Umbilical cord blood screening standard established based on CD3-CD56 + cell proportion and NK cell in-vitro amplification method
By establishing umbilical cord blood screening criteria based on the proportion of CD3-CD56+ cells and specific culture methods, the problem of unstable purity in NK cell in vitro culture was solved, the induction success rate and production efficiency of NK cells were improved, and the stability of cell products and the reliability of clinical applications were ensured.
Patent Information
- Application Number
- CN202511326700.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-17
- Publication Date
- 2025-12-09
AI Technical Summary
In the current technology, the induction success rate of NK cells in vitro culture is unstable, resulting in low purity of the final harvested NK cells, which cannot meet clinical or research needs and wastes human, material and time costs.
We established umbilical cord blood screening criteria based on the proportion of CD3-CD56+ cells. Samples with an initial CD3-CD56+ cell proportion ≥6% were screened for in vitro amplification, detected by flow cytometry, and cultured using specific culture media and reagents, including initial culture, medium change and replenishment, bag culture, and harvesting steps.
It significantly improves the success rate of NK cell induction, reduces production costs, enhances culture efficiency, ensures the stability of cell products and their clinical applications, and reduces treatment delays caused by batch failures.
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Figure CN121086983A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a screening criterion for umbilical cord blood based on the ratio of CD3-CD56+ cells, and a method for in vitro expansion of NK cells, belonging to the field of in vitro culture of NK cells. Background Technology
[0002] Natural killer cells (NK cells) are important immune cells in the body, possessing broad-spectrum anti-tumor and antiviral effects, and demonstrating great potential in immune regulation and tumor immunotherapy. Umbilical cord blood, due to its convenient collection and low immunogenicity, has become an ideal source for in vitro NK cell culture. The efficiency of its in vitro induction and expansion, and the quality of the final product, directly determine the safety and effectiveness of its clinical application.
[0003] In vitro induction culture of NK cells is a key step in their clinical application. The purity of NK cells after culture (measured by CD3+) is crucial. - CD56 + Cell ratio (as a marker) is a core indicator for measuring culture effectiveness, directly impacting the safety and efficacy of its clinical application. Currently, the in vitro culture of NK cells often faces the problem of unstable induction success rates, resulting in low purity (CD3+) of the final harvested NK cells. - CD56 + When the cell ratio is too low to meet clinical or research needs, it leads to a waste of human, material, and time resources. The initial state of the cells has a decisive influence on the final expansion efficiency and functional activity. Therefore, it is necessary to establish a CD3-based... - CD56 + The umbilical cord blood screening criteria based on cell ratio, and the in vitro expansion method of NK cells based on these criteria, are of great significance for improving production efficiency, increasing culture success rate, and ensuring the stability of cell products. Summary of the Invention
[0004] In view of the above-mentioned prior art, the present invention provides a screening standard for umbilical cord blood based on the ratio of CD3-CD56+ cells, and provides an in vitro expansion method for NK cells based on the screening standard.
[0005] This invention is achieved through the following technical solution:
[0006] A CD3-based - CD56 + The cell ratio selection criteria for cord blood used for in vitro NK cell expansion are as follows: CD3+ in cord blood mononuclear cells. - CD56 + Cell percentage ≥6%.
[0007] An in vitro expansion method for NK cells includes the following steps:
[0008] (i) After collecting umbilical cord blood, mononuclear cells are separated;
[0009] (ii) Flow cytometry was used to detect mononuclear cells and measure CD3. - CD56 + Initial cell ratio;
[0010] (III) Regarding CD3 - CD56 + Mononuclear cells with an initial proportion of ≥6% were cultured to harvest NK cells.
[0011] Furthermore, in step (iii), the specific method of cultivation is as follows:
[0012] (1) Initial culture: Mononuclear cells were resuspended in NK cell basal medium containing 200 U / mL IL-2 and 5% autologous inactivated plasma, and the cell density was controlled at 1.0 × 10⁶ cells / mL. 6 ~2.0×10 6 Add cells / mL, add A1 component of NK amplification reagent, transfer to T75 culture flask, place in an incubator, and incubate at 37℃ and 5% CO2.
[0013] (2) Medium change and replenishment: On the 3rd day of culture, centrifuge, discard the supernatant, and resuspend the cells in NK cell basal medium containing 200 U / mL IL-2 and 5% autologous inactivated plasma, and continue culturing; on the 4th to 6th days of culture, observe the cells daily, and add NK cell basal medium containing 200 U / mL IL-2 according to the color of the cell suspension or the cell volume, and add autologous inactivated plasma at a volume ratio of 1% to maintain the cell density at 1.0 × 10⁻⁶. 6 cells / mL;
[0014] (3) Transfer to a culture bag: On day 7 of culture, cells were transferred to a culture bag, and NK cell basal medium containing 200 U / mL IL-2 and 5% autologous inactivated plasma, as well as component A2 of the NK amplification reagent, were added. Culture continued. From day 8 to day 14 of culture, cells were observed daily. Based on the color of the cell suspension or the cell count, NK cell basal medium containing 200 U / mL IL-2 was added, and autologous inactivated plasma was added at a volume ratio of 1% to maintain a cell density of 1.0 × 10⁻⁶ cells / mL. 6 cells / mL;
[0015] (4) Harvest: On the 14th day of culture, collect the culture medium, centrifuge at 800g for 10min, discard the supernatant, add physiological saline to resuspend, centrifuge at 800g for 10min, discard the supernatant, and obtain NK cells.
[0016] Furthermore, the NK cell basal culture medium is selected from Corning KBM581 medium.
[0017] This invention posits that the initial state of cells has a decisive influence on the final expansion efficiency and functional activity of NK cells. Based on this, this invention studies the CD3+ of 82 umbilical cord blood mononuclear cells. - CD56 + Cell proportions and CD3 of NK cells used for in vitro expansion culture. - CD56 + Statistical analysis of cell proportions concluded that: if CD3+ is present in umbilical cord blood mononuclear cells... - CD56 + If the cell proportion is ≥6%, then CD3 in NK cells obtained by in vitro expansion culture can be obtained. - CD56 + The percentage of cells with a proportion ≥80% was extremely high, significantly higher than that of CD3 in umbilical cord blood mononuclear cells. - CD56 + Cell percentage <6% group. Therefore, this invention establishes a CD3-based... - CD56 + The cell ratio selection criteria for cord blood used for in vitro NK cell expansion are as follows: CD3+ in cord blood mononuclear cells. - CD56 + Cell percentage ≥6%.
[0018] The in vitro expansion method for NK cells of the present invention can significantly improve the success rate of NK cell induction and greatly reduce production costs: by early screening of low-potential samples (initial CD3). - CD56 ++ With a cell percentage of <6%, this method avoids ineffective culture of low-potential samples, saving on material, equipment, labor, and time costs; it also improves the stability of clinical supply, reducing treatment delays caused by batch failures and protecting patients' rights. This is of great significance for improving production efficiency, increasing culture success rates, and ensuring the stability of cell products. Attached Figure Description
[0019] Figure 1 CD3 counts of mononuclear cells in samples 1, 2, and 3 at initial and harvest time. - CD56 + A schematic diagram of the cell proportion detection results, where 1-1 represents the initial CD3 content of mononuclear cells in sample 1. - CD56 + The cell proportion detection results, 1-2 are the CD3 content of mononuclear cells at harvest from sample 1. - CD56 +Cell proportion detection results; 2-1 is the initial CD3 count of mononuclear cells in sample 2. - CD56 + The cell proportion detection results, 2-2 are the CD3 content of mononuclear cells at harvest from sample 2. - CD56 + Cell proportion detection results; 3-1 is the initial CD3 count of mononuclear cells in sample 3. - CD56 + The cell proportion detection results, 3-2 are the CD3 content of mononuclear cells at harvest from sample 3. - CD56 + Results of cell proportion detection.
[0020] Figure 2 CD3 counts of mononuclear cells in samples 4, 5, and 6 at initial and harvest time. - CD56 + A schematic diagram of the cell proportion detection results, where 4-1 represents the initial CD3 content of mononuclear cells in sample 4. - CD56 + The cell proportion detection results, 4-2 are the CD3 content of mononuclear cells at harvest from sample 4. - CD56 + Cell proportion detection results; 5-1 is the initial CD3 count of mononuclear cells in sample 5. - CD56 + The cell proportion detection results, 5-2 is the CD3 content of mononuclear cells at harvest from sample 5. - CD56 + Cell proportion detection results; 6-1 is the initial CD3 count of mononuclear cells in sample 6. - CD56 + The cell proportion detection results show that 6-2 represents the CD3 content of mononuclear cells from sample 6 at harvest. - CD56 + Results of cell proportion detection. Detailed Implementation
[0021] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.
[0022] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.
[0023] Research on experimental umbilical cord blood screening criteria
[0024] In existing technologies, the purity of NK cells cultured in vitro (measured by CD3+) is often a concern. - CD56 + This study addresses situations where the proportion of cells (as a marker) is too low to meet clinical or research needs. To resolve this technical issue, this experiment determined the initial CD3+ level in umbilical cord blood mononuclear cells. - CD56 + A critical value for cell proportion was determined to establish a screening criterion for umbilical cord blood, thereby improving the success rate of in vitro NK cell expansion, reducing ineffective culture, increasing production efficiency, and promoting standardization and proactive risk control in the cell culture process. The specific process is described below.
[0025] (I) In vitro isolation of umbilical cord blood mononuclear cells
[0026] (1) Umbilical cord blood collection: Umbilical cord blood is collected from the umbilical cord of a full-term healthy newborn and placed in a blood collection bag containing an anticoagulant. The blood is then separated within 24 hours after collection.
[0027] (2) Original blood aliquoting: Wipe the surface of the blood bag containing the umbilical cord blood sample with alcohol for disinfection, and use a 10mL pipette to inject the whole blood into a 50mL centrifuge tube, with each tube containing no more than 40mL of blood.
[0028] (3) Plasma separation and processing: After aliquoting the original blood, centrifuge at 800g for 10 min at room temperature. Transfer the supernatant plasma to a new 50mL centrifuge tube using a 10mL pipette. After sealing the plasma, place it in a water bath at 56℃ for 30 min to inactivate complement in the plasma. Then freeze it at -20℃ for 10 min, centrifuge at 1800g for 10 min, and transfer the supernatant plasma to a clean 50mL centrifuge tube.
[0029] (4) Ficoll density gradient centrifugation: After plasma separation, the remaining blood cells are diluted with physiological saline at a volume ratio of 1:1 and mixed by pipetting. The blood cell suspension is slowly loaded onto the Ficoll layer (keeping the layers clear), with a final volume of no more than 45 mL per tube, and centrifuged at 500 g for 20 min at room temperature.
[0030] (5) Collection of mononuclear cells: Use a 10mL pipette to aspirate most of the uppermost layer of liquid, and try to aspirate the second layer of boundary cell layer (white membrane layer), transfer it to a 50mL centrifuge tube, add physiological saline to make up to 45mL, invert and mix well, centrifuge at room temperature and 450g for 10min, discard the supernatant, and obtain umbilical cord blood mononuclear cell precipitate.
[0031] A total of 82 umbilical cord blood samples were collected in this experiment. The number of isolated mononuclear cells met the requirements, and the endotoxin, mycoplasma, and bacterial culture tests were all qualified.
[0032] (II) Culture of Umbilical Cord Blood Mononuclear Cells
[0033] The 82 mononuclear cells obtained above were cultured separately, and the steps were as follows:
[0034] (1) Initial culture: Mononuclear cells were resuspended in 30 mL of Corning KBM581 medium containing 200 U / mL IL-2 and 5% autologous inactivated plasma (i.e., 1.5 mL of autologous inactivated plasma and IL-2 were added to Corning KBM581 medium to achieve a concentration of 200 U / mL), and the cell density was controlled at 1.5 × 10⁻⁶ cells / mL. 6 The cells / mL were added to the A1 component of the NK amplification reagent, transferred to a T75 culture flask, and placed in an incubator for incubation at 37°C and 5% CO2. The NK amplification reagent is a commercially available product in the prior art, containing A1 and A2 components, and the one used in this invention was purchased from Hangzhou Zhongying Biomedical Technology Co., Ltd.
[0035] (2) Medium change and replenishment: On the 3rd day of culture, centrifuge at 350g for 5min, discard the supernatant, and resuspend the cells in 30mL of Korn KBM581 medium containing 200U / mL IL-2 and 5% autologous inactivated plasma, and continue culturing; on the 4th to 6th days of culture, observe the cells daily, and add Korn KBM581 medium containing 200U / mL IL-2 according to the color of the cell suspension or the cell volume, and add autologous inactivated plasma at a volume ratio of 1% to maintain the cell density at 1.0×10⁻⁶. 6 cells / mL.
[0036] (3) Transfer to a culture bag: On day 7 of culture, transfer the cells to a culture bag, add 30 mL of Korn Ferry KBM581 medium containing 200 U / mL IL-2 and 5% autologous inactivated plasma, and the A2 component of the NK amplification reagent, and continue culturing. From day 8 to day 14 of culture, observe the cells daily, and supplement with Korn Ferry KBM581 medium containing 200 U / mL IL-2 and autologous inactivated plasma at a volume ratio of 1% according to the color of the cell suspension or the cell mass to maintain the cell density at 1.0 × 10⁻⁶ cells / mL. 6 cells / mL.
[0037] (4) Harvest: On the 14th day of culture, collect the culture medium, centrifuge at 800g for 10min, discard the supernatant, add physiological saline to resuspend, centrifuge at 800g for 10min, discard the supernatant, and obtain NK cells.
[0038] (III) Phenotypic Detection of NK Cells
[0039] Phenotypic analysis was performed on 82 initial mononuclear cells and cultured NK cells, as follows:
[0040] (1) Sample processing: Transfer the cell sample into a flow cytometry tube using a 1 mL manual pipette, add 3 mL PBS and mix thoroughly. Centrifuge at 1600 rpm for 5 min, discard the supernatant, and resuspend the cells in an appropriate amount of PBS to adjust the cell concentration to 1.0 × 10⁻⁶. 7 cells / mL.
[0041] (2) Fluorescent labeling: Label flow cytometry tubes, labeling two tubes for each sample: tube 1: IgG-FITC / IgG-PE; tube 2: CD3-FITC / CD56-PE; add the corresponding fluorescent antibody to each tube, with each antibody amounting to 10 μl. Add 100 μl of the cell sample suspension to be tested to each tube.
[0042] (3) Gently rotate the mixture on a vortex mixer and incubate at room temperature in the dark for 15 minutes.
[0043] (4) Wash with 3 mL PBS, centrifuge at 1600 rpm for 5 min, discard the supernatant, add 500 μl PBS to suspend the precipitate, and mix thoroughly.
[0044] (5) On-machine testing, based on the same type comparison, CD3 analysis. - CD56 + Cell ratio.
[0045] (iv) Results
[0046] CD3 was found in mononuclear cells isolated from 82 umbilical cord blood samples. - CD56 + Fifteen cases had an initial cell percentage ≥8% (referred to as Group A), and 7% ≤ CD3 - CD56 + Eighteen cases had an initial cell percentage ≤8% (referred to as Group B), and 6% ≤ CD3 - CD56 + 23 cases had an initial cell percentage ≤7% (referred to as group C), CD3 - CD56 + There were 26 cases with an initial cell percentage of <6% (referred to as group D).
[0047] In harvested NK cells, CD3 - CD56 + A cell percentage of ≥80% is considered acceptable, CD3 - CD56 + Cells with a proportion <80% are considered unqualified; Induction success rate = Number of qualified cases / Total number of cases × 100%.
[0048] For Group A, there were 14 qualified cases and 1 unqualified case, with a success rate of 93.44%.
[0049] For Group B, there were 16 qualified cases and 2 unqualified cases, with a success rate of 88.89%.
[0050] For group C, there were 21 qualified cases and 2 unqualified cases, with a success rate of 91.31%.
[0051] For group D, there were 14 qualified cases and 12 unqualified cases, with a success rate of 53.85%.
[0052] The percentages of non-compliant cases in groups A, B, and C were 6.66%, 11.1%, and 8.69%, respectively, with no statistically significant difference between the groups (χ²). 2 =0.523, P=0.770); while the proportion of unqualified cases in group D was significantly higher than that in the first three groups (χ²=0.523, P=0.770). 2 =24.371, P<0.001). Further analysis of the induction success rate showed: Group A was 93.34% (14 / 15), Group B was 88.9% (16 / 18), Group C was 91.31% (21 / 23), and Group D was only 53.85% (14 / 26), suggesting that CD3 - CD56 + The induction success rate was significantly improved when the initial cell percentage was ≥6%. This indicates that CD3 - CD56 + An initial cell ratio of 6% can be used as a screening threshold or critical threshold.
[0053] Six mononuclear cells were randomly selected from umbilical cord blood and analyzed for their CD3 content. - CD56 + The initial cell ratio, and the CD3 content of NK cells obtained from culture and harvest. - CD56 + The cell ratios are shown in Table 1, and the flow cytometry results are as follows: Figure 1 , Figure 2 As shown.
[0054] Table 1. CD3 content of mononuclear cells at initial and harvest time - CD56 + Cell ratio
[0055]
[0056]
[0057] This experiment identified CD3+ in umbilical cord blood mononuclear cells. - CD56 +A critical initial cell percentage (6%) was established to create a screening criterion for umbilical cord blood used in the in vitro expansion of NK cells. The in vitro expansion method for NK cells established using this screening criterion can significantly improve culture efficiency and reduce culture and umbilical cord blood collection costs, providing important technical support for the standardized preparation and clinical application of NK cells.
[0058] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.
Claims
1. A method for in vitro expansion of NK cells, characterized in that, Includes the following steps: (i) After collecting umbilical cord blood, mononuclear cells are separated; (ii) Flow cytometry was used to detect mononuclear cells and measure CD3. - CD56 + Initial cell ratio; (III) Regarding CD3 - CD56 + Mononuclear cells with an initial proportion of ≥6% were cultured to harvest NK cells.
2. The method for in vitro expansion of NK cells according to claim 1, characterized in that, In step (iii), the specific method of cultivation is as follows: (1) Initial culture: Mononuclear cells were resuspended in NK cell basal medium containing 200 U / mL IL-2 and 5% autologous inactivated plasma, and the cell density was controlled at 1.0 × 10⁻⁶ cells / mL. 6 ~2.0×10 6 Add cells / mL, add A1 component of NK amplification reagent, transfer to T75 culture flask, place in an incubator, and incubate at 37℃ and 5% CO2; (2) Medium change and replenishment: On the 3rd day of culture, centrifuge, discard the supernatant, and resuspend the cells in NK cell basal medium containing 200 U / mL IL-2 and 5% autologous inactivated plasma, and continue culturing; from the 4th to the 6th day of culture, replenish NK cell basal medium containing 200 U / mL IL-2 daily, and add autologous inactivated plasma at a volume ratio of 1% to maintain the cell density at 1.0 × 10⁻⁶ cells / day. 6 cells / mL; (3) Transfer to a culture bag: On day 7 of culture, transfer to a culture bag, add NK cell basal medium containing 200 U / mL IL-2 and 5% autologous inactivated plasma, and the A2 component of the NK amplification reagent, and continue culturing; from day 8 to day 14 of culture, supplement NK cell basal medium containing 200 U / mL IL-2 daily, and add autologous inactivated plasma at a volume ratio of 1% to maintain a cell density of 1.0 × 10⁻⁶ cells / day. 6 cells / mL; (4) Harvest: On the 14th day of culture, collect the culture medium, centrifuge at 800g for 10min, discard the supernatant, add physiological saline to resuspend, centrifuge at 800g for 10min, discard the supernatant, and obtain NK cells.
3. The method for in vitro expansion of NK cells according to claim 2, characterized in that: The NK cell basal culture medium was selected from Corning KBM581 medium.
4. A CD3-based - CD56 + The cell ratio screening criteria for umbilical cord blood used for in vitro NK cell expansion are characterized by: CD3 in umbilical cord blood mononuclear cells - CD56 + Cell percentage ≥6%.