Tissue dissociation system and tissue dissociation method

Abstract

Disclosed are a tissue dissociation system and a tissue dissociation method. The tissue dissociation system comprises a tissue dissociator, a culture fluid bag, a sterile culture bag, and a tube welder. Specifically, the method comprises: placing a tissue to be dissociated in a sealed environment formed by a base and a sliding sleeve, and then using the tube welder to connect the culture fluid bag and the sterile culture bag to a dissociation operation chamber of the tissue dissociator; driving a dissociation piston rod to perform dissociation; after dissociation is completed, squeezing the culture fluid bag, such that the remaining culture fluid in the culture fluid bag flushes the dissociation operation chamber, and the dissociated tissue is flushed into the sterile culture bag; and finally using the sterile culture bag for culture. The entire dissociation process and the tissue transfer process after dissociation are both carried out in a closed environment, so that it is not necessary to provide a separate sterile environment for the dissociation process or the transfer process, thus making tissue dissociation more convenient and reducing environmental constraints, and also eliminating the risk of contamination of the dissociated tissue.

Description

Tissue dissociation system and tissue dissociation method

[0001] Priority application

[0002] This application claims priority to the Chinese invention patent application [application number 2023112451186] filed on September 25, 2023, entitled "A tissue dissociation system and a tissue dissociation method", which is incorporated by reference in its entirety. Technical Field

[0003] The present invention belongs to the technical field of primary cell separation, and in particular relates to a tissue dissociation system and a tissue dissociation method. Background Art

[0004] Primary cells are cells directly isolated and cultured from tissues or organs in the body. They are widely used in clinical medicine and treatment. For example, tumor-infiltrating lymphocytes (TILs) are isolated from tumor tissue. TILs are lymphocytes present in tumor tissue, lymph nodes, and other tissues containing metastatic tumors. TILs contain a high proportion of tumor-specific lymphocytes. After in vitro isolation, expansion, and activation, these lymphocytes have been used as a clinical treatment for adoptive cellular immunotherapy of tumors, achieving good clinical results in the treatment of malignant melanoma and other cancers.

[0005] The traditional tissue dissociation method is to transfer the specimens obtained from the clinic (for example, in a sterile operating room) to the laboratory, cut the tissue blocks with sterile scissors in a clean bench, and then chop them into small pieces with scissors or blades to allow the cells to disperse freely before subsequent culture and expansion. This method cannot avoid the risk of exposing tissue blocks, and there are problems of easy contamination and low efficiency. This makes the production of such cell therapy products require high environmental cleanliness and increase costs.

[0006] In order to avoid contamination, it is usually necessary to store tissue samples obtained from a sterile environment (such as an operating room) in a sealed container and transfer them to a laboratory or inspection room. Then, under another sterile operating environment (such as a large sterile operating device), the tissue sample is added to a tissue dissociation device for dissociation, and after dissociation, it is transferred to a culture dish under the sterile operating environment. However, this method requires a separate sterile operating environment, which will undoubtedly greatly increase costs, and not only makes the entire operation process more cumbersome, but also places high demands on the operators; and for some remote areas, or some laboratories or testing institutions or medical institutions with poor conditions, it is impossible to perform tissue dissociation and thus tissue culture because it is impossible to purchase large sterile operating equipment or provide a sterile operating environment.

[0007] Summary of the Invention

[0008] The purpose of the present invention is to provide a tissue dissociation system and a tissue dissociation method, which can reduce the risk of tissue contamination during the dissociation and transfer process by enabling the entire process of tissue dissociation, transfer and culture to be in a fully closed environment.

[0009] In order to solve the above-mentioned technical problems, the present invention specifically adopts the following technical solutions: The first aspect of the present invention is to provide a tissue dissociation system, which includes: a tissue dissociator, a culture medium bag, a sterile culture bag, and a connecting machine for connecting the liquid outlet hose of the culture medium bag and the liquid inlet hose of the sterile culture bag to the tissue dissociator respectively; in the initial state, the free ends of the liquid outlet hose and the liquid inlet hose (i.e., the ends not connected to the culture medium bag and the sterile culture bag) are in a closed state, wherein,

[0010] The tissue dissociator includes: a sliding sleeve, a base that can be detachably connected to one end of the sliding sleeve, and a dissociation piston rod that can move along the axial inner hollow of the sliding sleeve to the base. When the base is connected to the sliding sleeve, the base and the axial inner hollow of the sliding sleeve form a dissociation operation chamber for providing an axial movement path for the dissociation piston rod. The end of the dissociation piston rod close to the base is provided with a dissociation part for dissociating tissue; the dissociation operation chamber is connected to a liquid inlet pipe and a liquid outlet pipe, and the ends of the liquid inlet pipe and the liquid outlet pipe that are not connected to the dissociation operation chamber (or called free ends) are closed.

[0011] In some embodiments of the present invention, the liquid inlet pipe and the liquid outlet pipe are symmetrically arranged on both sides of the dissociation operation chamber, and the liquid inlet pipe and the liquid outlet pipe are inclined so that the extension lines of the center lines of each of the liquid inlet pipe and the liquid outlet pipe pass through the bottom of the dissociation operation chamber.

[0012] In some embodiments of the present invention, a return spring is sleeved on the disengagement piston rod; one end of the return spring abuts against the disengagement piston rod, and the other end abuts against a spring seat in the sliding sleeve.

[0013] In some embodiments of the present invention, a dissociation piston is provided at the end of the dissociation piston rod, and the dissociation portion is installed at the end of the dissociation piston; and the diameter of the dissociation piston is larger than the diameter of the dissociation piston rod.

[0014] In some embodiments of the present invention, a limiting step is provided at the upper end of the dissociation operation chamber.

[0015] In some embodiments of the present invention, an annular sealing ring is provided on the dissociation piston rod; and / or the upper end of the dissociation piston rod extends out of the sliding sleeve and is provided with a pressure plate.

[0016] In some embodiments of the present invention, the sliding sleeve is composed of two detachable upper and lower sections, and the dissociation piston rod is composed of two detachable upper and lower sections; and an elastic sealing sleeve is covered on the lower section of the dissociation piston rod.

[0017] In some embodiments of the present invention, the culture fluid bag includes a first chamber and a second chamber, the first chamber is connected to one end of the liquid outlet hose, and a separation membrane is provided between the first chamber and the second chamber, and the separation membrane can be opened under the action of an external force, so that the first chamber and the second chamber are connected, wherein the second preset volume of the culture fluid pre-stored in the first chamber is smaller than the third preset volume of the culture fluid pre-stored in the second chamber.

[0018] In some embodiments of the present invention, the sealing sleeve is in the shape of an inverted bowl, with its large end connected to the sliding sleeve and its small end connected to the lower dissociation piston rod, so that the connection between the dissociation piston rod and the sliding sleeve is closed.

[0019] The second aspect of the present invention is to provide a tissue dissociation method, which is based on the above-mentioned tissue dissociation system and includes the following steps: separating a base pre-placed in a sterile collection environment from a sliding sleeve, placing the obtained tissue to be dissociated on the base, and then connecting the base with the sliding sleeve so that the axial inner hollow of the base and the sliding sleeve forms a dissociation operation chamber for providing an axial movement path for the dissociation piston; using a connecting machine to connect a culture medium bag and a sterile culture bag to the liquid inlet pipe and the liquid outlet pipe on the dissociation operation chamber respectively, so that the culture medium bag and the sterile culture bag are connected to the dissociation operation chamber; squeezing the culture medium bag so that the culture medium A second preset volume of culture fluid stored in the bag enters the dissociation operation chamber through the liquid inlet tube; the dissociation piston rod is driven to dissociate the tissue to be dissociated, and during the dissociation process, the liquid outlet passage between the sterile culture bag and the dissociation operation chamber is always kept unobstructed; after the dissociation is completed, the culture fluid bag is squeezed so that the remaining culture fluid in the culture fluid bag flushes the dissociation operation chamber and flushes the dissociated tissue into the sterile culture bag; the liquid outlet passage between the sterile culture bag and the dissociation operation chamber is cut off, and the sterile culture bag is separated from the tissue dissociator, and after separation, the liquid inlet hose of the sterile culture bag is in a closed state.

[0020] In some embodiments of the present invention, the step of squeezing the second preset volume of culture fluid in the culture fluid bag into the dissociation operation chamber specifically includes the steps of: squeezing the first chamber in the culture fluid bag along the direction of the liquid outlet hose on the culture fluid bag, so that the second preset volume of culture fluid stored in the first chamber enters the dissociation operation chamber through the liquid outlet hose.

[0021] In some embodiments of the present invention, the step of squeezing the culture fluid bag after dissociation is completed so that the remaining culture fluid in the culture fluid bag flushes the dissociation operation chamber and flushes the dissociated tissue into the sterile culture bag specifically includes the steps of: squeezing the second chamber of the culture fluid bag so that the partition membrane between the first chamber and the second chamber opens, thereby allowing the third preset volume of culture fluid pre-stored in the second chamber to enter the first chamber; squeezing the first chamber multiple times along the direction of the liquid outlet hose so that the third preset volume of culture fluid enters the dissociation operation chamber through the liquid outlet hose to flush the dissociation operation chamber and flush the dissociated tissue into the sterile culture bag.

[0022] In some embodiments of the present invention, the step of squeezing the first chamber multiple times along the direction of the liquid outlet hose specifically includes: squeezing the first chamber so that the culture fluid enters the dissociation operation chamber; oscillating or shaking the tissue dissociator so that most of the tissue particles adhering to the wall of the dissociation operation chamber are detached from the wall and gathered at the bottom of the dissociation operation chamber; placing the tissue dissociator at an angle and squeezing the first chamber multiple times so that the culture fluid flushes the bottom of the dissociation operation chamber under the action of squeezing and gravity, so as to flush the dissociated tissue particles into the sterile culture bag.

[0023] The benefit of the present invention is that a part of the tissue dissociator of the present invention, for example, the base or the base and part of the sliding sleeve (see the embodiment) can be used as a disposable consumable. When in use, the base can receive clinically obtained specimens (tissue to be dissociated) in a sterile environment and immediately connect with the sliding sleeve to form a seal; then the culture medium bag and the sterile culture bag are connected to the originally closed liquid inlet pipe and liquid outlet pipe respectively through the connecting machine, and then the dissociation operation is performed in the dissociation operation chamber formed by the base and the sliding sleeve; and when the tissue dissociation is completed, the dissociation operation chamber is flushed with the culture medium in the culture medium bag, thereby flushing the dissociated tissue into the sterile culture bag. After the tissue transfer is completed, a heat sealer or other measures can be used to seal / block the liquid outlet between the sterile culture bag and the dissociation operation chamber, and the sterile culture bag can be separated from the tissue dissociator at the same time. The hose of the separated sterile culture bag is always kept in a closed state, so that sterile culture can be carried out directly in the culture bag without the need to set up a separate sealed container for culture. The entire process of dissociation and tissue transfer, and even the culture process, are carried out in a closed environment, without the need to provide a separate sterile environment, making tissue dissociation more convenient and reducing environmental constraints, while eliminating the risk of contamination of the dissociated tissue.

[0024] The entire process of dissociating and transferring tissues is carried out in a closed environment, without the need to provide a separate sterile environment. This makes tissue dissociation more convenient and reduces environmental constraints, thereby greatly reducing the requirements for operators and reducing costs. It also enables laboratories, medical institutions and testing institutions in remote areas or with poor economic conditions to perform tissue dissociation and culture. Compared with existing tissue dissociation devices, it is more flexible and has a wider range of adaptability.

[0025] Furthermore, in the present invention, a certain amount of sterile gas is pre-stored in the culture bag (of course, preferably, the volume of gas in the sterile culture bag is smaller than the capacity of the sterile culture bag, specifically, its volume only needs to allow the dissociation piston to move freely in the activity space defined by the sleeve and the dissociation operation chamber), thereby limiting the operating stroke of the dissociation piston in a sealed environment due to insufficient air pressure in the sealed space due to reasons such as misoperation.

[0026] Furthermore, in the present invention, the amount of culture fluid required for different operation processes, such as the dissociation process and the transfer process, is quantitatively stored in the two chambers of the culture fluid bag, thereby avoiding the situation in which too much culture fluid is pressed into the dissociation operation chamber due to unstable manual operation during the dissociation process, thereby weakening the force of the dissociation piston acting on the tissue, resulting in a longer dissociation operation time or even overflow of culture fluid; or too little culture fluid is pressed into the dissociation operation chamber due to excessive force acting directly on the tissue by the dissociation piston, thereby damaging the tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for the embodiments or the description of the prior art. In all drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the various elements or parts are not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without inventive work.

[0028] FIG1 is a front view of a tissue dissociator in a tissue dissociation system according to an exemplary embodiment of the present invention;

[0029] FIG2 is a cross-sectional view of a tissue dissociator in a tissue dissociation system according to an exemplary embodiment of the present invention;

[0030] FIG3 is a perspective view of a tissue dissociator in a tissue dissociation system according to an exemplary embodiment of the present invention;

[0031] FIG4 is a front view of a tissue dissociator in a tissue dissociation system according to another exemplary embodiment of the present invention;

[0032] FIG5 is a cross-sectional view of a tissue dissociator in a tissue dissociation system according to another exemplary embodiment of the present invention;

[0033] FIG6 is a front view of a tissue dissociator in a tissue dissociation system according to yet another exemplary embodiment of the present invention;

[0034] FIG7 is a cross-sectional view of a tissue dissociator in a tissue dissociation system according to yet another exemplary embodiment of the present invention;

[0035] FIG8 is a schematic diagram of the coordination between a tissue dissociator, a culture medium bag, and a sterile culture bag in a tissue dissociation system in an exemplary embodiment of the present invention;

[0036] FIG9 is a schematic flow chart of a tissue dissociation method according to an exemplary embodiment of the present invention.

[0037] Markings in the figure: 1 sleeve, 2 dissociation piston rod, 3 base, 4 liquid inlet pipe, 5 liquid outlet pipe, 6 pressure plate, 7 dissociation piston, 8 dissociation operating chamber, 81 piston activity space, 9 annular sealing ring, 10 return spring, 11 sealing sleeve, 101 upper sleeve, 102 lower sleeve, 201 upper dissociation piston rod, 202 lower dissociation piston rod; 21 first chamber, 22 second chamber, 23 liquid outlet hose, 24 separation membrane; 31 liquid inlet hose. DETAILED DESCRIPTION

[0038] To make the purpose, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0039] Herein, suffixes such as "module," "component," or "unit" used to represent elements are only used to facilitate description of the present invention and have no specific meaning. Therefore, "module," "component," or "unit" may be used interchangeably.

[0040] As used herein, terms such as "upper," "lower," "inner," "outer," "front," "back," "one end," and "the other end" indicate positions or locations based on those shown in the accompanying drawings. These terms are intended solely to facilitate and simplify the description of the present invention and are not intended to indicate or imply that the devices or components referred to must have, be constructed, or operate in a specific orientation. Therefore, they should not be construed as limitations on the present invention. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0041] As used herein, unless otherwise expressly specified or limited, the terms "installed," "provided with," and "connected" should be understood broadly. For example, "connected" may refer to a fixed connection, a detachable connection, or an integral connection; it may refer to a mechanical connection, a direct connection, an indirect connection via an intermediate medium, or internal communication between two components. Those skilled in the art will understand the specific meanings of the above terms in the present invention on a case-by-case basis.

[0042] Herein, "and / or" includes any and all combinations of one or more of the listed items. Herein, "plurality" means two or more, that is, it includes two, three, four, five, etc.

[0043] In this article, "sterile environment" or "sterile collection environment" refers to a clean operating environment with the highest cleanliness level, such as an operating room. In this article, "dissociation operating environment" refers to various clean operating environments with a lower cleanliness level (for example, the clean operating area of ​​a testing room) or a non-clean operating environment (for example, the office of a medical institution or testing institution).

[0044] Chinese patent CN201880042732.3 discloses a tissue dissociator comprising a blade holder having a blade; and a sample holder comprising a tissue actuator having a distal flexible stopper, wherein the tissue actuator is configured to shift along the longitudinal axis within the sample holder. The above-mentioned prior art only proposes a solution for dissociating tissue, and does not include a method for ensuring that the tissue is not contaminated during the dissociation and transfer process. In fact, the above-mentioned technology also needs to be operated in a sterile room.

[0045] In view of the above problems, the present invention provides a tissue dissociator, a tissue dissociation system including the tissue dissociator, and a tissue dissociation method based on the tissue dissociation system.

[0046] Embodiment 1: As shown in Figures 1 to 3, the tissue dissociator in this embodiment includes a sliding sleeve 1, a base 3 that can be detachably connected to one end of the sliding sleeve 1, and a dissociation piston rod 2 that can move along the axial inner space of the sliding sleeve 1, and a dissociation portion for dissociating tissue is provided at one end of the dissociation piston rod 2 close to the base 3; wherein, a dissociation chamber 8 with a top opening is provided in the base 3, and the dissociation chamber 8 is communicated with the axial inner space of the sliding sleeve 1, and a mounting groove is provided at one end of the axial inner space of the sliding sleeve 1 close to the base 3, and when the base 3 is in contact with the sliding sleeve 1, the dissociation portion 8 is provided with a dissociation portion 8 for dissociating tissue. When the sleeve 1 is connected, the top of the mounting groove cooperates with the opening of the dissociation chamber 8, thereby forming a dissociation operation chamber for providing an axial movement path for the dissociation piston rod 2; specifically, the maximum stroke of the dissociation piston 7 with a blade provided on the dissociation piston rod 2 within the dissociation operation space is the difference between the maximum height of the dissociation chamber 8 and the height of the dissociation piston 7 itself; the dissociation chamber 8 (that is, the dissociation operation chamber) is symmetrically connected with a liquid inlet pipe 4 and a liquid outlet pipe 5, and the ends of the liquid inlet pipe 4 and the liquid outlet pipe 5 that are not connected to the dissociation chamber 8 are in a closed state.

[0047] In the present invention, the ends of the inlet and outlet tubes 4 and 5 not connected to the dissociation chamber 8 are originally sealed to create a sealed environment. After the inlet and outlet tubes 4 and 5 are connected to the culture fluid bag and sterile culture bag, respectively, the culture fluid bag and sterile culture bag communicate with the dissociation operation chamber (i.e., the dissociation chamber 8), maintaining a sealed environment even after connection. To achieve this goal, in this embodiment, the inlet and outlet tubes 4 and 5 are plastic hoses (e.g., PVC tubes), which are connected to the culture fluid bag and sterile culture bag via a pipe adapter to achieve communication.

[0048] Of course, it is foreseeable that the culture medium bag and the sterile culture bag should also be equipped with a plastic (such as PVC) connector. For example, a plastic hose (such as a PVC tube) is provided on the culture medium bag as a liquid outlet hose, and a plastic hose (such as a PVC tube) is provided on the sterile culture bag as a liquid inlet hose, which is used to connect with the liquid inlet pipe 4 and the liquid outlet pipe 5 under the action of the connecting machine.

[0049] The connecting device in the present invention is a medical device that connects two pipes (e.g., the inlet pipe 4 on the dissociation chamber 8 and the outlet hose 23 on the culture medium bag; or the outlet pipe 5 on the dissociation chamber 8 and the inlet hose 31 on the sterile culture bag) in a dissociation operating environment (e.g., an environment with a low cleanliness level) and maintains the pipes in a constant state of communication. It mainly uses the principle of heat sealing to connect the two pipes. It is indispensable in the fields of blood collection and supply, clinical surgery, biopharmaceuticals, etc.

[0050] In this embodiment, the sleeve 1 and the base 3 are detachably connected, and the connection methods include but are not limited to thread, buckle, interference fit, etc., and the two are connected together during dissociation. When the two are separated, it is convenient to place and take out tissues.

[0051] After dissociation is complete, the culture medium bag must be squeezed to flush the dissociation chamber under a certain pressure and carry the dissociated tissue into the sterile culture bag. The inlet pipe 4 and the outlet pipe 5 are located on either side of the dissociation chamber 8. The inlet pipe 4 and the outlet pipe 5 are arranged at an angle, with the extension of their respective center lines passing through the bottom of the dissociation chamber 8. This ensures consistent pressure and more thorough flushing of the tissue throughout the dissociation chamber 8.

[0052] In this embodiment, a dissociation piston 7 is provided at the end of the dissociation piston rod 2, and the dissociation part is installed at the end of the dissociation piston 7; the diameter of the dissociation piston 7 is larger than the diameter of the dissociation piston rod 2, and a limiting step is provided at the upper end of the dissociation chamber 8 to prevent the dissociation piston rod 2 from slipping out of the sliding sleeve 1.

[0053] In order to prevent the tissue from overflowing from the dissociation chamber 8 during dissociation, an annular sealing ring 9 can also be provided on the dissociation piston rod 2 or the dissociation piston 7. Similarly, the annular sealing ring 9 can also prevent external impurities from entering the dissociation chamber and contaminating the tissue.

[0054] In some embodiments, the dissociation portion may be in the form of a blade array, but other forms are also possible. For example, the end of the dissociation piston 7 may be provided with textures or protrusions, making it uneven and rough, so that during the reciprocating motion of the dissociation piston rod 2, the textures or protrusions dissociate the tissue to be dissociated.

[0055] In order to facilitate the drive to dissociate the piston rod 2 movement, the upper end of the dissociation piston rod 2 extends out of the sliding sleeve 1 and is provided with a pressure plate 6. The shape of the pressure plate 6 is convenient for the operator to hold and push and pull the dissociation piston rod.

[0056] Example 2: As shown in Figures 4 and 5, the difference from Example 1 is that:

[0057] In this embodiment, a return spring 10 is sleeved on the disengagement piston rod 2; one end of the return spring 10 abuts against the disengagement piston rod 2, and the other end abuts against the spring seat in the sliding sleeve 1; accordingly, a receiving space for accommodating the return spring 10 is provided at the top of the sliding sleeve 1;

[0058] In this embodiment, the axial inner space of the sleeve 1 near one end of the base 3 is set as a piston activity space 81 that provides a partial movement path for the dissociation piston 7, and the dissociation chamber 8 of the base 3 (the height of the chamber wall of which is much smaller than the height of the chamber wall of the dissociation chamber in the above-mentioned embodiment 1) together form a dissociation operation chamber; that is, the difference between the sum of the height of the piston activity space 81 and the height of the chamber wall of the dissociation chamber 8 on the base 3 and the height of the dissociation piston 7 itself is the maximum stroke of the axial movement of the dissociation piston 7; and the liquid inlet pipe 4 and the liquid outlet pipe 5 are symmetrically arranged on both sides of the piston activity space 81; while pushing the dissociation piston rod 2, the reset spring 10 is compressed, and the reset spring 10 can reset the dissociation piston rod 2 when it rebounds, thereby facilitating operation.

[0059] Specifically, referring to Figure 5, the axial inner space of the sleeve 1 includes three sections, the first section serves as a spring accommodating chamber, the second section cooperates with the dissociation piston rod 2, and the third section serves as a piston activity space 81. Due to the setting of the spring accommodating chamber and the piston activity space 81, compared with the cooperation mode of the dissociation piston rod 2 and the sleeve 1 in Example 1, the part directly in contact with the axial inner space of the sleeve 1 is reduced, thereby greatly reducing the friction force in the process of pushing the dissociation piston rod 2.

[0060] Preferably, the cross section of the piston active space 81 is cylindrical, and its diameter is larger than the diameter of the corresponding axial inner space of the dissociation piston rod 2 above it.

[0061] Example 3: As shown in Figures 6 and 7, the differences from Examples 1 and 2 are:

[0062] In this embodiment, the sliding sleeve 2 is composed of two detachable upper and lower sections, namely the upper sliding sleeve 101 and the lower sliding sleeve 102; and the dissociation piston rod 2 is composed of two detachable upper and lower sections, namely the upper dissociation piston rod 201 and the lower dissociation piston rod 202.

[0063] Before dissociation, the lower sleeve 102, the lower dissociation piston rod 202, and the base 3 can be placed separately as consumables in a sterile collection environment, and the three are used to receive and seal the collected tissue to be dissociated, so that they can be assembled with the upper dissociation piston rod 201 and the upper sleeve 101 after long-distance transportation to the destination, and then the dissociation operation can be performed.

[0064] After the dissociation is completed, the lower sleeve 102 remains connected to the base 3, and the lower dissociation piston rod 202 is used to fill and seal the cavity of the lower sleeve 102. In this way, a closed sterile environment can be maintained, which is suitable for long-distance transportation and then transferred through culture medium bags and sterile culture bags.

[0065] In addition, to further improve the sealing performance, in this embodiment, an elastic sealing sleeve 11 is placed on the lower dissociation piston rod 202. The sealing sleeve 11 is in the shape of an inverted bowl, with its large end connected to the sliding sleeve 1 and its small end connected to the lower dissociation piston rod 202, so that the connection between the dissociation piston rod 2 and the sliding sleeve 1 is sealed.

[0066] Example 4: Based on any one of the tissue dissociators in Examples 1 to 3 above, the present invention also provides a tissue dissociation system, which includes the tissue dissociator, a culture fluid bag, a sterile culture bag, and a connecting machine for connecting the liquid outlet hose 23 of the culture fluid bag and the liquid inlet hose 31 of the sterile culture bag to the liquid inlet pipe 4 and the liquid outlet pipe 5 of the tissue dissociator.

[0067] In this embodiment, referring to Figure 8, the culture medium bag includes a first chamber 21 and a second chamber 22, and a liquid outlet hose 23 connected to the first chamber 21. A separation membrane 24 is provided between the first chamber 21 and the second chamber 22. The separation membrane 24 can be opened under the action of an external force, so that the first chamber 21 and the second chamber 22 are connected, wherein the second preset volume of the culture medium pre-stored in the first chamber 21 is smaller than the third preset volume of the culture medium pre-stored in the second chamber.

[0068] Furthermore, in some embodiments, a first preset volume of sterile gas is pre-stored in the sterile culture bag. By pre-storing a certain amount of sterile gas in the culture bag (of course, preferably, the volume of gas in the sterile culture bag is less than the capacity of the sterile culture bag, specifically, the volume is only sufficient to allow the dissociation piston to move freely within the movable space defined by the sliding sleeve and the dissociation chamber), the travel of the dissociation piston is limited in a sealed environment due to insufficient air pressure in the sealed space due to reasons such as misoperation.

[0069] Example 5: Referring to FIG9 , based on the above tissue dissociation system, the present invention further provides a tissue dissociation method, comprising:

[0070] S1 will be pre-placed in a sterile collection environment, the base 3 is separated from the sliding sleeve 1, and the obtained tissue to be dissociated is placed in the base 3. In some embodiments, when the tissue dissociator in the above-mentioned embodiment 1 or 2 is used, the base is a disposable consumable, and the sliding sleeve 1 and the dissociation piston rod 2 can be reused. Therefore, the base 3 can be equipped in a sterile environment for sampling, such as an operating room. Of course, it is foreseeable that the base 3 needs to be sealed with a sealing bag before use. After obtaining the tissue to be dissociated, the base 3 is taken out of the sealing bag and the tissue to be dissociated is placed in the dissociation chamber 8 of the base 3.

[0071] In other embodiments, when the tissue dissociator in the above-mentioned embodiment 3 is used, the base 3, the lower sleeve 102 and the lower dissociation piston rod 202 are all disposable consumables (of course, the culture medium bag and the sterile culture bag are also disposable consumables) and can be stored separately in a sterile collection environment, while the upper sleeve 101 and the upper dissociation piston rod 201 can be placed in a detection room or laboratory for repeated use, so there is no need to ensure that the base 3, the lower sleeve 102 and the lower dissociation piston rod 202 are arranged in equal proportions with the upper sleeve 101 and the upper dissociation piston rod 201, that is, one set of upper sleeve 101 and upper dissociation piston rod 201 can be used to assemble with multiple sets of base 3, lower sleeve 102 and lower dissociation piston rod 202 to perform operations such as tissue dissociation and transfer, further reducing costs.

[0072] S2 connects the base 3 to the sliding sleeve 1 in a sterile collection environment, so that a dissociation operation chamber for providing an axial movement path for the dissociation piston is formed between the base 3 and the axial inner space of the sliding sleeve 1 .

[0073] In some embodiments, when the tissue dissociator of the above-mentioned embodiment 1 is used, the dissociation operation chamber provides a movement path for the dissociation piston, and the sliding sleeve limits its maximum stroke along the movement path.

[0074] In other embodiments, when the tissue dissociator of the above-mentioned embodiment 2 is used, the piston active space 81 and the cavity wall of the dissociation chamber 8 jointly provide a movement path for the dissociation piston, and the maximum stroke along the movement path is limited by the top of the piston active space 81.

[0075] In other embodiments, when the tissue dissociator of the above-mentioned embodiment 3 is used, it is only necessary to connect the base 3 and the lower sleeve 102 in a sterile collection environment, and then connect the lower sleeve 102 and the upper sleeve 101, as well as the lower dissociation piston rod 202 and the upper dissociation piston rod 201.

[0076] When the base 3 is connected to the sliding sleeve 1, the liquid inlet pipe 4 and the liquid outlet pipe 5 are in the initial state, that is, the closed state, so the internal dissociation operation chamber forms a closed sterile environment without the risk of contamination.

[0077] S3 uses a connecting machine to connect the culture liquid bag and the sterile culture bag to the liquid inlet pipe 4 and the liquid outlet pipe 5 respectively, so that the culture liquid bag and the sterile culture bag are connected to the dissociation operation chamber.

[0078] In some embodiments, step S3 can be completed in a sterile collection environment; of course, in order not to occupy more space in the sterile collection environment, step S3 can also be completed not in the sterile collection environment, but anywhere outside the sterile collection environment. Accordingly, this anywhere can also be referred to as a dissociation operation environment. Of course, it will be understood by those skilled in the art that anywhere here does not include places with serious pollution, such as garbage stations or next to garbage bins. After the culture medium bag and the sterile culture bag are respectively connected to the liquid inlet pipe 4 and the liquid outlet pipe 5 by the takeover machine, the culture medium bag is connected to the liquid inlet pipe 4, and the sterile culture bag is connected to the liquid outlet pipe 5, so that the culture medium bag, the dissociation operation chamber, and the sterile culture bag form a passage.

[0079] In some embodiments, a first preset volume of sterile gas is pre-stored in the sterile culture bag, thereby enabling the free movement of the dissociated piston rod in a sealed environment.

[0080] In some embodiments, before connecting the hoses with the connecting machine, the hoses of the culture fluid bag and the sterile culture bag can be clamped with a tube clamp, or the liquid inlet and outlet tubes of the tissue dissociator can be clamped with a tube clamp.

[0081] S4 squeezes the culture solution of a second preset volume in the culture solution bag into the dissociation operation chamber, and then blocks the liquid inlet passage between the culture bag and the dissociation operation chamber.

[0082] In some embodiments, if the above-mentioned two-chamber culture medium bag is not used, the squeezing amount can be controlled manually.

[0083] In other embodiments, when using the above-mentioned two-chamber culture fluid bag, a second preset volume of culture fluid actually required for the dissociation process can be pre-stored in the first chamber of the culture fluid bag, thereby avoiding errors caused by manual operation (for example, if too much squeezing is applied, the force of the piston during the dissociation process may be weakened, thereby prolonging the dissociation time and even causing the culture fluid to overflow into the space above the piston; if too little squeezing is applied, the buffering effect is not significant, which may affect the dissociation effect). Specifically, the first chamber is squeezed in the direction of the liquid outlet hose, so that the second preset volume of culture fluid stored in the first chamber enters the dissociation operation chamber through the liquid outlet hose. As previously mentioned, since a partition membrane is provided between the two chambers, a tube clamp is not required to block the liquid inlet path between the culture fluid bag and the dissociation operation chamber.

[0084] S5 drives the dissociation piston rod 2 to dissociate the tissue to be dissociated. During dissociation, the dissociation portion on the dissociation piston rod 2 and the dissociation piston 7 dissociates the tissue to be dissociated. During the pushing and pulling of the dissociation piston rod 2, the gas in the dissociation operation chamber is discharged into the sterile culture bag, avoiding the influence of the air pressure in the dissociation operation chamber and ensuring smooth dissociation.

[0085] Of course, preferably, a certain amount of sterile gas can also be pre-stored in the sterile culture bag, and during the dissociation process, the volume of gas in the sterile culture bag is negatively correlated with the distance between the dissociation piston rod 2 and the bottom of the dissociation chamber. In other words, as the dissociation piston rod 2 is pushed toward the dissociation chamber, gas will enter the culture bag, causing the volume of gas in the culture bag to gradually increase. Correspondingly, as the dissociation piston rod 2 is pulled away from the dissociation chamber, gas will enter the dissociation operation chamber and the sliding sleeve 1 from the culture bag, and the volume of gas in the culture bag will gradually decrease.

[0086] After dissociation is complete, the culture bag is squeezed, allowing the remaining culture fluid in the bag to flush the dissociation chamber and transfer the dissociated tissue into the sterile culture bag. After dissociation is complete, the bag is squeezed manually or by other means, causing the culture fluid to spray into the dissociation chamber. The culture fluid has a certain pressure, which allows it to flush the dissociation chamber, flushing the tissue from the dissociation chamber walls and into the sterile culture bag along with the culture fluid, thus completing tissue dissociation and transfer in a closed environment.

[0087] In some embodiments, the second chamber is squeezed to open the partition membrane between the first chamber and the second chamber, thereby allowing the culture fluid in the second chamber to enter the first chamber; then the first chamber is squeezed multiple times along the direction of the liquid outlet hose, so that a second preset volume of culture fluid enters the dissociation operation chamber through the liquid outlet hose to flush the dissociation operation chamber and flush the dissociated tissue into the sterile culture bag.

[0088] Of course, in other embodiments, in order to reduce the residual tissue particles in the dissociation operation chamber, after squeezing the first chamber to allow the culture fluid to enter the dissociation operation chamber, the tissue dissociator can be oscillated or shaken to allow most of the tissue particles adhering to the wall of the dissociation operation chamber to detach from the wall and gather at the bottom of the dissociation chamber; then the tissue dissociator is placed at an angle (for example, when the tissue dissociator is tilted, the angle between the center line of the dissociation operation chamber and the horizontal line is 0°-45°; of course, specifically, it can be tilted toward the liquid outlet pipe side, or toward the liquid inlet pipe side, so as to flush the two side walls and bottom of the dissociation operation chamber), and the first chamber is squeezed multiple times so that the culture fluid flushes the bottom of the dissociation operation chamber under the action of squeezing and gravity, so as to flush the dissociated tissue particles into the sterile culture bag.

[0089] S7 isolates the liquid outlet passage between the culture bag and the dissociation chamber, and separates the sterile culture bag from the tissue dissociator, with the separated sterile culture bag being in a sealed state. In some embodiments, a heat sealer can be used to isolate the liquid outlet passage, automatically separating the sterile culture bag from the tissue dissociator while simultaneously sealing the liquid inlet hose, thereby sealing the sterile culture bag. This allows sterile culture to be performed directly in the sterile culture bag without the need for transfer.

[0090] It should be noted that, in this document, the terms "comprises," "includes," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a process, method, article, or apparatus comprising a series of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of other identical elements in the process, method, article, or apparatus comprising the element.

[0091] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are protected by the present invention.

Claims

1. A tissue dissociation system, characterized in that: include: A tissue dissociator, a culture fluid bag, a sterile culture bag, and a connecting machine for connecting the liquid outlet hose of the culture fluid bag and the liquid inlet hose of the sterile culture bag to the tissue dissociator respectively; In the initial state, the free ends of the liquid outlet hose and the liquid inlet hose are in a closed state, wherein: The tissue dissociator comprises: a sliding sleeve, a base detachably connected to one end of the sliding sleeve, and a dissociation piston rod movable along the axial inner hollow of the sliding sleeve to the base; when the base is connected to the sliding sleeve, the base and the axial inner hollow of the sliding sleeve form a dissociation operation chamber for providing an axial movement path for the dissociation piston rod; a dissociation portion for dissociating tissue is provided at one end of the dissociation piston rod close to the base; a liquid inlet pipe and a liquid outlet pipe are connected to the dissociation operation chamber, and the ends of the liquid inlet pipe and the liquid outlet pipe that are not connected to the dissociation operation chamber are closed; A dissociation piston is provided at the end of the dissociation piston rod, and the dissociation portion is installed at the end of the dissociation piston; the diameter of the dissociation piston is greater than the diameter of the dissociation piston rod; The sliding sleeve is composed of two detachable upper and lower sections, and the disengagement piston rod is composed of two detachable upper and lower sections; and an elastic sealing sleeve is sleeved on the lower section of the disengagement piston rod; The sealing sleeve is in the shape of an inverted bowl, with a large end connected to the inside of the sliding sleeve and a small end connected to the lower dissociation piston rod, so that the connection between the dissociation piston rod and the sliding sleeve is closed.

2. A tissue dissociation system according to claim 1, characterized in that: The liquid inlet pipe and the liquid outlet pipe are symmetrically arranged on both sides of the dissociation operation chamber, and the liquid inlet pipe and the liquid outlet pipe are inclinedly arranged so that the extension lines of the respective center lines of the liquid inlet pipe and the liquid outlet pipe pass through the bottom of the dissociation operation chamber.

3. A tissue dissociation system according to claim 1, characterized in that: A return spring is sleeved on the disengagement piston rod; one end of the return spring abuts against the disengagement piston rod, and the other end abuts against the spring seat in the sliding sleeve.

4. A tissue dissociator according to claim 1, characterized in that: The dissociation piston rod is provided with an annular sealing ring; and / or, the upper end of the dissociation piston rod extends out of the sliding sleeve and is provided with a pressure plate for operation.

5. A tissue dissociation system according to claim 1, characterized in that: The culture fluid bag includes a first chamber and a second chamber, the first chamber is connected to one end of the liquid outlet hose, a separation membrane is arranged between the first chamber and the second chamber, the separation membrane can be opened under the action of an external force, so that the first chamber and the second chamber are connected, wherein a second preset volume of the culture fluid pre-stored in the first chamber is smaller than a third preset volume of the culture fluid pre-stored in the second chamber.

6. A tissue dissociation method, applied to the tissue dissociation system according to any one of claims 1 to 5, characterized in that include: Separating the base pre-placed in the sterile collection environment from the sliding sleeve, placing the obtained tissue to be dissociated on the base, and then connecting the base with the sliding sleeve so that the axial inner hollows of the base and the sliding sleeve form a dissociation operation chamber for providing an axial movement path for the dissociation piston; Using a connecting machine to connect the culture liquid bag and the sterile culture bag to the liquid inlet pipe and the liquid outlet pipe on the dissociation operation chamber respectively, so that the culture liquid bag and the sterile culture bag are connected to the dissociation operation chamber; squeezing the culture fluid bag so that a second preset volume of culture fluid stored in the culture fluid bag enters the dissociation operation chamber through the liquid inlet tube; Driving the dissociation piston rod to dissociate the tissue to be dissociated, and during the dissociation process, the liquid outlet passage between the sterile culture bag and the dissociation operation chamber is always kept unobstructed; After the dissociation is completed, the culture liquid bag is squeezed so that the remaining culture liquid in the culture liquid bag flushes the dissociation operation chamber and flushes the dissociated tissue into the sterile culture bag; The liquid outlet passage between the sterile culture bag and the dissociation operation chamber is cut off, and the sterile culture bag is separated from the tissue dissociator, and after the separation, the liquid inlet hose of the sterile culture bag is in a closed state.

7. A tissue dissociation method according to claim 6, characterized in that: The step of squeezing a second preset volume of culture fluid in the culture fluid bag into the dissociation operation chamber specifically comprises the steps of: The first chamber in the culture fluid bag is squeezed in the direction of the liquid outlet hose on the culture fluid bag so that the first chamber The culture solution of a second preset volume stored in the chamber enters the dissociation operation chamber through the liquid outlet hose; and / or, The step of squeezing the culture fluid bag after the dissociation is completed so that the remaining culture fluid in the culture fluid bag flushes the dissociation operation chamber and flushes the dissociated tissue into the sterile culture bag specifically comprises the steps of: The second chamber of the culture fluid bag is squeezed to open the partition membrane between the first chamber and the second chamber, so that the culture fluid of a third preset volume pre-stored in the second chamber enters the first chamber; The first chamber is squeezed multiple times along the direction of the liquid outlet hose, so that the culture fluid of the third preset volume enters the dissociation operation chamber through the liquid outlet hose to flush the dissociation operation chamber and flush the dissociated tissue into the sterile culture bag.

8. A tissue dissociation method according to claim 7, characterized in that: The step of squeezing the first chamber multiple times along the direction of the liquid outlet hose specifically includes: squeezing the first chamber so that the culture solution enters the dissociation operation chamber; Oscillating or shaking the tissue dissociator so that most of the tissue particles adhering to the wall of the dissociation operation chamber are separated from the wall and gathered at the bottom of the dissociation operation chamber; The tissue dissociator is placed at an angle, and the first chamber is squeezed multiple times, so that the culture fluid flushes the bottom of the dissociation operation chamber under the action of squeezing and gravity, so as to flush the dissociated tissue particles into the sterile culture bag.

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