WO2012071760A1 - 生产免疫细胞的自动集成装置和方法 - Google Patents

生产免疫细胞的自动集成装置和方法 Download PDF

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Publication number
WO2012071760A1
WO2012071760A1 PCT/CN2010/080563 CN2010080563W WO2012071760A1 WO 2012071760 A1 WO2012071760 A1 WO 2012071760A1 CN 2010080563 W CN2010080563 W CN 2010080563W WO 2012071760 A1 WO2012071760 A1 WO 2012071760A1
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Prior art keywords
cell
culture
bottle
cells
rinsing
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PCT/CN2010/080563
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English (en)
French (fr)
Inventor
汪华
Original Assignee
Wang Hua
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Publication of WO2012071760A1 publication Critical patent/WO2012071760A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M37/00—Means for sterilizing, maintaining sterile conditions or avoiding chemical or biological contamination

Definitions

  • the invention belongs to the technical field of cell culture. Specifically, a medical cell production device and a production method thereof are disclosed.
  • immunocyte treatment is to in vitro culture, induce and multiply the lymphocytes of the patient with auto-cancerous efficacy, and then revert to the patient to enhance the body's own internal immune anti-cancer ability.
  • the purpose of treating and preventing tumors is to increase the cancer treatment to a high level of cellular immunotherapy, and has a good effect on treating tumors.
  • the object of the present invention is to overcome the deficiencies of the prior art and to provide an automated production device for medical cells, which can separate cells, induce culture and large-scale proliferation culture of each patient, and filter, rinse and sterilize cells.
  • the recycling process and the like are carried out in a completely sealed, laminar, sterile, separate system that ensures constant cell culture environmental conditions and is effective in preventing microbial contamination and cross-contamination by manual operations.
  • the invention drives the cell proliferation culture bag up and down reciprocating system, increases the movement of the cells in the culture bag, promotes the rapid proliferation of the cells, greatly increases the proportion of the effector cells, can meet the therapeutic needs of the patient, and has good cellular immunotherapy effect.
  • the invention also provides an automatic production method of medical cells, which fully realizes automation, has simple and convenient operation process, small operation error, good repeatability, conforms to GMP standards, and can be widely applied to different working environments and occasions such as medical and biopharmaceutical. .
  • the invention relates to a medical cell automatic production device, which comprises a laminar sterile sealed chamber, wherein the sealed chamber is provided with a sample bottle such as blood, and the sample bottle in the sealed chamber is sequentially connected to the sample by a connecting tube.
  • An isolated cell separation device, a cell rinsing device, a cell-inducing culture device, a cell proliferation culture device, a cell filtration, rinsing, and recovery device that filters, rinses, and recovers the cultured cells.
  • the cell separation device is a centrifuge with a closed accommodating cavity, and the accommodating cavity of the centrifuge is provided with a separation bag for holding and separating blood into plasma and lymphocytes. And a cell collection bag for collecting lymphocytes separated in the separation bag, wherein the port of the separation bag is further connected with a plasma collection bottle through a connecting tube. The output port of the cell collection bag is connected to the cell rinsing device through a connecting tube.
  • the cell rinsing device comprises a cell primary filtration rinse tube and a rinse bottle connected to the cell primary filter rinse tube, the cell primary filter rinse tube is placed behind the cell collection bag, and the cell primary filter rinse tube
  • the inlet is connected to the cell collection bag, and the outlet is respectively connected with a cell-inducing culture bottle and a waste liquid bottle, and a filter is arranged at the entrance of the primary filtration rinse tube of the cell to effectively prevent entry of large substances such as blood clots.
  • a filter cover is disposed at the outlet of the cell primary filtration rinse tube corresponding to the waste liquid to prevent loss of the rinsed cells; the connection tube of the rinse bottle port of the cell rinse device is equipped with a peristaltic pump for better rinsing cell.
  • the cell-inducing culture apparatus comprises at least one cell-inducing culture flask and a cell-inducing culture solution bottle provided above the cell-induced culture flask, and the respective cells are induced to be placed in a sealed cell-induced culture incubator.
  • the output port of the induction flask is opened, the suspension cells are placed in the proliferation culture bag, and then the outlet of the culture medium bottle and the inlet of the induction culture bottle are opened.
  • the adherent cells (such as dendritic cells, etc.) of the induction culture bottle are peeled off by the pipetting device and simultaneously transferred to the proliferation culture bag to increase the proportion of effector cells, and the cells induce a blown off-wall device with the peeling adhesion cells attached to the bottle.
  • the cell proliferation culture device is connected to an output end of a cell culture culture bottle, and the cell proliferation culture device includes at least one cell proliferation culture bag and a proliferation culture medium for simultaneously proliferating the culture bag of each cell.
  • the culture medium bottle is propagated, and the cell proliferation culture bag is placed in a sealed cell proliferation culture incubator.
  • the cell proliferation culture bag is suspended on a suspension frame, and the suspension frame is provided with a cell sediment prevention device for driving the cell proliferation culture bag to reciprocate up and down (or left and right) to promote the circulation of the suspension cells and prevent the suspension of the cells.
  • the cell filtration, rinsing and recovery device comprises a rinsing bottle connected to the output end of the cell proliferation culture bag through a connecting tube, a saline bottle, and a cell filtration rinsing tube for collecting the rinsed cells, the cells
  • a filter screen is arranged at the inlet of the filter rinsing tube, and a filter hood is arranged at the outlet of the cell filter rinsing tube corresponding to the waste liquid, and a secondary filter screen is arranged at the outlet of the cell filter rinsing tube corresponding to the finished effector cells.
  • the sample bottle, the rinsing bottle, the inducing broth bottle, the proliferating broth bottle, and the physiological saline bottle are respectively provided with a peristaltic pump on the liquid flow line, and each peristaltic pump is applied on the liquid flow line.
  • the pressure rather than acting directly on the liquid itself, causes the liquid to flow in the pipeline, and any part of the peristaltic pump has no contact with the liquid and is hygienic.
  • the rotational speed of the peristaltic pump can be controlled by a computer program or by a manual control button, which is convenient and reliable to use.
  • the liquid flow lines are provided with line clamps, and the computer and the operator can control the opening/closing of each line clamp to allow/prevent liquid from entering the respective lines.
  • the cell filtration rinse tube is provided with a baroreceptor, when the pressure exceeds the setting At the time of the value, it is suggested that the number of cells recovered in the cell filtration rinse tube reaches the upper limit, and the cell rinsing and cell recovery procedures can be entered.
  • the sealed chamber is further provided with a safety monitoring sensor, which includes a pipeline pressure sensor on the liquid flow pipeline, a pipeline flowmeter, is installed in the sample bottle, the rinse bottle, the induced culture fluid bottle, and the proliferation a liquid level detector on the culture medium bottle and the saline bottle, a temperature detector placed in the centrifuge, a humidity detector and a centrifugal balance detector, and a temperature detector in the cell-induced incubator and the cell proliferation incubator. Humidity detector, carbon dioxide concentration detector and liquid level detector.
  • the computer monitors it and initiates an alarm system if an abnormal condition occurs.
  • the line pressure sensor on the liquid flow line can also be classified into a negative pressure detecting sensor and a positive pressure detecting sensor.
  • the negative pressure detecting sensor is disposed on the output line of the sample liquid and the rinsing liquid.
  • the positive pressure detecting sensor is disposed on the discharge line of the cell to detect the smoothness of the cell discharge line. When the pressure in the line exceeds the set value, a warning alarm will occur.
  • the pipeline flowmeter is a non-contact metering device using an ultrasonic working principle, and may be an external clip type or an external stick type.
  • the pipe flow meter displays the amount of liquid flowing through the line, and when the liquid flow reaches the set value, the computer automatically shuts down the line.
  • the liquid level level detector is a liquid level level detector using an ultrasonic working principle for monitoring the liquid level in the air tube on the pipeline. When the liquid level is lower or higher than the detector, an alarm will be activated.
  • a cell culture monitoring system for monitoring the cell culture state is connected to the cell-inducing flask and the cell proliferation culture bag.
  • the cell culture monitoring system comprises a light-emitting device, an optical sensor and a cell output tube, wherein the cell culture monitoring system is independently and correspondingly disposed in a cell-inducing incubator and a cell proliferation incubator, and the cell output tube is correspondingly connected Inducing culture flasks and proliferation culture bags.
  • the cell culture monitoring system is configured to monitor the permeation amount of the culture solution in the culture process in each of the incubators (including the cell-induced culture incubator and the cell proliferation incubator), and when the cell concentration is increased, the light permeation amount is reduced, and the use of the cell culture monitoring system is reduced.
  • a monitoring method the computer monitors the amount of cells in the culture line. In addition, when there is microbial contamination, the amount of light is rapidly reduced and exceeds the upper limit, and the computer can monitor the contamination of the cell culture.
  • the front of the cell-induced incubator and the cell proliferation incubator is provided with a pipe interface window at the position of the cell culture monitoring system in the box, and the closed/open line and the control cell and the culture are set at the near interface.
  • the pipe clamp for liquid discharge, the interface window is used for the connection of the pipelines of each culture device and the extraction of cells and culture fluids.
  • the sealed chamber in order to provide an environment for sterilizing and suitable cell growth in a sealed chamber, the sealed chamber is provided with a laminar flow cleaning and disinfecting system for controlling the cleanliness, temperature and humidity of the sealed chamber, and a control induction incubator and a proliferation incubator. Incubator control system for temperature humidity and carbon dioxide concentration.
  • the liquid flow lines between the respective connected devices are connected by a connection hose having a good aseptic sealing property to ensure good sealing performance.
  • the method for producing a medical cell automated production device taking immune lymphocytes as an example, the specific steps are:
  • Inserting a patient sample Move the patient sample into the sample bottle (eg, blood is transferred to the sample bottle);
  • the preliminary treatment sample such as blood is separated by a cell separation device to separate a target cell such as a lymphocyte;
  • the target cells isolated and rinsed are subjected to induction culture by specific cells (such as CIK, NK cells, etc.) through a cell induction device;
  • Cell proliferation culture process cells in the cultured culture medium are subjected to a large-scale proliferation culture in a proliferation culture incubator, and cell sedimentation is prevented by preventing reciprocation of the cell sedimentation device;
  • the cell culture monitoring system that is, the photoelectric sensor is used to monitor the cell concentration, and when the set cell concentration is reached, it is suggested that cell dosing or cell filling is required. Enter the cell product recycling process;
  • the present invention is achieved by connecting a cell by separation, induction culture, large proliferation culture, cell filtration, rinsing, and recovery through a sterile hose and completely in a sterile, self-contained sealed chamber.
  • the infection of the cells in addition, the cell production environment is constant, the proportion of effector cells is greatly improved, which can meet the treatment needs of patients, and greatly improve the treatment of immune cells;
  • the method for producing medical cells according to the present invention has higher automation degree, simple and convenient operation process, small operation error, high repeatability, compliance with GMP standards, and can be widely applied to the method of artificially culturing cells. Different working environments and occasions such as medical and biopharmaceutical.
  • FIG. 1 is a schematic flow chart of a method for producing a medical cell according to the present invention
  • FIG. 2 is a schematic structural view of a medical cell automated production apparatus according to the present invention.
  • Figure 3 is a schematic view showing the arrangement of cell-induced culture flasks in the cell-induced culture tank of the present invention
  • Figure 4 is a layout diagram of a cell proliferation culture cell proliferation culture bag in the present invention.
  • Figure 5 is a schematic view showing the structure of a cell primary filtration rinse tube in the present invention.
  • Figure 6 is a schematic view showing the planar structure of the cell filtration rinse tube in the present invention.
  • Figure 7 is a schematic exploded perspective view of the cell filtration rinse tube of the present invention.
  • the specific steps are as follows:
  • the method for producing a medical cell automated production device according to the present invention, taking immune lymphocytes as an example, the specific steps are as follows:
  • Inserting a patient sample Move the patient sample into the sample bottle (eg, blood is transferred to the sample bottle);
  • the preliminary treatment sample such as blood is separated by a cell separation device to separate a target cell such as a lymphocyte;
  • the target cells isolated and rinsed are subjected to induction culture by specific cells (such as CIK, NK cells, etc.) through a cell induction device;
  • Cell proliferation culture process cells in the cultured cells are cultured in a proliferation culture box to prevent proliferation of cells, and prevent cell sedimentation by preventing reciprocation of the cell sedimentation device;
  • the cell culture monitoring system that is, the photoelectric sensor is used to monitor the cell concentration, and when the set cell concentration is reached, it is suggested that cell dosing or cell filling is required. Enter the cell product recycling process;
  • Cell filtration, rinsing and recovery process Open the output port of the proliferation culture bag, let the cells in the proliferation culture bag and the culture solution flow into the filtration and rinsing tube together, and the primary filter in the rinsing tube blocks the large non-cell mass Into the tube, the rinse net cover allows the culture liquid and the rinse liquid to be discharged into the waste liquid bottle to prevent the passage of the target cells; the baroreceptor monitors the pressure in the rinse tube, and when the pressure reaches the set value, enters the cell rinse program, and the pipeline flow meter monitors the culture Discharge of liquid and rinsing liquid When the amount of fluid or the amount of rinsing liquid reaches the set value, the computer terminates the cell filtration or rinsing procedure, opens the cell recovery outlet, recovers the cells to the product bag, obtains qualified effector cells, and returns these effector cells to the patient. To enhance the body's inherent immune and anti-cancer ability.
  • the medical cell automated production device of the present invention comprises a sealed sterile sealed chamber 1 in which a sample bottle 2 for extracting blood of a patient is provided, and a sample bottle in the sealed chamber 1 is provided. 2, a cell separation device 3 for separating blood, a cell rinsing device 4, a cell-inducing culture device 5, a cell proliferation culture device 6, and a filter, rinse, and recovery of the cultured cells are sequentially connected by a sterile connection hose. Cell filtration, rinsing and recovery unit 7.
  • the cell separation device 3 is a centrifuge 31 with a closed receiving cavity, and the inside of the cavity of the centrifuge is provided for holding and separating blood into plasma and lymphocytes.
  • a separation bag 32, and a cell collection bag 33 for collecting lymphocytes separated in the separation bag 32, and a port of the separation bag 32 is further connected to a plasma collection bottle 34 via a connection hose, the cell collection bag 33
  • the output port is connected to the cell-inducing culture device through a connecting hose, where the lighter plasma and the heavier lymphocytes are slowly layered by the centrifugation principle of the centrifuge, and will float on the surface.
  • the plasma is gradually collected into the plasma collection bottle 11, and then the heavier specific lymphocytes are collected into the cell collection bag 33, thereby completing the cell separation process, and the cell separation device 31 is also connected to the waste liquid collection tank.
  • the cell rinsing device 4 includes a cell primary filtration rinse tube 41 and a rinse bottle 42 connected to the cell primary filter rinse tube 41, the cell primary filter rinse tube 41 being placed behind the cell collection bag 33, and
  • the cell primary filtration rinse tube 41 is connected at its inlet to a cell collection bag 33 having a waste liquid discharge port and an outlet of a target cell: wherein, the cell outlet of the target cell and the cell of the cell-induced culture device 5
  • the induction culture bottle 51 is connected, and the waste liquid bottle 9 is connected to the waste liquid outlet, and a filter screen 43 is disposed at the entrance of the cell primary filtration rinse pipe 41, which can effectively prevent the entry of large substances such as blood clots;
  • a filter cover 44 is further disposed at the outlet of the cell primary filtration rinse tube 41 corresponding to the waste liquid, which can effectively prevent the loss of the rinsed cells; further, the connection tube of the rinse bottle 42 port of the cell rinse device is disposed.
  • the cell-inducing culture apparatus 5 includes a plurality of cell-inducing culture bottles 51, and is provided above each of the cell-inducing culture flasks 51 and is connected to each of the cell-inducing culture flasks 51 to induce a culture solution.
  • the bag 52, the induced culture solution bag 52 respectively injects an induction culture solution into each of the cell induction bottles 51, and the induced culture solution can effectively stimulate lymphocyte activation.
  • all the cell-inducing culture bottles 51 are placed in a sealed state. The cells are induced in the incubator 10 (as shown in Fig.
  • the cell culture flask 51 is connected to the cell proliferation culture device 6 via a connection hose at the output end.
  • the outlet of the induction flask is first opened, and the suspension cells are transferred to the cell proliferation culture bag 61 of the late cell proliferation device 6, and then the adherent cells (such as dendrites) are used. Cells, etc.)
  • the cell proliferation culture bag 61 of the cell proliferation device 6 increases the ratio of effector cells, and the cell-inducing bottle 51 is connected with a pipetting device for peeling off adhering cells, which mainly passes through the liquid by an external peristaltic pump or a reciprocating liquid suction device. The impulse is blown down by the cells adhering to the wall of the bottle, and the operation is carried out in a closed sterile space to minimize contamination and cross-contamination.
  • the cell proliferation culture apparatus 6 includes at least one cell proliferation culture bag 61, and a proliferation culture solution bag 62 for simultaneously providing a proliferation culture solution to each cell proliferation culture bag 61, and cell proliferation culture.
  • the bag 61 is placed in the cell proliferation incubator 20, and each cell proliferation culture bag 61 is suspended from the suspension frame 63.
  • the proliferation culture medium in the proliferation culture solution bag 62 can effectively promote the proliferation of effective lymphocytes, and the culture medium according to the desire The type of lymphocytes to be cultured varies.
  • the suspension frame 63 is provided with a driving cell proliferation culture bag 61 that reciprocates up and down (or left and right) to promote the circulation movement of the suspension cells and prevent
  • the cell precipitating device 64 for suspending cell sedimentation specifically drives the bottom of the cell proliferation culture bag 61 to reciprocate up and down (or left and right) by the cell-preventing device 64, thereby preventing sedimentation of suspended cells during continuous reciprocating motion. The role.
  • the cell filtration, rinsing and recovery device 7 of the present invention comprises a rinse bottle 71 connected to the output end of the cell proliferation culture bag 61 through a connecting tube, a saline bottle 72, and a pair.
  • the rinsed cells are filtered, and the collected cell filtration rinse tube 73 is provided with a primary filter 74 at the inlet of the cell filtration rinse tube 73, and a filter cover is arranged in the cell filtration rinse tube corresponding to the outlet of the waste liquid bottle 12. 75.
  • a secondary filter net 76 is disposed at the outlet of the cell filtration rinse tube 73 corresponding to the finished effector cells, and the qualified finished effect cells are obtained by stepwise filtration, and then the qualified finished effect cells are passed through the infusion bag 30. It is transmitted to the patient to enhance the body's own internal immune and anti-cancer ability, so as to effectively achieve the purpose of treating and preventing tumors, and the medicinal effect is good.
  • the cell filtration, rinsing and recovery device 7 of the present invention is provided with a baroreceptor to monitor the unobstructed condition of the cell rinsing tube, and automatically enters the rinsing program when the pressure reaches the set value.
  • the pipeline flow meter monitors the discharge amount of the culture solution and the rinse liquid. When the culture liquid amount or the rinse liquid amount reaches the set value, the computer terminates the cell filtration or rinsing process, opens the cell recovery outlet, and recovers the cells to the product bag to obtain the qualified.
  • the effector cell is finished.
  • the sample bottle 2 As shown in FIG. 2, in the present invention, the sample bottle 2, the rinse bottle 41, the induced culture liquid bottle 52, the proliferation culture liquid bottle 62, the rinse bottle 71, and the liquid flow line at the outlet of the physiological saline bottle 72 are correspondingly arranged.
  • the rotational speed of the peristaltic pump can be controlled by a computer program or by a manual control button, which is convenient and reliable to use.
  • the liquid flow lines are provided with line clamps, and the computer and the operator can control the opening/closing of each line clamp to allow/prevent liquid from entering the respective lines.
  • a safety monitoring sensor is further disposed in the sealed chamber 1, and the safety monitoring sensor includes a liquid.
  • a line pressure sensor and a line flow meter on the body flow line, a liquid level level detector mounted on the sample bottle 2, the rinse bottle 41, the induced culture liquid bottle 52, the proliferation culture liquid bottle 62, and the physiological saline bottle 72 The temperature detector, the humidity detector and the centrifugal balance detector in the centrifuge, and the cell-induced incubator 51, the temperature detector in the cell proliferation incubator 61, the humidity detector carbon dioxide concentration detector and the liquid level detector.
  • the above various safety monitoring sensors are connected with the computer control system. During the operation, the computer monitors them.
  • the line pressure sensor on the liquid flow line can also be divided into a negative pressure detecting sensor and a positive pressure detecting sensor.
  • the negative pressure detecting sensor is disposed on the output line of the sample liquid 2 and the rinsing bottle 41.
  • the positive pressure detecting sensor is disposed on the discharge line of the cell to detect the unobstructed condition of the cell discharge line. When the pressure in the line exceeds the set value, a warning alarm will occur.
  • the pipeline flowmeter uses the ultrasonic principle to calculate the output of the liquid.
  • the liquid level level detector is a liquid level level detector using an ultrasonic working principle for monitoring the liquid level in the air tube on the pipeline, and when the liquid level is lower than the detector, an alarm will be activated. Due to the use of various safety monitoring sensors, the safety performance is greatly improved, the use is more convenient, and the utility is good.
  • the cell-inducing culture bottle 51 and the cell proliferation culture bag 61 are connected to a cell culture monitoring system for monitoring the cell culture condition.
  • the cell culture monitoring system includes a photoinductor and a cell output tube, and the photosensor is composed of two components of an illuminator and a photoreceiver.
  • the cell culture monitoring systems are independently and correspondingly disposed in the cell-inducing incubator 10 and the cell proliferation incubator 20, and the cell output tubes are correspondingly coupled to the induction culture bottle 51 and the proliferation culture bag 61.
  • the cell culture monitoring system is used for monitoring the permeation amount of the culture solution in the culture process in each of the incubators (including the cell-induced culture incubator 10 and the cell proliferation incubator 20).
  • the computer monitors the amount of cells in the culture line.
  • the amount of light is rapidly reduced, and the computer can monitor the contamination of cell culture.
  • This cell culture monitoring system is easy to operate and easy to monitor cell culture.
  • the front portions of the cell-induced incubator 10 and the cell proliferation incubator 20 are provided with pipe interface windows 101 and 201 at positions corresponding to the cell culture monitoring system in the chamber, and the near interface is closed/
  • the open pipe and the pipe clamp for controlling the discharge of the cells and the culture liquid are used for the connection of the tubes of the respective culture devices and the extraction of the cells and the culture liquid.
  • the sealed chamber 1 in order to provide an environment for sterilizing and suitable cell growth in a sealed chamber, the sealed chamber 1 is provided with a laminar flow cleaning and disinfecting system for controlling the cleanliness, temperature and humidity of the sealed chamber, and the control induction incubator 10 and An incubator control system that proliferates the temperature and humidity of the incubator 20 and the concentration of carbon dioxide.
  • the liquid flow lines between the respective connected devices are connected by a connection hose having a good aseptic sealing property to ensure good sealing performance.
  • the various types of safety monitoring sensors and cell culture monitoring systems described in the present invention are not in direct contact with the liquid, and the occurrence of pollution can be avoided.
  • the medical cell automated production device of the present invention can make each patient's cell separation, induction culture and large-scale proliferation culture as well as cell filtration, rinsing and recovery processes in a completely sealed, laminar sterile independent system. This is done to ensure that the cell culture environment is constant and can effectively prevent microbial contamination and cross-contamination by manual operations.
  • the invention drives the cell proliferation culture bag up and down reciprocating system, increases the movement of the cells in the culture bag, promotes the rapid proliferation of the cells, greatly increases the proportion of effect cells, can meet the therapeutic needs of the patient, and has good cellular immunotherapy effect.
  • the medical cell automated production method is fully automated, the operation process is simple and convenient, the operation error is small, the repeatability is good, and the GMP standard is met, and the invention can be widely applied to different working environments and occasions such as medical and biopharmaceutical.

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Description

生产免疫细胞的自动集成装置和方法
技术领域
本发明属于细胞培养技术领域。 具体公开一种医用细胞生产装置及其生产方法。
背景技术
在医学技术领域， 免疫细胞治疗是将患者自体具有杀癌效力的淋巴细胞经体外培养， 诱 导和成千倍扩增后， 回输至患者体内， 以增强机体自身内在免疫抗癌能力， 从而达到治疗和 预防肿瘤的目的， 其能将癌症治疗提高至细胞免疫治疗的高水平， 对治疗肿瘤具有较好的成 效。
现有技术中， 一些用于免疫细胞治疗的医用细胞生产过程一般都是人工操作完成， 操作 过程繁琐， 操作过程易发生微生物污染和交叉污染， 不能确保整个过程的无菌化生产； 人工 培养方法， 对操作人员要求高， 可重复性低， 质量不稳定； 同时现有的医疗用细胞培养方法， 细胞诱导和扩增数量少， 效应细胞比例低， 细胞免疫治疗效果差， 很难普遍推广应用。
发明内容
本发明的目的是克服现有技术的不足， 提供一种医用细胞自动化生产装置， 该医用细胞 自动化生产装置可以使每一个患者的细胞分离、诱导培养及大规模增殖培养以及细胞的过滤、 漂洗和回收过程等过程在一个完全密封、 层流无菌的独立系统中完成， 这样确保细胞培养环 境条件恒定， 可有效地防止人工操作的微生物污染和交叉污染。 同时， 本发明通过驱动细胞 增殖培养袋上下往复运动系统， 增加了细胞在培养袋内运动， 促进了细胞的快速增殖， 效应 细胞比例大大提高， 可满足患者的治疗需要， 细胞免疫治疗效果好。
本发明同时还提供了一种医用细胞自动化生产方法， 其完全实现自动化， 操作过程简单 方便， 操作误差小， 重复性好， 符合 GMP标准， 能广泛适用于医疗和生物制药等不同工作环 境和场合。
为了实现上述第一个技术目的， 本发明是按以下技术方案实现的：
本发明所述的一种医用细胞自动化生产装置， 包括层流无菌的密封室， 所述密封室内设 有血液等样本瓶， 所述密封室内样本瓶后通过连接管依次连接有对样品进行细胞分离的细胞 分离装置、 细胞漂洗装置、 细胞诱导培养装置、 细胞增殖培养装置、 对培养后的细胞进行过 滤、 漂洗和回收的细胞过滤、 漂洗和回收装置。
在本发明中， 所述细胞分离装置为带有封闭容置空腔的离心机， 所述离心机的容置空腔 内部设有用于盛放并对血液进行分离成血浆和淋巴细胞的分离袋， 以及将分离袋内分离出的 淋巴细胞进行收集的细胞收集袋， 所述分离袋的端口处还通过连接管连接有血浆收集瓶， 所 述细胞收集袋的输出端口通过连接管连接所述的细胞漂洗装置。
在本发明中， 所述细胞漂洗装置包括细胞初级过滤漂洗管和与细胞初级过滤漂洗管连接 的漂洗瓶， 所述细胞初级过滤漂洗管置于细胞收集袋的后方， 且该细胞初级过滤漂洗管的入 口处连接于细胞收集袋， 其出口处分别连接有细胞诱导培养瓶和废液瓶， 所述细胞初级过滤 漂洗管的入口处设有一过滤网， 有效防止血凝块等大块物质的进入； 此外， 所述细胞初级过 滤漂洗管内对应于废弃液的出口处设有过滤罩， 防止漂洗细胞的丢失； 所述细胞漂洗装置的 漂洗瓶端口的连接管配有蠕动泵， 以较好地漂洗细胞。
在本发明中， 所述细胞诱导培养装置包括至少一细胞诱导培养瓶及设在细胞诱导培养瓶 上方的细胞诱导培养液瓶， 所述各细胞诱导培养瓶放置于密封的细胞诱导培养箱内。
在本发明中， 为了将诱导培养后的细胞转入增殖培养袋， 首先开放诱导培养瓶的输出口， 将悬浮细胞放入增殖培养袋， 然后开放增殖培养液瓶输出口和诱导培养瓶的入口， 通过吹打 装置剥离诱导培养瓶的贴壁细胞（如树突状细胞等）同时转移至增殖培养袋， 增加效应细胞比 例， 所述细胞诱导瓶上连接有剥离粘附细胞的吹打离壁装置， 其主要通过外加的往复液体抽 吸设备或蠕动泵通过液体的冲力将粘附在瓶壁的细胞吹下，由于操作在密闭的无菌空间进行， 可最大程度地防止污染和交叉污染。
此外， 在本发明中， 所述细胞增殖培养装置连接在细胞培养瓶的输出端， 所述细胞增殖 培养装置包括至少一个细胞增殖培养袋及用于对各细胞增殖培养袋同时提供增殖培养液的增 殖培养液瓶， 所述细胞增殖培养袋置放于密封的细胞增殖培养箱内。 所述细胞增殖培养袋悬 挂于悬挂架上， 所述悬挂架上设有驱动细胞增殖培养袋上下 （或左右） 往复运动的促使悬浮 细胞循环运动、 防止悬浮细胞沉淀的防止细胞沉淀装置。
在本发明中， 所述细胞过滤、 漂洗和回收装置包括通过连接管连接在细胞增殖培养袋输 出端的漂洗瓶、 生理盐水瓶、 以及对漂洗后的细胞进行收集的细胞过滤漂洗管， 所述细胞过 滤漂洗管的入口处设有一级过滤网，细胞过滤漂洗管内对应于废弃液的出口处设有过滤网罩， 细胞过滤漂洗管内对应于成品效应细胞的出口处设有二级过滤网。
作为上述技术的进一步改进， 所述样本瓶、 漂洗瓶、 诱导培养液瓶、 增殖培养液瓶和生 理盐水瓶的液体流动管路上均对应设有蠕动泵， 各蠕动泵通过在液体流动管路上施加压力而 不是直接作用于液体本身， 使液体在管路中流动， 蠕动泵的任何部位与液体均无任何接触， 清洁卫生。 而且， 所述蠕动泵的转速可由计算机程序控制或由手动控制键操作， 使用方便可 靠。 此外， 所述液体流动管路均设有管路钳， 计算机和操作人员可控制每个管路钳的打开 / 关闭， 容许 /防止液体进入相应管路。
作为上述技术的更进一步改进， 所述细胞过滤漂洗管设有压力感受器， 当压力超过设定 值时， 提示细胞过滤漂洗管内回收的细胞数达到上限， 可进入细胞漂洗和细胞回收程序。 在本发明中， 所述密封室内还设有安全监测传感器， 该安全监测传感器包括液体流动管 路上的管路压力传感器、 管路流量计， 安装于样本瓶、 漂洗瓶、 诱导培养液瓶、 增殖培养液 瓶和生理盐水瓶上的液面水平探测器、 置于离心机内的温度探测器、 湿度探测器及离心平衡 探测器， 以及细胞诱导培养箱、 细胞增殖培养箱内的温度探测器， 湿度探测器， 二氧化碳浓 度探测器和液面水平探测器。 在运行过程中， 计算机对其进行监测， 如果发生异常情况将启 动报警系统。
具体来说， 所述液体流动管路上的管路压力传感器还可以分为负压探测传感器和正压探 测传感器。 负压探测传感器设置于样本液和漂洗液的输出管路上， 当管内液体压力低于计算 机设定的下限值时， 将出现提示报警。 而正压探测传感器设置于细胞的排出管路上， 检测细 胞排出管路的通畅情况， 当管路内的压力超过设定值时， 将出现提示报警。
所述管路流量计为采用超声工作原理的非接触式计量装置， 可以是外夹式或外贴式。 管 路流量计可显示流过该管路的液体量， 当液体流量达到设定值时， 计算机可自动关闭该管路。
所述液面水平探测器为采用超声工作原理的液面水平探测器， 用于监测管路上空气管内 的液平面， 当液面低于或高于该探测器时， 将启动报警。
在本发明中， 为了更好地监控细胞的培养状况， 所述细胞诱导培养瓶及细胞增殖培养袋 上均连接有用于监控细胞培养状况的细胞培养监控系统。 所述细胞培养监控系统包括发光装 置， 光学传感器和细胞输出管， 所述细胞培养监控系统均独立且对应地设置于细胞诱导培养 箱和细胞增殖培养箱内， 且所述细胞输出管对应地连接于诱导培养瓶和增殖培养袋。 所述细 胞培养监控系统用于监测各培养箱 （包括细胞诱导培养箱和细胞增殖培养箱） 内培养过程中 培养液的通透量， 当细胞浓度增加时， 光的通透量减少， 利用这一监控方法， 计算机可监控 培养管路内的细胞含量。 此外， 当有微生物污染时， 光的通透量急速减少并超过上限值， 计 算机也可以监控细胞培养的污染状况。
此外， 为了便于观测， 所述细胞诱导培养箱和细胞增殖培养箱的前部对应于箱内的细胞 培养监控系统位置处设置管道接口窗，近接口处设置关闭 /开放管路和控制细胞和培养液排出 的管路钳， 该接口窗用于各培养装置管路的连通和细胞及培养液的抽取检查。
在本发明中， 为了提供密封室内无菌和适宜细胞生长的环境， 所述密封室内设有控制密 封室内洁净度、 温度和湿度的层流洁净及消毒系统， 以及控制诱导培养箱和增殖培养箱的温 度湿度和二氧化碳浓度的培养箱控制系统。
在本发明中， 各对应连接的装置之间的液体流动管路均通过无菌密封性能好的连接软管 连接， 以保证较好的密封性能。 为了实现上述第二个技术目的， 本发明是按以下技术方案实现的：
本发明所述的医用细胞自动化生产装置的生产方法， 以免疫淋巴细胞为例， 其具体步骤 是：
(1)置入患者样本： 将患者样本并移至样本瓶内 （如血液移至样本瓶）；
(2)细胞分离过程： 对初步处理的样本如血液通过细胞分离装置进行分离， 分离出目的细 胞如淋巴细胞；
(3)细胞漂洗过程： 将分离出的细胞通过细胞漂洗装置进行初步漂洗；
(4)细胞诱导培养过程： 对分离并漂洗后的目的细胞通过细胞诱导装置进行特定细胞（如 CIK, NK细胞等)诱导培养；
(5)细胞转入增殖培养袋过程： 开放诱导培养瓶的输出口， 首先将悬浮细胞放入增殖培养 袋， 然后开放增殖培养液瓶输出口和诱导培养瓶的入口， 通过吹打装置剥离诱导培养瓶的贴 壁细胞， 并将其转入增殖培养袋。
(6)细胞增殖培养过程：在增殖培养箱内对诱导培养后的细胞进行细胞大量增殖培养，并通 过防止细胞沉淀装置的往复运动防止细胞沉淀；
(7)培养细胞的监控过程： 通过设置于诱导培养箱和增殖培养箱内的细胞培养监控系统， 即光电感应器监控细胞浓度， 当达到设定的细胞浓度时， 提示需要进行细胞加液或进入细胞 产品回收过程；
(8)细胞过滤、 漂洗和回收过程： 开放增殖培养袋的输出口， 使增殖培养袋内的细胞和培 养液一同流入细胞过滤漂洗管， 细胞过滤漂洗管内的一级过滤网阻止大的非细胞团块进入管 内， 漂洗网罩容许培养液和漂洗液通过排入废液瓶， 阻止目的细胞通过； 压力感受器监控漂 洗管内压力， 当压力达到设定值时， 进入细胞漂洗程序； 管路流量计监控培养液和漂洗液的 排出量， 当培养液量或漂洗液量达到设定值时， 计算机终止细胞过滤或漂洗程序， 开放细胞 回收出口， 将细胞回收至产品袋， 获得合格的效应细胞成品。
与现有技术相比， 本发明的有益效果是：
(1)本发明由于将细胞的分离、 诱导培养、 大增殖培养、 细胞过滤、 漂洗和回收等过程之 间通过无菌的软管连接且完全在无菌的独立密封的密封室内完成， 避免了在各中间环节中细 胞的感染， 此外， 细胞生产环境恒定， 效应细胞比例大大提高， 可满足患者治疗需要， 大大 提高免疫细胞治疗；
(2)本发明所述的医用细胞生产方法与现有的人工培养细胞的方式相比， 自动化程度高， 操作过程简单方便， 操作误差小， 重复性高， 符合 GMP标准， 能广泛地适用于医疗和生物制 药等不同工作环境和场合。 附图说明
下面结合附图和具体实施例对本发明做详细的说明：
图 1是本发明所述的医用细胞生产方法流程示意图；
图 2是本发明所述的医用细胞自动化生产装置结构示意图；
图 3是本发明中细胞诱导培养箱内细胞诱导培养瓶排布示意图；
图 4是本发明中细胞增殖培养箱细胞增殖培养袋布置图；
图 5是本发明中细胞初级过滤漂洗管结构示意图；
图 6是本发明中细胞过滤漂洗管平面结构示意图；
图 7是本发明中细胞过滤漂洗管立体分解结构示意图。
具体实施方式
如图 1所示， 本发明所述的医用细胞自动化生产装置的生产方法， 具体步骤是： 本发明所述的医用细胞自动化生产装置的生产方法， 以免疫淋巴细胞为例， 其具体步骤 是：
(1)置入患者样本： 将患者样本并移至样本瓶内 （如血液移至样本瓶）；
(2)细胞分离过程： 对初步处理的样本如血液通过细胞分离装置进行分离， 分离出目的细 胞如淋巴细胞；
(3)细胞漂洗过程： 将分离出的细胞通过细胞漂洗装置进行初步漂洗；
(4)细胞诱导培养过程： 对分离并漂洗后的目的细胞通过细胞诱导装置进行特定细胞（如 CIK, NK细胞等)诱导培养；
(5)细胞转入增殖培养袋过程： 开放诱导培养瓶的输出口， 首先将悬浮细胞放入增殖培养 袋， 然后开放增殖培养液瓶输出口和诱导培养瓶的入口， 通过吹打装置剥离诱导培养瓶的贴 壁细胞， 并将其转入增殖培养袋。
(6)细胞增殖培养过程：在增殖培养箱内对诱导培养后的细胞进行细胞大量增殖培养，并通 过防止细胞沉淀装置的往复运动防止细胞沉淀；；
(7)培养细胞的监控过程： 通过设置于诱导培养箱和增殖培养箱内的细胞培养监控系统， 即光电感应器监控细胞浓度， 当达到设定的细胞浓度时， 提示需要进行细胞加液或进入细胞 产品回收过程；
(8)细胞过滤、 漂洗和回收过程： 开放增殖培养袋的输出口， 使增殖培养袋内的细胞和培 养液一同流入过滤和漂洗管， 漂洗管内的一级过滤网阻止大的非细胞团块进入管内， 漂洗网 罩容许培养液和漂洗液通过排入废液瓶， 阻止目的细胞通过； 压力感受器监控漂洗管内压力， 当压力达到设定值时， 进入细胞漂洗程序， 管路流量计监控培养液和漂洗液的排出量， 当培 养液量或漂洗液量达到设定值时， 计算机终止细胞过滤或漂洗程序， 开放细胞回收出口， 将 细胞回收至产品袋， 获得合格的效应细胞成品， 并将这些效应细胞回输至患者体内， 以增强 机体自身内在免疫抗癌能力。
以下结合本发明所述的医用细胞生产装置具体说明其生产过程。
如图 2所示， 本发明所述的医用细胞自动化生产装置， 包括密封无菌的密封室 1， 在该 密封室 1内设有抽取患者血液的样本瓶 2， 所述密封室 1内样本瓶 2后通过无菌的连接软管 依次连接有对血液进行分离的细胞分离装置 3、 细胞漂洗装置 4、 细胞诱导培养装置 5、 细胞 增殖培养装置 6、 对培养后的细胞进行过滤、 漂洗和回收的细胞过滤、 漂洗和回收装置 7。
具体由图 2可知， 所述细胞分离装置 3为带有封闭容置空腔的离心机 31， 所述离心机的 容置空腔内部设有用于盛放并对血液进行分离成血浆和淋巴细胞的分离袋 32， 以及将分离袋 32内分离出的淋巴细胞进行收集的细胞收集袋 33， 所述分离袋 32的端口处还通过连接软管 连接有血浆收集瓶 34， 所述细胞收集袋 33的输出端口通过连接软管连接有细胞诱导培养装 置， 此处利用离心机工作时的离心原理， 将血液中较轻的血浆与较重的淋巴细胞缓慢的分层， 并将浮在面上的血浆逐渐的收集至血浆收集瓶 11， 然后将比重较重的淋巴细胞收集至细胞收 集袋 33中， 从而完成细胞的分离过程， 同时细胞分离装置 31上还连接有废液收集箱。
在本发明中，所述细胞漂洗装置 4包括细胞初级过滤漂洗管 41和与细胞初级过滤漂洗管 41连接的漂洗瓶 42， 所述细胞初级过滤漂洗管 41置于细胞收集袋 33的后方， 且该细胞初级 过滤漂洗管 41在其入口处连接细胞收集袋 33， 所述细胞初级过滤漂洗管 41有废液排出口和 目的细胞的出口： 其中， 目的细胞出口处与细胞诱导培养装置 5的细胞诱导培养瓶 51连接， 废液出口处连接有废液瓶 9， 所述细胞初级过滤漂洗管 41的入口处设有一过滤网 43， 其能有 效防止血凝块等大块物质的进入； 此外， 所述细胞初级过滤漂洗管 41内对应于废弃液的出口 处还设有过滤罩 44， 其能有效地防止漂洗细胞的丢失； 此外， 所述细胞漂洗装置的漂洗瓶 42 端口的连接管配设有蠕动泵， 以较好地漂洗细胞。 然后将漂洗洁净的淋巴细胞进行后续的细 胞诱导培养过程及细胞增殖培养过程。
此外， 在本发明中， 如图 2所示， 所述细胞诱导培养装置 5包括若干细胞诱导培养瓶 51 及设在各细胞诱导培养瓶 51上方且与各细胞诱导培养瓶 51均连接诱导培养液袋 52， 所述诱 导培养液袋 52分别向各细胞诱导瓶 51内注入诱导培养液， 诱导培养液能有效地剌激淋巴细 胞活化， 如图 3所示， 所有细胞诱导培养瓶 51置于密封的细胞诱导培养箱 10内 （如图 3所 示）； 接着， 在细胞培养瓶 51的输出端通过连接软管连接所述细胞增殖培养装置 6连接。 在 细胞转入增殖培养袋 61的过程中， 首先开放诱导培养瓶的输出口， 将悬浮细胞转移至后期的 细胞增殖装置 6的细胞增殖培养袋 61， 然后为了将贴壁细胞 (如树突状细胞等）同时转移至后 期的细胞增殖装置 6的细胞增殖培养袋 61， 增加效应细胞比例， 所述细胞诱导瓶 51上连接 有剥离粘附细胞的吹打装置， 其主要通过外加的蠕动泵或往复液体抽吸设备通过液体的冲力 将粘附在瓶壁的细胞吹下， 由于操作在密闭的无菌空间进行， 可最大程度地防止污染和交叉 污染。
如图 2、 图 4所示， 所述细胞增殖培养装置 6包括至少一个细胞增殖培养袋 61及用于对 各细胞增殖培养袋 61同时提供增殖培养液的增殖培养液袋 62， 且细胞增殖培养袋 61放置于 细胞增殖培养箱 20中， 且各细胞增殖培养袋 61均对应悬挂于悬挂架 63上， 增殖培养液袋 62中增殖培养液能有效地促进有效淋巴细胞增殖， 增殖培养液根据欲需培养的淋巴细胞的种 类而有所不同。此外， 为了防止细胞增殖培养袋 61长时间静置使其内的细胞沉淀， 所述悬挂 架 63上设有驱动细胞增殖培养袋 61上下 （或左右） 往复运动的、 促使悬浮细胞循环运动、 防止悬浮细胞沉淀的防止细胞沉淀装置 64， 具体是通过该防细胞沉淀装置 64带动细胞增殖 培养袋 61的底部做上下（或左右）往复运动， 在不断地往复运动过程中， 起到防止悬浮细胞 沉淀的作用。
如图 2、 图 6、 图 7所示， 本发明所述细胞过滤、 漂洗和回收装置 7包括通过连接管连接 在细胞增殖培养袋 61输出端连的漂洗瓶 71、生理盐水瓶 72、 以及对漂洗后的细胞进行过滤、 收集的细胞过滤漂洗管 73， 所述细胞过滤漂洗管 73的入口处设有一级过滤网 74， 细胞过滤 漂洗管内对应于废弃液瓶 12的出口处设有过滤网罩 75， 细胞过滤漂洗管 73内对应于成品效 应细胞的出口处设有二级过滤网 76， 通过逐级过滤， 以获取合格的成品效应细胞， 然后将该 合格的成品效应细胞通过输液袋 30回输至患者体内以增强机体自身内在免疫抗癌能力，从而 有效地达到治疗和预防肿瘤的目的， 药用效果好。 进一步， 本发明所述细胞过滤、 漂洗和回 收装置 7上设置有压力感受器， 以监控细胞漂洗管的通畅状况， 当压力达到设定值时， 自动 进入漂洗程序。 管路流量计监控培养液和漂洗液的排出量， 当培养液量或漂洗液量达到设定 值时， 计算机终止细胞过滤或漂洗程序， 开放细胞回收出口， 将细胞回收至产品袋， 获得合 格的效应细胞成品。
如图 2所示， 本发明中， 所述样本瓶 2、漂洗瓶 41、诱导培养液瓶 52、增殖培养液瓶 62、 漂洗瓶 71和生理盐水瓶 72出口处的液体流动管路上均对应设有蠕动泵 40、 50、 60、 70、 80、 90等， 各蠕动泵通过在液体流动管路上施加压力而不是直接作用于液体本身， 使液体在管路 中流动， 蠕动泵的任何部位与液体均无任何接触， 清洁卫生。 而且， 所述蠕动泵的转速可由 计算机程序控制或由手动控制键操作， 使用方便可靠。 此外， 所述液体流动管路均设有管路 钳， 计算机和操作人员可控制每个管路钳的打开 /关闭， 容许 /防止液体进入相应管路。
此外， 在本发明中， 所述密封室 1 内还设有安全监测传感器， 该安全监测传感器包括液 体流动管路上的管路压力传感器和管路流量计、 安装于样本瓶 2、 漂洗瓶 41、 诱导培养液瓶 52、 增殖培养液瓶 62和生理盐水瓶 72上的液面水平探测器、 置于离心机内的温度探测器、 湿度探测器及离心平衡探测器， 以及细胞诱导培养箱 51、 细胞增殖培养箱 61 内的温度探测 器， 湿度探测器二氧化碳浓度探测器和液面水平探测器。 上述各种不同的安全监测传感器与 计算机控制系统连接， 在运行过程中， 计算机对其进行监测， 如果发生异常情况将启动报警 系统， 这样就能做到有效地提醒警示功能。 具体来说， 所述液体流动管路上的管路压力传感 器还可以分为负压探测传感器和正压探测传感器。 负压探测传感器设置于样本液 2和漂洗瓶 41的输出管路上， 当管内液体压力低于计算机设定的下限值时， 将出现提示报警。 而正压探 测传感器设置于细胞的排出管路上， 检测细胞排出管路的通畅情况， 当管路内的压力超过设 定值时， 将出现提示报警。 管路流量计利用超声原理计算液体的输出量， 当流出的液体量达 到设定值时， 计算机将自动关闭管路， 进入下一操作程序。 而所述液面水平探测器为采用超 声工作原理的液面水平探测器， 用于监测管路上空气管内的液平面， 当液面低于该探测器时， 将启动报警。 由于使用各安全监测传感器， 安全性能大大提高， 使用更加方便， 实用性好。
此外， 为了更好地监控细胞的培养状况， 所述细胞诱导培养瓶 51及细胞增殖培养袋 61 上均连接有用于监控细胞培养状况的细胞培养监控系统。 所述细胞培养监控系统包括光电感 应器和细胞输出管， 光电感应器由发光器和受光器两个元件组成。 所述细胞培养监控系统均 独立且对应地设置于细胞诱导培养箱 10和细胞增殖培养箱 20内， 且所述细胞输出管对应地 连接于诱导培养瓶 51和增殖培养袋 61。 该细胞培养监控系统用于监测各培养箱 （包括细胞 诱导培养箱 10和细胞增殖培养箱 20 ) 内培养过程中培养液的通透量， 当细胞浓度增加时， 光的通透量减少， 利用这一监控方法， 计算机可监控培养管路内的细胞含量。 此外， 当有微 生物污染时， 光的通透量急速减少， 计算机也可以监控细胞培养的污染状况。 该种细胞培养 监控系统操作方便， 易于监控细胞的培养情况。 在本发明中， 为了便于观测， 所述细胞诱导 培养箱 10和细胞增殖培养箱 20的前部对应于箱内的细胞培养监控系统位置处设置管道接口 窗 101和 201， 近接口处设置关闭 /开放管路和控制细胞和培养液排出的管路钳， 该接口窗用 于各培养装置管路的连通和细胞及培养液的抽取检查。
在本发明中， 为了提供密封室内无菌和适宜细胞生长的环境， 所述密封室 1 内设有控制 密封室内洁净度、温度和湿度的层流洁净及消毒系统， 以及控制诱导培养箱 10和增殖培养箱 20的温度湿度和二氧化碳浓度的培养箱控制系统。
在本发明中， 各对应连接的装置之间的液体流动管路均通过无菌密封性能好的连接软管 连接， 以保证较好的密封性能。 同时本发明所述的各类安全监测传感器和细胞培养监控系统 均不与液体直接接触， 可避免污染的发生。 本发明所述的医用细胞自动化生产装置可以使每一个患者的细胞分离、 诱导培养及大规 模增殖培养以及细胞的过滤、 漂洗和回收过程等过程在一个完全密封、 层流无菌的独立系统 中完成， 这样确保细胞培养环境条件恒定， 可有效地防止人工操作的微生物污染和交叉污染。 同时， 本发明通过驱动细胞增殖培养袋上下往复运动系统， 增加了细胞在培养袋内运动， 促 进了细胞的快速增殖， 效应细胞比例大大提高， 可满足患者的治疗需要， 细胞免疫治疗效果 好。 此外， 所述的医用细胞自动化生产方法， 其完全实现自动化， 操作过程简单方便， 操作 误差小， 重复性好， 符合 GMP标准， 能广泛适用于医疗和生物制药等不同工作环境和场合。

Claims

权 利 要 求 书
1 . 一种医用细胞自动化生产装置， 包括层流无菌的密封室， 所述密封室内设有样本瓶， 其特征在于： 所述密封室内样本瓶后通过连接管依次连接有对样品进行细胞分离的细胞分离 装置、 细胞漂洗装置、 细胞诱导培养装置、 细胞增殖培养装置、 对培养后的细胞进行过滤、 漂洗和回收的细胞过滤、 漂洗和回收装置。
2.根据权利要求 1所述的医用细胞自动化生产装置， 其特征在于： 所述细胞分离装置为 带有封闭容置空腔的离心机， 所述离心机的容置空腔内部设有用于盛放并对血液进行分离成 血浆和淋巴细胞的分离袋， 以及将分离袋内分离出的淋巴细胞进行收集的细胞收集袋， 所述 分离袋的端口处还通过连接管连接有血浆收集瓶， 所述细胞收集袋的输出端口通过连接管连 接所述的细胞漂洗装置。
3.根据权利要求 2所述的医用细胞自动化生产装置， 其特征在于： 所述细胞漂洗装置包 括细胞初级过滤漂洗管和与细胞初级过滤漂洗管连接的漂洗瓶， 所述细胞初级过滤漂洗管置 于细胞收集袋的后方， 且该细胞初级过滤漂洗管的入口处连接于细胞收集袋， 所述细胞初级 过滤漂洗管有废液排出口和目的细胞的出口，所述目的细胞出口处与细胞诱导培养装置连接， 所述废液出口处连接有废液瓶， 所述细胞初级过滤漂洗管的入口处设有一过滤网， 所述细胞 初级过滤漂洗管内对应于废弃液的出口处设有过滤罩。
4.根据权利要求 3所述的医用细胞自动化生产装置， 其特征在于： 所述细胞诱导培养装 置包括至少一细胞诱导培养瓶及设在细胞诱导培养瓶上方的细胞诱导培养液瓶， 所述细胞诱 导培养瓶放置于细胞诱导培养箱内。
5.根据权利要求 4所述的医用细胞自动化生产装置， 其特征在于： 所述细胞诱导培养瓶 上连接有剥离粘附细胞的吹打装置。
6.根据权利要求 5所述的医用细胞自动化生产装置， 其特征在于： 所述细胞增殖培养装 置连接在细胞诱导培养瓶的输出端， 所述细胞增殖培养装置包括至少一个细胞增殖培养袋及 用于对各细胞增殖培养袋同时提供增殖培养液的增殖培养液瓶， 所述细胞增殖培养袋置放于 细胞增殖培养箱内。
7.根据权利要求 6所述的医用细胞自动化生产装置， 其特征在于： 所述细胞增殖培养袋 悬挂于悬挂架上， 所述悬挂架上设有驱动细胞增殖培养袋往复运动的防止细胞沉淀装置。
8.根据权利要求 7所述的医用细胞自动化生产装置， 其特征在于： 所述细胞过滤、 漂洗 和回收装置包括通过连接管连接在细胞增殖培养袋输出端的漂洗瓶、 生理盐水瓶、 以及对漂 洗后的细胞进行收集的细胞过滤漂洗管， 所述细胞过滤漂洗管的入口处设有一级过滤网， 细 胞过滤漂洗管内对应于废弃液的出口处设有过滤网罩， 细胞过滤漂洗管内对应于成品效应细 胞的出口处设有二级过滤网。
9.根据权利要求 8所述的医用细胞自动化生产装置， 其特征在于： 所述样品瓶、漂洗瓶、 诱导培养液瓶、 增殖培养液瓶和生理盐水瓶的液体流动管路上设有蠕动泵。
10. 根据权利要求 9所述的医用细胞自动化生产装置， 其特征在于： 所述液体流动管路 均设有管路钳。
11.根据权利要求 10所述的医用细胞自动化生产装置， 其特征在于： 所述密封室内设有 安全监测传感器， 该安全监测传感器包括液体流动管路上的管路压力传感器和管路流量计， 样本瓶、 漂洗瓶、 诱导培养液瓶、 增殖培养液瓶和生理盐水瓶上安装的液面水平探测器； 离 心机内的温度探测器、 湿度探测器及离心平衡探测器； 以及细胞诱导培养箱、 细胞增殖培养 箱内的温度探测器， 湿度探测器， 二氧化碳浓度探测器和液面水平探测器。
12.根据权利要求 11所述的医用细胞自动化生产装置， 其特征在于： 所述液体流动管路 上的管路压力传感器分为负压探测传感器和正压探测传感器。
13.根据权利要求 11所述的医用细胞自动化生产装置， 其特征在于： 所述液面水平探测 器和管路流量计为采用超声工作原理的液面水平探测器和超声波流量计。
14.根据权利要求 6所述的医用细胞自动化生产装置， 其特征在于： 所述细胞诱导培养瓶 及细胞增殖培养袋上均连接有用于监控细胞培养状况的细胞培养监控系统。
15. 根据权利要求 14所述的医用细胞自动化生产装置， 其特征在于： 所述细胞培养监控 系统包括光电感应器和细胞输出管， 光电感应器由发光器和受光器两个元件组成； 所述细胞 培养监控系统分别地设置于细胞诱导培养箱和细胞增殖培养箱内， 且所述细胞输出管对应地 连接于诱导培养瓶和增殖培养袋。
16. 根据权利要求 15所述的医用细胞自动化生产装置， 其特征在于： 所述细胞诱导培养 箱和细胞增殖培养箱的前部对应于箱内的细胞培养监控系统位置处设置管道接口窗， 近接口 处设置关闭 /开放管路和控制细胞和培养液排出的管路钳。
17.根据权利要求 1所述的医用细胞自动化生产装置， 其特征在于： 所述密封室内设有控 制密封室内洁净度、 温度和湿度的层流洁净系统及消毒系统。
18.根据权利要求 1所述的医用细胞自动化生产装置的生产方法， 其特征在于： 具体生产 步骤是：
(1)置入患者样本： 将患者样本并移至样本瓶内；
(2)细胞分离过程： 对初步处理的样本通过细胞分离装置进行分离， 分离出目的细胞如淋 巴细胞； (3)细胞漂洗过程： 将分离出的细胞通过细胞漂洗装置进行初步漂洗；
(4)细胞诱导培养过程： 对分离并漂洗后的目的细胞通过细胞诱导装置进行特定细胞诱导 培养；
(5)细胞转入增殖培养袋过程： 开放诱导培养瓶的输出口， 首先将悬浮细胞放入增殖培养 袋， 然后开放增殖培养液瓶输出口和诱导培养瓶的入口， 通过吹打装置剥离诱导培养瓶的贴 壁细胞， 并将其转入增殖培养袋；
(6)细胞增殖培养过程：在增殖培养箱对诱导培养后的细胞进行细胞大量增殖培养，并通过 防止细胞沉淀装置的往复运动防止细胞沉淀；
(7)培养细胞的监控过程： 通过设置于诱导培养箱和增殖培养箱内的细胞培养监控系统， 即光电感应器监控细胞浓度， 当达到细胞培养标准时， 提示进入产品回收过程；
(8)细胞过滤、 漂洗和回收过程： 开放增殖培养袋的输出口， 对增殖培养后的淋巴细胞通 过过滤、 漂洗和回收装置进行细胞过滤、 漂洗和回收， 获得合格的效应细胞成品。
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CN113832028B (zh) * 2020-06-24 2024-08-16 上海医药集团生物治疗技术有限公司 一种全自动细胞培养系统及培养方法
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WO2022252003A1 (zh) * 2021-05-31 2022-12-08 北京永泰生物制品有限公司 密闭培养系统

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN2923718Y (zh) * 2005-07-21 2007-07-18 高春平 自动血细胞分离系统
CN101300340A (zh) * 2005-11-01 2008-11-05 迈世耐特股份公司 细胞培养装置、细胞培养方法、细胞培养程序及细胞培养系统

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4309815B2 (ja) * 2004-07-05 2009-08-05 株式会社日立製作所 細胞培養システム及び細胞培養方法
CN201883098U (zh) * 2010-12-01 2011-06-29 汪华 一种医用细胞自动化生产装置

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN2923718Y (zh) * 2005-07-21 2007-07-18 高春平 自动血细胞分离系统
CN101300340A (zh) * 2005-11-01 2008-11-05 迈世耐特股份公司 细胞培养装置、细胞培养方法、细胞培养程序及细胞培养系统

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