CN112662626A - Method for co-culturing natural killer cells by umbilical cord mesenchymal stem cells - Google Patents
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Abstract
The invention provides a method for co-culturing natural killer cells by umbilical cord mesenchymal stem cells, which utilizes umbilical cord mesenchymal stem cells with the same source as an umbilical cord blood supplier to co-culture with nucleated cells in umbilical cord blood, provides a microenvironment for promoting proliferation and differentiation for the nucleated cells in the umbilical cord blood, realizes the in-vitro large-scale amplification of the nucleated cells in the umbilical cord blood to NK cells and precursor cells thereof, reduces the demand of umbilical cord blood raw materials in the subsequent NK cell large-scale production, is applied to the industrialization of subsequent NK cell treatment products, contains hematopoietic stem cells capable of reconstructing human hematopoietic and immune systems in the umbilical cord blood, and can be used for hematopoietic stem cell transplantation. Therefore, cord blood has become an important source of hematopoietic stem cells, and particularly a source of hematopoietic stem cells without relationship to blood, and is also a very important human biological resource.
Description
Technical Field
The invention relates to a method for co-culturing natural killer cells by umbilical cord mesenchymal stem cells, belonging to the technical field of cells.
Background
For a long time, the traditional treatment means represented by surgery, radiotherapy and chemotherapy obtains better curative effect in the treatment of malignant tumor. However, these treatments are limited to different tumors and are associated with significant side effects. Therefore, the search for a therapeutic method which is less harmful to patients and can effectively control tumor growth and metastasis is an urgent need for clinical tumor treatment. With the rapid development and cross-penetration of related subjects such as tumor biology, immunology, molecular biology and the like, the research on tumor immunotherapy is rapidly advanced, and the tumor immune cell therapy established by taking the immunology principle as the basis and the cell biology technology as the method is gradually transited from laboratory research to effective and safe clinical application.
Natural killer cells (also called NK cells) are the main cells of natural immunity of the body, and NK cells are large granular lymphocytes in morphology, are derived from bone marrow, are third-class lymphocytes except T cells and B cells, and account for about 15% of all immune cells (white blood cell number) in blood. NK cells are nonspecific in recognizing target cells, and participate in the recognition process of NK cells through the action of intercellular adhesion molecule-1 (ICAM-1) on the surface of the target cells. In addition, NK cells kill target cells by secreting perforin, NK cytotoxic factors, TNF, and the like. Since the killing activity of NK cells is MHC-independent, antibody-independent, it is called natural killing activity. In recent years, research on NK cells and their anti-tumor function has become one of the hot contents of immunological and oncologic research. NK cell anti-tumor has been carried out a large amount of clinical tests in countries such as the United states and Japan, and shows good application prospect.
Natural killer cells (NK cells) function as innate immune cells, and their activation and killing is not MHC restricted nor pre-primed. Therefore, it is not limited to autologous use, and allogeneic NK immune cell therapy is receiving increasing attention.
NK cells are derived from bone marrow lymphoid stem cells, the differentiation and development of the NK cells depend on bone marrow and thymus microenvironment, and the NK cells are mainly distributed in bone marrow, peripheral blood, liver, spleen, lung and lymph nodes. The NK cells currently used in immunocytotherapy are mainly derived from donor peripheral blood, allogeneic cord blood and NK cell lines.
The traditional NK cell line is NK92 cell derived from peripheral blood of malignant non-Hodgkin lymphoma patient, which can maintain effective proliferation in the presence of low concentration IL-2, and has strong cytotoxic effect at the concentration of 100IU/ml IL-2. Like NK cells, NK-92 recognizes target cells without MHC restriction, kills tumor cells without prior sensitization, and produces a series of cytokines that regulate the acquired immunity of the body. Currently, induced pluripotent stem cells (iPS cells) are used as a source and are induced in vitro into NK cells, and the NK cells are generated continuously by using the characteristic of unlimited proliferation of iPSc in vitro. The recombinant human NK cell is used as an excellent cell source for allogeneic NK immune cell therapy. However, there is a great safety risk in both NK92 cell line and the induced differentiation into NK cell source by iPSc. First, the NK92 cell line is derived from tumor cells, can be proliferated indefinitely in vitro, and has a larger canceration risk. Therefore, irradiation inactivation is required before use, so that the long-term survival time of the patient in the body of the patient is limited, and the treatment effect is influenced. And iPSc is induced and differentiated into an NK cell source, so that the iPSc cell has the capability of unlimited proliferation and has the canceration risk. Secondly, iPSc is subjected to gene manipulation, and the tumorigenicity of the iPSc is possibly increased. Therefore, the NK cell source using cell line as allogeneic source has a large safety risk although it is low in mass production cost and stable in quality.
Adult peripheral blood is used as a source of NK cells, and a donor of the NK cells can be a receptor or other healthy adult donors, so that the technology is low in cost and flexible to realize. However, in tumor therapy, the recipient himself is generally a tumor patient, and there is a risk that the biological activity of the cells is low and the number of cells is small when the recipient himself is a cell source. Although the use of NK cells is not limited by MHC, certain immunogenicity still exists, immunological rejection can be caused in allogeneic use, whether autologous or allogeneic adult donors are NK cell sources, certain infectious disease risks exist, complete isolation operation is needed in the production process to avoid cross contamination, so that adult peripheral blood is used as the NK cell source, although the technical requirement is low and the production is more flexible, the quality of a final product is unstable and the quality control cost is higher due to the inconsistency of the cell sources, and the cells of each donor need to be produced and isolated as independent product batches, so that the production cost is increased without any change, but the biological source of umbilical cord blood is the newborn and the biological characteristics of the umbilical cord blood are inconsistent. Second, the total number of cells per donor is limited per unit of cord blood, limited by the volume of blood at the time of collection. Therefore, the key to the application of cord blood as a source of NK cells for immune cell therapy is to achieve large-scale production of cells.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a method for co-culturing natural killer cells by umbilical cord mesenchymal stem cells, so as to solve the problems in the background technology.
In order to achieve the purpose, the invention is realized by the following technical scheme: a method for co-culturing natural killer cells by umbilical cord mesenchymal stem cells, which utilizes umbilical cord mesenchymal stem cells with the same source as an umbilical cord blood supplier to co-culture with nucleated cells in umbilical cord blood, provides a microenvironment for promoting proliferation and differentiation for the nucleated cells in the umbilical cord blood, realizes the in-vitro large-scale amplification of the nucleated cells in the umbilical cord blood to NK cells and precursor cells thereof, reduces the demand on umbilical cord blood raw materials in the subsequent NK cell large-scale production, and is applied to the industrialization of subsequent NK cell treatment products.
Further, a method for culturing natural killer cells by umbilical cord mesenchymal stem cells comprises the following steps:
(1) separating and freezing umbilical cord blood nucleated cells;
(2) umbilical cord mesenchyme preparation and cryopreservation of the same donor;
(3) co-culturing and in-vitro amplifying the nucleated cells of the umbilical cord blood and the mesenchymal stem cells of the umbilical cord;
(4) sorting the amplified cord blood nucleated cells NK cells;
(5) in vitro large scale amplification and cryopreservation of purified NK cells.
Further, the separation and cryopreservation of the nucleated cells of the umbilical cord blood comprise the following steps:
(1) acquiring umbilical cord and umbilical cord blood of a newborn who normally gives birth from a hospital, and collecting the umbilical cord blood by using a 200ml disposable blood collection bag containing blood preservation solution;
(2) the sterility-ensuring environment and handling of the collection, and the confirmation that the biological mother and biological father-donor of the neonate are not at risk of infectious diseases before collection, including but not limited to the following infectious diseases: hepatitis B, hepatitis C, syphilis and HIV, and the collection process is free from bacterial, fungal and mycoplasma contamination;
(3) uniformly mixing the collected cord blood, transferring the cord blood into a 250ml centrifuge bottle, adding 1/4 amount of 6% hydroxyethyl starch sodium chloride injection, fully and uniformly mixing, suspending in a super clean bench, and standing for 45 min;
(4) sucking the upper layer of all suspension rich in nucleated cells, sampling, counting the number and the survival rate of the nucleated cells, centrifuging at 4 ℃ at 1500rpm/min for 8min, removing redundant centrifugal supernatant according to the counting result, and adjusting the volume of the cell suspension;
(5) adjusting the cell density to 1 x 10^6cells/ml, and if the volume of the cell centrifugation supernatant is insufficient, supplementing the volume corresponding to 6% hydroxyethyl starch sodium chloride injection;
(6) resuspended cells were treated according to cell suspension: adding DMSO into DMSO at a volume ratio of 9:1, and subpackaging into 5ml frozen storage tubes according to the production of 1.2 x 10^7cells in each batch;
(7) and (3) sequentially placing the subpackaged cells at 4 ℃ for 30min, at-20 ℃ for 2h and at-80 ℃ for 12h to complete program cooling, and placing the subsequent cells in a gas-phase liquid nitrogen tank for long-term storage.
Further, the umbilical cord mesenchyme preparation and cryopreservation of the same donor comprises the following steps:
(1) taking 10-20cm umbilical cord of a full-term infant collected under aseptic condition with the same umbilical cord blood as a newborn donor source, flushing with normal saline until the surface has no blood component residue, removing the surface amnion, two umbilical artery blood vessels and one umbilical vein blood vessel, shearing, adding a tissue block into a mesenchymal stem cell serum-free complete culture medium containing 1% double antibody, and then adding into a cell culture bottle for culture under the environment of 37 ℃, saturation humidity and 5% CO 2;
(2) the mesenchymal stem cell serum-free complete culture medium is a prepared StemRD Mgro-500MesenGro human mesenchymal stem cell serum-free culture medium;
(3) replacing half of fresh serum-free complete culture medium containing double antibody every 5 days during the first 3 periods of culture, when the cell fusion degree reaches 70-80%, digesting the cells with 0.25% of trypsin, filtering the cells by using a 100 mu m cell filter, brushing the cells by using physiological saline, and centrifugally recovering the cells;
(4) the recovered human umbilical cord mesenchymal stem cells are resuspended in serum-free complete culture medium without double antibodies and added into a T175 cell culture flask to be cultured under the environment of 37 ℃, saturation humidity and 5% CO 2;
(5) when the cell fusion degree of each generation reaches 90%, 0.25% of trypsin digests the cells, the cells are washed by physiological saline, centrifuged at 1100rpm for 5 minutes and recovered, the cells of the batch are suspended in a serum-free complete culture medium according to the proportion of 1 bottle to 3 bottles and are inoculated in T175 for continuous expansion for 1-5 generations, and the cells are cultured in an environment with 37 ℃ and saturated humidity and 5% CO 2;
(6) when the cell fusion degree reaches 90% after passage expansion of P5 generation, 0.25% of trypsin digests cells, after being washed by physiological saline, the cells are recovered by centrifugation at 1100rpm for 5 minutes, the recovered cells are collected by centrifugation at 1100rpm for 5 minutes after being re-suspended and counted by DMEM, and the cell density is adjusted to 1 x 10^7cells/ml according to the counting result and then re-suspended by mesenchymal stem cell frozen stock solution;
(7) the formula of the mesenchymal stem cell frozen stock solution is a DMEM basal medium: fetal bovine serum: DMSO is 6:3:1, the cell suspension after being resuspended is divided into 5ml freezing tubes according to the total number of 1.2 x 10^8 cells;
(8) and (3) sequentially placing the subpackaged cells at 4 ℃ for 30min, at-20 ℃ for 2h and at-80 ℃ for 12h to complete program cooling, and placing the subsequent cells in a gas-phase liquid nitrogen tank for long-term storage.
Further, the coculture in vitro amplification of the umbilical cord blood nucleated cells and the umbilical cord mesenchymal stem cells comprises the following steps:
(1) respectively recovering umbilical cord blood nucleated cells and umbilical cord mesenchymal stem cells from the same source donor, rapidly adding the corresponding cell freezing tube into a 10-time volume DMEM basic culture medium after melting in a water bath at 37 ℃, diluting and centrifuging at 1500rpm for 5min, removing centrifugal supernatant, and then resuspending in a co-culture complete culture medium;
(2) the formulation of the co-culture complete culture medium is a basic culture medium prepared by mixing StemRD Mgro-500MesenGro human mesenchymal stem cell serum-free culture medium and GIBCO AIM-V medium CTS serum-free culture medium according to the ratio of 1:2, and adding 300ng/ml FLT-3L, 300ng/ml SCF, 10ng/ml IL-3, 5ng/ml IL-7, 50IU/ml TNF-alpha, 100ng/ml GM-CSF, 5ng/ml TPO, 100ng/ml IL-6 and 40ng/ml IL-2;
(3) the initial culture volume of co-culture inoculation is 100ml, and the resuscitated umbilical cord blood nucleated cells and the P5 generation umbilical cord mesenchymal stem cells are directly cultured together in a suspension way according to the quantity ratio of 1:10, wherein the density of the umbilical cord blood nucleated cells is controlled to be more than 1 x 10 cells/ml;
(4) injecting the cells into a 2L cell culture bag for culture under the conditions of 37 ℃, saturation humidity and 5% CO2, and sampling and counting every 2 days in the culture process;
(5) supplementing fresh co-culture complete medium after sampling counting until the cell density is slightly between 1 x 10^6cells/ml and 1.5 x 10^6 cells/ml;
(6) after final replenishment to a 2L medium volume based on cell density, the cells were cultured for an additional 2 days, and all cell suspensions were harvested and centrifuged at 1500rpm for 5min to collect all cells and sampled for counting.
Further, the sorting of the expanded cord blood nucleated cells NK cells comprises the following steps:
(1) adjusting the cell density of the harvested co-cultured nucleated cells to 4 x 10^6cells/ml by using PBS according to counting results, slowly adding the cell density to the upper layer of the separating liquid of the Ficoll human peripheral blood lymphocytes, wherein the volume ratio of the Ficoll to the cell suspension is 1: 1;
(2) performing density gradient centrifugation, wherein the centrifugation conditions are 300g,20min and 20 ℃, and the centrifugation acceleration and deceleration are set as the minimum values allowed by the centrifuge;
(3) after centrifugation is finished, removing supernatant liquid to absorb cells of a middle leucocyte layer, washing and uniformly mixing the recovered cell suspension by using PBS with 4 times of volume, and centrifuging at the room temperature at 1100rpm for 5 min;
(4) collection of cell pellets cell density was adjusted by resuspension in PBS to 1 x 10 < SP > 7 </SP >/80 ul, and CD3 microspheres were added at 10 < SP > 7 </SP >/20 ul. After mixing, incubation is carried out for 15min at 4 ℃. PBS was added at 10^7/2 ml. Centrifuging at 1100rpm for 10 minutes, resuspending the precipitate to 500ul, and separating with autoMACS column to obtain CD 3-cells;
(5) the cells were washed 3 times with PBS, and CD56 microspheres were added in the same manner, mixed well and incubated at 4 ℃ for 15 min. Adding PBS according to 10^7/2ml, centrifuging for 10min at 1100rpm, resuspending the pellet to 500ul, and separating by using an autoMACS column to obtain CD3-CD56+ cells.
Further, the in vitro large-scale expansion and cryopreservation of the purified NK cells comprises the following steps:
(1) coating a T225 culture bottle with 20ml of monoclonal antibody at 4 ℃ for 24h before inoculating the purified NK cells, wherein the monoclonal antibody is 35ng/ml of CD25 monoclonal antibody, 5ug/ml of CD16 monoclonal antibody and 5ug/ml of CD56 monoclonal antibody is dissolved in PBS, and discarding a coating solution and brushing the coating solution with PBS before inoculating the purified NK cells;
(2) the initial culture volume of the purified NK cells is 50ml, and the controlled density of the purified NK cells is more than 2 x 10^6cells/ml and is suspended in GIBCO AIM-V medium CTS serum-free culture medium;
(3) 24h after cell inoculation, the culture bottle is supplemented with 50ml of NK stimulating medium and 10ml of 20% human serum albumin;
(4) the NK cell activation culture medium is based on a GIBCO AIM-V medium CTS serum-free culture medium, and 15ng/ml IL-18, 20ng/ml IL-15, 10ng/ml IL-7, 100IU/ml GM-CSF and 20ng/ml IL-12 are added;
(5) on day 3 of cell culture, the flask was supplemented with 50ml of NK stimulating medium and 5ml of 20% human serum albumin;
(6) on day 5 of cell culture, the culture flask was supplemented with 100ml of NK stimulating medium and 10ml of 20% human serum albumin;
(7) on the 7 th day of cell culture, after blowing out all cells in the culture bottle, sampling and counting, subpackaging the cells into 2L cell culture bags and supplementing the cells to the total volume of 600 ml;
(8) the NK cell amplification culture medium is a GIBCO AIM-V medium CTS serum-free culture medium added with 1000IU/ml IL-2;
(9) adding an NK cell amplification culture medium to the 9 th day of culture of the purified NK cells till the total volume is 1200 ml;
(10) adding NK cell amplification culture medium to the total volume of 2000ml on the 11 th day of purified NK cell culture;
(11) sampling and counting cell suspensions on 7 th, 9 th, 11 th and 14 th days of purified NK cell culture, using an NK cell amplification culture medium, and controlling the cell density after fluid infusion to be more than 1.5 x 10^6 cells/ml;
(12) after the culture is carried out for 3 days after the solution is supplemented to 2L volume, all cell suspensions are collected, centrifuged and washed to obtain all suspension cells, and the obtained cells are centrifuged for 5min at 1100rpm after counting;
(13) collecting cell sediment, using NK cell frozen stock solution to re-suspend and adjust the cell density to 2.5 x 10^7cells/ml according to counting results, and subpackaging the cell sediment into 25ml frozen stock bags, wherein each bag is provided with 4 x 10^8 cells;
(14) the volume ratio of the NK frozen stock solution is 20% human serum albumin: DMSO ═ 9: 1;
(15) performing cell cryopreservation on the cells subjected to cryopreservation subpackaging by using a Thermo programmed cooling instrument, and placing the subsequent cells in a gas-phase liquid nitrogen tank for long-term storage;
(16) the prepared NK cells are stored in a liquid nitrogen tank to be used for subsequent treatment;
further, the production method can scale up the production of each batch of cells in the same ratio according to the production conditions.
The invention has the beneficial effects that: cord blood, which is blood remaining in the placenta and umbilical cord after the fetus is delivered, the umbilical cord is ligated and detached, is usually discarded and used, and contains hematopoietic stem cells capable of reconstituting the human hematopoietic and immune systems, and can be used for hematopoietic stem cell transplantation. Therefore, cord blood has become an important source of hematopoietic stem cells, and particularly a source of hematopoietic stem cells without relationship to blood, and is also a very important human biological resource. The application of umbilical cord blood as the source of NK cells has the following advantages: (1) CD34+ hematopoietic stem cells contained in umbilical cord blood can be induced, differentiated and massively expanded in vitro to serve as a large-scale source of NK and precursor cells thereof; (2) the umbilical cord blood comes from the newborn, does not contact the external environment, and has low infection risk of human viruses and microorganisms; (3) umbilical cord blood is used as medical waste, and a stable cell source can be formed through umbilical cord blood banks in various places; (4) the cells in the cord blood are less immunogenic than the original cells and have a lower rejection risk.
Drawings
Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:
FIG. 1 is a schematic diagram showing the amplification factor of the co-cultured cells of nucleated cells of umbilical cord blood and mesenchymal stem cells of umbilical cord;
FIG. 2 is a schematic diagram showing in vitro expansion of purified NK cells of the present invention;
Detailed Description
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.
Example 1: according to the cell amplification curve of the co-culture of the umbilical cord blood nucleated cells and the umbilical cord mesenchymal stem cells, 5 umbilical cord blood nucleated cells and umbilical cord mesenchymal stem cells from different newborn donors are separated and cultured to P5 generations according to the specific embodiment of the invention, and the cells are temporarily frozen and stored in a liquid nitrogen tank after being prepared. According to the technical scheme of the invention, the umbilical cord blood nucleated cells 1.2 x 10^7cells and the P5 umbilical cord mesenchymal stem cells 1.2 x 10^8cells are recovered in each batch of water bath at 37 ℃. According to the cell counting result, the cells are inoculated into 100ml of co-culture complete medium, and the inoculation density is that the umbilical cord blood nucleated cells are 1.0 x 10^6cells/ml and the P5 generation umbilical cord mesenchymal stem cells are 1.0 x 10^7 cells/ml. The subsequent culture was carried out in a 2L cell culture bag under conditions of 37 deg.C, saturated humidity and 5% CO2, and the samples were sampled and counted every 2 days during the culture. Complete sampling and counting and then supplementing fresh cocultivation complete medium until the cell density is slightly between 1 x 10^6cells/ml and 1.5 x 10^6 cells/ml. After final replenishment to a 2L medium volume based on cell density, the cells were cultured for an additional 2 days, and all cell suspensions were harvested and centrifuged at 1500rpm for 5min to collect all cells and sampled for counting.
As a result: the 5 groups of co-cultured cells were subjected to cell counting, and the fold amplification of the cells was calculated as shown in FIG. 1
And (4) conclusion: the nucleated cells of the umbilical cord blood and the mesenchymal stem cells of the umbilical cord are co-cultured, and the cells in the culture bag are greatly amplified.
Example 2: collecting differentiation statistics after co-culturing the umbilical cord blood nucleated cells and the umbilical cord mesenchymal stem cells, recovering and washing 5 groups of umbilical cord blood nucleated cells obtained by separation and nucleated cells obtained by co-culturing and amplification in the first embodiment, washing with DMEM to adjust the cell density to 1 x 10^6cells/ml, uniformly mixing 100ul and 900ul Stemcell MethoCult human hematopoietic stem cell methyl cellulose culture medium, and inoculating the mixture into a 24-hole plate, wherein each group has 3 multiple holes. The 24-well plate was cultured at 37 ℃ under saturated humidity and 5% CO2 for 14 to 16 days, and the clone count was performed.
As a result: after 16 days of culture, the number of hematopoietic cell colonies in 24-well plates counted microscopically was as follows:
TABLE 1 statistics of colony differentiation after co-culture of nucleated cells of umbilical cord blood and mesenchymal stem cells of umbilical cord
And (4) conclusion: after the umbilical cord blood nucleated cells are co-cultured with the umbilical cord mesenchymal stem cells, the proliferation capacity and the differentiation capacity of the umbilical cord blood nucleated cells are greatly enhanced.
Example 3: after the cord blood nucleated cells are sorted, carrying out flow cytometry on phenotype statistics on flow cytometry, namely CD3-CD56+, sorting the nucleated cells obtained according to the first embodiment of the invention by using CD3-CD56+ MACS magnetic beads according to the technical scheme of the invention, and respectively carrying out statistics on cell purity of NK cell phenotype CD3-CD16+ CD56+ before and after sorting by using flow cytometry.
| Before sorting | After sorting | |
| Group 1 | 20.45％ | 98.77 |
| Group | ||
| 2 | 19.56％ | 97.53％ |
| Group 3 | 15.34％ | 94.35％ |
| Group 4 | 12.56％ | 97.12％ |
| Group 5 | 14.37％ | 96.51％ |
TABLE 2 comparison of NK cell purity before and after MACS CD3-CD56+ sorting
And (4) conclusion: MACS magnetic bead sorting can effectively purify NK cells and precursor cells thereof.
Example 4: and (3) carrying out in-vitro large-scale amplification proliferation curve on the NK cells, inoculating the purified NK cells obtained in the third embodiment into the coated T225 culture bottle according to the technical scheme of the invention, and adding the cytokines for stimulation the next day of inoculation. Then, according to the growth condition and density of the cells, fluid replacement is carried out to 2L volume, and the cells are counted and harvested after 3 days of culture.
As a result: the 5 groups of co-cultured cells were subjected to cell counting, and the fold expansion of the cells was calculated as shown in FIG. 2.
And (4) conclusion: after the umbilical cord blood-derived nucleated cells are co-cultured with umbilical cord mesenchymal stem cells and amplified in vitro, the umbilical cord blood-derived nucleated cells can be stimulated to be amplified in vitro in a large scale by various cytokines.
Example 5: and (3) performing flow type phenotype statistics on the NK cells after in-vitro large-scale amplification, recovering the NK cells prepared in the fourth embodiment, and performing flow cytometry detection on the recovered NK cells to respectively count the purities of different groups of NK cells.
| Before sorting | |
| Group 1 | 98.85 |
| Group | |
| 2 | 95.53％ |
| Group 3 | 96.22％ |
| Group 4 | 99％ |
| Group 5 | 96.82％ |
Table 3 shows the purity of in vitro amplified cord blood-derived NK cells
And (4) conclusion: the process of the invention can prepare high-purity NK cells in vitro.
Example 6: and detecting the killing capacity of the prepared NK cells, counting the subculture cell strain K562 cells, preparing a cell suspension of 1 x 10^5cells/mL, and adding the cell suspension into a 96-well plate, wherein each well is 50 mu l. Four cultured NK cells cultured according to the technical scheme of the invention are respectively subjected to the following effects: adding the target of 1:1, 10:1, 20:1 and 40:1 into a 96-well plate, simultaneously setting effector cells and target cells natural release holes, culture medium natural release holes, target cells maximum release holes and volume correction control, setting 3 multiple holes in each pore volume of 100 mu l, centrifuging for 4min at 250g, and incubating for 4h in an incubator at 37 ℃, 5% CO2 and 95% saturated humidity. In total, the killing ability of 5 groups of NK cells was examined. 10. mu.l of lysis solution was added to each well 45min before the end of the reaction. After the reaction, 50. mu.l of supernatant and 50. mu.l of LDH enzyme reaction solution were aspirated from each well and placed in another new 96-well plate, and reacted for 30min at room temperature in the dark, 50. mu.l of reaction stop solution was added, and the OD value was measured with an enzyme-linked immunosorbent assay. The natural killer activity was calculated. The formula is as follows: the% natural killer activity is (measurement tube OD value-target cell natural release tube OD value-effector cell natural release tube OD value)/(target cell maximum release tube OD value-target cell natural release tube OD value) × 100%.
As a result:
| effective target ratio | 1:1 | 10:1 | 20:1 | 40:1 |
| Group 1 | 15.37％ | 47.56％ | 61.33％ | 91.21 |
| Group | ||||
| 2 | 11.49％ | 48.40％ | 59.72％ | 85.45％ |
| Group 3 | 18.14％ | 41.78％ | 69.84％ | 87.49％ |
| Group 4 | 10.07％ | 39.84％ | 62.48％ | 89.32％ |
| Group 5 | 9.98％ | 45.58％ | 70.80％ | 92.75％ |
TABLE 4 in vitro killing ability of NK cells from umbilical cord blood of various donor sources in each group
And (4) conclusion: according to the technical scheme of the invention, the NK cell with a killing effect on the K562 cancer cell strain can be prepared and can be used for subsequent cancer cell treatment.
While there have been shown and described what are at present considered the fundamental principles and essential features of the invention and its advantages, it will be apparent to those skilled in the art that the invention is not limited to the details of the foregoing exemplary embodiments, but is capable of other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (8)
1. A method for culturing natural killer cells by umbilical cord mesenchymal stem cells is characterized by comprising the following steps: the method utilizes umbilical cord mesenchymal stem cells with the same source as an umbilical cord blood donor to perform co-culture with the nucleated cells in the umbilical cord blood, provides a microenvironment for promoting proliferation and differentiation for the nucleated cells in the umbilical cord blood, realizes the in-vitro large-scale amplification of the nucleated cells in the umbilical cord blood to NK cells and precursor cells thereof, reduces the requirement on umbilical cord blood raw materials in the subsequent large-scale production of the NK cells, and is applied to the industrialization of subsequent NK cell treatment products.
2. The method of claim 1, wherein the natural killer cell is co-cultured with the umbilical cord mesenchymal stem cell, and the method comprises the following steps: the method comprises the following steps:
(1) separating and freezing umbilical cord blood nucleated cells;
(2) umbilical cord mesenchyme preparation and cryopreservation of the same donor;
(3) co-culturing and in-vitro amplifying the nucleated cells of the umbilical cord blood and the mesenchymal stem cells of the umbilical cord;
(4) sorting the amplified cord blood nucleated cells NK cells;
(5) in vitro large scale amplification and cryopreservation of purified NK cells.
3. The method of claim 2, wherein the natural killer cell is co-cultured with the umbilical cord mesenchymal stem cell, and the method comprises the following steps: the separation and cryopreservation of the nucleated cells of the umbilical cord blood comprise the following steps:
(1) acquiring umbilical cord and umbilical cord blood of a newborn who normally gives birth from a hospital, and collecting the umbilical cord blood by using a 200ml disposable blood collection bag containing blood preservation solution;
(2) the sterility-ensuring environment and handling of the collection, and the confirmation that the biological mother and biological father-donor of the neonate are not at risk of infectious diseases before collection, including but not limited to the following infectious diseases: hepatitis B, hepatitis C, syphilis and HIV, and the collection process is free from bacterial, fungal and mycoplasma contamination;
(3) uniformly mixing the collected cord blood, transferring the cord blood into a 250ml centrifuge bottle, adding 1/4 amount of 6% hydroxyethyl starch sodium chloride injection, fully and uniformly mixing, suspending in a super clean bench, and standing for 45 min;
(4) sucking the upper layer of all suspension rich in nucleated cells, sampling, counting the number and the survival rate of the nucleated cells, centrifuging at 4 ℃ at 1500rpm/min for 8min, removing redundant centrifugal supernatant according to the counting result, and adjusting the volume of the cell suspension;
(5) adjusting the cell density to 1 x 10^6cells/ml, and if the volume of the cell centrifugation supernatant is insufficient, supplementing the volume corresponding to 6% hydroxyethyl starch sodium chloride injection;
(6) resuspended cells were treated according to cell suspension: adding DMSO into DMSO at a volume ratio of 9:1, and subpackaging into 5ml frozen storage tubes according to the production of 1.2 x 10^7cells in each batch;
(7) and (3) sequentially placing the subpackaged cells at 4 ℃ for 30min, at-20 ℃ for 2h and at-80 ℃ for 12h to complete program cooling, and placing the subsequent cells in a gas-phase liquid nitrogen tank for long-term storage.
4. The method of claim 2, wherein the natural killer cell is co-cultured with the umbilical cord mesenchymal stem cell, and the method comprises the following steps: the umbilical cord mesenchyme preparation and cryopreservation of the same donor comprises the following steps:
(1) taking 10-20cm umbilical cord of a full-term infant collected under aseptic condition with the same umbilical cord blood as a newborn donor source, flushing with normal saline until the surface has no blood component residue, removing the surface amnion, two umbilical artery blood vessels and one umbilical vein blood vessel, shearing, adding a tissue block into a mesenchymal stem cell serum-free complete culture medium containing 1% double antibody, and then adding into a cell culture bottle for culture under the environment of 37 ℃, saturation humidity and 5% CO 2;
(2) the mesenchymal stem cell serum-free complete culture medium is a prepared StemRD Mgro-500MesenGro human mesenchymal stem cell serum-free culture medium;
(3) replacing half of fresh serum-free complete culture medium containing double antibody every 5 days during the first 3 periods of culture, when the cell fusion degree reaches 70-80%, digesting the cells with 0.25% of trypsin, filtering the cells by using a 100 mu m cell filter, brushing the cells by using physiological saline, and centrifugally recovering the cells;
(4) the recovered human umbilical cord mesenchymal stem cells are resuspended in serum-free complete culture medium without double antibodies and added into a T175 cell culture flask to be cultured under the environment of 37 ℃, saturation humidity and 5% CO 2;
(5) when the cell fusion degree of each generation reaches 90%, 0.25% of trypsin digests the cells, the cells are washed by physiological saline, centrifuged at 1100rpm for 5 minutes and recovered, the cells of the batch are suspended in a serum-free complete culture medium according to the proportion of 1 bottle to 3 bottles and are inoculated in T175 for continuous expansion for 1-5 generations, and the cells are cultured in an environment with 37 ℃ and saturated humidity and 5% CO 2;
(6) when the cell fusion degree reaches 90% after passage expansion of P5 generation, 0.25% of trypsin digests cells, after being washed by physiological saline, the cells are recovered by centrifugation at 1100rpm for 5 minutes, the recovered cells are collected by centrifugation at 1100rpm for 5 minutes after being re-suspended and counted by DMEM, and the cell density is adjusted to 1 x 10^7cells/ml according to the counting result and then re-suspended by mesenchymal stem cell frozen stock solution;
(7) the formula of the mesenchymal stem cell frozen stock solution is a DMEM basal medium: fetal bovine serum: DMSO is 6:3:1, the cell suspension after being resuspended is divided into 5ml freezing tubes according to the total number of 1.2 x 10^8 cells;
(8) and (3) sequentially placing the subpackaged cells at 4 ℃ for 30min, at-20 ℃ for 2h and at-80 ℃ for 12h to complete program cooling, and placing the subsequent cells in a gas-phase liquid nitrogen tank for long-term storage.
5. The method of claim 2, wherein the natural killer cell is co-cultured with the umbilical cord mesenchymal stem cell, and the method comprises the following steps: the co-culture in-vitro amplification of the umbilical cord blood nucleated cells and the umbilical cord mesenchymal stem cells comprises the following steps:
(1) respectively recovering umbilical cord blood nucleated cells and umbilical cord mesenchymal stem cells from the same source donor, rapidly adding the corresponding cell freezing tube into a 10-time volume DMEM basic culture medium after melting in a water bath at 37 ℃, diluting and centrifuging at 1500rpm for 5min, removing centrifugal supernatant, and then resuspending in a co-culture complete culture medium;
(2) the formulation of the co-culture complete culture medium is a basic culture medium prepared by mixing StemRD Mgro-500MesenGro human mesenchymal stem cell serum-free culture medium and GIBCO AIM-V medium CTS serum-free culture medium according to the ratio of 1:2, and adding 300ng/ml FLT-3L, 300ng/ml SCF, 10ng/ml IL-3, 5ng/ml IL-7, 50IU/ml TNF-alpha, 100ng/ml GM-CSF, 5ng/ml TPO, 100ng/ml IL-6 and 40ng/ml IL-2;
(3) the initial culture volume of co-culture inoculation is 100ml, and the resuscitated umbilical cord blood nucleated cells and the P5 generation umbilical cord mesenchymal stem cells are directly cultured together in a suspension way according to the quantity ratio of 1:10, wherein the density of the umbilical cord blood nucleated cells is controlled to be more than 1 x 10 cells/ml;
(4) injecting the cells into a 2L cell culture bag for culture under the conditions of 37 ℃, saturation humidity and 5% CO2, and sampling and counting every 2 days in the culture process;
(5) supplementing fresh co-culture complete medium after sampling counting until the cell density is slightly between 1 x 10^6cells/ml and 1.5 x 10^6 cells/ml;
(6) after final replenishment to a 2L medium volume based on cell density, the cells were cultured for an additional 2 days, and all cell suspensions were harvested and centrifuged at 1500rpm for 5min to collect all cells and sampled for counting.
6. The method of claim 2, wherein the natural killer cell is co-cultured with the umbilical cord mesenchymal stem cell, and the method comprises the following steps: the expanded cord blood nucleated cell NK cell sorting method comprises the following steps:
(1) adjusting the cell density of the harvested co-cultured nucleated cells to 4 x 10^6cells/ml by using PBS according to counting results, slowly adding the cell density to the upper layer of the separating liquid of the Ficoll human peripheral blood lymphocytes, wherein the volume ratio of the Ficoll to the cell suspension is 1: 1;
(2) performing density gradient centrifugation, wherein the centrifugation conditions are 300g,20min and 20 ℃, and the centrifugation acceleration and deceleration are set as the minimum values allowed by the centrifuge;
(3) after centrifugation is finished, removing supernatant liquid to absorb cells of a middle leucocyte layer, washing and uniformly mixing the recovered cell suspension by using PBS with 4 times of volume, and centrifuging at the room temperature at 1100rpm for 5 min;
(4) collecting cell precipitates, using PBS to carry out resuspension to adjust the cell density to 1 x 10^7cells/80ul, adding CD3 microspheres according to 10^7cells/20ul, evenly mixing, then carrying out incubation for 15min at 4 ℃, adding PBS according to 10^7/2ml, carrying out centrifugation at 1100rpm for 10 subpackaging, carrying out resuspension to 500ul, and then using an autoMACS to carry out column separation to obtain CD 3-cells;
(5) washing cells with PBS 3 times, adding CD56 microsphere in the same way, mixing uniformly, incubating for 15min at 4 ℃, adding PBS according to 10^7/2ml, centrifuging for 10min at 1100rpm, suspending the precipitate to 500ul, and separating with autoMACS column to obtain CD3-CD56+ cells.
7. The method of claim 2, wherein the natural killer cell is co-cultured with the umbilical cord mesenchymal stem cell, and the method comprises the following steps: the in vitro large-scale amplification and cryopreservation of the purified NK cells comprises the following steps:
(1) coating a T225 culture bottle with 20ml of monoclonal antibody at 4 ℃ for 24h before inoculating the purified NK cells, wherein the monoclonal antibody is 35ng/ml of CD25 monoclonal antibody, 5ug/ml of CD16 monoclonal antibody and 5ug/ml of CD56 monoclonal antibody is dissolved in PBS, and discarding a coating solution and brushing the coating solution with PBS before inoculating the purified NK cells;
(2) the initial culture volume of the purified NK cells is 50ml, and the controlled density of the purified NK cells is more than 2 x 10^6cells/ml and is suspended in GIBCO AIM-V medium CTS serum-free culture medium;
(3) 24h after cell inoculation, the culture flask was supplemented with 50ml of NK stimulating medium and 10ml of 20% human serum albumin;
(4) the NK cell activation culture medium is based on a GIBCO AIM-V medium CTS serum-free culture medium, and 15ng/ml IL-18, 20ng/ml IL-15, 10ng/ml IL-7, 100IU/ml GM-CSF and 20ng/ml IL-12 are added;
(5) on day 3 of cell culture, the flask was supplemented with 50ml of NK stimulating medium and 5ml of 20% human serum albumin;
(6) on day 5 of cell culture, the culture flask was supplemented with 100ml of NK stimulating medium and 10ml of 20% human serum albumin;
(7) on the 7 th day of cell culture, after blowing out all cells in the culture bottle, sampling and counting, subpackaging the cells into 2L cell culture bags and supplementing the cells to the total volume of 600 ml;
(8) the NK cell amplification culture medium is a GIBCO AIM-V medium CTS serum-free culture medium added with 1000IU/ml IL-2;
(9) adding an NK cell amplification culture medium to the 9 th day of culture of the purified NK cells till the total volume is 1200 ml;
(10) adding NK cell amplification culture medium to the total volume of 2000ml on the 11 th day of purified NK cell culture;
(11) sampling and counting cell suspensions on 7 th, 9 th, 11 th and 14 th days of purified NK cell culture, using an NK cell amplification culture medium, and controlling the cell density after fluid infusion to be more than 1.5 x 10^6 cells/ml;
(12) after the culture is carried out for 3 days after the solution is supplemented to 2L volume, all cell suspensions are collected, centrifuged and washed to obtain all suspension cells, and the obtained cells are centrifuged for 5min at 1100rpm after counting;
(13) collecting cell sediment, using NK cell frozen stock solution to re-suspend and adjust the cell density to 2.5 x 10^7cells/ml according to counting results, and subpackaging the cell sediment into 25ml frozen stock bags, wherein each bag is provided with 4 x 10^8 cells;
(14) the volume ratio of the NK frozen stock solution is 20% human serum albumin: DMSO ═ 9: 1;
(15) performing cell cryopreservation on the cells subjected to cryopreservation subpackaging by using a Thermo programmed cooling instrument, and placing the subsequent cells in a gas-phase liquid nitrogen tank for long-term storage;
(16) the prepared NK cells were stored in a liquid nitrogen tank for subsequent therapeutic use.
8. The method of claim 1, wherein the natural killer cell is co-cultured with the umbilical cord mesenchymal stem cell, and the method comprises the following steps: the preparation method can adjust the production scale of each batch of cells according to the same scale enlargement according to the production conditions.
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