Method and system for evaluation of tissue samples - Patents.com

Abstract

Systems and methods for evaluation of live tissue samples are provided including well inserts that may be capable of maintaining an air-liquid interface for three-dimensional (3D) cell culture. The well plate insert may include a semi-permeable filter. The tissue sample is placed in the well and placed on top of the filter, where it can retrieve nutrients from the medium below the filter while still being exposed to air throughout the duration of the cell culture. A filter assembly may be used to float and support the top-placed tissue sample for transfer after slicing. An automated tissue slicer and handler may cut thin sections of the tissue sample and transfer them to the filter assembly. A tissue culture plate may receive the sections captured by the filter assembly, which are sent for subsequent processing.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is related to pending U.S. Provisional Patent Application No. 63 / 301,038, entitled "Automated Systems and Methods for Processing Biological Samples," filed January 19, 2022, and U.S. Provisional Patent Application No. 63 / 439,803, entitled "Methods and Systems for Evaluation of Tissue Samples," filed January 18, 2023. Each of these applications is incorporated herein by reference as if set forth in its entirety herein.

[0002] Field of the Disclosure The present disclosure relates generally to the evaluation of tissue samples, and more particularly to systems and methods for the evaluation of tissue samples. [Background technology]

[0003] Mammalian organs / tissues are composed of various cell types that have specific structures and cell-cell interactions and form three-dimensional (3D) structures called ecosystems. Current techniques for ex vivo mammalian tissue culture involve disruption and dissociation of the tissue ecosystem to obtain a set of cells that are used to initiate cell culture in two dimensions (2D). Although several techniques exist for 3D culture, such as organoids, hydrogels, and micro engineered 3D scaffolds, these techniques are artificial, not typical in naturally occurring animal / human tissue ecosystems, and are time-consuming and expensive.

[0004] Currently, mammalian tissues, such as surgical resections or biopsies, when transported to the laboratory with viability maintained for ex vivo culture, are placed in tubes, such as standard 5 ml, 15 ml, or 50 ml tubes, which may be filled with a liquid buffer solution and placed on ice or a cold pack for cooling. The buffer solution containing the tissue is then removed, and the tissue is picked up using forceps or other manual methods that may apply force to, disintegrate, or otherwise damage the tissue. In addition, movement caused by transportation can cause the tissue to break down into smaller pieces that are difficult to find and process. Currently, there are no methods used to minimize damage caused by fluid movement during transportation and handling, nor methods to ensure systematic and consistent handling of tissues in the laboratory.

[0005] Current methods for slicing live tissue that is neither fixed nor frozen into small sections use a vibratome or tissue chopper. The sample and slicing mechanism occur in a liquid buffer solution, and the released tissue sections float into the buffer and are manually picked up from the solution. Current methods do not utilize an automated means of handling the live tissue sections after cutting. Additionally, current devices do not allow for controlled sterilization of the mechanism that comes into contact with the sample. Summary of the Invention

[0006] An embodiment of the present disclosure may provide a system for processing a live tissue sample, the system comprising a funnel-shaped structure and a tube filled with a buffer solution, the funnel-shaped structure being inserted into the tube to transport the tissue sample. The tube may be a 5 ml tube, a 15 ml tube, or a 50 ml tube. The funnel-shaped structure may be a thin plastic mesh. The funnel-shaped structure may be solid and formed from plastic, metal, and / or a gel-like substance and may be separable into two parts, or the two parts may be hinged. The funnel-shaped structure may have an opening at a tapered portion of the funnel-shaped structure where the buffer solution can flow out or be sucked out while the tissue sample remains within the funnel-shaped structure.

[0007] Another embodiment of the present disclosure may provide a system for processing a live tissue sample, the system comprising at least one filter assembly comprising a filter and a ring made of a material connectable to an outer portion of the filter, the ring providing buoyancy for the filter to float on the surface of the fluid. The filter may be hydrophobic on the side not in contact with the ring and hydrophilic on the opposite side in contact with the ring. The filter may have a pore size of about 2 μm to about 10 μm. The ring may be formed from a mixture of biologically inert plastic, biologically inert wax, and / or wrapping film / tape.

[0008] Further embodiments of the present disclosure may provide a system for processing live tissue samples, the system comprising a well plate insert comprising at least one column and a semi-permeable filter attached to the bottom of each column, the semi-permeable filter capable of maintaining an air-liquid interface for tissue samples placed in the column, wherein when the well plate insert is placed in the column with liquid at the bottom, the well plate insert may be capable of removing culture medium without flooding the inner chamber of the well plate in which the live tissue sample is located. The semi-permeable filter may have a pore size of about 2 μm to about 10 μm. Particulates and / or nutrients in the culture medium may migrate upward through the semi-permeable filter without allowing the culture medium to permeate the air chamber in the well plate.

[0009] An embodiment of the present disclosure includes a system for processing a raw tissue sample, the system comprising a sample holder configured to receive the raw tissue sample, a bracket configured to receive the sample holder, an indexing mechanism configured to move the raw tissue sample in precise increments, and a blade operably connected to the sample holder and configured to cut the raw tissue sample. In some examples, the indexing mechanism comprises a linear actuator comprising a plunger configured to move the raw tissue sample toward the blade. In some cases, the raw tissue sample is moved about 200 micrometers to about 400 micrometers. The indexing mechanism may comprise a cutting surface, the cutting surface configured to receive a controlled amount of fluid sufficient to lubricate the cutting surface and the raw tissue sample. The delivered fluid may be in the form of a mist having at least one droplet. The system may also comprise a receptacle operably connected to the sample holder configured to receive a fragment of the raw tissue sample after the fragment is cut by the blade. In some examples, the receptacle is positioned to contact the fluid. In some cases, embodiments may include a user interface, and the system may be configured for automated operation with no more than one single live tissue sample. In this regard, single-use operation of the system and any or all of its components may include multiple rounds of use, but refers to use with a single live tissue sample and avoidance of any cross-contamination between different samples.

[0010] The embodiments of the present disclosure may include a method of processing a raw tissue sample, the method including providing a well plate with a filter assembly operably connected to a blade, cutting the raw tissue sample with the blade, and obtaining fragments of the raw tissue sample. In some cases, the embodiments may further include contacting the raw tissue sample with a fluid, the fluid configured to generate a droplet of fluid sufficient to lubricate the raw tissue sample, contacting the droplet with a filter assembly, and contacting the fragments of the raw tissue sample with the filter assembly contacted with the droplet. In some cases, the methods may include a user interface, and the system may be configured for automated operation with no more than one single raw tissue sample. In this regard, a single use operation of the system and any or all of its components may include multiple rounds of use, but refers to use with a single raw tissue sample and avoidance of any cross-contamination between different samples.

[0011] Further embodiments of the present disclosure may provide an automated tissue slicer and handler (ATSH) that includes a bracket that receives a sample holder having a tissue sample, such as a surgical resection or biopsy, embedded in a support structure such as agarose, an indexing mechanism for pushing the tissue sample out of the bottom of the sample holder in controlled increments, and a blade positioned below the sample holder that cuts a thin section from the bottom of the sample. The indexing mechanism may be a linear actuator that indexes the tissue by moving a plunger downward. The linear actuator may be capable of pushing a controlled amount of the embedded tissue sample out of the bottom of the sample holder. The linear actuator may index sections of the tissue sample that are 200 micrometers to 400 micrometers thick. There may be a slicing surface that receives a controlled amount of fluid to lubricate the cutting surface and keep the tissue sample from drying out. The controlled amount of fluid may be provided by applying a fine mist. The system may also include a receptacle located under the sample holder that can receive individual sections of the tissue sample as it is sliced ​​from the bottom of the tissue sample. The receptacle may be raised until it contacts a fluid that is applied to lubricate the cutting surface, after which the receptacle is lowered. A robotic arm can be positioned under the sample holder that can hold a well plate with liquid medium in each well, the well plate containing a filter assembly that floats above the liquid medium in each well and captures the sections by contacting the filter assembly with a droplet of buffer. A tissue holder, such as half of a small plastic funnel with a tip into which the sample settles, can be inserted inside the sample holder and swirled in the agarose to separate the tissue sample from the tissue holder and embed it in the agarose. The sample holder can have a first portion in which the tissue sample is embedded or encased in agarose, and a second portion that is part of the ATSH, which receives the first portion containing the embedded tissue sample, holds the first portion in place, and connects to the ATSH to prepare the tissue sample for slicing.It should be understood that in an embodiment of the disclosure, the embedding can be done in the first part so that the tip of the funnel can be inserted into the liquid agarose and swirled before the agarose cools and solidifies. Although the sample holder is described as a two-part assembly, there may be other embodiments of the disclosure in which more or less than two parts may be provided. The tissue sample may be indexed from the bottom of the tissue sample and pushed with a plunger from the top. The ATSH may also include a pump to aspirate sections of the tissue sample as they are sliced ​​from the sample. The ATSH may further include a removable reservoir of buffer that connects to a pump that can dispense buffer at a controlled continuous flow rate or in controlled discrete amounts for the purpose of lubricating the agarose as it is pushed out of the sample holder, or to deposit droplets on the exposed bottom of the sample to keep the sample moist, to keep the cutting surface clean, or to lubricate the slicing action, and an aspiration pump to collect the sections. It will be appreciated that the components exposed to the tissue sample may also be single-use disposable components, facilitating easy aseptic processing of the sample by removing the single-use components used with the previous sample and replacing them with new components for the new sample. These single-use components can include, but are not limited to, the agarose, the sample holder, the cutting blade, the filter assembly and well plate, the sample pusher that presses against the top of the sample, and the lubricating fluid.

[0012] Other embodiments of the present disclosure may provide a method of transferring a section of a tissue sample onto a filter assembly, the method including holding the tissue sample above a slicing blade disposed above a well plate containing the filter assembly, and using the slicing blade to cut a section from a bottom of the tissue sample. The method may also further include dispensing a lubricating fluid onto an exposed portion of the tissue sample to generate a droplet of the lubricating fluid to lubricate the tissue sample, raising the filter assembly into contact with the droplet, where the droplet is drawn into the filter assembly by surface tension, and after cutting the section, lowering the section onto the fitter assembly. [Brief description of the drawings]

[0013] The following description is made in conjunction with the accompanying drawings. [Figure 1] FIG. 1 illustrates a top isometric view of a well plate insert according to one embodiment of the present disclosure. [Diagram 2] FIG. 2 shows a bottom isometric view of a well plate insert according to one embodiment of the present disclosure. [Diagram 3] FIG. 3 illustrates a filter assembly and filter according to one embodiment of the present disclosure. [Figure 4] FIG. 4 illustrates a transfer process for transferring sections onto a filter assembly according to one embodiment of the present disclosure. [Diagram 5] FIG. 5 shows a free-standing automated apparatus in which an automated tissue slicer and handler is one component for performing treatment sensitivity assays according to one embodiment of the present disclosure. [Figure 6A] 6A-6B show top and side views of a filter assembly having a support structure below the filter according to one embodiment of the present disclosure. [Figure 6B] 6A-6B show top and side views of a filter assembly having a support structure below the filter according to one embodiment of the present disclosure. [Figure 6C] 6C-6E show top and side views of a filter assembly using an air pocket according to one embodiment of the present disclosure. [Figure 6D] 6C-6E show top and side views of a filter assembly using an air pocket according to one embodiment of the present disclosure. [Figure 6E] 6C-6E show top and side views of a filter assembly using an air pocket according to one embodiment of the present disclosure. [Figure 6F] 6F-6G show side and top views of an integrated well plate and filter assembly according to one embodiment of the present disclosure. [Figure 6G]6F-6G show side and top views of an integrated well plate and filter assembly according to one embodiment of the present disclosure. [Figure 6H] 6H-6K show top and side views of a floating filter assembly and a well plate according to one embodiment of the present disclosure. [Figure 6I] 6H-6K show top and side views of a floating filter assembly and a well plate according to one embodiment of the present disclosure. [Figure 6J] 6H-6K show top and side views of a floating filter assembly and a well plate according to one embodiment of the present disclosure. [Figure 6K] 6H-6K show top and side views of a floating filter assembly and a well plate according to one embodiment of the present disclosure. [Figure 7] FIG. 7 illustrates a sample transfer cassette (STC), placement of a tissue sample into the STC, and transfer to agarose, according to one embodiment of the present disclosure. [Figure 8A] 8A-8B illustrate a slicing and plating method according to an embodiment of the present disclosure. [Figure 8B] 8A-8B illustrate a slicing and plating method according to an embodiment of the present disclosure. [Figure 8C] FIG. 8C illustrates a method for tissue slicing according to one embodiment of the present disclosure. [Figure 9] FIG. 9 illustrates a method for tissue slicing and positioning according to one embodiment of the present disclosure. [Figure 10] FIG. 10 illustrates a workflow from STC to placement inside a tissue culture plate according to one embodiment of the present disclosure. [Figure 11] FIG. 11 illustrates an ATSH according to one embodiment of the present disclosure. [Figure 12A] 12A-12C show the system architecture for the handling process after tissue slicing and handling is completed in an embodiment of the present disclosure. [Figure 12B]12A-12C show the system architecture for the handling process after tissue slicing and handling is completed in an embodiment of the present disclosure. [Figure 12C] 12A-12C show the system architecture for the handling process after tissue slicing and handling is completed in an embodiment of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The embodiments of the present disclosure may provide automated systems and methods for processing biological samples, particularly live tissues. As described herein, "tissue" may be defined as a collection of physically attached cells, where physical attachment may include association in the extracellular matrix, as well as cell-cell adhesion via occlusive junctions, adherens junctions, desmosomes, anchoring junctions, and gap junctions. Tissue as used herein may include normal cells, pre-cancerous cells, and cancer cells. Additionally, "live" may be defined as metabolically active or viable, where live and dead cells may be differentiated via cell membrane permeable indicators and / or dyes. As used herein, "live tissue" is defined as tissue that contains living cells, although live tissue samples may also include dead cells. Also, "single-use" may be defined to include single-use, disposable, non-reusable, and / or non-renewable. This may include conditions related to avoiding cross-sample contamination, such as any pre- or post-use sterilization, decontamination, and / or disinfection, or minimization of any harmful or deleterious effects as defined by laws or regulatory standards. In this regard, single use may include multiple uses of the system with a single live tissue sample or repeated method steps.

[0015] In one embodiment of the present disclosure, a well insert may be provided that may be capable of maintaining an air-liquid interface for three-dimensional (3D) cell cultures, specifically harvested mammalian tissues, independent of external factors including, but not limited to, medium volume or evaporation. A well plate insert according to an embodiment of the present disclosure may include a 3 micron filter, which may be semi-permeable in that it may allow particulates / nutrients in the medium to move upward through the filter without allowing liquid to penetrate into the air chamber in the well plate. Although the filter is described as 3 micron, it is understood that there may be embodiments of the present disclosure in which the filter pore size is larger or smaller. The filter and the surrounding wall of the insert independently cannot allow liquid to enter the air chamber. A tissue sample may be placed in the well and placed on top of the filter located at the bottom of the well plate, where nutrients can be collected from the medium below the filter while still in contact with the air above the tissue throughout the duration of the tissue culture. In an embodiment of the present disclosure, the tissue sample may be 400 microns in thickness. However, it is understood that the thickness may be larger or smaller in some embodiments of the present disclosure.

[0016] FIG. 1 shows a top isometric view of a well plate insert 10 according to one embodiment of the present disclosure. More specifically, FIG. 1 shows the form factor of the insert oriented for a 24-well plate having a lid-like perimeter. While the insert 10 is shown for a 24-well plate, it should be understood that more or less wells may be provided without departing from the present disclosure. Holes 101 visible at the top of the lid 102 may extend through the lid 102, and semi-permeable filters may be attached to the bottom of each column (see FIG. 2).

[0017] 2 shows a bottom isometric view of a well plate insert 20 according to one embodiment of the present disclosure. As shown herein, the bottom of the column 201 may have a semi-permeable filter securely attached so that tissue may rest on top of the filter. When the insert 20 is placed into a well plate containing media, the insert can remove the media without flooding the inner chamber where the tissue is located.

[0018] The dimensions of the well plate may be adapted for 96, 48, 24, 12, and 6 well plates in the embodiments of the present disclosure. However, other dimensions, configurations, and numbers of wells may be provided without departing from the present disclosure. Of course, the walls of the well plate may not have any openings, such as holes or pores, to allow liquid to enter the chamber from the side, but may include holes / pores near the top above the liquid. The inner and outer diameters of each well insert column may be varied in the embodiments of the present disclosure, as well as the height of each column. It should also be understood that the filter pore size may be varied in the embodiments of the present disclosure to allow different particles to pass through.

[0019] A filter assembly may be used in the embodiments of the present disclosure. The filter assembly may have a 3 μm pore size filter that is hydrophobic on one side and hydrophilic on the opposite side. When placed hydrophobic side down on the medium / buffer solution, the filter assembly floats and can support a biopsy that can be placed on top. The 3 μm pore size may ensure that T cells do not pass through the filter. However, other size filters may be used depending on the tissue type and testing purpose without departing from the present disclosure. It should also be understood that there may be embodiments of the present disclosure where the filter itself does not need to be buoyant (i.e., one side does not need to be hydrophobic) and other methods of providing buoyancy may be utilized.

[0020] The periphery of the circular cut filter assembly may include a ring of Parafilm wrapping film or other equivalent non-toxic buoyant film winch attached to the filter assembly. Although the filter assembly is described as circular cut, other shapes may be used without departing from this disclosure. The wrapping film / tape may provide additional buoyancy (while being non-toxic) so that the filter may float to the top even when the filter assembly is handled. A filter according to an embodiment of the present disclosure may be a 5 mm circle with a ring of plastic such as polyethylene terephthalate (PET), which may be about 0.5 mm thick and 1 mm tall. This ring may be attached using a non-toxic adhesive that may be heated and bonded to the filter. However, another non-toxic mechanism for attaching the ring to the filter may be used without departing from this disclosure. The shape and arrangement of the filter may allow the biopsy to be exposed to air on the top side and nutrients that pass through the filter on the bottom side.

[0021] The filter assembly according to the embodiments of the present disclosure can maintain a gas / liquid interface, including during handling and / or processing of tissue. The filter assembly may allow for easier transfer of tissue slices from an automated slicer into the filter. As described herein, in the embodiments of the present disclosure, the filter assembly may float on top supported by the buoyant filter itself, as shown in Figures 6C-6E. More specifically, Figure 6C shows a top view of a buoyant filter assembly. The filter may be hydrophobic on one side, or the filter may be less dense than the liquid in which it may float. The filter may include a sealed air pocket or may be open to the liquid below the filter. Figure 6D shows a side view with an open air pocket 603 exposed to the liquid, and Figure 6E shows a side view with a sealed air pocket, where the filter shape may provide a sealed air pocket 603. Alternatively, the filter may be supported by a separate support structure with buoyancy.

[0022] 6A-6B show top and side views of a filter assembly having a support structure below the filter according to one embodiment of the present disclosure. The filter 601 may generally be supported from the bottom and may be held via a fastening mechanism, including but not limited to adhesive, thermal bonding, chemical bonding, clamping, and piercing. An outer ring 602 may be provided on the bottom of the filter that may be held via one of the fastening mechanisms. In another alternative, an item such as an outer ring may be provided on the top of the filter and held via one of the fastening mechanisms. FIG. 6A shows a bottom view of the filter assembly showing a filter bottom 601 with a plastic or other buoyant support structure 602 at the bottom, and FIG. 6B shows a side view showing the filter 601 on top of a support structure.

[0023] In a further embodiment of the present disclosure, a fixed filter can be placed on top of the well plate assembly as shown in Figure 4. These fixed filter assemblies can be assembled into an array as shown in Figures 1 and 2.

[0024] In further embodiments of the present disclosure, the filter may be incorporated into a well plate assembly such as that shown in the integrated well plate and filter assembly of Figures 6F-6G. As shown herein, a filter 604 may be bonded to a well plate 606 and the top of the buffer solution 605 may be open to air while the filter may prevent the buffer solution from leaking onto the top of the filter, such as in the side view shown in Figure 6F. This may allow for a supply of buffer to the area below the filter, such as a "water tower" which is the side where gravity may supply the buffer to the area below the filter. The water tower may be filled with enough buffer solution so that even if it evaporates, the fluid level is sufficient to maintain contact with the filter throughout the process. This may be a pressurized bag or other mechanism in embodiments of the present disclosure. Figure 6G shows a top view showing a supply of buffer solution 608 where the filter 609 covers the top of the buffer solution in the area it covers.

[0025] 6H-6K show top and side views of a floating filter assembly and well plate according to one embodiment of the present disclosure. More specifically, FIG. 6H shows a top view of a two-part assembly in which the filter assembly 610 floats above the fluid at the bottom of the wells in the well plate, and the filter assembly may include pockets or areas that allow liquid handling to occur, such as with a pipette or other liquid handling device, without hitting the filter. FIG. 61 shows a side view of the two-part assembly showing the filter 613 against the buffer / media 614. FIG. 6 shows a top view of an alternative design of the two-part assembly. Again, the filter assembly 610 is shown floating above the fluid at the bottom of the wells in the well plate, and the filter assembly 610 may include space 615 for pipetting without hitting the filter assembly. The filter assembly may include one or more nodules or protrusions 616 in the sidewall of the well plate that may limit the contact area with the filter assembly 610 to reduce friction sliding up and down the wall. FIG. 6K shows a top view of another alternative design of the two-part assembly. Again, the filter assembly 610 is shown floating on top of the fluid at the bottom of the well in the well plate, and the filter assembly 610 may include space 615 for pipetting without hitting the filter assembly. The filter assembly may include one or more posts 617 in the well to limit the contact area with the filter assembly to reduce friction of sliding up and down the walls. In each of the embodiments of Figures 6H-6K, the well plate may protect the floating filter assembly, such as a filter with an outer buoyancy ring, from pipette tips or other devices that may need to enter the well plate to perform liquid handling operations such as adding or removing media reagents. This can be done by adding a recess or alcove to the side of the area where the filter assembly resides. This recess or alcove may be where a pipette or other device enters to perform liquid handling. Different embodiments may exist within the area where the filter assembly resides.Figures 6J and 6K show a configuration in which the contact points where the walls of the well plate meet the filter assembly can be clearly defined. This can minimize friction, resulting in a more reliable raising and lowering of the filter assembly as liquid is added and removed while the filter floats on top.

[0026] FIG. 3 shows a filter assembly 304 and filter 302 according to one embodiment of the present disclosure. The filter assembly 304 may have an inner wall height of about 2.5 mm for a 200 μl medium / buffer solution volume in an 11 mm diameter well, the dimensions of a typical 48-well plate. The walls may aid in floating the filter assembly 304 even when it is handled and / or bumped. The walls may also provide a rigid surface to hold for manipulating the filter assembly 304 in embodiments of the present disclosure. The wall height may facilitate interaction with a droplet on the bottom of the sample to keep the sample from drying out while still lubricating the slicing action. This wall height may come into contact with the droplet, which may center the droplet and make controlled transfer of the biopsy section to the filter assembly easier. However, it should be understood that the wall height of the filter assembly may be reduced in embodiments of the present disclosure to allow for easier placement of the section.

[0027] As shown herein, the filter assembly 304 does not float, but sits on top of the well plate, and the depth of the filter assembly is deep enough to ensure that the filter 302 contacts the medium 303 at the bottom of the well, so that the filter 302 can be pressed into the surface of the medium 303. If the filter assembly 304 presses the filter 302 slightly (about 0.5 mm) down into the surface of the medium 303, the water pressure may be low enough that the medium 303 does not push or "bleed" through the filter 302. The filter 302 may remain in contact with the medium 303, and the medium 303 does not evaporate so much that the filter 302 loses contact with the medium 303. Thus, through the use of the filter assembly 304 in the embodiments of the present disclosure, evaporation may be reduced as the surface area of ​​the medium-to-air interface is reduced, which may reduce the variability of medium volume due to evaporation, or allow smaller volumes of liquid reagents to be used, thereby reducing costs and analytical variability.

[0028] By minimizing the surface area of ​​the filter 302 and the exposed top of the medium 303, evaporation can occur which can be as small as only about 0.2 mm of medium height can be lost over a 3 day incubation at 37° C. More specifically, Figure 3 shows 3 day evaporation of about 13 μl, which can be about 0.2 mm of medium height lost in embodiments of the present disclosure.

[0029] Although filter 302 and filter assembly 304 are shown in FIG. 3, it should be understood that there may be embodiments of the disclosure in which a filter fixed in place may be used, such that a filter assembly that floats on the surface of medium 303 may not be necessary. In other embodiments of the disclosure, an array or rack of wells that is connected and "does not float" may be used. This rack may facilitate lifting the filter assembly with the filter with the biopsy on top, while at the same time allowing easier liquid handling in downstream operations. This rack of interconnected fitter assemblies may be shallow (approximately 2.5 mm in height) and may be used like the individual filter assemblies described herein, such that the wall height allows interaction with the droplets to help center and facilitate controlled transfer of the sections.

[0030] FIG. 4 illustrates a transfer process for transferring sections onto a filter assembly 404 according to one embodiment of the present disclosure. As shown herein, the sample 401 may be held above the slicing blade 402 and then pushed down / indexed from above. A lubricating fluid may be dispensed onto the bottom / exposed portion of the sample 401 to lubricate the section, keep the exposed portion of the sample wet, or keep the slicing blade clean. When the filter assembly 404 is raised and contacts the droplet 403, surface tension may pull the droplet 403 into the filter assembly 404. The section may settle and then be lowered so that it may be on the filter assembly 404. As shown herein, the section may be cut from the bottom of the sample 401. The well plate containing the filter assembly 404 may be raised until the filter assembly 404 contacts the droplet 403. The filter assembly 404 may then be lowered.

[0031] Agarose is a gelatin-like substance that melts and solidifies at a particular temperature. An embodiment of the present disclosure uses a "low melting point" agarose 405 that melts at 40° C. This may allow the agarose 405 to be heated to its melting point, tissue such as from biopsy 401 is embedded in the agarose 405, and then cooled, solidifying and holding the biopsy in place. The 40° C. melting point means that when the sample is incubated at 37° C. (body temperature), the agarose is solid and can also be melted so that the biopsy can be embedded at a temperature low enough that the biopsy is viable and not altered in its behavior / response by being too hot.

[0032] 5 shows a free-standing automated device for performing a treatment sensitivity assay including steps performed according to one embodiment of the present disclosure. In an embodiment of the present disclosure, the portion of the device that performs the initial processing of the tissue is referred to as an automated tissue slicer and handler (ATSH). The portion of the device that processes the tissue after the ATSH can be used for liquid handling, plate reading, incubation, and / or reagent management for downstream processing of the assay.

[0033] FIG. 7 shows a hinged design of a sample transfer cassette (STC), which is a device that accepts the input of a tissue sample at the clinic after it is taken, and facilitates keeping the tissue more intact during transport to a facility that processes the tissue sample, making removal and handling of the sample easier and less stressful on the tissue at the processing facility. This processing at the facility may be the transfer of the tissue from the STC to agarose. In panel 701, the tissue sample, shown here as a biopsy, may be transferred to a 15 ml tube that holds a buffer solution, which may be referred to as the STC in embodiments of the present disclosure. This transfer process may be performed at the clinic. Once the tissue sample is placed in the STC, it may be transferred from the clinic to a laboratory.

[0034] Panel 702 shows the hinged funnel design of the STC according to one embodiment of the present disclosure. The STC can be used to aid in the transport of samples from the clinic to the laboratory. The STC can fit inside a container such as a standard 5 ml, 15 ml, or 50 ml tube containing a liquid for transporting the tissue, such as buffer, media, or PBS. The tissue may be dropped into the liquid and allowed to settle to the bottom of the removable tissue retaining strip. In the case of biopsy needles, they can be swirled in the solution to rinse the buffer from the needle into the solution. The tissue retaining strip may be formed from two pieces. They may be hinged, snapped together, and / or held together by a third part, such as a cap fitting or whirl of the transport container in an embodiment of the present disclosure. When the sample is ready for use, the tissue retaining strip can be pulled out of the transport container or tube. The retaining strip can then be pulled apart leaving the tissue on one part of the retaining strip. At this point, the tissue can be processed. In one embodiment, the portion of the retaining piece containing the biopsy sample can be inverted on the molten agarose bed or swirled inside a chamber containing agarose or other embedding material. The retaining piece can be in multiple shapes. In one embodiment, the retaining piece can be a funnel shape that tapers toward the bottom. However, the retaining piece can be a straight cylinder, triangle, rectangle, or other cross-sectional shape in other embodiments of the present disclosure. The retaining piece can have a mesh, filter, small holes, or porous material at the bottom so that when it is lifted from the transport container, the liquid solution can flow out or be sucked out by putting an absorbent material on the other side. The tissue is still held by the retaining piece.

[0035] Panel 703 shows the transfer of a biopsy from an STC to agarose in an embodiment of the present disclosure. Panel 704 shows a biopsy embedded in agarose in an embodiment of the present disclosure.

[0036] Generally, when a biopsy is performed, a technician places the needle in a buffer solution in a 5ml, 15ml, or 50ml tube and swirls it to flush the biopsy from the biopsy needle. An embodiment of the present disclosure may fit inside these tubes, leaving room for the technician to swirl the biopsy needle in the buffer solution and then rely on gravity to allow the biopsy to settle into the funnel / mesh. In some cases, such as with adipose tissue, the biopsy may float. However, in these situations, when the mesh / funnel is withdrawn, the liquid drains through the bottom and the tissue can be drawn into the funnel for downstream handling. The STC may fit inside a 15ml tube, which may be constructed from polypropylene (PP) plastic, and the biopsy needle may be inserted to release the biopsy into the buffer solution. The biopsy may settle into the tip of the hinged funnel piece. Once the sample is ready to be embedded, the hinged funnel can be withdrawn from the 15ml tube and excess liquid can be aspirated out. The funnel may be hinged so that the biopsy may be released and seated in one half of the funnel (FIG. 7, panel 702). The half containing the biopsy sample may be inserted inside a 5 mm sample holder containing melted agarose (FIG. 7, panel 703). The tip of the STC containing the tissue sample may be swung inside the melted agarose to allow the tissue to separate from the plastic funnel and become embedded in the agarose. After the tissue sample is in the agarose, the sample holder may be cooled to solidify the agarose, resulting in a fully embedded tissue sample or biopsy (FIG. 7, panel 704). It should be understood that tissue samples other than biopsies, such as surgical resections, may be shipped in an STC and receive many of the same benefits of less damaging transport and easier handling and processing in the laboratory or processing facility.

[0037] 8A-8B show a slicing and plating method according to an embodiment of the present disclosure. In an embodiment of the present disclosure, the slicing setup may be submerged in a bath so that the tissue may be sliced ​​from the top down. In another embodiment of the present disclosure, the tissue may be sliced ​​from the bottom up and pushed with a plunger, which may make a buffer bath impractical. Rather, drop lubrication may be used. In FIG. 8A, the capture and transport may be performed with a pump, which may gently aspirate as the slices come out of the slicer so that the slices are sorted and placed into the well plate. In this embodiment, slicing may be performed from the top down or bottom up. In FIG. 8B, the tissue may be captured by lifting the insert or capture container to capture the sample and gently trapping it on the inner portion of the insert. In this embodiment, slicing may be performed from the bottom up.

[0038] 8C illustrates a method for tissue slicing according to an embodiment of the present disclosure. Tissue slices may be captured by generating a flowing current of a buffer that can direct the tissue slices into a channel, filter, or collection vessel, where they may be collected using a suction pump or mechanical capture.

[0039] FIG. 9 illustrates a method for tissue slicing and placement, according to one embodiment of the present disclosure. The tissue sample may be released from the STC and embedded in agarose inside the smooth inner layer of the sample holder. The embedded core may then be placed inside the sample holder, which may be clamped onto the ATSH. The sample holder may be mounted on a bracket 902, and a linear actuator indexes the sample in increments of 200 microns to 400 microns with a disposable plunger 901. This assembly may be positioned to allow the vibrating blade to move forward and cut the tissue section. A robotic arm 903 holding the filter assembly / well plate may be positioned below the blade assembly 905 and sample holder 904. A buffer solution may be dripped to facilitate surface tension capture of the section, lubricate the section, and keep the exposed tissue moist. The filter assembly may be contained within a modified tissue culture well.

[0040] FIG. 10 illustrates the workflow from STC to placement in tissue culture plate according to one embodiment of the present disclosure. The tissue sample may be deployed in a 15 ml tube containing the STC. The STC 1001 may have a hinged design that may keep the tissue sample intact and align the pieces towards the tip. The tip of the STC may be inserted into the agarose filled core and swiveled to release and embed the sample. The embedded sample may be placed inside the second part of the sample holder 1102 to prepare it for slicing. The sample holder may be mounted on a bracket and a linear actuator may index the sample in increments of 200 microns to 400 microns using a disposable plunger. This assembly may be positioned to allow the vibrating blade to move forward and cut the tissue section. A robotic arm holding the filter assembly may be positioned below the blade and sample holder. A buffer solution may be dispensed in drops to facilitate capturing the section with surface tension. After capturing each section, it may be placed in a well (96 well plate). The plate may then pass through a liquid handling system and other processing steps.

[0041] FIG. 11 illustrates an ATSH according to one embodiment of the present disclosure. A linear actuator 1101 may be used to index sections of 200-400 microns thickness by moving a plunger 1102 downward with a resolution of 0.003125 mm. A robotic arm 1105 may hold a filter assembly 1106 used to capture the sections as they are cut using a blade assembly 1104 from a specimen holder 1103. The ATSH may include a tissue culture plate into which the filter assembly 1106 containing the tissue sections may be inserted. The wells of the tissue culture plate may be pre-filled with media. The well plate may be used to hold the sections that are held within the filter assembly and sent for further processing of liquid handling and incubation cycles. Although not shown, an infusion pump, syringe pump, or other type of pump may dispense a buffer solution dropwise onto the slicing surface to keep the exposed portion of the sample wet and lubricate the slicing process. This may also help capture the sections in a drop of buffer solution into the filter assembly.

[0042] The automated tissue handling and slicing described in these embodiments may eliminate steps that must be performed when the process is done manually. An automated means of handling the sample after it has been sliced ​​may be provided. As described herein, this may eliminate steps that must be performed when the process is done manually. Additionally, as described herein, single-use disposable elements may be used in the automated methods according to embodiments of the present disclosure. These single-use disposable elements may help avoid cross-contamination from one sample to the next, making sample processing easier and eliminating the currently time-consuming process of taking the slice assembly apart and wiping everything down with alcohol. And, facilitate the sale of the entire instrument or automated tissue slicer and handler at a lower price while selling the single-use components as a package (all single-use components needed to process one sample) or individually at a price that generates a continuing profit revenue stream.

[0043] 12A-12C show a system architecture for handling processing after tissue slicing and handling is completed in an embodiment of the present disclosure. FIG. 12A shows a system architecture for handling processing after tissue slicing and handling is completed, including a robot 1205, a fluorescent imager 1206, a cell culture incubator 1207, a BSC class A2 / B2 1208, a tissue slicer 1209, a cold block 1210, an I / O 1211, and / or a HEPA laminar flow 1212. FIG. 12B shows a configuration in which there is a central robot that moves samples between different components of the automation. As shown herein, one or more robot controllers may be utilized in conjunction with one or more application controllers that communicate with an automation panel and / or information management system via one or more interfaces to operate the robot, components of the robot such as robot grippers, field devices, barcode readers, and / or safety devices. FIG. 12C depicts a linear configuration in which samples are moved linearly back and forth between different components of the system, including primarily an incubator 1201 , an automated pipetter 1202 , a plate reader 1203 , and / or a reagent handler 1204 .

[0044] It should be understood that in the embodiment described in Figures 9 and 10, the sample may be transferred from the slicing operation to the tissue holding container through the use of gravity. The slicing operation is not lubricated and the sections will fall off on their own or be knocked off with the slightest tap or agitation. Although the benefits of lubrication may not be provided in this embodiment, the sections may still be transferred in a controlled manner.

[0045] In alternative embodiments, liquid can be added so that it can drip into the holding vessel. Liquid may need to be removed from the excess fluid used from the transport mechanism, but the sections can still be transported in a controlled manner. A vacuum can be used to suck up the sample on a surface, then transport the section to another area, then positive pressure can be used to release the section and push it through the positive pressure into the holding vessel. Transport can be done in a variety of ways, including but not limited to the use of a robotic arm and / or a moving conveyor belt. The robotic arm can include a vacuum at the end, and the moving conveyor belt can include a center section that pulls the vacuum as it moves.

[0046] In some embodiments where sections are cut from the top of the sample and the sample including the cut surface is submerged in the medium, a tray may be provided on the far side of the sample with mesh walls and a mesh bottom, so that as the sections are released they are pushed into the mesh area by the cutting or slicing action characteristics. The blade can then be tilted and lifted out of the medium leaving the sections in the mesh (like a net with some rigidity), and the net can then be lifted in an automated fashion.

[0047] There can be other embodiments in which the sections are pushed into a known area and then lifted out of the media bath and picked up by a vacuum, or the lifting surface is mobile and deposits the sections elsewhere. In some embodiments, the sample can be placed on its side and sliced ​​vertically with the sections coming out of the end of the sample. As they fall off, the sections can fall onto a surface in a domino-like fashion, which can then be used to transport the sections to a new location, or the surface onto which the sections fall can be the filter assembly itself. The surface can be porous to allow lubricating fluid to pass through or to wick up, leaving primarily the tissue sections on top.

[0048] In other embodiments, a shallow cap may be provided that is slightly deeper than the thickness of the section. The cap may be positioned at the bottom of the exposed sample to be sliced, leaving a gap large enough to allow the blade to go between it and the sample. Thus, when the blade finishes slicing, the section is inside the cap and can be moved to the side or lowered to transport the section for later processing. The cap configuration may be a filter assembly with sides large enough to capture the section. The cap may contain liquid inside to eliminate the need to add fluid to the bottom of the section during the slicing operation, or may have holes or gaps in the walls large enough to allow fluid to flow into the cutting area but small enough to prevent the section from exiting.

[0049] Cutting can be performed by a blade that oscillates back and forth in the X direction and then advances in a direction perpendicular to the X direction. The XY plane may be oriented substantially perpendicular to the sample. In some embodiments of the present disclosure, a rotating blade can be used. The blade can be made from one or more materials, including, but not limited to, steel, other metals, obsidian (volcanic glass), or other similar materials.

[0050] Some embodiments of the present disclosure may use a flow current of the medium as described herein. The net at the end where the sample enters may be at the end of a robot or part of a moving system and can then be used to transport the sample.

[0051] Further embodiments of the present disclosure may include a vision system that may direct a net or other restraining structure to pick up the floating sections after they are released from the top of the sample when slicing is performed in a configuration similar to that currently performed manually, slicing from the top and immersing the sample in medium. The agarose may be dyed or colored to make it easier to see when floating in the medium.

[0052] Other embodiments may include a small cap to cover the top of the sample that may be used while slicing the sample from the top down while the sample is submerged in the medium. The cap may have a slight suction and may be positioned so that the blade can go under the cap. The suction may draw the sample into the cap as it is sliced ​​and be removed upon completion of the slicing step and transported to another location.

[0053] Although the present disclosure and its advantages have been described in detail, it should be understood that various changes, substitutions, and alterations can be made herein without departing from the spirit and scope of the present disclosure as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the processes, machines, manufacture, compositions, means, methods, and steps described herein. As one skilled in the art will readily appreciate from this disclosure, any currently existing or later developed process, machine, manufacture, composition, means, method, or step that performs substantially the same function or achieves substantially the same result as the corresponding embodiments described herein may be utilized in accordance with the present disclosure. Thus, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions, means, methods, or steps.

Claims

1. 1. A system for processing a live tissue sample, comprising: a sample holder configured to receive the live tissue sample; a bracket configured to receive the sample holder; an indexing mechanism configured to move the live tissue sample in precise increments; a blade operatively connected to the sample holder and configured to cut the live tissue sample; A system comprising:

2. The system of claim 1 , wherein the indexing mechanism comprises a linear actuator comprising a plunger configured to move the live tissue sample toward the blade.

3. The system of claim 2 , wherein the live tissue sample is moved between about 200 micrometers and about 400 micrometers.

4. 10. The system of claim 1, further comprising a cutting surface configured to receive a controlled amount of fluid sufficient to lubricate the cutting surface and the raw tissue sample, and wherein the indexing mechanism is operably connected to the cutting surface.

5. The system of claim 4 , wherein the fluid is provided in the form of a mist having at least one droplet.

6. 5. The system of claim 4, further comprising a receptacle operably connected to the sample holder, the receptacle configured to receive a fragment of the raw tissue sample after the fragment has been severed by the blade.

7. The system of claim 6 , wherein the receptacle is positioned in contact with the fluid.

8. The system of any of claims 1 to 7, wherein the system further comprises a user interface and the device is configured for automated operation with no more than one of the live tissue samples.

9. 1. A method for processing a live tissue sample, comprising the steps of: providing a well plate comprising a filter assembly, the well plate being operably connected to a blade; cutting the raw tissue sample with the blade; obtaining a fragment of said live tissue sample; A method comprising:

10. contacting the raw tissue sample with a fluid configured to produce droplets of the fluid sufficient to lubricate the raw tissue sample; contacting the droplet with the filter assembly; contacting the fragment of the live tissue sample with the droplet that has been contacted with the filter assembly; The method of claim 9 further comprising:

11. 11. The method of any of claims 9 to 10, further comprising the step of providing a user interface operatively connected to a device configured to perform said steps of the method in an automated operation involving no more than one said live tissue sample.

12. 1. A system for processing a live tissue sample, comprising: A funnel-shaped structure, A tube filled with buffer solution Equipped with the funnel-shaped structure is configured for insertion into the tube and transport of the live tissue sample; system.

13. The system of claim 12, wherein the tube is sized for a volume of about 5 ml to 50 ml.

14. The system of claim 12 , wherein the funnel-shaped structure is a thin plastic mesh.

15. The system of claim 12 , wherein the funnel-shaped structure is solid, formed of plastic, and configured to separate and recombine into two portions.

16. The system of claim 15 , wherein the two portions are hinged.

17. 16. The system of claim 15, wherein the funnel-shaped structure has an opening at a tapered portion of the funnel-shaped structure, the opening configured to allow a buffer solution to flow out or be sucked out while leaving the live tissue sample within the funnel-shaped structure.

18. 1. A system for processing a live tissue sample, comprising: At least one filter assembly, A filter, a ring connected to an outer portion of the filter, the ring being configured to allow the filter to float on the surface of a fluid; At least one filter assembly comprising: A system comprising:

19. 20. The system of claim 18, wherein a portion of the ring in contact with the filter is hydrophilic and an opposing portion of the ring is hydrophobic.

20. 20. The system of claim 18, wherein the filter has a pore size of about 2 μm to about 10 μm.

21. The system of claim 18 , wherein the ring is formed of a biologically inert plastic.

22. 20. The system of claim 18, wherein the ring is formed from a mixture of biologically inert waxes.

23. The system of claim 18 , wherein the ring is a wrapping film / tape.

24. 1. A system for processing a live tissue sample, comprising:

1. A well plate insert comprising: At least one column; a semi-permeable filter attached to the bottom of each column, the semi-permeable filter configured to maintain an air-liquid interface for the live tissue sample after it is loaded into the column; A well plate insert comprising: Equipped with and after the well plate insert is placed into the column having liquid at the bottom, the well plate insert is configured to remove medium without flooding an inner chamber of the well plate insert in which the live tissue sample is located. system.

25. 25. The system of claim 24, wherein the semi-permeable filter has a pore size of about 2 μm to about 10 μm.

26. 25. The system of claim 24, wherein particulates and / or nutrients within the medium migrate upwardly through the semi-permeable filter without allowing the medium to enter an air chamber within the well plate insert.

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