Patent Application
20250180442
The present invention relates to method for the transfer and rearrangement of multiple separate biological samples from multiple separate first locations on, for example, a microscope slide, to multiple separate second locations, such as wells, on a receiving plate.
In medical diagnostics, as well as in research and development, there exists a need to be able to isolate rare cells, or small tissue samples, which occur in small numbers within larger solid tissue samples.
The rare cells or tissue samples are often surrounded within the tissue and while technology exists by which these cells can be cut from a sample, such as for example laser microdissection (LMD), the transfer of these rare cells is made in an one-by-one fashion, i.e. by picking up a given target rare cell, or a cluster of rare cells, from a substrate, and transferring it to a sample well. The rare cells are generally located on a microscope slide on which they are identified by optical, in particular fluorescence, microscopy and must be transferred to a dedicated receptacle for further analysis, for example genetic analysis.
For instance, 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 cap of the opened tube is contacted with the LMD sample, by gently and vertically pushing the transfer surface against the sample from above to adhere it to the inner surface of the microceninfuge cap. The cap is then pulled away with the sample adhered to it. Once this is done, the microcentrifuge tube is closed and centrifuged. Through the centrifugal force applied, the sample is released from the inner surface of the cap and drops to the well part of the microcentrifuge tube.
This method of transferring single cells is time and labor intensive and thus requires the use of a lot of material and equipment space. In addition to the aforementioned drawbacks, the cells are stored in individual receptacles, from which they may need to be individually transferred yet again into a container that can hold multiple samples in multiple wells for further analysis, such as for example a 96-well plate. That additional transfer step further increases the risk of losing at least part of the analytical material
It is therefore an aim of the present invention to provide a method, along with a device and apparatus, by which the above-mentioned complexity of the known transfer methods is reduced.
The present invention provides a method, a device and an apparatus that facilitate the transfer and rearrangement of multiple, spatially separate biological samples from multiple, spatially separate locations on a tissue sample, from the “native” pattern that is inherent to the tissue sample to multiple and spatially separate locations on a transfer device into a pattern that is a regular pattern, such as a rectangular or polar array.
It is a first object of the present invention to provide a method for the rearrangement and transfer of 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, said transfer device comprising at least M adhesive transfer areas, wherein the at least M adhesive transfer areas are formed of at least a flexible sheet material, and wherein N≥2 and N≥M, comprising the steps of, in this order,
One advantage of the method according to the first object of the present invention is that the biological samples can the removed and transferred to the transfer device directly without intermediate steps and that the single transfer device, thus loaded with multiple biological samples in a regular array, facilitates the downstream handling of the multiple biological samples.
It is understood that the method according to the first object of the present invention may be carried when the sample support plate is oriented such that the separate biological samples face down, in vertical direction, or when the sample support plate is oriented such that the separate biological samples face up, in vertical direction. The sample support plate may be flipped into 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 sample support plate that comprises the separate biological samples. Thus, when the sample support plate is oriented such that the separate biological samples face down, in vertical direction, the transfer device is positioned below the sample support plate, with the the at least M adhesive transfer areas facing the separate biological samples and when the sample support plate is oriented such that the separate biological samples face up, in vertical direction, the transfer device is positioned above the sample support plate, with the the at least M adhesive transfer areas facing the separate biological samples. It is noted that the adhesion of the biological samples to the adhesive transfer areas is such that it allows carrying out the method in either orientation.
In a preferred embodiment of the method according to the first object of the present invention N may be equal to M.
In a preferred embodiment of the method according to the first object of the present invention, the method further comprises the step of:
An advantage of the above preferred embodiment of the method according to the first object of the present invention is that by affixing an augmenting plate, the further transfer of the biological samples to a separate multi-well plate for down-stream analysis can be avoided, since the affixing of the augmenting plate leads to the “on-sample” formation of the side walls of a well around each of the biological samples. Each newly formed well can then receive liquids, such as for example lysis buffers, that enable further analysis of the biological samples. In a more preferred embodiment, the cross-sectional area and cross, sectional shape of the at least M perforations is essentially the same as the area and shape of the at least M adhesive transfer areas of the transfer device, in vertical direction. In an another more preferred embodiment, the cross-sectional area and cross-sectional shape of the at least M perforations is smaller than the area and shape of the at least M adhesive transfer areas of the transfer device, in vertical direction, in which case the area of the adhesive transfer area that surrounds the cross-sectional area and cross-sectional shape of the at least M perforations forms a sealing gasket between transfer device and augmenting plate. It is understood that the augmenting plate having at least M perforations to the transfer device, when affixed to the transfer device, forms a seal at the contact surfaces between the at least M adhesive transfer areas of the transfer device and the augmenting plate having at least M perforations. It is further understood that the augmenting plate having at least M perforations can be affixed to the transfer device, using suitable means such as mechanical fixation means, like for example clamps, screws, interlocking pins and recesses, latches, and so forth or other means such as glueing.
In preferred embodiment of the method according to the first object of the present invention, the augmenting plate may be formed of metal or of a synthetic thermoplastic polymer such as for example polyolefin or a polymer resin such as silicon resin.
In preferred embodiment of the method according to the first object of the present invention, the augmenting plate may comprise integrated caps to close the formed wells.
In preferred embodiment of the method according to the first object of the present invention, the method further comprises, after step e., the step of:
In a preferred embodiment of the method according to the first object of the present invention, the augmenting plate having at least M perforations may be equipped with a gasket, to enhance the seal between the augmenting plate and the transfer device. This may be particularly advantageous if the cross-sectional area and cross-sectional shape of the at least M perforations is greater than the area and shape of the at least M adhesive transfer areas of the transfer device, in vertical direction.
In another preferred embodiment of the method according to the first object of the present invention, the method further comprises the step of:
The method according to the first object of the present invention, when including step e., allows to potentially simplify the task of transferring multiple samples from multiple locations by performing roughly N contact and N pickup steps and one transfer step (2N+1), for the transfer of N samples, instead of N contact and N pickup steps and N transfer steps (3N) in the case of one-by-one transfer of N samples.
In preferred embodiment of the method according to the first object of the present invention, the receiver plate may comprise integrated caps for each of the wells of the received plate to close the wells.
In preferred embodiment of the method according to the first object of the present invention, the method further comprises, after step e., the step of:
In a preferred embodiment of the method according to the first object of the present invention, the method further comprises the step of, after step d., and preferably between step d. and e., 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 chosen among proteolytic enzymes, such as trypsin. In particular, this may facilitate releasing each of the separate biological samples, either simultaneously or sequentially, from the at least M adhesive transfer areas of the transfer device into the at least M wells of the receiver plate in step e. For instance, the use of a releasing solution allows for shorter centrifugation time or for lesser acceleration when releasing the sample from the transfer device in a centrifuge.
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 separate biological sample and the adhesive transfer area can be decreased in vertical direction, and in step c. the distance between the first location of one separate biological sample and the one adhesive transfer area is increased in vertical direction. When the transfer device is approached to the biological sample, the biological sample may be taken up via the extension/retraction of the sheet material of the adhesive transfer area more easily because less extension/retraction is necessary. Likewise, after the biological sample is taken up, the transfer device may be withdrawn to facilitate the repositioning within the horizontal plane for the next iteration of taking up the next biological sample.
However, the step of approaching/withdrawing the transfer device is optional, since the transfer device may be, and remain in proximity, in the vertical direction during the iteration of the method, provided the distance between the first location of one separate biological sample and the one adhesive transfer area in vertical direction is small enough such as to allow a successful taking up of the biological sample solely by the extension/retraction of the sheet material of the adhesive transfer area. For example, this may be the case for adhesive transfer areas, preferably circular ones, when the distance between the adhesive transfer area and the first location of the biological sample is smaller than the diameter of the adhesive transfer area, or in the case of a square adhesive transfer area when the distance between the adhesive transfer area and the first location of the biological sample is smaller than a side of the adhesive transfer area.
In general, when decreasing the distance, in vertical direction, between the first location of the separate biological sample and the adhesive transfer area, or when increasing the distance, in vertical direction, between the first location of the separate biological sample and the second location of the separate biological sample on the adhesive transfer area, the threshold is less than 3, 4 or 5 mm, meaning that when decreasing the distance, the distance is decreased to less than 3, 4 or 5 mm and that when increasing the distance the distance is increased to more than 3. 4 or 5 mm. However, the threshold may also be 6, 7, 8, 9, 10 or 15 m.
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 at least within the visible spectrum (VIS). When the flexible sheet material is optically transparent, various advantages are achieved depending on the configuration of the apparatus used to carry out the method. In one configuration, the apparatus includes an optical unit capable of optically identifying the biological samples across the flexible sheet from the side of the flexible sheet that faces away from the sample, i.e. from behind the flexible sheet material. In another configuration, the apparatus includes an optical subunit capable of illuminating the samples via a light source from the side of the flexible sheet that faces away from the sample i.e. from behind the flexible sheet material. In one configuration, the apparatus includes an optical unit that is capable of optically identifying the biological samples, via an optical sensor such as a camera, across the flexible sheet from the side of the flexible sheet that faces away from the sample, i.e. from behind the flexible sheet material as well as illuminating the samples, via a light source, from the side of the flexible sheet that faces away from the sample i.e. from behind the flexible sheet material.
In a preferred embodiment of the method according to the first object of the present invention, the extension the sheet material of the one adhesive transfer area of the M adhesive transfer areas is achieved by applying pneumatic, hydraulic or mechanical pressure. By applying pneumatic, hydraulic or mechanical pressure, the extension of the sheet material of the one adhesive transfer area of the M adhesive transfer areas can be controlled, preferably individually, both with respect to the degree of extension and the duration of extension, It is noted that the flexible sheet material will spring back into its original shape once the applied pressure is removed.
In a preferred embodiment of the method according to the first object of the present invention, the extension of the sheet material of the one adhesive transfer area of the M adhesive transfer areas is achieved by applying mechanical pressure via a plunger. A plunger has the advantage of not suffering from possible pneumatic or hydraulic leaks which may interfere with the correct operation of the method.
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 such as for example a light guide. The light guide may be configured to emit light towards the sample, across an optically transparent flexible sheet material forming the adhesive transfer areas. Thus, the plunger can advantageously fulfill both the task of extending the sheet material and that of illuminating the biological sample for better identification via an optical microscope. Alternatively, the light guide may be configured to receive light from the sample, across the sheet material in the adhesive transfer areas. In that case, the plunger can advantageously fulfil both the task of extending the sheet material and that of providing an image of the sample to an imaging device.
In a preferred embodiment of the method according to the first object of the present invention, the N separate biological samples are transferred simultaneously. The simultaneous transfer of the samples is simplified with respect to individually transferring each of the sample. The simultaneous transfer can be carried out by applying a centrifugal force or vibrations to the N separate biological samples. In order to achieve this, the transfer device can be affixed to the receiver plate such as for example a multi-well plate, and centrifuging the assembly of receiver plate and transfer device.
In a preferred embodiment of the method according to the first object of the present invention, the at least N separate biological samples in their first locations are arranged on a sample support plate, such as for example a microscope slide. The sample support plate such as a microscope slide may have a tissue section sample, such as for example a cryotome or microtome section, from which the biological sample is to be separated, applied onto it. In general, the tissue section sample will be covered with a synthetic polymer layer such as a laser sensitive layer such that the target biological sample can be cut out via laser microdissection. In most cases, thus, the spatially separate biological samples comprise a cell or cluster of cells and one or more synthetic polymer layers adhered to the cell or a cluster of cells
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 areas of the flexible sheet material are coated with an adhesive agent, such as for example a silicone resin. The adhesive may enhance the adhesion of the biological sample to the flexible sheet material and facilitate the taking up of the biological sample at the adhesive transfer areas.
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 chosen among polyolefins, polesters, polycarbonates or polyamides. Alternatively, the flexible sheet material comprises, or consists of, a elastomeric polymer material such as silicone resin, for example polydimethylsiloxane (PDMS). These polymer materials were found to have good mechanical properties, such as elasticity.
In a preferred embodiment of the method according to the first object of the present invention, the flexible sheet material and in particular the thermoplastic polymer material has a thickness of from 40 to about 400 micrometers, preferably of from 40 to about 200 micrometers. It has been found that the range of thickness allows for sufficient optical transmission and mechanical property, such as elasticity. Alternatively, the flexible sheet material and in particular the elastomeric polymer material has a thickness of from up to 5 mm, preferably of up to 2 mm.
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 blown film.
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 cyclic olefin copolymer or cyclic olefin polymer. COC and COP have good optical properties in VIS and UV, which is advantageous when the method uses light sources and optical microscopes to identify the biological samples, and exhibit a good resistance to tear or bursting. At the same time, it has been found that these polymers further exhibit good adhesion to the biological samples as—is, i.e. without a need to apply an adhesive agent.
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.
In a preferred embodiment of the method according to the first object of the present invention, the at least M wells on a receiver plate are M wells on a multi-well plate, preferably M wells on a 96-well plate, under the proviso that M is 96 or less.
In a preferred embodiment of the method according to the first object of the present invention, the at least M wells on a receiver plate are M wells on a multi-well plate, preferably M wells on a 384-well plate, under the proviso that M is 384 or less.
In a preferred embodiment of the method according to the first object of the present invention, the at least N separate biological samples are at least N separate laser microdissection samples, in particular laser microdissection samples of cryotome sections or microtome sections such as paraffin microtome sections.
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 the M adhesive transfer areas are defined by the overlap between the cross-sectional area of said perforations and the sheet material, wherein the perforations are preferably circular or polygonal and wherein the plate is made from a metal or a polymer.
The transfer device may be formed by a support plate having at least M perforations and where the sheet material is applied to a face of the support plate, in particular to the face directed at the biological samples. 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 perforations may comprise two plates having overlapping perforations, in vertical direction, the two plates having the flexible sheet material sandwiched between them. In this case the sheet material is essentially countersunk with respect to the surface of the plates of the transfer device, to a degree depending on the thickness of the plates.
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.
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 that of the second locations, i.e. for example in an array, and more preferably are arranged in an array that at least partially corresponds to, or overlaps with, an array of wells in a 6-, 12-, 24-, 48-, 96-, 384-well plate. For example, the adhesive surface areas may be arranged on the transfer device in an array 8×12 or an array of 16×24. The regular pattern on the transfer device may correspond to an array, preferably an array of 8×12 or an array of 16×24.
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, and in particular by at least M separate sheets of the flexible sheet material. For instance, the at least M adhesive transfer areas may be formed by a plurality of tabs of the flexible sheet material, with each of the tabs overlapping with one or multiple perforations of the plate of the transfer device. The tabs may be circular with a diameter that is larger than the diameter of the perforations in the plate of the transfer device, or may be essentially rectangular strips in which the length and breadth are each larger than the diameter of the perforations in the plate of the transfer device.
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 one continuous sheet of the flexible sheet material. In this case, the one continuous sheet of the flexible sheet material covers and overlaps with all the perforations of the support plate of the transfer device. When using one continuous sheet of the flexible sheet material, the transfer device can be more easily assembled when compared to the use of multiple tabs of flexible sheet material.
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 from a metal or a polymer and the panel is formed by one continuous sheet of the flexible sheet material. In this case, the one sheet of the flexible sheet material spans the area in which the adhesive surface areas are comprised.
It is a further object of the present invention to provide a transfer device for use in a method for the rearrangement and transfer of 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 said transfer device, and which is preferably for use in the method according to the above first object.
In a preferred embodiment of the transfer device according to the further object of the present invention, the transfer device comprises at least M adhesive transfer areas formed of at least one sheet of a flexible sheet material, wherein said transfer device comprises a support plate having at least M perforations and wherein the at least M adhesive transfer areas are defined by the overlap between the cross-sectional area of said perforations and the sheet material, wherein the perforations are preferably circular or polygonal, and wherein the plate is made from a metal or a polymer, or wherein said transfer device comprises a frame and a panel, wherein the frame is made from a metal or a polymer and the panel is formed by one continuous sheet of the flexible sheet material. Thus, on the transfer device, each of the at least M adhesive transfer areas may take up one biological sample (N equals M), or may take up more than one sample (N larger than M).
In a preferred embodiment of the transfer device according to the further object of the present invention, the entire area of the flexible sheet material or at least the adhesive transfer areas of the flexible sheet material are coated with an adhesive agent, such as for example a silicone resin. The adhesive may enhance the adhesion of the biological sample to the flexible sheet material and facilitate the taking up of the biological sample at the adhesive transfer areas.
In a preferred embodiment of the transfer device according to the further object of the present invention, the flexible sheet material comprises, or consists of, a thermoplastic polymer material chosen among polyolefins, polesters, polycarbonates or polyamides.
These polymer materials were found to have good mechanical properties, such as elasticity.
In a preferred embodiment of the transfer device according to the further object of the present invention, the flexible sheet material comprises, or consists of, a elastomeric polymer material chosen among silicon resins such as for example PDMS. These polymer materials were found to have good mechanical properties, such as elasticity.
In a preferred embodiment of the transfer device according to the further object of the present invention, the flexible sheet material has a thickness of from 40 to about 400 micrometers, preferably of from 40 to about 200 micrometers. It has been found that the range of thickness allows for sufficient optical transmission and mechanical property, such as elasticity.
It has been found that the range of thickness allows for sufficient optical transmission and mechanical property, such as elasticity. Alternatively, the flexible sheet material and in particular the elastomeric polymer material has a thickness of from up to 5 mm, preferably of up to 2 mm. It has been found that the range of thickness allows for sufficient optical transmission and mechanical property, such as elasticity.
In a preferred embodiment of the transfer device according to the further object of the present invention, the flexible sheet material is a cast film or blown film.
In a preferred embodiment of the transfer device according to the further object of the present invention, the flexible sheet material comprises, or consists of, a cyclic olefin copolymer or cyclic olefin polymer. COC and COP have good optical properties in VIS and UV, which is advantageous when the method uses light sources and optical microscopes to identify the biological samples, and exhibit a good resistance to tear or bursting. At the same time, it has been found that these polymers further exhibit good adhesion to the biological samples as—is, i.e. without a need to apply an adhesive agent.
In a preferred embodiment of the transfer device according to the further object of the present invention, the transfer device comprises a support plate having M perforations, the M adhesive transfer areas are essentially in a plane with a surface of the plate and wherein said surface of the plate preferably faces the at least N separate biological samples. In the case where M adhesive transfer areas are essentially in a plane with the surface of the support plate facing the N separate biological samples, the degree of extension/retraction needed to pick up a biological sample can be reduced when compared to the case where the flexible sheet material is countersunk with respect to the outer surface of the support plate, which provides for a more robust method of transfer and/or robust operation of the transfer device. Where the M adhesive transfer areas are essentially in a plane with the surface of the support plate, manufacturing of the transfer device is simplified since, at least in some embodiments, only one support plate is needed, in contrast to when the M adhesive transfer areas are countersunk, where at least two support plates are needed.
It is yet a further, and third object of the present invention to provide an apparatus to carry out the method for the rearrangement and transfer according to the first object of the present invention and preferably using a transfer device according to the further object of the present invention.
It is thus yet a third object of the present invention to provide an apparatus comprising:
In a preferred embodiment of the apparatus according to the third object of the present invention, the apparatus further comprises a fourth unit configured for applying a releasing solution comprising a releasing agent, preferably with an aqueous releasing solution comprising a releasing agent chosen among proteolytic enzymes, such as trypsin, to the M adhesive transfer areas of the transfer device.
Further embodiments of the invention are laid down in the dependent claims.
Preferred embodiments of the invention are described in the following with reference to the drawings, which are for the purpose of illustrating the present preferred embodiments of the invention and not for the purpose of limiting the same. In the drawings,
In the method according to the present invention, a number of spatially separate biological samples are essentially transferred from a number of spatially separate first locations to a number of spatially separate second locations, and in doing so are also rearranged from an irregular pattern that is inherent to and defined by the tissue sample toward a regular pattern that is defined by the downstream analysis the biological samples are subjected to. Generally, the number of spatially separate samples, first locations and second locations is the same, i.e. each individual biological sample is taken up and ultimately ends up in its individual well in a multi-well plate e. In this case, N is equal to M in the method of the present invention, or stated alternatively, only one biological sample is taken up per one adhesive transfer area. However, it may be advantageous to collect more than one spatially separate biological samples from more than one separate first locations, provided they share a common feature to be analyzed, into one and the same second location. In this case, N is larger than M in the method of the present invention, or stated alternatively, two or more biological samples are taken up per one adhesive transfer area.
The term “biological sample” refers to a sample comprising, or consisting of, a cell or clusters of cells, that can be found in a tissue or body fluid of an organism such as for example animals or plants.
The biological sample is usually prepared, as is known in the art, before being manipulated in the method according to the present invention.
In one embodiment, the biological sample essentially consists of a single cell or clusters of cells.
In another embodiment the biological sample comprises a cell or a cluster of cells and one or more synthetic polymer layers adhered the side of the sample that is opposite of the side of the sample facing the sample support plate, or stated alternatively, the side of the sample facing the transfer device or adhesive transfer area. The synthetic polymer layer is preferably a polyester layer such as for example PET or PEN.
In a particular embodiment of the method according to the present invention, as can be seen in
Upon achieving the overlap, the transfer device (1) is moved downwards, as can be seen in
Once the first biological sample (4) is picked up, the transfer device is moved upwards, as can be seen in
Once the first iteration is completed, the next iteration is carried out, as can be seen in
Once all adhesive transfer areas (3, 3, 3″, 3″) have taken up the biological sample (4, 4′,, 4″, 4″′) in their separate second locations on the transfer device, the transfer device (1) is positioned on top of a multi-well plate (6) in which wells such as to bring each one of the adhesive transfer areas (3, 3′, 3″, 3″′) into overlap, in vertical direction, with each one of the wells of the multi-well plate., as can be seen in
Each of the separate biological samples (4, 4, 4″, 4″′) are released simultaneously, from the separate second locations in the adhesive transfer areas (3, 3′, 3″, 3″′) into the wells on the multi-well plate (6), as can be seen from exploded view in
| Number | Date | Country | Kind |
|---|---|---|---|
| 22161427.4 | Mar 2022 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2023/055925 | 3/8/2023 | WO |
