CN115521910A - Method for separating peripheral blood mononuclear cells - Google Patents

Method for separating peripheral blood mononuclear cells Download PDF

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Publication number
CN115521910A
CN115521910A CN202211230922.2A CN202211230922A CN115521910A CN 115521910 A CN115521910 A CN 115521910A CN 202211230922 A CN202211230922 A CN 202211230922A CN 115521910 A CN115521910 A CN 115521910A
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bag
mononuclear cells
peripheral blood
standing
blood
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李政
钟桂强
韦棋
王清芳
陈辉
张芬
钟振忠
梁晓
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Shenzhen Beike Bio Technology Co ltd
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Shenzhen Beike Bio Technology Co ltd
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Abstract

The invention discloses a method for separating peripheral blood mononuclear cells, and relates to the technical field of cell separation. The invention provides a separation method, which adopts a separation device to separate peripheral blood mononuclear cells, and transfers a peripheral blood sample in a blood collection bag into a standing bag through a connecting piece by a sampling unit. Further, hydroxyethyl starch is added into the standing bag through the connecting piece by the sample adding unit, so that the hydroxyethyl starch and the blood are uniformly mixed. And then sealing the pipeline between the standing bag and the connecting piece, and hanging and standing the standing bag upside down to ensure that the transfer bag is positioned at the upper side of the standing bag. And when the supernatant and the precipitate in the standing bag are layered, slowly extruding the standing bag to transfer the supernatant into the transfer bag, thereby realizing the rapid separation of the peripheral blood mononuclear cells in the supernatant. The peripheral blood mononuclear cell separation method provided by the invention is used for separating peripheral blood mononuclear cells, and has the advantages of high separation efficiency, small damage to cells and high WBC recovery rate.

Description

Separation method of peripheral blood mononuclear cells
Technical Field
The invention relates to the technical field of cell separation, in particular to a method for separating peripheral blood mononuclear cells.
Background
Peripheral Blood Mononuclear Cells (PBMCs) mainly refer to lymphocytes and monocytes, are the most commonly used cells in immunological experiments, and have strong in vitro culture and amplification potential. At present, the treatment of mononuclear cells is widely researched and popularized at home and abroad, diseases are cultured, proliferated and activated in vitro, and then infused back into a human body to induce autologous antiviral immune response, and once the antiviral immunity of the human body is activated, antiviral substances are continuously generated to kill viruses. After being activated, most of T cells with the function of killing tumor cells become memory cells to be stored in lymphatic tissues, thereby providing long-term protection for thoroughly eliminating the tumor cells and preventing and treating metastasis and relapse. With the increase of age, the activity of the mononuclear cells in the peripheral blood in vivo is reduced, so that the mononuclear cells with higher activity are frozen in a young period, and the method has important significance for future immunotherapy.
At present, a ficoll method is mainly adopted for separating peripheral blood mononuclear cells, and the method has the following defects:
1. the operation is relatively complex, the layering speed needs to be accurately controlled, the requirement on the proficiency of operators is high, in addition, the manual open operation time of the sample is long, and the risk of introducing exogenous pollution is high;
2. multiple centrifugal operations are needed, and high-speed centrifugation easily causes damage to cells, so that the cell survival rate obtained by separation is low;
3. the recovery rate of the mononuclear cells is low, the operation time is long, and the method is not suitable for separation operation of batch peripheral blood.
Aiming at a series of defects existing in the separation of peripheral blood mononuclear cells by the ficoll method, a brand new separation method is urgently needed, and the technical problems that the conventional separation of the peripheral blood mononuclear cells is complex in operation, long in operation time, easy to damage the cells, low in cell recovery rate, high in risk of introducing exogenous pollution and the like are solved.
Disclosure of Invention
The invention aims to solve the technical problems that the existing separation method of peripheral blood mononuclear cells has the defects of complex operation, long operation time, easy damage to cells, low cell recovery rate and high risk of introducing exogenous pollution.
In order to solve the above problems, the present invention proposes the following technical solutions:
a separation method of peripheral blood mononuclear cells adopts a separation device to separate the peripheral blood mononuclear cells, wherein the separation device comprises a sampling unit, a sample adding unit, a connecting piece, a standing bag and a transfer bag; the sampling unit, the sample adding unit and the static bag are all connected with the connecting piece, and the connecting piece is used for communicating the static bag with the sampling unit and the sample adding unit; the standing bag is communicated with the transfer bag through a pipeline; the transfer bag is connected with a pipeline with a closed tail end;
the separation method comprises the following steps:
s1, disinfecting the surface of a blood collection bag by using disinfecting alcohol, transferring the blood collection bag into a normally opened biological safety cabinet, and slightly kneading the blood collection bag to fully and uniformly mix blood in the bag;
s2, sampling the blood in the blood collection bag by using a sampling unit, and transferring the blood from the blood collection bag to a standing bag through a connecting piece;
s3, adding hydroxyethyl starch into the sample adding unit, transferring the hydroxyethyl starch into a standing bag through a connecting piece, and fully and uniformly mixing the hydroxyethyl starch with blood;
s4, closing a communication pipeline between the connecting piece and the standing bag, hanging the standing bag upside down, and performing blood standing separation;
s5, when the ratio of the supernatant to the precipitate in the standing bag reaches 1;
s6, sterilizing the closed-end pipeline connected with the transfer bag by using sterilized alcohol, cutting the pipeline by using sterile scissors, and transferring the supernatant in the transfer bag into a centrifugal tube;
s7, sampling the centrifuge tube filled with the supernatant by using a pipette to calculate the number of cells and the survival rate;
s8, centrifuging the centrifugal tube of S7 at room temperature for 3-10min with the parameters of 200-500 g;
s9, removing the centrifugal supernatant, adding a pre-cooled cryopreservation solution at 4 ℃ in advance according to the counting result of S7 according to the preset cryopreservation density, uniformly mixing and subpackaging into a cryopreservation tube to obtain the cryopreservation tube filled with the mononuclear cells;
and S10, carrying out programmed cooling on the mononuclear cells which are sub-packaged in the freezing storage tube, and transferring to a liquid nitrogen tank below 150 ℃ below zero for storage after the cooling is finished.
The disinfecting alcohol is medical disinfecting alcohol, and the concentration of the disinfecting alcohol is 75% (volume fraction).
In one or more embodiments, step S8 of the present invention, the centrifugation parameter may be 200g, 300g, 400g, or 500g; the centrifugation time may be 3min, 4min, 5min, 7min, 9min, or 10min.
The further technical scheme is that the step between S1 and S2 further comprises: samples were taken by puncture from the open end of the blood collection bag using a disposable syringe for complete blood cell counting. In step S7 and the above sampling and counting operation, a 0.5ml blood sample is generally taken to count the number of cells.
The method further comprises the following step of weighing the blood collection bag before the step S1, obtaining the weight of the blood, and converting the weight unit of the blood into a volume unit through unit conversion.
In actual practice, blood to weight units are converted to volume units in a ratio of 1.03; for example, the weight of peripheral blood is recorded as g (grams), and its volume V = g/1.03 (milliliters).
The further technical scheme is that in the step S3, the addition is carried out according to the proportion that the volume ratio of the hydroxyethyl starch to the blood is 1.
The further technical scheme is that in the step S9, the preset freezing density is 1 × 10 7 ～4*10 7 One per ml.
The further technical scheme is that in the step S9, the frozen stock solution is prepared by mixing a basic immune culture medium, DMSO and human hemoglobin according to a volume ratio of 18.
The further technical scheme is that in the step S5, a slurry separating clamp is used for transferring the supernatant from the standing bag to the transferring bag.
The technical scheme is that the connector is a three-way pipe, the three-way pipe comprises three interfaces, and the three interfaces of the three-way pipe are respectively connected with the sampling unit, the sample adding unit and the standing bag.
The sampling unit comprises a puncture outfit, a communication pipeline between the sampling unit and the connecting piece is marked as a first liquid conveying pipe, and two ends of the first liquid conveying pipe are respectively connected with the puncture outfit and the connector of the three-way pipe.
In step S2, a puncture device of the sampling unit is used to sample blood, and the blood is transferred from the blood collection bag to the standing bag through the first fluid transfer tube and the connector by the puncture device.
The technical scheme is that a first switch is arranged on a communication pipeline between the sampling unit and the connecting piece and used for controlling the communication state of a pipeline between the sampling unit and the connecting piece. Namely, the first liquid conveying pipe is provided with a first switch for controlling the communication state of the first liquid conveying pipe.
The further technical proposal is that the tail end of the puncture outfit is a tip shaped like a triangle.
The technical scheme is that a communicating pipeline between the sample adding unit and the connecting piece is recorded as a second liquid conveying pipe, the sample adding unit comprises a rubber plug, the rubber plug is arranged at one end of the second liquid conveying pipe in a sealing mode, and the other end of the second liquid conveying pipe is communicated with an interface of the three-way pipe.
The further technical scheme is that in the step S4, the communication pipeline between the connecting piece and the standing bag is closed, specifically, the communication pipeline between the connecting piece and the standing bag is closed in a heat seal sealing mode.
In a more specific embodiment, the tubing between the connector and the resting pouch is heat sealed and then the tubing near the end of the connector is cut.
In specific implementation, a communication pipeline between the connecting piece and the standing bag is denoted as a third liquid conveying pipe, and two ends of the third liquid conveying pipe are respectively communicated with the standing bag and the connector of the three-way pipe. In the step S4, the pipeline for communicating the connector and the standing bag is closed, specifically, the third liquid conveying pipe is sealed by heat sealing, and further, the pipeline close to the connector end after sealing can be cut off.
The further technical scheme is that a second switch is arranged on a communication pipeline between the static bag and the transfer bag and used for controlling the communication state of the pipeline between the static bag and the transfer bag.
In specific implementation, a communication pipeline between the standing bag and the transfer bag is referred to as a fourth liquid conveying pipe, that is, a second switch is arranged on the fourth liquid conveying pipe and is used for controlling the communication state of the fourth liquid conveying pipe.
The still standing bag comprises a first inner cavity, the first inner cavity comprises a first side and a second side which are opposite, the inner diameter of the first inner cavity is gradually reduced from the first side to the second side, and the fourth liquid conveying pipe is connected to the second side.
The further technical proposal is that the separation device is irradiated and sterilized before use, so that the separation device is in a sterile state.
Compared with the prior art, the invention can achieve the following technical effects:
the invention provides a peripheral blood mononuclear cell separation method, which adopts a separation device to separate peripheral blood mononuclear cells, and transfers a peripheral blood sample in a blood collection bag into a standing bag through a connecting piece by a sampling unit. Further, hydroxyethyl starch is added into the standing bag through the connecting piece by the sample adding unit, so that the hydroxyethyl starch and the blood are uniformly mixed. And then sealing (for example, adopting a heat sealing mode) the pipeline between the standing bag and the connecting piece, and hanging and standing the standing bag upside down to enable the transfer bag to be positioned at the upper side of the standing bag. When the supernatant and the sediment in the standing bag are layered, the supernatant is transferred into the transfer bag by slowly extruding the standing bag, so that the peripheral blood mononuclear cells in the supernatant are quickly separated. The separation device has the advantages of simple structure and good tightness, so that the separation method is simple to operate, short in time, small in damage to cells and small in risk of exogenous pollution.
The method for separating peripheral blood mononuclear cells provided by the invention is used for separating the mononuclear cells, and has the advantages of high separation efficiency, small damage to cells and high WBC recovery rate.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
Fig. 1 is a schematic structural diagram of a separation device according to an embodiment of the present invention.
Reference numerals
The sampling unit 10, the sample adding unit 20, the connecting member 30, the standing bag 40, the transfer bag 50, the first switch 60, the third liquid delivery pipe 70, the fourth liquid delivery pipe 80, the second switch 90, the closed tube 100, the tee 31, the interface 32, the puncture outfit 11, the first liquid delivery pipe 12, the tip 111, the second liquid delivery pipe 21, the rubber plug 22, the first inner cavity 41, the first side 411, the second side 412 and the second inner cavity 51.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments. It is apparent that the embodiments to be described below are only a part of the embodiments of the present invention, and not all of them. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It will be understood that the terms "comprises" and/or "comprising," when used in this specification and the appended claims, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.
It is also to be understood that the terminology used in the description of the embodiments of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of embodiments of the invention. As used in the description of embodiments of the present invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.
Example 1
The embodiment of the invention provides a method for separating peripheral blood mononuclear cells, which is characterized in that a separation device is adopted to separate the peripheral blood mononuclear cells, and the structural schematic diagram of the separation device is shown in figure 1.
The separation device comprises a sampling unit 10, a sample adding unit 20, a connecting member 30, a resting bag 40 and a transfer bag 50. The connection relationship and structure of each component are specifically described as follows:
the sampling unit 10, the sample adding unit 20 and the resting bag 40 are all connected with the connecting piece 30, and the connecting piece 30 is used for communicating the resting bag 40 with the sampling unit 10 and the sample adding unit 20; the standing bag 40 is communicated with the transfer bag 50 through a pipeline; the transfer bag 50 is connected to a closed-end pipe, which is designated as a closed pipeline 100.
The separation device used in the embodiment of the present invention can add the peripheral blood sample in the blood collection bag to the static bag 40 through the connection member 30 by the sampling unit 10. Further, a preparation, such as hydroxyethyl starch, required for separating peripheral blood mononuclear cells is added to the static bag 40 through the connection member 30 by the sample adding unit 20 to be mixed with the blood. The tubing between the resting bag 40 and the connector 30 (i.e., the third liquid delivery tube 70) is then sealed (e.g., by heat sealing) and the resting bag 40 is left standing upside down so that the transfer bag 50 is positioned on the upper side of the resting bag 40. When the supernatant and the sediment in the standing bag 40 are layered, the supernatant is transferred into the transfer bag 50 by slowly squeezing the standing bag 40, so that the peripheral blood mononuclear cells in the supernatant are quickly separated.
Further, the connecting member 30 is a three-way pipe 31, the three-way pipe 31 includes three connectors 32, and the three connectors 32 of the three-way pipe 31 are respectively connected to the sampling unit 10, the sample adding unit 20, and the resting bag 40. The stationary bag 40 can be communicated with the sampling unit 10 and the sample adding unit 20 through a three-way pipe 31.
Further, the sampling unit 10 includes a puncture outfit 11, a communication pipeline between the sampling unit 10 and the connecting piece 30 is denoted as a first liquid conveying pipe 12, and both ends of the first liquid conveying pipe 12 are respectively communicated with the puncture outfit 11 and a port 32 of the three-way pipe 31.
In a specific separation method, a sample is taken by the puncture instrument 11 of the sampling unit 10, and blood is transferred from the blood collection bag into the stationary bag 40 through the first liquid transfer tube 12 and the connector 30 by the puncture instrument 11. The puncture outfit 11 is pricked into the opening end of the screw cap of the blood collection bag and is screwed down, and then the sampling of the peripheral blood sample can be realized. The first liquid delivery pipe 12 may be embodied as a silicone hose. The length of the first liquid delivery tube 12 may be specifically 20cm.
Further, a first switch 60 is disposed on a communication pipeline between the sampling unit 10 and the connecting member 30, and is used for controlling a pipeline communication state between the sampling unit 10 and the connecting member 30. That is, the first liquid conveying pipe 12 is provided with a first switch 60 for controlling the communication state of the first liquid conveying pipe 12. The first switch 60 is used to turn on or off the first liquid delivery tube 12. In this embodiment, the first switch 60 is specifically a water trap, but those skilled in the art may also use other switch devices, and the present invention is not limited thereto.
Further, the end of the puncture device 11 is a tip 111 with a triangular shape, similar to a needle shape, for being inserted into the open end of the cap of the blood collection bag.
Further, a communication pipeline between the sample adding unit 20 and the connecting member 30 is denoted as a second liquid conveying pipe 21, the sample adding unit 20 includes a rubber plug 22, the rubber plug 22 is hermetically arranged at one end of the second liquid conveying pipe 21, and the other end of the second liquid conveying pipe 21 is connected with an interface 32 of the three-way pipe 31. The preparation, such as hydroxyethyl starch, required for the isolation of peripheral blood mononuclear cells can be delivered into the resting bag 40 by syringe through the plug 22. The rubber plug 22 on the one hand seals the pipeline and on the other hand enables the preparation to be conveyed. The second liquid delivery pipe 21 may be embodied as a silicone hose.
In the separation method of the present invention, in the step S4, the communication pipeline between the connector 30 and the stationary bag 40 is closed, specifically, the communication pipeline between the connector 30 and the stationary bag 40 is closed by heat sealing after cutting.
In a specific implementation, a communication pipeline between the connector 30 and the standing bag 40 is referred to as a third liquid delivery pipe 70, and both ends of the third liquid delivery pipe 70 are respectively communicated with the standing bag 40 and the interface 32 of the three-way pipe 31. In the step S4, the communication pipeline between the connector 30 and the standing bag 40 is closed, specifically, the third liquid conveying pipe 30 is closed by adopting a heat seal mode. The third liquid delivery tube 70 may be embodied as a silicone hose. The length of the third liquid delivery pipe 70 may be specifically 20cm.
In specific implementation, after the third liquid conveying pipe is sealed in a heat seal mode, the pipeline close to the end of the connecting piece can be cut off, so that the separating device is simple, and subsequent operation is facilitated.
Further, a second switch 90 is disposed on a communication pipeline between the static bag 40 and the transfer bag 50, and is used for controlling a pipeline communication state between the static bag 40 and the transfer bag 50. In a specific implementation, a communication pipeline between the resting bag 40 and the transfer bag 50 is referred to as a fourth liquid delivery pipe 80, that is, a second switch 90 is arranged on the fourth liquid delivery pipe 80 and is used for controlling a communication state of the fourth liquid delivery pipe 80.
The fourth liquid delivery tube 80 is connected to the resting pouch 40 and the transfer pouch 50, respectively; the second switch 90 is disposed on the fourth liquid delivery pipe 80. The fourth fluid transfer tube 80 is used to communicate the resting bag 40 with the transfer bag 50. The second switch 90 is used to effect on/off control of the fourth liquid transport pipe 80. In this embodiment, the second switch 90 is specifically a pulley speed adjusting clamp, and the pulley speed adjusting clamp can also realize the control of the liquid flow. Other switching devices may be used by those skilled in the art and the present invention is not limited thereto. The fourth liquid delivery pipe 80 may be embodied as a silicone hose. The length of the fourth liquid delivery tube 80 may be specifically 20cm.
Further, the stationary bag 40 comprises a first inner cavity 41, the first inner cavity 41 comprises a first side 411 and a second side 412 which are opposite, the inner diameter of the first inner cavity 41 is gradually reduced from the first side 411 to the second side 412, and the fourth liquid delivery pipe 80 is connected to the second side 412. It can be seen that first lumen 41 is approximately triangular in shape and the sharp corner of the triangle (i.e., second side 412) connects to the fourth fluid delivery tube 80. Thereby, the sediment can be concentrated on the first side 411 of the first lumen 41, facilitating separation of the supernatant. The first lumen 41 may specifically have a capacity of 200ml.
Further, the transfer bag 50 includes a second lumen 51, the second lumen 51 being approximately square in shape. The volume of the second lumen 51 may be specifically 100ml.
The method for separating peripheral blood mononuclear cells provided by the invention is described in detail below, and comprises the following steps:
and S0, weighing the blood collection bag, obtaining the weight of the blood, and converting the weight unit of the blood into a volume unit through unit conversion. In actual practice, blood to weight units are converted to volume units in a ratio of 1.03; for example, the weight of peripheral blood is recorded as g (g), and its volume V = g/1.03 (ml).
S1, disinfecting the surface of the blood collection bag by using 75% alcohol, transferring the blood collection bag into a normally opened biological safety cabinet, and slightly kneading the blood collection bag to fully and uniformly mix blood in the bag.
S11, puncturing 0.5ml of blood sample from the open end of the blood collection bag by using a disposable syringe, and using the blood sample for whole blood cell counting.
S12, before the separation device is used, the separation device is irradiated and sterilized to be in an aseptic state, the separation device is checked and started, and the first switch and the second switch are closed tightly;
s2, sampling the blood in the blood collection bag by using a sampling unit, and transferring the blood from the blood collection bag to a standing bag through a connecting piece;
specifically, the puncture outfit is pricked into the opening end of the rotary cap of the blood collection bag and is screwed tightly; the blood collection bag is lifted, and the first switch is opened, so that peripheral blood in the blood collection bag flows into the standing bag.
S3, after all peripheral blood in the blood collection bag is transferred to the standing bag, the hydroxyethyl starch is added into the blood collection bag through a rubber plug by using a disposable syringe according to the weight percentage of the hydroxyethyl starch: transferring the blood into a standing bag through a connecting piece according to the proportion of 1;
s4, closing a communication pipeline between the connecting piece and the standing bag, hanging the standing bag upside down, and performing blood standing separation; the method specifically comprises the steps of sealing a pipeline between a connecting piece and a standing bag in a heat sealing mode, then cutting off the pipeline close to the end of the connecting piece, hanging the standing bag in an inverted mode, removing residual liquid on the standing bag, squeezing out bubbles, and carrying out standing separation.
S5, when the ratio of the supernatant to the precipitate in the standing bag reaches 1;
s6, sterilizing the closed-end pipeline connected with the transfer bag by using 75% alcohol, cutting the pipeline by using sterile scissors, and transferring the supernatant in the transfer bag into a centrifugal tube;
s7, taking 0.5ml of the centrifuge tube filled with the supernatant by using a pipette to count the number of cells and the survival rate;
s8, centrifuging the centrifugal tube of the S7 at room temperature for 5min with the parameter of 200 g;
s9, removing the centrifugal supernatant, and counting according to the counting result and 2 x 10 7 Adding a frozen stock solution which is pre-cooled at 4 ℃ in advance and prepared by mixing the basic immune culture medium, the DMSO and the human hemoglobin according to the volume ratio of 18.
And S10, carrying out programmed cooling on the mononuclear cells which are sub-packaged in the freezing storage tube, and transferring to a liquid nitrogen tank below 150 ℃ below zero for storage after the cooling is finished.
Comparative example 1
The traditional ficoll method is used for separating peripheral blood mononuclear cells, and the specific steps are as follows:
s1, transferring the peripheral blood in the blood collection bag into a 50ml centrifugal tube, and taking 0.5ml for whole blood counting.
S2, centrifuging the centrifuge tube filled with peripheral blood, wherein the centrifugation conditions are as follows: room temperature, 900g,15min;
and S3, removing the centrifuged supernatant, uniformly mixing the precipitate by using 0.9% sodium chloride injection, and metering to 30ml.
And S4, slowly paving the diluted cell sediment into a 50ml centrifuge tube added with 20ml of separation solution in advance, adding 30ml of separation solution into each tube, and ensuring that the interface of the separation solution and the diluted blood is clear.
S5, centrifuging the centrifugal tube of S4, wherein the centrifuging conditions are as follows: room temperature, 600g,15min.
S6, after the centrifugation is finished, sucking the mononuclear cell layer into a 50ml centrifuge tube, metering the volume to 50ml by using 0.9% sodium chloride injection, and mixing uniformly.
S7, centrifuging the centrifugal tube of S5, wherein the centrifuging conditions are as follows: at room temperature, 600g,10min.
And S8, after the centrifugation is finished, discarding the supernatant, merging the cell precipitates into 1 centrifuge tube, metering the volume to 50ml by using 0.9% sodium chloride injection, uniformly mixing, and taking 0.5ml to detect the cell number and the cell viability.
S9, centrifuging the centrifuge tube of S8, wherein the centrifuging conditions are as follows: at room temperature, 200g,5min.
S10, after the centrifugation is finished, discarding the supernatant, and counting according to the counting result, wherein the counting result is 2 × 10 7 Adding the pre-cooled freezing solution with the freezing density of each ml at 4 ℃ in advance, uniformly mixing and subpackaging into freezing tubes.
S11, carrying out programmed cooling on the mononuclear cells which are sub-packaged in the freezing tube, and transferring to a liquid nitrogen tank below 150 ℃ below zero for storage after the cooling is finished.
The following table 1 shows the specific experimental data obtained by separating 6 samples according to the separation methods provided in example 1 and comparative example 1.
TABLE 1 data for 6 samples separated according to the procedure of example 1 and comparative example 1
Figure BDA0003881266470000121
Figure BDA0003881266470000131
As can be seen from the comparison of the procedures, the method of comparative example 1 requires centrifugation of the blood sample for many times, which is a procedure that causes great damage to cells; 0.9 percent of sodium chloride is added into the solution for dilution and volume fixing for many times, and exogenous pollution is very easy to introduce. Meanwhile, the data of table 1 also show that:
1. the average WBC recovery rate of 6 samples separated by the method of example 1 is 77.37%, compared with that of comparative example 1, the recovery rate is greatly improved by nearly 4.5 times;
2. the average activity rate of 6 samples separated by the method of the embodiment 1 is 99.85 percent, which is improved by 21.33 percent compared with that of the comparative example 1;
3. the average operation time of 6 samples separated by adopting the method in the embodiment 1 is 60.17min, compared with the method in the comparative example 1, the time of each sample is saved by 72.53min, and the efficiency is improved by 54.66%;
4. the 6 samples separated by the method of the example 1 are negative to the anaerobe and fungus detection, but only 5 samples of the comparative example are negative to the anaerobe and fungus detection, which shows that the method of the example 1 is safer in operation process and less prone to pollution compared with the method of the comparative example 1.
Therefore, the method for separating the peripheral blood mononuclear cells provided by the invention is better than the traditional ficoll method in the comparative example 1, and has the advantages of simple operation, short operation time, small damage to the cells, low pollution risk and high recovery rate.
In the foregoing embodiments, the descriptions of the respective embodiments have respective emphasis, and for parts that are not described in detail in a certain embodiment, reference may be made to the related descriptions of other embodiments.
While the invention has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims (10)

1. A separation method of peripheral blood mononuclear cells is characterized in that a separation device is adopted to separate the peripheral blood mononuclear cells, and the separation device comprises a sampling unit, a sample adding unit, a connecting piece, a standing bag and a transfer bag; the sampling unit, the sample adding unit and the static bag are all connected with the connecting piece, and the connecting piece is used for communicating the static bag with the sampling unit and the sample adding unit; the standing bag is communicated with the transfer bag through a pipeline; the transfer bag is connected with a pipeline with a closed tail end;
the separation method comprises the following steps:
s1, disinfecting the surface of a blood collection bag by using disinfecting alcohol, transferring the blood collection bag into a normally opened biological safety cabinet, and slightly kneading the blood collection bag to fully and uniformly mix blood in the bag;
s2, sampling the blood in the blood collection bag by using a sampling unit, and transferring the blood from the blood collection bag to a standing bag through a connecting piece;
s3, adding hydroxyethyl starch into the sample adding unit, transferring the hydroxyethyl starch into a standing bag through a connecting piece, and fully and uniformly mixing the hydroxyethyl starch with blood;
s4, closing a communication pipeline between the connecting piece and the standing bag, hanging the standing bag upside down, and performing blood standing separation;
s5, when the ratio of the supernatant to the precipitate in the standing bag reaches 1;
s6, sterilizing the closed-end pipeline connected with the transfer bag by using sterilized alcohol, cutting the pipeline by using sterile scissors, and transferring the supernatant in the transfer bag into a centrifugal tube;
s7, sampling the centrifuge tube filled with the supernatant by using a pipette to calculate the number of cells and the survival rate;
s8, centrifuging the centrifuge tube of the S7 at room temperature for 3-10min with the parameters of 200-500 g;
s9, removing the centrifugal supernatant, adding a pre-cooled cryopreservation solution at 4 ℃ in advance according to the counting result of S7 according to the preset cryopreservation density, uniformly mixing and subpackaging into a cryopreservation tube to obtain the cryopreservation tube filled with the mononuclear cells;
and S10, carrying out programmed cooling on the mononuclear cells which are sub-packaged in the freezing storage tube, and transferring to a liquid nitrogen tank below 150 ℃ below zero for storage after the cooling is finished.
2. The method for separating peripheral blood mononuclear cells according to claim 1, further comprising, between steps S1 and S2: samples were taken by puncture from the open end of the blood collection bag using a disposable syringe for complete blood cell counting.
3. The method for separating peripheral blood mononuclear cells according to claim 1, further comprising, before the step S1, weighing the blood collection bag, obtaining a weight of the blood, and converting a weight unit of the blood into a volume unit by unit conversion.
4. The method for separating peripheral blood mononuclear cells according to claim 1, wherein in step S3, the addition is performed in such a manner that the ratio of the volume of hydroxyethyl starch to the volume of blood is 1.
5. The method for separating peripheral blood mononuclear cells according to claim 1, wherein the predetermined cryopreservation density is 1 x 10 in step S9 7 ～4*10 7 One per ml.
6. The method for separating mononuclear cells from peripheral blood according to claim 1, wherein in step S9, the frozen stock solution is prepared by mixing the basic immune culture medium, DMSO and human hemoglobin in a volume ratio of 18.
7. The method for separating peripheral blood mononuclear cells according to claim 1, wherein in the step S5, the supernatant is transferred from the standing bag to a transfer bag using a plasma clamp.
8. The method for separating peripheral blood mononuclear cells according to claim 1, wherein in step S4, the communication pipeline between the connector and the standing bag is sealed, specifically, the communication pipeline between the connector and the standing bag is sealed by heat sealing.
9. The method for separating peripheral blood mononuclear cells according to claim 1, wherein a first switch is provided in a communication pipe between the sampling unit and the connecting member for controlling a communication state of a pipe between the sampling unit and the connecting member;
and a second switch is arranged on a communication pipeline between the static bag and the transfer bag and used for controlling the communication state of the pipeline between the static bag and the transfer bag.
10. The method for separating peripheral blood mononuclear cells according to claim 1, wherein the separation device is sterilized by irradiation before use to be in a sterile state.
CN202211230922.2A 2022-09-30 2022-09-30 Method for separating peripheral blood mononuclear cells Pending CN115521910A (en)

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