CN108441407B - Automatic cell purification and cryopreservation device - Google Patents
Automatic cell purification and cryopreservation device Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention discloses an automatic cell purification and cryopreservation device, belongs to the technical field of biology, and aims to overcome the defects that at present, when a sample is separated, manual operation steps are frequent, cell pollution is easy to cause, cells are easy to damage, the biological effect is influenced, and the like. This automatic purification of cell freezes deposits device is including the purification case of freezing, liquid bag frame, the stop valve, the peristaltic pump, suspension separator and injection device, the left side of purification freezing the case is automatically controlled purification district, the right side of purification freezing the case is for freezing the district, be provided with a plurality of peristaltic pumps and a suspension separator on the middle part tank wall in automatically controlled purification district, the lower part in automatically controlled purification district is provided with injection device, it is equipped with the cell storage bag to hang on liquid bag frame, the infusion pipe of the bottom connection of cell storage bag is carried in the peristaltic pump through the stop valve clamp, it freezes deposits bag and heat sealing device to have placed on the mixing platform in advance in the box in freezing the district. The automatic cell purification and cryopreservation device provided by the invention realizes multifunctional automation, improves the operation accuracy and reduces the biological pollution risk.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a control device for automatic cell purification and program cryopreservation, which is used for cell separation, extraction and program cryopreservation.
Background
With the development of biological treatment technology, cell purification technology is gradually optimized and developed, the purpose of cell layering separation is achieved by controlling corresponding rotating speed of an original traditional centrifuge, and the whole set of sterile pipelines developed to date are centrifuged at high speed through a centrifuge cup to separate cell components to the edge, so that the required components are separated, but the separation speed is low, the noise is high, the cell loss is high, the manual intervention is high, and the preparation stability is poor.
The program-controlled cooling technology is to carry out program cryopreservation on a sample needing ultralow temperature cryopreservation by adopting a temperature control technology and a liquid nitrogen dispersion technology and safely and reliably controlling the temperature change rate, is widely applied to various fields of agriculture, medicine, basic biological research and the like, is used for cryopreservation of lymphocytes, tissue banks, stem cells, bone marrow cells, tumor cells, animal cells, plant cells, myocardial cells and the like, however, in the prior art, precooling in advance, cell cryopreservation pretreatment, transfer and other programs are needed, and due to plasma stratification, the detection probe is easy to be incompletely immersed into the plasma, so that the detection data is abnormal, the cryopreservation curve is not real, the cell viability is influenced, unnecessary waste of manpower, material resources and the like is caused, and the production cost is increased.
Therefore, there is still a need in the art for a cryopreservation device that can perform cell purification and cryopreservation from an integrated, intelligent manner, including selective enrichment of stem cells or other cells such as plasma, red blood cells, platelets, etc., while achieving automated separation and purification, maintaining sterility, cell viability, and regeneration and cell viability.
Disclosure of Invention
The invention aims to purify stem cells or other cells from a liquid biological product and automatically perform cryopreservation, overcomes the defects that the prior manual operation step is frequent when a sample is separated, the cell pollution is easy to cause, the cells are easy to be damaged and the biological effect is influenced, and provides an automatic cell purification cryopreservation device which reduces open operation and avoids the sample pollution, so as to improve the preparation efficiency and reduce the sample pollution and the cell survival rate.
The invention relates to an automatic cell purification and cryopreservation device which comprises a purification and cryopreservation box, a liquid bag frame, stop valves, peristaltic pumps, a suspension separation device and an injection device, wherein the left side of the purification and cryopreservation box is an electric control purification area, the right side of the purification and cryopreservation box is a cryopreservation area, the upper part of the electric control purification area is a display screen, the middle box wall of the electric control purification area is provided with a plurality of peristaltic pumps and a suspension separation device, the lower part of the electric control purification area is provided with the injection device, the side box wall of the electric control purification area is provided with a plurality of stop valves, and a connecting pipe channel is formed between the electric control purification area and the cryopreservation area;
the liquid bag rack is arranged on the top surface of the purification cryopreservation box, a cell storage bag is hung on the liquid bag rack, the bottom of the cell storage bag is communicated with one end of an infusion tube, the infusion tube is clamped in a peristaltic pump through a stop valve, the other end of the infusion tube is connected with an input port of the suspension separation device, an output port of the side wall of the bottom of the suspension separation device penetrates through a connecting tube channel through a first connecting tube to be communicated with the cryopreservation bag, and a liquid outlet of the injection device penetrates through the connecting tube channel through a second connecting tube to be communicated with the cryopreservation bag;
the freezing storage area is internally provided with a box body, a box door is arranged on the box body, a freezing storage bag and a heat sealing device are arranged on a pre-mixing platform in the box body of the freezing storage area, a first connecting pipe and a second connecting pipe penetrate through a heat sealing die of the heat sealing device, and purified cells in the freezing storage bag are frozen through liquid nitrogen in the freezing storage area.
The invention relates to a cell automatic purification separation and cryopreservation device, which mainly comprises an automatic cell purification separation part, a cell acquisition premixing part and a program cryopreservation part, wherein the automatic cell purification separation part is positioned at the left side of the automatic purification cryopreservation device, a cell premixing platform is positioned in a program cryopreservation area, a program control part of the program cryopreservation part is positioned above the left side of the automatic purification cryopreservation device and is provided with a liquid crystal touch display screen, a circuit board is connected with each micromotor, a push injection device and each sensor, an MCU chip on the circuit board outputs signals to control the micromotor to drive a peristaltic pump to step forward and reverse deflection, the action of the peristaltic pump and the action of the push injection device are accurately controlled, the directional flow of liquid flow of an infusion pipeline is controlled, the automatic cell purification separation part separates the liquid flow of cells through a micropore suspension separation device and conveys the liquid flow to the premixing part, and (4) performing program cryopreservation through the program cryopreservation part.
The automatic cell purification and cryopreservation device disclosed by the invention has the following beneficial effects:
1. according to the automatic cell purification and separation part, the liquid bags on the liquid bag rack are controlled by a preset program in the liquid crystal touch display screen to control the on-off of the infusion pipeline, and then are respectively pumped into the micropore suspension separation device and the cryopreservation bags on the premixing and homogenizing platform by the pressurization of the peristaltic pump and the injection device, so that the multifunctional automation is realized, the physical and material resources are greatly saved, the cell filtration efficiency is increased, the operation accuracy is improved, and the biological pollution risk is reduced.
2. A cell acquisition pre-mixing part is provided with a perspective switch door, a freezing bag connected with an automatic cell purification and separation part is placed on a pre-mixing platform in a cavity and shaken uniformly, a liquid crystal touch display screen is metered by a weighing sensor and separated by a heat seal pipeline, an MCU controls a liquid nitrogen controller to input liquid nitrogen into the cavity, and program freezing is carried out through parameter response of an ultralow temperature sensor. The purification and uniform mixing of blood components can be realized by different sets of plastic multi-connection bags under the action of the micro-pore suspension separation device, and the loss of cells caused by misoperation is avoided.
3. The program freezing part of the invention is used for infusing liquid containing freezing liquid and the like into a freezing bag on a pre-mixing platform in the cavity through an infusion pipeline for mixing uniformly, a liquid nitrogen controller inputs liquid nitrogen into the cavity from a liquid nitrogen output port, and program freezing is carried out through the parameter response of an ultralow temperature sensor. In a closed environment, the temperature of the inner cavity is controlled by a liquid nitrogen indirect program, and cells are quickly cooled, so that the influence of transferring and transferring the cells is avoided, and the protection of operators and the cells is ensured.
4. Compared with the prior art, the invention has the advantages of saving manpower and material resources, being accurate in operation and carrying out efficient filtration aiming at different cell characteristics.
Drawings
FIG. 1 is a schematic structural diagram of an automatic cell purification and cryopreservation apparatus according to the present invention;
FIG. 2 is a schematic structural diagram of a cryopreservation zone;
FIG. 3 is a schematic structural view of a suspension separation apparatus;
FIG. 4 is a schematic top view of a suspension separation apparatus.
Detailed Description
The first embodiment is as follows: the automatic cell purification and cryopreservation device comprises a purification and cryopreservation box 1, a liquid bag frame 5, stop valves 7, peristaltic pumps 8, a suspension separation device 9 and an injection device 10, wherein the left side of the purification and cryopreservation box 1 is an electronic control purification area, the right side of the purification and cryopreservation box 1 is a cryopreservation area, the upper part of the electronic control purification area is a display screen 6, a plurality of peristaltic pumps 8 and one suspension separation device 9 are arranged on the middle box wall of the electronic control purification area, the injection device 10 is arranged at the lower part of the electronic control purification area, a plurality of stop valves 7 are arranged on the side box wall of the electronic control purification area, and a connecting pipe channel 4 is arranged between the electronic control purification area and the cryopreservation area;
the liquid bag rack 5 is arranged on the top surface of the purification freezing storage box 1, a cell storage bag is hung on the liquid bag rack 5, the bottom of the cell storage bag is communicated with one end of an infusion tube 13, the infusion tube 13 is clamped in a peristaltic pump 8 through a stop valve 7, the other end of the infusion tube 13 is connected with an input port 9-5 of a suspension separation device 9, an output port 9-6 of the side wall of the bottom of the suspension separation device 9 penetrates through a connecting tube channel 4 through a first connecting tube to be communicated with the freezing storage bag, and a liquid outlet of a pushing injection device 10 penetrates through the connecting tube channel 4 through a second connecting tube to be communicated with the freezing storage bag;
the freezing storage area is internally provided with a pre-mixing platform 3, a freezing storage bag and a heat sealing device 12 are placed on the pre-mixing platform 3, a first connecting pipe and a second connecting pipe penetrate through a heat sealing die of the heat sealing device 12, and purified cells in the freezing storage bag are frozen through liquid nitrogen in the freezing storage area.
This embodiment freezes and is provided with the liquid nitrogen delivery outlet in the district, and the liquid nitrogen flows into through this delivery outlet and freezes in the district.
The second embodiment is as follows: the difference between the present embodiment and the first embodiment is that a weighing sensor is provided on the pre-mixing platform 3.
In the embodiment, the weight of the cell sap in the cryopreservation bag is measured through the weighing sensor, and the heat sealing device is controlled to heat seal the infusion pipeline through parameter setting and metering so as to separate the pipeline.
The third concrete implementation mode: the present embodiment is different from the first or second embodiment in that a temperature sensor is provided in the freezing region.
The fourth concrete implementation mode: this embodiment differs from one of the first to third embodiments in that the bolus device 10 is a micro syringe pump.
The fifth concrete implementation mode: the present embodiment is different from one of the first to fourth embodiments in that a plurality of shut valves 7 are vertically arranged.
The sixth specific implementation mode: this embodiment differs from one of the first to fifth embodiments in that the bolus device 10 is filled with a frozen stock solution.
The seventh embodiment: the difference between the present embodiment and one of the first to sixth embodiments is that the suspension separation device 9 comprises a cylindrical shell 9-1, a driving shaft 9-2, a cross impeller 9-3, a fiber filter membrane 9-4, an input port 9-5, an output port 9-6 and a waste cell discharge port 9-7, wherein the fiber filter membrane 9-4 is sleeved in the cylindrical shell 9-1 in a cylindrical shape, a filter cavity 9-8 is formed between the inner wall of the cylindrical shell 9-1 and the fiber filter membrane 9-4, the cross impeller 9-3 is arranged at the top in the shell of the cylindrical shell 9-1, the driving shaft 9-2 is connected with the cross impeller 9-3 and penetrates out of the top of the cylindrical shell 9-1, the input port 9-5 is arranged at the upper part of the shell side wall of the cylindrical shell 9-1 and is communicated with an inner cavity surrounded by the fiber filter membrane 9-4, the output port 9-6 is arranged at the lower part of the side wall of the shell of the cylindrical shell 9-1, and the waste cell discharge port 9-7 is vertically arranged on the bottom shell of the cylindrical shell 9-1.
The drive shaft in this embodiment is rotatory by the motor drive, and cell sap gets into the inner chamber that the fibrous filtration membrane encloses from the input port, and the vortex rotation makes the cell pass through the fibrous filtration membrane, gets into the cavity that fibrous filtration membrane and transparent shell formed and infuse to the cryopreserving bag through concentrated cell delivery outlet, and the delivery outlet is along the radial of cylinder shell, and abandonment cell discharge port communicates with each other with the fibrous filtration membrane inner chamber, and the abandonment cell can't pass through the fibrous filtration membrane and retrieve in the waste liquid bag through abandonment cell discharge port under the effect of rotation force.
The specific implementation mode is eight: the seventh embodiment is different from the seventh embodiment in that the waste cell discharging port 9-7 of the suspension separation apparatus 9 is communicated with the waste liquid collecting bag through the infusion tube 13.
The specific implementation method nine: the seventh embodiment is different from the seventh embodiment in that the fibrous filtration membrane 9-4 in the suspension separation device 9 is formed by stacking a plurality of layers of cellulose nanofibers.
The aperture of the fiber filter membrane of the embodiment can be made to be 2-10 microns so as to meet the requirement of filtering different cells such as red blood cells, white blood cells, granulocytes and the like.
Example (b): the automatic cell purification and cryopreservation device comprises a purification and cryopreservation box 1, a liquid bag frame 5, stop valves 7, peristaltic pumps 8, a suspension separation device 9 and an injection device 10, wherein the left side of the purification and cryopreservation box 1 is an electric control purification area, the right side of the purification and cryopreservation box 1 is a cryopreservation area, the upper part of the electric control purification area is a liquid crystal touch display screen 6, a plurality of peristaltic pumps 8 and one suspension separation device 9 are arranged on the middle box wall of the electric control purification area, the injection device 10 is arranged at the lower part of the electric control purification area, a plurality of stop valves 7 are arranged on the side box wall of the electric control purification area, and a connecting pipe channel 4 is arranged between the electric control purification area and the cryopreservation area;
the liquid bag rack 5 is arranged on the top surface of the purified freezing storage box 1, a cell storage bag is hung on the liquid bag rack 5, the bottom of the cell storage bag is communicated with one end of an infusion tube 13, the infusion tube 13 is clamped in a peristaltic pump 8 through a stop valve 7, the other end of the infusion tube 13 is connected with an input port 9-5 of a suspension separation device 9, an output port 9-6 of the side wall of the bottom of the suspension separation device 9 penetrates through a connecting tube channel 4 through a first connecting tube to be communicated with the freezing storage bag, a liquid outlet of an injection device 10 penetrates through the connecting tube channel 4 through a second connecting tube to be communicated with the freezing storage bag, and polyester fiber cotton is filled in the connecting tube channel 4 to isolate liquid nitrogen and preserve heat;
be provided with switch door 2 on the preceding tank wall in freezing district, placed freezing bag and heat sealing device 12 on the even platform 3 of mixing in advance in the box in freezing district, even platform 3 for shaking or wave the platform in advance, first connecting pipe and second connecting pipe pass the heat sealing die of heat sealing device 12, liquid nitrogen gets into through liquid nitrogen mouth 11 and makes the purification cell in the freezing bag freeze and deposit in freezing district.
The automatic purification process in this example was as follows:
a physiological saline bag (right side) is hung on a liquid bag frame 5, the bottom of the physiological saline bag is communicated with an infusion tube, the infusion tube is clamped in a peristaltic pump through a stop valve, the other end of the infusion tube is connected with an input port 9-5 of a suspension separation device 9, an output port 9-6 of the side wall of the bottom of the suspension separation device 9 is communicated with a waste liquid recovery bag through a third connecting tube and the peristaltic pump, and cleaning liquid is used for cleaning and filling the suspension separation device 9.
Secondly, a cell storage bag is hung on the liquid bag rack 5, the bottom of the cell storage bag is communicated with one end of an infusion tube 13, the infusion tube 13 is clamped in a peristaltic pump 8 through a stop valve 7, the other end of the infusion tube 13 is connected with an input port 9-5 of a suspension separation device 9, an output port 9-6 of the side wall of the bottom of the suspension separation device 9 penetrates through a connecting tube channel 4 through a first connecting tube to be communicated with the cryopreservation bag, and a liquid outlet of a push injection device 10 penetrates through the connecting tube channel 4 through a second connecting tube to be communicated with the cryopreservation bag;
thirdly, according to the second step, the output port 9-6 of the side wall of the bottom of the suspension separation device 9 penetrates through the connecting pipe channel 4 through the first connecting pipe to be communicated with the freezing bag, the output port 9-6 of the side wall of the bottom is connected in parallel with one path, and the incompletely purified cells are returned to the cell storage bag through the fourth connecting pipe, the peristaltic pump and the stop valve, and then enter the suspension separation device 9 again for purification and separation.
The automatic cryopreservation process in this embodiment is as follows:
firstly, at the beginning of an automatic purification process, a liquid nitrogen controller is controlled by an MCU (microprogrammed control Unit) to input liquid nitrogen into a cavity, program freezing is carried out through parameter response of an ultralow temperature sensor, and in the purification process, the freezing cavity is kept at 4 ℃ until a weighing sensor measures that the weight of cell fluid in a freezing bag accords with the freezing weight;
and secondly, when the weight of the cell fluid in the freezing bag is measured by a weighing sensor to accord with the freezing weight in the purification process, automatically stopping the purification process, flushing a pipeline, thermally sealing an infusion pipeline by a parameter setting and metering control heat sealing device to separate the pipeline, keeping the freezing bag on a pre-mixing platform in the cavity at 4 ℃ for 10 minutes, controlling a liquid nitrogen controller by an MCU (micro control unit) to input the liquid nitrogen into the cavity and keep the liquid nitrogen at-20 ℃ for 25 minutes, and finally, reducing the liquid nitrogen input into the cavity by the liquid nitrogen controller from the room temperature to below-80 ℃ at the speed of-1 to-3 ℃/minute and keeping the liquid nitrogen.
Claims (8)
1. The automatic cell purifying and freezing device is characterized by comprising a purifying and freezing box (1), a liquid bag frame (5), stop valves (7), peristaltic pumps (8), a suspension separation device (9) and an injection device (10), wherein the left side of the purifying and freezing box (1) is an electric control purifying area, the right side of the purifying and freezing box (1) is a freezing area, the upper part of the electric control purifying area is a display screen (6), the middle box wall of the electric control purifying area is provided with a plurality of peristaltic pumps (8) and a suspension separation device (9), the lower part of the electric control purifying area is provided with the injection device (10), the side box wall of the electric control purifying area is provided with a plurality of stop valves (7), and a connecting pipe channel (4) is arranged between the electric control purifying area and the freezing area;
the cell freezing and storing device comprises a liquid bag frame (5), a cell storage bag, an infusion tube (13), a peristaltic pump (8), a suspension separation device (9), a liquid injection device (10), a liquid injection device and a freezing and storing bag, wherein the liquid bag frame (5) is arranged on the top surface of a purifying freezing and storing box (1), the cell storage bag is hung on the liquid bag frame (5), the bottom of the cell storage bag is communicated with one end of the infusion tube (13), the infusion tube (13) is clamped and loaded in the peristaltic pump (8) through a stop valve (7), the other end of the infusion tube (13) is connected with an input port (9-5) of the suspension separation device (9), an output port (9-6) of the bottom side wall of the suspension separation device (9) penetrates through a connecting tube channel (4) through a first connecting tube to be communicated with the freezing and storing bag, and a liquid outlet of the injection device (10) penetrates through a connecting tube channel (4) through a second connecting tube to be communicated with the freezing and storing bag;
a door (2) is arranged on the front box wall of the freezing area, a freezing bag and a heat sealing device (12) are placed on a pre-mixing platform (3) in a box body of the freezing area, a first connecting pipe and a second connecting pipe penetrate through a heat sealing die of the heat sealing device (12), and purified cells in the freezing bag are frozen in the freezing area through liquid nitrogen;
the suspension separation device (9) comprises a cylindrical shell (9-1), a driving shaft (9-2), a cross impeller (9-3), a fiber filter membrane (9-4), an input port (9-5), an output port (9-6) and a waste cell discharge port (9-7), wherein the fiber filter membrane (9-4) is sleeved in the cylindrical shell (9-1) in a cylindrical shape, a filter cavity (9-8) is formed between the inner wall of the cylindrical shell (9-1) and the fiber filter membrane (9-4), the cross impeller (9-3) is arranged at the top in the shell of the cylindrical shell (9-1), the driving shaft (9-2) is connected with the cross impeller (9-3) and penetrates out of the shell of the cylindrical shell (9-1), and the input port (9-5) is arranged at the upper part of the side wall of the cylindrical shell (9-1) and surrounds the fiber filter membrane (9-4) The inner cavities are communicated, the output port (9-6) is arranged at the lower part of the side wall of the shell of the cylindrical shell (9-1), and the waste cell discharge port (9-7) is vertically arranged on the bottom shell of the cylindrical shell (9-1).
2. The automatic cell purifying and freezing device according to claim 1, wherein a weighing sensor is arranged on the pre-mixing platform (3).
3. The device according to claim 1, wherein a temperature sensor is disposed in the freezing zone.
4. The device according to claim 1, wherein the injection device (10) is a micro-syringe pump.
5. The automatic cell purification and cryopreservation device according to claim 1, wherein a plurality of stop valves (7) are vertically arranged.
6. The automatic cell purification cryopreservation device of claim 1 wherein the bolus device (10) contains cryopreservation fluid.
7. The automatic cell purification and cryopreservation device of claim 1, wherein the waste cell discharge port (9-7) of the suspension separation device (9) is communicated with the waste liquid recovery bag through an infusion tube (13).
8. The automatic cell purification and cryopreservation device of claim 1, wherein the fiber filter membrane (9-4) in the suspension separation device (9) is formed by stacking multiple layers of cellulose nanofibers.
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| CN201810283055.6A CN108441407B (en) | 2018-04-02 | 2018-04-02 | Automatic cell purification and cryopreservation device |
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| CN201810283055.6A CN108441407B (en) | 2018-04-02 | 2018-04-02 | Automatic cell purification and cryopreservation device |
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| CN204841174U (en) * | 2015-08-12 | 2015-12-09 | 宁波市远发生物工程有限公司 | A filter equipment for amino acid production |
| CN205687938U (en) * | 2016-05-09 | 2016-11-16 | 北京银丰鼎诚生物工程技术有限公司 | A kind of separation device for adipose-derived stem cells |
| CN105925475B (en) * | 2016-06-16 | 2018-02-09 | 芜湖中科医凌生命科技有限公司 | A kind of full-automatic concentration instrument |
| US11259520B2 (en) * | 2016-08-04 | 2022-03-01 | Fanuc Corporation | Stem cell manufacturing system, stem cell information management system, cell transport apparatus, and stem cell frozen storage apparatus |
| CN206467237U (en) * | 2016-12-15 | 2017-09-05 | 刘劼 | Blood filter device for being enriched with specific cells |
| CN206504516U (en) * | 2017-02-27 | 2017-09-19 | 银丰低温医学科技有限公司 | High-precision cooling system |
| CN207130277U (en) * | 2017-08-23 | 2018-03-23 | 湖南开启时代生物科技有限责任公司 | A kind of cell cultivates packaging system automatically |
| CN210163457U (en) * | 2019-06-23 | 2020-03-20 | 南方医科大学口腔医院 | A centrifugal washing device for adipose tissue stem cell osteogenic differentiation |
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2018
- 2018-04-02 CN CN201810283055.6A patent/CN108441407B/en active Active
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