CN114231485A - Immune cell in-vitro induced amplification and preservation method - Google Patents
Immune cell in-vitro induced amplification and preservation method Download PDFInfo
- Publication number
- CN114231485A CN114231485A CN202111273161.4A CN202111273161A CN114231485A CN 114231485 A CN114231485 A CN 114231485A CN 202111273161 A CN202111273161 A CN 202111273161A CN 114231485 A CN114231485 A CN 114231485A
- Authority
- CN
- China
- Prior art keywords
- immune cells
- culture
- immune
- heat preservation
- culture dish
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 210000002865 immune cell Anatomy 0.000 title claims abstract description 124
- 238000004321 preservation Methods 0.000 title claims abstract description 58
- 230000003321 amplification Effects 0.000 title claims abstract description 41
- 238000003199 nucleic acid amplification method Methods 0.000 title claims abstract description 41
- 238000000034 method Methods 0.000 title claims abstract description 27
- 238000000338 in vitro Methods 0.000 title claims abstract description 20
- 239000001963 growth medium Substances 0.000 claims abstract description 39
- 230000001939 inductive effect Effects 0.000 claims abstract description 15
- 210000003855 cell nucleus Anatomy 0.000 claims abstract description 8
- 238000004115 adherent culture Methods 0.000 claims abstract description 7
- 239000011248 coating agent Substances 0.000 claims abstract description 7
- 238000000576 coating method Methods 0.000 claims abstract description 7
- 210000005087 mononuclear cell Anatomy 0.000 claims abstract description 7
- 230000006698 induction Effects 0.000 claims abstract description 5
- 230000004936 stimulating effect Effects 0.000 claims abstract description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 32
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 15
- 238000007789 sealing Methods 0.000 claims description 14
- 239000011550 stock solution Substances 0.000 claims description 14
- 239000007640 basal medium Substances 0.000 claims description 12
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 10
- 108010002350 Interleukin-2 Proteins 0.000 claims description 10
- 230000005931 immune cell recruitment Effects 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 9
- 238000011084 recovery Methods 0.000 claims description 9
- 239000004017 serum-free culture medium Substances 0.000 claims description 9
- 108010002352 Interleukin-1 Proteins 0.000 claims description 8
- 210000004027 cell Anatomy 0.000 claims description 7
- 230000004069 differentiation Effects 0.000 claims description 7
- 239000000243 solution Substances 0.000 claims description 6
- 244000025254 Cannabis sativa Species 0.000 claims description 5
- 235000019996 baijiu Nutrition 0.000 claims description 5
- 229940119744 dextran 40 Drugs 0.000 claims description 5
- FZWBNHMXJMCXLU-BLAUPYHCSA-N isomaltotriose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O)O1 FZWBNHMXJMCXLU-BLAUPYHCSA-N 0.000 claims description 5
- 239000001397 quillaja saponaria molina bark Substances 0.000 claims description 5
- 229930182490 saponin Natural products 0.000 claims description 5
- 150000007949 saponins Chemical class 0.000 claims description 5
- 230000004913 activation Effects 0.000 claims description 4
- 239000012595 freezing medium Substances 0.000 claims description 4
- 238000007710 freezing Methods 0.000 claims description 3
- 230000008014 freezing Effects 0.000 claims description 3
- 239000011259 mixed solution Substances 0.000 claims description 3
- 210000004990 primary immune cell Anatomy 0.000 claims description 3
- 238000010257 thawing Methods 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 2
- 238000002474 experimental method Methods 0.000 abstract description 4
- 238000012864 cross contamination Methods 0.000 abstract description 3
- 239000002609 medium Substances 0.000 description 7
- 101000581981 Homo sapiens Neural cell adhesion molecule 1 Proteins 0.000 description 6
- 102000014150 Interferons Human genes 0.000 description 6
- 108010050904 Interferons Proteins 0.000 description 6
- 102100027347 Neural cell adhesion molecule 1 Human genes 0.000 description 6
- 229940079322 interferon Drugs 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 101000917858 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-A Proteins 0.000 description 4
- 101000917839 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-B Proteins 0.000 description 4
- 102100029185 Low affinity immunoglobulin gamma Fc region receptor III-B Human genes 0.000 description 4
- RWRDLPDLKQPQOW-UHFFFAOYSA-N Pyrrolidine Chemical compound C1CCNC1 RWRDLPDLKQPQOW-UHFFFAOYSA-N 0.000 description 4
- 210000004698 lymphocyte Anatomy 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 101710099301 Low affinity immunoglobulin gamma Fc region receptor III-A Proteins 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 230000000840 anti-viral effect Effects 0.000 description 2
- 238000005485 electric heating Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 238000009434 installation Methods 0.000 description 2
- 210000000822 natural killer cell Anatomy 0.000 description 2
- QYSPLQLAKJAUJT-UHFFFAOYSA-N piroxicam Chemical compound OC=1C2=CC=CC=C2S(=O)(=O)N(C)C=1C(=O)NC1=CC=CC=N1 QYSPLQLAKJAUJT-UHFFFAOYSA-N 0.000 description 2
- 229960002702 piroxicam Drugs 0.000 description 2
- 239000012679 serum free medium Substances 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- 241000700605 Viruses Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000008602 contraction Effects 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 210000004443 dendritic cell Anatomy 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000001360 synchronised effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Immunology (AREA)
- Hematology (AREA)
- Dentistry (AREA)
- Environmental Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides an in vitro induction amplification and preservation method of immune cells, which relates to the technical field of xx, and comprises the following operation steps: s1, carrying out adherent culture on the immune antibody in a culture dish to obtain an antibody coated culture dish; s2, stimulating immune cells to activate special culture medium, separating and preparing single cell nucleus, placing the single cell nucleus in the coating culture dish, and preparing immune cells after culture; s3, performing primary amplification culture on the mononuclear cells by using the immune cell amplification culture medium to obtain immune cells for inducing amplification. The user need not stretch into the inside of heat preservation room with the hand, avoids the user to be scalded, also avoids the heat preservation room to expose the time overlength in the interior air, produces cross contamination, reduces experiment safety risk, avoids leading to experimental data distortion, influences experimental data's accuracy.
Description
Technical Field
The invention relates to the technical field of immune cell in-vitro induced amplification, in particular to an immune cell in-vitro induced amplification and preservation method.
Background
The biological immune cell therapy is to induce self-antiviral immune response by in vitro culture, proliferation and activation and infusion back to the body, so that antiviral substances are continuously generated to kill viruses after human antiviral immunity is activated. The immune cells have strong recognition capability on tumor cells, bacteria and the like, like 'cellular missiles', can accurately 'shoot' the tumor cells or the bacteria, but cannot hurt 'innocent' normal cells, and is a very effective treatment mode.
In the prior art, the in vitro induction amplification culture system for immune cells is complicated, the immune effect is poor, and cross contamination is easy to generate in the process of actual experimental operation, so that data distortion is caused, and the safety risk is caused.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides an in vitro induced expansion and preservation method of immune cells.
The invention solves the technical problems through the following technical means: the method for inducing the expansion and preservation of immune cells in vitro comprises the following operation steps:
s1, carrying out adherent culture on the immune antibody in a culture dish to obtain an antibody coated culture dish;
s2, stimulating immune cells to activate special culture medium, separating and preparing single cell nucleus, placing the single cell nucleus in the coating culture dish, and preparing immune cells after culture;
s3, performing primary amplification culture on the mononuclear cells by using an immune cell amplification culture medium to obtain immune cells for inducing amplification;
s4, carrying out differentiation culture on the immune cells subjected to primary amplification by using a special induction culture medium for the immune cells to obtain differentiated immune cells;
s5, performing secondary activation and amplification culture on the differentiated primary amplified immune cells by using an immune cell activation culture medium to obtain secondary immune cells;
s6, freezing and storing the immune cells by using the immune cell freezing medium to obtain frozen and stored secondary immune cells; placing the frozen secondary immune cells in a constant-temperature water bath device for thawing, and mixing with a frozen recovery liquid of the primary immune cells to obtain a recovery cell mixed liquid;
and S7, centrifuging the revived cell mixed solution to obtain revived immune cells.
Further, the frozen resuscitation fluid comprises HAS, dextran 40 and FBS, and the basal medium is DMEM/F12 culture medium.
Further, the immune cell amplification culture medium is composed of 200U/mL IL-1, 350U/mL IL-2 and 0.01KE/mL piroxicam, and OpTzerTMCTTSTM serum-free culture medium is used as a basic culture medium.
Furthermore, the immune cell frozen stock solution is composed of DMSO, FBS, HAS + and 0.001g/mL of baijiu grass saponin R, and DMEM/F12 culture medium is used as a basic culture medium.
Furthermore, the water bath device comprises a water bath box, a heat preservation chamber is arranged in the water bath box, an electric heating wire for supplying heat is arranged on one side, close to the inner wall, of the heat preservation chamber, a culture dish is accommodated in the heat preservation chamber, and an accommodating cavity is accommodated in the heat preservation chamber and used for accommodating the culture dish; the bottom of water bath device is provided with drive assembly, drive assembly's top extends into the inside of heat preservation room for hold the inside axial lift of cavity along the heat preservation room to the drive.
Further, the interior bottom surface that holds the cavity is provided with the fixing base, the surface joint of fixing base has a plurality of culture dish, a plurality of the culture dish is array distribution, the fixed slot that is used for holding the culture dish is offered to the top surface of fixing base, the mounting groove has been seted up to the inner wall of fixed slot, mounting groove inside is fixed with the rubber strip, one side that the fixing base was kept away from to the rubber strip supports the surface of culture dish.
Further, the bottom fixedly connected with lifting seat that holds the cavity, be provided with the spacing subassembly that a plurality of has the flexible function of elasticity between the interior bottom surface of lifting seat and heat preservation room, a plurality of spacing subassembly is personally submitted the array and is distributed along the interior bottom surface of heat preservation room.
Furthermore, spacing subassembly includes the extension pipe of the indoor bottom surface of fixed connection heat preservation, extension spring has been held in the inside of extension pipe, extension spring's top is fixed with the connecting rod, the top of connecting rod and the bottom surface fixed connection of lifting seat, the bottom outside array distribution of connecting rod has spacing slider, spacing spout has been seted up for spacing slider's inner wall to the extension pipe, spacing slider slides along the inside of spacing spout.
Further, drive assembly is including the gear motor who is located the bottom of water bath device, gear motor's top is provided with output power's output shaft, the coaxial pivoted lifting screw rod in the top outside of output shaft, the inside that the water bath extends to the heat preservation room is run through on the top of lifting screw rod, the top outside screw thread of lifting screw rod has closed the lifting screwed pipe, the top fixed connection lifting seat of lifting screwed pipe.
Further, the outside fixedly connected with sealing ring of lifting screw rod, the seal groove has been seted up for the inner wall of sealing ring to the water bath, seal groove and sealing ring phase-match.
The invention has the beneficial effects that:
when the culture dish is subjected to induced amplification and cryopreservation resuscitation, the steps are fewer, the used materials are limited, the economic cost is low, the obtained immune cells are more in number and the immune effect brought by the immune cells is better through differentiation treatment, the accommodating cavity is driven to ascend and descend by the aid of the driving assembly, a user can take out the culture dish conveniently, the user does not need to stretch hands into the heat preservation chamber, the user is prevented from being scalded, the phenomenon that the heat preservation chamber is exposed in the air for too long time and generates cross contamination is avoided, the experiment safety risk is reduced, the experiment data distortion is avoided, and the accuracy of the experiment data is not influenced.
Drawings
FIG. 1 is a schematic front view of the water bath apparatus of the present invention;
FIG. 2 is a schematic sectional view of the water bath apparatus of the present invention;
FIG. 3 is a schematic cross-sectional view of the culture dish of the present invention;
FIG. 4 is an enlarged view of the structure at A in FIG. 2 according to the present invention;
FIG. 5 is an enlarged view of the structure at B in FIG. 2 according to the present invention;
FIG. 6 is a schematic view of the flow structure of the preparation method of the present invention.
In the figure: 10. a water bath device; 11. a heat preservation chamber; 12. a water bath tank; 13. an electric heating wire; 20. a drive assembly; 21. a reduction motor; 22. an output shaft; 23. lifting the screw rod; 24. lifting the threaded pipe; 25. a sealing groove; 26. a seal ring; 30. an accommodating cavity; 31. a fixed seat; 32. mounting grooves; 33. a rubber strip; 34. fixing grooves; 35. lifting the seat; 40. a culture dish; 50. a limiting component; 51. an extension spring; 52. a connecting rod; 53. an extension pipe; 54. a limiting slide block; 55. and a limiting sliding groove.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It will be understood that when an element is referred to as being "secured to" another element, it can be directly on the other element or intervening elements may also be present. When an element is referred to as being "connected" to another element, it can be directly connected to the other element or intervening elements may also be present.
Examples
As shown in fig. 6, the method for inducing expansion and preservation of immune cells in vitro of the present embodiment comprises the following steps:
A. preparation of immune cell activation medium
A1, configuring IFN-a1 type interferon containing 400U/mL, IL-2 containing 700U/mL, piroxicam containing 0.01KE, FBS containing 8 volume percent, to OpTzerTMCTTSTM serum-free medium.
A2, preparing IFN-a1 type interferon containing 650U/mL, IL-2 containing 840U/mL, pirrolipidem containing 0.012KE and FBS containing 10 volume percent, and taking SuperCultureTmL500 human lymphocyte serum-free culture medium as a basic culture medium.
A3, preparing IFN-a1 type interferon containing 700U/mL, IL-2 containing 1080U/mL, pirrolipidem containing 0.0144KE and FBS containing 12 volume percent, and taking SuperCultureTmL500 human lymphocyte serum-free culture medium as a basic culture medium.
B. Preparation of immune cell amplification medium
B1, preparing IL-1 containing 200U/mL, IL-2 containing 350U/mL and pirrolidine 0.01KE/mL, and taking OpTzerTMCTSTM serum-free culture medium as a basal medium.
B2, preparing IL-1 containing 200U/mL, IL-2 containing 420U/mL and pirrolipidem containing 0.012KE/mL, and taking a SuperCultureTmL500 human lymphocyte serum-free culture medium as a basic culture medium.
B3, preparing IL-1 containing 200U/mL, IL-2 containing 540U/mL and pirrolidine 0.0144KE/mL, and taking OpTzerTMCTTSTM serum-free culture medium as a basal medium.
C. Preparation of immune cell scale amplification culture medium
C1, preparing IL-1 containing 200U/mL and IL-2 containing 350U/mL, and taking OpTzerTMCTTSTM serum-free culture medium as a basal medium.
C2, preparing IL-1 containing 200U/mL and 420U/mL, and taking SuperCultureTmL500 human lymphocyte serum-free culture medium as a basic culture medium.
C3, IL-1 containing 200U/mL and IL-2 containing 540U/mL were prepared, and OpTzerTMCTTSTM serum-free medium was used as a basal medium.
D. Preparation of immune cell freezing medium
D1, 5 vol% DMSO +30 vol% FBS +3 vol% HAS +, and 0.001g/mL baijiu grass saponin R were prepared, and DMEM/F12 medium was used as a basal medium.
D2, 8 vol% DMSO +25 vol% FBS +5 vol% HAS +, 0.0012g/mL baijiu grass saponin R were prepared, and DMEM/F12 medium was used as the basal medium.
D3, preparing a baijiu grass saponin R containing 11 vol% of DMSO, 20 vol% of FBS, 7 vol% of HAS + and 0.00144g/mL, and taking DMEM/F12 culture medium as a basal medium.
E. Preparation of frozen resuscitation fluid
E1, prepared to contain 2 vol% HAS, 4 vol % dextran 40, 13 vol% FBS, DMEM/F12 medium as the basal medium.
E2, prepared to contain 3 vol% HAS, 3 vol% dextran 40, 15 vol% FBS, DMEM/F12 medium as the basal medium.
E3, prepared to contain 4 vol% HAS, 4 vol% dextran 40, 17 vol% FBS, DMEM/F12 medium as the basal medium.
Operation F1
S1, carrying out adherent culture on the CD16-2 antibody in a glass culture dish to prepare an antibody-coated culture dish;
s2, placing mononuclear cells of the natural killer cells obtained through separation into a coating culture dish according to a B-type immune cell activation culture medium which is prepared through A1 and stimulated by injected non-inactivated IFN-a1 type interferon, and performing immune cell activation culture for 24 hours to obtain activated immune cells;
s3, performing induced amplification culture on the activated immune cells by using the immune cell amplification culture medium prepared in the embodiment B1 to prepare immune cells induced to be amplified;
s4, when the expression of CD56+ CD16 antibody of the immune cell induced to expand exceeds 60%, carrying out differentiation culture to prepare a differentiated immune cell;
s5, when the expression of the CD56+ CD17 antibody of the differentiated immune cells exceeds 80%, performing amplification culture on the differentiated immune cells by using the immune cell scale amplification culture medium configured in the example C1 to prepare the scale-amplified immune cells, and keeping the number of the immune cells to be more than 4.5x106One passage every 2 days.
Operation F2
S1, carrying out adherent culture on the CD16-2 antibody in a glass culture dish to prepare an antibody-coated culture dish;
s2, placing mononuclear cells of dendritic cells obtained through separation into a coating culture dish according to a B-type immune cell activation culture medium which is prepared according to A2 and stimulated by injected non-inactivated IFN-a1 type interferon, and performing immune cell activation culture for 30 hours to obtain activated immune cells;
s3, performing induced amplification culture on the activated immune cells by using the immune cell amplification culture medium prepared in the embodiment B2 to prepare immune cells induced to be amplified;
s4, when the expression of CD56+ CD16 antibody of the immune cell induced to expand exceeds 65%, carrying out differentiation culture to prepare a differentiated immune cell;
s5, when the expression of the CD56+ CD16 antibody of the differentiated immune cells exceeds 84%, performing amplification culture on the differentiated immune cells by using the immune cell scale amplification culture medium configured in the example C2 to prepare the scale-amplified immune cells, and keeping the number of the immune cells to be more than 5.5x106One passage every 2.5 days.
Operation F3
S1, carrying out adherent culture on the CD16-2 antibody in a glass culture dish to prepare an antibody-coated culture dish;
s2, placing mononuclear cells of the natural killer cells obtained through separation into a coating culture dish according to a B-type immune cell activation culture medium which is prepared through A3 and stimulated by injected non-inactivated IFN-a1 type interferon, and performing immune cell activation culture for 36 hours to obtain activated immune cells;
s3, performing induced amplification culture on the activated immune cells by using the immune cell amplification culture medium prepared in the embodiment B3 to prepare immune cells induced to be amplified;
s4, when the expression of CD56+ CD16 antibody of the induced and amplified immune cells exceeds 70%, carrying out differentiation culture to prepare differentiated immune cells;
s5, when the expression of the CD56+ CD17 antibody of the differentiated immune cells exceeds 88%, performing amplification culture on the differentiated immune cells by using the immune cell scale amplification culture medium configured in the example C3 to prepare the scale-amplified immune cells, and keeping the number of the immune cells to be more than 6.5x106One passage every 3.0 days.
Operation F4
On the basis of F1, S5 is followed by:
s6, amplifying immune cells in scale according to 0.8x107The frozen stock solution of the immune cells prepared in the embodiment D1 is added in the amount of one/mL;
s7, adding the frozen recovery liquid prepared in the example E1 in an amount which is 3 times the weight of the immune cell frozen stock solution into the immune cell frozen stock solution, and then placing the immune cell frozen stock solution in a thermostatic water bath at 37.3 ℃ for heat preservation.
Operation F5
On the basis of F2, S5 is followed by:
s6, amplifying immune cells in scale according to 0.8x108The frozen stock solution of the immune cells prepared in the embodiment D2 is added in the amount of one/mL;
s7, adding the frozen recovery liquid prepared in the example E2 in an amount which is 4 times the weight of the immune cell frozen stock solution into the immune cell frozen stock solution, and then placing the immune cell frozen stock solution in a constant temperature water bath at 39.3 ℃ for heat preservation.
Operation F6
On the basis of F3, S5 is followed by:
s6, amplifying immune cells in scale according to 0.8x109The frozen stock solution of the immune cells prepared in the embodiment D3 is added in the amount of one/mL;
s7, adding the frozen recovery liquid prepared in the example E2 in an amount which is 5 times the weight of the immune cell frozen stock solution into the immune cell frozen stock solution, and then placing the immune cell frozen stock solution in a constant-temperature water bath at 41.3 ℃ for heat preservation.
The steps are simplified:
the method comprises the following steps: carrying out adherent culture on the immune antibody in a culture dish to obtain an antibody coated culture dish;
step two: stimulating immune cells to activate a special culture medium, separating and preparing single cell nucleuses, accommodating the single cell nucleuses in the coating culture dish, and preparing the immune cells after culturing;
step three: performing primary amplification culture on the mononuclear cells by using an immune cell amplification culture medium to obtain immune cells subjected to induced amplification;
step four: carrying out differentiation culture on the immune cells subjected to primary amplification by using a special induction culture medium for the immune cells to obtain differentiated immune cells;
step five: performing secondary activation and amplification culture on the differentiated primary amplified immune cells by using an immune cell activation culture medium to obtain secondary immune cells;
step six: freezing immune cells by using the immune cell freezing medium to obtain frozen secondary immune cells; placing the frozen secondary immune cells in a constant-temperature water bath device 10 for thawing, and mixing with a frozen recovery liquid of the primary immune cells to obtain a recovery cell mixed liquid;
step seven: and (4) centrifuging the recovered cell mixed solution to obtain recovered immune cells.
Referring to fig. 1 and 2, the water bath apparatus 10 includes a water bath 12, a heat preservation chamber 11 is opened inside the water bath 12, a heating wire 13 for supplying heat is disposed on one side of the heat preservation chamber 11 close to an inner wall, a culture dish 40 is accommodated inside the heat preservation chamber 11, and an accommodating cavity 30 for accommodating the culture dish 40 is accommodated inside the heat preservation chamber 11; the bottom end of the water bath device 10 is provided with a driving assembly 20, and the top end of the driving assembly 20 extends into the interior of the heat preservation chamber 11 and is used for axially lifting the driving accommodating cavity 30 along the interior of the heat preservation chamber 11.
During the use, when needing to take out the culture dish 40 that is located and holds cavity 30 inside, start drive assembly 20, the drive holds cavity 30 and drives culture dish 40 and goes up and down along the inside axial of heat preservation room 11, when holding cavity 30 and being in suitable height, takes out culture dish 40 from the inside of water bath device 10.
Through utilizing drive assembly 20 to drive and hold cavity 30 lift, the user of being convenient for takes out culture dish 40, and the user need not stretch into the inside of heat preservation room 11 with the hand, avoids the user to be scalded, also avoids heat preservation room 11 to expose the time overlength in the interior air, leads to suffering the pollution, influences test data's accuracy.
Referring to fig. 2, the inner bottom surface of the accommodating cavity 30 is provided with a fixing seat 31, the surface of the fixing seat 31 is clamped with a plurality of culture dishes 40, and the plurality of culture dishes 40 are distributed in an array.
During the use, utilize fixing base 31 and culture dish 40 joint together, can make between fixing base 31 and the culture dish 40 fixed mutually, improve culture dish 40's stability.
Referring to fig. 3, a fixing groove 34 for accommodating a culture dish 40 is formed in the top surface of the fixing seat 31, an installation groove 32 is formed in the inner wall of the fixing groove 34, a rubber strip 33 is fixed inside the installation groove 32, and one side of the rubber strip 33, which is far away from the fixing seat 31, abuts against the surface of the culture dish 40.
During the use, hold culture dish 40 in the inside of fixed slot 34, when the surface of rubber strip 33 support culture dish 40, the position of culture dish 40 is fixed, and rubber strip 33 possesses elasticity, receives when vibrations at culture dish 40, can play the effect of bradyseism to culture dish 40, improves culture dish 40's stability.
Referring to fig. 2, a lifting seat 35 is fixedly connected to the bottom end of the accommodating cavity 30, a plurality of limiting components 50 having elastic expansion and contraction functions are arranged between the lifting seat 35 and the inner bottom surface of the heat preservation chamber 11, and the plurality of limiting components 50 are distributed in an array along the inner bottom surface of the heat preservation chamber 11.
During the use, through installing the spacing subassembly 50 of a plurality of between fixing base 31 and heat preservation room 11, make and hold cavity 30 and can do the axial lift along the inside of heat preservation room 11, play limiting displacement to holding cavity 30.
Referring to fig. 5, the limiting assembly 50 includes an extension pipe 53 fixedly connected to the inner bottom surface of the insulating chamber 11, an extension spring 51 is accommodated in the extension pipe 53, a connecting rod 52 is fixed to the top end of the extension spring 51, and the top end of the connecting rod 52 is fixedly connected to the bottom surface of the lifting seat 35.
During the use, when needs go up and down to holding cavity 30, hold cavity 30 and drive connecting rod 52 and shift up, the in-process that connecting rod 52 shifted up, extension spring 51 is tensile gradually, and when holding cavity 30 and moving down, holding cavity 30 and promoting connecting rod 52 and move down, extension spring 51 contracts gradually.
Referring to fig. 5, the limiting sliding blocks 54 are distributed on the outer side of the bottom end of the connecting rod 52 in an array manner, a limiting sliding groove 55 is formed in the inner wall of the extending pipe 53 relative to the limiting sliding blocks 54, and the limiting sliding blocks 54 slide along the inside of the limiting sliding groove 55.
When the device is used, the limiting sliding block 54 is matched with the limiting sliding groove 55, so that the connecting rod 52 can be limited, and the connecting rod 52 is prevented from rotating.
Referring to fig. 2 and 4, the driving assembly 20 includes a speed reduction motor 21 located at the bottom end of the water bath apparatus 10, an output shaft 22 for outputting power is provided at the top end of the speed reduction motor 21, a lifting screw 23 coaxially rotates outside the top end of the output shaft 22, the top end of the lifting screw 23 penetrates through the water bath tank 12 and extends to the inside of the heat preservation chamber 11, a lifting threaded pipe 24 is screwed on the outside of the top end of the lifting screw 23, and the top end of the lifting threaded pipe 24 is fixedly connected to a lifting seat 35.
During the use, start gear motor 21, under gear motor 21's effect, output shaft 22 and lifting screw 23 synchronous rotation, under the screw drive effect, lifting screwed pipe 24 promotes lifting seat 35 and rises gradually, and when lifting screw 23 reversal, lifting screwed pipe 24 drives lifting seat 35 and descends.
Referring to fig. 5, a sealing ring 26 is fixedly connected to an outer side of the lifting screw 23, a sealing groove 25 is formed in an inner wall of the water bath tank 12 opposite to the sealing ring 26, and the sealing groove 25 is matched with the sealing ring 26.
By matching the sealing groove 25 with the sealing ring 26, the insulating chamber 11 can be sealed.
It is noted that, in this document, relational terms such as first and second, and the like, if any, are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (10)
1. The method for inducing, expanding and preserving immune cells in vitro is characterized in that: the method comprises the following operation steps:
s1, carrying out adherent culture on the immune antibody in a culture dish to obtain an antibody coated culture dish;
s2, stimulating immune cells to activate special culture medium, separating and preparing single cell nucleus, placing the single cell nucleus in the coating culture dish, and preparing immune cells after culture;
s3, performing primary amplification culture on the mononuclear cells by using an immune cell amplification culture medium to obtain immune cells for inducing amplification;
s4, carrying out differentiation culture on the immune cells subjected to primary amplification by using a special induction culture medium for the immune cells to obtain differentiated immune cells;
s5, performing secondary activation and amplification culture on the differentiated primary amplified immune cells by using an immune cell activation culture medium to obtain secondary immune cells;
s6, freezing and storing the immune cells by using the immune cell freezing medium to obtain frozen and stored secondary immune cells; placing the frozen secondary immune cells in a constant-temperature water bath device (10) for thawing, and mixing with a frozen recovery liquid of the primary immune cells to obtain a recovery cell mixed liquid;
and S7, centrifuging the revived cell mixed solution to obtain revived immune cells.
2. The method for inducing expansion and preservation of immune cells in vitro according to claim 1, wherein: the frozen resuscitation solution comprises HAS, dextran 40 and FBS, and DMEM/F12 culture medium is used as a basal medium.
3. The method for inducing expansion and preservation of immune cells in vitro according to claim 1, wherein: the immune cell amplification culture medium is composed of 200U/mL IL-1, 350U/mL IL-2 and 0.01KE/mL pirirobicin, and takes OpTzerer TMCTTSTM serum-free culture medium as a basic culture medium.
4. The method for inducing expansion and preservation of immune cells in vitro according to claim 1, wherein: the immune cell frozen stock solution is composed of DMSO, FBS, HAS + and 0.001g/mL of baijiu grass saponin R, and DMEM/F12 culture medium is taken as a basic culture medium.
5. The method for inducing expansion and preservation of immune cells in vitro according to claim 1, wherein: the water bath device (10) comprises a water bath box (12), a heat preservation chamber (11) is formed in the water bath box (12), heating wires (13) for supplying heat are arranged on one side, close to the inner wall, of the heat preservation chamber (11), a culture dish (40) is accommodated in the heat preservation chamber (11), and an accommodating cavity (30) for accommodating the culture dish (40) is accommodated in the heat preservation chamber (11); the bottom of water bath device (10) is provided with drive assembly (20), the top of drive assembly (20) extends into the inside of heat preservation room (11) for hold cavity (30) and go up and down along the inside axial of heat preservation room (11) to the drive.
6. The method for inducing expansion and preservation of immune cells in vitro as claimed in claim 5, wherein: the interior bottom surface that holds cavity (30) is provided with fixing base (31), the surface joint of fixing base (31) has a plurality of culture dish (40), a plurality of culture dish (40) are array distribution, fixed slot (34) that are used for holding culture dish (40) are offered to the top surface of fixing base (31), mounting groove (32) have been seted up to the inner wall of fixed slot (34), mounting groove (32) inside is fixed with rubber strip (33), one side that fixing base (31) were kept away from in rubber strip (33) supports the surface of culture dish (40).
7. The method for inducing expansion and preservation of immune cells in vitro according to claim 6, wherein: hold bottom fixedly connected with lifting seat (35) of cavity (30), be provided with spacing subassembly (50) that a plurality of has the flexible function of elasticity between the interior bottom surface of lifting seat (35) and heat preservation room (11), a plurality of spacing subassembly (50) are the array distribution along the interior bottom surface of heat preservation room (11).
8. The method for inducing expansion and preservation of immune cells in vitro according to claim 7, wherein: spacing subassembly (50) are including extension pipe (53) of bottom surface in fixed connection heat preservation room (11), extension pipe (53) inside has held extension spring (51), extension spring (51)'s top is fixed with connecting rod (52), the top of connecting rod (52) and the bottom surface fixed connection of lifting seat (35), the bottom outside array distribution of connecting rod (52) has spacing slider (54), spacing spout (55) have been seted up for the inner wall of spacing slider (54) in extension pipe (53), spacing slider (54) slide along the inside of spacing spout (55).
9. The method for inducing expansion and preservation of immune cells in vitro as claimed in claim 5, wherein: drive assembly (20) is including gear motor (21) that is located the bottom of water bath device (10), the top of gear motor (21) is provided with output power's output shaft (22), the coaxial pivoted lifting screw rod (23) in the top outside of output shaft (22), the inside that water bath case (12) extended to heat preservation room (11) is run through on the top of lifting screw rod (23), the top outside screw thread of lifting screw rod (23) has closed lifting screwed pipe (24) soon, the top fixed connection lifting seat (35) of lifting screwed pipe (24).
10. The method for inducing expansion and preservation of immune cells in vitro according to claim 9, wherein: the outside fixedly connected with sealing ring (26) of lifting screw rod (23), seal groove (25) have been seted up for the inner wall of sealing ring (26) in water bath (12), seal groove (25) and sealing ring (26) phase-match.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111273161.4A CN114231485A (en) | 2021-10-29 | 2021-10-29 | Immune cell in-vitro induced amplification and preservation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111273161.4A CN114231485A (en) | 2021-10-29 | 2021-10-29 | Immune cell in-vitro induced amplification and preservation method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114231485A true CN114231485A (en) | 2022-03-25 |
Family
ID=80743474
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111273161.4A Pending CN114231485A (en) | 2021-10-29 | 2021-10-29 | Immune cell in-vitro induced amplification and preservation method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114231485A (en) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20050013424A (en) * | 2003-07-28 | 2005-02-04 | 주식회사 포스코 | A water bath for reaction beaker |
| CN106591233A (en) * | 2016-12-28 | 2017-04-26 | 广州沙艾生物科技有限公司 | Method of extracorporeal induction, proliferation and cryopreservation of immune cells |
| CN106754704A (en) * | 2016-12-28 | 2017-05-31 | 华南生物医药研究院 | The method of the external evoked amplification of immunocyte |
| CN109069470A (en) * | 2016-03-21 | 2018-12-21 | 杨宁荪 | Specific Dihydrobenzofuran Lignans are in the purposes for inhibiting breast cancer cell transfer |
| WO2019103528A2 (en) * | 2017-11-24 | 2019-05-31 | 주식회사 차바이오랩 | Serum-free culture medium composition |
| CN111019892A (en) * | 2019-12-16 | 2020-04-17 | 杭州恩格生物医疗科技有限公司 | Immune cell in-vitro induction amplification method |
| CN113440332A (en) * | 2021-06-24 | 2021-09-28 | 西安交通大学医学院第一附属医院 | Tumor interventional thermotherapy device |
-
2021
- 2021-10-29 CN CN202111273161.4A patent/CN114231485A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20050013424A (en) * | 2003-07-28 | 2005-02-04 | 주식회사 포스코 | A water bath for reaction beaker |
| CN109069470A (en) * | 2016-03-21 | 2018-12-21 | 杨宁荪 | Specific Dihydrobenzofuran Lignans are in the purposes for inhibiting breast cancer cell transfer |
| CN106591233A (en) * | 2016-12-28 | 2017-04-26 | 广州沙艾生物科技有限公司 | Method of extracorporeal induction, proliferation and cryopreservation of immune cells |
| CN106754704A (en) * | 2016-12-28 | 2017-05-31 | 华南生物医药研究院 | The method of the external evoked amplification of immunocyte |
| WO2019103528A2 (en) * | 2017-11-24 | 2019-05-31 | 주식회사 차바이오랩 | Serum-free culture medium composition |
| CN111019892A (en) * | 2019-12-16 | 2020-04-17 | 杭州恩格生物医疗科技有限公司 | Immune cell in-vitro induction amplification method |
| CN113440332A (en) * | 2021-06-24 | 2021-09-28 | 西安交通大学医学院第一附属医院 | Tumor interventional thermotherapy device |
Non-Patent Citations (2)
| Title |
|---|
| 明洁: "化疗药物体外干预后MCF-7细胞株耐药基因表达情况及其在临床化学治疗中的意义", 中国优秀硕士学位论文全文数据库医药卫生科技辑, no. 05, pages 072 - 186 * |
| 杨森;南存金;木海琦;王怡君;苏红侠;陈映鹤;: "自体DC-CIK联合膀胱内吡柔比星灌注治疗对浅表性膀胱癌术后复发和生存的影响", 温州医学院学报, no. 11, pages 746 - 749 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20200131477A1 (en) | Medium system and method for ex vivo expansion of nk cells | |
| Shimizu et al. | Systemic administration of interleukin 2 enhances the therapeutic efficacy of dendritic cell-based tumor vaccines | |
| Mortimer et al. | The effect of therapeutic ultrasound on calcium uptake in fibroblasts | |
| Medawar | Tests by tissue culture methods on the nature of immunity to transplanted skin | |
| CN107929727A (en) | A kind of preparation method of new dendritic cell vaccine | |
| CN104938477A (en) | CIK (cytokine-induced killer) frozen stock solution and frozen preservation method | |
| CN105176927A (en) | Preparation method of cytotoxicity-enhanced efficient target killing NK/CIK (Natural Killer)/( Cytokine Induced Killer) cells | |
| WO1990002171A1 (en) | In vitro cell culture reactor | |
| CN109385374B (en) | Miniature cell culture device and cavity internal environment control method thereof | |
| CN104491853A (en) | Preparation method of human dendritic cell tumour vaccine | |
| CN104371973B (en) | A kind of serum free medium of immunocyte | |
| CN114231485A (en) | Immune cell in-vitro induced amplification and preservation method | |
| Hewlett et al. | Growth regulation of a murine lymphoma cell line by a 2‐mercaptoethanol or macrophage‐activated serum factor | |
| OJIMA et al. | A blood culture method for fish chromosomes | |
| CN107083362A (en) | A kind of autologous NK cells preparation method and the kit for implementing this method | |
| Zeng et al. | Biophysical studies on the differentiation of human CD14+ monocytes into dendritic cells | |
| CN110305843A (en) | Immunocyte of autoimmune disease and the preparation method and application thereof can be improved | |
| Dietz et al. | Optimizing preparation of normal dendritic cells and bcr-abl+ mature dendritic cells derived from immunomagnetically purified CD14+ cells | |
| CN107400660B (en) | A method of activation and amplification NKT cell | |
| Chang et al. | Immunotherapy with dendritic cells pulsed by autologous dactinomycin-induced melanoma apoptotic bodies for patients with malignant melanoma | |
| CN109486761A (en) | The cultural method of peripheral blood CTL cell | |
| CN103173410A (en) | Composition and method for stimulating dendritic cell maturation | |
| CN101321861B (en) | Method for generating dendritic cells employing decreased temperature | |
| CN107090434B (en) | Blood anticoagulation and preservation method for improving CIK cell culture effect | |
| KR102111283B1 (en) | Shaking culture method of immune cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |