Portable in vitro blood cell gene modification system

Abstract

A portable in vitro blood cell gene editing or modification system comprises: a fluid inlet (111) and a fluid outlet (112); processing units (1200, 1201, 1202, 1203), wherein the processing units (1200, 1201, 1202, 1203) are configured to perform gene editing or gene modification on target cells in the blood input through the fluid inlet (111) by gene delivery and are also configured to remove non-treatment-requiring substances in the treated blood, and the processing units (1200, 1201, 1202, 1203) are fluidically connected between the fluid inlet (111) and the fluid outlet (112).

Description

Technical Field

[0001] The present application relates to an in vitro blood cell therapy apparatus and system, in particular to an in vitro blood cell gene editing or gene modification system for treating diseases based on gene editing or gene modification technology. Background Art

[0002] Currently, cell therapy products based on gene editing or gene modification technology are required to be prepared in cell preparation factories that meet certified clean standards, such as chimeric antigen receptor T cell immunotherapy CAR-T (Chimeric Antigen Receptor T-Cell Immunotherapy), T cell receptor T cell immunotherapy (T-Cell Receptor T-Cell Immunotherapy, TCR-T), and synthetic T cell antigen receptor T cell immunotherapy (Synthetic T-Cell Antigen Receptor T-Cell Immunotherapy, STAR-T) and other different types of cell therapy products.

[0003] However, the preparation process for these cell products is lengthy, costly, and requires significant investment. Taking CAR-T as an example, the current mainstream process involves blood collection at a hospital, followed by transportation to a GMP-grade production facility for peripheral blood mononuclear lymphocyte (PBMC) isolation, T cell purification, T cell activation, and CAR gene transduction. This is followed by amplification, culture, concentration, filling, and cryopreservation. After quality assurance, the cells are then transported via a cold chain to the hospital for reinfusion into the patient. The entire process is complex, with numerous environmental exposure steps, leading to increased quality control and further increasing production costs. Therefore, there is an urgent need for an integrated, rapid cell preparation system that bypasses the complex process flow and instead directly performs rapid gene transduction on blood cells, thereby enabling the preparation of gene-modified cell products. These blood cells can include T cells, B cells, NK cells, macrophages, monocytes, and innate lymphoid cells. This system does not require complicated separation and activation steps. Instead, it directly performs real-time, online, rapid, and fully closed gene modification or gene delivery on PBMC cells after collecting them in vitro, thereby achieving rapid preparation of CAR-T, CAR-NK, TCR-T and other cell products, greatly shortening the preparation time of these cell products and reducing the preparation cost of gene-edited cell products. Summary of the Invention

[0004] In response to the above problems, this application aims to propose a novel genetically engineered cell therapy system, including equipment for collecting patient cells, cleaning PBMCs, injecting reagents, cleaning gene-edited cells, and supporting equipment for reinfusion of edited cells, as well as methods for PBMC separation, PBMC cell transduction, post-transduction PBMC cleaning, and dilution and reinfusion of new gene-modified lymphocyte products performed on this system. CAR-T, CAR-NK, TCR-T, etc. can all be produced using this system. Compared with traditional preparation methods in cell factories, there is no need to perform steps such as T cell separation, purification, activation, and amplification. Only one transfection step is required to complete the preparation of these cell products, thereby shortening the preparation cycle of gene-modified or gene-edited cell products, and thus shortening the treatment cycle, ensuring that patients can obtain cells for treatment in a timely manner, thereby increasing their chances of survival.

[0005] According to one aspect of the present application, an extracorporeal blood cell therapy apparatus is provided, which has a host, the host comprising:

[0006] case;

[0007] a fluid inlet and a fluid outlet provided in the housing;

[0008] a first processing unit located inside the housing, the first processing unit being configured to perform gene editing or gene modification on target cells in the blood input through the fluid inlet by gene delivery; and

[0009] a second processing unit located inside the housing, wherein the second processing unit is configured to remove substances not requiring treatment from the blood processed by the first processing unit;

[0010] The first processing unit and the second processing unit are fluidly connected in series between the fluid inlet and the fluid outlet in sequence.

[0011] Optionally, the first processing unit comprises a container for containing liquid, the fluid inlet is fluidically connected to the container, the container is fluidically connected to the second processing unit via a pipeline, and the second processing unit is fluidically connected to the fluid outlet.

[0012] Optionally, the first processing unit includes at least a first injection module and a second injection module that are independent of each other, the first injection module stores a first reagent that can be selectively injected into the container, and the second injection module stores a second reagent that can be selectively injected into the container.

[0013] Optionally, the first reagent comprises a buffer, and the second reagent comprises a gene delivery reagent.

[0014] Optionally, the gene delivery reagent is used to achieve transduction or transfection of target cells.

[0015] Optionally, at least one of the injection modules is detachably mounted within the housing.

[0016] Optionally, the second processing unit includes a filter, a first recovery end located downstream of the filter, and a second recovery end located upstream of the filter.

[0017] Optionally, the first processing unit further comprises a shaking mechanism, by means of which the container can be selectively shaken.

[0018] Optionally, a first switch that is selectively turned on and off is provided at the first recovery end, and a second switch that is selectively turned on and off is provided at the second recovery end.

[0019] Optionally, a recovery module is fluidly connected to the first recovery end via a pipeline so that liquid can be recovered from the second treatment unit, in particular from the downstream of the filter, when the first switch is in the on state; and / or, a pipeline is configured to fluidly connect the container to the second recovery end so that liquid can be recovered from the second treatment unit, in particular from the upstream of the filter, when the second switch is in the on state.

[0020] Optionally, the second treatment unit further includes a waste liquid outlet port located downstream of the filter so that the waste liquid filtered by the filter can be discharged from the second treatment unit; and an output port located at the filter that is different from the second recovery port so that the suspension retained by the filter can be discharged from the second treatment unit.

[0021] Optionally, a third switch that can be selectively turned on and off is provided at the waste liquid outlet end, and a fourth switch that can be selectively turned on and off is provided at the output end.

[0022] Optionally, the fluid outlet is fluidically connected to the output end, so that the suspension retained by the filter can be discharged into the fluid outlet when the fourth switch is in an on state.

[0023] Optionally, the container comprises a fluid outlet end, and a pipeline connecting the container to the second processing unit fluid is connected to the fluid outlet end.

[0024] Optionally, a fifth switch that is selectively turned on and off is provided at the fluid outlet end, so that the liquid in the container can flow to the second processing unit through the fluid outlet end only when the fifth switch is in an on state.

[0025] Optionally, the filter is a molecular sieve, a centrifugal filter device, a magnetic screening filter device, a chromatography column, or a filter membrane.

[0026] Optionally, the filter is configured such that target cells intended to be transduced or transfected are retained upstream of the filter.

[0027] Optionally, at least when the fifth switch is in the off state, blood can be input into the first processing unit, in particular, into the container of the first processing unit, through the fluid inlet.

[0028] Optionally, a sorting module is provided upstream of the first processing unit, the inlet of the sorting module is fluidly connected to the fluid inlet, and the outlet of the sorting module is fluidly connected to the first processing unit via a pipeline. The sorting module is configured to exclude substances other than the target cells from the blood before the blood is input into the first processing unit.

[0029] Optionally, the sorting module can exclude substances other than the target cells from the blood by physical or biological means.

[0030] Optionally, the extracorporeal blood cell therapy apparatus further includes a movable base and pillars provided on the base, wherein the pillars are configured to support the shell.

[0031] Optionally, the extracorporeal blood cell therapy apparatus further includes a display for displaying data monitored during the operation of the extracorporeal blood cell therapy apparatus; and an input device for setting operating parameters of the extracorporeal blood cell therapy apparatus.

[0032] Optionally, the gene delivery agent includes a viral vector or a non-viral vector.

[0033] Optionally, the non-therapeutic substance comprises an agent for gene delivery.

[0034] Optionally, the target cells include but are not limited to T cells, B cells, NK cells, macrophages, monocytes, or innate lymphoid cells.

[0035] According to another aspect of the present application, an extracorporeal blood cell therapy apparatus is provided, which has a host, the host comprising:

[0036] case;

[0037] a fluid inlet and a fluid outlet provided in the housing;

[0038] A processing unit is located inside the housing, wherein the processing unit is configured to perform gene editing or gene modification on target cells in the blood input through the fluid inlet by gene delivery and is also configured to remove non-treatment-requiring substances in the treated blood.

[0039] The processing unit is fluidly connected between the fluid inlet and the fluid outlet.

[0040] Optionally, the processing unit includes a container for containing liquid, and the processing unit also includes at least a first injection module and a second injection module independent of each other, the first injection module stores a first reagent that can be selectively injected into the container, and the second injection module stores a second reagent that can be selectively injected into the container.

[0041] Optionally, the first reagent comprises a buffer, and the second reagent comprises a gene delivery reagent.

[0042] Optionally, the gene delivery reagent is used to achieve transduction or transfection of target cells.

[0043] Optionally, at least one of the injection modules is detachably mounted within the housing.

[0044] Optionally, a filter is provided in the container, the container comprises a fluid input end fluidly connected to the fluid inlet; and a fluid output end fluidly connected to the fluid outlet, the fluid input end and the fluid output end are located upstream of the filter.

[0045] Optionally, the first injection module and / or the second injection module are configured to selectively inject the respective reagents into the container upstream of the filter.

[0046] Optionally, the first injection module and / or the second injection module are configured to selectively inject the respective reagents into the container downstream of the filter.

[0047] Optionally, a first switch that is selectively turned on and off is provided at the fluid output end, so that the liquid in the container can be discharged through the fluid outlet when the first switch is in an on state.

[0048] Optionally, the container further includes a waste liquid outlet port and a recovery port located downstream of the filter and independent of each other, a second switch that can be selectively turned on and off is provided at the recovery port, and a third switch that can be selectively turned on and off is provided at the waste liquid outlet port.

[0049] Optionally, a recovery module is fluidically connected to the recovery end via a pipeline, so that liquid can be recovered from the container, in particular, from the downstream of the filter when the second switch is in the on state.

[0050] Optionally, the processing unit further comprises a pressure difference generating device for selectively generating a pressure difference between upstream and downstream of the filter to force the liquid to flow through the filter.

[0051] Optionally, the filter is a molecular sieve, a centrifugal filter device, a magnetic screening filter device, a chromatography column, or a filter membrane.

[0052] Optionally, the filter is configured such that target cells intended to be transduced or transfected are retained upstream of the filter.

[0053] Optionally, the processing unit further comprises a shaking mechanism, by means of which the container can be selectively shaken.

[0054] Optionally, a sorting module is provided upstream of the processing unit, the inlet of the sorting module is fluidly connected to the fluid inlet, and the outlet of the sorting module is fluidly connected to the processing unit via a pipeline. The sorting module is configured to exclude substances other than the target cells from the blood before the blood is input into the processing unit.

[0055] Optionally, the sorting module can exclude substances other than the target cells from the blood by physical or biological means.

[0056] Optionally, when the first switch, the second switch, and the third switch are all in an off state, blood is input into the container through the fluid inlet.

[0057] Optionally, after the blood is input into the container, a buffer solution is selectively injected into the container via the first injection module to ensure that there is at least an amount of liquid sufficient to dilute the blood upstream of the filter; simultaneously or subsequently, the shaking mechanism is activated to selectively shake the container.

[0058] Optionally, the third switch is turned on to allow at least a portion of the liquid downstream of the filter to be discharged through the waste liquid outlet.

[0059] Optionally, when the first switch, the second switch, and the third switch are all in the off state, the transduction or infection reagent is selectively injected into the container via the second injection module so that there is an amount of liquid upstream of the filter that is sufficient to immerse the target cells to be transduced or transfected; simultaneously or subsequently, the shaking mechanism is activated to selectively shake the container.

[0060] Optionally, after a predetermined period of time since the transduction or infection reagent is injected, the second switch is turned on so that at least a portion of the liquid downstream of the filter can be output to the recovery module via the recovery end.

[0061] Optionally, after the liquid is recovered, the remaining liquid in the container is flushed with a buffer solution while the first switch, the second switch, and the third switch are all in an off state.

[0062] Optionally, flushing is accomplished as follows:

[0063] is selectively injected into the container via the first injection module to ensure that a predetermined amount of liquid is present upstream of the filter, and simultaneously or subsequently, the shaking mechanism is activated to selectively shake the container;

[0064] Then, the third switch is turned on to allow at least a portion of the liquid downstream of the filter to be discharged through the waste liquid outlet.

[0065] Optionally, the flushing is performed multiple times.

[0066] Optionally, after the last flushing is completed, the first switch is turned on to allow at least a portion of the liquid upstream of the filter to be discharged through the fluid outlet.

[0067] Optionally, the extracorporeal blood cell therapy apparatus further includes a movable base and pillars provided on the base, wherein the pillars are configured to support the shell.

[0068] Optionally, the extracorporeal blood cell therapy apparatus further includes a display for displaying data monitored during the operation of the extracorporeal blood cell therapy apparatus; and an input device for setting operating parameters of the extracorporeal blood cell therapy apparatus.

[0069] Optionally, the gene delivery agent includes a viral vector or a non-viral vector.

[0070] Optionally, the non-therapeutic substance comprises an agent for gene delivery.

[0071] Optionally, the target cells include but are not limited to T cells, B cells, NK cells, macrophages, monocytes, or innate lymphoid cells.

[0072] According to another aspect of the present application, a portable in vitro blood cell gene editing or modification system is provided, comprising:

[0073] a first processing unit configured to perform gene editing or gene modification on target cells in the blood introduced into the system by gene delivery; and

[0074] a second processing unit, wherein the second processing unit is configured to remove substances not requiring treatment from the blood processed by the first processing unit;

[0075] The first processing unit and the second processing unit are fluidically connected in series.

[0076] Optionally, the first processing unit comprises a container for containing liquid, and the container is fluidically connected to the second processing unit via a pipeline.

[0077] Optionally, the first processing unit includes at least a first injection module and a second injection module that are independent of each other, the first injection module stores a first reagent that can be selectively injected into the container, and the second injection module stores a second reagent that can be selectively injected into the container.

[0078] Optionally, the first reagent comprises a buffer, and the second reagent comprises a gene delivery reagent.

[0079] Optionally, the gene delivery reagent is used to achieve transduction or transfection of target cells.

[0080] Optionally, at least one of the injection modules is mounted in a detachable manner.

[0081] Optionally, the second processing unit includes a filter, a first recovery end located downstream of the filter, and a second recovery end located upstream of the filter.

[0082] Optionally, the first processing unit further comprises a shaking mechanism, by means of which the container can be selectively shaken.

[0083] Optionally, a first switch that is selectively turned on and off is provided at the first recovery end, and a second switch that is selectively turned on and off is provided at the second recovery end.

[0084] Optionally, a recovery module is fluidly connected to the first recovery end via a pipeline so that liquid can be recovered from the second treatment unit, in particular from the downstream of the filter, when the first switch is in the on state; and / or, a pipeline is configured to fluidly connect the container to the second recovery end so that liquid can be recovered from the second treatment unit, in particular from the upstream of the filter, when the second switch is in the on state.

[0085] Optionally, the second treatment unit further includes a waste liquid outlet port located downstream of the filter so that the waste liquid filtered by the filter can be discharged from the second treatment unit; and an output port located at the filter that is different from the second recovery port so that the suspension retained by the filter can be discharged from the second treatment unit.

[0086] Optionally, a third switch that can be selectively turned on and off is provided at the waste liquid outlet end, and a fourth switch that can be selectively turned on and off is provided at the output end.

[0087] Optionally, the fluid outlet is fluidically connected to the output end, so that the suspension retained by the filter can be discharged into the fluid outlet when the fourth switch is in an on state.

[0088] Optionally, the container comprises a fluid outlet end, and a pipeline connecting the container to the second processing unit fluid is connected to the fluid outlet end.

[0089] Optionally, a fifth switch that is selectively turned on and off is provided at the fluid outlet end, so that the liquid in the container can flow to the second processing unit through the fluid outlet end only when the fifth switch is in an on state.

[0090] Optionally, the filter is a molecular sieve, a centrifugal filter device, a magnetic screening filter device, a chromatography column, or a filter membrane.

[0091] Optionally, the filter is configured such that target cells intended to be transduced or transfected are retained upstream of the filter.

[0092] Optionally, at least when the fifth switch is in the off state, blood can be input into the first processing unit, in particular, into the container of the first processing unit, through the fluid inlet.

[0093] Optionally, a sorting module is provided upstream of the first processing unit, the inlet of the sorting module is fluidly connected to the fluid inlet, and the outlet of the sorting module is fluidly connected to the first processing unit via a pipeline. The sorting module is configured to exclude substances other than the target cells from the blood before the blood is input into the first processing unit.

[0094] Optionally, the sorting module can exclude substances other than the target cells from the blood by physical or biological means.

[0095] Optionally, the portable extracorporeal blood cell therapy system further includes a movable base and a support arranged on the base.

[0096] Optionally, the portable in vitro blood cell gene editing or modification system further includes a display for displaying data monitored during the operation of the in vitro blood cell therapy apparatus; and an input device for setting operating parameters of the in vitro blood cell therapy apparatus.

[0097] Optionally, the gene delivery agent includes a viral vector or a non-viral vector.

[0098] Optionally, the non-therapeutic substance comprises an agent for gene delivery.

[0099] Optionally, the target cells include but are not limited to T cells, B cells, NK cells, macrophages, monocytes, or innate lymphoid cells.

[0100] According to another aspect of the present application, a portable in vitro blood cell gene editing or modification system is also provided, comprising:

[0101] a fluid inlet and a fluid outlet;

[0102] a processing unit configured to perform gene editing or gene modification on target cells in the blood input through the fluid inlet by gene delivery and further configured to remove non-treatment-requiring substances in the treated blood,

[0103] The processing unit is fluidly connected between the fluid inlet and the fluid outlet.

[0104] Optionally, the processing unit includes a container for containing liquid, and the processing unit also includes at least a first injection module and a second injection module independent of each other, the first injection module stores a first reagent that can be selectively injected into the container, and the second injection module stores a second reagent that can be selectively injected into the container.

[0105] Optionally, the first reagent comprises a buffer, and the second reagent comprises a gene delivery reagent.

[0106] Optionally, the gene delivery reagent is used to achieve transduction or transfection of target cells.

[0107] Optionally, at least one of the injection modules is mounted in a detachable manner.

[0108] Optionally, a filter is provided in the container, the container comprises a fluid input end fluidly connected to the fluid inlet; and a fluid output end fluidly connected to the fluid outlet, the fluid input end and the fluid output end are located upstream of the filter.

[0109] Optionally, the first injection module and / or the second injection module are configured to selectively inject the respective reagents into the container upstream of the filter.

[0110] Optionally, the first injection module and / or the second injection module are configured to selectively inject the respective reagents into the container downstream of the filter.

[0111] Optionally, a first switch that is selectively turned on and off is provided at the fluid output end, so that the liquid in the container can be discharged through the fluid outlet when the first switch is in an on state.

[0112] Optionally, the container further includes a waste liquid outlet port and a recovery port located downstream of the filter and independent of each other, a second switch that can be selectively turned on and off is provided at the recovery port, and a third switch that can be selectively turned on and off is provided at the waste liquid outlet port.

[0113] Optionally, a recovery module is fluidically connected to the recovery end via a pipeline, so that liquid can be recovered from the container, in particular, from the downstream of the filter when the second switch is in the on state.

[0114] Optionally, the processing unit further comprises a pressure difference generating device for selectively generating a pressure difference between upstream and downstream of the filter to force the liquid to flow through the filter.

[0115] Optionally, the filter is a molecular sieve, a centrifugal filter device, a magnetic screening filter device, a chromatography column, or a filter membrane.

[0116] Optionally, the filter is configured such that target cells intended to be transduced or transfected are retained upstream of the filter.

[0117] Optionally, the processing unit further comprises a shaking mechanism, by means of which the container can be selectively shaken.

[0118] Optionally, a sorting module is provided upstream of the processing unit, the inlet of the sorting module is fluidly connected to the fluid inlet, and the outlet of the sorting module is fluidly connected to the processing unit via a pipeline. The sorting module is configured to exclude substances other than the target cells from the blood before the blood is input into the processing unit.

[0119] Optionally, the sorting module can exclude substances other than the target cells from the blood by physical or biological means.

[0120] Optionally, when the first switch, the second switch, and the third switch are all in an off state, blood is input into the container through the fluid inlet.

[0121] Optionally, after the blood is input into the container, a buffer solution is selectively injected into the container via the first injection module to ensure that there is at least an amount of liquid sufficient to dilute the blood upstream of the filter; simultaneously or subsequently, the shaking mechanism is activated to selectively shake the container.

[0122] Optionally, the third switch is turned on to allow at least a portion of the liquid downstream of the filter to be discharged through the waste liquid outlet.

[0123] Optionally, when the first switch, the second switch, and the third switch are all in the off state, the transduction or infection reagent is selectively injected into the container via the second injection module so that there is an amount of liquid upstream of the filter that is sufficient to immerse the target cells to be transduced or transfected; simultaneously or subsequently, the shaking mechanism is activated to selectively shake the container.

[0124] Optionally, after a predetermined period of time since the transduction or infection reagent is injected, the second switch is turned on so that at least a portion of the liquid downstream of the filter can be output to the recovery module via the recovery end.

[0125] Optionally, after the liquid is recovered, the remaining liquid in the container is flushed with a buffer solution while the first switch, the second switch, and the third switch are all in an off state.

[0126] Optionally, flushing is accomplished as follows:

[0127] is selectively injected into the container via the first injection module to ensure that a predetermined amount of liquid is present upstream of the filter, and simultaneously or subsequently, the shaking mechanism is activated to selectively shake the container;

[0128] Then, the third switch is turned on to allow at least a portion of the liquid downstream of the filter to be discharged through the waste liquid outlet.

[0129] Optionally, the flushing is performed multiple times.

[0130] Optionally, after the last flushing is completed, the first switch is turned on to allow at least a portion of the liquid upstream of the filter to be discharged through the fluid outlet.

[0131] Optionally, the portable in vitro blood cell gene editing or modification system further includes a movable base and a support arranged on the base.

[0132] Optionally, the portable in vitro blood cell gene editing or modification system further includes a display for displaying data monitored during the operation of the in vitro blood cell therapy apparatus; and an input device for setting operating parameters of the in vitro blood cell therapy apparatus.

[0133] Optionally, the gene delivery agent includes a viral vector or a non-viral vector.

[0134] Optionally, the non-therapeutic substance comprises an agent for gene delivery.

[0135] Optionally, the target cells include but are not limited to T cells, B cells, NK cells, macrophages, monocytes, or innate lymphoid cells.

[0136] According to another aspect of the present application, there is also provided an application of the aforementioned extracorporeal blood cell therapy apparatus or the aforementioned portable extracorporeal blood cell gene editing or modification system for blood treatment.

[0137] According to another aspect of the present application, a portable in vitro blood cell gene editing or modification system is provided, comprising:

[0138] a fluid inlet and a fluid outlet;

[0139] a processing unit configured to perform gene editing or gene modification on resting or non-activated target cells in the blood input through the fluid inlet by gene delivery and further configured to remove non-therapeutic substances in the treated blood cells,

[0140] The processing unit is fluidly connected between the fluid inlet and the fluid outlet.

[0141] Optionally, the processing unit includes a container for containing liquid, and the processing unit also includes at least a first injection module and a second injection module that are independent of each other, the first injection module stores a first reagent that can be selectively injected into the container, and the second injection module stores a second reagent that can be selectively injected into the container, so that the target cells in the blood input into the container are gene-edited or gene-modified through gene delivery without leaving the container, and non-treatment-required substances in the treated blood are removed.

[0142] Optionally, the first reagent comprises a buffer, and the second reagent comprises a gene delivery reagent.

[0143] Optionally, the gene delivery reagent is used to achieve transduction or transfection of target cells without separation, purification, activation or amplification.

[0144] Optionally, a filter is provided in the container, the container comprises a fluid input end fluidly connected to the fluid inlet; and a fluid output end fluidly connected to the fluid outlet, the fluid input end and the fluid output end are located upstream of the filter.

[0145] Optionally, the first injection module and / or the second injection module are configured to selectively inject the respective reagents into the container upstream of the filter.

[0146] Optionally, the first injection module and / or the second injection module are configured to selectively inject the respective reagents into the container downstream of the filter.

[0147] Optionally, the filter is a molecular sieve, a magnetic screening filter device, a chromatography column, or a filter membrane.

[0148] Optionally, the processing unit is a separation device for separating PBMCs using density gradient centrifugation, and the container of the processing unit includes a first container that can selectively rotate around a rotation axis.

[0149] Optionally, the container of the processing unit also includes a second container independent of the first container, Ficoll cell separation fluid is injected into the first container and as the first container rotates, different liquid component layers are generated in the radial direction relative to the rotation axis, the liquid containing the substance to be treated is drawn into the second container, and the second reagent is injected into the second container.

[0150] Optionally, the axis of rotation is the axis of rotation of the container itself.

[0151] Optionally, before the second reagent is injected into the first container, Ficoll cell separation fluid is injected into the first container and as the first container rotates, different liquid component layers are generated in the radial direction relative to the rotation axis, leaving only substances in the blood that need to be treated in the first container to mix with the second reagent that is subsequently injected.

[0152] Optionally, the gene delivery agent includes a viral vector or a non-viral vector.

[0153] Optionally, the non-viral vector comprises a synthetic vector or a biological vector; and / or, the viral vector comprises a retrovirus or a modified or mutant thereof, a lentivirus or a modified or mutant thereof, an adenovirus or a modified or mutant thereof, or an adeno-associated virus or a modified or mutant thereof.

[0154] Optionally, the synthetic carrier is a lipid nanoparticle (LNP) or a lipid polyplex (LPP), and the biological carrier is an extracellular vesicle.

[0155] Optionally, the target cells include but are not limited to T cells, B cells, NK cells, macrophages, monocytes, or innate lymphoid cells.

[0156] Optionally, a sorting module is provided upstream of the processing unit, the inlet of the sorting module is fluidly connected to the fluid inlet, and the outlet of the sorting module is fluidly connected to the processing unit via a pipeline. The sorting module is configured to exclude substances other than the target cells from the blood before the blood is input into the processing unit.

[0157] Optionally, the sorting module can exclude substances other than the target cells from the blood by physical or biological means.

[0158] Optionally, the portable in vitro blood cell gene editing or modification system has a host, the host has a shell, and the processing unit, the first injection module, the second injection module and the sorting module are arranged in the shell.

[0159] Optionally, the non-viral vector comprises a synthetic vector or a biological vector; and / or, the viral vector comprises a retrovirus or a modified or mutant thereof, a lentivirus or a modified or mutant thereof, an adenovirus or a modified or mutant thereof, or an adeno-associated virus or a modified or mutant thereof.

[0160] Optionally, the synthetic carrier is a lipid nanoparticle (LNP) or a lipid polyplex (LPP), and the biological carrier is an extracellular vesicle.

[0161] Optionally, the target cells include but are not limited to T cells, B cells, NK cells, macrophages, monocytes, or innate lymphoid cells.

[0162] Optionally, the gene delivery reagent includes a CAR gene to transduce or transfect T cells in PBMCs.

[0163] Optionally, the time for transducing or transfecting the target cells using the gene delivery agent without separation, purification, activation or amplification is 1 to 5 hours.

[0164] According to another aspect of the present application, there is provided an in vitro blood cell therapy method comprising:

[0165] Blood will be collected from the patient using an apheresis machine;

[0166] Injecting blood into the portable in vitro blood cell gene editing or modification system or in vitro blood cell therapy apparatus, thereby subjecting the blood to gene editing or gene modification of target cells in the injected blood by gene delivery without the blood leaving the portable in vitro blood cell gene editing or modification system or in vitro blood cell therapy apparatus, and further configuring the apparatus to remove substances not requiring treatment from the treated blood;

[0167] The treated fluid is returned to the patient.

[0168] Optionally, the portable in vitro blood cell gene editing or modification system or in vitro blood cell therapy device includes a container for containing liquid to receive the injected blood,

[0169] The first reagent can be selectively injected into the container, and / or the second reagent can be selectively injected into the container, so that the target cells in the blood injected into the container are gene-edited or gene-modified by gene delivery without leaving the container and non-therapeutic substances in the treated blood are removed.

[0170] Optionally, the first reagent comprises a buffer, and the second reagent comprises a gene delivery reagent.

[0171] Optionally, the gene delivery reagent is used to achieve transduction or transfection of target cells.

[0172] Optionally, a container that can be selectively rotated about a rotation axis serves as the container of the processing unit.

[0173] Optionally, the axis of rotation is the axis of rotation of the container itself.

[0174] Optionally, before the second reagent is injected into the container, Ficoll cell separation fluid is injected into the container and as the container rotates, different liquid component layers are generated in the radial direction relative to the rotation axis, leaving only the substances in the blood that need to be treated in the container to mix with the second reagent that is subsequently injected.

[0175] Optionally, the gene delivery agent includes a viral vector or a non-viral vector.

[0176] Optionally, the non-viral vector comprises a synthetic vector or a biological vector; and / or, the viral vector comprises a retrovirus or a modified or mutant thereof, a lentivirus or a modified or mutant thereof, an adenovirus or a modified or mutant thereof, or an adeno-associated virus or a modified or mutant thereof.

[0177] Alternatively, the synthetic carrier is a lipid nanoparticle (LNP) or a lipid polyplex (LPP), and the biological carrier is an extracellular vesicle. Alternatively, the target cells include but are not limited to T cells, B cells, NK cells, macrophages, monocytes, or innate lymphoid cells.

[0178] Optionally, the gene delivery reagent includes a CAR gene to transduce or transfect T cells in PBMCs.

[0179] Optionally, the time for transducing or transfecting the target cells using the gene delivery agent without separation, purification, activation or amplification is 1 to 5 hours.

[0180] According to another aspect of the present application, a method for in vitro gene editing or modification of blood cells is also provided, comprising:

[0181] collecting blood from the patient;

[0182] Under closed conditions, gene editing or gene modification is performed on resting or unactivated target cells in collected blood by gene delivery, and the method is also configured to remove non-therapeutic substances in the treated blood cells;

[0183] The treated fluid is returned to the patient.

[0184] Optionally, a first reagent and a second reagent are injected into the collected blood separately, wherein the first reagent includes a buffer and the second reagent includes a reagent for gene delivery.

[0185] Optionally, the gene delivery reagent is used to achieve transduction or transfection of target cells without separation, purification, activation or amplification.

[0186] Optionally, before the second reagent is injected, PBMCs in the blood are separated by density gradient centrifugation, and the second reagent is injected into the separated PBMC liquid.

[0187] Optionally, the gene delivery agent includes a viral vector or a non-viral vector.

[0188] Optionally, the non-viral vector comprises a synthetic vector or a biological vector; and / or, the viral vector comprises a retrovirus or a modified or mutant thereof, a lentivirus or a modified or mutant thereof, an adenovirus or a modified or mutant thereof, or an adeno-associated virus or a modified or mutant thereof.

[0189] Optionally, the synthetic carrier is a lipid nanoparticle (LNP) or a lipid polyplex (LPP), and the biological carrier is an extracellular vesicle.

[0190] Optionally, the target cells include but are not limited to T cells, B cells, NK cells, macrophages, monocytes, or innate lymphoid cells.

[0191] Optionally, the gene delivery reagent includes a CAR gene to transduce or transfect T cells in PBMCs.

[0192] Optionally, the time for transducing or transfecting the target cells using the gene delivery agent without separation, purification, activation or amplification is 1 to 5 hours.

[0193] By adopting the above-mentioned technical means of the present application, the extracorporeal blood cell therapy device can directly input and collect the patient's blood from the site, and directly perform gene editing of the target cells in the blood at the treatment site or bedside, which significantly shortens the treatment cycle compared with the traditional GMP cell preparation factory. In addition, since the various components inside the extracorporeal blood cell therapy device can be packaged in a manner that meets the medical cleanliness requirements during manufacturing, the possibility of any accidental infection during the treatment process is avoided. More importantly, since the extracorporeal blood cell therapy device can be used at the treatment site, the attending physician can change the treatment plan at any time according to the changes in the patient's condition and use the extracorporeal blood cell therapy device to implement the changed treatment plan, thereby increasing the patient's chance of survival. BRIEF DESCRIPTION OF THE DRAWINGS

[0194] The principles and various aspects of the present application will be more fully understood from the detailed description below in conjunction with the following drawings. It should be noted that the scales of the drawings may be different for the purpose of clarity, but this will not affect the understanding of the present application.

[0195] In the attached figure:

[0196] Figure 1 is a perspective view schematically illustrating an embodiment of an extracorporeal blood cell therapy apparatus according to the present application;

[0197] Figure 2 is a system block diagram schematically illustrating an embodiment of a host computer for an extracorporeal blood cell therapy apparatus;

[0198] Figure 3 is a system block diagram schematically illustrating another embodiment of a host computer for implementing an extracorporeal blood cell therapy apparatus;

[0199] Figure 4is a system block diagram schematically illustrating another embodiment of a host for implementing an extracorporeal blood cell therapy apparatus; and

[0200] Figure 5 is a system block diagram schematically illustrating another embodiment of a host computer for implementing an extracorporeal blood cell therapy apparatus;

[0201] Figure 6 is a system block diagram schematically illustrating another embodiment of a host computer for implementing an extracorporeal blood cell therapy apparatus;

[0202] Figure 7A Schematically shows a view of a centrifugal device according to an example of the present application;

[0203] Figure 7B Schematically shows a view of a centrifugal device according to another example of the present application;

[0204] Figures 8A to 8D Provided are transfection data indicator graphs for blood treated using an example of the in vitro blood cell therapy protocol of the present application; and

[0205] Figure 9 It is an experimental result diagram, which schematically shows that after rapid CAR gene transduction of blood cells using an example of the in vitro blood cell treatment scheme of the present application, the transduced CAR-T cells have the killing function. DETAILED DESCRIPTION

[0206] In the various figures of this application, features with the same structure or similar functions are indicated by the same reference numerals.

[0207] Figure 1An embodiment of an extracorporeal blood cell therapy apparatus 100 is schematically shown. The extracorporeal blood cell therapy apparatus 100 includes a host, which has a housing 110. For example, the housing 110 of the host can be substantially cubic or any other suitable shape. According to the present application, the extracorporeal blood cell therapy apparatus mainly uses gene editing / gene modification technology to process the patient's blood in vitro and return it to the patient's body. Therefore, the housing 110 of the host can accommodate those processing units, devices described in detail below and other devices that may be needed but are not described in the specification of this application for realizing the extracorporeal treatment of the patient's blood using gene editing / gene modification technology. A fluid inlet 111 and a fluid outlet 112 are provided on the housing 110, which are respectively used to input the patient's blood and output the blood processed by the extracorporeal blood cell therapy apparatus 100. For example, the fluid inlet 111 can be fluidically connected to the output end of a blood collection machine (not shown) equipped in the medical room, and the fluid outlet 112 can be fluidically connected to a device (not shown) for supplying blood to the patient's body. In the context of this application, the term "fluid connection" refers to a fluid-tight connection between two related features, such as a connection that can be achieved through (not shown) medical hoses and / or pipes; optionally, the connection can also be a detachable connection. The fluid inlet 111 and the fluid outlet 112 are positioned on the housing 110 of the host device to facilitate medical applications. For example, they can be located on the same surface of the housing 110 of the host device or on different surfaces.

[0208] For example, a display screen 141 may be provided on the surface of the main unit housing 110 for displaying data collected and / or processed by the extracorporeal blood cell therapy apparatus 100. A suitable input device 142, such as a keyboard, may also be provided on the surface of the main unit housing 110 to allow for setting parameters of the relevant processing units, devices, and / or components housed within the main unit housing 110 as needed. In alternative embodiments, the input device 142 may be omitted, and the display screen 141 may be provided as a touchscreen display.

[0209] For example, the extracorporeal blood cell therapy apparatus 100 may further include a base 180 for contacting the ground. Meanwhile, a support 170 is provided on the top surface of the base 180 for supporting the housing 110 of the main unit. Furthermore, a plurality of rollers 181, such as four rollers 181 (only three of which are shown in the figure), may be provided on the bottom surface of the base 180, which are respectively located around the base 180, thereby enabling the extracorporeal blood cell therapy apparatus 100 to be easily moved. Furthermore, a cable 190 with a plug electrically connected to a power supply device (not shown) provided in the housing 110 of the main unit can extend from the housing 110 of the main unit, wherein the power supply device can provide power for the operation of the relevant processing units, devices, and / or components as needed. The cable 190 can, for example, be connected to a dedicated power socket in a medical office, thereby providing power for the normal operation of the extracorporeal blood cell therapy apparatus 100 via the power supply device.

[0210] Those skilled in the art will appreciate that, in alternative embodiments, the base 180 and / or the support 170 may be omitted, so that the housing 110 of the extracorporeal blood cell therapy apparatus 100 can be placed directly on a table within the treatment room. For example, with the base 180 and the support 170, the extracorporeal blood cell therapy apparatus 100 can be configured as a portable therapy apparatus in a manner similar to a computer or server.

[0211] The relevant processing units, devices, and / or components contained in the housing 110 of the host constitute the main components of the host of the extracorporeal blood cell therapy device 100 . Figure 2 An embodiment of a host of an extracorporeal blood cell therapy apparatus 100 is schematically shown. Figure 2 In the illustrated embodiment, the extracorporeal blood cell therapy apparatus 100 , or more specifically, its host, includes a first processing unit 120 , a second processing unit 130 , and a control device 140 , which are arranged or integrated within a housing 110 of the host.

[0212] The first processing unit 120 may include, for example, a container 123 for containing liquid, a first injection module 121, and a second injection module 122. The container 123 has a fluid inlet port 123a, which is fluidically connected to the fluid inlet 111; and fluid inlet ports 123b and 123c, which are fluidically connected to the first injection module 121 and the second injection module 122, respectively. In addition, the container 123 also has a fluid outlet port 123d, which is fluidically connected to the second processing unit 130.

[0213] The control device 140 may include, for example, a computer processing unit, a memory, and a data storage unit. For example, the computer processing unit may be in the form of a single-chip microcomputer, an embedded computer chip, a computer, or the like, and is used to call and execute a program stored in the memory. During the execution of the program, the data obtained or processed by the control device 140 may be stored in the data storage unit for subsequent retrieval. The control device 140 is operatively connected to the first injection module 121 and the second injection module 122. In the context of this application, the term "control device operatively connected to a feature" means that the control device can be connected to the feature or a sub-feature within the feature and control the operation of the feature or the sub-feature within the feature. Taking the first processing unit 120 as an example, the control device 140 being operatively connected to the first processing unit 120 means that the control device 140 can connect the first injection module 121 and the second injection module 122 via corresponding control circuits (not shown) and control the operation of their liquid injection mechanisms (as described later) so that the corresponding liquid can be quantitatively injected into the container 123 as needed. Furthermore, the control device 140 is also operatively connected to a display screen 141 and an input device 142 .

[0214] The first processing unit 120 and the second processing unit 130 are fluidically connected between the fluid inlet 111 and the fluid outlet 112. The first processing unit 120 is configured to perform gene editing or gene modification on the target cells in the blood input through the fluid inlet 111 by gene delivery. For example, the gene delivery reagent includes a viral vector or a non-viral vector. For another example, the target cells include but are not limited to T cells, B cells, NK cells, macrophages, monocytes, or innate lymphoid cells. The gene delivery reagent is configured to achieve transduction or transfection of the target cells. In the context of this application, the term "transduction" refers to the process of introducing exogenous genes into eukaryotic cells or prokaryotic cells through a specific vector such as a recombinant viral vector to cause the corresponding gene recombination of the cell or the expression or function of the exogenous gene as needed; the term "transfection" refers to the process of recombinant viral vectors invading recipient cells to cause gene recombination or gene expression or function in the recipient cells. Here, a vector or viral vector refers to a self-replicating DNA molecule or a liquid or fluid containing such a DNA molecule that transfers a DNA fragment (target gene) to a recipient cell in genetic engineering recombinant DNA technology. For example, commonly used vectors include non-viral vectors and viral vectors. Non-viral vectors include synthetic vectors represented by lipid nanoparticles (LNP), lipid polyplexes (LPP), etc. and biological vectors represented by extracellular vesicles. Viral vectors include retroviruses or their modified bodies or mutants, lentiviruses or their modified bodies or mutants, adenoviruses or their modified bodies or mutants, or adeno-associated viruses (AAV) or their modified bodies or mutants, and also include nucleic acid vectors such as plasmids and phages. In the technical solution of the present application, the vector or viral vector used includes a wild type or mutant or a viral vector modified by a chemical or biological method, and the medium containing the vector or viral vector is screened to improve the transduction or transfection efficiency of the vector or viral vector to the recipient cell.

[0215] The fluid outlet end 123d of the container 123 is located at the lowest point of the container 123 in the direction of gravity, and the fluid inlet ends 123a, 123b, and 123c are higher than the fluid outlet end 123d by a certain distance. It is preferable not to affect the subsequent processing of blood cells. In this way, the blood cells transported through the fluid inlet 111 can be discharged into the container 123 via the fluid inlet end 123a. After undergoing a certain amount of processing, they can be discharged to the second processing unit 130 via the fluid outlet end 123d according to gravity. During the processing, liquid or fluid can be discharged into the container 123 from the first and second injection modules 121 and 122 via their respective fluid inlet ends 123b and 123c as needed. In addition, the container 123 can also be provided with an exhaust port (not shown) to facilitate the exhaust gas generated during the processing from the container 123 to the outside world.

[0216] Each injection module 121 or 122 may, for example, include a compartment and a liquid injection mechanism well known to those skilled in the art, such as an electric syringe or a metering pump, for quantitatively discharging the liquid or fluid contained in the compartment independently of each other via the fluid inlet port 123b or 123c. According to an embodiment of the present application, the container and / or compartment described above and / or below may be configured as any form of liquid or fluid that complies with medical standards, such as a tank, a bag, a bottle, etc. The container 123 and / or each injection module or at least one of them is detachably arranged in the first treatment unit 120, and / or each compartment is, for example, in the form of a tank, a bag, a bottle, etc., and is detachably arranged in the injection module 121 or 122, so that during the medical treatment process, they can be conveniently replaced or refilled according to actual needs.

[0217] Each compartment of the first and second injection modules 121, 122 can be loaded with different treatment reagents as needed, so that the corresponding treatment reagents can be injected into the container. For example, the compartment of the first injection module 121 can be configured to be able to load a buffer solution, and the compartment of the second injection module 122 can be configured to be able to load a gene delivery reagent for realizing transduction or transfection of target cells. In the context of the present application, gene delivery reagents mainly refer to reagents that help exogenous genes or target genes to be transduced into target cells such as T cells, B cells, NK cells or other types of cells, including viral (genetically modified or non-modified) vectors such as lentiviral vectors, adeno-associated viral vectors, plasmid vectors, lipid nanoparticles (LNP), cationic lipid complexes (LPX), lipid multipolymers (LPP), inorganic nanoparticles (INP) and other reagents; or non-viral vectors, such as transposons, etc. For example, a buffer solution can be used to dilute the blood supplied to the container 123 through the fluid inlet 111 or alternatively the interior of the container 123 can be rinsed before or after each treatment. For example, the buffer solution can be physiological saline, culture fluid, nutrient solution, culture medium, or other liquids that can be used in the applications of the present application. A switch K10 can be provided at the fluid outlet port 123d. By turning the switch K10 on and off, it can be controlled whether the liquid in the container 123 enters the second processing unit 130 through the fluid outlet port 123d. For example, the switch K10 can take any suitable form, such as a solenoid valve. The control device 140 is operatively connected to the switch K10, so that the switch K10 can be selectively turned on and off under the control of the control device 140.

[0218] The second processing unit 130 may include a filter 131, for example. The filter 131 may be implemented by using a filter membrane, molecular sieve, centrifugal filtration, chromatography, etc. Figure 2In the illustrated embodiment, the second processing unit 130 is described using a filter membrane as an example. However, those skilled in the art will appreciate that the principles of the present invention remain applicable even if the filter is implemented in other ways. For example, by selecting an appropriate pore size for the filter membrane serving as filter 131, it is possible to ensure that selected eukaryotic cells, prokaryotic cells, or recipient cells in a suspension of blood, blood-related cells, or other types of cells are retained by the filter membrane 131 of the second processing unit 130. Therefore, the second processing unit 130 includes an input port 130a located upstream of the filter 131 and fluidically connected to the fluid outlet port 123d of the container 123 via a line L10; a waste liquid outlet port 130b located downstream of the filter 131 for discharging filtered waste liquid from the second processing unit 130; and an output port 130c also located upstream of the filter 131 but downstream of the input port 130a for discharging the suspension retained by the filter 131. The second processing unit 130 is configured to remove non-treatment-required substances in the blood processed by the first processing unit, for example, the non-treatment-required substances include excess gene delivery reagents or other non-treatment-required substances.

[0219] In an alternative or additional embodiment, the second processing unit 130 may include a first recovery end 130d located downstream of the filter 131 and a second recovery end 130e located upstream of the filter 131. Switches K20, K30, K40, and K50 are each provided at the waste liquid outlet end 130b, the first recovery end 130d, the output end 130c, and the second recovery end 130e. For example, the switches K20, K30, K40, and K50 can be constructed in a manner similar to K10, and the control device 140 is operatively connected to the switches K20, K30, K40, and K50 so that, under the control of the control device 140, the switches K20, K30, K40, and K50 can be selectively switched on and off. In addition, according to the alternative or additional embodiment, the first processing unit 120 also includes a recovery module 125, which is provided with a compartment for storing liquid. The first recovery end 130d is fluidically connected to the recovery module 125, particularly its compartment, via a pipeline L20. The second recovery port 130e is connected to the container 123 via a pipeline L30, so that the suspension retained by the filter 131 during filtration can be returned to the container 123 via the pipeline L30. In an alternative embodiment, the pipeline L30 can also be directly returned to the upstream of the fluid inlet port 123a, so that the returned liquid can be discharged into the container 123 via the fluid inlet port 123a.

[0220] In addition, the first processing unit 120 may further include a shaking mechanism 124. The shaking mechanism 124 can be physically or mechanically connected to the container 123, thereby causing the container 123 to shake at a certain amplitude as needed. This facilitates repeated mixing of the buffer or carrier injected into the container 123 with the blood injected into the container 123 via the fluid inlet 111, thereby improving the efficiency of dilution, transduction, or transfection. It should be clear to those skilled in the art that although the shaking mechanism 124 is provided for the container 123, the fluid connections at the inlet ends 123a, 123b, 123c, and the outlet end 123d of the container 123 are not adversely affected during the shaking of the container 123. The shaking mechanism 124 can be implemented in any manner known to those skilled in the art. As just one example, the shaking mechanism 124 can include a connecting rod structure physically or mechanically connected to the container 123 and a motor that drives the connecting rod structure to move back and forth. The control device 140 is operatively connected to the shaking mechanism 124, particularly the motor thereof, so that under the control of the control device 140, the shaking mechanism 124 can cause the container 123 to shake with a certain amplitude. In an alternative embodiment, the shaking mechanism 124 can be independently controlled to operate.

[0221] The first treatment unit 120 and the second treatment unit 130 are fluidically connected to each other via a line L10. The second treatment unit 130 is configured to remove carriers and / or viruses from the liquid treated by the first treatment unit 120 (eg, discharged via the line L10).

[0222] It should be clear to those skilled in the art that a pumping device can be provided in pipelines L10 and / or L20 and / or L30 or optionally in any required pipeline (as described above or below), so that the movement of the liquid can be achieved by the operation of the pumping device without relying on gravity, and the control device 140 is operatively connected to the pumping device to control its operation.

[0223] The setting of the recovery end 130d enables the filtrate to be recycled and reused as needed, which is particularly important for the recovery of expensive or high-cost carriers. Figure 2 The following briefly describes the blood treatment process using the extracorporeal blood cell therapy apparatus 100. In the following process description, the relevant control programs may be implemented as coded programs stored in memory, accessible and executed by the control device 140. It should be understood that the following process is merely illustrative and non-limiting. In other words, after reading this specification, those skilled in the art will recognize that other feasible operating procedures using the extracorporeal blood cell therapy apparatus 100 are consistent with the objectives of this application and fall within the scope of this application.

[0224] First, the collected blood, blood-related cells, or other types of cell suspension are injected into the container 123 of the first processing unit 120 via the fluid inlet 111. At this point, switch K10 is off, and the first injection module 121 is activated to inject a certain amount of buffer into the container 123. Simultaneously, the shaking mechanism 124 is operated to shake the container 123, ensuring that the blood, blood-related cells, or other types of cell suspension are evenly mixed with the buffer, allowing components in the buffer, such as gene vectors or viral vectors, to fully contact the cells. Next, switch K10 is on, and the mixed liquid is input into the second processing unit 130 via the pipeline L10. At this point, switches K30, K40, and K50 of the second processing unit 130 are off, and switch K20 is on. Consequently, the filtrate from the filter 131 (located downstream of the filter 131) exits the second processing unit 130 via the waste liquid outlet 130b. By appropriately selecting the pore size of the filter membrane of filter 131, smaller, non-therapeutic substances can be filtered out of a suspension of blood, blood-related cells, or other types of cells. The eukaryotic cells or recipient cells required for transduction or transfection are retained in the remaining suspension upstream of filter 131. At this point, switches K20, K30, and K40 of the second processing unit 130 and switch K10 of the first processing unit 120 are turned off, and the remaining suspension in the second processing unit 130 is returned to container 123 via pipeline L30, for example, by activating a pumping device associated with pipeline L30. It will be apparent to those skilled in the art that the fluid inlet ports 123a, 123b, and 123c of container 123 and the fluid connection port between pipeline L30 and container 123 are preferably positioned above the liquid level in container 123 during transduction or transfection, or so as not to affect the transduction or transfection process within container 123.

[0225] In order to fully dilute the blood and exclude smaller substances that do not require treatment, the above steps can be repeated multiple times. Next, the second injection module 122 is started to inject a certain amount of reagent for transduction or transfection into the container 123 (the switch 10 is in the off state), and at the same time, the shaking mechanism 124 is operated to shake the container 123, thereby ensuring that the reagent is fully mixed with the liquid in the container 123. Then, you can wait for a certain time to allow the eukaryotic cells or recipient cells in the liquid in the container 123 to be fully transduced or transfected. For example, the time can be 2 hours or longer. During this period, the shaking mechanism 124 can be operated selectively, thereby helping to improve the efficiency of transduction or transfection.

[0226] Various sensors, such as pressure sensors and temperature sensors, may be disposed within the container 123 and / or within the pipeline. The control device 140 can be operatively connected to these sensors to determine whether the transduction or transfection process is normal or monitor the operating status of the therapeutic device 100 based on data measured by the sensors. Optionally, the measured data can be displayed on the display 141 for user monitoring.

[0227] After transduction or transfection has been completed (for example, determined by the passage of a predetermined time or monitoring of the operating status), the control device 140 instructs the switch K10 to be in the on state, so that the liquid that has been transduced or transfected is input from the first treatment unit 120 to the second treatment unit 130 via the pipeline L10.

[0228] Because the reagents used for transduction or transfection are relatively expensive, they need to be recycled. Thus, at least when the liquid that has been transduced or transfected is first input into the second processing unit 130 (or when the switch K10 is first turned on after the first processing unit 120 has completed the transduction or transfection process), the switches K20, K40, and K50 of the second processing unit 130 are turned off and the switch K30 is turned on. As a result, the filtrate of the input liquid after being filtered through the filter 131 still contains a high concentration of transduction or transfection vectors. The filtrate can then be collected into the recovery module 125 via the pipeline L20, and then further purified and reused.

[0229] Next, switches K30, K40, and K50 are turned off, while switches K10 and K20 are turned on. The first injection module 121 is activated to inject the buffer solution into the container 123 and into the second processing unit 130, thereby further flushing the suspension retained by the filter 131. The filtrate is then discharged through the waste liquid outlet 130b. After this flushing process has occurred multiple times, the first injection module 121 can be deactivated, and switches K20, K30, and K50 can be turned off while switch K40 is turned on, allowing the final suspension to be discharged from the fluid outlet 112 through the output end 130c.

[0230] In an alternative embodiment, during the flushing process, the suspension retained by the filter 131 can be returned to the container 123 via the pipeline L30, and the buffer solution can be injected again while the switch 10 is in the off state, and the container 123 can be shaken. The switch can then be turned on and filtered by the filter 131 of the second processing unit 130, thereby ensuring a better flushing effect.

[0231] In the extracorporeal blood cell therapy apparatus 100 of the present application, any part involving fluid connection is configured to comply with the mandatory national medical and health regulations. Compared with the traditional GMP cell preparation factory that requires maintaining the same high medical cleanliness, the cost of manufacturing the extracorporeal blood cell therapy apparatus 100 of the present application is obviously lower. In addition, the input blood is only processed inside the extracorporeal blood cell therapy apparatus 100, and there is no need for the connection between the various processes of the traditional GMP cell preparation factory, which greatly eliminates the possibility of accidental blood infection. More importantly, because the extracorporeal blood cell therapy apparatus 100 of the present application can be used directly at the treatment site where the patient is located, the attending physician can change the treatment plan on the spot in a targeted and purposeful manner according to the patient's condition, even by replacing the reagents or carriers used for treatment, which is beneficial to the patient's cure.

[0232] In an alternative or additional embodiment, the first processing unit 120 of the extracorporeal blood cell therapy device 100 can also be configured to have a third, fourth, fifth or more injection modules, each injection module storing different transduction or transfection reagents in advance as needed, so that the treatment plan can be conveniently switched according to changes in the patient's condition or different patients.

[0233] Figure 3 The main unit of an extracorporeal blood cell therapy apparatus 100 according to another embodiment of the present application is schematically shown. A first processing unit 1200 is arranged or integrated in a housing 110 of the main unit.

[0234] The first processing unit 1200 may include, for example, a container 1230 for containing liquid, a first injection module 121, and a second injection module 122. Here, the first injection module and the second injection module adopt the same Figure 2 The same reference numerals in the first injection module 121 and the second injection module 122 are used to indicate that Figure 3 The first injection module and the second injection module of the embodiment shown can be used as follows Figure 2 The embodiment or corresponding alternative or additional example is set in the manner described. Figure 3 The description of the first injection module 121 and the second injection module 122 can refer to the description of the first injection module 121 and the second injection module 122. Figure 2 Description.

[0235] Container 1230 has a fluid input end 1230a, which is fluidically connected to the fluid inlet 111; and fluid inlet ends 1230b and 1230c, which are fluidically connected to the first injection module 121 and the second injection module 122, respectively. A filter 1310 is provided within container 1230. For example, similar to filter 131, filter 1310 may be provided in the form of a filter membrane. Fluid input end 1230a, fluid inlet ends 1230b, and fluid inlet ends 1230c are located upstream of filter 1310. Container 1230 also has a fluid output end 1230d, which may be fluidically connected to fluid outlet 112 via a pipeline (not shown). A switch K100 may be provided at fluid output end 1230d. Switching switch K10 on and off controls whether the liquid within container 1230 can be discharged through fluid outlet 112.

[0236] A waste liquid outlet 1230e and a recovery port 1230f are provided at the bottom of the container 1230. The waste liquid outlet 1230e and the recovery port 1230f are located downstream of the filter 1310. The recovery port 1230f is fluidically connected to the recovery module 125 via a pipeline L100. For example, the recovery module 125 can be configured as follows: Figure 2 The recovery module in the illustrated embodiment is similarly configured. Switches K200 and K300 may be provided at the waste liquid outlet 1230e and the recovery terminal 1230f, respectively, so that their opening and closing can respectively allow or prohibit the liquid in the container 1230, particularly downstream of the filter 1310, from being discharged from the waste liquid outlet 1230e and the recovery terminal 1230f.

[0237] According to the embodiments of the present application, whether for Figure 2 The embodiment shown is still directed to Figure 3 In the illustrated embodiment or the embodiments described below, the output end 130c or 1230d located upstream of the filter 131 or filter 1310 can be positioned just above the filter 131 or 1310, allowing sufficient filtered suspended liquid to be discharged through the output end. According to a preferred embodiment, the portion of the container 1230 located downstream of the filter 1310 can be designed to gradually taper toward its bottom, for example, in the form of a bell-shaped mouth (with the larger end at the top and the smaller end at the bottom). This ensures that the filtrate after the liquid is filtered through the filter 1310 can be discharged more quickly.

[0238] To improve filtration efficiency, the first processing unit may further include a pressure differential generating device 126. For example, the pressure differential generating device 126 may be a negative pressure generating device located downstream of the filter 1310 (as shown in the figure) or a positive pressure generating device (not shown) located upstream of the filter 1310. When necessary, the pressure differential generating device 126 operates to generate a pressure differential from upstream to downstream of the filter 1310, thereby prompting the liquid to flow through the filter 1310 as quickly as possible, thereby shortening the time required for filtration.

[0239] and Figure 2 Similar to the embodiment shown, the host further includes a control device 140 provided or integrated in the housing 110 of the host. In addition, the first processing unit 1200 may further include a shaking mechanism 124. The description of the control device 140 and the shaking mechanism 124 may be found in the description of the embodiment shown. Figure 2 The description of the embodiment of Figure 3 In the illustrated embodiment, the control device 140 is operatively connected to the first processing unit 1200 , in particular, to the switches K100 , K200 , K300 and the pressure difference generating device 126 of the first processing unit 1200 .

[0240] It should be clear that various sensors, such as pressure sensors, temperature sensors, etc., can be set in the container 1230 and / or in the pipeline connected to the container. The control device 140 can be operatively connected to these sensors to determine whether the transduction or transfection process is normal or monitor the operating status of the therapeutic device 100 based on the data measured by the sensors.

[0241] The following schematic description uses Figure 3 The host computer's extracorporeal blood cell therapy apparatus 100 is shown as a user's preferred operating procedure. In the following process description, the relevant control program may be implemented as a coded program stored in memory for access and execution by the control device 140. It should be understood that the following process is merely illustrative and non-limiting. In other words, after reading this specification, those skilled in the art will recognize that other feasible operating procedures using the extracorporeal blood cell therapy apparatus 100 are consistent with the objectives of this application and fall within the scope of this application.

[0242] First, the collected patient blood is injected into the container 1230 of the first processing unit 1200 through the fluid inlet 111. At this time, the switches K100, K200, and K300 are in the off state. Simultaneously or subsequently, the first injection module 121 starts to inject a certain amount of buffer into the container 1230, and at the same time, the shaking mechanism 124 operates to shake the container 1230 to ensure that the blood and the buffer are evenly mixed, thereby achieving a suitable dilution effect. The amount of buffer injected should be such that the final mixed liquid level is at a certain height above the filter 1310, thereby ensuring that the dilution of the blood is achieved. In other words, the amount of liquid injected should ensure that there is enough liquid upstream of the filter 1310 to immerse the target cells.

[0243] Next, switch K200 is turned on, and the portion of the mixed liquid located downstream of filter 1310 (waste liquid) is discharged from container 1230 through waste liquid outlet 1230e, while the portion of the mixed liquid located upstream of filter 1310 (i.e., retained by filter 1310) remains within container 1230. Then, switch K200 is turned off, and first injection module 121 is activated again to inject a certain amount of buffer into container 1230. Simultaneously, shaking mechanism 124 is operated to cause container 1230 to shake. Next, switch K200 is turned on again, and the waste liquid is discharged from container 1230 through waste liquid outlet 1230e. For example, by appropriately selecting the pore size of the filter membrane of filter 1310, this dilution and flushing process can be repeated multiple times, thereby filtering out smaller, non-therapeutic substances in the blood.

[0244] Next, after the smaller, non-therapeutic substances in the blood have been appropriately removed, switches K100, K200, and K300 are all turned off. The second injection module 122 is activated to inject a certain amount of transduction or transfection reagent into container 1230. Simultaneously, the shaking mechanism 124 is operated to shake container 123, thereby ensuring thorough mixing of the reagent with the liquid in container 1230. Next, a predetermined period of time is allowed to pass, allowing the eukaryotic cells or recipient cells in the liquid in container 123 to be fully transduced or transfected. For example, this period can be two hours or longer. During this period, the shaking mechanism 124 can be selectively operated, thereby helping to improve transduction or transfection efficiency.

[0245] In such Figure 3 In the illustrated embodiment, the transduction or transfection process is completed in a container 1230 provided with a filter 1310, so the amount of reagent injected for transduction or transfection should ensure that the final liquid level is higher than a certain height of the filter 1310, so that the selected eukaryotic cells or recipient cells can be sufficiently immersed in the liquid mixed with the reagent.

[0246] After transduction or transfection is completed, the control device 140 instructs the switch K300 to be in the conducting state, so that the liquid downstream of the filter 1310 can enter the recovery module 125 through the recovery end 1230f through the pipeline L100 and be collected, so that it can be reused after further purification. In an alternative embodiment of the present application, the recovery module 125 can also be configured similarly to the second injection module 122, and has its own liquid injection mechanism such as an electric syringe or a metering pump to be able to quantitatively inject liquid into the container 1230, so that the recovered liquid (mainly composed of the reagent for transduction or transfection) can be reused in subsequent transduction or transfection.

[0247] Next, switches K100, K200, and K300 are turned off. Simultaneously or subsequently, first injection module 121 is activated to inject a certain amount of buffer into container 1230. Simultaneously, shaking mechanism 124 is operated to agitate container 1230, ensuring uniform mixing of the suspension upstream of filter 1310 and the buffer. Subsequently, switch K200 is turned on to drain the liquid downstream of filter 1310 out of container 1230. This process can be repeated multiple times to complete rinsing of the transduced or transfected cells.

[0248] After flushing is completed, a suspension still remains upstream of the filter 131, and the suspension contains cells that have been transduced or transfected and flushed. At this time, the switch K100 can be turned on and the switches K200 and K300 can be turned off, so that the suspension can be discharged from the container 1230 through the fluid outlet port 123d and then output from the fluid outlet 112.

[0249] like Figure 3 The illustrated embodiment further simplifies the host design of the extracorporeal blood cell therapy apparatus 100 , thereby ensuring a smaller instrument size and reducing the number of fluid connectors, thereby further reducing the manufacturing cost while achieving medical cleanliness.

[0250] Figure 4 The main unit of the extracorporeal blood cell therapy apparatus 100 according to another embodiment of the present application is schematically shown. Figure 4 The housing 110 of the host shown in FIG. 1 is provided with or integrated with a first processing unit 1201. In addition to the first processing unit 1201, as shown in FIG. Figure 4 The remainder of the embodiment shown (with Figure 3 Those features with the same reference numerals may refer to Figure 3 The first processing unit 1201 has most of the same features as the first processing unit 1200, so the description of those features with the same reference numerals can refer to the following. Figure 3The embodiment shown. The first treatment unit 1201 differs from the first treatment unit 1200 in that the fluid inlet ports 1230b and 1230c, which are fluidically connected to the first injection module 121 and the second injection module 122, are disposed downstream of the filter 1310 within the container 1230 of the first treatment unit 1201. Furthermore, switches K500 and K400 are disposed at the fluid inlet ports 1230b and 1230c, respectively, such that the on / off switching of the switches can control whether the first injection module 121 and the second injection module 122 are allowed to be injected into the container 1230 and, when the switches are off, prevent the liquid in the container 1230 from flowing back into the injection modules. It should be understood that in the embodiments of the present application, the fluid inlet ports and / or the switches disposed at the fluid inlet ports can be unidirectional, meaning that even when the switches are on, they only allow liquid to be fed into the container in one direction, preventing the liquid in the container from flowing into the associated injection modules.

[0251] According to Figure 4 In the embodiment shown, the first injection module 121 and the second injection module 122 inject the corresponding liquid into the container 1230 from the downstream of the filter 1310. The advantage of this is that, for example, when injecting the buffer solution, some cells are trapped in the filter pores of the filter membrane of the filter 1310 due to their size. Therefore, immersing the filter membrane with liquid from the downstream helps to avoid the possibility of damage to the target cells caused by the direct impact of the injected liquid.

[0252] In such Figure 4 When the host is operated as shown, first, the collected patient blood is injected into the container 1230 of the first processing unit 1201 through the fluid inlet 111. At this time, switches K100, K200, K300, K400, and K500 are in the off state. Then, the switch 500 is in the on state and the first injection module 121 is started to inject a certain amount of buffer into the container 1230. The switch 500 is again turned off. At the same time, the shaking mechanism 124 is operated to shake the container 1230 to ensure that the blood and buffer are evenly mixed, thereby achieving the appropriate infection and / or conduction effect. The amount of buffer injected should ensure that the final mixed liquid level is a certain height above the filter 1310, thereby ensuring that the blood is diluted.

[0253] Next, switch 500 is turned off and switch K200 is turned on. The portion of the mixed liquid located downstream of filter 1310 (waste liquid) is discharged from container 1230 through waste liquid outlet 1230e, while the portion of the mixed liquid located upstream of filter 1310 (i.e., retained by filter 1310) remains within container 1230. Then, switch K200 is turned off and switch 500 is turned on. The first injection module 121 is activated again to inject a certain amount of buffer into container 1230. Switch 500 is turned off again, and the shaking mechanism 124 is operated to shake container 1230. Next, switch K200 is turned on again, and the waste liquid is discharged from container 1230 through waste liquid outlet 1230e. For example, by appropriately selecting the pore size of the filter membrane of filter 1310, this dilution and flushing process can be repeated multiple times, thereby filtering out smaller, non-therapeutic substances in the blood.

[0254] Next, after the smaller, non-therapeutic substances in the blood have been appropriately removed, switches K100, K200, K300, and K500 are all turned off, and switch K400 is turned on. The second injection module 122 is activated to inject a certain amount of transduction or transfection reagent into container 1230. Switch 400 is again turned off, and the shaking mechanism 124 is operated to shake container 123, thereby ensuring thorough mixing of the reagent with the liquid in container 1230. Next, a certain amount of time is allowed to pass to allow the eukaryotic cells or recipient cells in the liquid in container 123 to be fully transduced or transfected. For example, this time period can be two hours or longer. During this period, the shaking mechanism 124 can be selectively operated to help improve transduction or transfection efficiency.

[0255] After transduction or transfection is completed, the control device 140 instructs the switch K300 to be in the conducting state, so that the liquid downstream of the filter 1310 can enter the recovery module 125 through the recovery end 1230f through the pipeline L100 and be collected, so that it can be reused after further purification. In the embodiment of the present application, the recovery module 125 can also be configured similarly to the second injection module 122, and has its own liquid injection mechanism, such as an electric syringe or a metering pump, which can quantitatively inject liquid into the container 1230, so that the recovered liquid (mainly composed of the reagent used for transduction or transfection) can be reused in subsequent transduction or transfection.

[0256] Next, switches K100, K200, K300, and K400 are turned off, and switch K500 is turned on. The first injection module 121 is activated to inject a certain amount of buffer into container 1230. Switch 500 is again turned off, and the shaking mechanism 124 is operated to shake container 1230, ensuring that the suspension upstream of filter 1310 and the buffer are evenly mixed. Subsequently, switch K200 is turned on, draining the liquid downstream of filter 1310 from container 1230. This process can be repeated multiple times to complete the flushing of the transduced or transfected cells.

[0257] After flushing is completed, a suspension still remains upstream of the filter 1310, and the suspension contains cells that have been transduced or transfected and flushed. At this time, the switch K100 can be turned on and the switches K200, K300, K400, and K500 can be turned off, so that the suspension can be discharged from the container 1230 through the fluid outlet port 123d and then output from the fluid outlet 112.

[0258] For example Figure 4 In the illustrated embodiment, it should be clear to those skilled in the art that only one of the fluid inlet of the first injection module 121 or the second injection module 122 in the container 1230 may be disposed upstream of the filter 1310 .

[0259] Figure 5 The main unit of an extracorporeal blood cell therapy apparatus 100 according to another embodiment of the present application is schematically shown. According to this embodiment, the main unit includes a first processing unit 1202 and a sorting module 127 located upstream of the first processing unit 1202, wherein the inlet of the sorting module 127 is fluidically connected to the fluid inlet 111, and the outlet of the sorting module 127 is fluidically connected to the first processing unit 1202 via a pipeline L80. Here, the first processing unit 1202 can be configured as follows: Figure 2 、 3 The first processing unit 120, 1200 or 1201 or their corresponding modifications are implemented, so they are not described in detail here. Figure 5In the illustrated embodiment, the function of the sorting module 127 is to pre-screen cells of interest for treatment using physical or biological means, and use the screened cell suspension as the input liquid for the first processing unit 1202, thereby improving the efficiency of transduction or transfection and ultimately improving the operating efficiency of the entire instrument. For example, in the case of physical means, a specific screening device such as a chromatography column, a filter membrane, or a molecular sieve can be used to first enrich and screen cells that meet the size requirements and require treatment as the input liquid for the first processing unit 1202. Of course, those skilled in the art will appreciate that specific biological reagents can also be selected in the sorting module 127 to achieve biological cell screening. Under the premise of biological screening, an injection unit can also be added to the first processing unit 1202 to inject a biological screening reagent into the container to achieve biological cell screening. Therefore, before the initial dilution of the blood, the biological screening reagent is injected into the container, so that the cells in the blood in the container that do not need to be treated are removed and the specific cells that need to be treated are retained. For example, according to the present application, the sorting module 127 can be in the form of a chromatography column for specific molecular markers or chelation or other biological or physical methods available in the art, so that after the patient's PBMC is collected from the apheresis machine, these cells are washed and enter the sorting device so that specific lymphocytes such as T cells, B cells, NK cells, etc. can be sorted out, and then gene editing or gene modification is performed by gene delivery using the corresponding functional units of the extracorporeal blood cell therapy device 100, such as the first processing unit 1202.

[0260] In the embodiments of the present application, the target cells to be treated, such as T cells, B cells, NK cells, etc., can be transduced or transfected without undergoing the separation or purification or activation or amplification treatment required in the prior art, which significantly shortens the time for gene editing or gene modification and significantly improves the efficiency of patient treatment.

[0261] Figure 6 The schematic diagram of the host of the extracorporeal blood cell therapy device 100 according to another embodiment of the present application is schematically shown. A first processing unit 1203 is provided or integrated in the housing 110 of the host. For example, the first processing unit 1203 may include a separation device for separating PBMC using density gradient centrifugation (as described below). The host also includes a cell separation fluid injection module 123, which can be connected to the fluid of the first processing unit 1203 in a manner similar to the first injection module 121 or the second injection module 122 described above, and can selectively inject cell separation fluid into the first processing unit 1203 under the control of the control device 140. In addition, Figure 6Other features shown in the figure, such as the fluid inlet 111, the fluid outlet 112, the first injection module 121, the second injection module 122, the shaking mechanism 124, the recovery module 125, the pressure difference generating device 126, the control device 140, the display screen 141 and the input device 142, etc., can be referred to the description of the embodiments described above or below.

[0262] Those skilled in the art will appreciate that PBMC separation using density gradient centrifugation can include Percoll density gradient centrifugation and Ficoll density gradient centrifugation. The following examples in the specification primarily describe the use of Ficoll density gradient centrifugation in the technical solution of this application, but do not exclude the use of Percoll density gradient centrifugation in the technical solution of this application.

[0263] According to an example of this application, Figure 7A As shown, the first processing unit 1203 may include a container 12031 for containing liquid. For example, the container 12031 has a rotation axis to define a rotation axis O, and the rotation axis O is substantially perpendicular to the ground after the extracorporeal blood cell therapy apparatus 100 is stabilized. For example, the rotation axis of the container can be selectively driven to rotate via a driving device (for example, including a motor) not shown in the figure under the control of the control device 140, such as forward rotation, reverse rotation and / or forward and reverse rotation at a certain frequency for a predetermined time. According to an alternative or additional embodiment of the present application, as Figure 6 As shown, the first processing unit 1203 may further include an independent container 12131, such as a culture container or culture chamber. For example, the transduction and / or transfection and / or washing and / or dilution of PBMCs can be completed independently in the independent container 12131. For another example, the independent container 12131 can serve as a temporary storage container to temporarily store PBMCs to be transduced and / or transfected and / or washed and / or diluted, and ensure that the transduction and / or transfection and / or washing and / or dilution of PBMCs can be completed only in the container 12031. For example, before each of the above-mentioned treatments is performed on the container 12031 or 12131, the container can be rinsed with a suitable liquid such as physiological saline. The shaking mechanism 124 can be configured to independently shake the container 12031 or 12131.

[0264] According to one example of the present application, a connecting tube 12032 may be provided at the top of the container 12031. The connecting tube 12032 can be connected to the top of the container 12031 in a manner that is not affected by the rotation of the container 12031, so that the multiple passages (hidden in the figure) provided in the connecting tube 12032 can have openings located at different radial positions relative to the rotation axis O in the container 12031 as needed, and the opposite corresponding openings of these passages can be connected to the fluid inlet 111, the fluid outlet 112, the first injection module 121, the second injection module 122, the cell separation solution injection module 123, the recovery module 125, and the container 12131, respectively. The cell separation solution injection module 123 is configured to inject Ficoll cell separation solution into the container 12031 as needed. In addition, liquid can be drawn from the container 12031 into the container 12131 or from the container 12131 into the container 12031 via the connecting tube 12032 as needed. When an independent container 12131 is set up, the first injection module 121, the second injection module 122, and the cell separation fluid injection module 123 can also be configured to be fluidically connected to the container 12131, and the fluid connection method can be set with reference to the method of connection with the container 12031.

[0265] According to a non-limiting example, after collected patient blood is injected into container 12031 of first processing unit 1203 via fluid inlet 111, first injection module 121 activates and injects a predetermined amount of buffer into container 12031. The blood and buffer are uniformly mixed by operating shaking mechanism 124 and / or rotating container 12031 forward and reverse at a predetermined frequency for a predetermined time, thereby achieving appropriate infection and / or conduction effects. Ficoll cell separation fluid is then injected into container 12031, and container 12031 is rotated at a predetermined speed about rotation axis O, thereby causing radial stratification of different components within the mixed solution due to the different densities of the liquid components along the radial direction of rotation axis O. Then, the radially layered components of the liquid are extracted using different passages arranged in the container 12031 relative to the rotation axis O. For example, some of the extracted liquid components can be sucked into the recovery module 125 for reuse, and some of the extracted liquid components can be directly discharged from the container 12031 as waste liquid, thereby ultimately leaving only the substances in the blood that require treatment in the container 12031. Alternatively and / or additionally, the liquid components to be transduced or transfected can be extracted into the container 12131. For example, if it is desired that the transduction or transfection process be performed in the container 12031 (for example, the container 12031 is used as a culture chamber), the liquid components to be transduced or transfected can be first extracted into the container 12131, and then the first injection module 121 can be configured to inject a certain amount of buffer into the container 12031 to flush the remaining liquid. Finally, the liquid components in the container 12131 are extracted back into the container 12031. For another example, if it is desired that transduction or transfection treatment be performed solely in the container 12131 , the reagents for transduction or transfection can be directly injected into the container 12131 via the second injection module 122 , and the container 12131 is then used as a culture chamber.

[0266] Next, the second injection module 122 is activated to inject a certain amount of transduction or transfection reagent into container 12031 or 12131. The shaking mechanism 124 is again operated and / or container 12031 is rotated forward and backward at a certain frequency for a predetermined time to ensure that the reagent is thoroughly mixed with the liquid in container 12031 or 12131. Next, a predetermined period of time is allowed to pass to allow the eukaryotic cells or recipient cells in the liquid in container 12031 or 12131 to be fully transduced or transfected. For example, the period of time may be two hours or longer. During this period, the shaking mechanism 124 and / or container 12031 may be selectively operated to help improve the transduction or transfection efficiency. After the transduction or transfection is complete, the post-transfection liquid components, particularly the PBMCs, may be diluted, for example, with a buffer solution. Centrifugation is then performed to further remove impurities from the post-transfection or transfected PBMC liquid by utilizing the density differences of the different components. For example, the liquid components after transduction or transfection are completed can be located in the container 12031 or drawn from the container 12131 into the container 12031, and then a certain amount of buffer solution is selectively injected into the container 12031 through the first injection module 121, and the container 12031 is started to rotate, so that the different components in the liquid are in different radial layering positions according to their different densities. Then, different passages arranged relative to the rotation axis O of the container 12031 are used to draw these radially layered components of the liquid. For example, beneficial liquid components (such as the required liquid components after transduction or transfection) can be collected through the fluid outlet 112 for subsequent treatment use, and other liquid components can be discharged as waste liquid.

[0267] According to another alternative example of the present application, Figure 7B As shown, the container 12031 can also be configured so that after rotational separation, the liquid components are separated into layers along the direction of gravity height due to the different densities of the components in the liquid. In this case, for example, a selective on-off switch (such as the first switch K100, the second switch K200, and the third switch K300 mentioned above) can be provided at the bottom of the container 12031, so that the liquids of different layers can be collected under the action of gravity by controlling the on-off of the switch. For example, these collected liquids can be directly discarded as waste liquids or used as final therapeutic substances or re-injected into the container 12031 or 12131 for further dilution, transduction, or transfection, depending on the components. It should be clear to those skilled in the art that Figure 5 The sorting module 127 shown can also be Figure 6 The first processing unit 1203 is configured as shown.

[0268] According to one embodiment of the present application, the containers 1230 , 12031 , and 12131 may be equipped with a heating or heat preservation device to ensure that a suitable temperature is maintained during PBMC cell culture.

[0269] The following describes an example of a method for transducing PBMC using the GFP or CAR gene using the device of the present application. Compared with the prior art, one of the advantages of the present application is that the target cells in the blood to be treated can be transduced or transfected without separation, purification, activation or amplification, thereby significantly saving the corresponding blood treatment time, achieving rapid pathological treatment, and significantly shortening the patient's disease treatment time. First, fresh anticoagulated whole blood or single blood can be injected into the first processing unit 1203 through the fluid inlet 111, for example, into the container 12031, and then a certain amount of buffer, such as physiological saline, is injected into the container 12031 through the first injection module 121 to dilute the blood to reduce the blood viscosity for later use. For example, the volume of the injected buffer: the volume of the blood to be diluted = 1:1. During this process, the container 12031 can be configured to rotate alternately forward and reverse at a certain speed to ensure more uniform mixing.

[0270] Then, a certain amount of Ficoll cell separation solution can be injected into the container 12031 via the cell separation solution injection module 123. For example, the injection ratio can be Ficoll cell separation solution volume: the aforementioned diluted blood volume = 1:2.

[0271] Next, the container 12031 is rotated at room temperature for a certain period of time, for example, 20 to 30 minutes, in a manner that generates a centrifugal force of 800 g. After or simultaneously with the centrifugation, the PBMC layer (i.e., the buffy coat layer) separated by centrifugation, and then the collected PBMCs can be stored or temporarily stored in the container 12131 as a liquid component to be transduced or transfected. Depending on the volume of blood injected via the fluid inlet 111, for example, the volume of the liquid of the collected PBMCs can be different. In one method example of the present application, for example, 5 ml of PBMC liquid can be collected and stored in the container 12131. For example, the above-mentioned sub-process of dilution, centrifugation, and collection of PBMCs can be repeated 1, 2, or more times to maximize the extraction of PBMCs to be processed in the blood.

[0272] Next, the GFP or CAR gene transduction or transfection of the PBMC is performed. For example, the transduction or transfection process can be completed in the container 12031 or in the container 12131. For example, a buffer such as a culture medium can be injected into the container 12031 or 12131 via the first injection unit 121, so that the cell concentration is adjusted to 1*10 6 ~1*10 8Then, the LNP-encapsulated GFP-mRNA or CAR-mRNA or the genetically modified / transformed AAV virus can be injected into the container 12031 or 12131 via the second injection unit 122 and incubated for 1 to 5 hours or other suitable time for transduction.

[0273] Then, the transduced or transfected liquid is added to container 12031 (if the transduction or transfection is completed in container 12131, the transduced or transfected liquid can be drawn into container 12031) with 5 to 15 times the volume of a buffer solution, such as physiological saline or culture fluid or culture medium, and container 12031 is rotated at room temperature for a certain time, such as 20 to 30 minutes, in a manner that generates a centrifugal force of 800g. Thereafter, the transduced or transfected PBMCs are drawn from a specific centrifugal layer for subsequent treatment. For example, this process of adding buffer and drawing from the transduced or transfected PBMCs using centrifugal layer (which can be regarded as a washing-centrifugation process) can be repeated multiple times, such as 2 to 3 times.

[0274] In response to the above-mentioned PBMC transduction or transfection test results, the inventors of the present application used GFP protein expression to detect the transduction or transfection efficiency, and the process is as follows.

[0275] The cell concentration of the PBMC solution after the washing and centrifugation treatment was adjusted to 1*10 6 ~5*10 6 Afterwards, cells were collected at 24 and 36 hours after culture for CD3 staining and T cell-specific GFP fluorescent protein expression was detected using flow cytometry.

[0276] In addition, based on the above-mentioned PBMC transduction or transfection test results, the inventors of the present application tested the killing function of the transduced or transfected CAR-T cells, and the process is as follows.

[0277] a) Place the transduced or transfected PBMC liquid (e.g., anti-CD19-CAR-mRNA transfected cell liquid) after the above-mentioned washing and centrifugation treatment in container 12031 and centrifuge at a speed of 1500 rpm for a certain period of time, for example, 5 minutes. Then, discard the supernatant, retain the cells, and add buffer to make the cell concentration approximately 2*10 6 pcs / ml.

[0278] b) At the same time, a Raji cell line stably expressing luciferase was obtained. The Raji-luciferase cells were centrifuged to remove impurities (at a speed of 1500 rpm, for example, for 5 minutes) and the cell concentration was adjusted to 1*10 6 pcs / ml.

[0279] c) The cells obtained in steps a) and b) were plated in duplicate at effector-target ratios of 1:1, 2:1, and 1:2, respectively. 100 μl of transduced or transfected PBMCs were added with 100 μl of Raji-luciferase (2:1); 50 μl of transduced or transfected PBMCs were added with 100 μl of Raji-luciferase (1:1); and 25 μl of transduced or transfected PBMCs were added with 100 μl of Raji-luciferase (1:2). The cells were seeded into a 96-well plate, and culture medium was added to 200 μl / well. The culture was continued for 24 hours.

[0280] d) Centrifuge (1500 rpm, e.g., 5 minutes), discard the supernatant, collect all cells into V-shaped wells, add 100 ml of lysis buffer, let stand at room temperature for 10 to 15 minutes, then centrifuge (3500 rpm, e.g., 20 minutes), and transfer 20 μl of supernatant to 8 strips / round-bottom 96-well plates. Add 50 μl of substrate / well and immediately measure the OD value.

[0281] e) Calculation: (1-test / NC)%=tumor lysis (cytotoxicity). Figures 8A to 8D The observation results after gene delivery using LNP, removal of impurities by centrifugation, and cell culture after transfection in container 12031 are shown respectively.

[0282] By using the simple and rapid CAR-T preparation method under closed conditions of the present invention, it has been found that LNPs with different optimized conditions can be removed after transfection within 5 hours. For the transfection efficiency of T cells in PBMC, after culturing for another 24 hours, GFP protein expression was observed regardless of whether FBS culture conditions or donor's own human serum culture conditions were used. This indicates that the LNP-encapsulated mRNA has a certain transfection efficiency for T cells within a short period of 5 hours, which can reach about 5% ( Figure 8A , Figure 8B ); After culturing for another 36 hours, the transfection expression efficiency can reach about 10% ( Figure 8C , Figure 8D ).

[0283] After testing the transfection efficiency, the inventors also tested the CAR-T killing function of PBMC cells transfected with the CAR gene. The results showed that the CAR-T produced by this closed system, when the transfection efficiency was about 5%, could achieve a killing efficiency of >30% on target cells at an effector-target ratio of 1:1. At an effector-target ratio of 2:1, the killing efficiency on target cells could reach >90%. This result shows that in this closed system, the CAR-T cells produced by this new method with simplified steps have strong killing activity against targeted tumor cells ( Figure 9 ).

[0284] The extracorporeal blood cell therapy device 100 of the present application can be used to construct a closed environment that facilitates extracorporeal blood cell treatment in a portable manner, and to construct an extracorporeal blood treatment method under such closed conditions that meet medical standards. The method includes, for example:

[0285] collecting blood from the patient;

[0286] Under closed conditions, target cells in the collected blood are gene-edited or gene-modified by gene delivery, and the method is also configured to remove non-therapeutic substances in the treated blood;

[0287] The treated fluid is returned to the patient.

[0288] It should be noted that the "closed conditions" referred to in this application may refer to conditions in which no human intervention is involved throughout the entire treatment process and that the treatment process meets medical standards. Those skilled in the art will appreciate that "closed conditions" are not limited to the extracorporeal blood cell therapy device described above. Other portable centrifugal devices that meet medical standards and specifications can also achieve the closed conditions required by the method of this application by connecting them to a specific container.

[0289] Although the filter is implemented as a filter membrane in the described embodiments, it will be clear to those skilled in the art that the embodiments described herein can be modified accordingly when other filtering methods are employed, while still achieving the purpose of the present application.

[0290] For example, in a Figure 2 In a possible modification of the illustrated embodiment, the filter 131 can be replaced with a centrifugal filtration device. Those skilled in the art will appreciate that centrifugal filtration involves adding a liquid to be filtered (in this case, a blood suspension) to a perforated drum containing a filter medium (e.g., a filter screen, filter cloth, etc.) using centrifugal force as the driving force. The cells to be treated are then retained on the filter medium by selecting an appropriate pore size, while the remaining liquid (unnecessary liquid) passes through the filter medium and is discharged, ultimately achieving the retention of the cells to be treated.

[0291] For example, in the case of Figure 2 In another possible modification of the embodiment shown, the filter 131 can be replaced with a chromatography column filtration device. The chromatography column mainly uses fillers with different pore sizes to perform targeted adsorption and filtration on particles of different sizes (here, cells).

[0292] For example, in the case of Figure 2 In another feasible modification of the embodiment shown, the filter 131 can be replaced with a magnetic screening filter device. This magnetic screening filter device mainly uses antibody magnetic beads to specifically mark the target cells, and then the cells marked with the magnetic beads are enriched and recovered by adsorption through a magnetic device after transduction or transfection. For example, in this modification, a unit for injecting specific antibody magnetic beads can be set, and the filter can be set as a magnetic adsorption device, such as a strong magnetic stand or a chromatography column with magnetic characteristics to replace the filter set in the second processing unit. When in use, after diluting the blood, the antibody magnetic beads are selectively injected, and the target cells will be characteristically marked by the antibody magnetic beads; then, after the transduction or transfection is completed, these marked target cells are enriched and collected by the magnetic adsorption device, and finally discharged from the second processing unit after flushing.

[0293] In the embodiments of the present application, the extracorporeal blood cell therapy device may also take any other suitable form. For example, under the premise of meeting medical and health requirements, the corresponding modules can be directly connected with medical hoses or pipes and / or data cables to form a portable extracorporeal blood cell gene editing or modification system. For example, in the case of forming a system, blood can be directly input into the first processing unit 120, and the processed blood can be output from the second processing unit 130.

[0294] Although specific embodiments of the present application are described in detail herein, they are provided for illustrative purposes only and should not be construed as limiting the scope of the present application. Furthermore, it will be apparent to those skilled in the art that the various embodiments described herein may be used in combination with one another. Various substitutions, variations, and modifications may be contemplated without departing from the spirit and scope of the present application.

Claims

1. A portable in vitro blood cell gene modification system comprising: a fluid inlet (111) and a fluid outlet (112); The processing units (1200, 1201, 1202, 1203) are configured to genetically modify resting or non-activated target cells in the blood input through the fluid inlet (111) by gene delivery and to remove substances not requiring treatment from the treated blood cells. The processing units (1200, 1201, 1202, 1203) are fluidically connected between the fluid inlet (111) and the fluid outlet (112), and the processing units (1200, 1201, 1202, 1203) include containers (1230, 12031) for containing liquid. The processing units (1200, 1201, 1202, 1203) further include at least a first injection module (121) and a second injection module (122) that are independent of each other, the first injection module (121) storing a first reagent that can be selectively injected into the container, and the second injection module (122) storing a second reagent that can be selectively injected into the container (1230, 12031), so that the target cells in the blood injected into the container are genetically modified by gene delivery without leaving the container. The invention also removes non-treatment-required substances in the treated blood. The first reagent includes a buffer solution, and the second reagent includes a gene delivery reagent. The gene delivery reagent is used to achieve transduction or transfection of target cells without separation, purification, activation or amplification. A filter (1310) is provided in the container (1230). The container (1230) includes a fluid input end (1230a), which is connected to the fluid inlet fluid; and a fluid output end (1230d), which is connected to the fluid outlet fluid. The fluid input end (1230a) and the fluid output end (1230d) are located upstream of the filter (1310). The first injection module (121) and the second injection module (122) are configured to selectively inject their respective reagents into the container (1230) downstream of the filter (1310).

2. The portable in vitro blood cell gene modification system according to claim 1, characterized in that: The filter (1310) is a molecular sieve, a magnetic screening filter device, a chromatography column, or a filter membrane.

3. The portable in vitro blood cell gene modification system according to claim 2, characterized in that: The gene delivery agent includes a viral vector or a non-viral vector.

4. The portable in vitro blood cell gene modification system according to claim 3, characterized in that: The non-viral vector includes a synthetic vector or a biological vector; and / or the viral vector includes a retrovirus or a modified or mutant thereof, a lentivirus or a modified or mutant thereof, an adenovirus or a modified or mutant thereof, or an adeno-associated virus or a modified or mutant thereof.

5. The portable in vitro blood cell gene modification system according to claim 4, characterized in that: The synthetic carrier is a lipid nanoparticle (LNP) or a lipid polyplex (LPP), and the biological carrier is an extracellular vesicle.

6. The portable in vitro blood cell gene modification system according to claim 5, characterized in that: The target cells include, but are not limited to, T cells, B cells, NK cells, macrophages, monocytes, or innate lymphoid cells.

7. The portable in vitro blood cell gene modification system according to claim 6, characterized in that: The gene delivery reagent includes a CAR gene to transduce or transfect T cells in PBMC.

8. The portable in vitro blood cell gene modification system according to claim 7, characterized in that: The time for transducing or transfecting target cells using the gene delivery agent without separation, purification, activation or amplification is 1 to 5 hours.

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