CN112501117B - Recovery method of umbilical cord blood mononuclear cells frozen at low temperature - Google Patents
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Abstract
The invention provides a recovery method of umbilical cord blood mononuclear cells frozen at low temperature, which comprises the following steps: placing the resuscitation washing liquid in an environment with the temperature of 37 ℃; putting the umbilical cord blood mononuclear cells frozen at low temperature into a water bath kettle at 37 ℃ for rewarming; transferring the umbilical cord blood mononuclear cells subjected to rewarming to a centrifuge tube; dividing the resuscitation washing liquid into a first resuscitation washing liquid and a second resuscitation washing liquid, wherein the volume ratio of the first resuscitation washing liquid to the second resuscitation washing liquid is 0.154-0.25; dropwise adding the first resuscitation washing liquid into a centrifuge tube at a constant speed, wherein the dropwise adding time is 160-170 seconds; dropwise adding the second resuscitation washing liquid into the centrifugal tube at a constant speed for 50-60 seconds to obtain a cell suspension; layering the cell suspension by centrifugation; the supernatant was removed. The invention has the beneficial effects that: by optimizing the operation method of cell recovery, the survival rate and recovery rate of the recovered cells are effectively improved and the recovery effect is greatly improved under the condition of not changing the formulas of the freezing solution and the washing solution.
Description
Technical Field
The invention relates to the technical field of cell recovery, in particular to a recovery method of umbilical cord blood mononuclear cells by low-temperature freezing.
Background
The cord blood mononuclear cell is a cell with a mononuclear cell separated from cord blood, is different from red blood cells without a cell nucleus, and mainly consists of a mononuclear cell and a lymphocyte. The separation method mainly comprises Ficoll (polysucrose-diatrizoate) -density gradient centrifugation method and HES (hydroxyethyl starch) sedimentation method.
The separated cord blood mononuclear cells are rich in CD34+ hematopoietic cells (EPCs) and CD14+ mononuclear cells, can release various cytokines, play roles in neuroprotection, angiogenesis and immunoregulation, and can be well used for treating nervous system diseases, such as neonatal hypoxic-ischemic encephalopathy, stroke and the like. Meanwhile, the cord blood mononuclear cells are also rich in T cells and NK cells, can be used for amplifying immune cells such as CIK, CAR-T, CAR-NK and the like, and are used for tumor immunotherapy.
The existing recovery method needs to put a cell freezing tube into a 37 ℃ water bath kettle, and quickly transfer cells into a 4 ℃ precooled washing solution after quick rewarming. And then centrifuging, and removing the supernatant after centrifugation to reduce the damage of DMSO on cells and reduce the side effect on human bodies after the return transfusion.
The mononuclear cells of the freshly recovered cord blood are fragile and have poor tolerance. On one hand, the recovered cells are quickly added into a 4 ℃ precooled washing solution, and the cells are subjected to large osmotic pressure change, so that the cells are easy to break; on the other hand, addition of a 4 ℃ pre-cooled wash solution followed by centrifugation, the externally applied centrifugal force will cause the fragile cells to rupture.
The existing recovery method is adopted to recover the mononuclear cells, and the recovery rate of the recovered cells and the recovery rate of the living cells are low.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: aiming at the defects of the prior art, the recovery operation method of the umbilical cord blood mononuclear cells frozen at low temperature is provided, and the survival rate and the recovery rate of the umbilical cord blood mononuclear cells frozen at low temperature can be obviously improved.
In order to solve the technical problems, the invention adopts the technical scheme that: a method for resuscitating cryopreserved umbilical cord blood mononuclear cells, comprising:
s1, placing the resuscitation washing liquid in an environment with the temperature of 37 ℃;
s2, putting the umbilical cord blood mononuclear cells frozen at the low temperature into a water bath kettle at 37 ℃ for rewarming;
s3, transferring the cord blood mononuclear cells subjected to rewarming to a centrifuge tube;
s4, dividing the resuscitation washing liquid into a first resuscitation washing liquid and a second resuscitation washing liquid, wherein the volume ratio of the first resuscitation washing liquid to the second resuscitation washing liquid is 0.154-0.25;
s5, dropwise adding the first resuscitation washing solution into the centrifuge tube at a constant speed, wherein the dropwise adding time is 160-170 seconds;
s6, dropwise adding the second resuscitation washing solution into the centrifuge tube at a constant speed for 50-60 seconds to obtain a cell suspension;
s7, layering the cell suspension by a centrifugal method;
and S8, removing the supernatant.
Further, the resuscitation washing solution is 0.9% sodium chloride injection.
Further, the resuscitation washing solution is a solution prepared by mixing 5% of human serum albumin and 10% of dextran-40 according to a ratio of 1: 1.
Further, in step S1, the resuscitation washing solution is placed in an environment at 37 ℃ for at least 6 hours.
Further, in step S2, the umbilical cord blood mononuclear cells frozen at low temperature are placed in a 37 ℃ water bath for rewarming for 2-3 minutes.
Further, in step S7, the cell suspension is centrifuged and layered with the centrifugation parameters of 290-310 g.
Further, in step S7, the time for performing centrifugal stratification process on the cell suspension is 9-11 minutes.
Further, in step S3, the cells are transferred from the cryopreservation tube to the centrifuge tube in the biosafety cabinet.
Further, the ambient temperature in the biosafety cabinet is 18-26 ℃.
The invention has the beneficial effects that: by optimizing the operation method of cell recovery, the survival rate and recovery rate of the recovered cells are effectively improved and the recovery effect is greatly improved under the condition of not changing the formulas of the freezing solution and the washing solution.
Drawings
The specific process of the present invention is detailed below with reference to the accompanying drawings:
FIG. 1 is a schematic flow chart of the resuscitation operation of the present invention;
FIG. 2 is a graph showing the average viable cell recovery of examples of the present invention and the average viable cell recovery of comparative example;
FIG. 3 is a graph showing the average cell-viability of examples of the present invention and the average cell-viability of comparative examples.
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 of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. 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 should be noted that the description of the invention relating to "first", "second", etc. is for descriptive purposes only and is not to be construed as indicating or implying any relative importance or implicit indication of the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In addition, technical solutions between various embodiments may be combined with each other, but must be realized by a person skilled in the art, and when the technical solutions are contradictory or cannot be realized, such a combination should not be considered to exist, and is not within the protection scope of the present invention.
Examples
Referring to fig. 1, a method for resuscitating umbilical cord blood mononuclear cells frozen at low temperature comprises:
s1, placing a resuscitation washing solution in an environment at 37 ℃ for not less than 6 hours to ensure that the temperature is 37 ℃, wherein the resuscitation washing solution can be 0.9% sodium chloride injection, and the effect is better if the solution is prepared by mixing 5% human serum albumin and 10% dextran-40 according to a ratio of 1: 1;
s2, putting the umbilical cord blood mononuclear cells frozen at the low temperature into a 37 ℃ water bath for rewarming for 2-3 minutes;
s3, transferring the cryopreservation tube to a biological safety cabinet with the environmental temperature of 18-26 ℃, and transferring the umbilical cord blood mononuclear cells subjected to rewarming from the cryopreservation tube to a centrifuge tube in the biological safety cabinet;
s4, dividing the resuscitation washing liquid into a first resuscitation washing liquid and a second resuscitation washing liquid, wherein the volume ratio of the first resuscitation washing liquid to the second resuscitation washing liquid is 0.154-0.25, specifically, the volume of the resuscitation washing liquid is 30mL, the volume of the first resuscitation washing liquid is 4-6mL, and the volume of the second resuscitation washing liquid is 24-26 mL;
s5, dropwise adding 4-6mL of first resuscitation washing liquid into the centrifuge tube at a constant speed, wherein the dropwise adding time is 160-170 seconds, in the step, cell damage can be caused by excessively high dropwise adding speed, and toxicity of DMSO to cells can be caused by excessively low dropwise adding speed;
s6, dropwise adding 26-24mL of second resuscitation washing liquid into the centrifuge tube at a constant speed for 50-60 seconds to obtain a cell suspension;
s7, layering the cell suspension by a centrifugation method, specifically, carrying out centrifugation layering treatment on the cell suspension by adopting the centrifugation parameters of 290-310g, wherein the treatment time is 9-11 minutes;
and S8, removing the supernatant to reduce the damage of DMSO to the cells and reduce the side effect of the DMSO to the human body after the return transfusion.
From the above description, the beneficial effects of the present invention are: by optimizing the operation method of cell recovery, the survival rate and recovery rate of the recovered cells are effectively improved and the recovery effect is greatly improved under the condition of not changing the formulas of the freezing solution and the washing solution.
Example 1
Collecting 100mL umbilical cord blood, separating by Ficoll density gradient centrifugation method, collecting mononuclear cells of umbilical cord blood, collecting, adding cryopreservation protective solution (blood plasma + 5% DMSO, viable cell density before cryopreservation is 1.79 × 107Cell count/mL, cell viability is 95.1%), then the cells resuspended in the frozen stock solution are added into 1.8mL of frozen stock tubes, each 1mL, and the cells are transferred into a liquid nitrogen tank for storage after programmed cooling;
taking out the frozen umbilical cord blood mononuclear cells from the liquid nitrogen tank after one month, and quickly rewarming the umbilical cord blood mononuclear cells in a 37 ℃ water bath until the cell solution is changed from a solid state to a liquid state for 2 minutes;
before the recovery operation, 1 bottle of 0.9% sodium chloride injection is placed in an environment at 37 ℃ for not less than 6 hours;
transferring the cryopreserved tube into a biological safety cabinet with constant temperature of 37 ℃, sucking cells into a 50mL centrifuge tube, taking a 5mL pipette to suck 5mL rewarming 0.9% sodium chloride injection, and slowly and uniformly dropping the mixture into the centrifuge tube containing the cells for 2 minutes and 40 seconds;
taking a 25mL pipette to suck 25mL of rewarming 0.9% sodium chloride injection, slowly adding the rewarming 0.9% sodium chloride injection into a centrifugal tube containing cells at a constant speed, and keeping the total time at 54 seconds to obtain cell suspension;
centrifuging the cell suspension at a centrifugal force of 300g for 10 minutes;
and after the centrifugal treatment is finished, removing supernatant, re-suspending the cell mass precipitated at the bottom of the centrifugal tube by using re-warmed 0.9% sodium chloride injection, fixing the volume to 30mL, and taking 0.5mL to detect the cell number and the cell viability.
Example 2
Collecting a 90mL umbilical cord blood, separating by Ficoll density gradient centrifugation method, collecting umbilical cord blood mononuclear cells, collecting, adding cryopreservation protective solution (blood plasma + 5% DMSO, viable cell density before cryopreservation is 1.91 × 10)7Cell count/mL, cell viability 92.3%), then adding the cells resuspended in the frozen stock solution into 1.8mL of frozen stock tubes, wherein each cell count is 1mL, and transferring the cells into a liquid nitrogen tank for storage after programmed cooling;
taking out the frozen umbilical cord blood mononuclear cells from the liquid nitrogen tank after one month, and quickly rewarming the umbilical cord blood mononuclear cells in a water bath kettle at 37 ℃ until the cell solution is changed from a solid state to a liquid state, wherein the rewarming time is 2 minutes and 15 seconds;
before the recovery operation, 1 bottle of 0.9% sodium chloride injection is placed in an environment at 37 ℃ for not less than 6 hours;
transferring the cryopreserved tube into a biological safety cabinet with constant temperature of 37 ℃, sucking cells into a 50mL centrifuge tube, taking a 5mL pipette to suck 5mL rewarming 0.9% sodium chloride injection, and slowly and uniformly dropping the mixture into the centrifuge tube containing the cells for 2 minutes and 50 seconds;
taking a 25mL pipette to suck 25mL of rewarming 0.9% sodium chloride injection, slowly adding the rewarming 0.9% sodium chloride injection into a centrifugal tube containing cells at a constant speed, wherein the total time is 50 seconds, so as to obtain a cell suspension;
centrifuging the cell suspension at a centrifugal force of 300g for 10 minutes;
and after the centrifugal treatment is finished, removing supernatant, re-suspending the cell mass precipitated at the bottom of the centrifugal tube by using re-warmed 0.9% sodium chloride injection, fixing the volume to 30mL, and taking 0.5mL to detect the cell number and the cell viability.
Example 3
Collecting 120mL umbilical cord blood, separating by Ficoll density gradient centrifugation method, collecting umbilical cord blood mononuclear cells, collecting, adding cryopreservation protective solution (blood plasma + 5% DMSO, viable cell density before cryopreservation is 1.92 × 10)7Cell count/mL, the cell viability is 94.3%), then the cells resuspended in the frozen stock solution are added into 1.8mL of frozen stock tubes, each 1mL, and the cells are transferred into a liquid nitrogen tank for storage after programmed cooling;
taking out the frozen umbilical cord blood mononuclear cells from the liquid nitrogen tank after one month, and quickly rewarming the umbilical cord blood mononuclear cells in a water bath kettle at 37 ℃ until the cell solution is changed from a solid state to a liquid state, wherein the rewarming time is 3 minutes;
before the recovery operation, 1 bottle of 0.9% sodium chloride injection is placed in an environment at 37 ℃ for not less than 6 hours;
transferring the cryopreserved tube into a biological safety cabinet with constant temperature of 37 ℃, sucking cells into a 50mL centrifuge tube, taking a 5mL pipette to suck 5mL rewarming 0.9% sodium chloride injection, and slowly and uniformly dropping the mixture into the centrifuge tube containing the cells for 2 minutes and 50 seconds;
taking a 25mL pipette to suck 25mL of rewarming 0.9% sodium chloride injection, slowly adding the rewarming 0.9% sodium chloride injection into a centrifugal tube containing cells at a constant speed, wherein the total time is 60 seconds, so as to obtain cell suspension;
centrifuging the cell suspension at a centrifugal force of 300g for 10 minutes;
and after the centrifugal treatment is finished, removing supernatant, re-suspending the cell mass precipitated at the bottom of the centrifugal tube by using re-warmed 0.9% sodium chloride injection, fixing the volume to 30mL, and taking 0.5mL to detect the cell number and the cell viability.
Comparative example 1
The specific recovery method of the low-temperature frozen cells in the embodiment 1 is as follows:
taking out the frozen umbilical cord blood mononuclear cells from the liquid nitrogen tank after one month, and quickly rewarming the umbilical cord blood mononuclear cells in a 37 ℃ water bath until the cell solution is changed from a solid state to a liquid state for 2 minutes;
before the recovery operation, 1 bottle of 0.9% sodium chloride injection is placed in an environment at 4 ℃ for not less than 6 hours;
transferring the cryopreserved tube into a biological safety cabinet, sucking out cells into a 50mL centrifuge tube, and adding 30mL of precooled 0.9% sodium chloride injection into the centrifuge tube in advance;
centrifuging the cell suspension at a centrifugal force of 300g for 10 minutes;
and after the centrifugal treatment is finished, removing the centrifugal supernatant, resuspending the cell mass precipitated at the bottom of the centrifugal tube by using precooled 0.9% sodium chloride injection, fixing the volume to 30mL, and taking 0.5mL to detect the cell number and the cell viability.
Comparative example 2
The specific recovery method of the low-temperature frozen cells in the embodiment 2 is as follows:
taking out the frozen umbilical cord blood mononuclear cells from the liquid nitrogen tank after one month, and quickly rewarming the umbilical cord blood mononuclear cells in a water bath kettle at 37 ℃ until the cell solution is changed from a solid state to a liquid state for 2 minutes and 15 seconds;
before the recovery operation, 1 bottle of 0.9% sodium chloride injection is placed in an environment at 4 ℃ for not less than 6 hours;
transferring the cryopreserved tube into a biological safety cabinet, sucking out cells into a 50mL centrifuge tube, and adding 30mL of precooled 0.9% sodium chloride injection into the centrifuge tube in advance;
centrifuging the cell suspension at a centrifugal force of 300g for 10 minutes;
and after the centrifugal treatment is finished, removing the centrifugal supernatant, resuspending the cell mass precipitated at the bottom of the centrifugal tube by using precooled 0.9% sodium chloride injection, fixing the volume to 30mL, and taking 0.5mL to detect the cell number and the cell viability.
Comparative example 3
The specific recovery method of the low-temperature frozen cells in the embodiment 3 is as follows:
taking out the frozen umbilical cord blood mononuclear cells from the liquid nitrogen tank after one month, and quickly rewarming the umbilical cord blood mononuclear cells in a 37 ℃ water bath until the cell solution is changed from a solid state to a liquid state for 3 minutes;
before the recovery operation, 1 bottle of 0.9% sodium chloride injection is placed in an environment at 4 ℃ for not less than 6 hours;
transferring the cryopreserved tube into a biological safety cabinet, sucking out cells into a 50mL centrifuge tube, and adding 30mL of precooled 0.9% sodium chloride injection into the centrifuge tube in advance;
centrifuging the cell suspension at a centrifugal force of 300g for 10 minutes;
after the centrifugation treatment is finished, removing the centrifugation supernatant, resuspending the cell mass precipitated at the bottom of the centrifuge tube by using precooled 0.9% sodium chloride injection, fixing the volume to 30mL, and taking 0.5mL to detect the cell number and the cell viability.
Examples 1-3 and comparative examples 1-3 both measured cell number and viability using AO/PI (acridine orange/propidium iodide) fluorescent staining according to the following principle: the AO dye can combine the cell nucleuses of all cells through an intact cell membrane to show green fluorescence, and the PI dye can only combine the cell nucleuses of dead cells through the intact cell membrane to show red fluorescence without staining the red cells, thereby eliminating the interference of the red cells and the like.
The method for calculating the recovery rate of the living cells comprises the following steps:
recovery rate of viable cells is the number of viable cells after recovery/the number of viable cells before cryopreservation.
TABLE 1
Referring to table 1, fig. 2 and fig. 3, it can be seen from the results of table 1, fig. 2 and fig. 3 that the recovery rate of viable cells after resuscitation is significantly different between the example and the comparative example, P is less than 0.05, the survival rate of the example and the comparative example is significantly different, and P is less than 0.001, which indicates that the recovery method can significantly improve the recovery rate of mononuclear cells and the cell survival rate of umbilical cord blood after resuscitation.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes performed by the present specification and drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (6)
1. A method for resuscitating cryopreserved umbilical cord blood mononuclear cells, comprising:
s1, placing a recovery washing solution in an environment at 37 ℃, wherein the recovery washing solution is 0.9% sodium chloride injection;
s2, putting the umbilical cord blood mononuclear cells frozen at the low temperature into a water bath kettle at 37 ℃ for rewarming;
s3, transferring the cord blood mononuclear cells subjected to rewarming to a centrifuge tube at the constant temperature of 37 ℃;
s4, dividing the resuscitation washing liquid into a first resuscitation washing liquid and a second resuscitation washing liquid, wherein the volume ratio of the first resuscitation washing liquid to the second resuscitation washing liquid is 0.154-0.25;
s5, dropwise adding the first resuscitation washing solution into the centrifuge tube at a constant speed, wherein the dropwise adding time is 160-170 seconds;
s6, dropwise adding the second resuscitation washing solution into the centrifuge tube at a constant speed for 50-60 seconds to obtain a cell suspension;
s7, layering the cell suspension by a centrifugal method;
and S8, removing the supernatant.
2. The method for resuscitating cryopreserved umbilical cord blood mononuclear cells according to claim 1, wherein: in step S1, the resuscitation wash is placed in an environment at 37 ℃ for at least 6 hours.
3. The method for resuscitating cryopreserved umbilical cord blood mononuclear cells according to claim 2, wherein: in step S2, the umbilical cord blood mononuclear cells frozen at the low temperature are placed in a 37 ℃ water bath for rewarming for 2-3 minutes.
4. The method for resuscitating cryopreserved umbilical cord blood mononuclear cells according to claim 3, wherein: in step S7, the cell suspension is centrifuged and layered with the centrifugation parameters of 290-310 g.
5. The method for resuscitating cryopreserved umbilical cord blood mononuclear cells according to claim 4, wherein: in step S7, the time for centrifugal stratification of the cell suspension is 9-11 minutes.
6. The method for resuscitating cryopreserved umbilical cord blood mononuclear cells according to claim 5, wherein: in step S3, the cells are transferred from the cryopreserved tube to a centrifuge tube in a biosafety cabinet.
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