CN115094037A - Composition used in cooperation with autologous plasma and having canine natural killer cell in-vitro activation function and application thereof - Google Patents

Composition used in cooperation with autologous plasma and having canine natural killer cell in-vitro activation function and application thereof Download PDF

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CN115094037A
CN115094037A CN202210846883.2A CN202210846883A CN115094037A CN 115094037 A CN115094037 A CN 115094037A CN 202210846883 A CN202210846883 A CN 202210846883A CN 115094037 A CN115094037 A CN 115094037A
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canine
human interleukin
cells
monoclonal antibody
composition
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CN115094037B (en
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赵献军
魏强
白春娜
田方杰
马樱琬
张翊华
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Baioupai Tianjin Biotechnology Co ltd
Northwest A&F University
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Baioupai Tianjin Biotechnology Co ltd
Northwest A&F University
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    • 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
    • C12N5/0646—Natural killers cells [NK], NKT cells
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2302—Interleukin-2 (IL-2)
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2315—Interleukin-15 (IL-15)
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/599—Cell markers; Cell surface determinants with CD designations not provided for elsewhere

Abstract

The invention relates to the field of cell culture, and discloses a composition with a canine natural killer cell in-vitro activation function and application thereof, wherein the composition is used in cooperation with autologous plasma. The composition contains glucocorticoid, human interleukin 2, human interleukin 15, CD335 monoclonal antibody, CD16 monoclonal antibody, human interleukin 2 with the content of 10-40IU and human interleukin 15 with the content of 0.6-2.4ng relative to each nanogram of the CD335 monoclonal antibody, can provide nutrient substances independent of fetal bovine serum serving as a culture medium, ensures that the prepared dog natural killer cells are safe and can be efficiently amplified, avoids the defects of pathogenicity, pollution, non-producibility and easy allergy caused by heterologous protein, exogenous virus and other pathogenic factors, is better applied to clinical cell therapy of pets, and reduces the risk of clinical treatment of pets.

Description

Composition used in cooperation with autologous plasma and having canine natural killer cell in-vitro activation function and application thereof
Technical Field
The invention relates to the field of in vitro cell culture, in particular to a composition which is used together with autologous plasma and has a function of in vitro activation of canine natural killer cells and application thereof.
Background
Natural killer cells (NK) are important immune cells in the body. Plays an important role in tumor immunotherapy, viral disease immunotherapy and anti-aging treatment. In clinical application, NK cells are obtained by culturing Peripheral Blood Mononuclear Cells (PBMC), and the NK cells can be amplified and purified by in vitro culture so as to meet the requirements of quantity and purity of clinical application.
The in vitro culture method of NK cells mainly comprises a feeder layer cell co-culture method and a simple cytokine stimulation method. The simple cytokine stimulation method is characterized in that corresponding cytokines are added under the condition of not using feeder layer cell co-culture, so that NK cells in PBMC are activated and proliferated. However, the culture system used in the simple cytokine stimulation method often depends on fetal bovine serum or calf serum. Fetal calf serum or calf serum is rich in nutrients necessary for cell growth and proliferation, such as growth factors and hormones, and can promote the survival, growth and proliferation of NK cells and maintain the function of NK cells.
Based on the advantages of fetal bovine serum or calf serum, the existing method for preparing clinical canine NK cells mostly depends on the fetal bovine serum or calf serum. However, the use of fetal bovine serum or calf serum still brings inconvenience to the preparation of canine NK cells meeting clinical requirements. First, the difference in biological activity, hormones and cytokines of fetal calf serum or calf serum of different batches results in unstable quality of clinically prepared canine NK cells, and standardized production is difficult to achieve. Secondly, the fetal calf serum or calf serum contains a large amount of heterologous proteins, which easily causes the allergic reaction of the inoculated dog to the cell products. Third, the possible presence of foreign contaminating viruses and pathogenic agents in fetal bovine serum or calf serum presents a clinical application risk.
Disclosure of Invention
The invention aims to overcome the problems that the natural killer cells using fetal calf serum or calf serum in the prior art have unstable quality and can not be produced in a standardized way, the allergic reaction of dogs is easy to cause, and viruses and pathogenic factors possibly bring exogenous pollution, and provides a composition with the function of in vitro activating the clinical dog natural killer cells by using autologous plasma and an in vitro culture method thereof. The prepared canine natural killer cells are safe and can be efficiently amplified, the defects of pathogenicity, pollution, non-mass production and easy allergy caused by heterologous proteins, exogenous viruses and other pathogenic factors are avoided, and the method is better applied to clinical cell therapy of pets.
In order to achieve the above object, a first aspect of the present invention provides a composition for in vitro activation of canine natural killer cells, for use with autologous plasma, the composition comprising a glucocorticoid, human interleukin 2, human interleukin 15, a CD335 monoclonal antibody, and a CD16 monoclonal antibody, wherein the content of human interleukin 2 is 10 to 40IU and the content of human interleukin 15 is 0.6 to 2.4ng per nanogram of the CD335 monoclonal antibody.
In a second aspect, the present invention provides a method for in vitro culturing canine natural killer cells using autologous plasma, the method comprising: the dog autologous plasma is separated to obtain peripheral blood mononuclear cells, and the peripheral blood mononuclear cells are mixed with the composition for activation culture.
By adopting the technical scheme, the preparation method for the full natural killer cells by using the canine autologous plasma is safe and efficient, the amplification multiple can reach more than 30 times, the formula disclosed by the invention can avoid the damage of fetal bovine serum or calf serum to the canine, the standard easy-to-handle effect is achieved, and the preparation method is further applied to clinical treatment of pets to reduce risks and reduce pathogenic factors.
Drawings
FIG. 1 is a photomicrograph of NK cells from a golden retriever at various times during in vitro activation;
FIG. 2 is a photomicrograph of NK cells from golden retriever dogs at various times during in vitro amplification;
FIG. 3 is a cell proliferation curve of NK cells of retriever auricles in vitro;
FIG. 4 is a graph of the change in phenotype of ex vivo expanded cells of NK cells from golden retriever dogs;
FIG. 5 is photomicrographs of Labrador retriever NK cells at various times during in vitro activation;
FIG. 6 is photomicrographs of in vitro expansion of NK cells of Labrador retriever dogs at various times;
FIG. 7 is a labrador retriever NK cell in vitro expansion cell proliferation curve;
FIG. 8 is a graph of the phenotypic changes of in vitro expanded cells of Labrador retriever NK cells;
FIG. 9 is a graph showing the results of purity determination after in vitro amplification of canine NK cells.
Detailed Description
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
In the present invention, the term "PBMC" used herein generally refers to peripheral blood mononuclear cells, unless otherwise specified. "NK cells" refers to natural killer cells. "IL-2" refers to human interleukin 2. "IL-15" refers to human interleukin 15. KE is an elimination rate constant that represents a fixed fraction or percentage of the amount of drug that can be eliminated per unit time. The unit IU of IL-2 is an international unit which detects the minimum amount of IL-2 stimulating CTLL-2 cell proliferation of 0.1ng/mL by a bioassay method and obtains a minimum titer unit with certain biological efficacy as a unit (u).
In a first aspect, the present invention provides a composition for use with autologous plasma, the composition having an in vitro activating function of canine natural killer cells, the composition comprising a glucocorticoid, human interleukin 2, human interleukin 15, CD335 monoclonal antibody and CD16 monoclonal antibody, wherein the human interleukin 2 is present in an amount of 10 to 40IU (e.g., 10IU, 11IU, 12IU, 13IU, 14IU, 15, 16IU, 18IU, 19IU, 20IU, 26IU, 28IU, 34IU, 36, 40IU or any value therebetween) per nanogram of the CD335 monoclonal antibody, and the human interleukin 15 is present in an amount of 0.6 to 2.4ng (e.g., 0.6ng, 0.61ng, 0.62ng, 0.63ng, 0.64ng, 0.65ng, 0.66ng, 0.7ng, 0.75ng, 0.8ng, 1ng, 1.2ng, 1.6ng, 1.8ng, 2ng, 2.4ng, or any value therebetween).
According to the invention, the glucocorticoid is preferably present in an amount of 0.06-0.3 μ g (e.g. 0.06 μ g, 0.065 μ g, 0.066 μ g, 0.067 μ g, 0.0068 μ g, 0.069 μ g, 0.07 μ g, 0.071 μ g, 0.072 μ g, 0.073 μ g, 0.074 μ g, 0.075 μ g, 0.076 μ g, 0.08 μ g, 0.085 μ g, 0.09 μ g, 0.1 μ g, 0.15 μ g, 0.2 μ g, 0.25 μ g, 0.3 μ g or any value therebetween) per nanogram of the CD335 monoclonal antibody, preferably in an amount of 1-4ng (e.g, 1.1ng, 1.2ng, 1.3ng, 1.4ng, 1.5ng, 8.ng, 2ng, 2.5ng, 3ng, 3.5ng, or any value therebetween) of the CD16 monoclonal antibody.
According to a more preferred embodiment of the invention, the glucocorticoid is present in an amount of 0.07 to 0.15 μ g, the human interleukin 2 is present in an amount of 10 to 20IU, the human interleukin 15 is present in an amount of 0.6 to 1.2ng and the CD16 monoclonal antibody is present in an amount of 1 to 2ng per nanogram of CD335 monoclonal antibody.
According to the present invention, there is no particular requirement for selection of the glucocorticoid, and any substance belonging to the glucocorticoid may be used, and at least one selected from prednisone, methylprednisolone, betamethasone, beclomethasone propionate, prednisolone, hydrocortisone, and dexamethasone is preferable, and hydrocortisone is more preferable.
According to the invention, the composition also comprises at least one of X-VIVO 15 culture medium, canine autologous plasma and inactivated group A Streptococcus (Streptococcus pyogenes), and more preferably comprises X-VIVO 15 culture medium, canine autologous plasma and inactivated group A Streptococcus at the same time.
According to a more preferred embodiment of the present invention, the volume ratio of the X-VIVO 15 medium to the canine autologous plasma is 9-50: 1 (e.g., 9: 1, 9.5: 1, 10.1: 1, 10.8: 1, 11.5: 1, 1.3: 1, 13.3: 1, 14.4: 1, 15.7: 1, 17: 1, 18: 1, 19: 1, 20: 1, 22: 1, 24: 1, 32.3: 1, 49: 1 or any value therebetween), preferably 8-20: 1.
according to a more preferred embodiment of the present invention, the amount of the CD335 monoclonal antibody is preferably 50-200ng such as 50ng, 51ng, 52ng, 53ng, 54ng, 55ng, 56ng, 58ng, 60ng, 65ng, 70ng, 75ng, 80ng, 90ng, 100ng, 150ng, 200ng or any value therebetween, relative to the total amount of 1mL of X-VIVO 15 medium and canine autologous plasma).
According to a more preferred embodiment of the invention, the inactivated group a streptococcus is present in an amount of 0-0.0016KE (e.g. 0.0001KE, 0.0002KE, 0.0003KE, 0.00035KE, 0.00036KE, 0.00037KE, 0.00038KE, 0.00039KE, 0.0004KE, 0.00041KE, 0.00042KE, 0.00043KE, 0.00044KE, 0.00045KE, 0.0005KE, 0.0006KE, 0.0008KE, 0.0010KE, 0.0012KE, 0.0014KE, 0.0016KE or any value therebetween), preferably 0.0004-0.0008KE per nanogram of CD335 monoclonal antibody.
According to a more preferred embodiment of the invention, the inactivated group a streptococcus is a group a streptococcus for injection, such as a lyophilisate of group a hemolytic streptococcus treated with penicillin (saperin), commercially available.
In a second aspect, the present invention provides a method for in vitro culturing canine natural killer cells using autologous plasma, the method comprising: the dog autologous plasma is separated to obtain peripheral blood mononuclear cells, and the peripheral blood mononuclear cells are mixed with the composition for activation culture.
According to the method of the present invention, the composition is preferably used in such an amount that the initial cell density of canine peripheral blood mononuclear cells in the mixed system is 3X 10 or more 6 Individual cells/mL.
According to the method of the present invention, the conditions for activating the culture preferably include: the temperature is 36-38 deg.C, and the saturated humidity is generally 41.8-46.3g/m 3 )，CO 2 The content is 5-10%. "saturated humidity" refers to the maximum amount of water vapor that can be contained in a unit volume of air, which varies with temperature.
According to the method of the present invention, preferably, the in vitro culture method further comprises isolating and expanding the cells after the activation culture. The separation may be carried out by a conventional method, for example, by centrifugation.
According to the method of the present invention, the amplification conditions preferably include: saturated humidity (generally 41.8-46.3 g/m) 3 ) At a temperature of 36-38 ℃ and CO 2 The content is 5-10%.
According to the method of the present invention, preferably, the amplification centrifugation conditions are: 250-400g, 5-15 min.
According to the method of the invention, the culture solution used for amplification preferably comprises human interleukin 2, human interleukin 15, X-VIVO 15 culture medium and canine autologous plasma.
According to the method, the volume ratio of the X-VIVO 15 culture medium to the canine autologous plasma in the culture solution used for amplification is preferably 9-50: 1 ((e.g., 9: 1, 10.1: 1, 11.5: 1, 13.3: 1, 15.7: 1, 17: 1, 18: 1, 19: 1, 20: 1, 22: 1, 24: 1, 32.3: 1, 49: 1 or any value therebetween)), more preferably 13.3 to 32.3: 1.
According to a more preferred embodiment of the present invention, the culture solution used for the amplification contains 1000-.
According to a more preferred embodiment of the present invention, the amount of human interleukin 15 in the culture medium used for amplification is 30-120ng (e.g., 30ng, 30.5ng, 31ng, 32ng, 33ng, 34ng, 35ng, 36ng, 38ng, 40ng, 45ng, 50ng, 60ng, 70ng, 80ng, 100ng, 120ng or any value therebetween), and more preferably 30-60ng, relative to the total amount of 1mL of X-VIVO 15 culture medium and canine autologous plasma.
The method further comprises the step of preparing the canine autologous plasma before activating the culture, and specifically can comprise removing blood cells to obtain plasma after blood collection. Blood can be collected by adopting a jugular vein blood collection method with minimum damage to dogs. The method has no special requirements on transportation and storage of blood after blood collection of the dog, and adopts a conventional method, wherein the anticoagulant is preferably heparin sodium and/or sodium citrate. The blood cells can be removed by centrifugation, and the supernatant is plasma.
According to the method of the present invention, the in vitro activation of NK cells further comprises the step of obtaining PBMC by separation before activation culture, and specifically may comprise blood cell precipitation and separation after removing plasma after blood collection. PBMC-derived material to reduce the damage to dogs, the lower layer blood cell pellet obtained after jugular vein blood collection and centrifugation was used. The separation needs to add the canine blood cell sediment into the sample diluent for dilution, then add the diluted blood cells on the liquid surface of the lymphocyte separation liquid, and carry out the first centrifugation. And adding the centrifuged second layer of annular milky white mononuclear cells (namely PBMC) into the cell cleaning solution, uniformly mixing, and centrifuging for the second time. The supernatant was discarded and the cells were resuspended by adding cell wash and centrifuged for a third time.
According to a more preferred embodiment of the invention, there is no particular requirement for the transport and storage of blood after blood collection in dogs, as described above.
According to a more preferred embodiment of the present invention, the amount of the canine blood cell pellet is not particularly limited, and may be measured based on the amount actually required.
According to the method of the invention, the PBMC is separated by adding a sample diluent, a lymphocyte separating medium (commercially available) and a cell washing solution, and an activating composition and an amplification culture solution to obtain a better amplification effect.
According to a more preferred embodiment of the invention, the sample diluent comprises physiological saline, canine autologous plasma and heparin sodium.
According to the method of the invention, the volume ratio of the physiological saline to the canine autologous plasma in the sample diluent is 49-200: 1 (e.g., 49: 1, 99: 1, 199: 1, or any value therebetween).
According to a more preferred embodiment of the present invention, the content of heparin sodium is 50 to 200. mu.g (e.g., 50. mu.g, 53. mu.g, 55. mu.g, 57. mu.g, 60. mu.g, 65. mu.g, 70. mu.g, 80. mu.g, 90. mu.g, 100. mu.g, 150. mu.g, 200. mu.g or any value therebetween), and more preferably 50 to 100. mu.g, relative to 1mL of physiological saline and canine autologous plasma in total.
According to the method of the present invention, the volume ratio of the blood cell sedimentation liquid to the sample diluent is 1: 1-2.
According to the method of the present invention, the volume ratio of the diluted blood cell sedimentation liquid to the lymphocyte separation liquid is 1: 1-1.5
According to a more preferred embodiment of the present invention, the cell washing solution comprises physiological saline and canine autologous plasma.
According to the method, the volume ratio of the physiological saline to the canine autologous plasma in the cell washing solution is 49-200: 1 (e.g., 49: 1, 99: 1, 199: 1, or any value therebetween).
According to the method, when the PBMCs are uniformly mixed, the adding amount of the cell cleaning solution is made up to 8-13mL based on the volume of the PBMCs.
According to the method of the present invention, the amount of the cell washing solution added during the cell resuspension is 8-13mL based on the volume of the whole cells.
According to the method of the present invention, the conditions of the first centrifugation can be 23-28 ℃, 400-500g, 20-30 min. The conditions for the second centrifugation can be 200-300g, 5-15 min.
According to a more preferred embodiment of the present invention, the PBMCs can be counted according to the experiment, and the resuspension centrifugation can be repeated for the PBMCs to achieve the absolute number of cells required for the experiment.
According to a most preferred embodiment of the invention, the method comprises the steps of:
the PMBC obtained by separation is mixed according to the weight ratio of 13.3-32.3: 1 volume ratio of the activating composition mixed with X-VIVO 15 culture medium, dog autologous plasma, 0.0004-0.0008KE/mL of sapelin, 0.07-0.15 mu g/mL of hydrocortisone, 10-20IU/mL of human interleukin 2, 0.6-1.2ng/mL of human interleukin 15, 1-2ng/mL of CD335 monoclonal antibody and 1-2ng/mL of CD16 monoclonal antibody, the amount of the activating composition added is that the cell density of PMBC is more than or equal to 3X 10 6 Individual cells/mL, after culturing until clumpy cell clones are observed, add the cells at a ratio of 13.3-32.3: mixing a liquid composition of an X-VIVO 15 culture medium and canine autologous plasma in a volume ratio of 1 with an amplification culture solution mixed with 1000-2000IU/mL human interleukin 2 and 30-60ng/mL interleukin 15 for amplification, and adding the dosage of the amplification culture solution to readjust the cell density to be more than or equal to 3X 10 6 Individual cells/mL. According to the preferred embodiment, the purity of the NK cells can be further improved, the NK cells can be proliferated more efficiently, the defect of using fetal bovine serum is avoided, and the damage to dogs is reduced.
In the invention, the 'X-VIVO 15 culture medium' is an immune cell serum-free culture medium, contains recombinant human insulin, recombinant human transferrin and albumin, and does not contain gentamicin and phenol red.
The present invention will be described in detail below by way of examples. In the following examples, cell count parameters were measured by a cell counter, and the obtained cell number was an absolute number; observing the cell growth state by an inverted microscope of Miaodi AE31, and taking a picture by a high-resolution microscope camera MOTICAM-10; centrifuging through a TDZ5-WS type centrifuge; culturing cells in a culture box of FORMA3111 (water jacket type); measuring an NK in-vitro amplification and proliferation curve by a cell counting method; NK in vitro amplification cell phenotype was detected by flow cytometry. X-VIVO 15 medium is a commercial product of Lonza corporation under the brand name BE02-054Q, sapelin is a commercial product of Shang Ji Luya pharmaceutical Co., Ltd, hydrocortisone is a commercial product of Guo Ji Sheng pharmaceutical Co., Ltd, human interleukin 2 is a commercial product of Shang quan hong Kong pharmaceutical Co., Ltd, human interleukin 15 is a commercial product of PEPTECH corporation under the brand name AF-200-15, CD16 monoclonal antibody is a commercial product of eBiosciences corporation under the brand name 16-0166-82, CD335 monoclonal antibody is a commercial product of eBioscience official 16-0037-81, lymphocyte separation liquid is a commercial product of Tianjin Yan biologicals scientific and technical responsibility Co., Ltd under the brand name LDS 7, and heparin sodium is a commercial product of SIGMA corporation under the brand name H3149. The above reagents except human interleukin 15 are stored at-20 deg.C, and the rest are stored at 4 deg.C.
Preparation of canine autologous plasma
Collecting blood of 5-10ml in the neck, and adding heparin sodium anticoagulant. And (3) centrifuging at the temperature of 23-28 ℃ for 15min at 750G. Taking the upper layer plasma, inactivating in 56 deg.C water bath for 30min, and shaking for 1 time every 5 min. Rapidly cooling the crushed ice box for 30min at 23-28 deg.C for 1000G for 10min, centrifuging, and collecting supernatant and storing at 4 deg.C.
Examples and comparative examples
Mixing an X-VIVO 15 culture medium and canine autologous plasma according to the volume percentage shown in Table 1 to obtain a total liquid composition of 10mL, and adding saproline, hydrocortisone, human interleukin 2, interleukin 15, a CD335 monoclonal antibody and a CD16 monoclonal antibody into the liquid composition to obtain an in vitro activating composition, wherein the using amount of each raw material is shown in Table 1, and the using amount of the saproline, the hydrocortisone, the IL-2, the IL-15, the CD335 and the CD16 in the Table 1 refers to the using amount of the X-VO VIVO 15 culture medium and the canine autologous plasma relative to the total amount of 1 mL.
According to the volume ratio of 19: 1 mixing X-VIVO 15 culture medium and dog autologous plasma to obtain a liquid composition of 50mL, and adding 1000IU/mL human interleukin 2 and 30ng/mL human interleukin 15 into the liquid composition to obtain an in vitro amplification culture solution.
Test example 1
Step 1: taking 5mL of lower layer blood cell sediment obtained by separating golden hair dog venous blood, adding a sample diluent (99 vol% of normal saline +1 vol% of dog autologous plasma, and then adding 1mL of normal saline and 50 mu g of heparin sodium relative to the total amount of dog autologous plasma) according to a volume ratio of 1:1, uniformly mixing, adding 5mL of lymphocyte separation liquid into a 15mL centrifuge tube, sucking 5mL of diluted blood cells, lightly applying the blood cells onto the liquid surface of the separation liquid, ensuring that the interface of the separation liquid cannot be damaged, performing centrifugation for 25min at 25 ℃ and 450 g. And (3) sucking the second annular milky white mononuclear cell layer into a new 15mL centrifuge tube, adding 10mL of cell cleaning solution (99 vol% of physiological saline and 1 vol% of canine autologous plasma), uniformly mixing the cells, mixing the cells at 250g, and centrifuging the mixture for 10 min. Discard the supernatant, add 5mL cell wash to resuspend the cells, 250g, centrifuge for 10 min.
And 2, step: activating: PBMCs were counted and cell density adjusted to 3X 10 by adding the in vitro activating composition of example 1 6 After each cell/mL, the cells were placed at 44g/m 3 Saturated humidity, 37 ℃ and 5% CO 2 The culture was performed in the incubator (1), and the day (0) was counted. The activation process of the cells was observed using an inverted microscope during the activation process on the 0 th day (a), the 1 st day (B), the 2 nd day (C), and the 3 rd day (D) using the in vitro activation composition of example 1, and the results obtained by photographing with a microscope camera are shown in fig. 1.
And step 3: amplification: cell counts from activated cultures were collected on day 3, 300g and centrifuged for 10 min. The in vitro amplification culture solution of example 1 was added to adjust the cell density to 3X 10 according to the counting results 6 cells/mL, at 44g/m 3 Saturated humidity, 37 ℃ and 5% CO 2 The cultivation was continued on days 4 to 18, and the growth state of the cells was observed every three days, photographed by a microscope camera, and counted. 300g, centrifuging for 10min, changing the liquid, sucking and discarding half of the supernatant, adding fresh dog NK amplification culture solution to adjust the cell density to 3 × 10 6 cells/mL, after resuspension continued to culture. The in vitro cell amplification process using the in vitro activating composition of example 1 followed by the in vitro amplification culture solution was observed using an inverted microscope and photographed by a microscope camera for the results obtained on day 1 (A) of the amplification day 15 (B), as shown in FIG. 2. The proliferation curve of NK cells amplified in vitro was plotted using cell counts in triplicate as shown in FIG. 3. The cell surface markers CD3, CD56 and CD16 were detected by flow cytometry, and the change in phenotype of NK in vitro expanded cells is shown in FIG. 4. See table 2 for changes in cell number.
Test examples 2 to 9
The procedure of test example 1 was followed except that the components and amounts of the in vitro activating compositions of examples 2 to 4 and comparative examples 1 to 5 were changed and the number of cells was changed as shown in Table 2.
Test example 10
The procedure of test example 1 was followed except that the in vitro amplification culture solution was directly added to the in vitro amplification culture without adding the in vitro activating composition of example 1 in step 2, and the change in the number of cells was shown in Table 2.
Test example 11
According to the method of test example 1, except that the canine breed was changed to labrador, the cell activation process was observed using an inverted microscope using the in vitro activation composition of example 1 on day 0 (a), day 1 (B), day 2 (C) and day 3 (D), and the results obtained by photographing with a microscope camera were shown in fig. 5. The in vitro cell amplification process using the in vitro activating composition of example 1 followed by the in vitro amplification culture solution on day 1 (A) and day 15 (B) was observed using an inverted microscope and photographed by a microscope camera to obtain the results shown in FIG. 6. The proliferation curve of NK cells amplified in vitro was plotted using cell counts in triplicate as shown in FIG. 7. The cell surface markers CD3, CD56 and CD16 were detected by flow cytometry, and the change of the phenotype of NK in vitro amplified cells is shown in FIG. 8.
Using a flow cytometer, the NK cell specific cell surface marker CD56 was detected, and the purity of NK cells after activation of canine PBMCs of two breeds, test example 1 golden retriever and test example 2 labrador retriever, using the in vitro activating composition of example 1 and addition of in vitro amplification culture medium for amplification, was shown in FIG. 9.
Table 1 in vitro activating composition formula table
Figure BDA0003753216030000121
TABLE 2 Absolute number of cells before and after activation and expansion
Figure BDA0003753216030000122
Figure BDA0003753216030000131
As can be seen from FIGS. 1 and 5, on the 0 th day of activation, the PBMCs mostly grow in suspension, the cells are round, the refractive index is high, and only a few monocytes grow adherently. On day 3 of activation, a few colonies of aggregated cells appeared, indicating that NK had been activated and accompanied by a small amount of proliferation.
As is clear from fig. 2 and 6, in the amplification process, the proliferation of NK cells was generally observed on day 1 after half-exchange, the proliferated NK cells grew in a typical grape bunch-like clonal shape, and the number of cells in the clone increased as the volume of the clone increased with the extension of the culture time.
As can be seen from FIGS. 3 and 7, NK cell proliferation rate was slow and cell number was slowly increased during the activation period and on the first 1 st to third days of expansion culture (i.e., the first 6 days of culture). However, from day 6 of culture, NK cells enter an exponential growth phase after the first three days of amplification culture in the presence of an amplification culture solution, and the cells rapidly proliferate, and the number of cells significantly increases. By the end of the culture, the number of cells was expanded by more than 30 times.
As can be seen from FIGS. 4 and 8, CD3+ contained about 70% of T cells and CD3-CD56+ and CD3-CD16+ contained about 1% of NK cells before PBMC culture. After the activating composition is added to activate NK cells to generate more CD56, the proportion of T cells of CD3+ is gradually reduced, the proportion of NK cells of CD3-CD56+ CD16+ is gradually increased, and the activation of clustered cell clones is found to be completed on the third day, an expanding solution is added to culture, until the culture is finished, the proportion of T cells of CD3+ is reduced to be less than 5%, and the proportion of NK cells of CD3-CD56+ and CD3-CD16+ is increased to be more than 80%.
As can be seen from FIG. 9, the purity of CD56+ NK cells was 80% or more in both of the two varieties of canine PBMC activation amplification cultures tested.
Although not shown, the changes in the microphotographs, proliferation curves, cell phenotypes, purity, and the like of the NK cell in test example 2 in the in vitro activation and expansion process were similar to those of FIGS. 1 to 9. Test examples 3 and 4 were similar in photomicrographs to FIGS. 1-9, with a similar trend in proliferation curve change and cell phenotype.
As can be seen from the results of table 2, example 1 and example 2 using the autologous plasma-containing xeno-free canine NK cell-activated culture of the present invention have similar or even better amplification efficiency and NK cell purity than comparative example 5 containing the fetal bovine serum-activated culture method, and exclude the significantly better clinical safety risk effect of xeno serum.
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.

Claims (10)

1. A composition for use in combination with autologous plasma and having an in vitro activation function of canine natural killer cells, wherein the composition comprises a glucocorticoid, human interleukin 2, human interleukin 15, a CD335 monoclonal antibody and a CD16 monoclonal antibody, wherein the content of human interleukin 2 is 10-40IU and the content of human interleukin 15 is 0.6-2.4ng per nanogram of the CD335 monoclonal antibody.
2. The composition of claim 1, wherein the glucocorticoid is present in an amount of 0.06-0.3 μ g and the CD16 monoclonal antibody is present in an amount of 1-4ng per nanogram of CD335 monoclonal antibody;
preferably, the glucocorticoid content is 0.07-0.15. mu.g, the human interleukin 2 content is 10-20IU, the human interleukin 15 content is 0.6-1.2ng and the CD16 monoclonal antibody content is 1-2ng per nanogram of CD335 monoclonal antibody.
3. The composition according to claim 2, wherein the glucocorticoid is at least one of prednisone, methylprednisolone, betamethasone, beclomethasone dipropionate, prednisolone, hydrocortisone and dexamethasone, preferably hydrocortisone.
4. The composition of claim 1, further comprising at least one of X-VIVO 15 medium, canine autologous plasma, and inactivated group a Streptococcus (Streptococcus pyogenes).
5. The composition of claim 4, wherein the volume ratio of the X-VIVO 15 medium to the canine autologous plasma is 9-50: 1;
and/or the content of the CD335 monoclonal antibody is 50-200ng relative to the total amount of 1mL of X-VIVO 15 culture medium and canine autologous plasma.
6. The composition according to claim 4, wherein the inactivated group A Streptococcus is present in an amount of 0-0.0016KE, preferably 0.0004-0.0008KE, per nanogram of CD335 monoclonal antibody;
and/or, the inactivated group a streptococcus is preferably sapelin.
7. A method for in vitro culturing canine natural killer cells by using autologous plasma, the method comprising: activated culture of canine autologous plasma isolated peripheral blood mononuclear cells mixed with the composition of any one of claims 1 to 6.
8. The method of claim 7, wherein the composition is used in an amount such that the initial cell density of canine peripheral blood mononuclear cells in the mixed system is 3 x 10 or more 6 Individual cells/mL;
and/or, the conditions for activating the culture comprise: saturated humidity, temperature of 36-38 deg.C, CO 2 The content is 5-10%.
9. The method of claim 7, wherein the in vitro culturing method further comprises isolating and expanding the cells after activation culture; the amplification conditions include: saturated humidity, temperature of 36-38 deg.C, CO 2 The content is 5-10%; the amplification is carried outThe culture solution contains human interleukin 2, human interleukin 15, X-VIVO 15 culture medium and dog autologous plasma.
10. The method according to claim 9, wherein the volume ratio of the X-VIVO 15 culture medium to the canine autologous plasma in the culture solution used for the amplification is 9-50: 1;
and/or, the content of the human interleukin 2 is 1000-; the content of human interleukin 15 is 30-120ng, preferably 30-60 ng.
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