Cytocentrifuge device, and cytocentrifugation, washing and culturing methods

Abstract

The present invention provides a cell centrifuge device and a method for cell centrifugation, washing and culturing. The cell centrifuge device includes a centrifuge cup (1) and a liquid pushing plate (2). The centrifuge cup (1) has a rotation axis. The centrifuge cup (1) is connected to a rotation drive mechanism (4) and is used to centrifuge a blood sample contained in the centrifuge cup (1) by rotating about the rotation axis driven by the rotation drive mechanism (4). One or more liquid pushing plates (2) are provided. The liquid pushing plate (2) is disposed in the centrifuge cup (1). When the centrifuge cup (1) rotates, the liquid pushing plate (2) is used to push the blood sample and rotate it synchronously along the same rotation direction as the centrifuge cup (1).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to Chinese Patent Application No. 202110761903.1, entitled "Cytocentrifugation Apparatus and Method for Cell Centrifugation, Washing and Cultivation," filed on July 6, 2021, and Chinese Patent Application No. 202121524433.9, entitled "Cytocentrifugation Apparatus," filed on July 6, 2021, the entire disclosures of which are incorporated herein by reference. The present invention relates to the technical field of medical devices, and in particular to a cytocentrifuge device and a method for cytocentrifugation, washing and culturing. [Background technology]

[0002] In the field of cell therapy, cells collected from living organisms are often cultured and then inoculated into patients. To ensure the therapeutic effect of collected and cultured cells, peripheral blood mononuclear cell (PBMC) sorting is required. To concentrate the cells to a cell concentration suitable for treatment, impurities and medium must be removed during the cell culture process. The method for peripheral blood mononuclear cell sorting is density gradient centrifugation, which involves sedimenting different particles in a blood sample at a constant rate under centrifugal force, ultimately forming distinct zones to achieve the purpose of sorting.

[0003] In the centrifugation process of existing centrifugal devices, the inner wall of the centrifugal device rotates the outer layer liquid close to the inner wall due to viscous resistance, and then the outer layer liquid rotates the inner layer liquid away from the inner wall due to viscous resistance, ultimately achieving a rotating state of the liquid inside the centrifugal device. When viscous resistance acts on cells, the cells are subjected to shear force. Because cells are easily damaged under shear force, their viability is affected. Summary of the Invention

[0004] The present invention provides a cytocentrifuge device and a method for cytocentrifugation, washing and culturing to solve the problem that existing centrifuge equipment is prone to shear damage to cells during the centrifugation process.

[0005] The present invention provides a cytocentrifuge device, which includes a centrifuge cup and a liquid pusher plate. The centrifuge cup has a rotation axis, is connected to a rotation drive mechanism, and is used to centrifuge a blood sample contained in the centrifuge cup by rotating about the rotation axis when driven by the rotation drive mechanism. One or more liquid pusher plates are provided, and are disposed within the centrifuge cup. When the centrifuge cup rotates, the liquid pusher plate is used to push the blood sample and rotate it synchronously in the same rotation direction as the centrifuge cup.

[0006] According to the cell centrifuge device provided by the present invention, one end of the liquid pusher plate is close to the rotation axis and the other end extends toward the inner wall of the centrifuge cup, the projection of the first side edge of the liquid pusher plate onto the plane on which the cup bottom of the centrifuge cup is located is linear or arc-shaped, the second side edge of the liquid pusher plate is perpendicular to the cup bottom of the centrifuge cup, and when the projection of the first side edge of the liquid pusher plate onto the plane on which the cup bottom of the centrifuge cup is located is linear, the first side edge of the liquid pusher plate extends along the radial direction of the centrifuge cup.

[0007] According to the cell centrifuge device provided by the present invention, a first gap is provided between one end of the liquid pushing plate that is close to the inner wall of the centrifuge cup and the inner wall, and one end of the liquid pushing plate that is close to the cup bottom of the centrifuge cup and the cup side are in close contact with each other.

[0008] According to the cell centrifuge device provided by the present invention, when there are a plurality of liquid pushing plates, the plurality of liquid pushing plates are uniformly distributed around the circumference of the rotation axis.

[0009] The cell centrifuge device of the present invention further includes a cup lid, which is matched with a cup mouth of the centrifuge cup, and which is provided with a first fluid-passing structure and a second fluid-passing structure, the first ends of which are disposed on the outer surface of the cup lid and distributed along the rotation axis, the second ends of which are disposed on the inner surface of the cup lid, the second fluid-passing structure including a fluid-passing tube and a fluid-passing passage, the fluid-passing tube being disposed within the centrifuge cup, the first ends of which communicate with the second ends of the first fluid-passing structure and the second ends of which are disposed at the cup bottom of the centrifuge cup, the fluid-passing passage being disposed within a casing wall of the cup bottom, the first ends of which communicate with the second ends of the fluid-passing tube and the second ends of the fluid-passing passage being disposed at the inner surface of the cup bottom and communicating with the internal cavity of the centrifuge cup.

[0010] According to the cell centrifuge device provided by the present invention, a third fluid-passing structure is provided within the casing wall of the cup bottom of the centrifuge cup, and a first end of the third fluid-passing structure is located on the outer surface of the cup bottom and distributed along the rotation axis, and a second end of the third fluid-passing structure is located on the inner surface of the cup bottom and communicates with the internal cavity of the centrifuge cup.

[0011] In the cell centrifuge device provided by the present invention, the first fluid passage structure includes a connecting pipe, which is distributed along the rotation axis, and which has a liquid passage and a gas passage in the connecting pipe, a first end of the liquid passage having a liquid connection port, a second end of the liquid passage communicating with the first end of the liquid passage port, a first end of the gas passage having a gas connection port, and a second end of the gas passage communicating with the internal cavity of the centrifuge cup.

[0012] According to the cell centrifuge device provided by the present invention, the cup cover has an air hole, at least one air hole is provided, and the inside of the air hole is used to provide an air permeable membrane.

[0013] The present invention further provides a cell centrifugation method for a cell centrifuge device, which comprises the steps of connecting a centrifuge cup to a rotary drive mechanism and adding a blood sample into the centrifuge cup, and turning on the rotary drive mechanism to rotate the centrifuge cup on its own axis, so that the liquid pushing plate pushes the blood sample and rotates synchronously with the centrifuge cup in the same direction as the centrifuge cup, thereby obtaining a cell suspension.

[0014] The present invention further provides a cell washing method for a cell centrifuge device, which includes the steps of: introducing saline into the centrifuge cup through a second fluid-passing structure of the centrifuge cup when centrifuging a blood sample with the cell centrifuge device, thereby washing the surface of cells in the blood sample with the added saline during the centrifugation process; and, after the blood sample has been centrifuged for a predetermined time, aspirating the liquid in the centrifuge cup through the second fluid-passing structure to discharge waste liquid used to wash the cells.

[0015] The present invention further provides a cell culture method for a cell centrifuge device, which includes the steps of adding a cell suspension containing cells to be cultured into a centrifuge cup, adjusting the cell density in the cell suspension to a first preset density, and adding antibodies or magnetic beads into the centrifuge cup to activate the cells, adding carbon dioxide gas with a preset concentration and a culture medium with a preset composition into the centrifuge cup, and placing the centrifuge cup containing the cell suspension and the culture medium in a preset constant temperature environment to culture the cells, detecting the cell density in the centrifuge cup, and turning off the rotation drive mechanism if the cell density is less than a second preset density to statically culture the cells, and turning on the rotation drive mechanism for a first preset time and then turning it off for a second preset time if the cell density is greater than the second preset density, and repeating this process to dynamically culture the cells.

[0016] The cell centrifuge device and cell centrifugation, washing, and culturing method provided by the present invention include a liquid pusher plate installed inside the centrifuge cup. When the centrifuge cup rotates, the blood sample inside the centrifuge cup rotates synchronously with the centrifuge cup due to the push of the liquid pusher plate. That is, there is no relative motion between the outer layer cells adjacent to the inner wall of the centrifuge cup and the inner layer cells adjacent to the rotation axis in the direction of rotation of the centrifuge cup. The power source for the rotation of the inner layer cells is no longer the viscous resistance between the outer layer cells and the inner layer cells, but the pushing force of the liquid pusher plate. This avoids the shear effect on the cells when viscous resistance acts on them and reduces shear damage to the cells during the centrifugation process. [Brief explanation of the drawings]

[0017] In order to further describe the technical solutions of the present invention or the prior art, the following will briefly describe the drawings necessary for describing the embodiments or the prior art. Of course, the drawings in the following description are only some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without performing creative work. [Figure 1] 1 is a structural schematic diagram of a cell centrifuge device provided by the present invention. [Figure 2] 1 is a schematic diagram of cells subjected to forces during a centrifugation process provided by the present invention. [Figure 3] FIG. 1 is a schematic diagram of cells subjected to force during centrifugation when a liquid push plate is provided, as provided by the present invention. [Figure 4] 1 is a schematic diagram of a first type of arrangement structure of the liquid pushing plate in the centrifugal cup provided by the present invention; FIG. [Figure 5] FIG. 2 is a schematic diagram of the second type of arrangement structure of the liquid pushing plate in the centrifugal cup provided by the present invention. [Figure 6] 1 is a schematic diagram of the top structure of a cytocentrifuge device provided by the present invention. FIG. [Figure 7] 7 is a first type cross-sectional structure schematic diagram provided by the present invention, taken along the AA direction in FIG. 6. FIG. [Figure 8]FIG. 7 is a second type of cross-sectional structure schematic diagram provided by the present invention, taken along the AA direction in FIG. 6. [Figure 9] 1 is a first structural schematic diagram of the cup lid provided by the present invention; FIG. [Figure 10] FIG. 2 is a second structural schematic diagram of the cup lid provided by the present invention. [Figure 11] FIG. 1 is a flow diagram of the cytocentrifugation method provided by the present invention. [Figure 12] FIG. 1 is a schematic diagram of the flow of the cell washing method provided by the present invention. [Figure 13] FIG. 1 is a schematic diagram of the flow of the cell culture method provided by the present invention. [Figure 14] FIG. 1 is a line diagram of cell counts during a cell culture process provided by the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0018] In order to make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be described clearly and completely below with reference to the accompanying drawings. Of course, the embodiments described herein are only a part, not all, of the embodiments of the present invention. All other embodiments that those skilled in the art can obtain based on the embodiments of the present invention without creative work should fall within the protection scope of the present invention.

[0019] The cell centrifugation device and the cell centrifugation, washing and culturing methods provided by the present invention will be described below with reference to FIGS.

[0020] As shown in Figures 1 to 10, this embodiment provides a cell centrifuge device. This cell centrifuge device includes a centrifuge cup 1 and a liquid push plate 2. The centrifuge cup 1 has a rotation axis. The centrifuge cup 1 is connected to a rotation drive mechanism 4 and is used to centrifuge a blood sample contained in the centrifuge cup 1 by rotating about the rotation axis when driven by the rotation drive mechanism 4. One or more liquid push plates 2 are provided. The liquid push plate is disposed within the centrifuge cup 1. When the centrifuge cup 1 rotates, the liquid push plate 2 is used to push the blood sample and rotate it in sync with the centrifuge cup 1 in the same rotation direction.

[0021] Specifically, in this embodiment, by providing a liquid pushing plate 2 inside the centrifuge cup 1, when the centrifuge cup 1 rotates, the blood sample inside the centrifuge cup 1 rotates synchronously with the centrifuge cup 1 due to the pushing of the liquid pushing plate 2. That is, in the direction of rotation of the centrifuge cup 1, there is no relative motion between the outer layer cells adjacent to the inner wall of the centrifuge cup 1 and the inner layer cells adjacent to the rotation axis. The power source for the rotation of the inner layer cells is no longer the viscous resistance between the outer layer cells and the inner layer cells, but the pushing force of the liquid pushing plate. This avoids the shearing effect on the cells when viscous resistance acts on them and reduces shear damage to the cells during the centrifugation process.

[0022] The blood sample shown in this example contains target cells to be extracted, and in order to facilitate the extraction of the target cells, the blood sample is centrifuged using a centrifuge to stratify the target cells and other particles in the blood sample.

[0023] As shown in Figure 2, the centrifugal cup 1 shown in this example performs a centrifugation operation on a blood sample without the liquid pusher plate 2. The dotted line represents the trajectory of cell movement within the blood sample. Due to viscous resistance between the liquid and the outer layer cells, which are adjacent to the inner wall of the centrifugal cup 1, the inner layer cells, which are adjacent to the rotation axis of the centrifugal cup 1, rotate. During this process, there is a delay in the rotation of the inner layer cells, meaning that there is relative movement between the outer layer cells and the inner layer cells in the direction of rotation of the centrifugal cup 1. When there is relative movement, both the outer layer cells and the inner layer cells are subjected to a shear force τ. The direction of the shear force τ is tangent to the movement trajectory. The shear force τ experienced by the outer layer cells is greater than that experienced by the inner layer cells, while the shear force τ experienced by the inner layer cells is smaller. When the shear force τ acts on the cells, it causes shear damage to the cells. Taking a single cell as an example, the flow rate of the blood sample is high on the side close to the inner wall of the cell and low on the side close to the rotation axis, so the cell is also subjected to radial pressure, and when this pressure acts on the cell, it causes shear damage to the cell.

[0024] As shown in Figure 3, the centrifugal cup 1 shown in this embodiment is equipped with a liquid pusher plate 2 to perform a centrifugal operation on a blood sample. The dotted line indicates the trajectory of cells in the blood sample. The outer and inner layer cells rotate synchronously under the pressure F of the liquid pusher plate. The power source for the inner layer cell rotation is no longer the viscous resistance between the outer and inner layer cells, so there is no delay in the rotation of the inner layer cells. In other words, there is no relative motion between the outer and inner layer cells in the direction of rotation of the centrifugal cup 1. Without relative motion, the outer and inner layer cells are not subjected to shear force, meaning that no shear damage is caused to the cells during the centrifugation process.

[0025] The specific number of liquid pushing plates 2 may be one, two, three, four, etc., and is not particularly limited in this specification.

[0026] Preferably, as shown in Figures 4 and 5, one end of the liquid pushing plate 2 shown in this embodiment is close to the rotation axis, and the other end extends toward the inner wall of the centrifugal cup 1, the projection of the first side edge of the liquid pushing plate 2 onto the plane on which the cup bottom of the centrifugal cup 1 is located is linear or arc-shaped, and the second side edge of the liquid pushing plate 2 is perpendicular to the cup bottom of the centrifugal cup 1.

[0027] Specifically, if the projection of the first side edge of the liquid pushing plate 2 onto the plane where the cup bottom of the centrifuge cup 1 is located is a straight line, the plate surface of the liquid pushing plate 2 that pushes and rotates the blood sample is a flat surface. If the projection of the first side edge of the liquid pushing plate 2 onto the plane where the cup bottom of the centrifuge cup 1 is located is an arc shape, the plate surface of the liquid pushing plate 2 that pushes and rotates the blood sample is an arc surface. Different shapes of liquid pushing plates are selected according to different concentrations of blood samples to ensure good centrifugal effect of the blood sample.

[0028] Preferably, the liquid pushing plate 2 and the cup bottom of the centrifugal cup 1 shown in this embodiment are fixedly connected, or the liquid pushing plate 2 and the cup bottom of the centrifugal cup 1 are detachably connected.

[0029] Specifically, by detachably connecting the liquid pushing plate 2 to the cup bottom of the centrifugal cup 1, cleaning of the centrifugal cup 1 and replacement of the liquid pushing plate 2 become easy.

[0030] The fixed connection in this embodiment includes adhesive bonding. The detachable connection includes pin connection or bolt connection. Alternatively, a locking structure is provided at the bottom of the centrifugal cup 1, and the liquid pushing plate is locked into the locking structure.

[0031] In one embodiment, as shown in Figures 1, 4, 5, 7 and 8, the liquid pushing plate 2 and the cup bottom are connected via a pin shaft 21. A pin shaft hole is opened in the liquid pushing plate 2 along the rotation axis direction, and a fixing hole is opened in the cup bottom. One end of the pin shaft 21 is connected to the pin shaft hole, and the other end of the pin shaft 21 is connected to the fixing hole, allowing the liquid pushing plate 2 and the cup bottom to be detachably connected. To ensure the stability of the connection, two pin shaft holes are opened, and the fixing holes are installed in one-to-one correspondence with the pin shaft holes.

[0032] In another embodiment, the cup bottom is provided with a first locking groove along the radial direction of the centrifugal cup 1, the liquid pushing plate 2 is locked in the first locking groove, and there is an interference fit between the liquid pushing plate 2 and the first locking groove. Alternatively, the inner wall of the centrifugal cup 1 is provided with a second locking groove along the rotation axis direction, the liquid pushing plate 2 is locked in the second locking groove, and there is an interference fit between the liquid pushing plate 2 and the second locking groove. Alternatively, the cup bottom and the inner wall of the centrifugal cup 1 are provided with a first locking groove and a second locking groove, respectively, the first locking groove and the second locking groove are installed in a one-to-one correspondence, the liquid pushing plate 2 is locked in the first locking groove and the second locking groove simultaneously, and there is an interference fit between the liquid pushing plate 2 and the first locking groove and between the liquid pushing plate 2 and the second locking groove.

[0033] Preferably, as shown in Figures 4, 7 and 8, a first gap is provided between one end of the liquid pushing plate 2 shown in this embodiment that is close to the inner wall of the centrifugal cup 1 and the inner wall. One end of the liquid pushing plate 2 that is close to the cup bottom of the centrifugal cup 1 and the cup bottom are in close contact with each other.

[0034] Specifically, during centrifugation of the centrifuge cup 1, the target cells in the blood sample gradually approach the inner wall of the centrifuge cup 1 and finally adhere to the inner wall of the centrifuge cup 1, allowing the target cells to pass through the first gap, preventing the target cells from being concentrated and accumulating. At the same time, the blood sample can pass through the first gap freely, ensuring that the liquid levels on both sides of the liquid pushing plate 2 are consistent.

[0035] It is not advisable to set the first gap too large. If the first gap is set too large, the liquid pushing plate 2 will not be able to achieve a good liquid pushing effect. The ratio of the first gap to the inner radius of the centrifugal cup 1 ranges from 1 / 600 to 1 / 20, and in this case, the ratio of the first gap to the inner radius of the centrifugal cup 1 may be 1 / 600, 1 / 300, 1 / 100, 1 / 60, 1 / 40, or 1 / 20. Of these, 1 / 60 is preferred.

[0036] Preferably, as shown in FIGS. 1, 4 and 5, a second gap is provided between one end of the liquid pushing plate 2 shown in this embodiment that is close to the rotation shaft and the rotation shaft.

[0037] Specifically, by providing a second gap between the liquid pushing plate 2 and the rotating shaft, a certain working space is ensured for attaching and detaching the liquid pushing plate 2. At the same time, the linear velocity of the inner layer cells near the rotating shaft during the centrifugal process is relatively small, and the installation of the second gap reduces the impact on the centrifugal effect of the blood sample.

[0038] The ratio of the second gap to the inner radius of the centrifugal cup 1 ranges from 1 / 200 to 1 / 5, and in this case, the ratio of the second gap to the inner radius of the centrifugal cup 1 may be 1 / 200, 1 / 100, 1 / 60, 1 / 30, 1 / 10 or 1 / 5. Of these, 1 / 30 is preferred.

[0039] Preferably, when there are multiple liquid push plates 2, the multiple liquid push plates 2 are uniformly distributed around the circumference of the rotation axis.

[0040] Specifically, the centrifugal cup 1 is divided into a number of relatively independent centrifugal zones by a number of liquid pushing plates 2, and the liquid pushing plates 2 are uniformly distributed along the circumferential direction of the rotation axis to ensure that the centrifugal effect of the blood sample in each centrifugal zone is the same.

[0041] In one embodiment, two liquid push plates 2 are provided, and the included angle between the two liquid push plates 2 is 180°.

[0042] In another embodiment, three liquid push plates 2 are provided, and the included angle between two adjacent liquid push plates 2 is 120°.

[0043] In another embodiment, as shown in FIGS. 1 and 4, four liquid push plates 2 are provided, and the angle between any two adjacent liquid push plates 2 is 90°.

[0044] Preferably, as shown in Figures 6, 7 and 8, the centrifugal apparatus shown in this embodiment further includes a cup lid 3. The cup lid 3 is matched with the cup mouth of the centrifugal cup 1. The cup lid 3 is provided with a first liquid-passing structure 31. A first end of the first liquid-passing structure 31 is disposed on the outer surface of the cup lid 3 and distributed along the rotation axis, and a second end of the first liquid-passing structure 31 is disposed on the inner surface of the cup lid 3.

[0045] Specifically, by providing the first fluid-passing structure 31 in the cup lid 3, it is possible to add a blood sample to the centrifuge cup 1 without opening the cup lid 3.

[0046] Preferably, as shown in Figures 1, 4, 5, 7 and 8, the centrifugal cup 1 shown in this embodiment is provided with a second liquid-passing structure 11. The second liquid-passing structure 11 includes a liquid-passing tube 111 and a liquid-passing passage 112. The liquid-passing tube 111 is disposed within the centrifugal cup 1. A first end of the liquid-passing tube 111 communicates with a second end of the first liquid-passing structure 31. A second end of the liquid-passing tube 111 is disposed at the cup bottom of the centrifugal cup 1. The liquid-passing passage 112 is disposed within the casing wall at the cup bottom. A first end of the liquid-passing passage 112 communicates with a second end of the liquid-passing tube 111. A second end of the liquid-passing passage 112 is disposed on the inner surface of the cup bottom and communicates with the internal cavity of the centrifugal cup 1.

[0047] Specifically, to prevent splashing when the blood sample is added to the centrifuge cup through the first fluid-passing structure 31, the second fluid-passing structure 11 is provided. This allows the blood sample to pass through the first fluid-passing structure 31, enter the fluid-passing tube 111, then enter the fluid-passing passage 112, and finally enter the internal cavity of the centrifuge cup 1 from the bottom of the cup, ensuring stability during the blood sample injection process. At the same time, the fluid-passing passage 112 is embedded in the casing wall at the bottom of the cup, preventing the fluid-passing passage 112 from occupying the available space in the internal cavity of the centrifuge cup. After the blood sample is centrifuged, a cell suspension is obtained, which can be sucked out via the second fluid-passing structure 11 and the first fluid-passing structure 31.

[0048] In one embodiment, as shown in Figures 7 and 8, the liquid passage pipe 111 is preferably arranged along the rotation axis to improve the stability of the centrifugal cup 1 during rotation.

[0049] Preferably, as shown in FIGS. 7 and 8, one end of the liquid pushing plate 2 shown in this embodiment that is close to the cup lid 3 is in close contact with the cup lid 3.

[0050] Specifically, by installing the liquid pushing plate 2 so that one end thereof adjacent to the cup lid 3 is in close contact with the cup lid 3, it is possible to prevent the blood sample located on one side of the liquid pushing plate 2 from entering the other side of the liquid pushing plate 2 through the one end thereof adjacent to the cup lid 3 during the process of centrifuging the blood sample.

[0051] Preferably, a third fluid-passing structure is provided in the casing wall of the centrifugal cup 1 shown in this embodiment. A first end of the third fluid-passing structure is disposed on the outer surface of the cup bottom and distributed along the rotation axis, and a second end of the third fluid-passing structure is disposed on the inner surface of the cup bottom and communicates with the internal cavity of the centrifugal cup.

[0052] Specifically, the third fluid-passing structure allows a blood sample to be injected from the cup bottom into the internal cavity of the centrifuge cup 1. In response, the cell suspension can be sucked out by the third fluid-passing structure.

[0053] Preferably, as shown in Figures 7, 8 and 9, the first liquid passage structure 31 shown in this embodiment includes a connecting pipe 311. The connecting pipe 311 and the liquid passage pipe 111 are distributed along the rotation axis. A liquid passage 3111 and a gas passage 3112 are provided in the connecting pipe 311. A liquid connection port 3113 is provided at a first end of the liquid passage 3111, and a second end of the liquid passage 3111 is connected to a first end of the liquid passage pipe 111. A gas connection port 3114 is provided at a first end of the gas passage 3112, and a second end of the gas passage 3112 is connected to the internal cavity of the centrifuge cup 1.

[0054] Specifically, when the centrifuge cup 1 shown in this embodiment is used for cell culture, culture medium can be poured into the centrifuge cup through the liquid connection port 3113 and a certain concentration of carbon dioxide can be poured into the centrifuge cup through the gas connection port 3114 without opening the cup lid 3.

[0055] Note that liquid passage 3111 can be used not only for introducing liquid but also for introducing gas, and similarly gas passage 3112 can be used not only for introducing gas but also for introducing liquid. Liquid passage 3111 and gas passage 3112 are independent of each other, and when introducing liquid into one of liquid passage 3111 or gas passage 3112, gas can be introduced into the other at the same time, and the two operations of introducing liquid and gas do not interfere with each other.

[0056] In one embodiment, the liquid passageway 3111 and the gas passageway 3112 are arranged side by side.

[0057] In another embodiment, as shown in FIGS. 7 and 9, the liquid passage 3111 is provided in the gas passage 3112, and the second end of the gas passage 3112 is disposed along the rotation axis and communicates with the internal cavity of the centrifuge cup 1.

[0058] In yet another embodiment, as shown in FIG. 8, the second end of the gas passage 3112 extends along the radial direction of the centrifuge cup 1 and communicates with the internal cavity of the centrifuge cup 1.

[0059] Preferably, as shown in Figures 6, 9 and 10, the cup lid 3 shown in this embodiment has an air vent 32, at least one air vent 32 is provided, and the inside of the air vent 32 is used to provide an air-permeable membrane.

[0060] Specifically, when the centrifuge cup 1 shown in this embodiment is used for cell culture, the gas-permeable membrane can prevent external bacteria from entering the centrifuge cup 1, and at the same time, the gas-permeable membrane allows gas exchange between the inside and outside of the centrifuge cup 1, ensuring normal respiration of the cells in the centrifuge cup 1.

[0061] The gas-permeable membrane is preferably a hydrophobic gas-permeable membrane.

[0062] Preferably, as shown in Figures 7, 8, 9 and 10, the cup lid 3 shown in this embodiment includes a top cover 33 and an edge 34, the edge 34 extends along the circumferential direction of the top cover 33, the cup lid 3 is arranged to cover the cup mouth of the centrifugal cup 1, and the inner surface of the edge 34 is in close contact with the outer wall of the centrifugal cup 1.

[0063] Specifically, the cup lid 3 is arranged to cover the cup mouth of the centrifugal cup 1, and the inner surface of the edge 34 is in close contact with the outer wall of the centrifugal cup 1, thereby ensuring airtightness between the cup lid 3 and the centrifugal cup 1.

[0064] Preferably, as shown in Figures 4 to 6, when cells are cultured using the centrifuge cup 1, a plurality of observation holes 5 are opened in the centrifuge cup 1 or the cup lid 3 in order to easily observe the culture status of the cells in the centrifuge cup 1. During cell culture, the observation holes 5 are always closed, and when observation is required, the observation holes 5 are opened and connected to an optical monitoring device to perform the observation.

[0065] Preferably, this embodiment further provides a cell centrifugation method for the cell centrifuge device, as shown in Figure 11. This cell centrifugation method includes the following steps 102 to 103.

[0066] In step 102, the centrifugal cup is connected to a rotation drive mechanism, and a blood sample is added to the centrifugal cup.

[0067] In step 103, the rotary drive mechanism is turned on, and the rotary drive mechanism drives the centrifugal cup to rotate on its own axis, so that the liquid pushing plate pushes the blood sample and rotates synchronously in the same direction as the centrifugal cup, thereby obtaining a cell suspension.

[0068] Preferably, before step 102, the method further comprises step 101 of adding a preset volume of separation liquid into the centrifuge cup.

[0069] Preferably, this embodiment further provides a cell washing method for a cell centrifuge device, as shown in Figure 12. This cell washing method includes the following steps 201 and 202.

[0070] In step 201, when a blood sample is centrifuged using a cell centrifuge device, saline is introduced into the centrifuge cup through the second fluid-passing structure of the centrifuge cup, and the added saline washes away the surface of the cells in the blood sample during the centrifugation process.

[0071] In step 202, after the blood sample has been centrifuged for a predetermined period of time, the liquid in the centrifuge cup is sucked out through the second liquid-passing structure to discharge the waste liquid that has washed the cells.

[0072] Specifically, the centrifuge cup shown in this example can wash cells during the centrifugation process, improving the efficiency of washing, and the cells obtained after washing are used for culture.

[0073] Preferably, this embodiment further provides a cell culture method for a cell centrifuge device, as shown in Figure 13. This cell culture method includes the following steps 301 to 303.

[0074] In step 301, a cell suspension containing cells to be cultured is added into a centrifuge cup, the density of the cells in the cell suspension is adjusted to a first preset density, and antibodies or magnetic beads are added into the centrifuge cup to activate the cells.

[0075] In step 302, carbon dioxide gas of a predetermined concentration and a culture medium of a predetermined composition are poured into the centrifuge cup, and the centrifuge cup containing the cell suspension and the culture medium is placed in a predetermined constant temperature environment to perform cell culture.

[0076] In step 303, the density of cells in the centrifuge cup is detected, and if the density of cells is less than a second preset density, the rotary drive mechanism is turned off and the cells are statically cultured; if the density of cells is greater than the second preset density, the rotary drive mechanism is turned on and continues for a first preset time, and then turned off for a second preset time, and so on, to dynamically culture the cells.

[0077] The preset carbon dioxide concentration range is 2% to 7%, and may be 2%, 3%, 4%, 5%, 6%, or 7%. The temperature range of the constant temperature environment is 0 to 40°C, and may be 0°C, 10°C, 19°C, 20°C, 30°C, 37°C, or 40°C. The temperature of the constant temperature environment needs to be adaptively adjusted depending on the characteristics of different cells.

[0078] Preferably, steps 304 to 305 are further included after step 303 shown in this embodiment.

[0079] In step 304, metabolic waste products of the cells in the centrifuge cup are expelled from the centrifuge cup.

[0080] Step 305, monitor and record cell count and cell viability.

[0081] Specifically, under dynamic culture, the liquid push plate can effectively stir the culture medium to thoroughly mix the cells and the culture medium, and at the same time, the liquid push plate can effectively stir the cells to avoid concentrated cell accumulation and improve the cell culture effect.

[0082] The preset components of the medium include inorganic salts, a carbon source, a nitrogen source, interleukins, and trace elements. After the cells are cultured for a certain period of time, the cell density increases, and the first preset density becomes smaller than the second preset density. The first preset density range is 0.2×10 6 pieces / ml~2.0×10 6 The second preset density range is 2.0 x 10 6 pieces / ml~5.0×10 6 A specific method for adjusting the density of the cells in the cell suspension to the first preset density is to add a medium to the cell suspension to dilute the density of the cells in the cell suspension to the first preset density.

[0083] The first preset time is preferably 1 s, and the second preset time is preferably 6 s. In this case, the dynamic culture process is such that one cycle period of the centrifuge cup is 7 s, and the centrifuge cup first rotates for 1 s and then stops for 6 s. Here, the specific values ​​of the first preset time and the second preset time are not limited and can be adaptively adjusted according to the cell density.

[0084] Preferably, in order to compare the degree of influence on cell culture results after centrifuging cells with and without a liquid pusher plate, a control experiment is conducted in this embodiment, with a total of two control groups, each with five experimental samples.

[0085] In the first control group, the cells are centrifuged with the liquid pusher plate in place, and in the second control group, the cells are centrifuged without the liquid pusher plate in place.

[0086] After the centrifugation was completed, the cell yield of each experimental sample in the two control groups was detected respectively, and the detection results are shown in Table 1.

[0087] Table 1. Cell yield statistics after centrifugation: JPEG0007776889000001.jpg20170

[0088] The cell yield of the first control group was much higher than that of the second control group. In the second control group, the absence of a liquid pusher plate resulted in a large amount of cells being subjected to shear damage during centrifugation, resulting in a lower cell yield.

[0089] The cells after centrifugation were further cultured, and the number of cells initially cultured in both the first and second control groups was 50 million. The cell counts were counted daily, and the statistical results are shown in FIG.

[0090] The statistical results on day 14 showed that the cell number in the first control group had increased from the initial 50 million to 108 billion.

[0091] The statistical results on day 7 show that the number of cells in the second control group has increased from the initial 50 million to 20 billion. The statistical results on day 12 show that the number of cells in the second control group was 20 billion. The statistical results on day 14 show that the number of cells in the second control group had decreased to 19 billion.

[0092] Therefore, although the cell yield in the second control group was approximately 83% of that in the first control group after centrifugation, it can be concluded that a large number of cells in the second control group suffered varying degrees of shear damage, and that the shear-damaged cells had reduced biological activity and could not be cultured normally. Therefore, by installing a liquid pressure plate in the centrifuge cup, it is possible to effectively reduce the shear damage suffered by cells during centrifugation and ensure the effectiveness of cell culture.

[0093] It should be noted that the above embodiments are only for explaining the technical solutions of the present invention, and are not intended to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art may modify the technical solutions described in the above embodiments or replace some of the technical features with equivalents. These modifications and replacements do not deviate from the essence of the corresponding technical solutions and the spirit and scope of the technical solutions of the embodiments of the present invention. Industrial Applicability

[0094] The present invention provides a cell centrifuge device and a method for centrifugation, washing, and culturing cells. The cell centrifuge device includes a centrifuge cup and a liquid pusher plate. The centrifuge cup has a rotation axis. The centrifuge cup is connected to a rotation drive mechanism and rotates about the rotation axis when driven by the rotation drive mechanism, thereby centrifuging a blood sample contained in the centrifuge cup. One or more liquid pusher plates are provided. The liquid pusher plate is disposed within the centrifuge cup. When the centrifuge cup rotates, the liquid pusher plate is used to push the blood sample and rotate it synchronously along the same rotation direction as the centrifuge cup. By providing a liquid pusher plate within the centrifuge cup, the blood sample in the centrifuge cup is pushed by the liquid pusher plate to rotate synchronously with the centrifuge cup during the centrifugation process, thereby preventing cells in the blood sample from being subjected to shearing during the centrifugation process and reducing shear damage to the cells during the centrifugation process. This has relatively good economic value and potential for application. [Explanation of symbols]

[0095] 1: Centrifugal cup 11:Second liquid passage structure 111:Liquid pipe 112:Liquid passage 2: Liquid push plate 21: Pin shaft 3: Cup lid 31: 1st liquid passage structure 311: Connecting pipe 3111:Liquid passage 3112: Gas passage 3113: Liquid connection port 3114: Gas connection port 32: Ventilation hole 33: Cap 34: Edge 4: Rotation drive mechanism 5: Observation hole

Claims

1. A cytocentrifuge device comprising a centrifuge cup and a fluid pusher plate, the centrifugal cup has a rotation axis, the centrifugal cup is connected to a rotation drive mechanism, and is used to centrifuge a blood sample contained in the centrifugal cup by rotating about the rotation axis by being driven by the rotation drive mechanism; a first gap is provided between one end of the liquid push plate that is close to the inner wall of the centrifuge cup and the inner wall; a second gap is provided between one end of the liquid push plate that is close to the rotation shaft and the rotation shaft; a ratio range of the first gap to the inner radius of the centrifuge cup is 1 / 600 to 1 / 20, and a ratio range of the second gap to the inner radius of the centrifuge cup is 1 / 200 to 1 / 5; one end of the liquid push plate that is close to the rotation shaft is not connected to the rotation shaft, and one end of the liquid push plate that is close to the cup bottom of the centrifuge cup is connected to the cup bottom; and when the centrifuge cup rotates, the liquid push plate is used to push the blood sample and rotate it synchronously in the same rotation direction as the centrifuge cup.

2. One end of the liquid pushing plate is close to the rotation axis, and the other end extends toward the inner wall of the centrifugal cup; the liquid pushing plate has a first side edge that is horizontal to a plane on which the cup bottom of the centrifugal cup is located, and a second side edge that is perpendicular to the plane on which the cup bottom of the centrifugal cup is located, The projection of the first side edge of the liquid pushing plate onto a plane on which the cup bottom of the centrifugal cup is located is a straight line or an arc; 2. The cell centrifuge device according to claim 1, wherein when the projection of the first side edge of the liquid pusher plate onto a plane on which the cup bottom of the centrifuge cup is located forms a straight line, the first side edge of the liquid pusher plate extends along the radial direction of the centrifuge cup.

3. 3. The cell centrifuge according to claim 2, wherein one end of the liquid pushing plate that is close to the cup bottom of the centrifuge cup and the cup bottom are in close contact with each other.

4. 3. The cell centrifuge according to claim 2, wherein when there are a plurality of said liquid pushing plates, said plurality of liquid pushing plates are uniformly distributed around the circumference of said rotation axis.

5. The centrifugal cup further includes a cup lid, the cup lid being matched with a cup opening of the centrifugal cup, the cup lid being provided with a first liquid-passing structure, and the centrifugal cup being provided with a second liquid-passing structure; a first end of the first fluid-passing structure disposed on an outer surface of the cup lid and distributed along the rotation axis; a second end of the first fluid-passing structure disposed on an inner surface of the cup lid; 4. The cell centrifuge device according to claim 3, wherein the second fluid-passing structure includes a fluid-passing tube and a fluid-passing passage, the fluid-passing tube being disposed within the centrifuge cup, a first end of the fluid-passing tube communicating with a second end of the first fluid-passing structure, a second end of the fluid-passing tube being disposed at a cup bottom of the centrifuge cup, the fluid-passing passage being disposed within a casing wall of the cup bottom, a first end of the fluid-passing passage communicating with a second end of the fluid-passing tube, and a second end of the fluid-passing passage being disposed on an inner surface of the cup bottom and communicating with an internal cavity of the centrifuge cup.

6. a third fluid-passing structure is provided within a casing wall at the cup bottom of the centrifugal cup; 4. The cell centrifuge device according to claim 3, wherein a first end of the third fluid-passing structure is disposed on an outer surface of the cup bottom and distributed along the rotation axis, and a second end of the third fluid-passing structure is disposed on an inner surface of the cup bottom and communicates with the internal cavity of the centrifuge cup.

7. the first fluid passage structure includes a connecting pipe, 6. The cell centrifuge apparatus according to claim 5, wherein the connecting pipe and the liquid passage pipe are distributed along the rotation axis, the connecting pipe has a liquid passage and a gas passage, a first end of the liquid passage is provided with a liquid connection port, a second end of the liquid passage is connected to the first end of the liquid passage pipe, a first end of the gas passage is provided with a gas connection port, and a second end of the gas passage is connected to the internal cavity of the centrifuge cup.

8. The cup lid has a vent hole, 6. The cell centrifuge according to claim 5, wherein at least one vent hole is provided, and the inside of the vent hole is used to provide a vent membrane.

9. A cell centrifugation method based on the cell centrifuge device of claim 1, connecting the centrifuge cup to a rotation drive mechanism and adding a blood sample into the centrifuge cup; and a step of turning on the rotary drive mechanism and using the rotary drive mechanism to drive the centrifuge cup to rotate on its own axis, so that the liquid pushing plate pushes the blood sample and rotates synchronously in the same rotational direction as the centrifuge cup, thereby obtaining a cell suspension.

10. A cell centrifugation method based on the cell centrifuge device according to claim 2, comprising: connecting the centrifuge cup to a rotation drive mechanism and adding a blood sample into the centrifuge cup; and a step of turning on the rotary drive mechanism and using the rotary drive mechanism to drive the centrifuge cup to rotate on its own axis, so that the liquid pushing plate pushes the blood sample and rotates synchronously in the same rotational direction as the centrifuge cup, thereby obtaining a cell suspension.

11. A cell centrifugation method based on the cell centrifuge device according to claim 3, comprising: connecting the centrifuge cup to a rotation drive mechanism and adding a blood sample into the centrifuge cup; and a step of turning on the rotary drive mechanism and using the rotary drive mechanism to drive the centrifuge cup to rotate on its own axis, so that the liquid pushing plate pushes the blood sample and rotates synchronously in the same rotational direction as the centrifuge cup, thereby obtaining a cell suspension.

12. A cell centrifugation method based on the cell centrifuge device according to claim 4, connecting the centrifuge cup to a rotation drive mechanism and adding a blood sample into the centrifuge cup; and a step of turning on the rotary drive mechanism and using the rotary drive mechanism to drive the centrifuge cup to rotate on its own axis, so that the liquid pushing plate pushes the blood sample and rotates synchronously in the same rotational direction as the centrifuge cup, thereby obtaining a cell suspension.

13. A cell washing method based on the cell centrifuge device of claim 1, comprising: When centrifuging the blood sample using the cell centrifuge, introducing physiological saline into the centrifuge cup through the second fluid-passing structure of the centrifuge cup, thereby washing the surface of the cells in the blood sample with the added physiological saline during the centrifugation process; and after the time for centrifuging the blood sample reaches a predetermined length of time, sucking out the liquid in the centrifuge cup through the second fluid passage structure and discharging the waste liquid used to wash the cells.

14. A cell washing method based on the cell centrifuge device of claim 2, comprising: When centrifuging the blood sample using the cell centrifuge, introducing physiological saline into the centrifuge cup through the second fluid-passing structure of the centrifuge cup, thereby washing the surface of the cells in the blood sample with the added physiological saline during the centrifugation process; and after the time for centrifuging the blood sample reaches a predetermined length of time, sucking out the liquid in the centrifuge cup through the second fluid passage structure and discharging the waste liquid used to wash the cells.

15. A cell washing method based on the cell centrifuge device of claim 3, When centrifuging the blood sample using the cell centrifuge, introducing physiological saline into the centrifuge cup through the second fluid-passing structure of the centrifuge cup, thereby washing the surface of the cells in the blood sample with the added physiological saline during the centrifugation process; and after the time for centrifuging the blood sample reaches a predetermined length of time, sucking out the liquid in the centrifuge cup through the second fluid passage structure and discharging the waste liquid used to wash the cells.

16. A cell washing method based on the cell centrifuge device according to claim 4, comprising: When centrifuging the blood sample using the cell centrifuge, introducing physiological saline into the centrifuge cup through the second fluid-passing structure of the centrifuge cup, thereby washing the surface of the cells in the blood sample with the added physiological saline during the centrifugation process; and after the time for centrifuging the blood sample reaches a predetermined length of time, sucking out the liquid in the centrifuge cup through the second fluid passage structure and discharging the waste liquid used to wash the cells.

17. A cell culture method based on the cell centrifuge device of claim 1, Adding a cell suspension containing cells to be cultured into a centrifuge cup, adjusting the density of cells in the cell suspension to a first preset density, and adding antibodies or magnetic beads into the centrifuge cup to activate the cells; a step of introducing carbon dioxide gas of a predetermined concentration and a culture medium of a predetermined composition into a centrifuge cup, and placing the centrifuge cup containing the cell suspension and the culture medium in a predetermined constant temperature environment to perform cell culture; 1. A cell culture method comprising: detecting a density of cells in a centrifuge cup; and, if the density of the cells is less than a second preset density, turning off a rotary drive mechanism and statically culturing the cells; and, if the density of the cells is greater than the second preset density, turning on the rotary drive mechanism, continuing for a first preset time, and then turning it off for a second preset time, repeating this process to dynamically culture the cells.

18. A cell culture method based on the cell centrifuge device of claim 2, comprising: Adding a cell suspension containing cells to be cultured into a centrifuge cup, adjusting the density of cells in the cell suspension to a first preset density, and adding antibodies or magnetic beads into the centrifuge cup to activate the cells; a step of introducing carbon dioxide gas of a predetermined concentration and a culture medium of a predetermined composition into a centrifuge cup, and placing the centrifuge cup containing the cell suspension and the culture medium in a predetermined constant temperature environment to perform cell culture; 1. A cell culture method comprising: detecting a density of cells in a centrifuge cup; and, if the density of the cells is less than a second preset density, turning off a rotary drive mechanism and statically culturing the cells; and, if the density of the cells is greater than the second preset density, turning on the rotary drive mechanism, continuing for a first preset time, and then turning it off for a second preset time, repeating this process to dynamically culture the cells.

19. A cell culture method based on the cell centrifuge device according to claim 3, Adding a cell suspension containing cells to be cultured into a centrifuge cup, adjusting the density of cells in the cell suspension to a first preset density, and adding antibodies or magnetic beads into the centrifuge cup to activate the cells; a step of introducing carbon dioxide gas of a predetermined concentration and a culture medium of a predetermined composition into a centrifuge cup, and placing the centrifuge cup containing the cell suspension and the culture medium in a predetermined constant temperature environment to perform cell culture; 1. A cell culture method comprising: detecting a density of cells in a centrifuge cup; and, if the density of the cells is less than a second preset density, turning off a rotary drive mechanism and statically culturing the cells; and, if the density of the cells is greater than the second preset density, turning on the rotary drive mechanism, continuing for a first preset time, and then turning it off for a second preset time, repeating this process to dynamically culture the cells.

20. A cell culture method based on the cell centrifuge device according to claim 4, Adding a cell suspension containing cells to be cultured into a centrifuge cup, adjusting the density of cells in the cell suspension to a first preset density, and adding antibodies or magnetic beads into the centrifuge cup to activate the cells; a step of introducing carbon dioxide gas of a predetermined concentration and a culture medium of a predetermined composition into a centrifuge cup, and placing the centrifuge cup containing the cell suspension and the culture medium in a predetermined constant temperature environment to perform cell culture; 1. A cell culture method comprising: detecting a density of cells in a centrifuge cup; and, if the density of the cells is less than a second preset density, turning off a rotary drive mechanism and statically culturing the cells; and, if the density of the cells is greater than the second preset density, turning on the rotary drive mechanism, continuing for a first preset time, and then turning it off for a second preset time, repeating this process to dynamically culture the cells.

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