CN106834227B - In vitro purification method of natural killer cells - Google Patents
In vitro purification method of natural killer cells Download PDFInfo
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- CN106834227B CN106834227B CN201611219859.7A CN201611219859A CN106834227B CN 106834227 B CN106834227 B CN 106834227B CN 201611219859 A CN201611219859 A CN 201611219859A CN 106834227 B CN106834227 B CN 106834227B
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Abstract
The invention discloses an in vitro purification method of natural killer cells, which comprises the following steps: providing a solution of CD3 antibody; loading the CD3 antibody solution into a clinical-grade culture container, and fully reacting to obtain the clinical-grade culture container with the surface coated with the CD3 antibody; loading the natural killer cell mixed culture to be purified into a clinical-grade culture container coated with a CD3 antibody, culturing under conventional culture conditions, and collecting supernatant; and centrifuging the supernatant, reserving the precipitate, and washing the precipitate to obtain the purified natural killer cells. The in vitro purification method of the natural killer cells utilizes a clinical-grade culture container coated with a CD3 antibody to bear and culture a mixed culture of the natural killer cells, CD3 positive hybrid cells in the mixed culture of the natural killer cells are combined with a CD3 antibody to be attached to the inner wall of the clinical-grade culture container, and after the culture is finished, supernatant in the clinical-grade culture container is collected to obtain the purified natural killer cells.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to an in-vitro purification method of natural killer cells.
Background
Natural Killer cells (NK cells), an important lymphocyte component in natural immune cells, play an important role in immune surveillance of tumor and pathogen intracellular infected cells. The in vitro amplification of NK cells is a key technology in the NK cell treatment technology and is widely concerned. The prior in vitro NK cell amplification technology has various types, and the prior main NK cell in vitro amplification technology is divided into two modes of feeder cell dependence and feeder cell independence. Wherein, the feeder cell independent NK cell in vitro amplification technology has higher safety, but the purity of the finally obtained cells is not high, and the consistent NK cell amplification purity is difficult to achieve due to the technical method limitation and donor individual difference. The purity of NK cells directly influences the effect of cell therapy, and the higher the purity, the better the curative effect.
The main hybrid cells in the NK cell mixed culture system, except for NK cells, are CD3 (clusteridiferentiation 3) positive cell populations. The traditional method for improving the purity of NK cells is MACS (magnetic activated Cell sorting) magnetic bead sorting technology. However, the MACS magnetic bead sorting technology can combine magnetic beads on the surface of NK cells, and the magnetic beads cannot be completely removed, so that potential clinical application hazards exist, and the MACS magnetic bead sorting technology is difficult to apply on a large scale.
Disclosure of Invention
Based on this, there is a need to provide a method for in vitro purification of natural killer cells with promising large-scale application.
An in vitro purification method of natural killer cells, comprising the following steps:
providing a solution of CD3 antibody;
loading the CD3 antibody solution into a clinical-grade culture container, and fully reacting to obtain the clinical-grade culture container with the surface coated with the CD3 antibody;
loading the natural killer cell mixed culture to be purified into the clinical-grade culture container coated with the CD3 antibody, culturing for 2-4 h under conventional culture conditions, and collecting supernatant in the clinical-grade culture container coated with the CD3 antibody; and
and centrifuging the supernatant, reserving cell sediment, and washing the cell sediment to obtain the purified natural killer cells.
In one embodiment, the solute of the CD3 antibody solution is CD3 monoclonal antibody or CD3 polyclonal antibody;
the concentration of the CD3 antibody solution is 100 ng/mL-400 ng/mL.
In one embodiment, the solvent of the CD3 antibody solution is PBS buffer or NaCl solution with a concentration of 0.9g/100 mL.
In one embodiment, the clinical grade culture vessel is a T175 cell culture flask with an untreated culture surface.
In one embodiment, in the operation of obtaining the clinical-grade culture container coated with the CD3 antibody after the full reaction, the reaction time is 8-24 h, and the reaction temperature is 2-8 ℃.
In one embodiment, the temperature of the conventional culture conditions is 37 ℃ and 5% CO2。
In one embodiment, in the operation of centrifuging the supernatant and retaining the cell pellet, the rotation speed of the centrifugation is 600rpm to 1000rpm, and the time of the centrifugation is 5min to 10 min.
In one embodiment, the washing the cell pellet is performed by: resuspending the cell pellet with NaCl solution with concentration of 0.9g/100mL, then centrifuging at 600-1000 rpm for 5-10 min, and keeping the washed cell pellet.
The in vitro purification method of the natural killer cells utilizes a clinical-grade culture container coated with a CD3 antibody to bear and culture a mixed culture of the natural killer cells, CD3 positive hybrid cells in the mixed culture of the natural killer cells are combined with a CD3 antibody to be attached to the inner wall of the clinical-grade culture container, and after the culture is finished, supernatant in the clinical-grade culture container is collected to obtain the purified natural killer cells. Compared with the traditional MACS magnetic bead sorting technology, the in-vitro purification method of the natural killer cells can not combine foreign matters such as magnetic beads on the surfaces of the purified natural killer cells, and has a prospect of large-scale application.
Drawings
FIG. 1 is a flow chart of a method for in vitro purification of natural killer cells according to one embodiment;
FIG. 2 is a graph showing a comparison of cell purity of natural killer cells before and after purification in example 1.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with examples are described in detail below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, but rather should be construed as broadly as the present invention is capable of modification in various respects, all without departing from the spirit and scope of the present invention.
One embodiment of the method for in vitro purification of natural killer cells as shown in fig. 1 comprises the following steps:
s10, providing a CD3 antibody solution.
The solute of the CD3 antibody solution may be CD3 monoclonal antibody or CD3 polyclonal antibody.
The concentration of the CD3 antibody solution is 100 ng/mL-400 ng/mL.
Preferably, the concentration of the CD3 antibody solution is 100ng/mL to 250 ng/mL.
Particularly preferably, the concentration of the CD3 antibody solution is 200 ng/mL.
The solvent of the CD3 antibody solution is PBS buffer solution (pH is 7.2-7.4) or normal saline.
The physiological saline solution was 0.9g/100mL of NaCl solution.
S20, loading the CD3 antibody solution obtained from S10 into a clinical-grade culture container, and fully reacting to obtain the clinical-grade culture container with the surface coated with the CD3 antibody.
The clinical grade culture vessel may be a T175 cell culture flask with an untreated culture surface.
In the operation of obtaining a clinical-grade culture vessel coated with the CD3 antibody after the sufficient reaction, the reaction time is 8 to 24 hours (preferably 12 hours), and the reaction temperature is 2 to 8 ℃ (preferably 4 ℃).
S30, loading the natural killer cell mixed culture to be purified into the clinical-grade culture container coated with the CD3 antibody obtained in the S20, culturing for 2-4 h under the conventional culture condition, and collecting the supernatant in the clinical-grade culture container coated with the CD3 antibody.
Before adding the natural killer cell mixed culture to be purified, the solution of the CD3 antibody in a clinical-grade culture container coated with the CD3 antibody on the surface is removed, and then the natural killer cell mixed culture to be purified is loaded.
The temperature of the conventional culture conditions was 37 ℃ and 5% CO2。
S40, centrifuging the supernatant obtained in the step S30, reserving cell sediment, and washing the cell sediment to obtain the purified natural killer cells.
And in the operation of centrifuging the supernatant and keeping the cell sediment, the rotating speed of the centrifugation is 600 rpm-1000 rpm, and the time of the centrifugation is 5 min-10 min.
The operation of washing the cell pellet was: resuspending the cell pellet with physiological saline (NaCl solution with concentration of 0.9 g/mL), then centrifuging at 600-1000 rpm for 5-10 min, and keeping the washed cell pellet.
The in vitro purification method of the natural killer cells utilizes a clinical-grade culture container coated with a CD3 antibody to bear and culture a mixed culture of the natural killer cells, CD3 positive hybrid cells in the mixed culture of the natural killer cells are combined with a CD3 antibody to be attached to the inner wall of the clinical-grade culture container, and after the culture is finished, supernatant in the clinical-grade culture container is collected to obtain the purified natural killer cells. Compared with the traditional MACS magnetic bead sorting technology, the in-vitro purification method of the natural killer cells can not combine foreign matters such as magnetic beads on the surfaces of the purified natural killer cells, and has a prospect of large-scale application.
In addition, the in vitro purification method of the natural killer cells utilizes the simple and easy antibody adhesion effect to amplify CD3 in the NK cell culture system in vitro+The non-target cells are selectively removed through short-time incubation so as to improve the purity of NK cells, obtain clinical NK cells with better quality and good safety, and reduce the cost to below 20 percent (separation is 1 multiplied by 10) of the traditional MACS-based immunomagnetic bead cell sorting technology8cells approximately 100 yuan per square).
The following are specific examples.
The examples, which are not specifically illustrated, employ drugs and equipment, all of which are conventional in the art. The experimental procedures, in which specific conditions are not indicated in the examples, are usually carried out according to conventional conditions, such as those described in the literature, in books, or as recommended by the manufacturer of the kits.
Example 1
The CD3 monoclonal antibody (purchased from Miltenyi Biotec) was directly soluble added to 1 XPBS (PhosphateBuffer solution) to make a solution of CD3 monoclonal antibody at a concentration of 100 ng/mL.
The CD3 monoclonal antibody solution is loaded in a T175 cell culture flask with a culture surface which is not subjected to tissue treatment, and the T175 cell culture flask with the surface coated with the CD3 monoclonal antibody is obtained after reaction for 12 hours at 4 ℃.
Removing the CD3 monoclonal antibody solution in a T175 cell culture bottle with the culture surface which is not subjected to tissue treatment and is coated with the CD3 monoclonal antibody, uniformly mixing 30mL of NK cell mixed culture to be purified, putting the mixed culture into the T175 cell culture bottle with the culture surface which is not subjected to tissue treatment and is coated with the CD3 monoclonal antibody, and carrying out the steps of culturing at 37 ℃ and 5% CO2After 3 hours of incubation, the supernatant from the culture surface of the non-tissue treated T175 cell culture flask was collected.
The supernatant was centrifuged at 1000rpm for 5min, and the cell pellet was retained. Resuspending the cell pellet with physiological saline (NaCl solution with concentration of 0.9g/100 mL), then centrifuging at 600rpm for 510min, and reserving the washed cell pellet, namely the NK cell with improved purity.
Flow assay was performed on NK cells before and after purification, and the results are shown in fig. 2.
As can be seen from FIG. 2, the purity of NK cells before purification was 22.7%, the purity after purification could reach 73.8%, and the yield of NK cells was calculated to be 64%.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (6)
1. An in vitro purification method of natural killer cells, which is characterized by comprising the following steps:
providing a solution of CD3 antibody;
loading the CD3 antibody solution into a clinical-grade culture container, and fully reacting to obtain the clinical-grade culture container with the surface coated with the CD3 antibody, wherein the clinical-grade culture container is a T175 cell culture bottle with the culture surface not subjected to tissue treatment, the reaction time is 8-24 h, and the reaction temperature is 2-8 ℃;
loading the natural killer cell mixed culture to be purified into the clinical-grade culture container coated with the CD3 antibody, culturing for 2-4 h under conventional culture conditions, and collecting supernatant in the clinical-grade culture container coated with the CD3 antibody; and
and centrifuging the supernatant, reserving cell sediment, and washing the cell sediment to obtain the purified natural killer cells.
2. The method for purifying natural killer cells in vitro as claimed in claim 1, wherein the solute of the CD3 antibody solution is CD3 monoclonal antibody or CD3 polyclonal antibody;
the concentration of the CD3 antibody solution is 100 ng/mL-400 ng/mL.
3. The method for in vitro purification of natural killer cells according to claim 1 or 2, wherein the solvent of the CD3 antibody solution is PBS buffer or NaCl solution with concentration of 0.9g/100 mL.
4. The method for purifying natural killer cells in vitro as claimed in claim 1, wherein the temperature of the conventional culture conditions is 37 ℃ and 5% CO2。
5. The method for purifying natural killer cells in vitro as claimed in claim 1, wherein in the operation of centrifuging the supernatant and retaining the cell pellet, the rotation speed of the centrifugation is 600rpm to 1000rpm, and the time of the centrifugation is 5min to 10 min.
6. The method for purifying natural killer cells in vitro as claimed in claim 1, wherein said washing of said cell pellet is performed by: resuspending the cell pellet with NaCl solution with concentration of 0.9g/100mL, then centrifuging at 600-1000 rpm for 5-10 min, and keeping the washed cell pellet.
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| CN103756963A (en) * | 2012-12-13 | 2014-04-30 | 上海柯莱逊生物技术有限公司 | Method used for in vitro proliferation of NK cells |
| CN104204194A (en) * | 2011-12-22 | 2014-12-10 | 财团法人牧岩生命工学研究所 | Method for producing natural killer cells, natural killer cells produced thereby, and composition for treating cancers and infectious diseases containing the same |
| CN104357390A (en) * | 2014-10-15 | 2015-02-18 | 深圳源正细胞医疗技术有限公司 | Method for simultaneous and efficient amplification of CD<3+>CD<56+>CIK cells and CD<3->CD<56+>NK cells |
| CN105238754B (en) * | 2015-11-20 | 2016-08-17 | 山东兴瑞生物科技有限公司 | A kind of high proliferation power and the extracorporeal culturing method of High Fragmentation power NK cell |
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| CN102676453A (en) * | 2011-03-17 | 2012-09-19 | 中国医学科学院肿瘤研究所 | Method for culturing natural killer (NK) and/or natural killer T (NKT) cells |
| CN104204194A (en) * | 2011-12-22 | 2014-12-10 | 财团法人牧岩生命工学研究所 | Method for producing natural killer cells, natural killer cells produced thereby, and composition for treating cancers and infectious diseases containing the same |
| CN103756963A (en) * | 2012-12-13 | 2014-04-30 | 上海柯莱逊生物技术有限公司 | Method used for in vitro proliferation of NK cells |
| CN104357390A (en) * | 2014-10-15 | 2015-02-18 | 深圳源正细胞医疗技术有限公司 | Method for simultaneous and efficient amplification of CD<3+>CD<56+>CIK cells and CD<3->CD<56+>NK cells |
| CN105238754B (en) * | 2015-11-20 | 2016-08-17 | 山东兴瑞生物科技有限公司 | A kind of high proliferation power and the extracorporeal culturing method of High Fragmentation power NK cell |
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Inventor after: Yi Jihui Inventor after: Li Yangxi Inventor after: Jiang Haida Inventor after: Xu Chunlian Inventor after: Mao Kanlang Inventor before: Li Yangxi Inventor before: Jiang Haida Inventor before: Yi Jihui Inventor before: Xu Chunlian Inventor before: Mao Kanlang |
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