CN108441407B - Automatic cell purification and cryopreservation device - Google Patents

Abstract

An automatic cell purification and cryopreservation device belongs to the field of biotechnology, and aims to solve the defects of frequent manual operation steps when separating samples, which easily leads to cell contamination, and cells are easily damaged to affect biological effects. The cell automatic purification and freezing device includes a purification freezing box, a liquid bag rack, a stop valve, a peristaltic pump, a suspension separation device and a bolus injection device. The left side of the purification freezing box is an electronically controlled purification area. The right side is the cryopreservation area. There are multiple peristaltic pumps and a suspension separation device on the central box wall of the electronically controlled purification area. The lower part of the electronically controlled purification area is provided with a bolus device, and cells are hung on the liquid bag rack. The storage bag, the infusion tube connected to the bottom of the cell storage bag is clamped in the peristaltic pump through the stop valve, and the freezing bag and the heat sealing device are placed on the pre-mixing platform in the box of the freezing area. The cell automatic purification and cryopreservation device of the present invention realizes multifunctional automation, improves operation accuracy and reduces the risk of biological contamination.

Description

A cell automatic purification cryopreservation device

technical field

The invention belongs to the field of biotechnology, and in particular relates to a control device for automatic cell purification and program cryopreservation, used for cell separation, extraction and program cryopreservation.

Background technique

With the development of biological therapy technology, cell purification technology has also been gradually optimized and developed, from the original traditional centrifuge through the control of the corresponding speed to achieve the purpose of cell stratified separation, to the current development of a complete set of sterile pipelines through the centrifugal cup at high speed Centrifugation separates the cellular components to the edge, thereby separating the desired components, but the separation speed is slow, the noise is high, the cell loss is high, the human intervention is more, and the preparation stability is poor.

Program-controlled cooling technology uses temperature control technology and liquid nitrogen dispersion technology to safely and reliably control the temperature change rate for program freezing of samples that need ultra-low temperature freezing. It is widely used in various fields such as agriculture, medicine, and basic biological research. It is suitable for cryopreservation of lymphocytes, tissue banks, stem cells, bone marrow cells, tumor cells, animal and plant cells, cardiomyocytes, etc. However, pre-cooling in advance, cell cryopreservation pre-treatment and transfer procedures are required in current use. It is easy to see that the detection probe is not completely immersed in the plasma, resulting in abnormal detection data, which in turn leads to unrealistic cryopreservation curves, affects cell viability, causes unnecessary waste of manpower and material resources, and increases production costs.

Therefore, the field also needs to carry out cell purification and cryopreservation in an integrated and intelligent manner, including selective enrichment of stem cells or other cells such as plasma, red blood cells, platelets, etc., while realizing automated separation and purification, maintaining sterility, cell viability and Cryopreservation device for regeneration and cell viability.

SUMMARY OF THE INVENTION

The purpose of the present invention is to purify stem cells or other cells from liquid biological products and freeze them by automatic procedure, and overcome the defects of frequent manual operation steps when separating samples at present, which easily lead to cell contamination, and cells are easily damaged to affect biological effects. An automatic cell purification and cryopreservation device that reduces open operation and avoids sample contamination, so as to improve preparation efficiency, reduce sample contamination and cell viability.

The cell automatic purification and freezing device of the present invention includes a purification freezing box, a liquid bag rack, a stop valve, a peristaltic pump, a suspension separation device and a bolus injection device. The left side of the purification freezing box is an electronically controlled purification area. The purification freezing box The right side is the cryopreservation area, the upper part of the electronically controlled purification area is the display screen, a plurality of peristaltic pumps and a suspension separation device are arranged on the central box wall of the electronically controlled purification area, and the lower part of the electronically controlled purification area is provided with a bolus injection In the device, a plurality of stop valves are arranged on the side box wall of the electronically controlled purification area, and a connecting pipe channel is opened between the electronically controlled purification area and the cryopreservation area;

The liquid bag rack is arranged on the top surface of the purification freezing box, and a cell storage bag is hung on the liquid bag rack. The bottom of the cell storage bag is connected with one end of an infusion tube, and the infusion tube is clamped to the peristaltic pump through a stop valve. Inside, the other end of the infusion tube is connected to the input port of the suspension separation device, the output port of the bottom side wall of the suspension separation device is communicated with the freezing bag through the first connecting tube through the connecting tube channel, and the outlet of the bolus device is connected to the freezing bag. The liquid port is communicated with the freezing bag through the second connecting pipe through the connecting pipe channel;

An opening and closing door is arranged on the front box wall of the freezing area, and a freezing bag and a heat sealing device are placed on the pre-mixing platform in the box of the freezing area. The first connecting pipe and the second connecting pipe pass through the heat sealing mold of the heat sealing device. , the purified cells in the freezing bag are cryopreserved by liquid nitrogen in the cryopreservation area.

The present invention relates to an automatic cell purification, separation, and cryopreservation device. The device mainly includes an automatic cell purification and separation part, a cell acquisition premixing part and a (program) cryopreservation part. The automatic cell purification and separation part is located in the automatic purification and cryopreservation part. On the left side of the device, the cell premixing platform is located in the program cryopreservation area, and the program control part of the program cryopreservation part is located on the upper left side of the automatic purification cryopreservation device. The injection device and each sensor are connected, the MCU chip on the circuit board outputs the signal, controls the micromotor to drive the peristaltic pump to move forward and reverse, accurately controls the movement of the peristaltic pump and the bolus device, and controls the directional flow of the infusion pipeline. , the automatic cell purification and separation part separates the cell liquid flow through the microporous suspension separation device, transports it to the pre-mixing part, and performs programmed freezing through the programmed freezing part.

The cell automatic purification and cryopreservation device of the present invention includes the following beneficial effects:

1. The automatic cell purification and separation part of the present invention, the liquid bag on the liquid bag rack, is controlled by the cut-off valve through the internal prefabricated program of the liquid crystal touch screen to control the on-off of the infusion pipeline, and then pressurized by the peristaltic pump and the bolus device. , respectively pumped into the microporous suspension separation device and the freezing bag on the premixing platform to realize multi-functional automation, which greatly saves physical and material resources, thereby increasing cell filtration efficiency, improving operation accuracy and reducing biological pollution. risk.

2. The cell acquisition and premixing part of the present invention is provided with a see-through switch door, the freezing bag connected with the automatic cell purification and separation part is placed on the premixing platform inside the cavity and shaken, and the liquid crystal touch display screen passes through the weighing sensor. The measuring and heat sealing pipelines are separated, and the MCU controls the liquid nitrogen controller to input liquid nitrogen into the cavity, and program freezing is performed through the parameter response of the ultra-low temperature sensor. The blood components can be purified and mixed together under the action of the micro-aperture suspension separation device through different sets of plastic multi-unit bags, and the loss of cells caused by misoperation can be avoided.

3. In the cryopreservation part of the program of the present invention, liquids such as cryopreservation liquid are infused into the freezing bag on the pre-mixing platform inside the cavity through the infusion pipeline, and the liquid nitrogen controller transfers the liquid nitrogen from the liquid nitrogen to the freezing bag. The output port is input into the cavity, and the program freezing is performed by the parameter response of the ultra-low temperature sensor. In a closed environment, the temperature of the lumen is controlled by an indirect program of liquid nitrogen, and the cells are rapidly cooled to avoid the influence of transfer and transfer of cells, and ensure the protection of operators and cells.

4. Compared with the prior art, the present invention has the advantages of saving manpower and material resources, accurate operation, and high-efficiency filtration for different cell characteristics.

Description of drawings

Fig. 1 is the structural schematic diagram of the cell automatic purification and cryopreservation device of the present invention;

Fig. 2 is the structural representation of cryopreservation area;

Fig. 3 is the structural representation of the suspension separation device;

FIG. 4 is a schematic top view of the suspension separation device.

Detailed ways

Embodiment 1: The cell automatic purification and cryopreservation device in this embodiment includes a purification cryobox 1, a liquid bag rack 5, a stop valve 7, a peristaltic pump 8, a suspension separation device 9 and a bolus injection device 10. The purification cryobox 1 The left side of the electronic control purification area is the electronically controlled purification area, the right side of the purification freezing box 1 is the freezing storage area, the upper part of the electronically controlled purification area is the display screen 6, and a plurality of peristaltic pumps 8 and A suspension separation device 9, a bolus injection device 10 is arranged in the lower part of the electronically controlled purification area, a plurality of shut-off valves 7 are arranged on the side tank wall of the electronically controlled purification area, and there is a connection between the electronically controlled purification area and the cryopreservation area. pipe channel 4;

The liquid bag rack 5 is arranged on the top surface of the purification freezing box 1, and a cell storage bag is hung on the liquid bag rack 5. The bottom of the cell storage bag is connected with one end of an infusion tube 13, and the infusion tube 13 passes through a stop valve. 7 is clamped in the peristaltic pump 8, the other end of the infusion pipe 13 is connected with the input port 9-5 of the suspension separation device 9, and the output port 9-6 of the bottom side wall of the suspension separation device 9 passes through the first connecting pipe. is communicated with the freezing bag through the connecting pipe channel 4, and the liquid outlet of the bolus injection device 10 is communicated with the freezing bag through the connecting pipe channel 4 through the second connecting pipe;

An opening and closing door 2 is arranged on the front box wall of the freezing area, and a freezing bag and a heat-sealing device 12 are placed on the pre-mixing platform 3 in the box of the freezing area. The first connecting pipe and the second connecting pipe pass through the heat-sealing device. 12 heat-sealed molds, and the purified cells in the cryopreservation bag were cryopreserved by liquid nitrogen in the cryopreservation area.

In this embodiment, a liquid nitrogen output port is provided in the cryopreservation area, and the liquid nitrogen flows into the cryopreservation area through the output port.

Embodiment 2: The difference between this embodiment and Embodiment 1 is that a load cell is provided on the pre-mixing platform 3 .

In this embodiment, the weight of the cell fluid in the cryopreservation bag is measured by a weighing sensor, and the heat-sealing device is controlled by a given parameter to measure and control the heat-sealing and infusion pipeline to separate the pipeline.

Embodiment 3: The difference between this embodiment and Embodiment 1 or 2 is that a temperature sensor is provided in the freezing storage area.

Embodiment 4: The difference between this embodiment and one of Embodiments 1 to 3 is that the bolus injection device 10 is a micro-injection pump.

Embodiment 5: The difference between this embodiment and one of Embodiments 1 to 4 is that a plurality of shut-off valves 7 are arranged vertically.

Embodiment 6: The difference between this embodiment and one of Embodiments 1 to 5 is that the bolus injection device 10 is filled with a freezing solution.

Embodiment 7: The difference between this embodiment and one of Embodiments 1 to 6 is that the suspension separation device 9 includes a cylindrical casing 9-1, a drive shaft 9-2, a cross impeller 9-3, a fiber filter membrane 9-4, The input port 9-5, the output port 9-6 and the waste cell discharge port 9-7, the fiber filter membrane 9-4 is sleeved in the cylindrical shell 9-1 in a cylindrical shape, and the inner wall of the cylindrical shell 9-1 is connected to the fiber filter. A filter cavity 9-8 is formed between the membranes 9-4, a cross impeller 9-3 is arranged on the inner top of the cylindrical shell 9-1, and the drive shaft 9-2 is connected with the cross impeller 9-3 and is transmitted from the cylindrical shell 9-1. The top of the shell is passed out, the input port 9-5 is arranged on the upper part of the side wall of the cylindrical shell 9-1 and communicates with the inner cavity enclosed by the fiber filter membrane 9-4, and the output port 9-6 is arranged on the cylindrical shell 9-1. In the lower part of the side wall of the shell of -1, the waste cell discharge port 9-7 is vertically arranged on the bottom shell of the cylindrical shell 9-1.

In this embodiment, the drive shaft is driven to rotate by a motor, and the cell fluid enters the cavity enclosed by the fiber filter membrane from the input port, and the vortex rotation makes the cells pass through the fiber filter membrane, enter the cavity formed by the fiber filter membrane and the transparent shell, and The concentrated cells are infused into the freezing bag through the output port. The output port is along the radial direction of the cylindrical shell. The waste cell discharge port is communicated with the inner cavity of the fiber filter membrane. The waste cells cannot pass through the fiber filter membrane and are discarded under the action of rotational force. The cell discharge port is recovered into the waste bag.

Embodiment 8: The difference between this embodiment and Embodiment 7 is that the waste cell discharge port 9-7 of the suspension separation device 9 is communicated with the waste liquid recovery bag through the infusion tube 13.

Embodiment 9: The difference between this embodiment and Embodiment 7 is that the fiber filter membrane 9-4 in the suspension separation device 9 is formed by stacking multiple layers of cellulose nanofibers.

The pore size of the fiber filtration membrane in this embodiment can be made to be between 2 and 10 microns, so as to meet the needs of filtration of different cells such as red blood cells, white blood cells, and granulocytes.

Example: The cell automatic purification and cryopreservation device in this example includes a purification cryobox 1, a liquid bag rack 5, a stop valve 7, a peristaltic pump 8, a suspension separation device 9 and a bolus injection device 10. The side is the electronically controlled purification area, the right side of the purification freezing box 1 is the freezing storage area, the upper part of the electronically controlled purification area is the liquid crystal touch screen 6, and a plurality of peristaltic pumps 8 are arranged on the central box wall of the electronically controlled purification area. And a suspension separation device 9, a bolus device 10 is provided in the lower part of the electronically controlled purification area, a plurality of stop valves 7 are provided on the side box wall of the electronically controlled purification area, and there are openings between the electronically controlled purification area and the cryopreservation area. connecting pipe channel 4;

The liquid bag rack 5 is arranged on the top surface of the purification freezing box 1, and a cell storage bag is hung on the liquid bag rack 5. The bottom of the cell storage bag is connected with one end of an infusion tube 13, and the infusion tube 13 passes through a stop valve. 7 is clamped in the peristaltic pump 8, the other end of the infusion pipe 13 is connected with the input port 9-5 of the suspension separation device 9, and the output port 9-6 of the bottom side wall of the suspension separation device 9 passes through the first connecting pipe. It is communicated with the freezing bag through the connecting tube channel 4, and the liquid outlet of the bolus injection device 10 is communicated with the freezing bag through the second connecting tube through the connecting tube channel 4, and the connecting tube channel 4 is filled with polyester fiber cotton. To isolate liquid nitrogen and keep warm;

An opening and closing door 2 is arranged on the front box wall of the freezing area, and a freezing bag and a heat sealing device 12 are placed on a pre-mixing platform 3 in the box of the freezing area. The pre-mixing platform 3 is a shaking or shaking platform , the first connecting pipe and the second connecting pipe pass through the heat-sealing mold of the heat-sealing device 12, and the liquid nitrogen enters the freezing area through the liquid nitrogen port 11 to freeze the purified cells in the freezing bag.

In the present embodiment, the automatic purification process is as follows:

1. A physiological saline bag (right side) is hung on the liquid bag holder 5. The bottom of the physiological saline bag is connected with an infusion tube. The infusion tube is clamped in the peristaltic pump through the stop valve. The other end of the infusion tube is connected to the peristaltic pump. The input port 9-5 of the suspension separation device 9 is connected, and the output port 9-6 of the bottom side wall of the suspension separation device 9 is communicated with the waste liquid recovery bag through the third connecting pipe through the peristaltic pump and the stop valve, and the cleaning solution is mixed with each other. The suspension separation device 9 is cleaned and filled.

2. A cell storage bag is hung on the liquid bag holder 5. The bottom of the cell storage bag is connected with one end of the infusion tube 13. The infusion tube 13 is clamped in the peristaltic pump 8 through the stop valve 7. The other end is connected with the input port 9-5 of the suspension separation device 9, and the output port 9-6 of the bottom side wall of the suspension separation device 9 is communicated with the freezing bag through the first connecting pipe through the connecting pipe channel 4, and the bolus injection is carried out. The liquid outlet of the device 10 is communicated with the freezing bag through the second connecting pipe through the connecting pipe channel 4;

3. According to the second step, the output port 9-6 of the bottom side wall of the suspension separation device 9 is connected to the freezing bag through the first connecting pipe through the connecting pipe channel 4, and the output port 9-6 of the bottom side wall is connected in parallel All the way, the incompletely purified cells are returned to the cell storage bag through the fourth connecting pipe through the peristaltic pump and the stop valve, and then enter the suspension separation device 9 for purification and separation.

In the present embodiment, the automatic freezing process is as follows:

1. At the beginning of the automatic purification process, the MCU controls the liquid nitrogen controller to input the liquid nitrogen into the cavity, and performs programmed freezing through the parameter response of the ultra-low temperature sensor. Before weighting, keep 4°C in the freezing chamber;

2. During the purification process, when the weight of the cell fluid in the cryopreservation bag measured by the weighing sensor matches the cryopreservation weight, the automatic purification process is stopped and the pipeline is flushed. At this time, place the freezing bag on the premixing platform inside the chamber and keep it at 4°C for 10 minutes, then the MCU controls the liquid nitrogen controller to input the liquid nitrogen into the chamber and keep it at >-20°C for 25 minutes. The nitrogen controller inputs liquid nitrogen into the cavity at a speed of -1 to -3°C/min from room temperature to below -80°C and maintains it.

Claims (8)

1. A cell automatic purification cryopreservation device is characterized in that the cell automatic purification cryopreservation device comprises a purification cryobox (1), a liquid bag rack (5), a stop valve (7), a peristaltic pump (8), a mixing The suspension separation device (9) and the bolus injection device (10), the left side of the purification freezing box (1) is the electronically controlled purification area, the right side of the purification freezing box (1) is the freezing storage area, and the The upper part is a display screen (6), a plurality of peristaltic pumps (8) and a suspension separation device (9) are arranged on the middle box wall of the electronically controlled purification zone, and a bolus injection device (10) is arranged at the lower part of the electronically controlled purification zone. , a plurality of shut-off valves (7) are arranged on the side box wall of the electronically controlled purification area, and a connecting pipe channel (4) is opened between the electronically controlled purification area and the cryopreservation area; The liquid bag rack (5) is arranged on the top surface of the purification freezing box (1), a cell storage bag is hung on the liquid bag rack (5), and the bottom of the cell storage bag is connected with one end of the infusion tube (13) , the infusion tube (13) is clamped in the peristaltic pump (8) through the stop valve (7), and the other end of the infusion tube (13) is connected with the input port (9-5) of the suspension separation device (9), The output port (9-6) of the bottom side wall of the suspension separation device (9) is communicated with the freezing bag through the first connecting pipe through the connecting pipe channel (4), and the liquid outlet of the bolus injection device (10) is connected to the freezing bag through the first connecting pipe. Two connecting pipes are communicated with the freezing bag through the connecting pipe channel (4); An opening and closing 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 the pre-mixing platform (3) in the box of the freezing area, the first connecting pipe and the second The connecting tube passes through the heat-sealing mold of the heat-sealing device (12), and the purified cells in the freezing bag are cryopreserved by liquid nitrogen in the cryopreservation area; The suspension separation device (9) comprises a cylindrical shell (9-1), a drive shaft (9-2), a cross impeller (9-3), a fiber filter membrane (9-4), an input port (9-5), The output port (9-6) and the waste cell discharge port (9-7), the fiber filter membrane (9-4) is sleeved in the cylindrical shell (9-1) in a cylindrical shape, and the A filter cavity (9-8) is formed between the inner wall and the fiber filter membrane (9-4), a cross impeller (9-3) is arranged on the inner top of the cylindrical shell (9-1), and the drive shaft (9-2) is connected to the inner top of the cylindrical shell (9-1). The cross impeller (9-3) is connected and protrudes from the top of the cylindrical shell (9-1), and the input port (9-5) is arranged on the upper part of the shell side wall of the cylindrical shell (9-1) and is connected with the fiber filter. The inner cavity enclosed by the membrane (9-4) communicates with each other, 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 at the side wall of the cylindrical shell (9-1). on the bottom case of the cylindrical housing (9-1). 2. A cell automatic purification and cryopreservation device according to claim 1, characterized in that a load cell is provided on the pre-mixing platform (3). 3. A cell automatic purification and cryopreservation device according to claim 1, characterized in that a temperature sensor is arranged in the cryopreservation area. 4. A cell automatic purification and cryopreservation device according to claim 1, characterized in that the bolus device (10) is a micro-injection pump. 5. A cell automatic purification and cryopreservation device according to claim 1, characterized in that a plurality of shut-off valves (7) are arranged vertically. 6. A cell automatic purification and cryopreservation device according to claim 1, characterized in that the bolus injection device (10) is equipped with a cryopreservation solution. 7. A cell automatic purification and cryopreservation device according to claim 1, characterized in that the waste cell discharge port (9-7) of the suspension separation device (9) passes through the infusion tube (13) and the waste liquid recovery bag connected. 8. A cell automatic purification and cryopreservation device according to claim 1, characterized in that 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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