CN112899146B - Full-automatic cell separation system - Google Patents
Full-automatic cell separation system Download PDFInfo
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- CN112899146B CN112899146B CN202110110528.4A CN202110110528A CN112899146B CN 112899146 B CN112899146 B CN 112899146B CN 202110110528 A CN202110110528 A CN 202110110528A CN 112899146 B CN112899146 B CN 112899146B
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- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
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- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
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- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502753—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
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- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
- C12M33/04—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by injection or suction, e.g. using pipettes, syringes, needles
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- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
- C12M33/12—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by pressure
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- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
- C12M33/14—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus with filters, sieves or membranes
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- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
- C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
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- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/40—Means for regulation, monitoring, measurement or control, e.g. flow regulation of pressure
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- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/04—Cell isolation or sorting
Abstract
The invention relates to the technical field of medical equipment, in particular to a full-automatic cell separation system. The system comprises: the cell separation module is used for realizing separation of target cells; the sample injection module is used for injecting a sample into the cell separation module; the air pressure control module is used for controlling the pressure difference of two sides of the filter membrane in the micro-fluidic chip; the negative pressure suction module is used for sucking filtrate of the sample after passing through the filter membrane; the circuit control module is connected with the sample injection module, the air pressure control module and the negative pressure suction module and controls the work of the sample injection module, the air pressure control module and the negative pressure suction module. The full-automatic cell separation system provided by the invention can realize full-automatic separation of target cells, has high control precision, high enrichment rate, simplicity and high efficiency, brings convenience to biomedical research and has very important effect.
Description
Technical Field
The invention relates to the technical field of medical equipment, in particular to a full-automatic cell separation system.
Background
Currently, methods for cell separation fall into two main categories: 1) Based on the cell surface specific antigen properties, by antigen-antibody binding separation. Such methods are stable, but rely on the specificity of antibodies to cell surface antigens; 2) Aiming at physical characteristics of cells, including cell size, mechanical property, electrical property and the like, the cells are separated by physical methods such as filtration, centrifugation, dielectrophoresis and the like. Compared with the method based on cell surface markers, the method of physical separation has relatively low requirement, simpler operation and higher flux. However, how to increase the sensitivity is a key to this type of approach, since the variability of these physical properties from cell to cell is small.
Filtration by a microporous filter of a certain size in combination with a difference in cell size is more common in cell sorting. For example, circulating Tumor Cells (CTCs) in blood may be sorted by filtration through a filter membrane. CTC is approximately 14-26 μm in diameter, red blood cells are approximately 6-8 μm in diameter, and white blood cells are approximately 8-20 μm in diameter. When the microporous filter membrane with the diameter of 8 mu m is used for filtration, CTC can be trapped due to the difference of cell size and cell rheological property, and blood cells can flow away along with the buffer solution, so that the enrichment purpose is achieved. Rarecell, screenCell et al demonstrate the feasibility of this approach using a cylindrical microporous filter membrane with a diameter of 8 μm. Although the method is simple, convenient and quick, in the using process, the influence of pressure difference generated by cell blocking in the sorting process cannot be fully considered. Although the flow rate remains consistent, the instantaneous negative pressure may be far higher than the normal range due to the reduced number of effective holes, which easily causes loss of cells and damages cell structures, resulting in reduced cell activity, and poor cell sorting efficiency and sensitivity.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems existing in the prior art. Therefore, the invention provides a full-automatic cell separation system which can control the real-time pressure difference more accurately, so that the cell separation efficiency is higher, the sensitivity is better, and the influence on the cell activity is smaller.
Specifically, the technical scheme adopted by the invention is as follows.
An object of one aspect of the present invention is to provide a fully automated cell separation system comprising:
the cell separation module comprises a microfluidic chip, wherein the microfluidic chip comprises an upper cover plate, a filter membrane and a lower cover plate; the upper cover plate is provided with: the device comprises a suction through hole, a gas leakage through hole and a sample injection through hole; the lower cover plate is provided with: the cavity corresponds to the sample injection through hole, and the first microfluidic channel and the second microfluidic channel; one end of the first micro-fluid channel is communicated with the air leakage through hole, and the other end of the first micro-fluid channel is communicated with the cavity; one end of the second micro-fluid channel is communicated with the suction through hole, and the other end of the second micro-fluid channel is communicated with the cavity; the filter membrane is arranged between the sample injection through hole and the cavity;
the sample injection module comprises a sample injection needle which can move along the X-axis and Z-axis directions and is used for injecting a sample or a reagent into the cell separation module;
the air pressure control module is communicated with the cell separation module through the air leakage through hole and is used for monitoring and controlling the pressure difference at two sides of the filter membrane;
the negative pressure suction module is communicated with the cell separation module through the suction through hole and is used for sucking filtrate of the sample after passing through the filter membrane;
the circuit control module is connected with the sample injection module, the air pressure control module and the negative pressure suction module and controls the work of the sample injection module, the air pressure control module and the negative pressure suction module.
When the full-automatic cell separation system works, a sample containing cells to be separated is automatically or manually added into the cell separation module, the air pressure control module is started, the negative pressure suction module is started, the chamber of the microfluidic chip, the first microfluidic channel and the second microfluidic channel are in a negative pressure state, at the moment, pressure differences exist on two sides of the filter membrane, the sample is rapidly filtered under the action of the pressure differences, target cells cannot pass through the filter membrane due to large volume, and filtrate is pumped out by the negative pressure suction module through the second microfluidic channel, so that the rapid separation of the target cells is realized.
According to some embodiments of the invention, the sample injection module further comprises a plunger pump, the plunger pump being connected to the sample injection needle. Preferably, the sample injection module further comprises a first liquid pipe, and the plunger pump is connected with the sample injection needle through the first liquid pipe. The plunger pump is matched with the sampling needle, so that a sample or a reagent can be pumped into the sampling needle, and the sample or the reagent can be pushed out of the sampling needle.
According to some embodiments of the invention, the sample injection needle is provided with a liquid level detection function.
According to some embodiments of the invention, the sample injection module further comprises a high-throughput reagent module, the high-throughput reagent module being connected to the plunger pump. And a valve is further arranged between the large-flux reagent module and the plunger pump. Preferably, the plunger pump is connected to the high-throughput reagent module by a second fluid tube. The large-flux reagent module is used for storing reagents with larger dosage such as buffer solution, cleaning solution and the like, and the extraction and the sample injection of the reagents in the large-flux reagent module are realized through the switching of the valve and the drawing of the plunger pump.
According to some embodiments of the invention, the sample injection module further comprises a second cleaning tank, and the second cleaning tank is connected with the plunger pump through a second liquid pipe. The second cleaning tank is used for cleaning the pipeline.
According to some embodiments of the invention, the high throughput reagent module comprises a buffer, a wash solution, or the like.
According to some embodiments of the invention, the sample injection module further comprises a micro reagent module, and the sample injection is performed on the extraction of the reagent in the micro reagent module by driving a plunger pump. The plunger pump is used for realizing that buffer solution is filled in the pump body and the sample injection needle through switching of the valve and pulling of the plunger pump, and then the sample injection needle is used for extracting the trace reagent through the plunger pump after detecting the liquid level of the reagent in the trace reagent module, and at the moment, the sample injection needle and the plunger pump are filled with the buffer solution, so that the precision of extracting the trace reagent is extremely high.
According to some embodiments of the invention, the micro reagent module comprises a micro reagent container and a first wash tank, the first wash tank being connected to a waste liquid tank. Preferably, the first cleaning tank is connected with the waste liquid tank through a diaphragm pump. The first cleaning tank is used for cleaning the sample injection needle.
According to some embodiments of the invention, the micro reagent module comprises a pretreatment liquid, a blocking liquid, an antibody, a nuclear reagent, and the like.
According to some embodiments of the invention, the air pressure control module comprises an air pressure sensor and an air release valve, wherein the air pressure sensor is provided with two air vents, one air vent is communicated with the atmosphere, and the other air vent is connected with the air release valve and the air release through hole through a three-way luer connector. The air pressure sensor can monitor the air pressure below the filter membrane, when the air pressure is higher than a preset threshold value, the air release valve is opened to release air, so that the negative pressure below the filter membrane is ensured to be within a certain range, the target cells can not be pumped away due to the overlarge negative pressure, and the high enrichment rate of the target cells is ensured.
The air pressure sensor in the air pressure control module can monitor the real-time pressure difference of the sample separation process and can be used for judging the time node of the flow. The air release valve is used for protecting target cells from leakage and damage caused by overlarge pressure difference, and is automatically opened when the pressure difference exceeds a certain threshold value, so that the capture efficiency and the activity rate of the target cells are improved. For example, when the upper filtrate is almost completely filtered, the upper and lower pressure differential will continue to rise, trapping cells that are also immobilized on the filter. If the negative pressure suction module continues to provide negative pressure, the internal pressure difference of the chamber can be increased sharply until the tension is broken because the filtrate in the chamber cannot break through the surface tension of the liquid. The large pressure differential at this point can result in a very severe loss rate and activity of the captured cells of interest. Therefore, the air release valve needs to be opened in time, so that the interior of the microfluidic chip is directly communicated with air, redundant filtrate in the cavity flows out of the microfluidic chip along with the air from the first microfluidic channel, the cavity and the second microfluidic channel, and the cavity does not have any pressure difference exceeding the bearing range of cells during the period.
According to some embodiments of the invention, the negative pressure suction module comprises a syringe pump connected to the suction through-hole.
According to some embodiments of the invention, the negative pressure suction module comprises a syringe pump, the syringe pump comprises a syringe and a three-way Y-shaped rotary valve head, a Y-shaped inlet end of the three-way rotary valve head is connected with the syringe, one end of two outlet ends is connected with the suction through hole, and the other end is connected with a waste liquid barrel. When the outlet end is switched to be connected with the suction through hole, waste liquid can be pumped; when the outlet end is switched to be connected with the waste liquid barrel, waste liquid can be pumped into the waste liquid barrel.
According to some embodiments of the invention, the cell separation module further comprises a clamping device (for example, a clamping device of patent CN 208177479U) which can tightly fit the upper cover plate and the lower cover plate.
According to some embodiments of the invention, the lower surface of the upper cover plate and the upper surface of the lower cover plate are further provided with mutually bonded silica gel films. Further, the bonding may be chemical bonding or thermal bonding. The sealing performance of the upper cover plate and the lower cover plate when being closely adhered can be ensured to a greater extent through the bonding effect between the silica gel films.
According to some embodiments of the invention, the cell separation module further comprises a sample container connected to the sample introduction through hole.
According to some embodiments of the invention, the circuit control system includes a control circuit board, and a display screen coupled to the control circuit board.
The invention also provides a method for separating cells by the fully automatic cell separation system, which comprises the following steps:
s1, starting a control program of the circuit control system;
s2, manually injecting a sample into a sample container of the microfluidic chip or driving the sample injection needle to inject a reagent into the sample container of the microfluidic chip;
s3, starting the negative pressure suction module, and extracting filtrate in the microfluidic chip;
and S4, the air pressure control module monitors the air pressure below the filter membrane of the micro-fluidic chip, when the air pressure is higher than a preset threshold value, the air release valve is opened to reduce the pressure, and the negative pressure suction module continues to suck for a period of time and then closes the air release valve.
According to some embodiments of the invention, after the filtration is completed, the step of washing the cells of interest with a buffer is further included.
According to some embodiments of the invention, the bleed valve is opened to reduce pressure when the air pressure value rises steeply or exceeds-1 kPa.
According to some embodiments of the invention, the bleeder is opened to reduce pressure when the air pressure exceeds-1 kPa during flushing with buffer.
It is an object of a further aspect of the present invention to provide a use of a fully automated cell separation system as described above for cell separation.
It is an object of another aspect of the present invention to provide the use of a fully automated cell separation system as described above in cell detection.
It is an object of another aspect of the present invention to provide the use of a fully automated cell separation system as described above in cell counting.
The invention has at least the following beneficial effects:
the full-automatic cell separation system provided by the invention can realize full-automatic separation of target cells, can monitor the internal pressure difference of the microfluidic chip in real time, avoid cell selection omission or damage caused by overlarge pressure difference, can rapidly and efficiently realize separation of the target cells, improve the efficiency of physical filtration and increase the purity of separation of the target cells, and can also be used for realizing counting and detection of the cells, and has the advantages of simple method and low cost. The full-automatic cell separation system has high control precision, high enrichment rate, simplicity and high efficiency, brings convenience to biomedical research and has very important effect.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 is a schematic diagram of a fully automated cell separation system according to one embodiment of the present invention;
FIG. 2 is a schematic diagram of a sample injection module according to an embodiment of the present invention;
FIG. 3 is a schematic diagram showing the connection relationship among a cell separation module, a negative pressure suction module and a pneumatic control module according to an embodiment of the present invention;
FIG. 4 is a schematic diagram of a microfluidic chip according to one embodiment of the present invention;
FIG. 5 is a schematic view of the structure of the upper and lower cover plates according to an embodiment of the present invention;
FIG. 6 is a schematic diagram of a fully automated cell separation system according to one embodiment of the present invention;
wherein, the correspondence between the reference numerals and the component names in the figures is:
the cell separation module 110, the microfluidic chip 111, the sample container 112, the upper cover plate 1101, the filter membrane 1102, the lower cover plate 1103, the suction through hole 1104, the air leakage through hole 1105, the sample injection through hole 1106, the chamber 1107, the first microfluidic channel 1108 and the second microfluidic channel 1109;
the sample injection module 120, the sample injection needle 121, the plunger pump 122, the first liquid pipe 1221, the second liquid pipe 1222, the large-flux reagent module 123, the micro reagent module 124, the micro reagent container 1241, the first cleaning tank 1242, the second cleaning tank 125, the waste liquid tank 126, the valves 5 to 11 and the diaphragm pump 31;
the air pressure control module 130, the air pressure sensor 131 and the air release valve 13-20;
a negative pressure suction module 140, syringe pumps 21-28;
the circuit control module 150 controls the circuit board 151 and the display screen 152.
Detailed Description
Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to like or similar elements or elements having like or similar functions throughout. The embodiments described below by referring to the drawings are illustrative only and are not to be construed as limiting the invention.
In the description of the present invention, it should be understood that references to orientation descriptions such as upper, lower, front, rear, left, right, etc. are based on the orientation or positional relationship shown in the drawings, are merely for convenience of description of the present invention and to simplify the description, and do not indicate or imply that the apparatus or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus should not be construed as limiting the present invention.
In the description of the present invention, a number means one or more, a number means two or more, and greater than, less than, exceeding, etc. are understood to not include the present number, and above, below, within, etc. are understood to include the present number. The description of the first and second is for the purpose of distinguishing between technical features only and should not be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated or implicitly indicating the precedence of the technical features indicated.
In the description of the present invention, unless explicitly defined otherwise, terms such as arrangement, installation, connection, etc. should be construed broadly and the specific meaning of the terms in the present invention can be reasonably determined by a person skilled in the art in combination with the specific contents of the technical scheme.
Example 1
Describing a fully automated cell separation system 100 according to an embodiment of the present invention with reference to fig. 1 to 6, the fully automated cell separation system 100 may include: the cell separation module 110 comprises a microfluidic chip 111, wherein the microfluidic chip 111 comprises an upper cover plate 1101, a filter membrane 1102 and a lower cover plate 1103; the upper cover 1101 is provided with: a suction through hole 1104, a venting through hole 1105, and a sample introduction through hole 1106; the lower cover 1103 is provided with: a chamber 1107 corresponding to the sample introduction through hole 1106, a first microfluidic channel 1108 and a second microfluidic channel 1109; wherein one end of the first microfluidic channel 1108 communicates with the venting through-hole 1105 and the other end communicates with the chamber 1107; one end of the second microfluidic channel 1109 communicates with the suction through-hole 1104, and the other end communicates with the chamber 1107; filter membrane 1102 is disposed between sample introduction port 1106 and chamber 1107; the sample injection module 120 comprises a sample injection needle 121, wherein the sample injection needle 121 can move along the X axis and the Z axis and is used for injecting a sample or a reagent into the cell separation module 110; the air pressure control module 130 is communicated with the cell separation module 110 through the air leakage through hole 1105 and is used for monitoring and controlling the pressure difference of two sides of the filter membrane 1102; a negative pressure suction module 140, which is communicated with the cell separation module 110 through a suction through hole 1104, and is used for sucking filtrate of the sample after passing through the filter membrane 1102; the circuit control module 150 is connected with the sample injection module 120, the air pressure control module 130 and the negative pressure suction module 140 and controls the operation thereof.
When the fully automatic cell separation system 100 works, a sample or reagent containing cells to be separated is added into the cell separation module 110 through the sample injection module 120, the air pressure control module 130 is started, the negative pressure suction module 140 is started, the chamber 1107, the first microfluidic channel 1108 and the second microfluidic channel 1109 of the microfluidic chip 111 are in a negative pressure state, at the moment, pressure differences exist on two sides of the filter membrane 1102, the sample is rapidly filtered under the action of the pressure differences, target cells cannot pass through the filter membrane 1102 due to large volume, and filtrate is pumped out by the negative pressure suction module 140 through the second microfluidic channel 1109, so that the rapid separation of the target cells is realized.
Further, the sample injection module 120 further includes a plunger pump 122, and the plunger pump 122 is connected to the sample injection needle 121. Further, the sample injection module 120 further includes a first liquid pipe 1221, and the plunger pump 122 is connected to the sample injection needle 121 through the first liquid pipe 1221. The plunger pump 122 is matched with the sampling needle 121, so that a sample or a reagent can be pumped into the sampling needle 121, and the sample or the reagent can be pushed out of the sampling needle 121. Further, the sample injection needle 121 has a liquid level detection function.
Further, the sample injection module 120 further includes a large flux reagent module 123, and the large flux reagent module 123 is connected to the plunger pump 122. A valve 5 is also provided between the large throughput reagent module 123 and the plunger pump 122. Further, the plunger pump 122 is connected to the large flux reagent module 123 by a second liquid tube 1222. The large-flux reagent module 123 is used for storing reagents such as buffer solution and cleaning solution, and the extraction and sample injection of the reagents in the large-flux reagent module 123 are realized through the switching of the valve 5 and the drawing of the plunger pump 122.
Further, the sample injection module 120 further includes a second cleaning tank 125, and the second cleaning tank 125 is connected to the plunger pump 122 through a second liquid pipe 1222. The second cleaning tank 125 is used for cleaning the pipeline and the corresponding luer connector. Further, a valve 7 is provided between the plunger pump 122 and the second cleaning tank 125 for controlling the communication between the second liquid pipe 1222 and the second cleaning tank 125.
Further, the sample injection module 120 further includes a micro reagent module 124, and the sample injection needle 121 is driven by the plunger pump 122 to extract the reagent in the micro reagent module 124. The plunger pump 122 is switched by the valve 6 and the plunger pump 122 is pulled to fill the buffer solution in the plunger pump 122 and the sample injection needle 121, and then the sample injection needle 121 detects the liquid level of the reagent in the micro reagent module 124 and then the plunger pump 122 is used for extracting the micro reagent, at this time, the sample injection needle 121 and the plunger pump 122 are filled with the buffer solution, so that the accuracy of extracting the micro reagent is extremely high. Further, the micro reagent module 124 includes a micro reagent container 1241 and a first rinse tank 1242, the first rinse tank 1242 being connected to the waste liquid tank 126. Further, a diaphragm pump 31 is provided between the first cleaning tank 1242 and the waste liquid tank 126. The first wash tank 1242 is used for washing needles. Further, the micro reagent module 124 includes reagents such as a sample to be treated, a pretreatment reagent, a blocking reagent, an antibody, a nuclear stain, and the like.
Further, the air pressure control module 130 includes an air pressure sensor 131 and air release valves 13 to 20 (the number of the air release valves can be selected according to the actual situation, and one or more air release valves can be provided), the air pressure sensor 131 has two air vents, one air vent is communicated with the atmosphere, and the other air vent is connected with the air release valves 13 to 20 and the air release through hole 1105 through a three-way luer connector. The air pressure sensor 131 can monitor the air pressure below the filter membrane 1102, when the air pressure is higher than a preset threshold value, the air release valve is opened to release air, so that the negative pressure below the filter membrane 1102 is ensured to be within a certain range, the target cells can not be pumped away due to the overlarge negative pressure, and the high enrichment rate of the target cells is ensured. The air pressure sensor 131 in the air pressure control module 130 can monitor the real-time pressure difference of the sample separation process and can be used to determine the time node of the flow. The air release valves 13-20 are used for protecting target cells from leakage and damage caused by overlarge pressure difference, and when the pressure difference exceeds a certain threshold value, the air release valves 13-20 are automatically opened, so that the capture efficiency and the activity rate of the target cells are improved. For example, when the air pressure is increased abruptly, the sample is filtered almost completely, and the filtrate in the chamber 1107 is still unable to be pumped under the surface tension. If the negative pressure suction module 140 continues to provide negative pressure, the filtrate in the chamber 1107 will cause the pressure differential in the chamber to rise sharply, as it cannot break through the liquid surface tension, until the tension is broken through. The large pressure differential at this point can result in a very severe loss rate and activity of the captured cells of interest. Therefore, the air release valves 13-20 need to be opened in time to enable the interior of the microfluidic chip 111 to be directly communicated with air, and redundant filtrate in the cavity 1107 flows out of the microfluidic chip 111 along with the air from the first microfluidic channel 1108, the cavity 1107 and the second microfluidic channel 1109, so that the cavity 1107 does not have any pressure difference exceeding the bearing range of cells.
Further, the negative pressure suction module 140 includes syringe pumps 21 to 28 (the number of syringe pumps may be selected according to the actual situation, and may be one or more), and the syringe pumps 21 to 28 are connected to the suction through hole 1104. Further, the syringe pumps 21 to 28 comprise a syringe and a three-way Y-shaped rotary valve head, the Y-shaped inlet end of the three-way rotary valve head is connected with the syringe, one end of the two outlet ends is connected with the suction through hole 1104, and the other end is connected with the waste liquid barrel 126. When the outlet end is switched to be connected with the suction through hole 1104, waste liquid can be pumped; when the outlet port is switched to connect with the waste liquid tank 126, waste liquid can be pumped into the waste liquid tank 126. Syringe pumps 21-28 are used to pump filtrate under filter membrane 1102 into the waste liquid tank.
Further, the cell separation module 110 further includes a clamping device (for example, a clamping device using patent CN 208177479U) that can tightly attach the upper cover 1101 and the lower cover 1103 of the microfluidic chip 111.
Further, the lower surface of the upper cover plate 1101 and the upper surface of the lower cover plate 1103 are also provided with silicone films bonded to each other. Further, the bonding may be chemical bonding or thermal bonding. The sealability of the upper cover plate 1101 and the lower cover plate 1103 when they are closely adhered can be ensured to a greater extent by the bonding action between the silicone films.
Further, the cell separation module 110 further includes a sample container 112, and the sample container 112 is connected to the sample introduction hole 1106.
Further, the circuit control system 150 includes a control circuit board 151, and a display screen 152 connected to the control circuit board 151.
Example 2
A method for separating cells in a fully automated cell separation system 100 comprising the steps of:
s1, starting a control program of the circuit control system 150;
s2, driving a sample injection needle 121 to inject a sample into the microfluidic chip 111;
s3, starting a negative pressure suction module 140, and sucking out filtrate in the microfluidic chip 111;
and S4, the air pressure control module 130 monitors the air pressure below the filter membrane 1102 of the microfluidic chip 111, and when the air pressure is higher than a preset threshold value, the air pressure is released and reduced.
Further, after the completion of the filtration, a step of washing the objective cells with a buffer solution is included.
According to some embodiments of the invention, the air release valve is opened when the air pressure exceeds-1 kPa during flushing with buffer.
Example 3
Some working procedures of the fully automatic cell separation system:
1. the initialization process comprises the following steps: x axis and Z axis reset, valve 7 and 11 are opened, syringe pumps 21-28 reset, X axis moves to the cleaning tank, Z axis moves to the bottom of the cleaning tank, valve 6 is opened, plunger pump 122 resets, waiting for 3s, and valve 6 is closed.
2. And (3) filling a pipeline: the valves 5 and 10 are opened, the injection pumps 21 to 28 pump 20mL of PBS, wait for 3 seconds, the valves 5 and 10 are closed, the valve 6 is opened, the plunger pump 122 pumps 7mL of PBS to the cleaning tank, the diaphragm pump 31 is opened, 5 seconds of waste liquid is pumped, the Guan Gemo pump 31 is pumped, the valve 6 is closed, the Z axis rises to the obstacle avoidance height, and the air release valves 13 to 20 are closed.
3. The infiltration process comprises the following steps: the X axis moves to select the corresponding channel position, the Z axis descends to 3mL of the sample container 112, the valve 6 is opened, the plunger pump 122 adds 2mLPBS into the sample container 112 for 3s, the valve 6 is closed, the Z axis ascends to the obstacle avoidance height, the X axis moves to the cleaning tank for 1s; the injection pump 21-28 starts pumping liquid (speed is 1 mL/min), and after pumping liquid for 1min, the injection pump 21-28 is stopped strongly, the air release valve 13-20 is opened, waiting for 30s, and the air release valve 13-20 is closed.
4. The capturing process comprises the following steps: confirming the addition of a sample (5 mL whole blood+5 mL ISET), pumping liquid (500 mu L/min) by the injection pumps 21-28 through the pumping through holes 1104, recording the highest pressure value Pmax at the 13-20 ends of each air release valve in 18min, judging the channel with the pressure value at the 13-20 ends of the air release valves smaller than-1 Kpa, opening the corresponding air release valves 13-20, waiting for 30s, and strongly stopping the injection pumps 21-28 to close the air release valves 13-20.
5. Capturing and cleaning: x axis moves to the channel of the sample injection through hole 1106, Z axis descends to 3mL in the sample container 112, the valve 6 is opened, the plunger pump 122 pushes 2mL PBS to the sample container 112, the valve 6 is closed, the Z axis ascends to the obstacle avoidance height, the X axis returns to the cleaning tank, the injection pumps 21-28 pump liquid (500 mu L/min) through the suction through hole 1104, the channel of the pressure value at the end of the air release valve 13-20 is judged to be smaller than-1 Kpa, the corresponding air release valve 13-20 is opened for 5 seconds, the injection pumps 21-28 are stopped strongly, the air release valve 13-20 is closed, the cleaning is repeated for 2 times, the injection pumps 21-28 are reset after the cleaning of the 2 nd time is finished, the injection pumps 21-28 are stopped strongly for 10 seconds.
6. The pretreatment process comprises the following steps: the X axis moves to the position of the pretreatment reagent, the Z axis descends to the bottom of the reagent tube, the valve 6 is opened for 1s, the plunger pump 122 pumps 150 mu L of pretreatment liquid for 3s, the valve 6 is closed, the Z axis ascends to the obstacle avoidance height, the X axis moves to the position of the sample container 112, the Z axis descends to the position 5mm above the filter membrane 1102, the valve 6 is opened for 1s, the plunger pump 122 pushes 150 mu L of pretreatment liquid for 3s, the valve 6 is closed, the Z axis ascends to the obstacle avoidance height, the X axis moves to the cleaning tank, the pretreatment liquid is incubated for 10min, the injection pumps 21-28 pump liquid (200 mu L/min) through the suction through hole 1104, the channel of judging that the pressure value of the end of the air release valve 13-20 is smaller than-1 Kpa is opened, the corresponding air release valve 13-20 is closed for 5s, the injection pumps 21-28 are stopped strongly, and the valves 13-20 are closed.
7. Pretreatment cleaning process: the X axis moves to the position 5mm above the filter membrane 1102, the Z axis descends to the position of the sample container 112, the valve 6 is opened, the plunger pump 122 pushes 300 mu L of PBS to the sample container 112, the valve 6 is closed, the Z axis ascends to the barrier-avoiding height, the X axis returns to the cleaning tank, the injection pumps 21-28 pump liquid (200 mu L/min) through the suction through holes 1104, the channels with the pressure values of the ends of the air release valves 13-20 smaller than-1 Kpa are judged, the corresponding air release valves 13-20 are opened, the waiting time is 5 seconds, the injection pumps 21-28 are stopped strongly, the air release valves 13-20 are closed, and the needle cleaning is synchronously carried out during incubation.
8. The sealing process comprises the following steps: the X axis moves to the position of sealing reagent, the Z axis descends to the bottom of the reagent tube, the valve 6 is opened for 1s, the plunger pump 122 pumps 150 mu L of sealing liquid for 3s, the valve 6 is closed, the Z axis ascends to the obstacle avoidance height, the X axis moves to the position of the sample container 112, the Z axis descends to the position 5mm above the filter membrane 1102, the valve 6 is opened for 1s, the plunger pump 122 pushes 150 mu L of sealing liquid sample container 112 for 3s, the valve 6 is closed, the Z axis ascends to the obstacle avoidance height, the X axis moves to the cleaning tank, the cleaning tank is closed for 30min, the injection pumps 21-28 pump liquid (200 mu L/min) through the suction through hole 1104, the channels with the pressure values of the ends of the air release valves 13-20 smaller than-1 Kpa are judged, the corresponding air release valves 13-20 are opened for 5s, the injection pumps 21-28 are stopped strongly, and the air release valves 13-20 are closed.
9. Antibody process: the Z axis rises to the obstacle avoidance height, the X axis moves to the antibody reagent position, the valve 6 is opened, the plunger pump 122 firstly pumps 300 mu L of air (ensures that the antibody is not diluted), waiting for 3 seconds, the valve 6 is closed, the Z axis descends to the bottom of the reagent tube, the valve 6 is opened, the plunger pump 122 pumps 170 mu L of antibody, waiting for 3 seconds, the Z axis ascends to the obstacle avoidance height, the valve 6 is closed, the X axis moves to the sample container 112, the Z axis descends to the position 5mm above the filter membrane 1102, the valve 6 is opened, waiting for 1s, the plunger pump 122 pumps 170 mu L of antibody, waiting for 3 seconds, the valve 6 is closed, the Z axis ascends to the obstacle avoidance height, the X axis moves to the cleaning tank, the Z axis descends to the needle cleaning height, the valve 6 is opened, the plunger pump 122 opens 500 mu L of liquid to the cleaning tank, waiting for 3 seconds, the valve 6 is ascended to the obstacle avoidance height, the Z axis is 60 minutes, the syringe pumps 21-28 pump liquid (200 mu L/min) through the suction through hole 1104, the air valves 13-20 end pressure values are smaller than-1 Kpa, and the corresponding air valves 13-20 Kpa are closed, and the air valves are closed and 20-20 are closed and forced and stopped.
10. Antibody cleaning process: the X axis moves to the position of the sample container 112, the Z axis descends to the position 5mm above the filter membrane 1102, the valve 6 is opened, the plunger pump 122 pushes 300 mu LPBS to the sample container 112, the valve 6 is closed, the Z axis ascends to the barrier-avoiding height, the X axis returns to the cleaning tank and is immersed for 5min, the injection pumps 21-28 pump liquid (200 mu L/min) through the suction through holes 1104, the pressure value of the ends of the air release valves 13-20 is judged to be smaller than the channel of-1 Kpa, the corresponding air release valves 13-20 are opened for 5s, the injection pumps 21-28 stop strongly, the air release valves 13-20 are closed, and the needle washing is synchronously carried out during incubation.
11. The nuclear dyeing process comprises the following steps: the X axis moves to the position of the nuclear dyeing reagent, the Z axis descends to the bottom of the reagent tube, the valve 6 is opened for 1s, the plunger pump 122 pumps 150 mu L of the nuclear dyeing reagent, waiting for 3s, the valve 6 is closed, the Z axis ascends to the barrier-avoiding height, the X axis moves to the position of the sample container 112, the Z axis descends to the position 5mm above the filter membrane 1102, the valve 6 is opened for 1s, the plunger pump 122 pumps 150 mu L of the nuclear dyeing reagent to the sample container 112, waiting for 3s, the valve 6 is closed, the Z axis ascends to the barrier-avoiding height, the X axis moves to the cleaning tank, the nuclear dyeing incubation is carried out for 5min, the injection pumps 21-28 pump liquid (200 mu L/min) through the suction through holes 1104, the corresponding air release valves 13-20 are opened for 5s, the injection pumps 21-28 are stopped strongly, and when the air release valves 13-20 are closed, the needle washing incubation is synchronously carried out.
12. And (3) nuclear dyeing and cleaning: the X axis moves to the position of the sample container 112, the Z axis descends to the position 5mm above the filter membrane 1102, the valve 6 is opened, the plunger pump 122 pushes 300 mu L of PBS to the sample container 112, the valve 6 is closed, the Z axis ascends to the barrier-avoiding height, the X axis returns to the cleaning tank, the injection pumps 21-28 pump liquid (200 mu L/min) through the suction through holes 1104, the channels with the pressure values of the ends of the air release valves 13-20 smaller than-1 Kpa are judged, the corresponding air release valves 13-20 are opened, waiting for 5 seconds, the injection pumps 21-28 stop strongly, and the air release valves 13-20 are closed.
13. And (3) pipeline cleaning: x axis moves to the cleaning tank, Z axis descends to the bottom of the cleaning tank, valve 6 is opened, plunger pump 122 pushes liquid 10mL, wait 3s, valve 6 is closed, pump 31 is opened, wait 5s, guan Beng 31, plunger pump 122 empties waste liquid, wait 3s of air pressure balance, valves 6, 7 and 9 are opened, syringe pumps 21-28 turn to pump head No. 2, pump 10mL of liquid, wait 3s, turn to pump head No. 3, push liquid out, wait 3s, valve 9 is closed, valve 11 is opened, syringe pumps 21-28 turn to pump head No. 2, pumping 10mL of liquid, waiting for 3s, turning to the position 3 of the pump head, pushing out the liquid, waiting for 3s, closing the valve 11, opening the valve 8, turning the syringe pumps 21-28 to the position 2 of the pump head, pumping 10mL of liquid, waiting for 3s, turning to the position 3 of the pump head, pushing out the liquid, waiting for 3s, closing the valve 8, opening the valve 10, turning the syringe pumps 21-28 to the position 2 of the pump head, pumping 10mL of liquid, waiting for 3s, turning to the position 3 of the pump head, pushing out the liquid, waiting for 3s, closing the valve 10, and closing the valve 6.
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various modifications and variations of the present invention will be apparent to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (7)
1. A fully automated cell separation system, comprising:
the cell separation module comprises a microfluidic chip, wherein the microfluidic chip comprises an upper cover plate, a filter membrane and a lower cover plate; the upper cover plate is provided with: the device comprises a suction through hole, a gas leakage through hole and a sample injection through hole; the lower cover plate is provided with: the cavity corresponds to the sample injection through hole, and the first microfluidic channel and the second microfluidic channel; one end of the first micro-fluid channel is communicated with the air leakage through hole, and the other end of the first micro-fluid channel is communicated with the cavity; one end of the second micro-fluid channel is communicated with the suction through hole, and the other end of the second micro-fluid channel is communicated with the cavity; the filter membrane is arranged between the sample injection through hole and the cavity;
the sample injection module comprises a sample injection needle which can move along the X-axis and Z-axis directions and is used for injecting a sample or a reagent into the cell separation module; the sample injection module further comprises a plunger pump, and the plunger pump is connected with the sample injection needle; the sample injection module further comprises a first liquid pipe, and the plunger pump is connected with the sample injection needle through the first liquid pipe; the sample injection needle is provided with a liquid level detection function; the sample injection module further comprises a large-flux reagent module, and the large-flux reagent module is connected with the plunger pump; a valve is further arranged between the large-flux reagent module and the plunger pump; the plunger pump is connected with the large-flux reagent module through a second liquid pipe; the large-flux reagent module comprises buffer solution and cleaning solution; the sample injection module further comprises a micro reagent module, and the sample injection is realized by driving the plunger pump to extract the reagent in the micro reagent module; the trace reagent module comprises pretreatment liquid, sealing liquid, antibodies and nuclear dyeing reagents;
the air pressure control module is communicated with the cell separation module through the air leakage through hole and is used for monitoring and controlling the pressure difference of the two sides of the filter membrane in the micro-fluidic chip; the air pressure control module comprises an air pressure sensor and an air release valve, wherein the air pressure sensor is provided with two air vents, one air vent is communicated with the atmosphere, and the other air vent is connected with the air release valve and the air release through hole;
the negative pressure suction module is communicated with the cell separation module through the suction through hole and is used for sucking filtrate of the sample after passing through the filter membrane; the negative pressure suction module comprises a syringe pump, and the syringe pump is connected with the suction through hole;
the circuit control module is connected with the sample injection module, the air pressure control module and the negative pressure suction module and controls the work of the sample injection module, the air pressure control module and the negative pressure suction module.
2. The fully automated cell separation system of claim 1, wherein the cell separation module further comprises a clamping device that can tightly fit the upper cover plate and the lower cover plate.
3. The fully automated cell separation system of claim 1, wherein the cell separation module further comprises a sample container, the sample container being connected to the sample introduction port.
4. The fully automated cell separation system of claim 1, wherein the circuit control system comprises a control circuit board and a display screen coupled to the control circuit board.
5. A method of isolating cells in a fully automated cell isolation system according to any one of claims 1 to 4, comprising the steps of:
s1, starting a control program of the circuit control system;
s2, manually injecting a sample into a sample container of the microfluidic chip or driving the sample injection needle to inject a reagent into the sample container of the microfluidic chip;
s3, starting the negative pressure suction module, and extracting filtrate in the microfluidic chip;
and S4, the air pressure control module monitors the air pressure below the filter membrane of the micro-fluidic chip, when the air pressure is higher than a preset threshold value, the air release valve is opened to reduce the pressure, and the negative pressure suction module continues to suck for a period of time and then closes the air release valve.
6. The method of separating cells according to claim 5, wherein the air release valve is opened to reduce the pressure when the air pressure value is steeply increased or exceeds-1 kPa.
7. Use of a fully automated cell separation system according to any one of claims 1 to 4 for cell detection and/or cell counting.
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| JP2008076306A (en) * | 2006-09-22 | 2008-04-03 | Sumitomo Bakelite Co Ltd | Micro flow channel device |
| TW201333190A (en) * | 2012-01-05 | 2013-08-16 | Hitachi Chemical Co Ltd | Cell trapping device |
| CN106754720A (en) * | 2016-11-14 | 2017-05-31 | 中国科学院重庆绿色智能技术研究院 | A kind of circulating tumor cell enrichment and micro-imaging sample producing device |
| CN108841727A (en) * | 2018-05-31 | 2018-11-20 | 东南大学 | CAR-T automatic cell preparation system based on micro-fluidic chip |
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| FR2957672B1 (en) * | 2010-03-22 | 2013-03-15 | Novacyt | AUTOMATIC METHOD AND AUTOMATE FOR PREPARING AND ANALYZING A PLURALITY OF CELLULAR SUSPENSIONS |
| DE102011076228A1 (en) * | 2011-05-20 | 2012-11-22 | Siemens Ag | Arrangement and method for filtration |
| US20160237397A1 (en) * | 2015-01-09 | 2016-08-18 | Aviva Biosciences Corporation | Methods and devices for breaking cell aggregation and separating or enriching cells |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2008076306A (en) * | 2006-09-22 | 2008-04-03 | Sumitomo Bakelite Co Ltd | Micro flow channel device |
| TW201333190A (en) * | 2012-01-05 | 2013-08-16 | Hitachi Chemical Co Ltd | Cell trapping device |
| CN106754720A (en) * | 2016-11-14 | 2017-05-31 | 中国科学院重庆绿色智能技术研究院 | A kind of circulating tumor cell enrichment and micro-imaging sample producing device |
| CN108841727A (en) * | 2018-05-31 | 2018-11-20 | 东南大学 | CAR-T automatic cell preparation system based on micro-fluidic chip |
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