CN114015653A - Cryopreserved umbilical cord blood-derived NK (Natural killer) cell and preparation method thereof - Google Patents
Cryopreserved umbilical cord blood-derived NK (Natural killer) cell and preparation method thereof Download PDFInfo
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Abstract
The invention discloses NK cells derived from cryopreserved umbilical cord blood and a preparation method and application thereof, and particularly comprises the steps of dissolving and recovering the cryopreserved umbilical cord blood, extracting mononuclear cells in a washing solution containing 6% of human serum albumin, 10% of dextran and 9% of trehalose as a component protective agent, and then carrying out induced culture on the NK cells. The preparation method solves the problems of small induction quantity and low purity of NK cells from cryopreserved umbilical cord blood, and can fully utilize cryopreserved human umbilical cord blood.
Description
Technical Field
The invention relates to the technical field of blood products, in particular to NK cells derived from cryopreserved umbilical cord blood and a preparation method and application thereof.
Background
NK cells are natural immune effector cells with a natural killing function, are mainly differentiated and developed in a bone marrow microenvironment and are distributed in bone marrow, peripheral blood, liver, spleen, lung and lymph nodes. NK cells do not need specific antigen stimulation, can recognize self and non self through surface receptors, directly kill tumor cells and target cells infected by viruses, and are the first line of defense of organisms against pathogens and tumor cells.
NK cells have no HLA restriction, and researches show that HLA semi-compatible cells have stronger tumor killing effect. Therefore, the culture of allogeneic NK cells has extremely high clinical value. Compared with peripheral blood, the umbilical cord blood has the advantages of convenient material acquisition, small harm and low pollution probability, and is the best NK cell preparation material. However, the application range of the umbilical cord blood is wide, and the application direction is not very determined at the initial storage stage, so the umbilical cord blood is firstly frozen and stored.
At present, the number, purity and activity of NK cells induced after the recovery of cryopreserved umbilical cord blood cannot meet the requirements of clinical application, and a great space is provided.
Disclosure of Invention
Aiming at the problems in the related art, the invention provides the NK cell derived from cryopreserved umbilical cord blood and the preparation method and application thereof, aims to overcome the technical problems in the prior related art, and aims to solve the problem that the number, purity and activity of the NK cell induced after the conventional cryopreserved umbilical cord blood is recovered cannot meet the clinical application requirements.
In order to achieve the above object, the present invention provides a technical solution,
in a first aspect, the present invention provides a method for preparing cryopreserved umbilical cord blood-derived NK cells, comprising the steps of,
the method comprises the following steps: quickly transferring the cryopreserved umbilical cord blood to a preheated water bath kettle at 37 ℃ for dissolution and resuscitation;
step two: transferring the recovered umbilical cord blood into a 250mL centrifuge bottle, adding a prepared washing solution containing a protective agent, fixing the volume to 200mL, centrifuging at 1200rpm for 5min, and washing to remove components of the frozen stock solution;
step three: centrifuging, removing supernatant, adding the lower layer cells into a washing solution containing a protective agent, mixing uniformly, slowly adding the mixture into a centrifugal tube filled with Ficoll in advance according to the volume ratio of 2:1, performing centrifugation by lifting 2 and reducing 1 at 2000rpm for 20min to extract mononuclear cells;
step four: sucking the middle leucocyte layer, adding a washing solution containing a protective agent, centrifuging at 1500 rpm for 8min, and washing for 2-3 times to obtain relatively pure cord blood mononuclear cells;
step five: the mononuclear cells were adjusted to a concentration of 2-4X 10 using NK cell basal medium6The concentration of the solution is/mL, autologous or allogeneic plasma with the volume fraction of 4-8%, 150-250IU/mL IL-2 and trophoblast cells are added for NK cell induction culture, and the result is marked as D0;
step six: centrifuging at 1500 rpm for 8min for D3 days, washing for 2-3 times, removing original culture medium, and changing the culture medium under the same conditions using D0;
step seven: adding NK cell basic culture medium for supplementing daily for D4-D7 days, and regulating cell concentration to 0.8-2 × 106Adding autologous or allogeneic plasma with the volume fraction of 4-8% and 150-250IU/mL IL-2 for NK cell induction;
step eight: adding NK cell basic culture medium for supplementing liquid in D8 days, and adjusting cell concentration to 0.8-2 × 106Adding autologous or allogeneic plasma with the volume fraction of 1-3, 150IU/mL IL-2 and trophoblast A2 cells for NK cell induced expansion;
step nine: adding NK cell basic culture medium for supplementing daily for D9-D13 days, and regulating cell concentration to 0.8-2 × 106Adding autologous or allogeneic plasma with the volume fraction of 1-3%, 150IU/mL and IL-2, and continuing to perform NK cell induced amplification;
step ten: cells were harvested on day D14, counted and examined.
Preferably, the NK cells are derived from cryopreserved umbilical cord blood, and the cryopreserved umbilical cord blood is cryopreserved umbilical cord blood which is preserved for less than 20 years by liquid nitrogen.
Preferably, the washing solution containing the protective agent comprises PBS buffer solution, human serum albumin, dextran and trehalose.
Preferably, the concentration of the mononuclear cells is 3X 106/mL。
Preferably, the autologous or allogeneic plasma concentration is 7% after the D0-D7 days.
Preferably, the autologous or allogeneic plasma concentration is 2% after the D8-D13 days.
Preferably, the IL-2 is added at a concentration of 200 IU/mL.
Preferably, the cell concentration is adjusted to 1.5X 10 in D4-D13 days6/mL。
In a second aspect, the present invention provides cryopreserved umbilical cord blood-derived NK cells prepared by the method of the first aspect.
In a third aspect, the present invention provides a washing solution containing a protecting agent in the preparation of NK cells according to the first aspect, comprising the following components: PBS buffer solution, human serum albumin, dextran, and trehalose;
the volume fraction of the human serum albumin added is 6 percent;
the volume fraction of dextran addition was 10%;
the mass volume fraction of trehalose is 9%;
the main reagent component is PBS buffer solution.
Compared with the prior art, the invention has the beneficial effects that:
(1) the invention relates to NK cells derived from cryopreserved umbilical cord blood and a preparation method and application thereof, wherein the NK cells are derived from the cryopreserved umbilical cord blood, and the number of the induced NK cells can reach 3 multiplied by 109The activity can reach more than 90 percent, the purity can reach more than 80 percent, and all indexes can meet the market application requirements;
(2) the cryopreserved umbilical cord blood used in the invention has sufficient time for pathogen detection compared with fresh blood, so that the pollution risk is reduced, and the safety of cells is improved;
(3) the invention relates to a cryopreserved umbilical cord blood-derived NK cell and a preparation method and application thereof, wherein the added protective agent components are all clinically available reagents, so that the safety of the NK cell is further ensured;
(4) the invention relates to a cryopreserved umbilical cord blood-derived NK cell, a preparation method and application thereof.
Drawings
FIG. 1 is a graph showing the results of flow assay in example 1 of the present invention;
FIG. 2 is a graph showing the results of the flow assay in example 2 of the present invention;
FIG. 3 is a graph showing the results of the flow assay in example 3 of the present invention;
fig. 4 is a graph of NK cell killing activity against K562 cells of the invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to 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.
Example one
A method for preparing cryopreserved umbilical cord blood-derived NK cells comprises the following steps,
1. taking a blood sample: the blood is guaranteed to be the cryopreserved umbilical cord blood which is preserved for less than 20 years by liquid nitrogen;
2. the preparation of NK cells is carried out,
the method comprises the following steps: quickly transferring the cryopreserved umbilical cord blood to a preheated water bath kettle at 37 ℃ for dissolution and resuscitation;
step two: transferring the recovered human umbilical cord blood into a 250mL centrifuge bottle, adding a prepared washing solution containing a protective agent, fixing the volume to 200mL, centrifuging at 1200rpm for 5min, and washing to remove components of a frozen stock solution;
step three: centrifuging, removing supernatant, adding the lower layer cells into a washing solution containing a protective agent, mixing uniformly, slowly adding the mixture into a centrifugal tube filled with Ficoll in advance according to the volume ratio of 2:1, performing centrifugation by lifting 2 and reducing 1 at 2000rpm for 20min to extract mononuclear cells;
step four: sucking the middle leucocyte layer, adding a washing solution containing a protective agent, centrifuging at 1500 rpm for 8min, and washing for 2-3 times to obtain relatively pure cord blood mononuclear cells;
step five: the cells were adjusted to 2X 10 concentration using NK cell basal medium6Adding autologous or allogenic plasma with volume fraction of 4%, 150IU/mL IL-2 and trophoblast cells into the mixture at a concentration of/mL, and performing NK cell induction culture, wherein the concentration is marked as D0;
step six: d3 centrifuging at 1500 rpm for 8min, washing for 2-3 times, removing original culture medium, and changing the culture medium under the same conditions using D0;
step seven: adding NK cell basic culture medium every day for supplementing liquid and adjusting cell concentration to 0.8 × 10 for D4-D7 days6Adding autologous or allogenic plasma with volume fraction of 4% and 150IU/mL IL-2 for NK cell induction;
step eight: adding NK cell basic culture medium for supplementing liquid in D8 days, and adjusting cell concentration to 0.8 × 106Adding autologous or allogeneic plasma with the volume fraction of 1%, 150IU/mL IL-2 and trophoblast A2 cells into the mixture for inducing and amplifying NK cells;
step nine: adding NK cell basic culture medium every day for supplementing liquid and adjusting cell concentration to 0.8 × 10 for D9-D13 days6Adding autologous or allogeneic plasma with the volume fraction of 1% and 150IU/mL IL-2, and continuing to perform NK cell induced amplification;
step ten: collecting cells in D14 days, counting, and detecting;
step eleven: taking NK cells and K562 cells for mixed culture, and detecting the in-vitro tumor killing effect by using a CCK-8 kit.
Example two
A method for preparing cryopreserved umbilical cord blood-derived NK cells comprises the following steps,
1. taking a blood sample: the blood is guaranteed to be the cryopreserved umbilical cord blood which is preserved for less than 20 years by liquid nitrogen;
2. the preparation of NK cells is carried out,
the method comprises the following steps: quickly transferring the cryopreserved umbilical cord blood to a preheated water bath kettle at 37 ℃ for dissolution and resuscitation;
step two: transferring the recovered human umbilical cord blood into a 250mL centrifuge bottle, adding a prepared washing solution containing a protective agent, fixing the volume to 200mL, centrifuging at 1200rpm for 5min, and washing to remove components of a frozen stock solution;
step three: centrifuging, removing supernatant, adding the lower layer cells into a washing solution containing a protective agent, mixing uniformly, slowly adding the mixture into a centrifugal tube filled with Ficoll in advance according to the volume ratio of 2:1, performing centrifugation by lifting 2 and reducing 1 at 2000rpm for 20min to extract mononuclear cells;
step four: sucking the middle leucocyte layer, adding a washing solution containing a protective agent, centrifuging at 1500 rpm for 8min, and washing for 2-3 times to obtain relatively pure cord blood mononuclear cells;
step five: the cells were adjusted to 3X 10 concentration using NK cell basal medium6Adding 7% autologous or allogeneic plasma, 200IU/mL IL-2 and trophoblast cells by volume fraction at the concentration of/mL, and performing NK cell induction culture, wherein the concentration is marked as D0;
step six: d3 centrifuging at 1500 rpm for 8min, washing for 2-3 times, removing original culture medium, and changing the culture medium under the same conditions using D0;
step seven: adding NK cell basic culture medium every day for supplementing liquid for D4-D7 days, and adjusting cell concentration to 1.5 × 106Adding 7% autologous or allogeneic plasma in volume fraction and 200IU/mL IL-2 for NK cell induction;
step eight: adding NK cell basic culture medium for supplementing liquid in D8 days, and adjusting cell concentration to 1.5 × 106Adding 2% autologous or allogeneic plasma, 200IU/mL IL-2 and trophoblast A2 cells by volume fraction, and performing NK cell induced amplification;
step nine: adding NK cell basic culture medium every day for supplementing liquid for D9-D13 days, and adjusting cell concentration to 1.5 × 106Per mL, and adding the volume2 percent of autologous or allogeneic plasma and 200IU/mL IL-2 are divided, and NK cell induced amplification is continuously carried out;
step ten: collecting cells in D14 days, counting, and detecting;
step eleven: taking NK cells and K562 cells for mixed culture, and detecting the in-vitro tumor killing effect by using a CCK-8 kit.
EXAMPLE III
A method for preparing cryopreserved umbilical cord blood-derived NK cells comprises the following steps,
1. taking a blood sample: the blood is guaranteed to be the cryopreserved umbilical cord blood which is preserved for less than 20 years by liquid nitrogen;
2. the preparation of NK cells is carried out,
the method comprises the following steps: quickly transferring the cryopreserved umbilical cord blood to a preheated water bath kettle at 37 ℃ for dissolution and resuscitation;
step two: transferring the recovered human umbilical cord blood into a 250mL centrifuge bottle, adding a prepared washing solution containing a protective agent, fixing the volume to 200mL, centrifuging at 1200rpm for 5min, and washing to remove components of a frozen stock solution;
step three: centrifuging, removing supernatant, adding the lower layer cells into a washing solution containing a protective agent, mixing uniformly, slowly adding the mixture into a centrifugal tube filled with Ficoll in advance according to the volume ratio of 2:1, performing centrifugation by lifting 2 and reducing 1 at 2000rpm for 20min to extract mononuclear cells;
step four: sucking the middle leucocyte layer, adding a washing solution containing a protective agent, centrifuging at 1500 rpm for 8min, and washing for 2-3 times to obtain relatively pure cord blood mononuclear cells;
step five: the cells were adjusted to 4X 10 concentration using NK cell basal medium6Adding autologous or allogenic plasma with volume fraction of 8%, 250IU/mL IL-2 and trophoblast cells into the mixture at a concentration of/mL, and performing NK cell induction culture, wherein the concentration is recorded as D0;
step six: d3 centrifuging at 1500 rpm for 8min, washing for 2-3 times, removing original culture medium, and changing the culture medium under the same conditions using D0;
step seven: adding NK cell basic culture medium every day for supplementing liquid and adjusting cell concentration to 2 × 10 for D4-D7 days6/mL, and adding 8% volume fraction of autologousOr allogeneic plasma and 250IU/mL IL-2, performing NK cell induction;
step eight: adding NK cell basic culture medium for supplementing liquid in D8 days, and adjusting cell concentration to 2 × 106Adding autologous or allogeneic plasma with the volume fraction of 3%, 250IU/mL IL-2 and trophoblast A2 cells into the mixture for inducing and amplifying NK cells;
step nine: adding NK cell basic culture medium every day for supplementing liquid and adjusting cell concentration to 2 × 10 for D9-D13 days6Adding autologous or allogeneic plasma with the volume fraction of 3% and 250IU/mL IL-2, and continuing to perform NK cell induced amplification;
step ten: collecting cells in D14 days, counting, and detecting;
step eleven: taking NK cells and K562 cells for mixed culture, and detecting the in-vitro tumor killing effect by using a CCK-8 kit.
Results of the experiment
(1) Number of NK cells: NK cells were harvested after induction culture for 14 days, and the number of cells was counted, and the results are shown in Table 1.
TABLE 1 NK cell number
As can be seen from Table 1, under the resuscitation inducing conditions described herein, the number of NK cells is more than 9 times of 10, and the amplification factor is about 50-70 times, so that the requirement of clinical application can be met.
(2) Purity of NK cells: NK cells are harvested, induced and cultured for 14 days, the purity of the cells is detected by flow, and the data result is shown in table 2; the flow assay results are shown in FIGS. 1-3.
TABLE 2 NK cell purity
As can be seen from Table 2, the purity of NK cells is above 80% under the resuscitation induction condition, and the requirement of clinical application can be met.
(3) NK cell survival rate: NK cells were harvested and cultured for 14 days, and the cell viability was measured, the results are shown in Table 3.
TABLE 3 NK cell viability
| Group of | Example 1 | Example 2 | Example 3 |
| NK cell survival Rate | 94% | 99% | 99% |
As can be seen from Table 3, the NK cell survival rate is above 90% under the resuscitation induction condition, which can meet the clinical application requirement.
(4) And (3) detecting the killing activity of the K562 cells before and after the NK cells are frozen: the NK cells of the above examples were selected and resuspended to 1X 10 using the basal medium, respectively6Per mL, for standby; k562 cells were selected as target cells and resuspended to 5X 10 in basal medium4Per mL, for standby;
adding NK cells and k562 cells in a 96-well plate according to effective target ratio of 5:1, 10:1 and 20:1, respectively, controlling total volume to 200uL, placing at 37 deg.C and 5% CO2Culturing in a condition culture box;
the killing rate calculation formula is as follows:
NK cell killing rate = [1- (experimental cell well a value-effector cell well a value)/target cell well a value ] × 100%.
After 24h, adding a CCK-8 reagent, placing in an incubator for 2-4h, and detecting by using a microplate reader, wherein the specific result is shown in figure 4.
As can be seen from FIG. 4, the killing rate of the NK cells induced by the method on k562 is kept above 70%, and the method has an obvious killing effect.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (9)
1. A preparation method of cryopreserved umbilical cord blood-derived NK cells is characterized by comprising the following steps:
the method comprises the following steps: quickly transferring the cryopreserved umbilical cord blood to a preheated water bath kettle at 37 ℃ for dissolution and resuscitation;
step two: transferring the recovered umbilical cord blood into a 250mL centrifuge bottle, adding a prepared washing solution containing a protective agent, fixing the volume to 200mL, centrifuging at 1200rpm for 5min, and washing to remove components of the frozen stock solution;
step three: centrifuging, removing supernatant, adding washing solution into lower layer cells, mixing, slowly adding into a centrifuge tube filled with Ficoll according to the volume ratio of 2:1, centrifuging at 2000rpm for 20min by lifting 2 to 1 to extract mononuclear cells;
step four: sucking the middle leucocyte layer, adding a washing solution containing a protective agent, centrifuging at 1500 rpm for 8min, and washing for 2-3 times to obtain relatively pure cord blood mononuclear cells;
step five: adjusting the concentration of the mononuclear cells to 2-4X 10 by using NK cell basal medium6The concentration of the solution is/mL, autologous or allogeneic plasma with the volume fraction of 4-8%, 150-250IU/mL IL-2 and trophoblast cells are added for NK cell induction culture, and the result is marked as D0;
step six: d3 centrifuging at 1500 rpm for 8min, washing for 2-3 times, removing original culture medium, and changing the culture medium under the same conditions using D0;
step seven: adding NK cell medium every day for D4-D7 daysSupplementing basic culture medium with cell concentration of 0.8-2 × 106Adding autologous or allogeneic plasma with the volume fraction of 4-8% and 150-250IU/mL IL-2 for NK cell induction;
step eight: adding NK cell basic culture medium for supplementing liquid in D8 days, and adjusting cell concentration to 0.8-2 × 106Adding autologous or allogeneic plasma with the volume 1-3 times that of the NK cell basal medium, 150IU/mL IL-2 and trophoblast A2 cells for NK cell induced amplification;
step nine: adding NK cell basic culture medium for supplementing daily for D9-D13 days, and regulating cell concentration to 0.8-2 × 106Adding autologous or allogeneic plasma with the volume fraction of 1-3% and 150IU/mL IL-2, and continuing to perform NK cell induced amplification;
step ten: cells were harvested on day D14, counted and examined.
2. The method according to claim 1, wherein the cryopreserved umbilical cord blood-derived NK cells are cryopreserved umbilical cord blood that has a liquid nitrogen preservation time of less than 20 years.
3. The method according to claim 1, wherein the washing solution containing the protecting agent comprises: PBS buffer solution, human serum albumin, dextran and trehalose.
4. The method according to claim 1, wherein the mononuclear cell concentration is 2X 106/mL。
5. The method for preparing cryopreserved umbilical cord blood-derived NK cells according to claim 1, wherein the autologous or allogeneic plasma concentration is 4% after the D0-D7 days.
6. The method for preparing cryopreserved umbilical cord blood-derived NK cells according to claim 1, wherein the autologous or allogeneic plasma concentration is 1% after the D8-D13 days.
7. The method for preparing cryopreserved umbilical cord blood-derived NK cells according to claim 1, wherein the IL-2 is added at a concentration of 150 IU/mL.
8. The method of claim 1, wherein the cell concentration is adjusted to 0.8X 10 in D4-D13 days6/mL。
9. A cryopreserved umbilical cord blood-derived NK cell, wherein the cryopreserved umbilical cord blood-derived NK cell is prepared by the preparation method according to any one of claims 1 to 8.
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