CN110129267B - Resuscitation fluid and method for cryopreserving human peripheral blood mononuclear cells - Google Patents
Resuscitation fluid and method for cryopreserving human peripheral blood mononuclear cells Download PDFInfo
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2302—Interleukin-2 (IL-2)
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Abstract
The application relates to a resuscitation fluid and a method for cryopreserving human peripheral blood mononuclear cells, wherein the resuscitation fluid contains supernatant of human peripheral blood mononuclear cell culture fluid and optional culture medium of human peripheral blood mononuclear cells. The resuscitation method of the application comprises the steps of preparing a cell suspension by using the resuscitation fluid immediately after thawing frozen human peripheral blood mononuclear cells and culturing. Compared with the cells recovered by the prior method, the activity of the cells is obviously improved, and the death number of the cells is obviously reduced.
Description
Technical Field
The application relates to a resuscitation fluid and a method for cryopreserving single nucleated cells of human peripheral blood.
Background
Peripheral Blood Mononuclear Cells (PBMCs) are a class of any unexpected peripheral blood cells with rounded nuclei. These cells include lymphocytes (T cells, B cells, NK cells) and monocytes, whereas erythrocytes and platelets, due to the absence of nuclei, do not belong to this class, and granulocytes (including neutrophils, basophils and eosinophils) possess a multi-lobed nucleus, nor do they belong to this class. PBMC can be induced to differentiate into various immune cells in vitro, such as Cytokine-induced killer Cells (CIK), dendritic cells (Dendritic cells), natural killer cells (NK) and the like, which are now used in biotherapy. In addition, various T cells engineered by gene engineering have wide application prospects in the treatment of cancers.
At present, domestic cellular immunotherapy mostly draws blood and extracts immune cells after patients are ill, but the cellular activity and cell proliferation of the immune cells after the patients are definitely weaker than those of the healthy period, so that the therapeutic effect is poor. Therefore, it is particularly important to store immune cells during the healthy period and to ensure the activity and proliferation capacity of immune cells after resuscitation.
Cell cryopreservation is one of the main methods for long-term preservation of cells. Currently, the most common technique for cell cryopreservation is liquid nitrogen cryopreservation. By using this method, the cells are preserved in liquid nitrogen at-196 ℃ at low temperature, so that the cells can be temporarily separated from the growth state and the cell characteristics can be preserved, so that the cells can be recovered for experiments when needed. For patients, if the immune cells can be frozen in advance and immediately resuscitated and returned when needed, the immune cell treatment effect can be more effectively exerted, and more treatment schemes are provided for the patients.
Resuscitating immune cells will directly affect the viability and cellular activity of the later stage immune cells. The application is provided for solving the technical problems of lower survival rate, more cell death, poor amplification capability and the like of PBMC after resuscitation caused by the existing resuscitation method.
Disclosure of Invention
The application aims to provide a method for resuscitating human peripheral blood mononuclear cells, which can improve the activity of the human peripheral blood mononuclear cells after resuscitating and maintain higher cell proliferation capacity.
Specifically, the method for resuscitating human peripheral blood mononuclear cells comprises the steps of thawing frozen human peripheral blood mononuclear cells, immediately mixing the thawed human peripheral blood mononuclear cells with resuscitating liquid, centrifuging to obtain precipitate, and adding resuscitating liquid to prepare cell suspension; wherein the resuscitation fluid contains the supernatant of the human peripheral blood mononuclear cell culture fluid.
In one or more embodiments, the supernatant is prepared as follows: mixing human peripheral blood mononuclear cells with a culture medium thereof to prepare a cell suspension, culturing for 8-20 hours, preferably 14-20 hours, and collecting a culture solution supernatant, namely the supernatant; preferably, the culture supernatant is collected and then centrifuged further to collect the supernatant.
In one or more embodiments, the cell concentration in the cell suspension is 1×10 5 -1×10 8 Individual cells/mL, preferably 1X 10 6 -1×10 7 Individual cells/mL, more preferably 1X 10 6 -4×10 6 Individual cells/mL.
In one or more embodiments, the medium used for culturing with human peripheral blood mononuclear cells to prepare the supernatant isCTS TM Serum-free cell culture medium.
In one or more embodiments, the resuscitation fluid contains the supernatant and a medium of human peripheral blood mononuclear cells.
In one or more embodiments, the medium of the human peripheral blood mononuclear cells is CTS TM Serum-free cell culture medium.
In one or more embodiments, the volume ratio of the supernatant to the medium of the human peripheral blood mononuclear cells is 0.1:1-5:1.
in one or more embodiments, the volume ratio of the supernatant to the medium of the human peripheral blood mononuclear cells is 1:2-2:1.
in one or more embodiments, the volume ratio of the supernatant to the medium of the human peripheral blood mononuclear cells is 1:1.5-1:3.
in one or more embodiments, the resuscitation fluid consists of the supernatant and a medium of human peripheral blood mononuclear cells; the volume percentage of the supernatant is 5-70%, preferably 10-40%, more preferably 30-40%, and the volume percentage of the culture medium is 30-95%, preferably 60-80%, more preferably 60-70%.
In one or more embodiments, the thawing comprises rapidly thawing the container containing the cryopreserved human peripheral blood mononuclear cells at a temperature of 37 ℃.
In one or more embodiments, the method comprises adding 1-3 times of the volume of the resuscitation fluid into the container after the solid in the container is completely melted into liquid, adding 3-5 times of the volume of the resuscitation fluid after uniform mixing, centrifuging, discarding the supernatant, and adding the resuscitation fluid to prepare the cell suspension.
In one or more embodiments, the method further comprises exposing the resulting suspension to 37 ℃, 5% co 2 Is a step of culturing under the condition of (2).
The application also provides a resuscitation fluid containing the supernatant of human peripheral blood mononuclear cell culture fluid.
In one or more embodiments, the supernatant is prepared as follows: mixing human peripheral blood mononuclear cells with a culture medium thereof to prepare a cell suspension, culturing for 8-20 hours, preferably 14-20 hours, and collecting a culture supernatant to obtain the supernatant.
In one or more embodiments, the cell concentration in the cell suspension is 1×10 5 -1×10 8 Individual cells/mL, preferably 1X 10 6 -1×10 7 Individual cells/mL, more preferably 1X 10 6 -4×10 6 Individual cells/mL.
In one or more embodiments, the preparation of the supernatant further comprises; centrifuging the culture solution supernatant, and collecting the supernatant to obtain the supernatant.
In one or more embodiments, for culturing with human peripheral blood mononuclear cellsThe medium for culturing to prepare the supernatant isCTS TM Serum-free cell culture medium.
In one or more embodiments, the resuscitation fluid contains the supernatant and a medium of human peripheral blood mononuclear cells.
In one or more embodiments, the medium of the human peripheral blood mononuclear cells is CTS TM Serum-free cell culture medium.
In one or more embodiments, the volume ratio of the supernatant to the medium of the human peripheral blood mononuclear cells is 0.1:1-5:1.
in one or more embodiments, the volume ratio of the supernatant to the medium of the human peripheral blood mononuclear cells is 1:2-2:1.
in one or more embodiments, the volume ratio of the supernatant to the medium of the human peripheral blood mononuclear cells is 1:1.5-1:3.
in one or more embodiments, the resuscitation fluid consists of the supernatant and a medium of human peripheral blood mononuclear cells; the volume percentage of the supernatant is 5-70%, preferably 10-40%, more preferably 30-40%, and the volume percentage of the culture medium of the human peripheral blood mononuclear cells is 30-95%, preferably 60-80%, more preferably 60-70%.
The application also provides a kit which contains the supernatant of the human peripheral blood mononuclear cell culture solution and optionally the culture medium of the human peripheral blood mononuclear cells.
In one or more embodiments, the kit contains a supernatant of human peripheral blood mononuclear cell culture broth and a culture medium of human peripheral blood mononuclear cells, the supernatant being provided in separate packages from the culture medium or both in a mixture.
In one or more embodiments, the supernatant is prepared as follows: mixing human peripheral blood mononuclear cells with a culture medium thereof to prepare a cell suspension, culturing for 8-20 hours, preferably 14-20 hours, and collecting a culture supernatant to obtain the supernatant.
In one or more embodiments, the cell concentration in the cell suspension is 1×10 5 -1×10 8 Individual cells/mL, preferably 1X 10 6 -1×10 7 Individual cells/mL, more preferably 1X 10 6 -4×10 6 Individual cells/mL.
In one or more embodiments, the medium used for culturing with human peripheral blood mononuclear cells to prepare the supernatant isCTS TM Serum-free cell culture medium.
In one or more embodiments, the volume ratio of the supernatant to the medium of the human peripheral blood mononuclear cells in the mixture is 0.1:1-5:1, preferably 1:2-2:1 or 1:1.5-1:3.
in one or more embodiments, the volume percentage of the supernatant is 5-70%, preferably 10-40%, more preferably 30-40%, and the volume percentage of the medium is 30-95%, preferably 60-80%, more preferably 60-70%.
In one or more embodiments, the mixture contains, or consists of, 30-40% of the supernatant and 60-70% of the medium, by volume percent.
Drawings
Fig. 1: the cell number dynamic change line graph of the cell resuscitating method and the cell resuscitating method in the prior art is cultured for 48 hours.
Fig. 2: the cell viability dynamic change line graph of the cell in the recovery method and the existing recovery method is recovered and cultured for 48 hours.
Fig. 3: the cell number dynamic change line graph of the application is cultured for 4 days after the PBMC is frozen by the cell resuscitation serum-free freezing method and the existing resuscitation method.
Fig. 4: the cell viability dynamic change line graph of the application is cultured for 4 days after the PBMC is frozen by the cell resuscitation serum-free freezing method and the existing resuscitation method.
Fig. 5: the cell viability dynamic change line graph of the application is cultured for 2 days after the cell is thawed by the serum cryopreservation method for the resuscitation method and the existing resuscitation method.
Fig. 6: the cell number dynamic change line graph of the cell resuscitator with the serum cryopreservation method is cultured for 2 days after the PBMC is cryopreserved.
Fig. 7: the resuscitation method and the prior resuscitation method of the application are used for cell resuscitation, and the cryopreserved PBMC cells are subjected to microscopic examination after 48 hours of culture.
Fig. 8: the dynamic change line graph of the number of human peripheral blood mononuclear cells that were not frozen and thawed was cultured using the resuscitators a or D obtained in table 1 as a medium.
Fig. 9: the recovery solution A or D obtained in Table 1 was used as a medium to culture a dynamic change in the number of human peripheral blood mononuclear cells that had not been frozen and thawed.
A, B, C and D in FIGS. 3-7 are consistent with the nomenclature of the resuscitation fluid of Table 1.
Detailed Description
It is understood that within the scope of the present application, the above-described technical features of the present application and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute a preferred technical solution.
The application uses the supernatant of the human peripheral blood mononuclear cell culture solution as a part or all of the resuscitation fluid for the resuscitation of the frozen human peripheral blood mononuclear cells, and compared with the cells recovered by the prior method, the activity of the human peripheral blood mononuclear cell culture solution is obviously improved, and the cell death number is obviously reduced. In certain embodiments, the supernatant is the supernatant of the cell culture broth prior to cryopreservation of human peripheral blood mononuclear cells.
Thus, the method of resuscitating human peripheral blood mononuclear cells of the present application comprises the step of using the supernatant of the human peripheral blood mononuclear cell culture broth as part or all of the resuscitating fluid for suspending the thawed human peripheral blood mononuclear cells.
Typically, human peripheral blood mononuclear cells are cultured using media conventional in the art for human peripheral blood mononuclear cells. The human peripheral blood mononuclear cells can be fresh human peripheral blood mononuclear cells, or human peripheral blood mononuclear cells cultured for a period of time, or human peripheral blood mononuclear cells after frozen and thawed. Preferably, the human peripheral blood mononuclear cells are from healthy individuals.
The culture can be carried out under conventional culture conditions of human peripheral blood mononuclear cells, for example, at 37℃and 5% CO 2 Is carried out under the condition of (2). The incubation time may be 8-20 hours, for example 14-20 hours. In certain embodiments, the cell concentration may be 1X 10 when cultured 5 -1×10 8 Within a range of individual cells/mL, for example, 1X 10 6 -1×10 7 Within a range of individual cells/mL, or 1X 10 6 -4×10 6 Within a range of individual cells/mL.
The medium may be a conventional medium for human peripheral blood mononuclear cells. In a preferred embodiment, the application usesCTS TM Serum-free cell culture medium cultures human peripheral blood mononuclear cells to prepare the supernatant.
After the culture is finished, collecting the supernatant of the cell culture, and preparing a part or all of the supernatant which is used as the resurrection liquid. The supernatant may be filtered, and/or centrifuged, if desired, to obtain a supernatant as described herein. If it is required to be used in a short period of time, the supernatant can be stored at 4deg.C; can be stored at-20deg.C for 6-12 months and at-80deg.C for more than one year.
The resuscitation fluid may consist of the supernatant, or may contain the supernatant and is commonly used for culturing human peripheral blood mononuclear cellsA base. Preferably, the medium for the human peripheral blood mononuclear cells in the resuscitation fluid is the same medium used for culturing human peripheral blood mononuclear cells to prepare the supernatant. In certain embodiments, the medium used to culture human peripheral blood mononuclear cells to prepare the supernatants described herein is CTS TM Serum-free cell culture medium. In certain embodiments, the medium contained in the resuscitation fluid is +.>CTS TM Serum-free cell culture medium. In certain embodiments, the resuscitation fluid of the present application contains a composition comprising +.>CTS TM Supernatant prepared by culturing human peripheral blood mononuclear cells in serum-free cell culture medium and +.>CTS TM Serum-free cell culture medium.
When the resuscitation fluid contains the supernatant and medium described herein, the volume ratio of supernatant to medium of the human peripheral blood mononuclear cells may be 0.1:1-5:1, such as 1:2-2:1. in certain embodiments, the volume ratio of supernatant to medium of the human peripheral blood mononuclear cells is preferably 1:1.5-1:3. in certain embodiments, the volume percentage of the supernatant is 5-70%, preferably 10-40%, more preferably 30-40%, and the volume percentage of the medium of human peripheral blood mononuclear cells is 30-95%, preferably 60-80%, more preferably 60-70%.
Preferred resuscitation solutions contain 30-70% of said supernatant and 30-70% of said medium of human peripheral blood mononuclear cells by volume percent; more preferred resuscitation fluids contain 30-40% supernatant and 60-70% medium for human peripheral blood mononuclear cells.
In general, during resuscitation, frozen human peripheral blood mononuclear cells to be thawed are first thawed rapidly at 37 ℃, during which time the container containing the cells is continuously shaken to rapidly thaw the frozen cells into a liquid. Preferably, the frozen cell mass within the container is rapidly thawed to a liquid within 2 minutes. A small amount of the resuscitation fluid of the present application was then immediately added. An appropriate amount of resuscitation fluid of the present application may be added based on the volume of thawed cells, e.g., the amount of resuscitation fluid added may typically be 1-2 times the volume of thawed fluid. Blowing and mixing uniformly, adding recovery liquid with the volume being 3-5 times, centrifuging, discarding supernatant, adding a proper amount of recovery liquid into cell sediment to prepare suspension, thereby finishing recovery of the frozen human peripheral blood mononuclear cells. The amount of resuscitation fluid added at this time is such that the cell density in the resulting cell suspension meets conventional cell culture requirements. For example, the amount of resuscitation fluid added may be such that the cell density in the resulting cell suspension is 1X 10 5 -1×10 8 Within a range of individual cells/mL. Thus, 1-50mL or even more of resuscitation fluid can be added depending on the amount of cells actually thawed. The cell suspension can be placed under conventional culture conditions (such as 37deg.C, 5% CO) 2 ) Culturing
Therefore, the method for resuscitating human peripheral blood mononuclear cells comprises the steps of thawing frozen human peripheral blood mononuclear cells, immediately mixing the thawed human peripheral blood mononuclear cells with resuscitating liquid, centrifuging to obtain precipitate, and adding resuscitating liquid to prepare cell suspension.
Human peripheral blood mononuclear cells can be cultured using the resuscitation fluid described herein as a medium. Culturing can be carried out under conventional culturing conditions (e.g., 37℃and 5% CO 2 ) Is carried out. In the culture, cell stimulating factors such as IL-2 and CD137 which are conventionally used for culturing human peripheral blood mononuclear cells can be added into the resuscitating liquid. The concentration of such cytokines in the medium is well known in the art, and the total concentration of cytokines may be in the range of 10-100IU/mL, for example, depending on the cell density.
Thus, in certain embodiments, the application also includes a method of culturing human peripheral blood mononuclear cells comprising the step of culturing human peripheral blood mononuclear cells using the resuscitation fluid of the application as a medium. The human peripheral blood mononuclear cells may be resuscitated cells or isolated from fresh blood.
In certain embodiments, the methods of the application for resuscitating human peripheral blood mononuclear cells comprise the step of culturing the resuscitated cells using a resuscitation fluid as described herein as a medium supplemented with a cell stimulating factor.
The application also provides a resuscitation fluid containing the supernatant of human peripheral blood mononuclear cell culture fluid. The supernatant was prepared as described in any of the previous embodiments.
In certain embodiments, the medium used for culturing with human peripheral blood mononuclear cells to prepare the supernatant isCTS TM Serum-free cell culture medium.
In certain embodiments, the resuscitation fluid contains the supernatant and a medium of human peripheral blood mononuclear cells. Preferably, the medium used for culturing the mononuclear cells of human peripheral blood to prepare the supernatant is the same medium as that used in the resuscitation fluid. In certain embodiments, the medium of human peripheral blood mononuclear cells in the resuscitation fluid may beCTS TM Serum-free cell culture medium.
In the resuscitation fluid, the volume ratio of the supernatant to the culture medium of human peripheral blood mononuclear cells may be 0.1:1-5:1, such as 1:2-2:1 or 1:1.5-1:3. in certain embodiments, the resuscitation fluid consists of a supernatant and a medium of human peripheral blood mononuclear cells; the volume percentage of the supernatant is 5-70%, preferably 10-40%, more preferably 30-40%, and the volume percentage of the medium of human peripheral blood mononuclear cells is 30-95%, preferably 60-80%, more preferably 60-70%. Preferred resuscitation solutions contain 30-70% of said supernatant and 30-70% of said medium of human peripheral blood mononuclear cells by volume percent; more preferred resuscitation fluids contain 30-40% supernatant and 60-70% medium for human peripheral blood mononuclear cells.
The application also provides a medium for human peripheral blood mononuclear cells, the medium comprising the resuscitation fluid and optionally a cytokine as described in any one of the embodiments herein. As previously described, the cytokine may be a cytokine conventionally used in human peripheral blood mononuclear cell culture, such as IL-2, and preferably may also include CD137. The concentration of such cell stimulating factors in the medium is well known in the art and may be, for example, in the range of 10-100IU/mL, preferably 50IU/mL, depending on the cell density.
The application also includes the use of the supernatant or resuscitation fluid described herein to resuscitate human peripheral blood mononuclear cells.
The application also provides a kit which contains the supernatant of the human peripheral blood mononuclear cell culture solution and optionally the culture medium of the human peripheral blood mononuclear cells. The supernatant in the kit is the supernatant described in any one of the preceding embodiments; the medium of the human peripheral blood mononuclear cells may be a medium conventionally used in the art for the culture of human peripheral blood mononuclear cells.
The kits of the application may be used for resuscitation of frozen cells, and/or for further culture of cells after resuscitation. In certain embodiments, the kit may also contain other reagents and/or instrumentation necessary for thawing.
The kit for resuscitation typically contains at least the supernatant of the human peripheral blood mononuclear cell culture broth, and optionally also the culture medium of human peripheral blood mononuclear cells. The supernatant and the culture medium of the human peripheral blood mononuclear cells can be independently packaged, and are mixed according to the volume ratio when in use; alternatively, the culture medium of the supernatant and the human peripheral blood mononuclear cells may be provided in the form of a mixture. When provided as a mixture, the volume ratio of supernatant in the mixture to medium of human peripheral blood mononuclear cells was 0.1:1-5:1, such as 1:2-2:1 or 1:1.5-1:3. alternatively, in certain embodiments, the volume percent of the supernatant is 5-70%, preferably 10-40%, more preferably 30-40%, and the volume percent of the medium of human peripheral blood mononuclear cells is 30-95%, preferably 60-80%, more preferably 60-70%. In a more preferred embodiment, the mixture contains 30-70% by volume of said supernatant and 30-70% by volume of said medium of human peripheral blood mononuclear cells, or 30-40% by volume of said supernatant and 60-70% by volume of said medium of human peripheral blood mononuclear cells, or consists of 30-40% by volume of said supernatant and 60-70% by volume of said medium of human peripheral blood mononuclear cells.
The kit for culturing typically contains the resuscitation fluid described in any of the embodiments herein, and may optionally also contain a cell stimulating factor commonly used in human peripheral blood mononuclear cell cultures. The resuscitation fluid may be the only supernatant described herein, or a medium further comprising human peripheral blood mononuclear cells. Thus, the kit generally contains the supernatant described herein, a medium of human peripheral blood mononuclear cells, and optionally a cytokine. The supernatant and the culture medium of human peripheral blood mononuclear cells may be packaged separately, and used in a mixture in the volume ratios described herein, or provided as a mixture, the volumes of both of which are as described in any of the embodiments herein. The cell stimulating factor may be packaged separately or provided as a mixture with the supernatant, the culture medium of human peripheral blood mononuclear cells, or the mixture, where the concentration of the cell stimulating factor in the mixture is such that the concentration of the cell stimulating factor in the final formulated culture medium is that conventionally used in the art for human peripheral blood mononuclear cell culture.
When the kit of the application is used for resuscitation and culture of human peripheral blood mononuclear cells, the medium may generally contain the supernatant described herein, the medium of human peripheral blood mononuclear cells, and optionally a cell stimulating factor. The culture medium of the supernatant and human peripheral blood mononuclear cells may be packaged separately as described above or provided as a mixture of both. When included, the cell stimulating factor is preferably packaged separately for addition to the cell culture medium when it is desired to culture the cells.
The application will be illustrated by way of specific examples. It should be understood that these examples are illustrative only and are not intended to limit the scope of the application. The methods and materials used in the examples are those conventional in the art unless otherwise indicated.
Example 1: culture of human peripheral blood mononuclear cells and collection of resuscitation base fluid
After separating mononuclear cells of human peripheral blood by lymphocyte separation, 50mL of the mixture was addedCTS TM Serum-free cell culture Medium to prepare cell suspension (1×10) 6 -4×10 6 Individual cells/mL), 150cm into the vessel 2 In a flask, then placed at 37℃in 5% CO 2 Culturing in a cell culture box for 14-20h.
After culturing the PBMC overnight, cell supernatants were collected from the flasks, and the supernatants were dispensed into 50mL centrifuge tubes and centrifuged at 1000-1500rpm for 5-10min. The supernatant was collected as a resuscitation base.
After the collected resuscitation basic liquid is sucked by a disposable syringe, the resuscitation basic liquid is filtered by a 0.22 mu m filter head, and the filtrate is collected as the resuscitation basic liquid and is placed in a refrigerator for refrigeration. The product can be used for a short period at 4deg.C, stored for 6-12 months at-20deg.C, and stored for more than one year at-80deg.C.
Example 2: resuscitation of human peripheral blood mononuclear cells
The cell box is taken out from the liquid nitrogen tank, the freezing tube is quickly put into a water bath kettle which is preheated to 37 ℃ for quick thawing, and the liquid in the tube is quickly thawed by continuous shaking. After about 1-2min, the liquid in the freezing tube is completely melted, and the volume of the melted liquid is 1mL. Taking out the freezing tube, wiping the outer wall of the freezing tube with alcohol cotton balls, and taking into an ultra-clean bench. Opening the cover of the freezing storage tube, and adding 1mL meter into the freezing storage tube1 or D, blowing and mixing uniformly, transferring into a 15mL centrifuge tube, adding the resuscitation fluid with the volume corresponding to the volume of the melted fluid, and centrifuging at 1000-1500rpm for 5-8min. After centrifugation, the supernatant was aspirated. Adding 3mL of corresponding resuscitating solution into a centrifuge tube, blowing to obtain cell suspension, and standing at 37deg.C and 5% CO 2 Culturing in a cell culture box.
Table 1: volume (mL) ratio of resuscitation basic solution to AIM culture medium
Example 3: contrast of resuscitated cell viability
The cell suspensions prepared in example 2 were counted according to 2.4X10 6 A six-well plate was inoculated with a cell density of/mL, and the resuscitated base solution obtained in example 1 (resuscitated solution D of Table 1 supplemented with 50IU/mL final concentration of IL-2) was used at 37℃with 5% CO 2 The cells were cultured overnight in an incubator. After overnight culture, the cells were mixed by pipetting, taking a small amount of cell suspension, adding into the phenol blue, mixing, and transferring into a cell counting plate using a Countess TM The full-automatic cell counter of II FL carries out cell counting and cell viability determination.
Control group: frozen human peripheral blood mononuclear cells were removed from liquid nitrogen and used in accordance with the resuscitation method of example 2CTS TM Serum-free cell culture medium (resuscitation fluid a of table 1) was used for rapid resumption of cells. After subsequent preparation of the cell suspension, counting was carried out according to 2.4X10 6 Cell density/mL in six well plates, use +.>CTS TM Serum-free cell culture medium (supplemented with final concentration of 50IU/mL of cell stimulating factor IL-2) at 37deg.C and 5% CO 2 The cells were cultured overnight in an incubator. After overnight incubation, the mixture was blown with a pipetteHomogenizing cells, taking small amount of cell suspension, adding into phenol blue, mixing, transferring into cell counting plate, and adopting Countess TM The full-automatic cell counter of II FL carries out cell counting and cell viability determination.
The comparison of the two results is shown in fig. 1 and fig. 2, and it is obvious from fig. 1 that the cell number of the mononuclear cells of the human peripheral blood cultured for 48 hours after being resuscitated by the resuscitated method of the application is far higher than that of the control group. FIG. 2 shows that the cell viability recovered by the present application was maintained at 90% or more throughout the 48h culture, while the cell viability of the control group was gradually decreased. This shows that the resuscitating method of the application has higher cell activity and strong proliferation capability after resuscitating.
Example 4: different cryopreservation methods cryopreserved cells are resuscitated via the resuscitation method of the application
At present, the cell freezing and preserving mainly adopts low-temperature liquid nitrogen for preserving. The preservation method is divided into serum-containing and serum-free cryopreservation. In the embodiment, the resuscitation method is adopted to revive the cells frozen by the two freezing methods.
1. Serum-free cryopreservation and resuscitation of cells
(1) Cell serum-free cryopreservation solution formula: using commercial serum-free cell cryopreservation solutions such as ScienCell Serum Free-Cell Freezing Medium, CELLBANCER 2 or WakoSerum-free cell cryopreservation solution.
(2) Freezing and preserving: mixing the prepared cell serum-free frozen stock solution with PBMC (different from the donor of PBMC of example 1) and adjusting cell concentration to 2-3×10 7 And each mL.
(3) The cell suspension is split into 2mL frozen tubes, and the cell frozen tubes are put into a program cooling box and stored at-80 ℃ overnight. The cells were then transferred to liquid nitrogen for long term storage.
(4) The cryopreserved cells of step (3) were recovered as in example 2, and cell counts and cell viability were determined as described in example 3.
As shown in fig. 3 and 4, after the PBMCs were cryopreserved without serum, the number of cells recovered and cultured by the recovery solution was far greater than that of the existing recovery method (group a) in the observation of 4 days after recovery by the recovery method provided by the present application; especially in group B, the ratio of 1mL of resuscitation basic solution to 2mL of culture medium is adopted, and the cell number and the cell activity rate are far higher than those of the existing method.
2. Serum cryopreservation and resuscitation of cells
(1) The cell has the formula of serum frozen stock: according to cell serum-free culture mediumCTS TM The culture medium, serum and DMSO (dimethyl sulfoxide) are mixed uniformly in a volume ratio of 7:2:1.
(2) Freezing and preserving: mixing the prepared cell serum-containing frozen stock solution with PBMC (donor different from that of the PBMC used in the serum-free frozen stock of example 1) precipitate cells, and adjusting cell concentration to 2-3×10 7 And each mL.
(3) The cell suspension is split into 2mL frozen tubes, and the cell frozen tubes are put into a program cooling box and stored at-80 ℃ overnight. The cells were then transferred to liquid nitrogen for long term storage.
(4) The cryopreserved cells of step (3) were recovered as in example 2, and cell counts and cell viability were determined as described in example 3.
Results as shown in figures 5, 6 and 7, after the PBMCs were cryopreserved with serum, the number and cell viability of the resuscitated and cultured cells was far greater than that of the prior art resuscitation method (group a) in the 5 day observation after resuscitating via the resuscitation method provided by the present application. This shows that the resuscitation method of the application has the ability to promote the growth of human peripheral blood mononuclear cells after resuscitation for different cryopreserved PBMC methods, thereby improving the viability thereof.
Example 5: culture of human peripheral blood mononuclear cells
In this example, human peripheral blood mononuclear cells which were not frozen and thawed were cultured using the resuscitators obtained in Table 1 as a medium. Separating mononuclear cells of human peripheral blood by lymphocyte separating liquid, sampling and counting,two donor cells were taken 5X 10 separately 6 、3×10 6 The individual cells are transferred into a centrifuge tube and centrifuged at 1000-1200rpm for 5-10min, and the supernatant is discarded. Cell suspensions (supplemented with 50IU/ml final concentration of cell stimulating factor IL-2) were prepared using the resuscitator A or D of Table 1 and inoculated into 6-well plates at 37℃with 5% CO 2 Culturing in a cell culture box.
After culturing for 1-5 days, blowing and beating the mixed cells by a liquid transfer device, taking a small amount of cell suspension, adding the table phenol blue for mixing uniformly, then transferring into a cell counting plate, adopting a Countess TM The full-automatic cell counter of II FL carries out cell counting and cell viability determination.
The results of two donor experiments are shown in figures 8 and 9. The results show that when human peripheral blood mononuclear cells are cultured by using resuscitation fluid D ("AIM-V"), cell death is significantly reduced and cell proliferation is promoted, as compared with resuscitation fluid A ("resuscitation fluid").
Claims (7)
1. A resuscitation fluid comprising a supernatant of human peripheral blood mononuclear cell culture fluid and 50IU/mL of IL-2;
or the resuscitation fluid contains supernatant of human peripheral blood mononuclear cell culture fluid, 50IU/mL of IL-2 and culture medium of human peripheral blood mononuclear cells, and the volume ratio of the supernatant of the human peripheral blood mononuclear cell culture fluid to the culture medium of the human peripheral blood mononuclear cells is (1-2): (2-1) the medium of human peripheral blood mononuclear cells is AIM-V serum-free cell medium;
wherein, the supernatant of the human peripheral blood mononuclear cell culture solution is prepared from the following steps: preparing a cell suspension containing human peripheral blood mononuclear cells and a culture medium thereof, culturing for 14-20 hours, centrifuging at 1000-1500rpm for 5-10min, and collecting a supernatant of the culture solution, namely the supernatant of the human peripheral blood mononuclear cell culture solution, wherein the cell concentration in the cell suspension is 1X 10-6-4X 10-6 cells/mL, and the culture medium of the human peripheral blood mononuclear cells is AIM-V serum-free cell culture medium.
2. A method of resuscitating cryopreserved human peripheral blood mononuclear cells, the method comprising: thawing frozen human peripheral blood mononuclear cells, immediately mixing the thawed human peripheral blood mononuclear cells with the resuscitation fluid of claim 1, centrifuging to obtain cell pellets, adding the resuscitation fluid of claim 1 to prepare a cell suspension, and culturing in a 5% CO2 cell incubator at 37 ℃.
3. The method of claim 2, wherein thawing comprises rapidly thawing the cryopreserved human peripheral blood mononuclear cells at a temperature of 37 ℃.
4. A method according to claim 3, comprising adding 1-3 times the volume of the resuscitation fluid after the cell mass to be frozen has completely melted into a fluid, adding 3-5 times the volume of the resuscitation fluid after mixing, centrifuging, discarding the supernatant, and adding the resuscitation fluid to produce the cell suspension.
5. The method of claim 4, wherein the method further comprises: culturing cells from the cell suspension using the resuscitation fluid.
6. The use of the resuscitation fluid of claim 1, for resuscitation of cryopreserved human peripheral blood mononuclear cells.
7. A kit comprising the resuscitation fluid of claim 1.
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