CN110331081B - Cell washing container, cell collecting and washing method and system - Google Patents
Cell washing container, cell collecting and washing method and system Download PDFInfo
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- 238000005406 washing Methods 0.000 title claims abstract description 81
- 238000000034 method Methods 0.000 title claims abstract description 18
- 239000007788 liquid Substances 0.000 claims abstract description 80
- 239000012528 membrane Substances 0.000 claims abstract description 36
- 239000006285 cell suspension Substances 0.000 claims abstract description 23
- 238000001914 filtration Methods 0.000 claims abstract description 19
- 238000011010 flushing procedure Methods 0.000 claims abstract description 19
- 239000003761 preservation solution Substances 0.000 claims abstract description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 9
- 239000002504 physiological saline solution Substances 0.000 claims description 23
- 239000002699 waste material Substances 0.000 claims description 19
- 239000007787 solid Substances 0.000 claims description 14
- 238000001816 cooling Methods 0.000 claims description 3
- 238000004062 sedimentation Methods 0.000 abstract description 9
- 238000005119 centrifugation Methods 0.000 abstract description 6
- 230000005779 cell damage Effects 0.000 abstract description 3
- 208000037887 cell injury Diseases 0.000 abstract description 3
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 238000005086 pumping Methods 0.000 abstract 1
- 241000204031 Mycoplasma Species 0.000 description 9
- 230000000694 effects Effects 0.000 description 8
- 239000000463 material Substances 0.000 description 7
- 239000004695 Polyether sulfone Substances 0.000 description 6
- 229920006393 polyether sulfone Polymers 0.000 description 6
- 238000004321 preservation Methods 0.000 description 6
- 230000006378 damage Effects 0.000 description 5
- 230000003115 biocidal effect Effects 0.000 description 4
- 230000001413 cellular effect Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000036512 infertility Effects 0.000 description 3
- 239000011148 porous material Substances 0.000 description 3
- 238000009825 accumulation Methods 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000004743 Polypropylene Substances 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- -1 polypropylene Polymers 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
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Abstract
The invention discloses a cell washing container, a cell collecting and washing method and a cell collecting and washing system, wherein the method comprises pretreatment; the cell washing container is internally provided with a filtering membrane to divide the inner cavity of the cell washing container into an upper cavity and a lower cavity, and the negative pressure device enables the upper cavity to form negative pressure so that cell suspension to be washed is positioned in the upper cavity; pumping and filtering the liquid in the upper cavity into the lower cavity; adding normal saline to the upper chamber to resuspend the cells; and after repeated times until the liquid meets the standard, quantitatively adding the precooled cell preservation solution into the upper cavity and resuspending the cells to finish cell washing. The cell collecting and flushing system comprises a cell flushing container, a normal saline container, a cell suspension container, an air filter, a first negative pressure device and a second negative pressure device. The invention has the beneficial effects that: through the mode that negative pressure suction combines filtration membrane to replace cell suspension in order to accomplish cell enrichment and washing, not only wash fastly, compare the mode of traditional centrifugation and natural sedimentation moreover enrichment and wash efficiently higher, it is littleer to the cell damage simultaneously.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to a cell washing container, a cell collecting and washing method and a cell collecting and washing system.
Background
With the advance of science and technology, cell therapy is gradually developed to clinic, and the development of cell culture and process technology is also promoted. After large-scale culture, cells must be enriched and the culture medium, nutrient solution, etc. must be washed out while maintaining their activity. The existing cell washing method usually adopts a centrifugation mode and a natural sedimentation mode, the centrifugation mode has high efficiency but great damage to cells, and the natural sedimentation mode has small damage to cells but very low washing efficiency, so how to efficiently enrich and wash the cells and minimize the damage to the cells is a problem to be solved urgently.
Disclosure of Invention
The invention aims to solve the problems of low cell enrichment and washing efficiency and great damage to cells in the existing method and device, and provides a cell washing container which comprises a tank body, wherein a filter for dividing an inner cavity of the tank body into an upper cavity and a lower cavity is arranged in the tank body; the tank body is externally connected with a liquid input pipeline, a negative pressure access pipeline and a waste liquid collecting pipeline;
the filter is bowl-shaped, the bottom of the bowl is a solid disc with a non-porous structure, and a filtering membrane is arranged on the side wall of the bowl;
the end part of the liquid input pipeline in the tank body is close to the solid disc; the negative pressure access pipeline access end is located a tank body top, and the waste liquid collection pipeline access end is located a tank body bottom.
Further, the vertical distance between the end part of the liquid input pipeline in the tank body and the solid disc is less than 1 CM.
Furthermore, the filtering membrane is made of polyether sulfone materials, and the aperture is 300 nm.
The invention also provides a cell collecting and washing system, which comprises a cell washing container, a normal saline container, a cell suspension container, an air filter, a reversing valve, a first negative pressure device and a second negative pressure device;
the cell washing container comprises a tank body, wherein a filter for dividing an inner cavity of the tank body into an upper cavity and a lower cavity is arranged in the tank body; the tank body is externally connected with a liquid input pipeline, a negative pressure access pipeline and a waste liquid collecting pipeline; the filter is bowl-shaped, the bottom of the bowl is a solid disc with a non-porous structure, and a filter membrane is arranged on the side wall of the bowl; the end part of the liquid input pipeline in the tank body is close to the solid disc; the access end of the negative pressure access pipeline is positioned at the top of the tank body, and the access end of the waste liquid collecting pipeline is positioned at the bottom of the tank body;
the physiological saline container, the cell suspension container and the air filter are respectively connected with the reversing valve and are connected with the cell washing container through the reversing valve and the liquid input pipeline;
the negative pressure access pipeline is connected with the first negative pressure device, and the waste liquid collecting pipeline is connected with the second negative pressure device.
Further, the filtering membrane is made of polyether sulfone materials, and the aperture is 200-400 nm.
Further, the device also comprises a cell preservation solution container, an effluent collecting container, a first valve and a second valve; the cell preservation liquid container is communicated with the liquid input pipeline through a reversing valve; the effluent liquid collecting container is communicated with a waste liquid collecting pipeline or a second negative pressure device or a lower cavity; the first valve is arranged on the negative pressure access pipeline; the second valve is arranged on the waste liquid collecting pipeline.
The invention also provides a HepG2 cell collecting and washing method, which comprises the following steps:
s1, adding physiological saline pre-cooled to 0-4 ℃ into the cell washing container through a pipeline to wet a filtering membrane which is arranged in the cell washing container and divides a cavity of the cell washing container into an upper cavity and a lower cavity, and pre-cooling the pipeline and the cell washing container to realize pretreatment;
s2, quantitatively adding the cell suspension to be washed into the upper cavity of the cell washing container after the pretreatment in the step S1, and forming negative pressure of 100-300 mmHg in the upper cavity through a negative pressure device to enable the cell suspension to be washed to be in the upper cavity;
s3, releasing the negative pressure of the upper cavity, and forming a negative pressure of 100-300 mmHg in the lower cavity through a negative pressure device so that the liquid in the upper cavity is pumped and filtered into the lower cavity at a flow rate of 0.5-3L/min; meanwhile, adding physiological saline pre-cooled to 0-4 ℃ into the upper cavity 1.3 at the flow rate of 0.5-3L/min, so that the liquid in the upper cavity 1.3 keeps dynamic balance and lasts for 1-5 min;
s4, stopping adding the physiological saline into the upper chamber 1.3 to remove the dynamic balance of the liquid in the upper chamber 1.3 until the liquid in the upper chamber 1.3 is filtered into the lower chamber 1.4, removing the negative pressure in the lower chamber, quantitatively adding the physiological saline which is precooled to 0-4 ℃ into the upper chamber, forming the negative pressure of 100-300 mmHg in the upper chamber through a negative pressure device, and enabling the physiological saline to be at 0.5 ℃. (mm Hg)
Dispersing cells in the upper cavity at a flow rate of 3L/min and resuspending the cells;
and S5, repeating the steps S3 and S4 until the liquid pumped and filtered in the lower cavity meets the standard, quantitatively adding a cell preservation solution pre-cooled to 0-4 ℃ into the upper cavity, and resuspending the cells to finish cell washing.
Further, in step S4, pre-cooled saline is added into the upper chamber in a variable angle or position manner.
The invention has the beneficial effects that: the cell suspension is replaced by combining the negative pressure suction with the filtering membrane to complete cell enrichment and flushing, so that the flushing speed is high, the enrichment and flushing efficiency is higher compared with the traditional centrifugation and natural sedimentation modes, and the cell damage is smaller.
Drawings
FIG. 1 is a schematic diagram of the cell collection and washing system of the present invention.
FIG. 2 is a schematic sectional view of the cell washing container of the present invention.
Fig. 3 is a schematic perspective view of the can body in fig. 2.
Fig. 4 is a schematic perspective view of the filter of fig. 2.
Fig. 5 is a partially cut-away perspective view of fig. 4.
In the figure, 1. cell washing container; 1.1. a tank body; 1.2. a cover body; 1.3. an upper chamber; 1.4. a lower cavity; 2. a physiological saline container; 3. a cell suspension container; 4. an air filter; 5. a first negative pressure device; 6. a second negative pressure device; 7. a filter; 7.1. a solid disc; 7.2. a filtration membrane; 7.3. a side bracket; 8. a cell preservation fluid container; 10. an effluent collection vessel; 11. a first valve; 12. a second valve; 13. a diverter valve; 14. negative pressure is connected into a pipeline; 15. a waste liquid collection pipeline; 16. a liquid input pipeline.
Detailed Description
The invention is described in further detail below with reference to the figures and specific embodiments.
The cell washing container shown in fig. 2-5 comprises a tank body, wherein a cover body 1.2 is further arranged on the tank body 1.1; a filter 7 which divides the inner cavity of the tank body 1.1 into an upper cavity and a lower cavity (comprising an upper cavity 1.3 and a lower cavity 1.4) is arranged in the tank body 1.1; the tank body 1.1 is externally connected with a liquid input pipeline 16, a negative pressure access pipeline 14 and a waste liquid collecting pipeline 15.
The filter 7 is preferably bowl-shaped, the bottom of the bowl is a solid disc 7.1 with a non-porous structure, and a filter membrane 7.2 is arranged on the side wall of the bowl; the filter membrane 7.2 is made of polyether sulfone material and has a pore diameter of 300 nm. More preferably, the bowl side wall is also provided with a side bracket 7.3 for supporting the filter membrane 7.2.
The end of the liquid input pipeline 16 in the tank body 1.1 is arranged close to the solid disc 7.1; the negative pressure access pipeline 14 is connected to the end at the top of the tank body 1.1, and the waste liquid collecting pipeline 15 is connected to the end at the bottom of the tank body 1.1. The negative pressure access pipeline 14 and the liquid input pipeline 16 are communicated with the upper cavity 1.3, and the waste liquid collecting pipeline 15 is communicated with the lower cavity 1.4. The end of the liquid feed line 16 in the tank 1.1 is at a vertical distance of less than 1CM from the solid disc 7.1.
The vessel 1.1 and the cover 1.2 are preferably screwed together, so that the cover 1.2 can be rotated, and the position of the liquid feed line 16 can be changed, so that cell clumps can be blown off efficiently. The cells are preferably HepG2 cells.
The cell collection and washing system shown in fig. 1-5 comprises a cell washing container 1, a physiological saline container 2, a cell suspension container 3, an air filter 4, a first negative pressure device 5 and a second negative pressure device 6.
The cell washing container 1 comprises a tank body 1.1, wherein a filter for dividing the inner cavity of the tank body 1.1 into an upper cavity and a lower cavity is arranged in the tank body; the tank body 1.1 is externally connected with a liquid input pipeline 16, a negative pressure access pipeline 14 and a waste liquid collecting pipeline 15; the filter is bowl-shaped, the bottom of the bowl is a solid disc 7.1 with a non-porous structure, and a filter membrane 7.2 is arranged on the side wall of the bowl; the end of the liquid input pipeline 16 in the tank body 1.1 is arranged close to the solid disc 7.1; the inlet end of the negative pressure inlet pipeline 14 is positioned at the top of the tank body 1.1, and the inlet end of the waste liquid collecting pipeline 15 is positioned at the bottom of the tank body 1.1;
the physiological saline container 2, the cell suspension container 3 and the air filter 4 are respectively connected with a reversing valve 13 and are connected with the cell washing container 1 through the reversing valve 13 and a liquid input pipeline 16;
the negative pressure access pipeline 14 is connected with the first negative pressure device 5, and the waste liquid collecting pipeline 15 is connected with the second negative pressure device 6.
The cell collecting and flushing system also comprises a cell preservation solution container 8, an effluent collecting container 10, a first valve 11 and a second valve 12; the cell preservation liquid container 8 is communicated with a liquid input pipeline 16 through a reversing valve 13; the effluent liquid collecting container 10 is communicated with the waste liquid collecting pipeline 15 or the second negative pressure device 6 or the lower cavity 1.4; the first valve 11 is arranged on the negative pressure access pipeline 14; the second valve 12 is arranged in the waste liquid collecting line 15.
The cell collection and flushing system can also comprise a cell preservation container which is communicated with the first negative pressure device 5 or the negative pressure access pipeline 14. The cell preservation container is used for storing the washed cells (containing the cell preservation solution).
The first negative pressure device 5 or the second negative pressure device 6 is a negative pressure vacuum pump, and if the arrangement structure or the selected type of the negative pressure vacuum pump is reasonable, the first negative pressure device 5 and the second negative pressure device 6 can share the same negative pressure vacuum pump, and certainly, the matching of corresponding valves and pipelines is also needed; the first valve 11 or the second valve 12 is preferably a solenoid valve, and likewise the direction valve 13 is preferably a solenoid valve.
Pre-cooled physiological saline is stored in the physiological saline container 2; the cell suspension container 3 is used for storing the cell suspension to be washed; the cell preservation solution container 8 stores therein a cell preservation solution. Effluent collection vessel 10 is used to collect the liquid flowing from lower chamber 1.4.
The containers such as the cell washing container 1, the physiological saline container 2, the cell suspension container 3, the cell preservation solution container 8, the cell preservation container, and the effluent collecting container 10 may be rigid tank-like containers, box-like containers, tubular containers, and the like, or may be flexible bag-like containers or sac-like containers.
As shown in fig. 2 and 3, in the present embodiment, the cell washing container 1 is a pot-shaped container, which includes a pot body 1.1 and a cover body 1.2; one end of the negative pressure inlet line 14 and one end of the liquid inlet line 16 pass through the cover 1.2 and are fixed in a sealing manner. The cover 1.2 can be transparent, and the size of the tank body 1.1 can be divided into a plurality of sizes in order to meet different requirements.
In order to improve the flushing efficiency, the cover body 1.2 and the tank body 1.1 are connected by threads, and the length of the threads is such that the cover body 1.2 can rotate more than one turn relative to the tank body 1.1, because one end of the negative pressure access pipeline 14 and one end of the liquid input pipeline 16 penetrate through the cover body 1.2 to the tank body 1.1, when the cover body 1.2 rotates, the positions of the negative pressure access pipeline 14 and one end of the liquid input pipeline 16 can be changed, and the negative pressure access pipeline and the liquid input pipeline rotate along the circumferential direction relative to the tank body 1.1, so that pre-cooled physiological saline can be added into the upper chamber 1.3 in a position-changing manner, and cell agglomerates can be effectively blown away under the action of negative pressure. Of course, such circumferential rotation causes the negative pressure inlet line 14 and the liquid inlet line 16 to be twisted to some extent, so that the number of rotations is not easily too long, preferably from one to two rotations, and is not a continuous rotation, for example, first one rotation clockwise and then one rotation counterclockwise.
In order to further improve the washing efficiency and reduce the cell damage, the end of the liquid input pipeline 16 positioned in the tank body 1.1 can be changed in angle, so that the incident angle of the pre-cooled physiological saline for dispersing the cells is adjusted. The angle of the liquid input pipeline 16 can be changed by adding an angle sensor on the liquid input pipeline 16, arranging an electric push rod connected with the liquid input pipeline 16 below the cover body 1.2, pushing the tail end of the liquid input pipeline 16 through the electric push rod to change the inclination angle of the liquid input pipeline, and measuring the angle of the liquid input pipeline through the angle sensor to realize the regulation and control of the inclination angle.
The filtering membrane 7.2 is made of polyether sulfone material, and the aperture is 200-400 nm. The filter membrane 7.2 comprises at least one inclined or convex surface. That is, the filtering membrane 7.2 of this embodiment may be an uneven membrane, because the flat membrane has a smaller membrane area, and is easy to cause cell accumulation, and is also inconvenient for the flow of physiological saline to realize the washing of cells, therefore, the filtering membrane 7.2 is provided with at least one inclined surface or convex surface, which can effectively increase the membrane area, and is convenient for the flow of liquid, thereby improving the washing efficiency. Preferably, the multiple filter membranes 7.2 are connected and shaped into a hollow truncated cone-shaped or tuck-shaped or cup-shaped or funnel-shaped or bowl-shaped filter with a large upper end and a small lower end through a bracket 17; the bracket 17 is made of polypropylene material.
The shape of the filter may be as shown in fig. 4, 5. The inclined surface or the convex surface of the filtering membrane 7.2 can be a hollow truncated cone-shaped or tuck-shaped or cup-shaped or funnel-shaped or bowl-shaped filter with a large upper end and a small lower end, and the bottom surface of the filter is a non-filtering surface (namely the solid disc 7.1 of the cell washing container).
In order to verify the washing effect, the applicant selects a plurality of HepG2 cell suspensions to be washed with the same volume and sample data, and respectively adopts a traditional centrifugal washing system, a natural sedimentation washing system and the washing system provided by the invention to carry out a plurality of washing experiments; whereinThe cells were cultured with the cytopore microcarriers in 14L fermentors with 10L culture medium, 10 at a time 10 The above HepG2 cells.
In this washing experiment, the volume of the cell washing container was 2L, and the bottom area was 80cm 2 The bowl volume of the filter is about 200cm 3 The detection shows that the antibiotic residue before washing is 94ug/ml, the BSA residue is 46mg/ml, and the cell activity is 95%. The cells to be washed in each placement are about 1X 10 10 And (4) one cell.
The system controls the negative pressure to be 100-300 mmHg, the negative pressure suction flow rate to be 0.5-3L/min, and in order to better verify the experimental result, the negative pressure is controlled to be 100mmHg in the flushing experiment, the negative pressure suction flow rate is controlled to be 1L/min, and the precooling temperature is uniformly controlled to be 0 ℃; the experimental result data are as follows:
after washing the cell suspension to be washed 3 times by adopting the centrifugal washing system, the liquid content data in the upper cavity 1.3 of the cell washing container 1 are as follows:
| number of |
1 time of | 2 times (one time) | 3 times of |
| Time consuming | 5min | 5min | 5min |
| Has sterility | Sterile | Sterile | Sterile |
| Presence or absence of mycoplasma | Mycoplasma free | Mycoplasma free | Mycoplasma free |
| Residual antibiotic amount | 2.7ug/ml | 1.7ug/ml | 0.7ug/ml |
| Residual amount of BSA | 190ng/ml | 65ng/ml | 39ng/ml |
| Cellular activity | 85％ | 83％ | 80％ |
After the cell suspension to be washed is washed for 3 times by adopting the natural sedimentation washing system, the liquid content data in the upper cavity 1.3 of the cell washing container 1 are as follows:
after the system is adopted and a filtering membrane with the aperture of 200nm is selected for 7.2 flushing for 3 times, the liquid content data in the upper cavity 1.3 of the cell flushing container 1 are as follows:
| number of |
1 time of | 2 times (one time) | 3 times of |
| Time consuming | 5min | 5min | 5min |
| Has sterility | Sterile | Sterile | Sterile |
| Presence or absence of mycoplasma | Mycoplasma free | Mycoplasma free | Mycoplasma free |
| Residual antibiotic amount | 2.2ug/ml | 1.6ug/ml | 0.6ug/ml |
| Residual amount of BSA | 200ng/ml | 59ng/ml | 38ng/ml |
| Cellular activity | 94％ | 92％ | 91％ |
After the system is adopted and a filtering membrane with the aperture of 300nm is selected for 7.2 times of flushing for 3 times, the liquid content data in an upper cavity 1.3 of the cell flushing container 1 are as follows:
| number of |
1 time of | 2 times (one time) | 3 times of |
| Time consuming | 5min | 5min | 5min |
| Has sterility | Sterile | Sterile | Sterile |
| Presence or absence of mycoplasma | Mycoplasma free | Mycoplasma free | Mycoplasma free |
| Residual antibiotic amount | 2.4ug/ml | 1.8ug/ml | 0.6ug/ml |
| Residual amount of BSA | 267ng/ml | 62ng/ml | 39ng/ml |
| Cellular activity | 94％ | 92％ | 91％ |
After the system is adopted and a filtering membrane with the pore diameter of 400nm is selected for 7.2 times of flushing for 3 times, the liquid content data in an upper cavity 1.3 of the cell flushing container 1 are as follows:
from the data in the tables, it can be seen that the filter membrane 7.2 made of polyethersulfone material has pore sizes of 200nm, 300nm and 400nm, and the contents of endotoxin, antibiotics and BSA in the liquid in the upper chamber 1.3 of the cell washing container 1 are obviously reduced after three times of washing. The change of the aperture only influences the washing effect to a certain degree, and the cell activity after 3 times of washing is far higher than that of the traditional centrifugation and sedimentation method. The system is time consuming to flush and centrifuge but is more time efficient than sedimentation. Therefore, the system has higher cell enrichment and washing efficiency and less damage to cells compared with the traditional centrifugation and natural sedimentation modes.
Aiming at the cell collecting and washing system provided by the invention, the invention provides a HepG2 cell collecting and washing method applicable to the system, which comprises the following steps:
s1, adding physiological saline pre-cooled to 0-4 ℃ into the cell washing container 1 through a pipeline to wet a filtering membrane 7.2 which is arranged in the cell washing container 1 and divides the cavity of the cell washing container into an upper cavity 1.3 and a lower cavity 1.4, and pre-cooling the pipeline and the cell washing container 1 to realize pretreatment;
s2, quantitatively adding cells in the cell suspension to be washed into the upper cavity 1.3 of the cell washing container 1 after the pretreatment in the step S1, and forming negative pressure of 100-300 mmHg in the upper cavity 1.3 through a negative pressure device to enable the cell suspension to be washed to be in the upper cavity 1.3;
s3, releasing the negative pressure of the upper cavity 1.3, and forming a negative pressure of 100-300 mmHg in the lower cavity 1.4 through a negative pressure device so that the liquid in the upper cavity 1.3 is pumped and filtered into the lower cavity 1.4 at a flow rate of 0.5-3L/min; meanwhile, adding physiological saline pre-cooled to 0-4 ℃ into the upper cavity 1.3 at the flow rate of 0.5-3L/min, so that the liquid in the upper cavity 1.3 keeps dynamic balance and lasts for 1-5 min; in the embodiment, the dynamic balance time is preferably 2 min;
s4, stopping adding the physiological saline into the upper cavity 1.3 to remove the dynamic balance of the liquid in the upper cavity 1.3, removing the negative pressure in the lower cavity 1.4 after the liquid (90% or more) in the upper cavity 1.3 is pumped and filtered into the lower cavity 1.4, quantitatively adding the physiological saline which is precooled to 0-4 ℃ into the upper cavity 1.3, forming the negative pressure of 100-300 mmHg in the upper cavity 1.3 through a negative pressure device, and dispersing the cells in the upper cavity 1.3 by the physiological saline at the flow rate of 0.5-3L/min and re-suspending the cells;
and S5, repeating the steps S3 and S4 until the liquid pumped and filtered into the lower cavity 1.4 meets the standard, adding a cell preservation solution pre-cooled to 0-4 ℃ into the upper cavity 1.3 in a fixed amount, and resuspending the cells to finish cell washing.
In the method, the filter membrane 7.2 is made of polyether sulfone material, and the aperture is preferably 300 nm. The filter membrane 7.2 is fixed in the cell washing container 1. The filter membrane 7.2 is shaped or fixed by at least one inclined or convex surface. This arrangement of the filter membrane 7.2 will facilitate cell washing, in particular to avoid cell accumulation and improve fluid flow.
To improve the washing efficiency and cell viability, pre-cooled saline is added to the upper chamber 1.3 in a variable angle or position manner in step S4. For example, a tube with one end extending into the upper chamber 1.3 can be angled or can be rotated relative to the filter membrane 7.2. The variable angle or position can be implemented by other devices, such as a motor, a push rod, and the like, and can also be implemented manually.
The way of releasing the negative pressure of the upper chamber 1.3 in the step S3 includes closing the negative pressure device communicated with the upper chamber 1.3 and communicating the upper chamber 1.3 with the atmosphere through the air filter 4; the way of releasing the negative pressure in the lower chamber 1.4 in step S4 includes closing the negative pressure device communicating with the lower chamber 1.4.
The negative pressure device in the method is preferably a negative pressure vacuum pump, if the arrangement structure or the selected type of the negative pressure vacuum pump is reasonable, the negative pressure device communicated with the upper cavity 1.3 and the negative pressure device communicated with the lower cavity 1.4 can be the same negative pressure vacuum pump, and certainly, the matching of a corresponding valve (such as a three-way valve) and a pipeline is also required; the valve in the present method is preferably a solenoid valve.
Claims (2)
1. A method for collecting and flushing HepG2 cells by using a cell collecting and flushing system, wherein the cell collecting and flushing system comprises a cell flushing container (1), a normal saline container (2), a cell suspension container (3), an air filter (4), a reversing valve (13), a first negative pressure device (5) and a second negative pressure device (6); the cell washing container (1) comprises a tank body (1.1), and a filter (7) for dividing an inner cavity of the tank body (1.1) into an upper cavity and a lower cavity is arranged in the tank body; the tank body (1.1) is externally connected with a liquid input pipeline (16), a negative pressure access pipeline (14) and a waste liquid collecting pipeline (15); the filter is bowl-shaped, the bottom of the bowl is a solid disc (7.1) with a non-porous structure, and a filter membrane (7.2) is arranged on the side wall of the bowl; the filter membrane comprises at least one inclined surface or convex surface; the end of the liquid input pipeline (16) in the tank body (1.1) is arranged close to the solid disc (7.1); the access end of the negative pressure access pipeline (14) is positioned at the top of the tank body (1.1), and the access end of the waste liquid collecting pipeline (15) is positioned at the bottom of the tank body (1.1); the physiological saline container (2), the cell suspension container (3) and the air filter (4) are respectively connected with the reversing valve (13) and are connected with the cell washing container (1) through the reversing valve (13) and the liquid input pipeline (16); the negative pressure access pipeline (14) is connected with the first negative pressure device (5), and the waste liquid collecting pipeline (15) is connected with the second negative pressure device (6);
the method is characterized in that: the method comprises the following steps:
s1, adding physiological saline pre-cooled to 0-4 ℃ into the cell washing container (1) through a pipeline to wet a filtering membrane (7.2) which is arranged in the cell washing container (1) and divides a cavity of the cell washing container into an upper cavity (1.3) and a lower cavity (1.4), and pre-cooling the pipeline and the cell washing container (1) to realize pretreatment;
s2, quantitatively adding the cell suspension to be washed into the upper cavity (1.3) of the cell washing container (1) after the pretreatment of the step S1 is completed, and forming 100-300 mmHg negative pressure in the upper cavity (1.3) through a negative pressure device so that the cell suspension to be washed is positioned in the upper cavity (1.3);
s3, releasing the negative pressure of the upper cavity (1.3), and forming a negative pressure of 100-300 mmHg in the lower cavity (1.4) through a negative pressure device so that the liquid in the upper cavity (1.3) is pumped and filtered into the lower cavity (1.4) at a flow rate of 0.5-3L/min; meanwhile, physiological saline pre-cooled to 0-4 ℃ is added into the upper cavity (1.3) at the flow rate of 0.5-3L/min, so that the liquid in the upper cavity (1.3) keeps dynamic balance and lasts for 1-5 min;
s4, stopping adding the normal saline into the upper cavity (1.3) to remove the dynamic balance of the liquid in the upper cavity (1.3), removing the negative pressure of the lower cavity (1.4) after the liquid in the upper cavity (1.3) is pumped and filtered into the lower cavity (1.4), quantitatively adding the normal saline which is precooled to 0-4 ℃ into the upper cavity (1.3), forming the negative pressure of 100-300 mmHg in the upper cavity (1.3) through a negative pressure device, and dispersing the cells in the upper cavity (1.3) at the flow rate of 0.5-3L/min and re-suspending the cells;
s5, repeating the steps S3 and S4 until the liquid pumped and filtered into the lower cavity (1.4) meets the standard, adding a cell preservation solution pre-cooled to 0-4 ℃ into the upper cavity (1.3) in a fixed amount, and resuspending the cells to finish cell washing.
2. The method for collection and washing of HepG2 cells as claimed in claim 1, wherein: in step S4, the pre-cooled saline solution is added to the upper chamber (1.3) in a variable angle or position manner.
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| CN115369021B (en) * | 2022-08-15 | 2024-08-16 | 安徽省立医院(中国科学技术大学附属第一医院) | Multipurpose flow cell washing instrument, control method and application |
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