CN114561352A - Method for separating mononuclear cells from blood donation complete blood collection device - Google Patents
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Abstract
The invention discloses a method for separating peripheral blood mononuclear cells from a blood collection device for blood donation without compensation, which comprises the steps of clamping pipe walls at two sides by using pipe clamps under the aseptic condition, cutting pipe orifices at the upper end and the lower end of the pipe walls at the two sides by using aseptic scissors, and reserving a long pipe on the pipe walls at the two sides; the syringe is used for washing the forward washing and reverse washing white filter with washing liquid, liquid obtained by reverse washing is collected for centrifugation, supernatant is absorbed and discarded after centrifugation, and the supernatant is added into the other two centrifuge tubes filled with equal amount of Ficoll after the cells are resuspended by normal saline for continuous centrifugation; and (4) after the centrifugation is finished, absorbing the white membrane layer above the Ficoll into a new centrifuge tube, supplementing normal saline, absorbing and discarding the supernatant after the centrifugation, and manually counting by adopting a blood counting chamber after the normal saline is re-suspended. The method can aseptically extract mononuclear cells from a leukocyte filter of an uncompensated blood donation blood collection channel, culture the mononuclear cells in vitro, and take the cells cultured in vitro as DC-CIK treatment cells.
Description
Technical Field
The invention relates to a method for separating peripheral blood mononuclear cells from a blood collection device for blood donation without compensation, in particular to a laboratory sterile operation technology for obtaining a large number of human peripheral blood mononuclear cells in a large scale, which is used for subsequent cell therapy and other purposes, and belongs to the technical field of cell therapy products.
Background
In the last decade, cell therapy is rapidly developed as the leading edge of medical development, the most important development direction of cell therapy is that DC-CIK (dendritic cell-cytokine induced killer cell) is returned to the treatment of cancer cells in the body, activated immune cells are utilized to eliminate the cancer autologous cells, Dendritic Cells (DC) with strongest functions in vivo and antigen presenting cells from peripheral blood are co-cultured with the CIK cells to obtain CLK cells stimulated by the DC cells, the CIK cells are re-infused for many times to kill residual tumor cells in the body of a patient to inhibit the recurrence and metastasis of tumors, and the re-infused CIK cells can also improve the immune function of the body. This method employs a passive immunotherapy protocol, but has specificity for tumor cell killing, monoclonal antibodies alone or in combination with other agents, activated T lymphocytes specifically kill tumor cells, and the like. The adoptive cell immunotherapy is to utilize CIK cells, wherein CIK is cells which are induced in vitro by a plurality of cytokines and CD3-Ab and greatly expanded and have the activity of killing tumors, is CD3+ CD56+ heterogeneous cells with NK and T cell types, is a main immune cell therapy means at present, and has the main characteristics that: firstly, tumor cells are killed and killed selectively, and normal cells are not damaged; secondly, the killing effect is good, and the efficiency is more than 50 times of NK and LAK; and thirdly, the broad-spectrum anti-tumor can also kill drug-resistant tumor cells efficiently.
The DC-CIK therapeutic cells with enough quantity can be acquired from tumor patients, generally the acquisition quantity is larger and is more than 100ml, some tumor patients cannot tolerate the acquisition of peripheral blood in practical operation, the greater reason is that the activity of peripheral blood mononuclear cells of patients in the end stage of tumor is poor, and the peripheral blood mononuclear cells cannot be amplified to reach the treatment magnitude in vitro culture.
The present clinical blood transfusion is divided into component and whole blood transfusion, wherein the whole blood transfusion is suitable for patients with a large amount of blood loss and blood replacement, a blood collection device for blood donation without compensation is composed of a collection bag, a white filter, a transfer bag I, a transfer bag II and an anticoagulant storage bag, the whole blood of the blood donation without compensation is firstly collected into the collection bag, then the whole blood with mononuclear cells filtered out is transferred into the transfer bag I through the white filter, most of blood plasma in the transfer bag I is transferred into the transfer bag II through a centrifugal and full-automatic device, and then the anticoagulant in the anticoagulant storage bag is added into the transfer bag I according to the proportion to form leukocyte-removed suspended red blood cells for clinical use, as shown in figure 1. The applicant recovered over forty donor filters and, through repeated experiments, established a complete set of methods for extracting mononuclear cells from the filters and successfully expanding them to the magnitude required for cell therapy.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for separating peripheral blood mononuclear cells from a uncompensated blood donation whole blood collection device aiming at the defects in the prior art, and provides a method for recovering and sterilizing a leukocyte filter and extracting the mononuclear cells, which can aseptically extract the mononuclear cells from the leukocyte filter of an uncompensated blood donation blood collection channel, culture the mononuclear cells in vitro and take the cells cultured in vitro as DC-CIK treatment cells.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method of separating peripheral blood mononuclear cells from a gratuitous blood donation whole blood collection device, comprising the steps of:
(1) separating and filtering white:
placing the blood donation whole blood collection device in a biological safety cabinet, and spraying alcohol on the surface of the white filter and the pipe walls at two sides of the white filter to perform surface disinfection; clamping the pipe walls at two sides by using a pipe clamp, and cutting the pipe orifices at the upper end and the lower end of the pipe walls at two sides by using sterile scissors, wherein a long pipe is reserved on each of the pipe walls at two sides;
(2) forward and reverse flushing extraction white filter:
the white blood filter is suspended in the forward direction, flushing liquid passes through the long tube to wash the white blood filter in the forward direction by using the syringe, bubbles are prevented from being generated, a clean container is used for receiving waste liquid flowing out from the lower end of the white blood filter, pollution is avoided, the waste liquid is discarded after washing is finished, and the forward washing is in the direction of filtering white blood cells from whole blood and cannot wash the white blood cells (mononuclear cells); reversely suspending the leukocyte filter, reversely flushing the flushing liquid through a long tube by using an injector, and collecting cell sap by using two centrifuge tubes;
(3) isolation of mononuclear cells:
centrifuging the cell sap in the two centrifuge tubes in the step (2), sucking and discarding supernatant after centrifugation, and carrying out heavy suspension on cells by using normal saline to obtain cell suspension; respectively adding equal amount of Ficoll into the other two centrifuge tubes, respectively dripping the cell suspensions of the two centrifuge tubes into the Ficoll, and continuously centrifuging; after the centrifugation is finished, the supernatant is sucked and the tunica albuginea layer above the Ficoll is sucked into a new centrifuge tube;
(4) counting:
supplementing physiological saline into the centrifuge tube with the leukocyte layer in the step (3), sucking and discarding supernatant after centrifugation, manually counting by adopting a blood counting cell plate after the physiological saline is resuspended, and obtaining 1 × 10 for each leukocyte8～1.5×108After late-stage in vitro cell culture and staining, the ratio of living cells is found to be more than 90%; in subsequent experiments, it has been verified that mononuclear cells separated from the leukocyte filtering disc have the same cell viability as mononuclear cells separated from fresh blood, and can meet the experimental requirements.
In the above technical scheme, in the step (1), the alcohol is 75% alcohol.
In the above technical scheme, in the step (1), the length of the long tube is at least 15 cm.
In the above technical solution, in the step (2), the forward suspension of the white filter disc means that a long tube reserved after a side tube communicated with the collection bag is cut off is positioned above the white filter disc, and the flushing liquid is flushed forward by flushing downwards from the long tube.
In the above technical scheme, in the step (2), the injector is a 50mL injector, and the white filter disc is positively washed by 100mL of washing liquid twice.
In the above technical solution, in the step (2), the reversely suspended white filter disc means that a long pipe reserved after a side pipe communicated with the transfer bag I is cut off is positioned above the white filter disc, and the flushing liquid is flushed from the long pipe downwards to be reversely flushed.
In the above technical scheme, in the step (2), the syringe is a 50mL syringe, the leukocyte filtering disc is reversely washed by 100mL washing liquid twice, and 2 50mL centrifuge tubes are used for collecting cell suspension.
In the technical scheme, in the step (2), the flushing fluid is a mixed solution of DMEM-LG culture solution and fetal bovine serum, and the volume ratio of the DMEM-LG culture solution to the fetal bovine serum is 4: 1.
In the above technical scheme, in the step (3), the cell sap in the two centrifuge tubes is balanced by weight and then centrifuged, and if the difference between the cell volumes of the two centrifuge tubes is large, the cells of the two centrifuge tubes can be uniformly mixed.
In the technical scheme, in the step (3), a horizontal rotor low-temperature centrifuge is used for centrifuging, when cell sap in two centrifuge tubes is centrifuged, the temperature is controlled to be 18-20 ℃, 400g is used for centrifuging for 10min, after the centrifugation is finished, supernatant is sucked and discarded, and 15mL of physiological saline is used for resuspending cells to obtain cell suspension.
In the technical scheme, in the step (3), 15mL of Ficoll is respectively added into the other two centrifuge tubes, and a horizontal rotor low-temperature centrifuge is used for centrifuging, wherein the temperature is controlled at 18-20 ℃ and 800g, and the centrifuging is carried out for 25 min.
In the technical scheme, in the step (3), the centrifuge tube is carefully taken out of the centrifuge after the centrifugation is finished, liquid in the centrifuge tube is layered, the phenomenon that the layering is damaged by violent vibration is avoided, 10mL of supernatant is absorbed by a suction tube, a white membrane layer above Ficoll is carefully absorbed into a new 50mL centrifuge tube, and the transfer of the Ficoll is reduced as much as possible.
In the technical scheme, in the step (4), physiological saline is added into the centrifuge tube with the leucocyte layer in the step (3) to 40mL, the pipette is gently mixed uniformly, the mixture is centrifuged at 650g at room temperature for 10min, the supernatant is discarded after the centrifugation is finished, the mixture is resuspended by 40mL of physiological saline, and a blood cell counting plate is manually counted.
Compared with the prior art, the method has the following characteristics:
1. the source of the cells is sufficient, taking the blood station in Qingdao city as an example, about 13 million people collect the whole blood in 2022, and 13 million white filters provide mononuclear cells, which can greatly promote the development of the technology.
2. The waste utilization and the gratuitous blood donation white filter disc are treated as medical waste after the blood preparation process, and if the waste utilization and the gratuitous blood donation white filter disc are used as the source of cell therapy, the waste can be avoided and the gratuitous blood donation white filter disc can be used as seed cells of the cell therapy. 3. The enrichment degree of single nuclear cells in the leukocyte filter is high, and compared with the single nuclear cells obtained by extracting 50ML of peripheral blood of a cell provider in the prior art, the enrichment degree of the single nuclear cells in the leukocyte filter is 5-8 times that of the single nuclear cells obtained by extracting 50ML of peripheral blood of the cell provider.
Drawings
Fig. 1 is a blood collection device for blood donation without compensation (wherein 1 is a collection bag, 2 is a white filter, 3 is a transfer bag I, 4 is a transfer bag II, and 5 is an anticoagulant storage bag);
FIG. 2-1 is a schematic view of a forward flush extraction of leukocytes from the leukocyte filter, wherein 6 is a tube clamp, 7 is sterile scissors, and the downward arrow represents the forward flush;
FIG. 2-2 is a schematic diagram of a backwash extraction filter mononuclear cell, wherein 6 is a tube clamp, 7 is sterile scissors, and the upward arrow represents the backwash;
FIG. 3-1 is a schematic view of the case where Ficoll is added without centrifugation, A represents a cell suspension layer, and B represents a Ficoll layer;
FIG. 3-2 is a schematic view after centrifugation with Ficoll added, B represents a Ficoll layer, C represents a physiological saline layer, D represents a tunica albuginea layer, and E represents a cell layer;
FIG. 4 is a schematic diagram of in vitro cell culture and staining at a late stage in example 1.
Detailed Description
The following detailed description of the embodiments of the present invention is provided, but the present invention is not limited to the following description:
the blood collection device for blood donation without compensation is composed of a collection bag, a white filter, a transfer bag I, a transfer bag II and an anticoagulant storage bag, as shown in figure 1: the whole blood of a gratuitous blood donor is firstly collected into a collection bag, then the whole blood with mononuclear cells filtered out is transferred into a transfer bag I through a filter, most of blood plasma in the transfer bag I is transferred into a transfer bag II through centrifugal and full-automatic equipment, and then anticoagulant in an anticoagulant storage bag is added into the transfer bag I according to the proportion to form leukocyte-removed suspended red blood cells for clinical use. The applicant has recovered over forty donor filters and, after repeated experiments, has established a complete set of methods for extracting mononuclear cells from the filters and successfully expanding them to the order of magnitude required for cell therapy, which is illustrated below with reference to specific examples:
example 1:
a method of separating peripheral blood mononuclear cells from a gratuitous blood donation whole blood collection device, comprising the steps of:
(1) separating and filtering white:
placing the blood collection device for blood donation without compensation in a biological safety cabinet, and spraying 75% alcohol on the surface of the white filter and the pipe walls at two sides of the white filter for surface disinfection; the pipe clamps 6 are used for clamping the pipe walls at the two sides, the upper end pipe orifice and the lower end pipe orifice of the pipe walls at the two sides are cut off by sterile scissors 7, and a long pipe is reserved on the pipe walls at the two sides, and the length of the long pipe is at least 15 cm.
(2) Forward and reverse washing extraction and white filter:
the long tube reserved after cutting off the side tube communicated with the collection bag is positioned above, as shown in figure 2-1, the white filter is suspended in the forward direction with the arrow direction facing downwards, a 50mL syringe is used, a white filter disc is washed by 100mL washing liquid in the forward direction twice, bubbles are avoided, a clean container is used for receiving waste liquid flowing out from the lower end, pollution is avoided, the waste liquid is discarded after washing is finished, and forward washing is in the direction of filtering white blood cells by whole blood, so that white blood cells (single nuclear cells) cannot be washed away;
the long tube reserved after cutting off the side tube communicated with the transfer bag I is positioned above, as shown in figure 2-2, the leukocyte filter is reversely suspended with the arrow direction upward, the leukocyte filter disc is reversely washed with 100mL of washing liquid twice by using a 50mL syringe, and the cell suspension is collected by using 2 50mL centrifuge tubes.
In the step, the flushing fluid is a mixed solution of DMEM-LG culture solution and fetal calf serum, and the volume ratio of the DMEM-LG culture solution to the fetal calf serum is 4: 1.
(3) Isolation of mononuclear cells:
balancing the cell sap in the two centrifuge tubes in the step (2) according to weight, then centrifuging, and mixing and equally dividing the cells in the two centrifuge tubes if the cell amount difference between the two centrifuge tubes is large; centrifuging by using a horizontal rotor low-temperature centrifuge, controlling the temperature at 18-20 ℃, 400g, centrifuging for 10min, sucking and discarding supernatant after centrifugation is finished, and re-suspending cells by using 15mL of physiological saline to obtain cell suspension;
adding 15mL of Ficoll into the other two centrifuge tubes respectively, and dripping the cell suspensions of the two centrifuge tubes into the Ficoll respectively, as shown in figure 3-1; centrifuging by using a horizontal rotor low-temperature centrifuge at the temperature of 18-20 ℃ for 25min at 800 g; carefully taking out the centrifuge tube from the centrifuge after the centrifugation is finished, wherein the liquid in the centrifuge tube is layered as shown in figure 3-2, the phenomenon that the layering is damaged by violent vibration is avoided, 10mL of supernatant is sucked by a suction tube, and the white membrane layer above the Ficoll is carefully sucked into a new 50mL centrifuge tube, so that the transfer of the Ficoll is reduced as much as possible.
(4) Counting:
supplementing physiological saline into the centrifuge tube with the leukocyte membrane layer in the step (3) to 40mL, gently mixing the centrifuge tube with the physiological saline, centrifuging for 10min at room temperature of 650g, discarding supernatant after centrifugation, resuspending the supernatant with 40mL of physiological saline, and manually counting by a blood counting plate; each white disc can acquire 1 × 108～1.5×108The cells, after the late in vitro culture and staining, as shown in FIG. 4, were found to have a viable cell ratio above 90%; in subsequent experiments, it has been verified that mononuclear cells separated from the leukocyte filtering disc have the same cell viability as mononuclear cells separated from fresh blood, and can meet the experimental requirements.
The above examples are only for illustrating the technical concept and features of the present invention, and are not intended to limit the scope of the present invention. All equivalent changes or modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.
Claims (10)
1. A method of separating peripheral blood mononuclear cells from a gratuitous blood donation whole blood collection device, comprising the steps of:
(1) separating and filtering white:
placing the blood donation whole blood collection device in a biological safety cabinet, and spraying alcohol on the surface of the white filter and the pipe walls at two sides of the white filter to perform surface disinfection; clamping the pipe walls at two sides by using a pipe clamp (6), and cutting the pipe orifices at the upper and lower ends of the pipe walls at two sides by using sterile scissors (7), wherein a long pipe is reserved on each of the pipe walls at two sides;
(2) forward and reverse flushing extraction white filter:
the white blood filter is positively suspended, a syringe is used for enabling flushing fluid to pass through the long tube to positively flush the white blood filter, bubbles are prevented from being generated, a clean container is used for receiving waste liquid flowing out of the lower end of the white blood filter, pollution is avoided, the waste liquid is discarded after flushing is finished, and positive flushing is in the direction of filtering white blood cells from whole blood and cannot flush the white blood cells; reversely suspending the leukocyte filter, reversely flushing the flushing liquid through a long tube by using an injector, and collecting cell sap by using two centrifuge tubes;
(3) isolation of mononuclear cells:
centrifuging the cell sap in the two centrifuge tubes in the step (2), sucking and discarding supernatant after centrifugation, and carrying out heavy suspension on cells by using normal saline to obtain cell suspension; respectively adding equal amount of Ficoll into the other two centrifuge tubes, respectively dripping the cell suspensions in the two centrifuge tubes into the Ficoll, and continuously centrifuging; after the centrifugation is finished, the supernatant is sucked and the tunica albuginea layer above the Ficoll is sucked into a new centrifuge tube;
(4) counting:
supplementing physiological saline into the centrifuge tube with the white blood membrane layer in the step (3), absorbing and discarding supernatant after centrifugation, manually counting by adopting a blood cell counting plate after the physiological saline is re-suspended, and obtaining 1X 10 white blood cells from each white blood disk8～1.5×108After late-stage in vitro cell culture and staining, the ratio of living cells is found to be more than 90%; in subsequent experiments, it has been verified that mononuclear cells separated from the leukocyte filtering disc have the same cell viability as mononuclear cells separated from fresh blood, and can meet the experimental requirements.
2. The method according to claim 1, wherein in step (1), the alcohol is 75% alcohol; the length of the long pipe is at least 15 cm.
3. The method according to claim 1, wherein in the step (2), the forward suspension of the straining plate means that a long tube reserved after cutting off a side tube communicating with the collection bag is located above, and the washing liquid is flushed forward from the long tube by flushing downwards; the injector is a 50mL injector, and the white filter disc is positively washed by 100mL of washing liquid in two times.
4. The method as claimed in claim 1, wherein in the step (2), the reversed suspending and white filtering plate means that a long pipe reserved after the cutting of the side pipe communicated with the transfer bag I is positioned above, and the washing liquid is reversed by washing downwards from the long pipe; the syringe is a 50mL syringe, the white filter disc is reversely washed by 100mL washing liquid twice, and 2 50mL centrifuge tubes are used for collecting cell suspension.
5. The method according to claim 1, wherein in the step (2), the washing solution is a mixture of DMEM-LG culture solution and fetal bovine serum, and the volume ratio of DMEM-LG culture solution to fetal bovine serum is 4: 1.
6. The method of claim 1, wherein in step (3), the cellular fluids in the two centrifuge tubes are balanced by weight and then centrifuged, and if the difference between the cell amounts in the two centrifuge tubes is large, the cells in the two centrifuge tubes can be mixed and then equally divided.
7. The method of claim 1, wherein in the step (3), a horizontal rotor low-temperature centrifuge is used for centrifugation, the temperature of the cell fluid in two centrifuge tubes is controlled to be 18-20 ℃, 400g is used for centrifugation for 10min, the supernatant is discarded after the centrifugation is finished, and 15mL of physiological saline is used for resuspending the cells to obtain the cell suspension.
8. The method of claim 1, wherein in step (3), 15mL of Ficoll is added into each of the other two centrifuge tubes, and the mixture is centrifuged by using a horizontal rotor low temperature centrifuge at 18-20 ℃ and 800g for 25 min.
9. The method of claim 1, wherein in step (3), the centrifuge tube is carefully removed from the centrifuge after centrifugation is complete, the liquid in the centrifuge tube is stratified, violent shaking is avoided to destroy the stratification, 10mL of supernatant is removed by pipette, and the white membrane layer above the Ficoll is carefully pipetted into a new 50mL centrifuge tube to minimize Ficoll transfer.
10. The method of claim 1, wherein in step (4), the centrifuge tube with the buffy coat layer in step (3) is supplemented with physiological saline to 40mL, the pipette is gently mixed, the mixture is centrifuged at room temperature of 650g for 10min, the supernatant is discarded after the centrifugation is finished, the mixture is resuspended in 40mL physiological saline, and the count is manually performed by a hemocytometer.
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