3D printed mask for lysis at a location on a tissue section

Abstract

Some embodiments relate to a method for separating tissue from a tissue section on a tissue slide. The method may include: applying a 3D-printed mask on top of the tissue section, the mask including a barrier defining a cavity surrounding a region of interest on the tissue section, the cavity being open on a side facing the region of interest on the tissue section; dispensing a separation liquid into the cavity and allowing the separation liquid to separate tissue at the region of interest; and aspirating the separation liquid with the separated tissue from the cavity and transferring the liquid for further processing.

Description

Technical Field

[0001] The presently disclosed subject matter relates to methods for separating tissue from a tissue section on a tissue slide, methods for staining tissue from a tissue section on a tissue slide, separation apparatus, a 3D printing mask arranged for application on top of a tissue section, and computer-readable media. Background Art

[0002] International patent application WO2020131070, entitled “Method of treating a sample,” which is incorporated herein by reference, discloses a method for treating an isolated area of a sample with a liquid. The known method disclosed therein comprises the following steps:

[0003] - creating an isolated area of the sample by means of a first liquid reservoir, said first liquid reservoir enclosing the distal end of a second liquid reservoir, said isolated area of the sample being sealed relative to the rest of the sample;

[0004] - placing said isolated area of said sample in liquid connection with said second liquid reservoir;

[0005] - dispensing liquid from the second liquid reservoir into the first liquid reservoir, thereby generating a liquid flow in a first direction at the isolated area on the sample; and

[0006] - drawing the liquid from the first liquid reservoir into the second liquid reservoir, thereby generating a liquid flow in a second direction at the isolated area on the sample.

[0007] For example, Figure 9 of the above patent application shows a microscope image of a formalin-fixed paraffin-embedded (FFPE) section of a tissue. The corresponding description gives an example of processing a slide containing a tissue section. Summary of the Invention

[0008] Existing methods for separating tissue from tissue sections can be improved. For example, a barrier or mask can be applied directly to the tissue to block separation liquid (such as lysis buffer). The separation liquid can be distributed in the cavity formed by the mask. Using this method, separation time is less critical because separation can be completed in parallel. After separation, the liquid can be collected to carry out downstream processing.

[0009] For example, a method for separating tissue from a tissue section on a tissue slide may include applying a 3D printing mask to the top of the tissue section. The 3D printing mask can be directly printed on the tissue section. The mask may include a barrier that defines a cavity surrounding the region of interest on the tissue section. A separating liquid may be dispensed into the cavity and later aspirated from the cavity together with the separated tissue. Then, desired further processing may be applied to the separated tissue, such as genomic analysis, storage tissue, etc. Tissue sections are sometimes also referred to as tissue slices.

[0010] Distribution and aspiration can use the pipetting tip arranged at the motorized pipetting arm. The pipetting tip can be arranged so that the separating liquid is distributed to the cavity, or is aspirated from the cavity. Interestingly, between distribution and aspiration, the pipetting arm and / or the pipetting tip can be applied in other ways. For example, after distribution, the pipetting arm can be moved away from the cavity. For example, the pipetting arm can be used to process the next region of interest, for example, the next region of interest on the same tissue slice or different tissue slices, possibly even the next region of interest on different tissue slides. Another advantage is that the tissue slice can be thicker than conventional tissue slices.

[0011] The barriers defining the cavity can be arranged to match the edges of the region of interest. Thus, the cavity defined by the barriers can be irregularly shaped, for example, to match the contours of the region of interest. Similarly, the area and / or volume of the cavity can be flexibly adapted to the current separation, for example, from very small to very large regions of interest.

[0012] Interestingly, the 3D mask can be printed directly on the tissue section. This is advantageous because it allows enhanced automation, increased throughput, and improved alignment with the region of interest. For example, the position of the 3D mask application (e.g., printing) can be determined from a first image of the stained tissue section, the region of interest being visible on the first image and / or indicated on the first image or indicated relative to the first image. An image registration algorithm can be applied to register the first image of the tissue section with the second image on which separation is to be performed and the 3D mask is to be applied. For example, the coordinates of the region of interest identified in the image of the stained tissue section can be converted by registration into the coordinates of the region of interest in the image of the unstained tissue section on which the 3D mask application and tissue separation will be performed. The stained tissue section is typically stained as a whole, although this is not necessary. Conventional staining methods can be used to apply staining, such as H&E staining.

[0013] Even the design of the 3D mask may be obtained from the first image, possibly with the aid of intermediate determinations of the 2D design. The 2D mask and / or the 3D mask may be designed manually. For example, the 2D design may be done manually, for example in a user interface; for example, on top of a picture of a stained slice. The 2D design may be generated automatically, for example by a computer, for example from the first image. For example, the mask design algorithm may be configured to draw a barrier around the indicated region of interest. The region of interest may be an input to the mask design algorithm, or may be obtained from a marker associated with the stained tissue section, or may be obtained from a machine learning algorithm. The mask design algorithm may be configured to add optional features to the 3D design (such as a slope at the outside of the 3D mask). For example, the mask design algorithm may optionally be configured to connect multiple regions of interest to the support structure.

[0014] A variety of positive effects can be obtained using one embodiment. For example, using a 3D mask means that a separation chamber of predetermined size, such as a so-called tip extender, is not required. The shape of the predetermined separation chamber is usually not suitable for the shape of the region of interest. Typically, the tip extender is circular, while the region of interest is not circular. Therefore, the region of interest will need to be repeatedly detached using many tip extenders, after which the entire region of interest may still not be completely separated. Therefore, using a printed 3D mask can reduce the number of consumables required. Similarly, reagent costs can be reduced. Aggregation problems associated with repeated separations at multiple locations are also avoided.

[0015] The throughput can be significantly increased. This is true for a single region of interest because repeated separations (e.g., so-called lysis cycles) can be avoided. Alternatively, a single separation period can be performed in which the entire region of interest is separated. The throughput is increased even more when the parallelism allowed by the 3D mask is utilized. While the first region of interest is being separated (e.g., while lysis is being performed in the first chamber), the pipette tip can be dispensed into the second chamber corresponding to the second region of interest. Separation takes some time—a complete separation can take 8 minutes or even more—and the opportunity to work in parallel can significantly increase the throughput.

[0016] Because the shape of the 3D printed mask can be matched to the contours of the region of interest, separation of unwanted tissue (e.g., healthy tissue) or omission of desired tissue (e.g., tumor tissue) is avoided. This is especially true near the edge of a tissue slide, where the predetermined shape of the tip extender may extend beyond the boundaries of the tissue slide. The printed mask can be applied close to the edge while still having a large surface area.

[0017] The separation device and / or the 3D printing device and / or the 3D mask generating device is an electronic device, which may include a computer, for example to control the 3D printing and / or separation process.

[0018] On the one hand, there are separation methods (e.g., including separation of tissue using a 3D printed mask), 3D printing methods (e.g., including 3D printing of a 3D mask arranged for use in the separation method), and 3D design methods (e.g., including generation of 2D and / or 3D mask designs for use in the 3D printing method).

[0019] On the one hand, there are staining methods (e.g., comprising staining tissue using a 3D printed mask), 3D printing methods (e.g., comprising 3D printing of a 3D mask arranged for use in the staining method), and 3D mask design methods (e.g., comprising generating 2D and / or 3D mask designs for use in the 3D printing method).

[0020] An embodiment of these methods can be implemented on a computer as a computer-implemented method, or implemented in dedicated hardware, or a combination of the two. Executable code for an embodiment of the method can be stored on a computer program product. Examples of computer program products include memory devices, optical storage devices, integrated circuits, servers, online software, and the like. Preferably, the computer program product includes non-transitory program code stored on a computer-readable medium for performing an embodiment of the method when the program product is executed on a computer.

[0021] In one embodiment, the computer program comprises computer program code adapted to perform all or part of the steps of an embodiment of the method when the computer program is run on a computer.Preferably, the computer program is embodied on a computer readable medium.

[0022] Another aspect of the presently disclosed subject matter is a method of making a computer program downloadable. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Further details, aspects and embodiments will be described by way of example only with reference to the accompanying drawings. The elements in the drawings are illustrated for simplicity and clarity and are not necessarily drawn to scale. In the drawings, elements corresponding to elements already described may have the same reference numerals. In the drawings,

[0024] Figure 1 An example of an embodiment of a separation device is schematically shown;

[0025] Figure 2 An example of an embodiment of a separation device is schematically shown;

[0026] Figure 3a An example of one embodiment of a 3D printed mask applied to a tissue section is schematically shown in side view;

[0027] Figure 3b An example of one embodiment of two 3D printed masks applied to a tissue section is schematically shown in a top view;

[0028] Figure 4a Schematically shows an example of a diagram of a stained tissue section with a marked region of interest;

[0029] Figure 4b Schematically illustrates an example of one embodiment of a 2D design for a 3D printed mask;

[0030] Figure 4c An embodiment of a 3D printed mask is shown;

[0031] Figure 5 An example of one embodiment of a method for separating tissue is schematically illustrated;

[0032] Figure 6a schematically illustrates a computer-readable medium having a writable portion, the writable portion comprising a computer program according to an embodiment;

[0033] Figure 6b Schematically shown is a representation of a processor system according to one embodiment.

[0034] Reference Signs List

[0035] The following list of reference numerals and abbreviations corresponds to Figures 1 to 4c ,as well as Figures 6a to 6b , and the lists are provided to facilitate the explanation of the drawings and should not be construed as limiting the claims.

[0036] 100 Separation Equipment

[0037] 120 slides

[0038] 125 slide holder

[0039] 130 pipette tips

[0040] 131 Pipetting Arm

[0041] 140 Separation Unit

[0042] 151 First Camera

[0043] 152 Second Camera

[0044] 160 3D printing unit

[0045] 161 3D printing arm

[0046] 210 Separation Equipment

[0047] 230 processor system

[0048] 240 Memory

[0049] 250 communication interface

[0050] 300 tissue sections

[0051] 301 3D printed barrier

[0052] 302 cavity

[0053] 1000, 1001 Computer readable medium

[0054] 1010 Writable part

[0055] 1020 Computer Programs

[0056] 1110 Integrated Circuit

[0057] 1120 processing units

[0058] 1122 Memory

[0059] 1124 ASIC

[0060] 1126 Communication Components

[0061] 1130 Interconnect

[0062] 1140 processor system DETAILED DESCRIPTION

[0063] While the presently disclosed subject matter is susceptible of embodiment in many different forms, one or more specific embodiments thereof are shown in the drawings and will herein be described in detail, it being understood that this disclosure is to be considered as an exemplification of the principles of the presently disclosed subject matter and is not intended to be limited to the specific embodiments shown and described.

[0064] Hereinafter, for ease of understanding, the elements of the embodiments are described in operation. However, it will be apparent that each element is arranged to perform the function described as being performed by the element.

[0065] Furthermore, the presently disclosed subject matter is not limited solely to the embodiments, but also comprises every other combination of features described herein or recited in mutually different dependent claims.

[0066] Figure 1Schematically illustrated is an example of one embodiment of a separation apparatus 100. Separation apparatus 100 is one example of a separation apparatus that may be used in or with embodiments, such as embodiments of a separation method or a 3D printing method or separation apparatus.

[0067] Figure 1 1 shows a slide 120. Slide 120 is arranged for tissue sections to be applied to the slide surface. Portions of the tissue section are to be separated for further analysis, for example, to extract biomolecules, such as DNA material, from the tissue. For example, the tissue can be a biopsy. Slide 120 is shown on top of slide holder 125. For example, slide holder 125 can be arranged with a heating element for heating the tissue section on slide 120, for example, during separation, such as during lysis.

[0068] The separation device 100 includes a pipetting arm 131 having a pipetting tip 130 at the end of the pipetting arm. The pipetting arm is sometimes referred to as a pipetting channel. The slide surface of the slide 120 faces the pipetting tip 130. The pipetting tip 130 is movable and can be configured to move the pipetting tip 130 to a specific, defined position on the tissue slice. Typically, the pipetting arm 131 is motorized and is arranged to be controlled by a computer program. The program can instruct the pipetting arm 131 and the pipetting tip 130 to separate at one or more positions on the tissue slice. For example, the pipetting arm 131 can be part of a robotic arm that is arranged to move the tip 130 to the desired position on the tissue slice.

[0069] The pipetting tip 130 is configured to enable controlled exposure of chemicals to tissue at a defined location. The tip can also allow for dynamic liquid forces at that location to further promote separation of tissue slices at that location. For example, shear forces can be applied to the tissue by the liquid.

[0070] A location on the tissue slice (e.g., a portion, region, or area of the tissue slice) includes material to be separated (e.g., to be separated). The location is also referred to as an area of interest (AoI). The separation unit 140 can be configured to move the pipetting tip 130 to the location on the tissue slice, supply liquid to the pipetting tip 130, aspirate liquid from the pipetting tip 130, and so on.

[0071] For example, separating liquid can be a lysis liquid. For example, lysis liquid can be a lysis buffer (lysing buffer). Lysing liquid is applied to the region of interest at tissue section place and can cause the tissue in this region to be cracked. Lysing liquid and lysis tissue are then called lysate together.

[0072] The separating liquid can comprise an enzyme that is applicable to separation, and the enzyme can be cracked, but this is not necessary. The enzyme can be or can not be a synthetic enzyme. Except enzyme or as an alternative to enzyme, a chaotropic salt can be used. In one embodiment, the separating liquid can comprise a lysis buffer (for example, comprising a lysis buffer of proteinase K). The separating liquid can be applicable to the proteome based on liquid chromatography-mass spectrometry (LC-MS).

[0073] The separation device 100 also includes a 3D printing unit 160. The 3D printing unit 160 includes a 3D printing arm 161 for printing a 3D mask. The 3D mask can be applied to the tissue slice by printing the 3D mask directly on top of the tissue slice. Alternatively, the 3D mask can be printed separately and then placed on top of the tissue slice. Figure 1 The 3D printed arm is shown approaching a tissue slide 120 on top of a holder 125. This can be the same holder as used for the separation unit 140. However, preferably, the 3D printed part of the separation device 100 has a separate slide holder 125.

[0074] The 3D printing mask includes a barrier that defines a cavity surrounding the region of interest on the tissue slice. The cavity is open on the side facing the region of interest on the tissue slice and is arranged to receive a separation liquid from a pipette tip 130. The cavity includes an inlet for receiving the separation liquid. For example, the cavity can be at least partially open on the side opposite to the tissue slice. For example, the cavity can be connected to a liquid conduit that is in turn arranged to receive the separation liquid from the pipette tip 130. The cavity is also referred to as a separation chamber. If lysis is used, this can be referred to as a lysis chamber.

[0075] 3D printing can be accomplished using additive manufacturing. For example, the 3D printing material can be a plastic that can be melted during printing. The 3D printing material can also be a highly viscous liquid or gel. In the latter case, the 3D printing material can be dispensed via a pipette tip (e.g., a pipette tip located on the pipetting arm 131, such as tip 130). In this way, a separate 3D printing unit can be avoided. Another option is to use a 3D material comprising two components. The components react on a glass slide to become solid. The components can be dispensed from separate pipette tips or from the same tip.

[0076] Once the 3D printing mask is applied to the region of interest, a separating liquid is dispensed into the chamber. Depending on the configuration of the separation device 100, the slide 120 with the 3D printing mask applied can be moved from the 3D printing component of the device 100 to the separating component, such as from the 3D printing unit 160 to the separating unit 140; for example, this can be done using a slide moving mechanism. Instead of moving the slide, these units can alternatively be moved.

[0077] In some embodiments, the tissue of the region of interest is separated by separation liquid. Then, separation liquid is allowed to separate the tissue at the region of interest. Typically, this will occur in discrete culture modules or culture chambers. When separation is fully carried out, separation liquid is sucked out from the cavity together with the separated tissue, for example, by pipetting tip 130. If cracking is used, the lysis liquid sucked out is called lysate. Then, liquid can be transferred to further process (for example, genomic analysis), is transferred to separation liquid collection container to further process (for example, purification) or the like.

[0078] For example, separation may include moving a motorized pipetting tip to the region of interest, dispensing separation liquid into a cavity at the region of interest via the pipetting tip 131 at the end of the pipetting tip, and subsequently aspirating the separated material. The separation liquid in the separation chamber may be heated to a separation temperature.

[0079] Further information and examples of cleavage can be found, for example, in International Patent Publications WO2020131072 and WO2020132394, both of which are incorporated herein by reference. However, these publications use a tip extender, which is not required when using a 3D printed mask.

[0080] Separating tissue in this way, especially in an automated or partially automated manner, is advantageous because it is fast and reliable. Furthermore, using 3D printed masks has many advantages, such as higher throughput, allowing for irregularly shaped areas of intermediates, smaller regions of interest, and parallel processing of multiple locations.

[0081] For example, in a separation iteration, a separation liquid can be provided to a separation chamber and, after a period of time (e.g., after incubation), aspirated back with the separated material. The time the separation liquid is in the chamber, as well as other factors, has an impact on the amount of material separated from the tissue slide.

[0082] Camera 151 may be included in the separation device 100 to take images of tissue slices before and / or during tissue separation (e.g., lysis). Typically, the region of interest is limited to the stained tissue slice, or is limited relative to the stained tissue slice, and separation occurs at the same region but at an unstained tissue slice (e.g., a thin slice of the same tissue that is different from the stained tissue slice). The image of the stained slice and the image of the unstained slice can be registered so that the position defined relative to the stained tissue can be converted to the position on the unstained slice for separation. The same situation is true for 3D printing. Typically, a 3D mask is limited relative to the stained tissue slice (e.g., a picture of the stained tissue slice), but the 3D mask is applied to the unstained tissue slice (e.g., the 3D mask is printed on the unstained tissue slice or is placed on the unstained tissue slice). The 3D printing unit 160 can also use image registration to convert the region of interest or design, etc. from the stained tissue slice to the unstained tissue slice for applying the mask.

[0083] In staining tissue slices, at least the region of interest is stained, however, for the above purposes, typically the entire tissue section is stained. For example, conventional staining, such as H&E staining, can be used.

[0084] Moving the pipetting tip and / or 3D printing tip to and from the region of interest can be accomplished with the aid of a movable arm (e.g., a motorized arm). For example, a robotic arm can be used. In one embodiment, camera 151 can be used to guide the arm toward the region of interest, although this is not required. In one embodiment, camera 152 can be used to guide the printing arm toward the region of interest, although this is not required.

[0085] The slide 120 may include one or more fiducials to help locate a defined location in the camera image. The cameras 151, 152 and / or the fiducials may be used by guidance software configured to guide the pipetting arm 131 and the printing arm 161 to a region of interest.

[0086] Figure 2 An example of an embodiment of a separation device 210 is schematically shown. For example, Figure 2 The separation device 210 can be used to separate tissue from a tissue section. Figure 2 The separation device 210 may be used to control a separation unit (eg, a pipetting arm) and / or a 3D printing unit (eg, a 3D printing arm). The separation device 210 may include a processor system 230, a memory 240, and a communication interface 250.

[0087] Memory 240 may include local memory, such as a local hard drive or electronic memory. Memory 240 may include non-local memory, such as cloud memory. In the latter case, memory 240 may include a memory interface to the non-local memory.

[0088] The separation device 210 can communicate internally, with other systems, external storage, input devices, output devices, and / or one or more sensors, via a computer network. The computer network can be the Internet, an intranet, a LAN, a WLAN, or the like. The computer network can be the Internet. The system includes a connection interface that is configured to communicate within or outside the system as needed. For example, the connection interface can include a connector, such as a wired connector (e.g., an Ethernet connector, an optical connector, etc.), or a wireless connector (e.g., an antenna, such as a Wi-Fi, 4G, or 5G antenna).

[0089] In the separation device 210, the communication interface 250 can be used to send or receive digital data. For example, the communication interface 250 can receive an image of a stained tissue section, possibly with a marker indicating a region of interest. For example, the communication interface 250 can be used to receive commands and instructions. For example, the communication interface 250 can be used to transmit the results of a tissue separation analysis.

[0090] The execution of the separation device 210 can be implemented in a processor system, such as one or more processor circuits (e.g., microprocessors), an embodiment of which is shown herein. The processor system may include one or more GPUs and / or CPUs. The separation device 210 may include multiple processors, which may be distributed in different locations. For example, the separation device 210 may utilize cloud computing.

[0091] Figure 1 Functional units are shown, which may correspond to functional units of a processor system. For example, Figure 1 210 (e.g., stored in electronic memory of the separate device 210) and executable by a microprocessor of the separate device 210). In a hybrid embodiment, the functional units are implemented partially in hardware (e.g., as a coprocessor, such as an image processor) and partially in software stored and executed on the separate device 210.

[0092] A possible workflow of the device 210 and / or 100 is as follows:

[0093] 1. Receive tissue slides containing unstained tissue sections,

[0094] 2. Obtain the region of interest on the unstained tissue section,

[0095] 3. 3D print a 3D mask that matches the region of interest. 3D printing can be performed directly on tissue sections.

[0096] 4. Dispense the separation liquid into the cavity defined by the 3D mask and cover the region of interest,

[0097] 5. Allow the separation fluid to separate the tissue in the area of interest,

[0098] 6. Aspirate the separation fluid with the separated tissue from the cavity,

[0099] 7. Transfer the liquid for further processing.

[0100] Each of these terms can be controlled by a computer program. Many variations and refinements are possible for these steps. Not all of these steps are required. For example, instead of 3D printing the mask, the mask can be 3D printed on a different machine and received in printed form. Additional details are discussed below.

[0101] Figure 3a An example of an embodiment of a 3D printing mask applied to a tissue slice is schematically shown in a side view. Shown is a glass slide 120 on which a tissue slice with an area of interest is placed for separation. On the tissue slice, a barrier defining a cavity 302 is 3D printed. For example, a separation liquid (e.g., a lysis reagent) can be distributed within the cavity. The tissue slice and the separation liquid are then allowed to separate, such as by culturing. For example, the lysis reagent can be heated to an appropriate temperature so that liquid separation is performed. For example, humidity can be controlled during culturing, such as at approximately 95% rH. After a period of time, the liquid (e.g., lysate) can be collected, for example, using a pipette tip. Finally, the liquid can be transferred for further processing (e.g., by being placed in a microwell), or sent to other equipment, etc.

[0102] Figure 3b An example of one embodiment of two 3D-printed masks applied to a tissue slice 300 is schematically shown in a top view. Diagram 300 shows two regions of interest 302, each of which is surrounded by a 3D-printed barrier. Both regions are allowed to be cultured in parallel. Note that both regions of interest are irregularly shaped, yet can be completely separated.

[0103] Figure 4aAn example of an image of a stained tissue section with a marked region of interest is schematically shown. For example, a region of interest can be marked by a physician. For example, a region of interest may represent a suspected tumor requiring specific investigation. In this example, the region of interest has been indicated with a marker. The image of the marked stained tissue indicates that tissue is to be separated from the unstained tissue.

[0104] Figure 4b An example of one embodiment of a 2D design for a 3D printed mask is schematically shown. Figure 4b 2D design from Figure 4a Note that Figure 4a The black mark in has been transferred to (indicated in white) Figure 4b The cavity is surrounded by a barrier, which in turn is connected to the support structure. Areas where separation is not desired, not yet filled by the barrier or support structure, are filled. This prevents unintentional separation at those locations and provides additional strength to the mask.

[0105] Figure 4c One embodiment of a 3D printed mask is shown. Figure 4c A photo of a printed mask is shown. Note that on the outside of the mask, the outer barrier of the 3D mask is provided with beveled edges. These ensure a better seal between the mask and the tissue. Figure 4c The 3D printing mask shown in includes many beneficial but optional features. For example, multiple cavities, ramps, and supports between cavities are optional features.

[0106] Using a 3D mask to separate tissue can have several effects. For example, the entire region of interest can be separated at once, rather than having to separate it point by point. In addition, the shape of the region of interest can be highly irregular and does not need to conform to the fixed shape of the pipette tip.

[0107] Using fewer separation cycles is faster and also reduces the need for consumables, reagents, etc. A single separation using a tip extender that applies flow at one point is faster than a separation using a 3D mask that does not apply flow. The difference may be 2 or 3 times. However, separation using a 3D mask will not block the pipetting arm and can be completed in parallel, for example, in an incubator. In addition, the region of interest can be separated within one cycle without having to cycle multiple times at multiple points. The resulting throughput using a 3D mask is higher.

[0108] Being able to separate irregular regions means that the amount of healthy tissue in the separation region can be minimized, and missing tumor tissue outside the separation region, for example, can be avoided. Using background lysis methods to separate regions of interest close to the edge of the slide requires even more cycles because a smaller spot is used. However, as long as the region of interest is approximately one barrier away from the edge, the location of the region of interest does not pose any problem when using a 3D printed mask. Since separation requires fewer cycles and multiple regions can be separated in parallel, the net throughput can be very high.

[0109] A number of embodiments and variations for separating tissue from tissue sections are discussed below.

[0110] In one embodiment, a separation device (e.g., separation device 100 or 210) or a method for separating tissue is configured to receive a slide having a tissue section applied to the slide surface. For example, the slide can be a glass slide, or some other suitable material. At least one tissue slide comprising an unstained tissue section is received. Embodiments can receive multiple tissue sections, possibly on multiple slides. Embodiments can also receive stained tissue sections, for example, to guide the separation and application of a 3D mask.

[0111] In one embodiment, tissue section is paraffin-treated and / or formalin-fixed. These are important applications because formalin fixes tissue (e.g., protein). Paraffin-treated and formalin-fixed tissue is also referred to as FFPE tissue. However, there is no need to be limited to FFPE tissue. In one embodiment, tissue section is fresh frozen tissue.

[0112] Regions of interest are obtained on unstained tissue sections. There are different ways to achieve this. Note that multiple regions of interest can be defined.

[0113] In one embodiment, the region of interest can be defined by the user. For example, the user can define the region of interest by defining the coordinates indicating the region of interest. For example, the separation device can be configured to image the slide before separation, display the image in a user interface, and the user interface is configured to enable the user to define the region of interest. For example, the user can indicate the coordinates or position in the image in the user interface, etc. In one embodiment, the region of interest is defined by the user, for example, through a user interface. For example, the separation device can be configured to image the tissue section before separation, display the image in a user interface, and the user interface is configured to enable the user to define the position. Instead of the image of the tissue section to be separated, an image of a stained section of the same tissue can be used, for example, an image presented to the user.

[0114] One or more locations can be defined by, for example, an algorithm running in a tracking device or a separation device. Regions of interest can be defined by an algorithm. For example, an algorithm can define locations where separation will occur. For example, an algorithm can define one or more regions of interest where the tissue appearance deviates from a regular tissue. For example, a neural network can identify locations of suspicious tissue (e.g., deviating from a standard). For example, an image recognition algorithm can be trained on images that include regions of interest.

[0115] In one embodiment, an image of a stained tissue section is obtained. The stained tissue section is a thin slice of the same tissue that is different from the tissue section used for separation. For example, the stained tissue section can be received on a glass slide. A camera of the device can be used to photograph the stained tissue section, such as camera 151 or 152. For example, the image can be received in digital form at the separation device, such as from an external camera.

[0116] Images of stained tissue sections can be used to identify regions of interest. For example, as indicated above, the images can be presented to the user in a user interface, where the user can indicate the regions of interest. For example, a touch screen can be used, where the regions can be colored.

[0117] In one embodiment, the region of interest is indicated on a stained tissue section, on a transparent (e.g., glass) cover of a stained tissue section, or on a picture of a stained tissue section. For example, marking can be done physically with a marker. Such marking Figure 4a The marking can be done in a color that is easy to identify the number. For example, Figure 4a In , hematoxylin and eosin (h&e) staining was used; in Figure 4a In , the markers are black while the stained tissue is purple, making the markers easily identifiable by both human operators as well as image recognition algorithms.

[0118] Once the regions of interest are obtained, whether from the stained image, obtained through the user interface, or obtained by marking, they can be used to generate a 2D design from the identified regions of interest. The 2D design can be generated not only manually but also automatically. The design can be automatically generated, but can be modified by a human operator if necessary. For example, an automated design can place a barrier around the identified regions of interest. A support structure (e.g., connecting cavities) can be applied between the regions of interest. Larger areas between the cavities can be filled with material.

[0119] Once a 2D design is available, it can be converted into a 3D design, for example, a design suitable for 3D printing. For example, the 2D design can be expanded in the z-direction. It is not necessary to raise all parts of the 2D design by the same amount. For example, the barrier can be raised to a level depending on the desired amount of separation liquid that the cavity should accommodate. As an example, a height of 3 mm can be used. The support structure can have a lower height. Slicing software can be used to guide 3D printing based on the 3D design. The height can be indicated as a grayscale value in the 2D design of the mask.

[0120] 3D masks can now be printed. Interestingly, a 3D mask can be obtained that matches the region of interest. In this way, healthy tissue can be excluded from the separation section, while all or nearly all non-healthy tissue can be included. For example, in one embodiment, different cavities are assigned to tumor tissue and wild-type tissue. The two types of tissue can be extracted separately.

[0121] Advantageously, 3D printing can be done directly on the unstained tissue sections. This is not necessary, for example 3D printing can be done separately and applied to the tissue sections after printing. For example, a 3D mask can be printed in an external printer and manually placed on the tissue sections before separation. Although 3D printing directly on the tissue is preferred, placing an external 3D printed mask may still be advantageous for predetermined shapes; for example, an external 3D printed mask allows even smaller regions of interest to be separated, as well as separating multiple regions of interest, optionally in parallel. For example, in the case of many small regions of interest to be separated, manually placing a discrete 3D printed mask may even increase throughput and yield.

[0122] Different 3D printing technologies are suitable. For example, 3D printing can be an additive manufacturing technology. 3D printing can include extruding molten 3D printing material layer by layer to build a 3D mask. This can include extruding 3D printing material layer by layer on a tissue slice. For example, fused deposition modeling (FDM) can be used to accomplish 3D printing. A variety of 3D printing materials can be used, particularly 3D materials that do not interact with the separating liquid. For example, the 3D material can be, for example, polylactic acid (PLA). However, any suitable 3D printing material (e.g., plastic) can be used for printing. In one embodiment, medical-grade polypropylene (PP) can be used for 3D printing; for example, a medical variant of PLA filament. In addition to plastics, the mask material can be a wax with a higher melting point, a grease such as a chemically inert substance, or a silicone filler.

[0123] For example, 3D printing can be performed using a resin printer. For example, the 3D technology can be an SLA 3D printer, such as an LCD-based SLA printer. For example, the 3D printing material can be a photosensitive resin.

[0124] The 3D printing material can be a molten plastic. In one embodiment, the 3D printing material can be a highly viscous liquid or a gel. As an example, the gel can be a silicone gel.

[0125] An advantage of this approach is that the 3D mask can be dispensed by a pipetting tip (eg, a pipetting tip attached to the pipetting arm 130, such as the pipetting tip 131. This avoids the need for a separate 3D printer.

[0126] The 3D mask includes a barrier that defines a cavity surrounding a region of interest on the tissue slice. The cavity is open on the side facing the region of interest on the tissue slice. This allows the separation liquid to process the tissue in the region of interest. The barrier causes the separation liquid to remain in the region of interest rather than flowing out to the rest of the tissue slice.

[0127] The 3D mask is arranged to receive a separation liquid, such as a lysis liquid. For example, in one embodiment, the 3D mask includes an inlet that is arranged to allow the separation liquid to be introduced into the cavity through a pipette tip. For example, the cavity can be at least partially open on the side opposite to the tissue slice; the pipette arm can move toward the partially open side and introduce the separation liquid. For example, in the 3D mask, one or more conduits can be printed to transport the separation liquid to and from the pipette tip. For example, multiple cavities can each be provided with an inlet; multiple inlets can be connected to a single inlet for the mask.

[0128] The cavity may only have 1mm 2 area, although larger areas are possible, such as 1250mm 2 The area of the barrier can be as small as, for example, 1 mm, or even larger. The maximum diameter of the cavity in the 2D direction can be as small as 1 mm, but larger diameters are also possible, for example 20 mm or even larger. The height of the barrier depends on the amount of separation liquid to be used, which in turn can depend on the time the separation will take. As an example, a height of 3 mm can be used. However, lower or higher barriers are possible. The barrier can be as thin as the strength of the 3D material allows. As an example, a thickness between 1 mm and 2 mm can be used. Higher or lower values are possible. The desired height can be indicated as a grayscale value in the 2D design, although this is not necessary.

[0129] In one embodiment, the outer side of the barrier is provided with inclined supports. Figure 4cSuch a stent is shown at the outer edge of the mask. For example, the stent can be twice as thick as the barrier at the side of the tissue. For example, the stent can be about 5mm wide. Towards the top of the barrier, away from the tissue, the barrier can become thinner. The advantage of a wide stent (particularly, at the outer edge of the membrane) is that a better seal of the cavity is obtained.

[0130] In one embodiment, the barrier defining the cavity matches the edge of the region of interest. The shape of the cavity can follow the shape of the region of interest and can be irregular. For example, the 2D shape of the region of interest can be an irregular polygon or a shape defined by splines, etc. In one embodiment, 3D printing includes obtaining a 3D design of a mask, and guiding the 3D printing according to the 3D design of the mask.

[0131] In one embodiment, the 3D mask is printed directly onto the tissue section on top of a glass slide, for example using FDM printing. Printing directly onto the tissue is not required, but is advantageous. It provides a better seal for the cavity and avoids the additional step of placing the mask onto the tissue. In one embodiment, applying the 3D printed mask includes 3D printing the 3D printed mask directly onto the tissue section.

[0132] Some tissue sections are accompanied by a layer of paraffin. When printing directly on a tissue section, an advantageous additional step is to first remove the paraffin layer. Any conventional dewaxing method can be used, both manually and automatically. For example, one can apply a dewaxing scheme using a pipette tip. In one embodiment, at least a portion of the tissue section to which the 3D printing mask is to be applied is dewaxed. Note that the dewaxing liquid does not need to be confined to the area of interest for dewaxing. Larger areas or even the entire tissue section can be dewaxed. For example, xylene can be applied to the tissue section for dewaxing; other dewaxing methods are known in the art. The advantage of dewaxing is that the 3D printing mask is better attached to the tissue section. For example, in one embodiment, dewaxing is followed by FDM printing. Experiments have shown that dewaxing significantly improves the adhesion between the 3D printing mask printed on the tissue section and the tissue section.

[0133] The advantage of using 3D mask is that, compared with the advanced and sophisticated expander technology, each zone needs still less lysate.Particularly, this means that the unstained tissue section that is used to separate can be much thicker than the stained tissue section.For example, in one embodiment, the tissue section that is used to separate is at least 2 times thick of the stained tissue section, is at least 4 times thick of the stained tissue section, or is at least 8 times thick of the stained tissue section.

[0134] For example, typically, stained tissue sections can be, for example, 4 to 5 microns thick. Unstained tissue sections used for separation can be much thicker; for example, between 10 and 40 microns thick; for example, 10 microns or greater thickness, or 20 microns or greater, etc.

[0135] Once the 3D mask has been applied, such as printed on the tissue or printed outside the tissue but placed on it, a separating liquid is dispensed into the cavity defined by the 3D mask, with the separating liquid covering the area of interest. The separating liquid is then allowed to separate the tissue.

[0136] An advantageous choice of separation liquid is a lysis liquid, such as a lysis buffer.Lysing the tissue section with a lysis liquid results in the formation of a lysate comprising lysed tissue material.

[0137] For example, to dispense and / or aspirate the separating liquid, a pipette tip disposed on a motorized pipetting arm can be moved to a cavity, where the separating liquid can be dispensed directly into the cavity. It is also possible to fluidically connect to the cavity; for example, in a 3D mask, a liquid channel connected to one or more cavities can be printed. The liquid channel can be arranged to receive the separating liquid from the pipette tip.

[0138] In one embodiment, different separation fluids can be distributed to different regions of interest, such as different cavities. For example, different tissue types can be separated using separation fluids optimized for specific tissues. For example, different separation fluids can include different enzymes or different concentrations, etc.

[0139] Once separation liquid has been provided, the tissue at the region of interest is allowed to be separated. The pipetting tip can not only control the exposure of tissue to chemicals, but can also be configured to allow liquid forces such as shearing forces at the pipetting tip (such as in the separation chamber, such as in the cavity). During separation, applying shearing forces in the cavity can shorten the separation time, but the disadvantage is that it will take up the pipetting tip. However, this is not necessary. In fact, the advantage of using a 3D printed mask is that no pipetting tip is needed during separation. For example, the pipetting arm can move away from the cavity during separation of the tissue at the region of interest, and then return to the cavity to aspirate the separation liquid from the cavity. The pipetting arm can return once or multiple times during separation, such as for applying shearing forces, such as for mixing the separation liquid. However, this is not necessary. The pipetting tip can only be located at the cavity twice, such as for distributing the separation liquid for the first time, and for aspirating the separation liquid together with the separated tissue once.

[0140] In one embodiment, separating liquid can be sucked off, and is replaced with a new batch of separating liquid in the same chamber. However, typically, this is not necessary, because the volume of the separating chamber and the time for separation can be controlled. Therefore, in one embodiment, separating liquid is only assigned to the chamber once. For example, before or after sucking off the lysate from the chamber, the lysing liquid in the chamber can not be replaced. For example, the chamber is only filled once with lysate. For example, the lysate is only sucked off once from the chamber. In one embodiment, during the cracking period, there is no replacement of lysing liquid.

[0141] The tissue slide can be left in the pipetting unit of the separation machine. The slide, along with the 3D mask and the separation liquid in its cavity, can be placed in an incubator, where the separation liquid can perform tissue separation. The incubation chamber can have a controlled temperature and / or controlled humidity. For example, the temperature can be controlled to 56°C and / or the humidity can be controlled to 95%. The required temperature and / or humidity can depend on the separation liquid and tissue type.

[0142] For example, one or more 3D masks can define multiple cavities on a tissue section. Separation liquid can be distributed to multiple cavities, allowing separations to be performed in parallel in multiple cavities. This means that a single pipette tip can serve multiple cavities, such as multiple regions of interest, where separations are performed in parallel. Using this approach, higher throughput can be achieved without increasing the number of pipette tips and pipetting arms, while maintaining or even improving the separation accuracy of the region of interest.

[0143] Multiple chambers performing separations in parallel can be on the same tissue section on the same tissue slide, but even higher throughput is possible when multiple tissue slides are combined. For example, in one embodiment, multiple 3D printed masks are applied to multiple tissue sections on multiple tissue slides. Separation fluid is dispensed into the chambers across the resulting multiple tissue sections. Multiple tissue slides can be placed in an incubation chamber, where the separation fluid is allowed to separate the tissues.

[0144] The tissue slides can be collected in a stacker and placed in a culture chamber as a stacker. The stacker can also be in the culture chamber where the slides are stacked.

[0145] For example, separation methods may include:

[0146] - applying one or more 3D masks to the tissue slice, said one or more 3D masks defining one or more cavities,

[0147] - distributing the separation liquid to one or more chambers,

[0148] - Place the 3D tissue slide in the culture chamber,

[0149] - Repeated application, distribution and placement,

[0150] - retrieving the tissue slide from the culture chamber and aspirating the separation liquid from the chamber or chambers,

[0151] -Repeat retrieval and aspiration.

[0152] For example, separation methods may include:

[0153] - applying one or more 3D masks to the tissue slice, said one or more 3D masks defining one or more cavities,

[0154] - distributing the separation liquid to one or more chambers,

[0155] - Place the 3D tissue slides in the stacker,

[0156] - Repeated application, distribution and placement,

[0157] - placing the stacker in the culture chamber,

[0158] - obtaining said stacker from a culture chamber,

[0159] - retrieving tissue slides from the stacker and aspirating separation liquid from the one or more chambers,

[0160] -Repeat retrieval and aspiration.

[0161] Instead of forming the stacker outside the culture chamber, the stacker can be formed inside the chamber. This has the advantage that the slides can be placed immediately inside the chamber, but has the disadvantage that more slide handling machinery is required inside the chamber.

[0162] For example, the plurality of printing chambers can be filled with lysis buffer at room temperature. The tissue slides are placed in an incubation chamber, such as part of a stacker, for a period of time, such as about an hour. The incubator can be maintained at about 56°C, a temperature at which separation of the liquid becomes effective, for example. Humidity can also be controlled, such as at 95% RH.

[0163] During the separation of the tissue at the region of interest, the temperature of the tissue slice is increased. This can be done in an incubation chamber, but can also be done with a slide holder having a heating element (e.g. in a pipetting chamber).

[0164] The separation at the defined position on the tissue section can optionally include heating the separation liquid of the separation chamber to a separation temperature. For example, heating can be completed by a heating element at a side of the tissue section opposite to the pipetting tip. Heating can accelerate the separation.

[0165] Once the separation is complete or has been fully performed, the separation liquid with the separated tissue, such as the lysate, can be aspirated from the cavity. The aspirated separation liquid can then be transferred for further processing. This can be in the same separation machine or in a different analysis machine. For example, further processing can include flushing the lysate into an MP well, a microplate, etc. Further processing can include storing the liquid, such as temporarily or for long-term storage. For example, further processing can include:

[0166] - Genomic analysis of isolated tissues, and / or

[0167] - Extracting biomolecules, such as one or more of nucleic acids, proteins, lipids and hormones, from the isolated tissue.

[0168] The separation device can be configured to, for example, extract biomolecules, such as one or more of nucleic acids, proteins, lipids, and hormones, from the separated tissue material. For example, the separation device can be configured to aspirate the separated tissue material (e.g., separation liquid) and place it in a separation liquid collection container. For example, the container can be a well, a tube (particularly a microcentrifuge tube), a microplate, etc. The separation device can include, for example, a container located inside the separation unit 140, etc. The separation device can be configured to further process the separation liquid, such as purifying, obtaining specific biomolecules, etc. The separation device can also be arranged for separation liquids to be collected and to be processed by different machines.

[0169] Interestingly, 3D masks can also be applied for specific staining. In this case, the tissue at the region of interest is not separated but stained. For example, such staining can be applied to immunohistochemistry (IHC), for example based on antibodies. Free-formed cavities can be used to perform several stainings on one slice. For example, a grid of wells (e.g., cavities) can be printed on the region of interest, and each well can have a different antibody stain assigned to it. In one embodiment, multiple staining patterns can be used repeatedly; as an example, one pattern can use 4 stainings.

[0170] In one embodiment, the different staining liquids may include different stains or different antibodies. For example, two regions of interest on a tissue section may be provided with different staining liquids. In one embodiment of a method for staining tissue from a tissue section on a tissue slide, the method comprises: applying a 3D printed mask on top of the tissue section, the mask comprising a barrier defining a cavity surrounding the region of interest on the tissue section, the cavity being open on a side facing the region of interest on the tissue section; dispensing a staining liquid into the cavity; allowing the staining liquid to stain the tissue at the region of interest; and aspirating the staining liquid from the cavity.

[0171] For example, the 3D mask for staining method can be drawn from initial staining tissue slice, and initial staining tissue slice is the different slices of same tissue, for example, the slice to be stained by the method for an embodiment.Initial staining can be conventional staining, for example, without using 3D mask.In initial staining, at least region of interest is visible (for example, stained), for example, to guide distribution and 3D mask design, printing and / or application. Typically, whole initial tissue slice will be stained.

[0172] The details of distributing, designing, and applying the 3D mask as described herein can also be applied to the staining method. Specifically, the 3D mask can be printed directly on the tissue section. As described herein, the staining method can be implemented in a separation machine. The separation device can be specifically used as a staining device.

[0173] In different embodiments of the separate machines, the communication interface can be selected from different alternatives. For example, the interface can be a network interface with a local area network or a wide area network (e.g., the Internet), a storage interface with an internal data storage or an external data storage, an application programming interface (API), etc.

[0174] The separation machine may have a user interface, which may include well-known elements such as one or more buttons, a keyboard, a display, a touch screen, etc. The user interface may be arranged to accommodate user interactions for configuring the system. The user interactions may include one or more of the following: defining one or more regions of interest, confirming a proposed defined position, starting separation, verifying the separation process, etc.

[0175] Typically, the separate machine includes a microprocessor that executes appropriate software stored in the system; for example, the software may have been downloaded and / or stored in a corresponding memory, for example, a volatile memory (such as RAM) or a non-volatile memory (such as flash memory). Alternatively, the system may be implemented in whole or in part in programmable logic, such as a field programmable gate array (FPGA). The system may be implemented in whole or in part as a so-called application-specific integrated circuit (ASIC), such as an integrated circuit (IC) that is customized for its specific use. For example, the circuitry may be implemented in CMOS, for example using a hardware description language such as Verilog, VHDL, etc. In particular, the system may include circuitry for evaluating neural networks.

[0176] The processor circuit can be implemented in a distributed form, for example, as multiple sub-processor circuits. Memory can be distributed across multiple distributed sub-memories. Some or all of the memory can be electronic memory, magnetic memory, etc. For example, the memory can have volatile components or non-volatile components. Portions of the memory can be read-only.

[0177] Figure 5One embodiment of a separation method 500 for separating tissue from a tissue section on a tissue slide is schematically illustrated. The method 500 comprises:

[0178] - applying (510) a 3D printed mask on top of the tissue slice, the mask comprising a barrier defining a cavity surrounding a region of interest on the tissue slice, the cavity being open at a side facing the region of interest on the tissue slice,

[0179] - dispensing (520) a separating liquid into the cavity and allowing the separating liquid to separate the tissue at the region of interest,

[0180] - Aspirating (530) the separated liquid with the separated tissue from the cavity and forwarding the liquid for further processing.

[0181] A computer may implement some or all elements of method 500. For example, a computer may control the application of a 3D printed mask, such as by controlling the printing of the 3D mask onto the tissue. For example, a computer may control the dispensing and aspiration of a separation fluid, such as by controlling the movement of a pipetting arm and the motion of a tip. The computer may be configured to generate a 2D design and / or a 3D design of a 3D mask for use in separation.

[0182] Many different ways of performing the method are possible, as will be apparent to those skilled in the art. For example, the order of the steps may be performed in the order shown, but the order of the steps may be varied, or some steps may be performed in parallel. Furthermore, other method steps may be inserted between steps. Such inserted steps may represent refinements of the method, as described herein, or may be unrelated to the method. For example, some steps may be performed at least partially in parallel. Furthermore, a given step may not be fully completed before starting the next step.

[0183] The embodiment of the method can be performed using software, which includes instructions for causing a processor system to perform method 500. The software may only include those steps taken by a specific sub-entity of the system. The software can be stored in a suitable storage medium (such as a hard disk, floppy disk, memory, optical disk, etc.). The software can be sent as a signal wired or wirelessly, or using a data network (such as the Internet). The software can be downloaded and / or used remotely on a server. The embodiment of the method can be performed using a bitstream, which is arranged to configure programmable logic (such as a field programmable gate array (FPGA)) to perform the method.

[0184] It will be understood that the presently disclosed subject matter also extends to computer programs, particularly computer programs on or in a carrier, suitable for implementing the presently disclosed subject matter. The program can be in the form of source code, object code, code intermediate source, and object code (such as partially compiled form), or any other form suitable for use in an embodiment of the present method. An embodiment relevant to a computer program product includes computer-executable instructions corresponding to each processing step of at least one method stated. These instructions can be subdivided into subroutines and / or stored in one or more files, which can be linked statically or dynamically. Another embodiment relevant to a computer program product includes computer-executable instructions corresponding to at least one of each device, unit, and / or part of the system and / or product stated.

[0185] Figure 6a A computer-readable medium 1000 having a writable portion 1010 and a computer-readable medium 1001 also having a writable portion are shown. Computer-readable medium 1000 is shown in the form of an optically readable medium. Computer-readable medium 1001 is shown in the form of electronic memory (in this case, a memory card). Computer-readable media 1000 and 1001 can store data 1020, where the data can represent instructions that, when executed by a processor system, cause the processor system to perform an embodiment of a separation method and / or a 3D printing method according to one embodiment. Data 1020 can be embodied on computer-readable medium 1000 as a physical mark or through magnetization of computer-readable medium 1000. However, any other suitable embodiments are also contemplated. Furthermore, it will be understood that although computer-readable medium 1000 is shown here as an optical disk, computer-readable medium 1000 can be any suitable computer-readable medium, such as a hard disk, solid-state memory, flash memory, etc., and can be non-recordable or recordable. Computer program 1020 includes instructions for causing the processor system to perform the described lysis method and / or tracking method.

[0186] For example, computer-implemented (e.g., computer-controlled operations) may include: operating a pipette tip (e.g., dispensing and aspirating separate liquids), operating a pipette arm and / or a pipette tip (e.g., moving a pipette arm), 3D printing, controlling temperature, obtaining a 2D design and / or a 3D design for a 3D printed mask, and other embodiments such as those mentioned herein.

[0187] Figure 6b A processor system 1140 according to one embodiment of a separation device and / or one embodiment of a 3D printing device is shown schematically. The processor system includes one or more integrated circuits 1110. The architecture of the one or more integrated circuits 1110 is schematically shown in FIG. Figure 6b. The circuit 1110 includes a processing unit 1120 (e.g., a CPU) for running a computer program component to execute a method according to one embodiment and / or implement its modules or units. The circuit 1110 includes a memory 1122 for storing programming code, data, etc. A portion of the memory 1122 may be read-only. The circuit 1110 may include a communication element 1126, such as an antenna, a connector, or both. The circuit 1110 may include an application-specific integrated circuit 1124 for executing some or all of the processing defined in the method. The processor 1120, the memory 1122, the application-specific integrated circuit 1124, and the communication element 1126 may be connected to each other via an interconnect 1130 (e.g., a bus). The processor system 1110 may be arranged for contact and / or contactless communication, using an antenna and / or a connector, respectively.

[0188] For example, in one embodiment, the processor system 1140 (e.g., a tracking device or a cracking device) may include a processor circuit and a memory circuit, wherein the processor is arranged to execute software stored in the memory circuit. For example, the processor circuit may be an Intel Core i7 processor, an ARM Cortex-R8, or the like. The memory circuit may be a ROM circuit or a non-volatile memory (e.g., flash memory). The memory circuit may be a volatile memory (e.g., SRAM memory). In the latter case, the device may include a non-volatile software interface (e.g., a hard disk, a network interface, etc.) arranged to provide the software.

[0189] Although device 1140 is shown as including one of each described component, multiple components may be repeated in multiple embodiments. For example, the processor may include multiple microprocessors that are configured to independently perform the methods described herein, or are configured to perform steps or subroutines of the methods described herein, such that the multiple processors cooperate to implement the functionality described herein. In addition, in the case where device 1140 is implemented in a cloud computing system, the various hardware components may belong to separate physical systems. For example, the processor may include a first processor in a first server and a second processor in a second server.

[0190] In one advantageous embodiment, the 3D printed mask comprises paraffin, for example, is printed from paraffin. This is particularly advantageous if the tissue is paraffin-embedded and the 3D mask is printed directly onto the tissue. Printing the 3D mask from paraffin onto the paraffin-embedded tissue ensures a good connection between the mask and the tissue without the need for dewaxing.

[0191] In one embodiment, a 3D printed mask is printed directly onto a tissue section, wherein the tissue section is embedded in a first paraffin wax, and the 3D printed mask includes a second paraffin wax. The second paraffin wax is different from the first paraffin wax. The first paraffin wax and the second paraffin wax are selected such that the melting point of the first paraffin wax is lower than the melting point of the second paraffin wax. For example, in one embodiment, the melting point of the first paraffin wax is lower than the melting point of the second paraffin wax, and the 3D printed mask is printed directly onto the tissue section. All melting points are considered at 1 standard atmosphere.

[0192] The first paraffin wax having a lower melting point than the second paraffin wax means that separation can be effectively performed after applying the 3D printing mask by heating the tissue slide to a separation temperature between the melting point of the first paraffin wax and the melting point of the second paraffin wax (for example, a separation temperature at a point midway between these melting point temperatures, between 10% and 90% of the temperature difference of the melting difference).

[0193] In some embodiments, the first paraffin wax of the embedding tissue section is melted, and the second paraffin wax of the cavity surrounding the region of interest will not melt. At this point, a separation liquid (such as a lysis buffer) can be introduced into the cavity formed by the second paraffin wax. Because the wall formed by the second paraffin wax remains intact, the wall will accommodate the separation liquid in the cavity. Because the paraffin wax of the embedded tissue section melts, the first paraffin wax can not hinder the tissue from being separated by the separation liquid. For more effective separation, a sufficient amount of detergent can be added to the separation liquid.

[0194] Typically, before the tissue sections are embedded in paraffin, they have previously been subjected to appropriate processing, such as one or more of fixation, dehumidification, and cleaning. In particular, the tissue sections can be FFPE tissue, for example, formalin-fixed, paraffin-embedded tissue. Latex, dimethyl sulfoxide, and proprietary "plasticizers" can optionally be included in the formulation to modify the texture and ductility of the final tissue sample. See, for example, Stephen M. Hewitt et al., "Tissue Handling and Specimen Preparation in Surgical Pathology Issues Concerning the Recovery of Nucleic Acids From Formalin-Fixed, Paraffin Embedded Tissue," for an example of the use of paraffin in tissue sections; the paper is incorporated herein by reference.

[0195] Paraffin waxes have different melting points. For example, a paraffin wax with a low melting point (eg, up to 65°C, eg, in the range of from 55°C up to 63°C) can be used as the first paraffin wax to embed tissue sections.

[0196] As an example, in one embodiment, the melting point of the first paraffin wax is at least 5 degrees Celsius lower than the melting point of the second paraffin wax. The 5 degree temperature difference facilitates heating the tissue section and the mask to the separation temperature.

[0197] For example, the first paraffin wax may have a melting point in the range of 55-63°C, and the second paraffin wax may have a melting point of at least 60°C, for example, the second paraffin wax may have a melting point of at least 100°C. For example, the second paraffin wax may have a melting point in the range of 60-250°C.

[0198] For example, the commercially available 3D printing filament "print2cast" has a melting point of 117 degrees Celsius. Muslim Mukhtarkhanov's paper, "Process Parameter Optimization for 3D Printed Investment," incorporated herein by reference, lists various options for 3D printing parts made from wax filaments. The paper also lists options for suitable 3D printing technologies. Specifically, Table 2 lists commercially available moldable filaments suitable for use in FDM (fused deposition modeling).

[0199] In practice, the melting point of the second paraffin wax can be significantly higher than that of the first paraffin wax. For example, in one embodiment, the second paraffin wax has a melting point exceeding 100°C, or even exceeding 150°C, or even exceeding 200°C. Such a high melting point can degrade RNA or DNA in the tissue section at the point where the 3D mask was printed. However, this is not a problem because the degradation is limited to a small area near the 3D printed mask.

[0200] In one embodiment, the 3D printer of the separation device is configured to apply a 3D printing mask comprising a second paraffin wax directly on top of a tissue section arranged on a tissue slide, the tissue section being embedded in a first paraffin wax, the first paraffin wax having a lower melting point than the second paraffin wax. In one embodiment, the 3D printing mask is arranged to be applied on top of a tissue section embedded in a first paraffin wax, the first paraffin wax having a lower melting point than the second paraffin wax.

[0201] In one embodiment, a 3D printed mask comprising a second paraffin wax is mixed with a staining liquid instead of a separating liquid.

[0202] Several exemplary examples of separation using paraffin masks are given below.

[0203] Example 1. A method for separating tissue from a tissue section on a tissue slide, the tissue section being paraffin-embedded in a first paraffin wax, the method comprising:

[0204] - printing a 3D printing mask directly on top of the tissue slice, the mask comprising a barrier film defining a cavity surrounding a region of interest on the tissue slice, the cavity being open at a side facing the region of interest on the tissue slice, the 3D printing mask comprising a second paraffin wax, the first paraffin wax having a lower melting point than the second paraffin wax,

[0205] - dispensing a separating liquid into the cavity and allowing the separating liquid to separate the tissue at the region of interest,

[0206] - Aspirating the separated fluid with the separated tissue from the cavity and transferring the fluid for further processing.

[0207] Embodiment 2. The method of embodiment 1, wherein the separation liquid is dispensed and / or aspirated from a pipette tip arranged at a motorized pipetting arm, the arm being arranged to move the pipette tip to the chamber.

[0208] Embodiment 3. The method according to any one of embodiments 1 to 2, comprising:

[0209] - heating the tissue slide by a heating element for heating the tissue section on the tissue slide to a temperature between the melting point of the first paraffin wax and the melting point of the second paraffin wax during separation, such as lysis.

[0210] Embodiment 4. The method of any one of embodiments 1 to 3, comprising placing the tissue slide in an incubation chamber, wherein the separation liquid is heated and allowed to separate the tissue.

[0211] Embodiment 5. The method of any one of embodiments 1 to 4, wherein the first paraffin layer is not removed from the tissue section before applying a 3D printing mask on top of the tissue section.

[0212] Embodiment 6. The method of any one of embodiments 1 to 5, wherein the 3D mask is printed by means of fused deposition modeling using paraffin wax filaments.

[0213] Embodiments using paraffin wax may be combined with other embodiments, eg, as claimed or described herein.

[0214] The following numbered clauses are not claims, but include contemplated and non-limiting embodiments:

[0215] Clause 1. A method (500) for separating tissue from a tissue section on a tissue slide, the method comprising:

[0216] - applying (510) a 3D printed mask on top of the tissue slice, the mask comprising a barrier defining a cavity surrounding a region of interest on the tissue slice, the cavity being open at a side facing the region of interest on the tissue slice,

[0217] - dispensing (520) a separating liquid into the cavity and allowing the separating liquid to separate the tissue at the region of interest,

[0218] - Aspirating (530) the separation liquid with the separated tissue from the cavity and forwarding it for further processing.

[0219] Clause 2. The method of clause 1, wherein the separation liquid is dispensed and / or aspirated from a pipetting tip arranged at a motorized pipetting arm, the arm being arranged to move the pipetting tip to the chamber.

[0220] Clause 3. The method of Clause 2, wherein the pipetting arm is configured to move away from the cavity during separation of tissue at the region of interest and to later return to the cavity to aspirate the separation liquid from the cavity.

[0221] Clause 4. The method of any preceding clause, wherein the plurality of cavities are defined by one or more 3D printed masks applied to the tissue slice,

[0222] The separating liquid is distributed to a plurality of chambers, the distribution among the plurality of chambers being carried out in parallel.

[0223] Clause 5. The method of any preceding clause, comprising applying a plurality of 3D printed masks to a plurality of tissue sections on a plurality of tissue slides, and dispensing a separation liquid into cavities on the resulting plurality of tissue sections, the method further comprising:

[0224] - Placing a plurality of tissue slides in a culture chamber, wherein the separation liquid is allowed to separate the tissue.

[0225] Item 6. The method of Item 5, wherein the plurality of tissue slides are collected in a stacker of tissue slides, and the stacker is placed in a culture chamber.

[0226] Clause 7. The method of any of the preceding clauses, wherein the separation liquid is dispensed into the cavity only once.

[0227] Clause 8. The method of any preceding clause, wherein a barrier defining the cavity matches an edge of a region of interest.

[0228] Clause 9. The method of any of the preceding clauses, wherein the cavity is at least partially surrounded by an inclined support.

[0229] Clause 10. The method of any of the preceding clauses, wherein the cavity has an irregular cavity shape.

[0230] Clause 11. The method of any of the preceding clauses, wherein applying the 3D printing mask comprises 3D printing the 3D printing mask directly onto the tissue section.

[0231] Clause 12. A method as described in any of the preceding clauses, wherein the paraffin layer is removed from at least a portion of the tissue section before applying the 3D printing mask on top of the tissue section, the 3D printing mask being applied on the at least a portion of the tissue section.

[0232] Clause 13. The method of any of the preceding clauses, comprising obtaining a 2D design of the mask, converting the 2D design into a 3D design, and 3D printing the 3D design to obtain the 3D mask.

[0233] Clause 14. The method of clause 13, comprising:

[0234] - obtaining a picture of a stained tissue section, the stained tissue section being a different slice of the same tissue section than the tissue section used for the separation,

[0235] - Identify regions of interest on stained tissue sections,

[0236] -Generate a 2D design from the identified region of interest.

[0237] Clause 15. The method of any of the preceding clauses, wherein the tissue section used for separation is at least twice as thick as the stained tissue section, at least four times as thick as the stained tissue section, or at least eight times as thick as the stained tissue section.

[0238] Clause 16. The method of any preceding clause, wherein the further processing comprises:

[0239] - Genomic analysis of isolated tissues, and / or

[0240] - Extracting biomolecules, such as one or more of nucleic acids, proteins, lipids and hormones, from the isolated tissue.

[0241] Clause 17. The method of any one of the preceding clauses, wherein the tissue section is a FFPE tissue section.

[0242] Clause 18. The method of any of the preceding clauses, comprising increasing the temperature of the tissue section at the region of interest during tissue separation.

[0243] Clause 19. The method of any of the preceding clauses, wherein the method is at least partially computer-implemented.

[0244] Clause 20. The method of any of the preceding clauses, wherein the separation liquid comprises a lysis liquid that lyses tissue at the region of interest.

[0245] Clause 21. A separation device comprising:

[0246] - a tissue support arranged to receive a tissue slide,

[0247] a 3D printer for applying a 3D printed mask onto a tissue section arranged on a tissue slide, the mask comprising a barrier defining a cavity surrounding a region of interest on the tissue section, the cavity being open at a side facing the region of interest on the tissue section,

[0248] - a pipetting tip which is arranged at the motorized pipetting arm and is arranged to move the pipetting tip to the cavity for dispensing the separation liquid to the cavity and for aspirating the liquid from the cavity.

[0249] Item 22. A 3D printing mask arranged for application on top of a tissue slice, the mask comprising a barrier defining a cavity surrounding a region of interest on the tissue slice, the cavity being open on a side facing the region of interest on the tissue slice and at least partially open on a side opposite the tissue slice, the cavity being arranged for receiving a separation liquid for separating tissue at the region of interest.

[0250] Clause 23. A transitory or non-transitory computer-readable medium (1000) comprising data (1020) representing instructions that, when executed by a processor system, cause the processor system to perform the method of any of clauses 1-20.

[0251] In a variation of the above clause, staining is used instead of separation. For example, in one embodiment, a method for staining tissue from a tissue section on a tissue slide is provided. The staining method comprises:

[0252] - applying a 3D printed mask on top of the tissue slice, the mask comprising a barrier defining a cavity surrounding the region of interest on the tissue slice, the cavity being open at a side facing the region of interest on the tissue slice,

[0253] - dispensing a staining liquid into the cavity and allowing the staining liquid to stain the tissue at the area of interest,

[0254] - Aspirating the dyeing liquid from the cavity.

[0255] It should be noted that the above-mentioned embodiments illustrate rather than limit the presently disclosed subject matter, and that those skilled in the art will be able to design many alternative embodiments.

[0256] In the claims, any reference signs placed between brackets shall not be construed as limiting the claims. The use of the verb "comprise" and its variations does not exclude the presence of elements or steps other than those stated in the claim. The article "a" or "an" preceding an element does not exclude the presence of a plurality of such elements. When an expression such as "at least one" is placed before a list of elements, it means selecting all elements or any subset thereof from the list. For example, the expression "at least one of A, B, and C" should be understood to include only A, only B, only C, both A and B, both A and C, both B and C, or all of A, B, and C. The presently disclosed subject matter can be implemented by hardware comprising several different elements, as well as by a suitably programmed computer. In a device claim that lists several components, several of these components may be implemented by the same item of hardware. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.

[0257] In the claims, reference numerals in parentheses refer to reference symbols in the exemplary embodiment drawings or formulas of the embodiments, thereby improving the intelligibility of the claims. These reference numerals should not be construed as limiting the claims.

Claims

1. A method for separating tissue from a tissue section on a tissue slide, the method comprising: - applying a 3D printing mask on top of the tissue slice, the mask comprising a barrier defining a cavity surrounding a region of interest on the tissue slice, the cavity being open at a side facing the region of interest on the tissue slice, wherein applying the 3D printing mask comprises 3D printing the 3D printing mask directly on the tissue slice, - dispensing a separating liquid into the cavity and allowing the separating liquid to separate the tissue at the region of interest, - Aspirating the separated liquid with the separated tissue from the cavity and transferring the liquid for further processing.

2. The method according to claim 1, wherein the separation liquid is dispensed and / or aspirated from a pipette tip, the pipette tip being arranged at a motorized pipetting arm, the arm being arranged to move the pipette tip to the chamber. 3 . The method of claim 2 , wherein the pipetting arm is configured to move away from the cavity during separation of tissue at the region of interest and to later return to the cavity to aspirate the separation liquid from the cavity.

4. The method of claim 1 , wherein the plurality of cavities are defined by one or more 3D printed masks applied to the tissue slice, The separation liquid is distributed to the plurality of chambers, the separation in the plurality of chambers being performed in parallel.

5. The method according to claim 1, comprising: applying a plurality of 3D printed masks to a plurality of tissue sections on a plurality of tissue slides, and dispensing a separation liquid into the resulting cavities on the plurality of tissue sections, the method further comprising: - placing the plurality of tissue slides in an incubation chamber, wherein the separation liquid is allowed to separate the tissue. The method of claim 1 , wherein the cavity is at least partially surrounded by an angled support. The method according to claim 1 , wherein the cavity has an irregular cavity shape.

8. The method of claim 1 , wherein a paraffin layer is removed from at least a portion of the tissue section before applying the 3D printing mask on top of the tissue section, the 3D printing mask being applied on the at least a portion of the tissue section.

9. The method of claim 1, comprising obtaining a 2D design of the mask, converting the 2D design into a 3D design, and 3D printing the 3D design to obtain the 3D mask.

10. The method according to claim 9, comprising: - obtaining a picture of a stained tissue section, which is a different slice of the same tissue than the tissue section used for the separation, - identifying a region of interest on said stained tissue section, -Generate a 2D design from the identified region of interest.

11. The method of claim 1, wherein the tissue section used for separation is at least twice as thick as the stained tissue section, at least four times as thick as the stained tissue section, or at least eight times as thick as the stained tissue section.

12. The method of claim 1, wherein the further processing comprises: - genomic analysis of the isolated tissue, and / or - extracting biomolecules, such as one or more of nucleic acids, proteins, lipids and hormones from said isolated tissue.

13. The method of claim 1, wherein the method is at least partially computer-implemented.

14. The method of claim 1, wherein: - the tissue sections are FFPE tissue sections, and / or The separation liquid comprises a lysis liquid, which lyses the tissue at the region of interest.

15. The method according to claim 1, wherein the tissue section is paraffin-embedded in a first paraffin wax, the 3D printing mask comprises a second paraffin wax, the melting point of the first paraffin wax is lower than the melting point of the second paraffin wax, and the 3D printing mask is directly printed on the tissue section.

16. A method for staining tissue from a tissue section on a tissue slide, the method comprising: - applying a 3D printing mask on top of the tissue slice, the mask comprising a barrier defining a cavity surrounding a region of interest on the tissue slice, the cavity being open at a side facing the region of interest on the tissue slice, wherein applying the 3D printing mask comprises 3D printing the 3D printing mask directly on the tissue slice, - dispensing a staining liquid into the cavity and allowing the staining liquid to stain the tissue at the region of interest, - Aspirating the dyeing liquid from the cavity.

17. A separation device comprising: - a tissue support arranged to receive a tissue slide, a 3D printer for applying a 3D printed mask on top of a tissue section arranged on the tissue slide, the mask comprising a barrier defining a cavity surrounding a region of interest on the tissue section, the cavity being open at a side facing the region of interest on the tissue section, -processor system, - a pipetting tip arranged at a motorized pipetting arm, the motorized pipetting arm being arranged to move the pipetting tip to the cavity for dispensing the separation liquid to the cavity and aspirating the liquid from the cavity.

18. A 3D printing mask arranged to be applied on top of a tissue slice, the mask comprising a barrier defining a cavity surrounding an area of interest on the tissue slice, the cavity being open on a side facing the area of interest on the tissue slice and at least partially open on a side opposite the tissue slice, the cavity being arranged to receive a separation liquid for separating tissue at the area of interest, wherein the 3D printing mask is 3D printed directly onto the tissue slice.

19. A transitory or non-transitory computer-readable medium comprising data representing instructions which, when executed by the processor system, cause the separation device defined in claim 17 to perform the method according to any one of claims 1 to 16.

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