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CN-121991892-A - Preparation method of human placenta-derived NK cells

CN121991892ACN 121991892 ACN121991892 ACN 121991892ACN-121991892-A

Abstract

The invention relates to the technical field of immune cells, in particular to a preparation method of human placenta-derived NK cells. The preparation method comprises the following steps of S1, placenta tissue separation, S2, placenta tissue digestion, S3, mononuclear cell separation, S4.NK cell separation and S5.NK cell amplification culture.

Inventors

  • LV YIN

Assignees

  • 艾伯维(天津)生物科技有限公司

Dates

Publication Date
20260508
Application Date
20260311

Claims (7)

  1. 1. A method for preparing human placenta-derived NK cells, comprising the steps of: s1, placenta tissue separation, namely taking placenta tissue, shearing, centrifuging and discarding supernatant to obtain placenta tissue homogenate; S2, digesting placenta tissues, namely performing digestion treatment and resuspension on the placenta tissue homogenate obtained in the step S1 to obtain a cell suspension; S3, separating mononuclear cells, namely separating the cell suspension obtained in the step S2 by using lymphocyte separation liquid to obtain placenta mononuclear cells; S3, NK cell sorting, namely, sorting the NK cells by using the placenta mononuclear cells obtained in the step S3 to obtain placenta-derived NK cells of CD56 + CD3 - ; s4, NK cells are amplified and cultured, namely the NK cells obtained in the step 3 are resuspended by using a complete culture medium, inoculated into a culture bottle coated by a CD16 antibody, and the complete culture medium is supplemented; The complete culture medium is prepared by adding 500-800 IU/mL of IL-2, 20-40 ng/mL of IL-12, 5-10 ng/mL of IL-18, 10-20 ng/mL of IL-21, 5-10 ng/mL of IL-21 and 2-3 mg/mL of porous microspheres into a serum-free culture medium of a basic culture medium Lonza X-VIVO 15; The preparation method of the porous microsphere comprises the following steps: (1) Cleaning flos Lonicerae, oven drying, pulverizing, adding into deionized water, heating for extraction, concentrating, precipitating with ethanol, deproteinizing by Sevage method, dialyzing, and lyophilizing to obtain flos Lonicerae extract; (2) Dissolving the honeysuckle extract obtained in the step (1) in deionized water, adding sodium periodate, stirring, adding glycol, dialyzing, and freeze-drying to obtain an oxidized honeysuckle extract; (3) Adding epsilon-polylysine into DMSO, adding succinic anhydride, stirring, dialyzing, and freeze-drying to obtain modified polylysine; (4) Adding the modified polylysine obtained in the step (3) into DMSO, adding CDI, stirring, adding DMAP and resveratrol, stirring, dialyzing, and freeze-drying to obtain EPL-RSV; (5) Mixing paraffin and span 80 uniformly to obtain an oil phase, adding the oxidized honeysuckle extract obtained in the step (2), EPL-RSV and polyethylene glycol obtained in the step (4) into PBS buffer solution, mixing uniformly to obtain a water phase, dripping the water phase into the oil phase, stirring, centrifuging, washing the precipitate, drying, filtering, and freeze-drying to obtain the porous microspheres.
  2. 2. The method according to claim 1, wherein in step S2, the complex enzyme solution is serum-free DMEM medium containing type I collagenase, type IV collagenase, dnase I.
  3. 3. The method of producing human placenta-derived NK cells according to claim 2, wherein in step (2), the mass ratio of sodium periodate to honeysuckle extract is 1:2-3.
  4. 4. The method of producing human placenta-derived NK cells according to claim 3, wherein the mass ratio of polylysine to succinic anhydride in step (3) is 8-10:1.
  5. 5. The method of claim 4, wherein in step (4), the mass ratio of the modified polylysine, CDI, DMAP, and resveratrol is 10:0.8:1:3.
  6. 6. The method of claim 5, wherein in step (5), the mass ratio of the oxidized honeysuckle extract, EPL-RSV and polyethylene glycol 4000 is 2-4:3-5:1-2.
  7. 7. The method of claim 6, wherein in step (5), the volume ratio of the aqueous phase to the oil phase is 1:3-5.

Description

Preparation method of human placenta-derived NK cells Technical Field The invention relates to the technical field of immune cells, in particular to a preparation method of human placenta-derived NK cells. Background Natural killer cells (Natural KILLER CELL, NK cells) are core effector cells of the innate immune system of the organism, can specifically identify and kill tumor cells, virus-infected cells and abnormal proliferation cells without antigen pre-sensitization, have the function of immunoregulation, and are one of immune cells with the most application potential in the fields of tumor immunotherapy, antiviral infection therapy and autoimmune disease therapy. NK cells have the advantage that they can recognize and kill tumor cells without antigen sensitization and MHC restriction, and play an important role in immune responses such as anti-tumor and early antiviral of the body. But NK cells occupy a low proportion and a small quantity in vivo, which limits the application of NK cells. At present, an NK cell in vitro amplification system mainly comprises the following steps of firstly adopting immunomagnetic bead cell sorting to obtain purified NK cells from peripheral blood mononuclear cells, adding factors in vitro for amplification culture, secondly adopting a feeder cell method to amplify primary NK cells, and thirdly, stimulating the amplification of NK cells by a simple cytokine combination. The first method needs to extract a large amount of peripheral blood of a patient, and can only amplify tens of times, the second method can obtain a large amount of high-purity NK cells, but external cell line pollution is easy to introduce, and the third method is good in safety, high in clinical application value, and low in amplification factor and unstable. In addition, the conventional two-dimensional culture space has low utilization rate and poor uniformity of the environment where cells are located, so that the cell expansion efficiency is difficult to improve. Disclosure of Invention Aiming at the defects of the prior art, the invention provides a preparation method of human placenta-derived NK cells. The invention is realized by the following technical scheme: A method for preparing human placenta-derived NK cells, comprising the steps of: S1, placenta tissue separation, namely repeatedly flushing fresh placenta tissue with PBS buffer solution containing 1% penicillin-streptomycin for 3-5 times under aseptic condition, peeling placenta amniotic membrane, cutting the placenta chorion tissue into fragments of 1-3 mm 3, washing the placenta tissue with the PBS buffer solution for 2-3 times, centrifuging at 4 ℃ and 500 g for 5min, and discarding supernatant to obtain placenta tissue homogenate; S2, digesting the placenta tissue, namely, adopting a compound enzyme solution to digest the placenta tissue homogenate obtained in the step S1, immediately adding an equal volume of DMEM culture medium containing 10% fetal bovine serum to terminate digestion after digestion, filtering with a 200-mesh screen, collecting filtrate, washing with PBS buffer for 2 times, re-suspending with PBS buffer, and adjusting the cell concentration to 1X 10 7/mL to obtain a cell suspension; S3, separating mononuclear cells, namely slowly superposing the cell suspension obtained in the step S2 above the liquid level of the equal volume of human lymphocyte separation liquid along the pipe wall, centrifuging at 4 ℃ and 500 g to 20 min, sucking mononuclear cells in an intermediate tunica media layer, washing 2 times with PBS buffer, centrifuging at 4 ℃ and 600 g to 8 min, and discarding the supernatant to obtain placenta mononuclear cells; S3, NK cell sorting, namely, selecting the placenta mononuclear cells obtained in the step S3, and carrying out NK cell sorting by an immunomagnetic bead negative selection method by using an NK cell negative selection kit to obtain placenta-derived NK cells of CD56 +CD3-; S4, NK cells obtained in the step S3 are resuspended by using a complete medium, the cell concentration is regulated to be 1 multiplied by 10 6/mL, inoculated into a culture bottle which is coated with 5-10 mug/mL of CD16 antibody in advance, placed into a 37 ℃ and 5% CO 2 incubator for culture, the complete medium is supplemented every 2-3 days, the cell concentration is maintained to be 1-1.5X10 6/mL, and cells are collected after 14-d of culture. Further, the specific operation of the digestion treatment in the step S2 is that placenta tissue homogenate and compound enzyme solution are mixed according to the volume ratio of 1:3-5, and are placed in a constant temperature incubator with the temperature of 37 ℃ and the CO 2% for shaking digestion for 30-40 min, and the mixture is gently and evenly mixed once every 10 min. In step S2, the compound enzyme solution is serum-free DMEM medium containing type I collagenase, type IV collagenase and DNase I, wherein the final concentration of the type I collagenase is 0.8-1.2 mg/mL