tissue separation

Abstract

The present invention relates to a sample processing bag 10 for use in a tissue separation device 200 for tissue separation within the sample processing bag 10 . The clamp assembly 60, having the clamp mechanism 72 and the base support 70, is configured to hold the sample processing bag 10 adjacent to the base support 70 during a tissue separation operation so that the action of the tissue separation device 200 is performed on the sample processing bag 10 to separate any tissue within the sample processing bag 10.

Description

Technical Field

[0001] The present invention generally relates to the use of an improved configuration and method for the separation of cell samples in a closed space.

Background Art

[0002] In various fields of medicine and biology, it is necessary to collect tissue samples and separate cell samples into cell aggregates and single cells for further processing. The number of applications for the acquisition and separation of cell samples is enormous and may include cell extraction, for example: a) “Primary cells” can be extracted from tissues such as the liver, and “primary cells” can be used for downstream applications such as single-cell cytometry or single-cell flow cytometry and assays generally referred to as high-throughput screening. b) Tumor infiltrating lymphocytes (TILs) may be extracted from tumor tissue and used based on autologous cell therapy. c) Umbilical cord tissue may be used for the extraction of mesenchymal stem cells. d) Tumors can be excised and tumor cells can be analyzed for neoantigens. e) Tissues can metastasize and cells can be observed. By doing so, so-called cell multi-omics (e.g., proteomics, genomics, epigenetics) can be investigated for many purposes including personalized therapy.

[0003] In many applications, it is desirable to maintain as many healthy cells as possible and to maintain a clean and aseptic state.

[0004] In this application, terms such as sealed, aseptic, or sterile mean that the biological material is separated from the surrounding environment, but does not necessarily mean that there is no bioburden or other contamination, simply that there is not much bioburden or other contamination, and if any, it does not have a significant impact on the usefulness or survival rate of the separated substance.

[0005] From International Publication 2018 / 130845, a technique for tissue separation of cells is known. Similarly, U.S. Patent Publication 6439759 is known as a technique for tissue separation of cells, and the configuration considered in U.S. Patent Publication 6439759 describes a mixing apparatus that includes an internal baffle to assist in mixing sealed bags of material without temperature control.

[0006] To address the shortcomings of the aforementioned prior art, International Publication 2020 / 177920 was also developed with the aim of separating cells by considering more parameters than previously considered, and also to improve the performance of the separation, freezing, and thawing processes.

[0007] To avoid misunderstanding, all the contents of the previously mentioned documents, International Publication 2018 / 130845, U.S. 6439759, and International Publication 2020 / 177920, are also incorporated herein by this reference to the maximum extent permitted. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] International release 2018 / 130845 [Patent Document 2] U.S. 6439759 [Patent Document 3] International release 2020 / 177920 [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] However, prior art still has various shortcomings, particularly regarding the need to process multiple tissue samples sequentially under substantially identical processing conditions in order to provide comparable samples for further analysis. [Means for solving the problem]

[0010] Therefore, the present invention is defined and provided by the appended claims.

[0011] Accordingly, the present invention provides a sample processing bag for tissue separation in a sample processing bag and for use with a crushing device, comprising a clamp assembly that is detachably combined with a crushing device, and at least one leg for crushing the sample processing bag. The clamp assembly comprises a clamp mechanism configured to hold the sample processing bag adjacent to a base support during a tissue separation operation such that at least one leg assembly acts on the sample processing bag to separate any tissue in the sample processing bag, and a base support configured in conjunction with the clamp mechanism. [Effects of the Invention]

[0012] By providing the clamp assemblies and sample processing bags described above, various embodiments of the present invention facilitate, for example, better handling of multiple separated samples, or the separation and processing of multiple separated samples that occur after separation under substantially identical conditions. [Brief explanation of the drawing]

[0013] The present invention will now be described in more detail with reference to the attached drawings. [Figure 1] Figure 1 shows a front view of a crushing device for separating cells into cell aggregates or individual cells within a sample containment section, and the crushing device may be used in various embodiments of the present invention. [Figure 2] Figures 2A to 2C show an embodiment of a sample storage section for use in conjunction with the apparatus shown in Figure 1. [Figure 3] Figure 3 shows an embodiment of a sample housing clamp assembly for use in conjunction with the crushing device apparatus of Figure 1 and / or the sample housing of Figures 2A to 2C. [Figure 4] Figures 4A and 4B show sample processing bags held in place by a clamp assembly according to various embodiments of the present invention. [Modes for carrying out the invention]

[0014] The embodiments of the present invention will be described in detail below. Figure 1 shows a front view of a crushing device 200 for separating cells into cell aggregates or individual cells within a sample containment section. The crushing device 200 can be used in various embodiments of the present invention.

[0015] In this embodiment, the sample containment section comprises a flat and relatively thin sample processing bag 10 that is generally sterile, at least initially. The crushing device apparatus 200 includes a housing 210 formed from an assembly of components that may be detachably inserted into a temperature control device, such as a controlled rate of temperature change, like freezing, thawing, or heating, a commercially available device known as Via Freeze®, for example, commercially available from Life Science's Cytiva®.

[0016] In reality, the housing 210 includes a cover and is not shown in Figure 1. When in use, the crushing device 200 and the sample processing bag 10 provide a sealed system for separating cells within the system (animal or human cells, such as needle biopsies). The resulting cell suspension does not require the separated sample to be moved out of the sample processing bag 10 and can be frozen for subsequent analysis while the cells are suspended.

[0017] The crushing device 200 includes a crushing mechanism 220. Two crushing legs 234, 236 are powered by a 24-volt DC electric motor 214 that has a rotary encoder to provide feedback to a controller for monitoring and controlling the speed of the crushing operation, and periodically and alternately perform the crushing operation. The DC electric motor 214 drives a camshaft 224 through a toothed belt 222. The camshaft 224 includes a set of cams 230, 232 with an offset angle of 180 degrees. In this case, each cam draws a cycloid shape to provide a single oscillation of the cam follower. Each cam 230, 232 is elastic and can move an assembly of cam followers, including follower wheels 225, 227 that move in联动, spring followers 226, 228, and the axes 221, 223 of the follower wheels that are in a relationship of transmitting power to the spring followers 226, 228, so that they overlap the outer shape of the cam.

[0018] Each carriage 226, 228 slides on a linear guide 229, and each leg 234, 236 is connected to the carriage. When each cam of each cam follower assembly rotates against the driving force of a return spring 231 by a motor, the leg moves away from the crushing condition and is pushed up in turn by one of each follower wheel while riding on the contour of the cam. When the cam further rotates and the contour of the cam retreats, the spring 231 integrated with each follower assembly applies a downward force to the leg and the assembly to apply a crushing force. As a result, the crushing force is limited by the spring constants of the related follower assembly and the spring 231, and is not limited by the force of the driving motor.

[0019] It should be noted that "联动" in the original text seems to be an incorrect or unclear expression. It is translated as "联动" here for the time being, and it may need to be further corrected according to the actual situation.The force applied to the bag is limited by spring 231 during use because the crushing mechanism moves legs 234, 236 upward and the spring pushes legs 234, 236 back downward. This satisfies the following: a. The motor cannot move (regardless of the size or tissue of the tumor). b. The sample is not compressed by excessive force and the bag does not split. c. The maximum pressure applied to the bag is lower than the pressure tested during bag manufacturing. d. The receiving area of the bag connected by the hinge can receive the sample bag and any clamp used without pre-positioning legs 234, 236. In other words, since the sample area connected by the hinge is closed with respect to legs 234, 236, when receiving bag 10, legs 234, 236 can take any position, and when the area connected by the hinge to legs 234, 236 is closed, any sample can be compressed by legs 234, 236 as needed.

[0020] The crushing device 200 may further include a flexible sealing membrane extending from the device housing 210 to the upper part of the two legs 234, 236. Also, the flexible sealing membrane provides a dust seal between the fluid resistance and the bottom of legs 234, 236 and the rest of the crushing mechanism 220. Such a structure suppresses the contamination process, so the compressed bag 10 must be separated during use. While the use of the membrane is preferred, there is a risk that the legs slide over a seal such as a lip seal attached to a partition separating the bag area and mechanism 220, thereby obtaining a similar mechanism that should be required to suppress the contamination process.

[0021] The crushing device 200 further includes a heat transfer plate. This heat transfer plate is connected by a hinge to one side of the housing 210, making it easier to insert and remove the bag 10 to be crushed. The heat transfer plate may include a temperature sensor capable of monitoring and recording the temperature of the receiving area of ​​the bag and plate, such as a controller, including a quality controller.

[0022] Each leg 234, 236 can be adjusted in height relative to the heating plate of the crushing device 200, and its operation can also be monitored by the controller. Therefore, even if the rotary encoder indicates that the motor is rotating, mechanical failures such as a malfunction of the toothed belt 222 can still be detected by the controller, and appropriate actions such as issuing a warning can be taken.

[0023] The crushing device 200 is further formed such that the housing 210 can slide into a control-rate freezer (not shown) having a freezer lid in place.

[0024] Figures 2A and 2C show embodiments of a sample container for use in conjunction with the crushing device apparatus 200 of Figure 1. More specifically, the sample container may comprise a sample processing bag 10. The sample processing bag 10 comprises a plurality of separate chambers 12 within the sample processing bag 10. The processing bag 10 is preferably made of a polymer material. In various embodiments, it is advantageous that the sample processing bag 10 can have contents inside and be heat-sealed at various positions.

[0025] Figure 2A shows a plan view of the sample processing bag 10. Figure 2B shows a view of the sample processing bag 10 along the direction indicated as A in Figure 2A. Figure 2C shows a view of the sample processing bag 10 along the direction indicated as B in Figure 2A.

[0026] Each chamber 12 is accessible through an opening 14 in each bag and is also connected to an access port 16. The opening 14 should be sufficiently large; for example, a diameter of approximately 10 mm or more is preferable to accommodate, if necessary, samples that have been broken into small fragments and inserted into the chamber 12 by syringe. The opening 14 may be sealed with a heat seal once the sample has been introduced into each chamber 12. During use, various materials (e.g., samples and reagents such as separating enzymes and separation samples) may be inserted into and / or extracted from the chamber 12 via the access port 16, and the access port 16 may be further sealed when not in use.

[0027] The sample processing bag 10 generally has a flat structure, and in various embodiments, several additional measures are provided to allow tissue samples to fit inside the sample processing bag 10, so the sample processing bag 10 may have a thickness of up to 12 mm. The structure for one sample processing bag 10 may use two layers of polymer material (e.g., ethylene vinyl acetate (EVA)) sealed at each point around the chamber 12.

[0028] In this embodiment, the three independent chambers 12 are provided within the polymer material between each chamber 12 and can be further separated independently of each other by separating the chambers 12 along perforations or precuts. For example, the precuts 18 may be provided as a single elongated cut, or as a plurality of precuts provided between the chambers 12 that are substantially in the same straight line, whether or not the perforations provided between the chambers 12 are aligned.

[0029] Each chamber 12 may be adapted to process tissue samples having a mass of 1 gram or less (compared to previous designs where the tissue sample mass was 16 grams or less). Therefore, the minimum volume of the minimum number of chambers required may be as little as 2 ml (compared to a minimum volume of 5 ml in various well-known systems). Thus, compared to conventional systems, it is possible to process smaller samples requiring fewer reagents, etc., and smaller samples require fewer initial biopsies or similar, resulting in fewer demands on consumables.

[0030] Another advantage of providing independently separable chambers 12 is that the user can choose one or more chambers 12 to use at any point when processing the contents of the chambers 12, thus providing flexible processing. Furthermore, the contents of each chamber 12 can be processed under substantially identical conditions (temperature, freezing, or crushing time, etc.), and the chambers 12 are separable after processing. Subsequently, if necessary, one or more separable chambers 12 may be processed and stored for future reference, so that the user can be confident that the results of subsequent processing are based on substantially the same crushed initial sample.

[0031] Figure 3 shows an embodiment of a sample containment clamp assembly 60 for use with the crushing device apparatus 200 of Figure 1 and / or the sample processing bag 10 of Figures 2A to 2C.

[0032] The sample processing bag 10 can be secured to the clamp assembly 60, for example, either before or after heat sealing. Once the sample processing bag 10 is secured to the clamp assembly 60, it can be placed inside the crushing device 200 to separate any tissue within the sample processing bag 10. For example, the leg assemblies 234 and 236 of the crushing device 200 may act on the sample processing bag 10 to separate any tissue within it.

[0033] The clamp assembly 60 comprises a base support 70 and a clamp mechanism 72 used in conjunction with it to hold a sample processing bag 10 adjacent to the base support 70 during tissue separation operations. The clamp assembly 60 comprises an upper bar 62 and a lower bar 64, which can be fixed together by a pair of screws 66. The lower bar 64 is located at the distal end 78 of the base support 70 and may optionally be integrally formed with the distal end 78. When the screws 66 are tightened, the upper bar 62 and the lower bar 64 are restricted to contact. A portion of the sample processing bag 10 may be held between the upper bar 62 and the lower bar 64 by the upper bar 62 and the lower bar 64.

[0034] Furthermore, the base support 70 has a raised end 74 for supporting at least one access port 74 of the sample processing bag 10 on the base support 70. In this embodiment, three recesses 76 are provided on the raised end 74.

[0035] Any or all of the base support 70, clamping mechanism 72, and / or screw 66 may be formed from a polymer material. For example, polyamide may be used. Any or all of such polymer materials may be formed using a wide range of techniques, such as selective laser sintering (SLS): additive manufacturing techniques such as molding, casting, and processing.

[0036] The upper bar 62 has a tapering recess (not shown), and when the upper bar 62 and lower bar 64 are tightened, the upper bar 62 is positioned in a shape 61 that is supplementally wedge-shaped. The recess and wedge concentrate the clamping force at the apex of the wedge-shaped configuration 61, providing a higher clamping force than that which could be obtained by a flat mounting surface. For greater clamping force, the wedge-shaped configuration 61 further has a small channel 67 at the apex of the wedge-shaped configuration 61, and in the upper bar 62, the small channel 67 contacts the upper bar 62 by the use of a supplemental raised configuration (not shown). A heat seal may be provided for additional safety, but the clamping force may be sufficient to negate the need for a heat seal in the sample processing bag 10. The clamping force is further enhanced by the thickness and rigidity of the upper bar 62 and lower bar 64, which do not easily bend, so that the clamping force acting by the screw 66 is sustained.

[0037] During use, the sample processing bag 10 can be placed on the clamp assembly 60 such that it is positioned below the two legs 234, 236, and when placed in this manner, it can slide into the receiving area of ​​the crushing device 200. The sealed sample processing bag 10 may be supplied with tissue suspended in an aqueous solution, and the tissue may contain digestive enzymes such as collagenase and protease that promote the breakdown of the tissue previously introduced into the sample processing bag 10 via the opening 14.

[0038] The sample processing bag 10 may be placed in close proximity to the electric heating plate, or it may be heated from an external heat source up to approximately 35°C to accelerate the rate of tissue degradation. Furthermore, a single sample processing bag 10 may be used, and if necessary, digestive enzymes can be introduced through one or more access ports 16 of the sample processing bag 10 during or before separation.

[0039] The heat transfer plate may be used to transfer thermal energy into the sample processing bag 10 by heating the underside of the heat transfer plate in order to supply a desired temperature in the sample processing bag 10 for enzymatic action. The heat transferred to the sample processing bag 10 can be conveniently provided by an electrically heated heating plate, or by an electrically heated element on or within the heat transfer plate.

[0040] The amount of separation depends on a vast number of parameters, such as the size, density, and elasticity of the initial tissue sample, and therefore the separation time and crushing speed vary considerably. Crushing that is too long or too violent can lead to a decrease in cell viability. Therefore, the motor unit speed and separation cycle can be controlled.

[0041] One option is to determine the process time according to a lookup table that includes the power rate and time required to separate similar samples. Another option is to measure the power and instantaneous power over the time required to perform the separation process, or measure the force or pressure on another part of the heat transfer plate or clamping mechanism, or stop the separation process after a predetermined threshold is reached, which may indicate that the samples have been sufficiently separated. As the power / force / pressure decreases, the separation is getting closer to completion.

[0042] Another option is to measure the light absorption passing through the sample processing bag 10: the greater the absorption, the closer the sample is to completing the separation. Once the separation is complete, the contents of the sample processing bag 10 can be moved, and the cells or other components of interest can be separated or frozen in the crushing device 200 and kept in the fresh sample processing bag 10. Alternatively, all separated material can be left in the sample processing bag 10 (or one or more separate chambers 12) and then frozen. Therefore, an antifreeze may be introduced into the chamber 12 to be frozen via the access port 16.

[0043] While the sample processing bag 10 is being cooled, in order to control ice formation and avoid supercooling of the sample, the sample processing bag 10 can be kneaded by the legs 234 and 236 in the manner described above, albeit at a slower rate than separation, in order to control ice nucleation and thereby increase the viability of cells after thawing.

[0044] If necessary for use, the separated frozen sample in the sample processing bag 10 can be rapidly thawed by further external heat from the heat transfer plate and / or by partially immersing the crushing device 200 in a warmed water bath maintained at 37°C, after which the antifreeze is removed. In either case, while the sample processing bag 10 is thawing, it can be kneaded by the legs 234, 236. If enzymes are still present, they can be removed, if necessary, by filtration, as well as the removal of the antifreeze. Generally, enzyme activity stops at low temperatures, so during low-temperature storage, enzymes have little to no effect on cells.

[0045] All processing operations, such as heating, separation, cooling, freezing, and subsequent thawing, may occur within a similarly sealed sample processing bag 10, or they may be performed within a single device. Performing processing operations within a single device not only provides efficient time and space, but also allows a single record to capture everything that happens to the sample during the process, such as temperature, cycle, separation rate, and freezing protocol, reducing the chance of errors such as the sample spending excessive time in an uncontrollable environment between processing devices.

[0046] Furthermore, since the sample processing bag 10 is attached to the clamp assembly 60, the sample processing bag 10 can be easily removed from the crushing device 200, which in turn makes it easier to handle and clean the samples after use of the crushing device 200.

[0047] Figures 4A and 4B show the sample processing bag 10, which is held in place by a clamp assembly 60, as in various embodiments of the present invention.

[0048] The sample processing bag 10 is heat-sealed at its end 20 after each sample is added to its respective chamber 12 via the access port 14. In this case, the sample processing bag 10 comprises three chambers 12, most of which are along the entire length of the chambers 12 and separated by each pre-cut 18 extending between the chambers 12.

[0049] Once the sample processing bag 10 is heat-sealed, the sample processing bag 10 is fixed in close proximity to the end 20 at the distal end 78 of the base support 70. The sample processing bag 10 is held in a cavity formed in the base support 70 of the clamp assembly 60, and the clamp mechanism 72 is used to secure the sample processing bag 10 to the distal end 78. Subsequently, each access port 16 of the sample processing bag 10 is positioned in each recess 76 provided in the raised end 74 of the base support 70.

[0050] Therefore, the sealed sample processing bag 10 may be held in the clamp assembly 60, or both can be easily moved, stored and / or inserted into the crushing device 200. Furthermore, the individual chambers 12 may be easily separated from each other, and the individual chambers 12 previously together were in substantially the same processing state.

[0051] Thus, various aspects and embodiments of the present invention have been shown. However, the present invention is not to be considered limited by the embodiments shown above and may be modified within the scope of the appended claims, as will be immediately apparent to those skilled in the art.

[0052] For example, although the embodiments shown herein illustrate a base support including a raised end that supports at least one access port of a sample processing bag, those skilled in the art will understand that alternative modifications are also possible. For instance, a substantially planar base support structure may be provided to have one or more clips supported on the structure of each planar base support for engaging with the corresponding access port. Many other possible modifications are also obvious to those skilled in the art. [Explanation of symbols]

[0053] 10 ···Sample processing bags 12... Chambers 14 ···Opening 16 ···Access Port 60 ··· Clamp Assembly 70 ···Base support 72... Clamping mechanism 74 ···Elevated end 76 ··· recessed area 78 ···Distal end 200... Crushing device 234,236 ···Leg Assembly

Claims

1. A crushing device (200) for separating tissue in a sample processing bag (10), A clamp assembly (60) that is detachably combined with the crushing device (200) for crushing a sample processing bag (10), A crushing device apparatus (200) comprises a base support (70) and a clamping mechanism (72) configured to hold the sample processing bag (10) adjacent to the base support (70) during a tissue separation operation, wherein at least one of the leg assemblies (234, 236) acts on the sample processing bag (10) to separate any tissue within the sample processing bag (10), and the clamping assembly (60) comprises: The sample processing bag (10) has a plurality of separate bag chambers (12) therein, each of which is accessible through a corresponding opening (14) and / or access port (16). Each of the bag chambers (12) is separable and separated by a polymer material with perforations and / or pre-cuts provided between them. Crushing device (200).

2. The crushing device apparatus (200) according to claim 1, comprising a receiving area provided at the bottom of the chassis, having a support plate for supporting the base support (70) of the clamp assembly (60) adjacent to the support plate.

3. The crushing device (200) according to claim 2, wherein the support plate is equipped with an electric heating plate.

4. The crushing device apparatus (200) according to claim 3, wherein the temperature of the heat transfer plate for heating and / or cooling the clamp assembly (60) can be controlled.

5. The crushing device (200) according to any one of claims 1 to 4, wherein the sample processing bag (10) comprises three bag chambers (12).

6. A clamp assembly (60) for a crushing device (200) that separates tissue inside a sample containment section, comprising a base support (70) and a clamp mechanism (72) configured to hold a sample processing bag (10) adjacent to the base support (70), The clamp assembly (60) is detachably attached to the crushing device (200). The crushing device (200) includes at least one leg assembly (234, 236) for crushing the sample processing bag (10), The at least one leg assembly (234, 236) acts on the sample processing bag (10) to separate any tissue within the sample processing bag (10), The sample processing bag (10) has a plurality of separate bag chambers (12) therein, each of which is accessible through a corresponding opening (14) and / or access port (16). A clamp assembly (60) in which each of the bag chambers (12) is separable and separated by a perforated and / or pre-cut polymer material provided between them.

7. The clamp assembly (60) according to claim 6, wherein the base support (70) includes a raised end (74) for supporting at least one access port (16) of the sample processing bag (10).

8. The clamp assembly (60) according to claim 7, wherein the raised end portion (74) has at least one recess (76) inside it.

9. The clamp assembly (60) according to any one of claims 6 to 8, wherein the clamp mechanism (72) is provided at the distal end (78) of the base support (70).

10. A sample processing bag (10) for use with a tissue separation device (200), having a plurality of separated bag chambers (12) inside, each accessible via an opening (14) and an access port (16) connected to the bag chamber (12), The tissue separation device (200) is A clamp assembly (60) that is detachably combined with the crushing device (200) for crushing the sample processing bag (10), comprising at least one leg assembly (234, 236) and the crushing device (200), The device comprises a base support (70) and a clamping mechanism (72) configured to hold the sample processing bag (10) adjacent to the base support (70) during tissue separation operations, and a clamping assembly (60) in which at least one leg assembly (234, 236) acts on the sample processing bag (10) to separate any tissue within the sample processing bag (10), A sample processing bag (10) in which each of the bag chambers (12) is separable and separated by a polymer material with perforations and / or pre-cuts provided between them.

11. The sample processing bag (10) according to claim 10, wherein the opening (14) is heat-sealable.

12. The sample processing bag (10) according to claim 10 or 11, wherein the sample processing bag (10) comprises three bag chambers (12).

13. The sample processing bag (10) according to any one of claims 10 to 12, wherein the sample processing bag (10) is in a sterile state.

14. The sample processing bag (10) according to any one of claims 10 to 13, comprising two polymer material layers sealed at each point around the chamber (12) of the sample processing bag (10).

15. The sample processing bag (10) according to claim 14, wherein the polymer material comprises ethylene vinyl acetate (EVA).

16. A sample processing bag (10) according to any one of claims 10 to 15, configured for use in cryopreservation of separated tissue.

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