A PBMC isolation displacement method
By combining a single-inlet, two-outlet centrifuge cup technology with a tubular detector and a layered interface sensor, the problems of red blood cell residue and low recovery rate in existing PBMC separation methods have been solved, achieving efficient PBMC separation and washing replacement, which is suitable for processing large-volume samples.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SINO BIOCAN (SHANGHAI) BIOTECH LTD
- Filing Date
- 2023-07-05
- Publication Date
- 2026-07-03
AI Technical Summary
Existing PBMC separation methods are inefficient when processing large-volume samples and suffer from problems such as residual red blood cells and low recovery rates. In particular, the stratification state is unstable when using piston-type centrifuge cups, and residual red blood cells cannot be removed when using 1-in-1-out centrifuge cups.
Using a centrifuge cup with one inlet and two outlets, gradient liquid is pumped in and blood samples are separated through the combined use of the inner and outer ports. Combined with tubing detection and layer interface sensors, efficient separation and replacement of PBMCs are achieved, reducing red blood cell residue and improving recovery rate and purity.
It achieves efficient separation and washing/replacement of large-volume PBMCs, improves PBMC recovery rate and purity, reduces red blood cell residue, has high processing efficiency, and is suitable for processing large-volume samples.
Smart Images
Figure CN116855451B_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of cell separation technology, and in particular to a method for separating and replacing PBMCs. Background Technology
[0002] PBMCs are mononuclear cells found in peripheral blood, including lymphocytes and monocytes. The primary method for separating PBMCs is Ficoll-hypaque density gradient centrifugation. This method utilizes the differences in volume, morphology, and specific gravity between peripheral blood mononuclear cells and other peripheral blood cells. Red blood cells and polymorphonuclear leukocytes have a specific gravity of approximately 1.092, mononuclear cells 1.075-1.090, and platelets 1.030-1.035. By using a nearly isotonic solution (Ficoll gradient solution) between 1.075 and 1.092, density gradient centrifugation is performed, causing cells of a specific density to distribute according to the corresponding density gradient, thus separating various blood cells from mononuclear cells. Operators typically use manual or automated equipment, based on the principle of density gradient distribution, to add blood (peripheral blood, cord blood, or apheresis blood) to the separation solution for centrifugation.
[0003] Currently, automated cell separation equipment for preparing PBMCs mainly relies on piston-type centrifuge cups or single-in-single-out centrifuge cups. However, certain problems often arise during PBMC separation. When using piston-type centrifuge cups, large sample volumes cannot be handled. Furthermore, the process of pushing out plasma before collecting PBMCs causes changes in stratification both axially and radially during the upward pushing process, affecting the aliquoting and reducing the recovery rate. Conversely, with single-in-single-out centrifuge cups, after draining red blood cells and plasma, the PBMCs to be collected remain in the cup, resulting in red blood cell residue. This residue, due to its high density, cannot be completely removed even by subsequent washing and replacement, leading to low recovery efficiency. Therefore, this application proposes a PBMC separation and replacement method to solve the aforementioned technical problems. Summary of the Invention
[0004] The main objective of this application is to provide a PBMC separation and replacement method, which aims to solve the technical problem that existing PBMC separation methods have poor PBMC collection performance.
[0005] To achieve the above objectives, this application proposes a PBMC separation and replacement method, including a separation stage and a replacement stage. Both the separation stage and the replacement stage employ a centrifuge cup with one inlet and two outlets, and the centrifuge cup is provided with an inner port, an outer port, and a central port.
[0006] The separation phase includes the following steps:
[0007] S11. The gradient liquid is pumped into the centrifuge cup from the outer port, and then the blood sample is pumped into the centrifuge cup from the outer port. The centrifuge cup rotates and discharges the inner plasma from the center port.
[0008] S12. Perform centrifugal separation while continuously pumping gradient liquid into the external port;
[0009] S13. After centrifugation, red blood cells are discharged from the inner opening and then from the outer opening.
[0010] S14. Collect the product liquid and gradient liquid and discharge them from the inner port into the product bag to obtain the liquid to be replaced, and then discharge the waste liquid from the outer port.
[0011] S15. Pump the liquid to be replaced into the centrifuge cup through the inner port to start the replacement stage. After the replacement is completed, discharge the washing waste liquid to obtain the PBMC product liquid.
[0012] In one embodiment, the steps of pumping the gradient liquid into the centrifuge cup from the outer port, then pumping a blood sample into the centrifuge cup from the outer port, rotating the centrifuge cup, and discharging the inner plasma layer from the central port include:
[0013] The gradient liquid is pumped into the centrifuge cup from the outer port, and the centrifuge cup rotates to form a gradient liquid column layer inside the centrifuge cup;
[0014] After the gradient liquid column layer is formed, the blood sample in the sample bag is pumped into the centrifuge cup from the outer opening. The centrifuge cup rotates continuously, causing the plasma to move towards the center layer of the centrifuge cup. After the centrifuge cup is filled, the inner plasma layer is discharged from the center opening.
[0015] In one embodiment, the step of discharging red blood cells from the inner opening after centrifugation and then discharging red blood cells from the outer opening includes:
[0016] First, red blood cells are discharged from the inner port into the waste liquid bag, and the status of liquid discharged from the centrifuge cup is detected by the pipeline detector. When the red blood cells inside the inner port layer are discharged and the gradient liquid is discharged, the discharge of waste liquid from the inner port ends.
[0017] Then, some red blood cells are discharged from the external opening into the waste liquid bag.
[0018] In one embodiment, after step S13 and before step S14, the method further includes:
[0019] The residual red blood cells in the common pipeline between the sample bag, the waste liquid bag and the product bag are washed into the waste liquid bag using a gradient liquid.
[0020] The remaining red blood cells in the common pipeline connected to the centrifuge cup are then washed into the waste bag using a gradient liquid.
[0021] In one embodiment, the step of collecting the product liquid and gradient liquid and discharging them from the inner port into the product bag to obtain the liquid to be replaced, and then discharging the waste liquid from the outer port, includes:
[0022] The stratification state is determined by detecting the stratification interface between the gradient liquid and PBMC using a pipeline detector or a stratification interface sensor. Based on the stratification state, the amount of gradient liquid to be discharged into the waste bag is determined. Then, the remaining gradient liquid in the centrifuge cup and the product liquid are discharged together from the inner port into the product bag to obtain the liquid to be replaced.
[0023] After the replacement fluid is collected, all the liquid in the centrifuge cup is discharged from the outer port into the waste liquid bag.
[0024] In one embodiment, after step S14 and before step S15, the method further includes:
[0025] S141. Add washing solution to the centrifuge cup to clean the centrifuge cup, and then drain the liquid in the centrifuge cup into the waste liquid bag after cleaning; repeat step S141.
[0026] In one embodiment, the step of pumping the liquid to be replaced into the centrifuge cup from the inner port includes:
[0027] Step S151: Pump the liquid to be replaced into the centrifuge cup after the treatment in step S141 through the inner port; add washing liquid into the product bag to clean the product bag and discharge the liquid after cleaning the product bag into the centrifuge cup; repeat step S151.
[0028] In one embodiment, the replacement phase includes the following steps:
[0029] S21. The first washing liquid is continuously pumped into the centrifuge cup from the outer port and the centrifuge cup is filled. Then the first washing liquid is discharged from the center port to continuously wash and replace the liquid to be replaced.
[0030] S22. After continuous washing and replacement, the centrifuge cup is mixed and then separated by centrifugation to obtain cell fluid concentrated in the outer layer of the centrifuge cup and washing waste liquid concentrated in the inner layer of the centrifuge cup.
[0031] S23. After centrifugation, the washing waste liquid is discharged from the inner port, and the cell fluid concentrated in the outer layer of the centrifuge cup is retained in the centrifuge cup to obtain PBMC product liquid.
[0032] In one embodiment, after the step of discharging the washing waste liquid from the inner port and retaining the cell fluid concentrated on the outer layer of the centrifuge cup, the method further includes:
[0033] The second washing solution is continuously pumped into the centrifuge cup from the outer port and the centrifuge cup is filled. Then the second washing solution is discharged from the center port for continuous washing and replacement.
[0034] Repeat steps S22-S23.
[0035] In one embodiment, after step S23, the method further includes:
[0036] Shake off any remaining cells from the centrifuge cup wall and mix with the PBMC product solution.
[0037] This application achieves the separation and replacement of PBMCs using a centrifuge cup with one inlet and two outlets, such as... Figure 4 As shown, in the separation stage, the gradient solution is first pumped into the centrifuge cup from the outer port, followed by the blood sample. The centrifuge cup rotates continuously, causing red blood cells to accumulate on the outermost layer, while PBMCs and plasma move towards the inner layer. Once the centrifuge cup is full, the innermost plasma is discharged from the center port. Centrifugation then proceeds. To ensure thorough separation of PBMCs from the red blood cell layer, the gradient solution is continuously pumped in at a low speed from the outer port during centrifugation. This carries PBMCs out of the red blood cell layer, improving PBMC recovery. After centrifugation, the red blood cell waste liquid is first discharged from the inner port. Once all red blood cells within the inner port layer are discharged and reach the gradient solution, the liquid is then transferred to the outer port for further discharge. This keeps the red blood cell layer away from the inner port, facilitating subsequent discharge from the inner port. When collecting PBMC product solution from the inner port, contamination with red blood cells can be avoided. Then, begin collecting the product solution and gradient solution. For some blood samples, the separation is good, with little PBMC mixed in the gradient solution layer. More gradient solution can be discharged into the waste bag, and the remaining gradient solution and PBMC can be collected into the product bag. Collecting less gradient solution in this case reduces the number of subsequent washing and replacement cycles. However, for some blood samples, there is no clear stratification interface between PBMC and gradient solution after separation; that is, PBMC is mixed in the gradient solution layer. In this case, only a small amount of gradient solution needs to be discharged into the waste bag, and most of the gradient solution and PBMC should be collected together into the product bag. This achieves the goal of collecting as much PBMC product solution as possible, with the stratification state as shown in the image. Figure 12As shown (left: PBMCs are in good stratification, between the gradient solution and plasma; right: PBMCs are mixed in the gradient solution when stratification is poor), after product collection, all waste liquid in the centrifuge cup is discharged from the outside. Then, the replacement solution is pumped into the centrifuge cup from the inside for washing and replacement, removing as much waste liquid as possible, increasing the dilution ratio, and improving cell purity. This application can be used for the separation and processing of large-volume PBMCs, with continuous inflow and outflow, and can process sample volumes exceeding the volume of one centrifuge cup. It achieves PBMC separation and washing / replacement in the same cup, resulting in high processing efficiency. Furthermore, it locates the stratification position of PBMCs, enabling targeted collection and improving PBMC recovery rate. The PBMC purity is high, and the residual red blood cells are greatly reduced, avoiding the influence of red blood cells on PBMC collection. Attached Figure Description
[0038] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.
[0039] Figure 1 This is a schematic diagram of the center port of the centrifuge cup in the PBMC separation and replacement method described in the embodiments of this application;
[0040] Figure 2 This is a schematic diagram of the inner opening of the centrifuge cup in the PBMC separation and replacement method described in the embodiments of this application;
[0041] Figure 3 This is a schematic diagram of the outer opening of the centrifuge cup in the PBMC separation and replacement method described in the embodiments of this application;
[0042] Figure 4 This is a flowchart illustrating the separation stage of the PBMC separation and replacement method described in the embodiments of this application;
[0043] Figure 5 This is a flowchart illustrating the replacement stage of the PBMC separation and replacement method described in the embodiments of this application;
[0044] Figure 6 This is a schematic diagram of the liquid flow path for the gradient liquid in the separation stage of the PBMC separation and replacement method described in this application embodiment;
[0045] Figure 7 This is a schematic diagram of the liquid flow diagram for adding blood samples during the separation stage of the PBMC separation and replacement method described in this application embodiment;
[0046] Figure 8This is a schematic diagram of the liquid flow path for the waste liquid discharged from the external outlet during the separation stage of the PBMC separation and replacement method described in this application embodiment;
[0047] Figure 9 This is a schematic diagram of the liquid flow path for the waste liquid discharged from the inner port during the separation stage of the PBMC separation and replacement method described in this application embodiment;
[0048] Figure 10 This is a schematic diagram of the liquid flow path for the product liquid discharged from the inner port during the separation stage of the PBMC separation and replacement method described in this application embodiment;
[0049] Figure 11 This is a schematic diagram of the continuous flow washing and displacement fluid path of the PBMC separation and displacement method described in the embodiments of this application;
[0050] Figure 12 This is a schematic diagram of the layered state during the separation stage of the PBMC separation and replacement method described in this application embodiment.
[0051] The realization of the purpose, functional features and advantages of this application will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation
[0052] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.
[0053] Existing PBMC separation and replacement methods are based on piston-type centrifuge cups or one-in-one-out centrifuge cups. When separating PBMCs using piston-type centrifuge cups, it is impossible to handle large sample volumes. Furthermore, the process of pushing out plasma and then collecting PBMCs causes changes in the stratification state in both the axial and radial directions during the upward pushing process, which affects the dispensing state and reduces the recovery rate. When separating PBMCs using one-in-one-out centrifuge cups, after the red blood cells and plasma are drained, the PBMCs to be collected are left in the cup, resulting in the problem of red blood cell residue. Moreover, the high density of the residual red blood cells means that they cannot be removed even by subsequent washing and replacement, resulting in low recovery efficiency.
[0054] To address the technical problems existing in the current PBMC separation and replacement methods, embodiments of this application provide a PBMC separation and replacement method, including a separation stage and a replacement stage. Both the separation stage and the replacement stage employ a centrifuge cup with one inlet and two outlets, and the centrifuge cup has an inner outlet, an outer outlet, and a central outlet.
[0055] The separation phase includes the following steps:
[0056] S11. The gradient liquid is pumped into the centrifuge cup from the outer port, and then the blood sample is pumped into the centrifuge cup from the outer port. The centrifuge cup rotates and discharges the inner plasma from the center port.
[0057] S12. Perform centrifugal separation while continuously pumping gradient liquid into the external port;
[0058] S13. After centrifugation, red blood cells are discharged from the inner opening and then from the outer opening.
[0059] S14. Collect the product liquid and gradient liquid and discharge them from the inner port into the product bag to obtain the liquid to be replaced, and then discharge the waste liquid from the outer port.
[0060] S15. Pump the liquid to be replaced into the centrifuge cup through the inner port to start the replacement stage. After the replacement is completed, discharge the washing waste liquid to obtain the PBMC product liquid.
[0061] This application achieves the separation and replacement of PBMCs using a centrifuge cup with one inlet and two outlets, such as... Figure 4 As shown, in the separation stage, the gradient solution is first pumped into the centrifuge cup from the outer port, followed by the blood sample. The centrifuge cup rotates continuously, causing red blood cells to accumulate on the outermost layer, while PBMCs and plasma move towards the inner layer. Once the centrifuge cup is full, the plasma from the innermost layer is discharged from the center port. Centrifugation is then performed. To ensure thorough separation of PBMCs from the red blood cell layer, the gradient solution is continuously pumped in at a low speed from the outer port during centrifugation. This carries PBMCs out of the red blood cell layer, improving the PBMC recovery rate. After centrifugation, the red blood cell waste liquid is first discharged from the inner port. Once all red blood cells within the inner port layer are discharged, the solution is transferred to the outer port to discharge the remaining red blood cells. This ensures that the red blood cell layer... Keep the product solution away from the inner opening to avoid contamination by red blood cells when collecting PBMC product solution from the inner opening later. Then begin collecting the product solution and gradient solution. For some blood samples, the separation is good, and there is little PBMC mixed in the gradient solution layer. In this case, a small amount of gradient solution and PBMC can be collected together into the product bag. Collecting less gradient solution in this case reduces the number of subsequent washing and replacement cycles. However, for some blood samples, there is no clear stratification interface between PBMC and gradient solution after separation, meaning PBMC is mixed in the gradient solution layer. In this case, most of the gradient solution and PBMC need to be collected together into the product bag to collect as much PBMC product solution as possible. The stratification state is as follows: Figure 12As shown, after collecting the product, all waste liquid in the centrifuge cup is discharged from the outer port; then, the replacement solution is pumped into the centrifuge cup from the inner port for washing and replacement, removing as much waste liquid as possible, increasing the dilution ratio, and improving cell purity. This application can be used for the separation and processing of large-volume PBMCs, with continuous inflow and outflow, processing sample volumes exceeding the volume of one centrifuge cup. It achieves PBMC separation and washing / replacement within the same cup, resulting in high processing efficiency and significantly reducing the residue of red blood cells, avoiding the impact of red blood cells on PBMC collection, improving PBMC recovery rate, and achieving high PBMC purity.
[0062] In specific implementation methods, such as Figures 1-3 As shown, the central opening of the centrifuge cup is connected to the central layer of the centrifuge cup, which is the very center of the centrifuge cup. The outer opening of the centrifuge cup is connected to the outer layer of the centrifuge cup, which is the area inside the centrifuge cup near the cup wall. The inner opening of the centrifuge cup is connected to the inner layer of the centrifuge cup, which is the area between the central layer and the outer layer inside the centrifuge cup.
[0063] As one possible implementation of this application, the step of pumping the gradient liquid into the centrifuge cup from the outer port, then pumping a blood sample into the centrifuge cup from the outer port, and rotating the centrifuge cup to discharge the inner plasma layer from the central port includes:
[0064] The gradient liquid is pumped into the centrifuge cup from the outer port, and the centrifuge cup rotates to form a gradient liquid column layer inside the centrifuge cup;
[0065] After the gradient liquid column layer is formed, the blood sample in the sample bag is pumped into the centrifuge cup from the outer opening. The centrifuge cup rotates continuously, causing the plasma to move towards the center layer of the centrifuge cup. After the centrifuge cup is filled, the inner plasma layer is discharged from the center opening.
[0066] This application pumps a gradient liquid into the centrifuge cup from the external port, and the liquid flow is as follows: Figure 6 As shown, after the centrifuge cup rotates and forms a gradient liquid column layer, the blood sample is then pumped in. The liquid flow is as follows: Figure 7 As shown, under the rotation of the centrifuge cup, the red blood cells in the blood sample are concentrated on the wall of the centrifuge cup, while PBMCs and plasma move towards the central layer of the centrifuge cup. After the centrifuge cup is filled, the innermost plasma layer can be discharged through the central opening. For large sample volumes, the innermost plasma layer is removed first to facilitate the subsequent separation of PBMCs.
[0067] As one possible implementation of this application, the step of discharging red blood cells from the inner opening after centrifugation and then discharging red blood cells from the outer opening includes:
[0068] First, red blood cells are discharged from the inner port into the waste liquid bag, and the status of liquid discharged from the centrifuge cup is detected by the pipeline detector. When the red blood cells inside the inner port layer are discharged and the gradient liquid is discharged, the discharge of waste liquid from the inner port ends.
[0069] Then, some red blood cells are discharged from the external opening into the waste liquid bag.
[0070] To avoid the influence of red blood cells on the collection of PBMCs, this application first discharges the red blood cell waste liquid from the inner port after centrifugation. The liquid flow process is as follows: Figure 9 As shown, the status of the liquid discharged from the centrifuge cup is detected by a pipeline detector. After the red blood cells inside the inner layer are discharged, when they reach the gradient liquid, they are transferred to the outer outlet to discharge some of the red blood cell waste liquid. The liquid flow is as follows. Figure 8 As shown, this design allows the red blood cell layer to be kept away from the inner inlet, preventing the contamination of red blood cells during subsequent collection of PBMC product solution from the inner inlet. The amount of red blood cells discharged depends on the cell stratification state; a good stratification state allows for the discharge of more red blood cells, while a poor stratification state results in the discharge of fewer red blood cells. Specifically, this application can also use a stratification interface sensor to identify the state of the liquid discharged from the centrifuge cup.
[0071] As one possible implementation of this application, after step S13 and before step S14, the method further includes:
[0072] The residual red blood cells in the common pipeline between the sample bag, the waste liquid bag and the product bag are washed into the waste liquid bag using a gradient liquid.
[0073] The remaining red blood cells in the common pipeline connected to the centrifuge cup are then washed into the waste bag using a gradient liquid.
[0074] To prevent residual red blood cells in the tubing from entering the product bag, this application uses a gradient liquid to clean residual red blood cells in the common tubing between the sample bag, waste bag, and product bag, as well as the common tubing connected to the centrifuge cup, thereby reducing the amount of red blood cells mixed in.
[0075] As one possible implementation of this application, the step of collecting the product liquid and gradient liquid and discharging them from the inner port into the product bag to obtain the liquid to be replaced, and then discharging the waste liquid from the outer port, includes:
[0076] The stratification state is determined by detecting the stratification interface between the gradient liquid and PBMC using a pipeline detector or a stratification interface sensor. Based on this stratification state, the amount of gradient liquid to be discharged into the waste bag is determined. Then, the remaining gradient liquid in the centrifuge cup, along with the product liquid, is discharged from the inner port into the product bag. The liquid flow path is as follows: Figure 10 As shown, the solution to be replaced is obtained;
[0077] After the replacement fluid is collected, all the liquid in the centrifuge cup is discharged from the outer port into the waste liquid bag.
[0078] To improve the recovery rate of PBMCs, this application uses a pipeline detector or a layer interface sensor to identify the layer interface between the gradient liquid and PBMCs to determine the layering state. If the separation is good, less PBMCs are mixed in the gradient liquid layer, allowing more gradient liquid to be discharged into the waste bag. The remaining gradient liquid and PBMCs are then collected into the product bag. In this case, collecting less gradient liquid reduces the number of subsequent washing and replacement cycles. However, for some blood samples, there is no obvious layer interface between PBMCs and the gradient liquid after separation, meaning that PBMCs are mixed in the gradient liquid layer. In this case, only a small amount of gradient liquid needs to be discharged into the waste bag, while most of the gradient liquid and PBMCs are collected together into the product bag. This achieves the goal of collecting as much PBMC product liquid as possible. Finally, all waste liquid in the centrifuge cup is discharged from the external port, improving the purity of the PBMC product.
[0079] As one possible implementation of this application, after step S14 and before step S15, the method further includes:
[0080] S141. Add washing solution to the centrifuge cup to clean the centrifuge cup, and then drain the liquid in the centrifuge cup into the waste liquid bag after cleaning; repeat step S141.
[0081] This step prepares for subsequent washing and replacement. First, the residual red blood cells in the centrifuge cup are removed to prevent them from mixing into the product solution and affecting the subsequent incubation and transduction efficiency. Therefore, washing solution is added to the centrifuge cup, mixed well, and then the washing liquid is discharged into the waste bag. This process is repeated several times to achieve the purpose of removing the red blood cells completely.
[0082] As one possible implementation of this application, the step of pumping the liquid to be replaced into the centrifuge cup from the inner port includes:
[0083] Step S151: Pump the liquid to be replaced into the centrifuge cup after the treatment in step S141 through the inner port; add washing liquid into the product bag to clean the product bag and discharge the liquid after cleaning the product bag into the centrifuge cup; repeat step S151.
[0084] After cleaning the centrifuge cup, drain the liquid to be replaced from the product bag into the centrifuge cup to prepare for subsequent washing and replacement. To reduce the loss of PBMC in the product bag, add washing liquid to the product bag after emptying it to clean it, and then drain it into the centrifuge cup. Repeat this process multiple times to clean the product bag thoroughly and improve the recovery rate of PBMC.
[0085] As one possible implementation of this application, the replacement stage includes the following steps:
[0086] S21. The first washing liquid is continuously pumped into the centrifuge cup from the outer port and the centrifuge cup is filled. Then the first washing liquid is discharged from the center port to continuously wash and replace the liquid to be replaced.
[0087] S22. After continuous washing and replacement, the centrifuge cup is mixed and then separated by centrifugation to obtain cell fluid concentrated in the outer layer of the centrifuge cup and washing waste liquid concentrated in the inner layer of the centrifuge cup.
[0088] S23. After centrifugation, the washing waste liquid is discharged from the inner port, and the cell fluid concentrated in the outer layer of the centrifuge cup is retained in the centrifuge cup to obtain PBMC product liquid.
[0089] like Figure 5 As shown, in the replacement stage of this application, the liquid to be replaced is pumped into the centrifuge cup from the inner port, and then the first washing liquid is continuously pumped into the centrifuge cup from the outer port until the centrifuge cup is full. After that, the first washing liquid is discharged from the center port, and continuous washing and replacement are performed. The liquid flow is as follows: Figure 11 As shown, the replacement solution is located in the inner layer of the centrifuge cup. The first washing solution enters the centrifuge cup from the outer port, allowing it to penetrate the cell layer from the outer layer to the center layer. After the first washing solution fills the centrifuge cup, it is discharged from the center port, achieving a continuous flow washing and replacement process. The first washing solution is then thoroughly mixed with the cell solution to achieve uniform dilution of the residual culture medium. The mixture of the first washing solution and cell solution is then centrifuged, concentrating the cell solution in the outer layer of the centrifuge cup and the washing waste solution in the inner layer. The separated washing waste solution is then discharged from the inner port, leaving the cell solution concentrated in the outer layer of the centrifuge cup. This process further reduces volume and removes waste solution on top of continuous flow washing. By removing as much waste solution as possible, cell purity is improved, dilution ratio is increased, replacement time is shortened, and replacement efficiency is improved. This avoids the problem of conventional replacement stages where washing solution is discharged directly without penetrating the cell layer, failing to achieve both volume reduction and cell washing and replacement. It also reduces the damage to PBMC products caused by prolonged washing and replacement.
[0090] As one possible implementation of this application, after the step of discharging the washing waste liquid from the inner port and retaining the cell fluid concentrated on the outer layer of the centrifuge cup, the method further includes:
[0091] The second washing solution is continuously pumped into the centrifuge cup from the outer port and the centrifuge cup is filled. Then the second washing solution is discharged from the center port for continuous washing and replacement.
[0092] Repeat steps S22-S23.
[0093] Preferably, the first washing solution is replaced with the second washing solution to repeatedly wash the cell fluid, thereby further increasing the dilution ratio of the waste liquid so as to discharge as much waste liquid as possible and improve the purity of the cells.
[0094] As one possible implementation of this application, after step S23, the method further includes:
[0095] Shake off any remaining cells from the centrifuge cup wall and mix with the PBMC product solution.
[0096] Preferably, since the washing and replacement process and the volume reduction process are both carried out under centrifugal separation, the cells will adhere to the wall of the centrifuge cup under the action of centrifugal force. The centrifuge cup can be accelerated and stopped by centrifuging to shake off the adhered cells and mix them with the cell product liquid.
[0097] The above description is merely an optional embodiment of this application and does not limit the patent scope of this application. Any equivalent structural transformations made based on the inventive concept of this application and the contents of the specification and drawings of this application, or direct / indirect applications in other related technical fields, are included within the patent protection scope of this application.
Claims
1. A PBMC isolation displacement method, comprising an isolation stage and a displacement stage, characterized in that, Both the separation stage and the replacement stage use a centrifuge cup with one inlet and two outlets, and the centrifuge cup is provided with an inner port, an outer port and a center port; The separation phase includes the following steps: S11. The gradient liquid is pumped into the centrifuge cup from the outer port, and then the blood sample is pumped into the centrifuge cup from the outer port. The centrifuge cup rotates and discharges the inner plasma from the center port. S12. Perform centrifugal separation while continuously pumping gradient liquid into the external port; S13. After centrifugation, red blood cells are discharged from the inner opening and then from the outer opening. S14. Collect the product liquid and gradient liquid and discharge them from the inner port into the product bag to obtain the liquid to be replaced, and then discharge the waste liquid from the outer port. S15. Pump the liquid to be replaced into the centrifuge cup from the inner port to start the replacement phase; The replacement phase includes the following steps: S21. The first washing liquid is continuously pumped into the centrifuge cup from the outer port and the centrifuge cup is filled. Then the first washing liquid is discharged from the center port to continuously wash and replace the liquid to be replaced. S22. After continuous washing and replacement, the centrifuge cup is mixed and then separated by centrifugation to obtain cell fluid concentrated in the outer layer of the centrifuge cup and washing waste liquid concentrated in the inner layer of the centrifuge cup. S23. After centrifugation, the washing waste liquid is discharged from the inner port, and the cell fluid concentrated in the outer layer of the centrifuge cup is retained in the centrifuge cup to obtain PBMC product liquid.
2. The PBMC isolation displacement method of claim 1, wherein, The steps of pumping the gradient liquid into the centrifuge cup from the outer port, then pumping the blood sample into the centrifuge cup from the outer port, rotating the centrifuge cup, and discharging the inner plasma layer from the center port include: The gradient liquid is pumped into the centrifuge cup from the outer port, and the centrifuge cup rotates to form a gradient liquid column layer inside the centrifuge cup; After the gradient liquid column layer is formed, the blood sample in the sample bag is pumped into the centrifuge cup from the outer opening. The centrifuge cup rotates continuously, causing the plasma to move towards the center layer of the centrifuge cup. After the centrifuge cup is filled, the inner plasma layer is discharged from the center opening.
3. The PBMC separation and replacement method according to claim 2, characterized in that, The steps of centrifuging and then removing red blood cells from the inner orifice and then from the outer orifice include: First, red blood cells are discharged from the inner port into the waste liquid bag, and the status of liquid discharged from the centrifuge cup is detected by the pipeline detector. When the red blood cells inside the inner port layer are discharged and the gradient liquid is discharged, the discharge of waste liquid from the inner port ends. Then, some red blood cells are discharged from the external opening into the waste liquid bag.
4. The PBMC separation and replacement method according to claim 3, characterized in that, After step S13 and before step S14, the method further includes: The residual red blood cells in the common pipeline between the sample bag, the waste liquid bag and the product bag are washed into the waste liquid bag using a gradient liquid. The remaining red blood cells in the common pipeline connected to the centrifuge cup are then washed into the waste bag using a gradient liquid.
5. The PBMC separation and replacement method according to claim 1, characterized in that, The steps of collecting the product liquid and gradient liquid and discharging them from the inner port into the product bag to obtain the liquid to be replaced, and then discharging the waste liquid from the outer port, include: The stratification state is determined by detecting the stratification interface between the gradient liquid and PBMC using a pipeline detector or a stratification interface sensor. Based on the stratification state, the amount of gradient liquid to be discharged into the waste bag is determined. Then, the remaining gradient liquid in the centrifuge cup and the product liquid are discharged together from the inner port into the product bag to obtain the liquid to be replaced. After the replacement fluid is collected, all the liquid in the centrifuge cup is discharged from the outer port into the waste liquid bag.
6. The PBMC separation and replacement method according to claim 1, characterized in that, After step S14 and before step S15, the method further includes: S141. Add washing solution to the centrifuge cup to clean the centrifuge cup, and then drain the liquid in the centrifuge cup into the waste liquid bag after cleaning; repeat step S141.
7. The PBMC separation and replacement method according to claim 6, characterized in that, The step of pumping the liquid to be replaced into the centrifuge cup from the inner port includes: Step S151: Pump the liquid to be replaced into the centrifuge cup after the treatment in step S141 through the inner port; add washing liquid into the product bag to clean the product bag and discharge the liquid after cleaning the product bag into the centrifuge cup; repeat step S151.
8. The PBMC separation and replacement method according to claim 1, characterized in that, After the steps of discharging the washing waste liquid from the inner port and retaining the cell fluid concentrated on the outer layer of the centrifuge cup, the method further includes: The second washing solution is continuously pumped into the centrifuge cup from the outer port and the centrifuge cup is filled. Then the second washing solution is discharged from the center port for continuous washing and replacement. Repeat steps S22-S23.
9. The PBMC separation and replacement method according to claim 1, characterized in that, After step S23, the method further includes: Shake off any remaining cells from the centrifuge cup wall and mix with the PBMC product solution.