwards until the distance between the adhesive transfer area (3'') and the biological sample (4'') is restored. [Figure 8]The transfer device (1) is shown being moved in a horizontal plane such that a further adhesive transfer area (3'') defined by the hole (2') and a further biological sample (4'') at least partially overlap vertically, the biological sample (4'') being located on a sample support plate (5). [Figure 9] The transfer device (1) is shown after being moved downward to vertically decrease the distance between the adhesive transfer region (3'') and the further biological sample (4''). Once the adhesive transfer region (3'') is within proximity of the further biological sample (4''), the sheet material of the adhesive transfer region (3'') is stretched towards the biological sample (4'') in its first position to contact and adhere to the biological sample (4'') and then retracted to lift the biological sample (4''*) from its first position, as shown in more detail for the adhesive transfer region (3) and biological sample (4) in FIG. [Figure 10] The transfer device (1) is shown after being moved upwards to increase the distance vertically between the adhesive transfer area (3'') and the first position of the first biological sample (4''*). [Figure 11] The transfer device (1) is shown being moved in a horizontal plane such that a further adhesive transfer area (3''') defined by holes (2''') and a further biological sample (4''') at least partially overlap vertically, the biological sample (4''') being located on a sample support plate (5). [Figure 12]The transfer device (1) is shown after being moved downwards to vertically decrease the distance between the adhesive transfer region (3''') and the further biological sample (4'''). Once the adhesive transfer region (3''') is within proximity of the further biological sample (4'''), the sheet material of the adhesive transfer region (3''') is stretched towards the biological sample (4''') in its first position to contact and adhere to the biological sample (4'''), and then retracted to lift the biological sample (4'''*) from its first position, as shown in more detail for the adhesive transfer region (3) and biological sample (4) in Figure 2. At this point, all of the adhesive transfer regions (3, 3', 3'', 3'') have taken up the biological samples (4, 4', 4'', 4''') and the biological samples are in separate second positions on the transfer device. [Figure 13] 1 shows a transfer device (1) having all adhesive transfer areas (3, 3', 3'', 3''') incorporating biological samples (4, 4', 4'', 4''') and positioned on top of a multiwell plate having four wells (7, 7', 7'', 7'''') in which the biological samples are released from distinct second locations on the transfer device, causing each of the adhesive transfer areas (3, 3', 3'', 3''') to vertically overlap with the wells (7, 7', 7'', 7''') of the multiwell plate. Note that the four biological samples, which were originally randomly distributed in the tissue sample, are now rearranged in a regular pattern on the transfer device, i.e., a 2 x 2 rectangular array, at their second locations. [Figure 14] An exploded view shows the simultaneous release of biological samples (4, 4', 4'', 4''') from their separate secondary locations in the adhesive transfer regions (3, 3', 3'', 3''') into the wells of a multi-well plate. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0064] In the method according to the invention, several spatially distinct biological samples are essentially transferred from several spatially distinct first locations to several spatially distinct second locations, and in so doing are also rearranged from the irregular pattern that is specific to and determined by the tissue sample to the regular pattern that is determined by the downstream analysis that the biological samples undergo. In general, the number of spatially distinct samples, first locations and second locations are the same, i.e., each individual biological sample is captured and ends up in its individual well in the multi-well plate e. In this case, N is equal to M in the method according to the invention, in other words, only one biological sample is captured per adhesive transport area. However, if the characteristics to be analyzed are common, it may be advantageous to collect two or more spatially distinct biological samples from two or more distinct first locations to one and the same second location. In this case, N is greater than M in the method according to the invention, in other words, two or more biological samples are captured per adhesive transport area.

[0065] The term "biological sample" refers to a sample containing or consisting of a cell or cluster of cells that can be found in the tissue or body fluid of a living organism, for example an animal or plant.

[0066] Biological samples are typically prepared before being treated with the methods according to the present invention, as is known in the art.

[0067] In one embodiment, the biological sample consists essentially of a single cell or a cluster of cells.

[0068] In another embodiment, the biological sample comprises cells or clusters of cells and one or more synthetic polymer layers attached to the side of the sample opposite the side of the sample facing the sample support plate, in other words the side of the sample facing the transfer device or adhesive transfer region. The synthetic polymer layer is preferably a polyester layer, for example PET or PEN.

[0069] In a particular embodiment of the method according to the invention, as seen in Figure 1, a transfer device (1) formed by a plate having M holes (2, 2', 2'', 2''') is placed in a position in a horizontal plane such that a first adhesive transfer area (3) defined by a first hole (2) and a first biological sample (4) are vertically overlapped, said biological sample being located on a sample support plate (5). In general, it will be understood that overlapping can be achieved either by positioning the transfer plate in the horizontal plane or by positioning the sample support plate in the horizontal plane.

[0070] Once overlap is achieved, the transfer device (1) is moved downwards to decrease the distance between the adhesive transfer region (3) and the first biological sample (4) in the vertical direction, as seen in Figure 2. Once the adhesive transfer region (3) is within proximity of the first biological sample (4), the sheet material of the adhesive transfer region (3) is stretched towards the first biological sample (4) at its first position so as to contact and adhere to the first biological sample (4), and then the first biological sample (4) is moved toward the sheet material of the adhesive transfer region (3) so as to contact and adhere to the first biological sample (4). * ) from its first position. It will be appreciated that, in general, when the transfer apparatus is positioned above the sample support plate as in the illustrated configuration, the transfer apparatus (1) may be moved downwards or the sample support plate (5) may be moved upwards to decrease the distance between the adhesive transport region (3) and the first biological sample (4) in the vertical direction. Similarly, in general, when the transfer apparatus is positioned below the sample support plate, the transfer apparatus (1) may be moved upwards or the sample support plate (5) may be moved downwards to decrease the distance between the adhesive transport region (3) and the first biological sample (4) in the vertical direction.

[0071] Once the first biological sample (4) is lifted, the transfer device is moved upwards to increase the distance between the adhesive transfer area (3) and the first biological sample (4) in the vertical direction, as seen in FIG. 3. This allows the transfer device (1) or the lifted biological sample (4) to be moved upwards.* This allows for repositioning of the transfer apparatus for the next iteration without risk of collision between the adhesive transport area (3) and the biological sample (5). It will be appreciated that, in general, when the transfer apparatus is positioned above the sample support plate as in the illustrated configuration, the transfer apparatus (1) may be moved upwards or the sample support plate (5) may be moved downwards to increase the distance between the adhesive transport area (3) and the first biological sample (4) in the vertical direction. Similarly, in general, when the transfer apparatus is positioned below the sample support plate, the transfer apparatus (1) may be moved downwards or the sample support plate (5) may be moved upwards to increase the distance between the adhesive transport area (3) and the first biological sample (4) in the vertical direction.

[0072] As can be seen from Figures 4 to 7, once the first iteration is completed, the next iteration is performed. As can be seen in Figure 4, the transfer device (1) formed by a plate with M holes (2, 2', 2'', 2''') is then positioned in a horizontal plane such that the further adhesive transfer area (3') defined by the holes (2') overlaps in the vertical direction with the further biological sample (4'), which is located on the sample support plate (5). Then, as can be seen in Figure 5, the transfer device (1) is moved downwards to reduce the distance between the adhesive transfer area (3') and the further biological sample (4') in the vertical direction. As shown in Figure 6, once the adhesive transfer area (3') is in the vicinity of the further biological sample (4'), the sheet material of the adhesive transfer area (3') is stretched towards the biological sample (4') in its first position so as to contact and adhere to the biological sample (4') and then the biological sample (4') is transferred to the sample support plate (5). * Once the biological sample (4) has been lifted, the transfer tool is moved upwards again to increase the distance between the adhesive transfer area (3) and the biological sample (4) in the vertical direction, as seen in FIG. 7. This causes the transfer tool (1) or the lifted biological sample (4) to move upwards, as seen in FIG. 8. * , 4' *This allows repositioning of the transfer tool to begin the next iteration without risk of collision between the adhesive transfer regions (3, 3', 3'', 3''') and the biological sample (5). In Figures 8-12, two more iterations are performed until all adhesive transfer regions (3, 3', 3'', 3''') have taken up the biological sample (4, 4', 4'', 4''').

[0073] Once all of the adhesive transfer regions (3, 3', 3'', 3''') have loaded the biological samples (4, 4', 4'', 4''') into their distinct second locations on the transfer device, the transfer device (1) is positioned on top of the multiwell plate (6) such that each of the adhesive transfer regions (3, 3', 3'', 3''') vertically overlaps each of the wells of the multiwell plate, as shown in FIG. 13. Note that on the transfer device, the four biological samples that were originally randomly distributed in the tissue sample are now rearranged into a regular pattern, i.e., a 2 x 2 rectangular array, at their distinct second locations on the transfer device.

[0074] As can be seen from the exploded view of FIG. 14, each of the separate biological samples (4, 4', 4'', 4''') are simultaneously released from separate second locations within the adhesive transfer regions (3, 3', 3'', 3''') into wells on a multiwell plate (6). [Explanation of symbols]

[0075] 1 Transfer equipment 2 holes 3 Adhesive transfer area 4. Biological samples 5 Sample Support Plate 6. Tissue Samples 7 wells

Claims

1. 1. A method for repositioning and transferring N distinct biological samples from N distinct first locations arranged in an irregular pattern to M distinct second locations on a transfer device arranged in a regular pattern, the transfer device including at least M adhesive transfer regions, the at least M adhesive transfer regions being formed from at least one flexible sheet material, wherein N≧2 and N≧M; a) positioning one adhesive transfer region among the at least M adhesive transfer regions so as to overlap one distinct biological sample among the N distinct biological samples in its first position in a vertical direction, and optionally decreasing the distance between the first position of the distinct biological sample and the one adhesive transfer region in a vertical direction before or after the positioning step; b) stretching the flexible sheet material of the one adhesive transfer region of the at least M adhesive transfer regions so that the flexible sheet material of the one adhesive transfer region of the M adhesive transfer regions contacts the one distinct biological sample of the N distinct biological samples at its first location and adheres the one distinct biological sample of the N distinct biological samples to the flexible sheet material of the one adhesive transfer region of the M adhesive transfer regions; c) shrinking the flexible sheet material of the one adhesive transfer region of the at least M adhesive transfer regions to remove the one distinct biological sample of the N distinct biological samples from its first location and transfer the one distinct biological sample of the N distinct biological samples to its second location, optionally increasing the vertical distance between the first location of the one distinct biological sample and the second location of the one distinct biological sample on the one adhesive transfer region after said shrinking step; d. repeating steps a through c, in that order, for each remaining one of the N distinct biological samples individually, so as to remove each remaining one of the N distinct biological samples from its first location and transfer each remaining one of the N distinct biological samples to its second location; the flexible sheet material is optically transparent at least within the visible spectrum (VIS); The method of claim 1, wherein stretching the flexible sheet material of the one adhesive transfer region among the M adhesive transfer regions is accomplished by applying mechanical pressure using a plunger, the plunger comprising an optically transparent material.

2. The method of claim 1 , wherein the extending plunger is made of an optically transparent material.

3. 3. The method of claim 1 or 2, wherein the at least N distinct biological samples in their first positions are arranged on a sample support plate, such as a microscope slide.

4. 3. The method of claim 1 or 2, wherein the at least M adhesive transfer regions of the flexible sheet material are coated with an adhesive, such as a silicone resin, and / or the flexible sheet material comprises or consists of an elastomeric polymer material, such as a silicone resin, and / or the flexible sheet material comprises or consists of a thermoplastic polymer material selected from polyolefins, polyesters, polycarbonates or polyamides, and / or the flexible sheet material has a thickness of from 40 to about 400 micrometers, preferably from 40 to about 200 micrometers, and / or the flexible sheet material is a cast film or a blown film.

5. e. attaching an extension plate having at least M holes to the transfer device so that cross-sectional areas of the at least M holes vertically overlap the at least M adhesive transfer regions of the transfer device to form at least M wells, preferably containing biological samples at the bottom thereof, wherein sidewalls of each of the at least M wells are defined by inner walls of the at least M holes in the extension plate and bottoms of the at least M wells are defined by the at least M adhesive transfer regions; The method of claim 1 further comprising:

6. e. positioning the transfer device so that the at least M adhesive transfer regions vertically overlap at least M wells on a receiver plate having at least M wells and the at least N distinct biological samples face the at least M wells of the receiver plate, optionally decreasing the vertical distance between the at least M adhesive transfer regions of the transfer device and the bottoms of the at least M wells of the receiver plate, and simultaneously or sequentially releasing each of the distinct biological samples from the at least M adhesive transfer regions of the transfer device into the at least M wells of the receiver plate; The method of claim 1 further comprising:

7. 7. The method of claim 5 or 6, wherein after step d, and preferably between steps d and e, the N separate biological samples on the at least M adhesive transfer regions of the transfer device are treated with a release solution comprising a release agent, preferably an aqueous release solution comprising a release agent selected from proteolytic enzymes such as trypsin.

8. 7. The method of claim 6, wherein the at least M wells on the receiver plate are M wells on a multiwell plate, preferably M wells on a 96-well plate, with the proviso that M is 96 or less, or the at least M wells on the receiver plate are M wells on a multiwell plate, preferably M wells on a 384-well plate, with the proviso that M is 384 or less.

9. 9. The method of any one of claims 1, 2, 5, 6, and 8, wherein the at least N distinct biological samples are laser microdissected samples.

10. 9. The method of any one of claims 1, 2, 5, 6 and 8, wherein the transfer device comprises a plate having at least M holes and supporting the sheet material, the M adhesive transfer areas being defined by the overlap of cross-sectional areas of the holes and the sheet material, the holes preferably being circular or polygonal, and the plate being made of metal or polymer.

11. 9. The method of any one of claims 1, 2, 5, 6 and 8, wherein the at least M adhesive transfer regions are formed by a plurality, particularly up to M, separate sheets of the flexible sheet material.

12. 10. An apparatus configured to carry out a method for transferring N distinct biological samples at N distinct first locations to M distinct second locations on a receiving plate using a transfer device according to claim 5 or 6, comprising: i. a first unit configured to optically detect the N distinct biological samples at the N distinct first locations; ii. A second unit configured to perform steps a. to c. according to claim 5 or 6 for each of the N distinct biological samples detected in step i, the second unit comprising a mechanical subunit having a plunger capable of stretching and contracting the sheet material of the N adhesive transfer regions, the stretching of the sheet material of the adhesive transfer regions being performed by applying mechanical pressure using the plunger, the plunger comprising an optically transparent material; iii. A third unit configured to attach an extension plate to the transfer device according to the method of claim 5 or to position the transfer device on a receiver plate according to claim 6.

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