Method for transferring and rearranging biological samples

Abstract

A method of rearranging and transferring N individual biological samples from N individual first locations arranged in an irregular pattern to M individual second locations arranged in a regular pattern on a transfer apparatus comprising at least M adhesive transfer regions, wherein the at least M adhesion transfer regions are formed from at least one sheet of flexible sheet material, and wherein N > = 2 and N > = M.

Description

Technical Field

[0001] The present invention relates to a method of transferring and rearranging a plurality of individual biological specimens from a plurality of individual first locations, such as on a microscope slide, to a plurality of individual second locations, such as wells, on a receiving plate. Background Art

[0002] In medical diagnostics as well as in research and development, there is a need to be able to separate rare cells or small tissue samples that are present in small numbers within a larger solid tissue sample.

[0003] These rare cells or tissue samples are usually encapsulated in tissues, and although existing technologies can cut these cells from the sample, such as laser microdissection (LMD), the transfer of these rare cells is performed in a one-by-one manner, that is, by picking up a given target rare cell, or a rare cell cluster, from the matrix and transferring it to a sample well. Rare cells are usually located on a microscope slide, on which they are identified by optical microscopy, especially fluorescence microscopy, and must be transferred to a specialized container for further analysis, such as genetic analysis.

[0004] For example, it is known to transfer LMD samples using the inner surface of a microcentrifuge tube cap or Eppendorf as a transfer surface. Using a robotic tube holder, the transfer surface on the inner surface of the opened tube cap is brought into contact with the LMD sample, and the sample is made to adhere to the inner surface of the microcentrifuge tube cap by gently pushing the transfer surface vertically onto the sample from above. The cap with the attached sample is then pulled out. Once completed, the microcentrifuge tube is closed and centrifuged. By the applied centrifugal force, the sample is released from the inner surface of the tube cap and drips into the well of the microcentrifuge tube.

[0005] This method of transferring single cells is time-consuming and labor-intensive, requiring the use of a large amount of materials and equipment space. In addition to the above disadvantages, the cells are stored in individual containers, and it may be necessary to transfer them again individually to a container that can accommodate multiple samples in multiple wells for further analysis, such as a 96-well plate. This additional transfer step increases the risk of losing at least part of the analyzed material.

[0006] It is therefore an object of the present invention to provide a method as well as a device and an apparatus by means of which the complexity of the above-mentioned known transfer methods can be reduced. Summary of the invention

[0007] The present invention provides a method, apparatus and device to facilitate the transfer and rearrangement of multiple spatially separate biological samples from multiple spatially separate locations on a tissue sample, and from the inherent "native" pattern of the tissue sample to multiple spatially separate locations on a transfer device, into a regular pattern, such as a rectangular or polar array.

[0008] The first object of the present invention is to provide a method for rearranging and transferring N individual biological samples from N individual first positions arranged in an irregular pattern to M individual second positions arranged in a regular pattern on a transfer device, wherein the transfer device comprises at least M adhesive transfer areas, wherein the at least M adhesive transfer areas are formed by at least one piece of flexible sheet material, and wherein N≥2 and N≥M. The method comprises the following steps in sequence:

[0009] a. positioning the bonding transfer area of ​​at least M bonding transfer areas so that the bonding transfer area overlaps with the individual biological sample in its first position of the N individual biological samples in the vertical direction, and optionally before or after the above positioning step, reducing the distance between the first position of the individual biological sample and the bonding transfer area in the vertical direction,

[0010] b. stretching the flexible sheet material of the at least M adhesive transfer areas so that the flexible sheet material of the adhesive transfer area contacts the individual biological samples in the N individual biological samples in their first position, and causing the individual biological samples in the N individual biological samples to adhere to the flexible sheet material of the adhesive transfer area in the M adhesive transfer areas,

[0011] c. retracting the flexible sheet material of at least M adhesive transfer areas to remove the individual biological samples of the N individual biological samples from their first positions and transfer the individual biological samples of the N individual biological samples to their second positions on the transfer device, and optionally after the retracting step, increasing the distance in the vertical direction between the first position of the individual biological sample and the second position of the individual biological sample on the adhesive transfer area,

[0012] d. Repeating steps a to c for each of the remaining individual biological samples of the N individual biological samples, respectively, to remove each of the remaining individual biological samples of the N individual biological samples from its first position and to transfer each of the remaining individual biological samples of the N individual biological samples to its second position.

[0013] One advantage of the method according to the first object of the invention is that biological samples can be directly removed and transferred to a transfer device without intermediate steps and thus a single transfer device loaded with multiple biological samples in a regular array facilitates downstream processing of multiple biological samples.

[0014] It should be understood that the method according to the first object of the present invention can be implemented when the sample support plate is oriented so that the individual biological sample is facing downward in the vertical direction, or when the sample support plate is oriented so that the individual biological sample is facing upward in the vertical direction. The sample support plate can be flipped to the preferred orientation of the sample support plate. However, independently of the orientation of the sample support plate, the transfer device is positioned on the side of the sample support plate containing the individual biological sample. Therefore, when the sample support plate is oriented so that the individual biological sample is facing downward in the vertical direction, the transfer device is positioned below the sample support plate, and at least M adhesive transfer areas face the individual biological sample; and when the sample support plate is oriented so that the individual biological sample is facing upward in the vertical direction, the transfer device is positioned above the sample support plate, and at least M adhesive transfer areas face the individual biological sample. It should be noted that the adhesion of the biological sample to the adhesive transfer area makes it possible to implement the method in either direction.

[0015] In a preferred embodiment of the method according to the first object of the present invention, N can be equal to M.

[0016] In a preferred embodiment of the method according to the first object of the present invention, the method further comprises the following steps:

[0017] e. Fixing a reinforcing plate having at least M perforations to a transfer device so that the cross-sectional area of ​​the at least M perforations overlaps with the at least M adhesive transfer areas of the transfer device in a vertical direction to form at least M wells including biological samples, preferably including biological samples at the bottom of the M wells, wherein the side walls of each of the at least M wells are at least partially and preferably entirely defined by the inner walls of the at least M perforations of the reinforcing plate, and the at least M adhesive transfer areas at least partially define the bottom of the at least M wells.

[0018] The advantage of the above preferred embodiment of the method according to the first object of the present invention is that by fixing the reinforcement plate, further transfer of the biological sample to a separate multi-well plate for downstream analysis can be avoided, because the fixing of the reinforcement plate results in the "on-sample" morphology of the side walls of the well formed around each of the biological samples. Each newly formed well can then receive a liquid, such as a lysis buffer, thereby enabling further analysis of the biological sample. In a more preferred embodiment, in the vertical direction, the cross-sectional area and cross-sectional shape of at least M perforations are substantially the same as the area and shape of at least M adhesive transfer areas of the transfer device. In another more preferred embodiment, in the vertical direction, the cross-sectional area and cross-sectional shape of at least M perforations are smaller than the area and shape of at least M adhesive transfer areas of the transfer device, in which case the area of ​​the adhesive transfer area surrounding the cross-sectional area and cross-sectional shape of at least M perforations forms a sealing gasket between the transfer device and the reinforcement plate. It should be understood that when the reinforcement plate having at least M perforations is fixed to the transfer device, a seal is formed on the contact surface between the at least M adhesive transfer areas of the transfer device and the reinforcement plate having at least M perforations. It should also be understood that the reinforcement plate having at least M perforations can be fixed to the transfer device using suitable means (for example, mechanical fixing devices such as clamps, screws, interlocking pins and recesses, latches, or other means such as gluing).

[0019] In a preferred embodiment of the method according to the first aspect of the invention, the reinforcing plate may be made of metal or a synthetic thermoplastic polymer such as a polyolefin or a polymer resin such as a silicone.

[0020] In a preferred embodiment of the method according to the first object of the invention, the reinforcing plate may comprise an integrated cap for closing the well formed.

[0021] In a preferred embodiment of the method according to the first object of the present invention, the method further comprises the steps of:

[0022] f. Closing the well formed in step e, preferably with an integrated cap of a cap bar or cap pad or reinforcement plate.

[0023] In a preferred embodiment of the method according to the first object of the present invention, the reinforcing plate with at least M perforations can be provided with a gasket to improve the sealing between the reinforcing plate and the transfer device. It may be particularly advantageous if the cross-sectional area and cross-sectional shape of the at least M perforations in the vertical direction are larger than the area and shape of the at least M adhesive transfer areas of the transfer device.

[0024] In another preferred embodiment of the method according to the first object of the present invention, the method further comprises the steps of:

[0025] e. Positioning the transfer device so that at least M adhesive transfer areas overlap with at least M wells on a receiving plate having at least M wells in a vertical direction and at least N individual biological samples face the at least M wells of the receiving plate, optionally reducing the vertical distance between the at least M adhesive transfer areas of the transfer device and the bottom of the at least M wells of the receiving plate, and simultaneously or sequentially releasing each of the individual biological samples from the at least M adhesive transfer areas of the transfer device into the at least M wells of the receiving plate.

[0026] According to the first object method of the present invention, when step e is included, for transferring N samples, the method allows the task of transferring multiple samples from multiple locations to be potentially simplified by performing approximately N contact steps, N picking steps and one transfer step (2N+1), without having to perform N contact steps, N picking steps and N transfer steps (a total of 3N) as in the case of transferring N samples one by one.

[0027] In a preferred embodiment of the method according to the first aspect of the invention, the receiving plate may comprise an integrated cap for each of the wells of the receiving plate to close the well.

[0028] In a preferred embodiment of the method according to the first object of the present invention, the method further comprises the steps of:

[0029] f. Close the wells of the receiving plate, preferably using cap strips or cap pads or integrated caps on the receiving plate.

[0030] In a preferred embodiment of the method according to the first object of the present invention, the method further comprises the following step after step d, and preferably between step d and step e: treating the N individual biological samples on at least M adhesive transfer areas of the transfer device with a release solution comprising a release agent, preferably an aqueous release solution comprising a release agent selected from a proteolytic enzyme (e.g., trypsin). In particular, this may help to release each of the individual biological samples from the at least M adhesive transfer areas of the transfer device to at least M wells of the receiving plate simultaneously or sequentially in step e. For example, when releasing the sample from the transfer device in a centrifuge, the use of a release solution can reduce the centrifugation time, or reduce the acceleration.

[0031] In a preferred embodiment of the method according to the first object of the present invention, in step a, the distance between the first position of the individual biological sample and the adhesive transfer area in the vertical direction can be reduced, and in step c, the distance between the first position of an individual biological sample and an adhesive transfer area in the vertical direction can be increased. When the transfer device is close to the biological sample, it is easier to pick up the biological sample by extending / retracting the sheet material of the adhesive transfer area because less extension / retraction is required. Similarly, after picking up the biological sample, the transfer device can be withdrawn so as to be repositioned in the horizontal plane for the next iteration of picking up the next biological sample.

[0032] However, the step of approaching / withdrawing the transfer device is optional, as the transfer device may be in the vertical orientation and remain in the vicinity during the iterations of the method, as long as the distance between the first position of an individual biological sample and an adhesive transfer area in the vertical direction is short enough to allow successful picking up of the biological sample by extension / retraction of the sheet material of the adhesive transfer area alone. For example, for an adhesive transfer area, preferably a circular adhesive transfer area, this may be the case when the distance between the adhesive transfer area and the first position of the biological sample is less than the diameter of the adhesive transfer area, or in the case of a square adhesive transfer area, this may be the case when the distance between the adhesive transfer area and the first position of the biological sample is less than the side length of the adhesive transfer area.

[0033] Typically, when the distance between the first position of the separate biological sample and the adhesive transfer area in the vertical direction is reduced, or when the distance between the first position of the separate biological sample and the second position of the separate biological sample on the adhesive transfer area in the vertical direction is increased, the threshold value is less than 3mm, 4mm or 5mm, that is, when the distance is reduced, the distance is reduced to less than 3mm, 4mm or 5mm, and when the distance is increased, the distance is increased to more than 3mm, 4mm or 5mm. However, the threshold value may also be 6mm, 7mm, 8mm, 9mm, 10mm or 15mm.

[0034] In a preferred embodiment of the method according to the first object of the present invention, the flexible sheet material is further optically transparent, preferably transparent at least in the visible spectrum (VIS) range. When the flexible sheet material is optically transparent, various advantages can be achieved according to the configuration of the device for implementing the method. In one configuration, the device includes an optical unit that can optically identify the biological sample through the flexible sheet from the side of the flexible sheet facing away from the sample (i.e. from behind the flexible sheet material). In another configuration, the device includes an optical subunit that can illuminate the sample from the side of the flexible sheet facing away from the sample (i.e. from behind the flexible sheet material) by a light source. In one configuration, the device includes an optical unit that can optically identify the biological sample through the flexible sheet from the side of the flexible sheet facing away from the sample (i.e. from behind the flexible sheet material) by an optical sensor such as a camera, and can also illuminate the sample from the side of the flexible sheet facing away from the sample (i.e. from behind the flexible sheet material) by a light source.

[0035] In a preferred embodiment of the method according to the first object of the present invention, the stretching of the sheet material of one of the M bond transfer areas is achieved 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 bond transfer areas can be controlled, preferably individually, both in terms of the degree of stretching and in terms of the duration of stretching. It should be noted that once the applied pressure is removed, the flexible sheet material will rebound to its original shape.

[0036] 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 bond transfer areas is achieved by applying mechanical pressure using a plunger. The advantage of the plunger is that no pneumatic or hydraulic leaks that could interfere with the correct operation of the method occur.

[0037] In a preferred embodiment of the method according to the first object of the present invention, the plunger preferably comprises or consists of an optically transparent material (e.g., a light guide). The light guide can be configured to emit light to the sample through the optically transparent flexible sheet material forming the adhesive transfer area. Therefore, the plunger can advantageously accomplish the task of stretching the sheet material and the task of illuminating the biological sample for better identification using an optical microscope. Alternatively, the light guide can be configured to receive light from the sample through the sheet material of the adhesive transfer area. In this case, the plunger can advantageously accomplish the task of stretching the sheet material and the task of providing an image of the sample to the imaging device.

[0038] In a preferred embodiment of the method according to the first object of the present invention, N individual biological samples are transferred simultaneously. The simultaneous transfer of samples is simplified relative to transferring each of the samples individually. The simultaneous transfer can be implemented by applying centrifugal force or vibration to the N individual biological samples. To achieve the simultaneous transfer, the transfer device can be fixed on a receiving plate (e.g., a multi-well plate), and the assembly of the receiving plate and the transfer device can be centrifuged.

[0039] In a preferred embodiment of the method according to the first object of the present invention, at least N separate biological samples are arranged in their first position on a sample support plate (e.g., a microscope slide). The sample support plate (e.g., a microscope slide) may be coated with a tissue section sample (e.g., a frozen section or a microscopic section) from which the biological sample needs to be sorted. 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. Therefore, in most cases, the spatially separate biological sample includes a cell or a cell cluster and one or more synthetic polymer layers adhered to the cell or cell cluster.

[0040] 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 adhesion transfer area of ​​the flexible sheet material is coated with an adhesive (e.g., silicone resin). The adhesive can enhance the adhesion of the biological sample to the flexible sheet material and facilitate the picking up of the biological sample in the adhesion transfer area.

[0041] In a preferred embodiment of the method according to the first object of the present invention, the flexible sheet material comprises or consists of a thermoplastic polymer material selected from polyolefins, polyesters, polycarbonates or polyamides. Alternatively, the flexible sheet material comprises or consists of an elastic polymer material such as silicone (e.g. polydimethylsiloxane (PDMS)). These polymer materials have good mechanical properties, such as elasticity.

[0042] In a preferred embodiment of the method according to the first object of the present invention, the thickness of the flexible sheet material and in particular the thermoplastic polymer material is from 40 microns to about 400 microns, preferably from 40 microns to about 200 microns. It has been found that this thickness range allows sufficient optical transmission and mechanical properties such as elasticity. Alternatively, the thickness of the flexible sheet material and in particular the elastomeric polymer material is at most 5 mm, preferably at most 2 mm.

[0043] 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.

[0044] In a preferred embodiment of the method according to the first object of the present invention, the flexible sheet material comprises or consists of a cycloolefin copolymer or a cycloolefin polymer. COC and COP have good optical properties under VIS and UV, which is advantageous in methods for identifying biological samples using a light source and an optical microscope, and exhibit good tear or rupture resistance. At the same time, it has been found that these polymers also exhibit good adhesion to biological samples, i.e., no adhesive is required.

[0045] In a preferred embodiment of the method according to the first object of the present invention, N is at least 6, 12, 24, 48, 96 or 384.

[0046] In a preferred embodiment of the method according to the first object of the present invention, the at least M wells on the receiving plate are M wells on a multi-well plate, and under the condition that M is 96 or less, preferably M wells on a 96-well plate.

[0047] In a preferred embodiment of the method according to the first object of the present invention, the at least M wells on the receiving plate are M wells on a multi-well plate, and under the condition that M is 384 or less, preferably M wells on a 384-well plate.

[0048] In a preferred embodiment of the method according to the first object of the invention, the at least N individual biological samples are at least N individual laser microdissected samples, in particular laser microdissected samples of frozen sections or microsections such as paraffin sections.

[0049] 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 perforations and supporting the sheet material, wherein M bonding transfer areas are defined by the overlap between the cross-sectional area of ​​the perforations and the sheet material, wherein the perforations are preferably circular or polygonal and wherein the plate is made of metal or polymer.

[0050] The transfer device can be formed by a support plate with at least M perforations, and wherein the sheet material is added to the surface of the support plate, in particular applied to the surface facing the biological sample. In this case, the sheet material is substantially flush with the surface of the plate of the transfer device. Alternatively, the transfer device formed by the support plate with at least M perforations may include two plates with overlapping perforations in the vertical direction, with a flexible sheet material sandwiched between the two plates. In this case, the sheet material is substantially countersunk relative to the surface of the plate of the transfer device, and the degree of countersunk depends on the thickness of the plate.

[0051] In a preferred embodiment of the method according to the first object of the present invention, the at least M perforations have the same size and / or shape.

[0052] In a preferred embodiment of the method according to the first object of the present invention, the adhesive surface area is arranged on the transfer device in a regular pattern corresponding to the second position, i.e., for example, arranged in an array, and more preferably arranged in an array that corresponds or overlaps at least partially with an array of wells on a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, a 96-well plate, or a 384-well plate. For example, the adhesive surface area can be arranged on the transfer device in an 8×12 or 16×24 array. The regular pattern on the transfer device can correspond to an array, preferably to an 8×12 or 16×24 array.

[0053] In a preferred embodiment of the method according to the first object of the present invention, the at least M adhesive transfer areas are formed by a plurality of separate flexible sheet materials, in particular by at least M separate flexible sheet materials. For example, the at least M adhesive transfer areas can be formed by a plurality of convex pieces of flexible sheet material, wherein each of the convex pieces overlaps one or more perforations on the plate of the transfer device. The convex piece can be circular, with a diameter greater than the diameter of the perforations on the plate of the transfer device, or can be a substantially rectangular strip, with a length and a width greater than the diameter of the perforations on the plate of the transfer device.

[0054] In a preferred embodiment of the method according to the first object of the present invention, the at least M adhesive transfer areas are formed by a continuous piece of flexible sheet material. In this case, the continuous piece of flexible sheet material covers all holes on the support plate of the transfer device and overlaps with these holes. Compared with using multiple pieces of flexible sheet material, when using a continuous piece of flexible sheet material, the transfer device can be assembled more conveniently.

[0055] In a preferred embodiment of the method according to the first object of the present invention, the transfer device comprises a frame and a panel, wherein the frame is preferably made of metal or polymer, and the panel is formed by a continuous piece of flexible sheet material. In this case, the piece of flexible sheet material spans the area containing the bonding surface area.

[0056] Another object of the present invention is to provide a transfer device for rearranging and transferring N separate biological samples from N separate first positions arranged in an irregular pattern to M separate second positions arranged in a regular pattern on the transfer device, and the transfer device is preferably used in the method according to the above-mentioned first object.

[0057] In a preferred embodiment of the transfer device according to another object of the present invention, the transfer device comprises at least M adhesive transfer areas formed by at least one piece of flexible sheet material, wherein the transfer device comprises a support plate having at least M perforations, and wherein the M adhesive transfer areas are defined by the overlap between the cross-sectional area of ​​the perforations and the sheet material, wherein the perforations are preferably circular or polygonal, and wherein the plate is made of metal or polymer, or the transfer device comprises a frame and a panel, wherein the frame is made of metal or polymer, and the panel is formed by a continuous piece of flexible sheet material. Therefore, on the transfer device, each of the at least M adhesive transfer areas can pick up a biological sample (N is equal to M), or can pick up more than one sample (N is greater than M).

[0058] In a preferred embodiment of the transfer device according to another 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 (e.g., silicone resin). The adhesive can enhance the adhesion of the biological sample to the flexible sheet material and facilitate the picking up of the biological sample in the adhesive transfer area.

[0059] In a preferred embodiment of the transfer device according to another object of the present invention, the flexible sheet material comprises or consists of a thermoplastic polymer material selected from polyolefins, polyesters, polycarbonates or polyamides. These polymer materials have good mechanical properties such as elasticity.

[0060] In a preferred embodiment of the transfer device according to another object of the present invention, the flexible sheet material comprises or consists of an elastic polymer material selected from silicone (eg PDMS) These polymer materials have good mechanical properties such as elasticity.

[0061] In a preferred embodiment of the transfer device according to another object of the present invention, the thickness of the flexible sheet material is from 40 microns to about 400 microns, preferably from 40 microns to about 200 microns. It has been found that this thickness range allows sufficient optical transmission and mechanical properties such as elasticity.

[0062] It has been found that this thickness range allows sufficient optical transmission and mechanical properties, such as elasticity. Alternatively, the thickness of the flexible sheet material and in particular the elastomeric polymer material is up to 5 mm, preferably up to 2 mm. It has been found that this thickness range allows sufficient optical transmission and mechanical properties, such as elasticity.

[0063] In a preferred embodiment of the transfer device according to another object of the present invention, the flexible sheet material is a cast film or a blown film.

[0064] In a preferred embodiment of the transfer device according to another object of the present invention, the flexible sheet material comprises or consists of a cycloolefin copolymer or a cycloolefin polymer. COC and COP have good optical properties under VIS and UV, which is advantageous in methods for identifying biological samples using a light source and an optical microscope, and exhibit good tear or rupture resistance. At the same time, it has been found that these polymers also exhibit good adhesion to biological samples, i.e., no adhesive is required.

[0065] In a preferred embodiment of the transfer device according to another object of the invention, the transfer device comprises a support plate with M perforations, the M adhesive transfer areas being substantially in the same plane as the surface of the plate, and wherein the surface of the plate is preferably facing the at least N individual biological samples. Compared to the case where the flexible sheet material is countersunk relative to the outer surface of the support plate, in the case where the M adhesive transfer areas are substantially in the same plane as the surface of the support plate facing the N individual biological samples, the degree of extension / retraction required to pick up the biological sample can be reduced, which provides a more robust transfer method and / or robust operation of the transfer device. When the M adhesive transfer areas are substantially in the same plane as the surface of the support plate, the manufacture of the transfer device is simplified, because at least in some embodiments, only one support plate is required, compared to at least two support plates when the M adhesive transfer areas are countersunk.

[0066] Another object of the present invention, namely the third object, is to provide an apparatus to implement the method of rearranging and transferring according to the first object of the present invention, and preferably use the transfer device according to another object of the present invention.

[0067] A third object of the present invention is to provide a device comprising:

[0068] i. a first detection unit, which is configured to perform optical detection on the N individual biological samples in their respective individual first positions, preferably a microscope or fluorescence microscope unit,

[0069] ii. a second positioning unit configured to perform steps a to d of the method according to the invention on the N individual biological samples detected in step i, such as a linear robot or a robotic arm, the second positioning unit being equipped with a pneumatic, hydraulic or mechanical subunit capable of extending and retracting the sheet material of the adhesive transfer area, the subunit being preferably connected to an optical subunit capable of illuminating the sample by a light source optically coupled to a transparent plunger of the mechanical subunit,

[0070] iii. The third unit is configured as

[0071] A. Fix the reinforcement plate to the transfer equipment, or

[0072] B. Positioning the transfer device relative to the multi-well plate.

[0073] In a preferred embodiment of the device according to the third purpose of the present invention, the device also includes a fourth unit, which is configured to apply a release solution containing a release agent, preferably together with an aqueous release solution containing a release agent selected from a proteolytic enzyme (such as trypsin), to the M bonding transfer areas of the transfer device.

[0074] Further embodiments of the invention are specified in the dependent claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] The preferred embodiments of the present invention are described below with reference to the accompanying drawings, which are used to illustrate the preferred embodiments of the present invention and are not used to limit the preferred embodiments of the present invention.

[0076] Figure 1 A transfer device (1) is shown which is formed by a plate (P) with four perforations (2, 2', 2", 2"'), each perforation being covered with a flexible sheet material, wherein a first adhesive transfer area (3) is positioned such that the first adhesive transfer area (3) defined by the first perforation (2) at least partially overlaps with a first biological sample (4) in a vertical direction, wherein the biological sample (4) is located on a sample support plate (5). The biological sample (4, 4', 4", 4"') is irregularly distributed within a tissue sample (6) on the sample support plate (5).

[0077] Figure 2 The transfer device (1) is shown moving downward after alignment to reduce the distance in the vertical direction between the adhesive transfer area (3) and the first biological sample (4). Once the adhesive transfer area (3) is located near the first biological sample (4), the sheet material of the adhesive transfer area (3) extends toward the first biological sample (4) at a first position of the first biological sample (4) to contact and adhere to the first biological sample (4), and then retracts to pick up the first biological sample (4*) from the first position of the first biological sample (4*).

[0078] Figure 3 The transfer device (1) is shown moving upwards to increase the distance in the vertical direction between the adhesive transfer area (3) and the first location of the first biological sample (4*).

[0079] Figure 4 The transfer device (1) is shown moving in a horizontal plane so that another adhesive transfer area (3') defined by perforations (2') at least partially overlaps another biological sample (4') in a vertical direction, wherein the biological sample (4') is located on a sample support plate (5).

[0080] Figure 5The transfer device (1) is shown moving downwards to reduce the distance in the vertical direction between the adhesive transfer area (3') and the further biological sample (4').

[0081] Figure 6 It is shown that once the adhesive transfer area (3') is located near another biological sample (4'), the sheet material of the adhesive transfer area (3') will extend toward the biological sample (4') at a first position of the biological sample (4') to contact and adhere to the biological sample (4'), and then retract to pick up the biological sample (4'*) from the first position of the biological sample (4'*), as shown in FIG. Figure 2 The adhesive transfer area (3) and the biological sample (4) are shown in more detail.

[0082] Figure 7 The transfer device (1) is shown moving upwards in order to again increase the distance between the adhesive transfer area (3") and the biological sample (4") in the vertical direction.

[0083] Figure 8 The transfer device (1) is shown moving in a horizontal plane so that another bonding transfer area (3") defined by a hole (2') at least partially overlaps with another biological sample (4") in a vertical direction, wherein the biological sample (4") is located on a sample support plate (5).

[0084] Fig. 9 The transfer device (1) is shown after it has been moved downward to reduce the distance in the vertical direction between the adhesive transfer area (3") and another biological sample (4"). Once the adhesive transfer area (3") is located near another biological sample (4"), the sheet material of the adhesive transfer area (3") extends toward the biological sample (4") at the first position of the biological sample (4") to contact and adhere to the biological sample (4"), and then retracts to pick up the biological sample (4"*) from the first position of the biological sample (4"), as shown in FIG. Figure 2 The adhesive transfer area (3) and the biological sample (4) are shown in more detail.

[0085] Fig.10 It shows the situation after the transfer device (1) has been moved upwards to increase the distance in the vertical direction between the adhesive transfer area (3") and the first location of the first biological sample (4"*).

[0086] Fig.11 The transfer device (1) is shown moving in a horizontal plane so that another adhesive transfer area (3'"') defined by a hole (2'"') at least partially overlaps with another biological sample (4'"') in a vertical direction, wherein the biological sample (4'"') is located on a sample support plate (5).

[0087] Fig.12The transfer device (1) is shown after it has been moved downward to reduce the distance in the vertical direction between the adhesive transfer area (3') and another biological sample (4'). Once the adhesive transfer area (3') is located near another biological sample (4'), the sheet material of the adhesive transfer area (3') extends toward the biological sample (4') at a first position of the biological sample (4') to contact and adhere to the biological sample (4'), and then retracts to pick up the biological sample (4'*) from the first position of the biological sample (4'*), as shown in FIG. Figure 2 The adhesive transfer area (3) and the biological sample (4) are shown in more detail. At this point, all adhesive transfer areas (3, 3', 3", 3"') have picked up the biological sample (4, 4', 4", 4"'), and the biological samples are all located in a single second position on the transfer device.

[0088] Fig.13 A transfer device (1) is shown, all of which have adhesive transfer areas (3, 3', 3", 3"') picked up biological samples (4, 4', 4", 4"'), and the transfer device is positioned on top of a multi-well plate having 4 wells (7, 7', 7", 7"') so that each of the adhesive transfer areas (3, 3', 3", 3"') overlaps with a well (7, 7', 7", 7"') of the multi-well plate in a vertical direction, wherein the biological samples will be released from a separate second position on the transfer device into these wells. It is noted that on the transfer device, the 4 biological samples that were previously irregularly distributed in the tissue sample are rearranged into a regular pattern, i.e., a 2x2 rectangular array, at the second position on the transfer device.

[0089] Fig.14 An exploded view of biological specimens (4, 4', 4", 4"') being simultaneously released from their individual second locations at adhesive transfer areas (3, 3', 3", 3"') into wells of a multi-well plate is shown. DETAILED DESCRIPTION

[0090] In the method according to the present invention, several spatially separate biological samples are basically transferred from several spatially separate first positions to several spatially separate second positions, and in the process, several spatially separate biological samples are also rearranged from the irregular pattern inherent to the tissue sample and defined by the tissue sample to the regular pattern defined by the downstream analysis to be experienced by the biological sample. Usually, for the several spatially separate samples, the first position and the second position are the same, that is, each single biological sample is picked up and eventually arrives in its single well on the multi-well plate e. In this case, N is equal to M in the method of the present invention, or in other words, each bonding transfer area only picks up one biological sample. However, it may be advantageous to pick up more than one spatially separate biological sample from more than one single first position to the same second position, as long as they have common features to be analyzed. In this case, N is greater than M in the method of the present invention, or in other words, each bonding transfer area picks up two or more biological samples.

[0091] The term "biological sample" refers to a sample containing or consisting of cells or cell clusters that can be found in the tissue or body fluid of an organism such as an animal or plant.

[0092] As is known in the art, biological samples are typically prepared prior to being performed according to the methods of the invention.

[0093] In one embodiment, the biological sample consists essentially of single cells or clusters of cells.

[0094] In another embodiment, the biological sample comprises cells or cell clusters and one or more synthetic polymer layers adhered to a side of the sample opposite to the side of the sample facing the sample support plate, or in other words, the side of the sample facing the transfer device or adhesive transfer area. The synthetic polymer layer is preferably a polyester layer, such as PET or PEN.

[0095] In a specific embodiment of the method according to the present invention, Figure 1 As shown, a transfer device (1) formed by a plate having M perforations (2, 2', 2", 2"') is positioned in a horizontal plane so that a first adhesive transfer area (3) defined by a first perforation (2) overlaps with a first biological sample (4) in a vertical direction, wherein the biological sample is located on a sample support plate (5). It should be understood that the overlap can generally be achieved by positioning the transfer plate in a horizontal plane or positioning the sample support plate in a horizontal plane.

[0096] like Figure 2As shown, after the overlap is achieved, the transfer device (1) is moved downward to reduce the distance between the adhesive transfer area (3) and the first biological sample (4) in the vertical direction. Once the adhesive transfer area (3) is located near the first biological sample (4), the sheet material of the adhesive transfer area (3) extends toward the first biological sample (4) at the first position of the first biological sample (4) to contact and adhere to the first biological sample (4), and then retracts to pick up the first biological sample (4*) from the first position of the first biological sample (4*). It should be understood that when the transfer device is arranged above the sample support plate as shown in the figure, the transfer device (1) can usually be moved downward, or the sample support plate (5) can be moved upward to reduce the distance between the adhesive transfer area (3) and the first biological sample (4) in the vertical direction. Similarly, when the transfer device is located below the sample support plate, the transfer device (1) can usually be moved upward, or the sample support plate (5) can be moved downward to reduce the distance between the adhesive transfer area (3) and the first biological sample (4) in the vertical direction.

[0097] like Figure 3 As shown, once the first biological sample (4) is picked up, the transfer device moves upward to increase the distance in the vertical direction between the adhesive transfer area (3) and the first biological sample (4). The transfer device can then be repositioned for the next iteration without the risk of collision between the transfer device (1) or the picked biological sample (4*) and the biological sample (5). It should be understood that when the transfer device is arranged above the sample support plate as shown, the transfer device (1) can usually be moved upward, or the sample support plate (5) can be moved downward, to increase the distance in the vertical direction between the adhesive transfer area (3) and the first biological sample (4). Similarly, when the transfer device is located below the sample support plate, the transfer device (1) can usually be moved downward, or the sample support plate (5) can be moved upward, to increase the distance in the vertical direction between the adhesive transfer area (3) and the first biological sample (4).

[0098] After the first iteration is completed, the next iteration is performed, such as Figure 4-7 As shown. Figure 4 As shown, the transfer device (1) formed by a plate with M perforations (2, 2', 2", 2"') is then positioned on a horizontal plane so that another adhesive transfer area (3') defined by the perforations (2') overlaps with another biological sample (4') in the vertical direction, wherein the biological sample (4') is located on the sample support plate (5). Then, as shown in Figure 5 As shown, the transfer device (1) is moved downward to reduce the distance in the vertical direction between the adhesive transfer area (3') and the other biological sample (4'). Figure 6As shown, once the adhesive transfer area (3') is located near another biological sample (4'), the sheet material of the adhesive transfer area (3') extends toward the biological sample (4') at the first position of the biological sample (4') to contact and adhere to the biological sample (4'), and then retracts to pick up the biological sample (4') from the first position of the biological sample (4'). Figure 7 As shown, once the biological sample (4) is picked up, the transfer device is moved upwards to increase the distance between the adhesive transfer area (3) and the biological sample (4) in the vertical direction again. Figure 8 As shown, the transfer device can be repositioned to start the next iteration without the risk of collision between the transfer device (1) or the picked-up biological sample (4*, 4'*) and the biological sample (5). Figure 8-12 Two further iterations are performed until all the adhesive transfer regions (3,3',3",3"') have picked up the biological specimens (4,4',4",4"').

[0099] like Fig.13 As shown, once all the adhesive transfer areas (3, 3', 3", 3"') have picked up the biological samples from the second positions of the biological samples (4, 4', 4", 4"') onto the transfer device, the transfer device (1) is positioned on top of the multi-well plate (6), and the wells in the multi-well plate are such that each of the adhesive transfer areas (3, 3', 3", 3"') overlaps with each of the wells of the multi-well plate in the vertical direction. It should be noted that on the transfer device, the four biological samples that were previously irregularly distributed in the tissue sample are rearranged into a regular pattern, i.e., a 2x2 rectangular array, at their respective individual second positions on the transfer device.

[0100] like Fig.14 As shown in the exploded view of FIG. 1 , each of the individual biological samples ( 4 , 4 ′, 4 ″, 4 ′″) is simultaneously released from an individual second location in the adhesive transfer area ( 3 , 3 ′, 3 ″, 3 ′″) into a well of a multi-well plate ( 6 ).

[0101] Reference numerals list

[0102] 1 Transfer device 5 Sample support plate

[0103] 2 Puncture 6 Tissue Samples

[0104] 3 Adhesion transfer area 7 wells

[0105] 4 Biological samples

Claims

1. A method for rearranging and transferring N individual biological samples from N individual first positions arranged in an irregular pattern to M individual second positions arranged in a regular pattern on a transfer device, wherein the transfer device comprises at least M adhesive transfer areas, wherein the at least M adhesive transfer areas are formed by at least one piece of flexible sheet material, and wherein N≥2 and N≥M, the method comprising the following steps in sequence: a. positioning the bonding transfer area of ​​the at least M bonding transfer areas so that the bonding transfer area overlaps with the individual biological sample in the first position of the N individual biological samples in the vertical direction, and optionally before or after the above positioning step, reducing the distance between the first position of the individual biological sample and the bonding transfer area in the vertical direction, b. stretching the flexible sheet material of the at least M adhesive transfer areas so that the flexible sheet material of the adhesive transfer areas of the M adhesive transfer areas contacts the individual biological samples in the N individual biological samples in their first position, and causing the individual biological samples in the N individual biological samples to adhere to the flexible sheet material of the adhesive transfer areas in the M adhesive transfer areas, c. retracting the flexible sheet material of the at least M adhesive transfer areas to remove the individual biological samples of the N individual biological samples from their first positions and transfer the individual biological samples of the N individual biological samples to their second positions, and optionally after the retracting step, increasing the distance in the vertical direction between the first position of the individual biological sample and the second position of the individual biological sample on the adhesive transfer area, d. repeating steps a to c for each of the remaining individual biological samples in the N individual biological samples, respectively, to remove each of the remaining individual biological samples in the N individual biological samples from its first position and to transfer each of the remaining individual biological samples in the N individual biological samples to its second position, wherein the flexible sheet material is optically transparent at least in the visible spectrum (VIS) range, Wherein stretching the flexible sheet material of one of the M bond transfer regions is achieved by applying mechanical pressure using a plunger, wherein the plunger comprises an optically transparent material.

2. A method according to any one of the preceding claims, wherein: A plunger comprised of an optically transparent material is extended.

3. A method according to any one of the preceding claims, wherein: At least N individual biological samples are arranged in their first position on a sample support plate, such as a microscope slide.

4. A method according to any one of the preceding claims, wherein: At least M bonding transfer areas of the flexible sheet material are coated with an adhesive, such as silicone, and / or wherein the flexible sheet material comprises or consists of an elastic polymer material, such as silicone, and / or wherein the flexible sheet material comprises or consists of a thermoplastic polymer material selected from polyolefins, polyesters, polycarbonates or polyamides, and / or wherein the flexible sheet material has a thickness of 40 microns to about 400 microns, preferably a thickness of 40 microns to about 200 microns, and / or wherein the flexible sheet material is a cast film or a blown film.

5. The method according to claim 1, further comprising the steps of: e. Fixing a reinforcing plate having at least M perforations to a transfer device so that the cross-sectional area of ​​the at least M perforations overlaps with the at least M adhesive transfer areas of the transfer device in a vertical direction to form at least M wells, preferably including biological samples at the bottom of the at least M wells, wherein the side walls of each of the at least M wells are defined by the inner walls of the at least M perforations of the reinforcing plate, and the bottoms of the at least M wells are defined by the at least M adhesive transfer areas.

6. The method according to claim 1, further comprising the steps of: e. Positioning the transfer device so that at least M adhesive transfer areas overlap with at least M wells on a receiving plate having at least M wells in a vertical direction and at least N individual biological samples face the at least M wells of the receiving plate, optionally reducing the vertical distance between the at least M adhesive transfer areas of the transfer device and the bottom of the at least M wells of the receiving plate, and simultaneously or sequentially releasing each of the individual biological samples from the at least M adhesive transfer areas of the transfer device into the at least M wells of the receiving plate.

7. The method according to claim 5 or 6, wherein: After step d, and preferably between step d and step e, the N individual biological samples on at least M adhesive transfer areas 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 a proteolytic enzyme, such as trypsin.

8. A method according to claim 6 or claim 7 when dependent on claim 6, wherein: At least M wells on the receiving plate are M wells on a multi-well plate, preferably, under the condition that M is 96 or less, at least M wells on the receiving plate are M wells on a 96-well plate, or wherein at least M wells on the receiving plate are M wells on a multi-well plate, preferably, under the condition that M is 384 or less, at least M wells on the receiving plate are M wells on a 384-well plate.

9. The method according to any one of the preceding claims, wherein the at least N individual biological samples are laser microdissected samples.

10. The method according to any one of claims 1 to 9, wherein the transfer device comprises a plate having at least M perforations and supporting the sheet material, wherein M bonding transfer areas are defined by the overlap between the cross-sectional area of ​​the perforations and the sheet material, wherein the perforations are preferably circular or polygonal and wherein the plate is made of metal or polymer.

11. The method according to any of the preceding claims, wherein the at least M bond transfer areas are formed from a plurality of separate pieces of flexible sheet material, and in particular from up to M separate pieces of flexible sheet material.

12. An apparatus configured to transfer N individual biological samples at N individual first positions to M individual second positions on a receiving plate using a transfer device according to the method of any one of claims 1 to 11, the apparatus comprising: i. a first unit configured to perform optical detection on N individual biological samples at N individual first positions, ii. a second unit configured to perform steps a to c according to any one of claims 1 to 11 for each of the N individual biological samples detected in step i, the second unit being equipped with a mechanical subunit having a plunger capable of extending and retracting the sheet material of the N bond transfer areas, wherein the extension of the sheet material of the bond transfer areas is achieved by applying mechanical pressure using the plunger, the plunger comprising an optically transparent material, iii. A third unit configured to fix the reinforcing plate to the transfer device according to the method of claim 5 or configured to position the transfer device on the receiving plate according to the method of claim 6.

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