CN109125717B - Autologous whole cell vaccine formula for treating chronic diseases and preparation method thereof - Google Patents
Autologous whole cell vaccine formula for treating chronic diseases and preparation method thereof Download PDFInfo
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- C12N2501/20—Cytokines; Chemokines
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The invention discloses an autologous whole cell vaccine formula for treating chronic diseases, which comprises autologous cells, a monoclonal antibody, a cytokine, a cell lysis agent and normal saline. The invention also discloses a preparation method of the autologous whole cell vaccine for treating chronic diseases, which comprises the steps of collection, separation, extraction, culture, amplification, collection, lysis, purification and preparation. The present invention relates to a vaccine prepared by using variant cells in the blood of human body, it can stimulate the body to produce specific immunoreaction aimed at variant antigen, and can produce immune cell and monoclonal antibody with specific killing action, these cells and antibody can circularly recognize and kill the variant cells carrying variant antigen in vivo, and can remove the metabolic waste produced by variant cells so as to attain the goal of preventing disease recurrence and raising life quality.
Description
Technical Field
The invention relates to the technical field of immune cell therapy, in particular to an autologous whole cell vaccine formula for treating chronic diseases and a preparation method thereof.
Background
The whole-cell vaccine can safely and effectively activate the immune system and stimulate the body to generate natural killer cells. It takes about one week for the immune response to reach the variant cells, so as to generate the primary reaction of the specific monoclonal antibody, and it takes about two weeks for the primary reaction of the specific monoclonal antibody of the variant cells to reach the secondary reaction of the specific monoclonal antibody of the variant cells.
At present, there is no obvious breakthrough in the preparation of autologous whole cell vaccines for treating chronic diseases, and therefore, the formula and the preparation method of the autologous whole cell vaccine for treating chronic diseases are provided for solving the problems.
Disclosure of Invention
The invention aims to provide an autologous whole cell vaccine formula for treating chronic diseases and a preparation method thereof, so as to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme: an autologous whole cell vaccine formula for treating chronic diseases comprises the following components in parts by weight: 5 parts of autologous cells, 2 parts of monoclonal antibodies, 2 parts of cytokines, 1 part of cell lysis agent and normal saline;
wherein the monoclonal antibody comprises 1 part of monoclonal antibody CD3 and 1 part of monoclonal antibody CD56, and the cytokine comprises 1 part of IL-2 and 1 part of IL-15.
A method for preparing autologous whole cell vaccine for treating chronic diseases comprises the following steps:
the method comprises the following steps: collecting peripheral blood from a human body suffering from chronic diseases;
step two: separating, separating peripheral blood and extracting autoimmune cells;
step three: extracting, centrifuging, extracting cells, and washing the cells;
step four: culturing, adding monoclonal antibody, serum and cell factor to culture cell;
step five: amplifying, and supplementing serum, monoclonal antibody and cell factor to culture cells;
step six: collecting, culturing for half a month, and collecting cells;
step seven: lysing, with a cell lysis agent;
step eight: purifying to obtain semi-finished whole-cell vaccine;
step nine: preparing a whole-cell vaccine finished product;
as a modification of the present invention, the volume of peripheral blood collection in the first step is 100 ml.
As an improvement of the invention, in the second step, a centrifuge is adopted for centrifugal treatment, the rotating speed is 2000rpm, and the centrifugal time is 20 minutes.
As an improvement of the invention, the monoclonal antibody added in the step four is the monoclonal antibody CD3, the cytokine is IL-2, and the monoclonal antibody is dissolved in 500ml of culture medium and cultured in a 5.0% CO incubator at the culture temperature of 37 ℃.
As an improvement of the invention, the monoclonal antibody supplemented in the fifth step is the monoclonal antibody CD56, and the cytokine is IL-15.
As an improvement of the invention, the collection is carried out in a centrifugation mode in the step six, the rotation speed of the centrifuge is 1500rpm, and the centrifugation time is 10 minutes.
As an improvement of the invention, the cell lysis agent adopted in the seventh step is proteinase K, and the cell lysis mode is that the cell is cracked by shaking the shaking table for 2 hours.
And as an improvement of the invention, a centrifuge is adopted for purification in the step eight, the rotation speed of the centrifuge is 15000rpm, and the centrifugation time is 1 hour.
Compared with the prior art, the invention has the beneficial effects that: the present invention relates to a vaccine prepared by using variant cells in the blood of human body, it can stimulate the body to produce specific immunoreaction aimed at variant antigen, and can produce immune cell and monoclonal antibody with specific killing action, these cells and antibody can circularly recognize and kill the variant cells carrying variant antigen in vivo, and can remove the metabolic waste produced by variant cells so as to attain the goal of preventing disease recurrence and raising life quality.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the attached tables in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. 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.
The invention provides a technical scheme that: an autologous whole cell vaccine formula for treating chronic diseases comprises the following components in parts by weight: 5 parts of autologous cells, 2 parts of monoclonal antibodies, 2 parts of cytokines, 1 part of cell lysis agent and normal saline;
wherein the monoclonal antibody comprises 1 part of monoclonal antibody CD3 and 1 part of monoclonal antibody CD56, and the cytokine comprises 1 part of IL-2 and 1 part of IL-15.
The invention also provides a preparation method of the autologous whole cell vaccine for treating chronic diseases, and the technical scheme of the invention is further explained by combining specific embodiments.
Example one
A method for preparing autologous whole cell vaccine for treating chronic diseases comprises the following steps:
the method comprises the following steps: collecting peripheral blood from human body with chronic diseases about 50 ml; centrifuging the collected blood in a centrifuge at 1500rpm for 8 minutes, sucking the upper serum out and transferring into another centrifuge tube, inactivating the plasma in a 56 ℃ water bath for 30 minutes, centrifuging again at 1000rpm for 8 minutes, transferring the treated supernatant into another centrifuge tube, and placing at 4 ℃ in an environment;
step two: separating, namely separating peripheral blood to extract autologous cells; diluting the obtained plasma whole blood by 2 times by adding physiological saline, diluting the blood which is precipitated or diluted well, slowly adding the blood into a liquid containing lymphocyte separation liquid, adding the lymphocyte separation liquid according to the proportion of 1:1, and then centrifuging, wherein the rotating speed of a centrifuge is 1800rpm, and the centrifuging time is 20 minutes;
step three: extracting, after centrifugation, obviously seeing white rings, slightly inserting a suction tube into the white rings to suck out cells, putting the white rings into another test tube, adding a proper amount of PBS (phosphate buffer solution) or physiological saline, then centrifuging, wherein the rotating speed of a centrifuge is 1000rpm, the centrifuging time is 10 minutes, discarding supernate, washing the supernate with a proper amount of PBS or physiological saline once, centrifuging again, the rotating speed of the centrifuge is 1000rpm, the centrifuging time is 10 minutes, blowing up the cells with a serum-free culture medium for the third time, blowing the cells uniformly, and then putting the cells into a culture bottle;
step four: culturing by dissolving 1 count of cytokine and 1 count of monoclonal antibody in 500ml of culture medium, adding 30ml of culture medium into each culture flask, adding 1% patient serum, placing at saturated humidity and 37 deg.C, and charging 5.0% CO2Culturing in an incubator;
step five: expanding, continuously culturing for 3 days, pouring the cultured immunocytes for 3 days into a cell culture bag, pouring the rest culture medium and serum, and adding 1 cytokine and 1 sheetThe cloned antibody was dissolved in another 500ml flask of culture medium, poured into a cell culture bag, and continuously charged with 5.0% CO at 37 deg.C2Culturing in an incubator;
step six: collecting, namely collecting cells after culturing for 8 days, slowly pouring the cells in a culture bag or a culture bottle into a centrifuge cup, centrifuging at the rotating speed of 1000rpm for 10 minutes, discarding the supernatant, collecting the cells, washing the cells with physiological saline for 3 times, centrifuging again, at the rotating speed of 1000rpm for 10 minutes, and diluting the centrifuged and washed cells with 10ml of physiological saline for later use;
step seven: cracking, preparing 100mg of protease K into a working solution of 20mg/ml, adding 30ul of the working solution into 10ml of cell suspension, and placing the cell suspension in a shaking table to shake for half an hour to crack cells;
step eight: purifying, namely purifying the inactivated cells under high-speed centrifugation to ensure that the cells are fully cracked and then centrifuged, wherein the rotating speed of a centrifuge is 9000rpm, the centrifugation time is 1 hour, and the obtained cell precipitates are semi-finished whole cells;
step nine: the preparation is prepared by blowing the precipitate with normal saline to obtain suspension, i.e. whole cell vaccine, and subpackaging into 0.5ml injection.
Example two
A method for preparing autologous whole cell vaccine for treating chronic diseases comprises the following steps:
the method comprises the following steps: collecting peripheral blood from human body with chronic diseases, and collecting about 120 ml; centrifuging the collected blood in a centrifuge at 3000rpm for 8 minutes, sucking the upper serum out and transferring into another centrifuge tube, inactivating the plasma in a 56 ℃ water bath for 30 minutes, centrifuging again at 2800rpm for 8 minutes, transferring the treated supernatant into another centrifuge tube, and placing at 4 ℃ in an environment;
step two: separating, namely separating peripheral blood to extract autologous cells; diluting the obtained plasma whole blood by 2 times with physiological saline, diluting the blood which is precipitated or diluted well, slowly adding the blood into a liquid containing lymphocyte separation liquid according to the proportion of 1:1, and then centrifuging, wherein the rotating speed of a centrifuge is 3000rpm, and the centrifuging time is 20 minutes;
step three: extracting, after centrifugation, obviously seeing white rings, slightly inserting a suction tube into the white rings to suck out cells, putting the white rings into another test tube, adding a proper amount of PBS (phosphate buffer solution) or physiological saline, then centrifuging, wherein the rotating speed of a centrifuge is 2000rpm, the centrifuging time is 10 minutes, discarding supernate, washing the supernate with a proper amount of PBS or physiological saline once, centrifuging again, the rotating speed of the centrifuge is 2000rpm, the centrifuging time is 10 minutes, blowing up the cells with a serum-free culture medium for the third time, blowing the cells uniformly, and then putting the cells into a culture bottle;
step four: culturing by dissolving 3-count cytokine and 300ul monoclonal antibody in 500ml culture medium, adding 30ml culture medium into each culture flask, adding 5% patient serum, placing at saturated humidity, 37 deg.C and 5.0% CO2Culturing in an incubator;
step five: amplifying, after continuously culturing for 9 days, pouring the immune cells cultured for 9 days into a cell culture bag, pouring the rest culture medium and serum into the cell culture bag, dissolving 3 cytokines and 300ul monoclonal antibody in another 500ml culture medium bottle, pouring into the cell culture bag, and continuously filling 5.0% CO at 37 deg.C2Culturing in an incubator;
step six: collecting, namely collecting cells after culturing for 20 days, slowly pouring the cells in a culture bag or a culture bottle into a centrifuge cup, centrifuging at the rotating speed of 3000rpm for 5 minutes, discarding supernatant, collecting the cells, washing the cells with physiological saline for 3 times, centrifuging again at the rotating speed of 3000rpm for 5 minutes, and diluting the centrifuged and washed cells with 10ml of physiological saline for later use;
step seven: cracking, preparing 100mg of protease K into a working solution of 20mg/ml, adding 100ul of the working solution into 10ml of cell suspension, and shaking in a shaking table for 4 hours to crack cells;
step eight: purifying, namely purifying the inactivated cells under high-speed centrifugation to ensure that the cells are fully cracked and then centrifuged, wherein the rotating speed of a centrifuge is 25000rpm, the centrifugation time is 1 hour, and the obtained cell precipitates are semi-finished whole cells;
step nine: the preparation is prepared by blowing the precipitate with normal saline to obtain suspension, i.e. whole cell vaccine, and subpackaging into 1.5ml injection.
EXAMPLE III
A method for preparing autologous whole cell vaccine for treating chronic diseases comprises the following steps:
the method comprises the following steps: collecting peripheral blood from human body with chronic diseases, and collecting 100 ml; centrifuging the collected blood in a centrifuge at 2300rpm for 8 minutes, sucking the upper serum out and transferring into another centrifuge tube, inactivating the plasma in a 56 ℃ water bath for 30 minutes, centrifuging again at 1800rpm for 8 minutes, transferring the treated supernatant into another centrifuge tube, and placing at 4 ℃ in the environment;
step two: separating, namely separating peripheral blood to extract autologous cells; diluting the obtained plasma whole blood by 2 times with physiological saline, diluting the blood which is precipitated or diluted well, slowly adding the blood into a liquid containing lymphocyte separation liquid according to the proportion of 1:1, and then centrifuging, wherein the rotating speed of a centrifuge is 2000rpm, and the centrifuging time is 20 minutes;
step three: extracting, after centrifugation, obviously seeing white rings, slightly inserting a suction tube into the white rings to suck out cells, putting the white rings into another test tube, adding a proper amount of PBS (phosphate buffer solution) or physiological saline, then centrifuging, wherein the rotating speed of a centrifuge is 1500rpm, the centrifuging time is 10 minutes, discarding supernate, washing the supernate with a proper amount of PBS or physiological saline once, centrifuging again, the rotating speed of the centrifuge is 1500rpm, the centrifuging time is 10 minutes, blowing up the cells with a serum-free culture medium for the third time, blowing the cells uniformly, and then putting the cells into a culture bottle;
step four: culturing, dissolving 2 immune cell inducing and proliferating agent and 1 immune cell regulator in 500ml culture medium, adding 30ml culture medium into each culture bottle, adding 5% patient serum into each culture bottle, and culturing in an incubator with saturated humidity, 37 deg.C and 5.0% CO;
step five: expanding, continuously culturing for 5 days, pouring the cultured immunocytes into a cell culture bag, adding the rest culture medium and serum, dissolving 1 st immunocyte proliferation promoter and 1 st immunocyte regulating stimulator in 500ml culture medium, pouring into cell culture bag, continuously culturing at 37 deg.C under the action of 5.0% CO2Culturing in an incubator;
step six: collecting, namely collecting cells after culturing for 14 days, slowly pouring the cells in a culture bag or a culture bottle into a centrifuge cup, centrifuging at the rotating speed of 1500rpm for 10 minutes, discarding the supernatant, collecting the cells, washing the cells with physiological saline for 3 times, centrifuging again, at the rotating speed of 1500rpm for 10 minutes, and diluting the centrifuged and washed cells with 10ml of physiological saline for later use;
step seven: cracking, preparing 100mg of protease K into a working solution of 20mg/ml, adding 50ul of the working solution into 10ml of cell suspension, and shaking in a shaking table for 2 hours to crack cells;
step eight: purifying, namely purifying the inactivated cells under high-speed centrifugation to ensure that the cells are fully cracked and then centrifuged, wherein the rotating speed of a centrifuge is 15000rpm, the centrifugation time is 1 hour, and the obtained cell precipitates are semi-finished whole cells;
step nine: the preparation is prepared by blowing the precipitate with normal saline to obtain suspension, i.e. whole cell vaccine, and subpackaging into 1.5ml injection.
TABLE one Whole cell vaccine injection two weeks later, blood glucose Change in diabetic type II patients
(blood glucose concentration: mmol/L)
Blood glucose change in diabetic type II patients four weeks after injection of the Whole cell vaccine
(blood glucose concentration: mmol/L)
According to the three embodiments, the invention is a vaccine prepared by utilizing the variant cells in the blood of a human body, which can stimulate the body to generate specific immune response against the variant antigen, generate immune cells and monoclonal antibodies with specific killing effect, and the cells and the antibodies can recognize and kill the variant cells carrying the variant antigen in the in vivo circulation, and eliminate metabolic waste generated by the variant cells.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (5)
1. A preparation method of autologous whole cell vaccine for treating diabetes II, which is characterized in that,
the autologous whole cell vaccine formula for treating diabetes II comprises the following components in parts by weight: 5 parts of autologous cells, 2 parts of monoclonal antibodies, 2 parts of cytokines, 1 part of cell lysis agent and normal saline;
wherein the monoclonal antibody comprises 1 part of anti-CD 3 monoclonal antibody and 1 part of anti-CD 56 monoclonal antibody, and the cytokine comprises 1 part of IL-2 and 1 part of IL-15;
the preparation method of the autologous whole cell vaccine for treating the diabetes II comprises the following steps:
the method comprises the following steps: collecting peripheral blood from a human having diabetes type II;
step two: separating, namely separating peripheral blood to extract autologous cells; adding physiological saline for diluting by 2 times, slowly adding the diluted blood into lymphocyte separation liquid according to the proportion of 1:1, and then centrifuging, wherein the rotating speed of a centrifuge is 2000rpm, and the centrifuging time is 20 minutes;
step three: extracting, centrifuging, extracting cells, and washing the cells;
step four: culturing, adding monoclonal antibody, serum, and cytokine to culture cells, wherein the added monoclonal antibody is anti-CD 3 monoclonal antibody, and the cytokine is IL-2, dissolving in 500ml culture medium, and culturing at 37 deg.C and 5.0% CO2Culturing in an incubator;
step five: amplifying, and supplementing serum, monoclonal antibody and cell factor to culture cells, wherein the supplemented monoclonal antibody is anti-CD 56 monoclonal antibody, and the cell factor is IL-15;
step six: collecting, culturing for half a month, and collecting cells;
step seven: cracking, namely cracking by using a cell cracking agent, wherein the cell cracking agent is proteinase K, and the cracking mode is that the cells are cracked by shaking a table for 2 hours;
step eight: purifying to obtain semi-finished whole-cell vaccine;
step nine: preparing the preparation into a whole-cell vaccine finished product.
2. The method of claim 1, wherein the preparation of the autologous whole cell vaccine for the treatment of type II diabetes is characterized by: the volume of peripheral blood collection in step one was 100 ml.
3. The method of claim 1, wherein the preparation of the autologous whole cell vaccine for the treatment of type II diabetes is characterized by: and in the second step, a centrifugal machine is adopted for centrifugal treatment, the rotating speed is 2000rpm, and the centrifugal time is 20 minutes.
4. The method of claim 1, wherein the preparation of the autologous whole cell vaccine for the treatment of type II diabetes is characterized by: and collecting in a centrifugation mode in the sixth step, wherein the rotation speed of the centrifuge is 1500rpm, and the centrifugation time is 10 minutes.
5. The method of claim 1, wherein the preparation of the autologous whole cell vaccine for the treatment of type II diabetes is characterized by: and step eight, purifying by adopting a centrifugal machine, wherein the rotating speed of the centrifugal machine is 15000rpm, and the centrifugation time is 1 hour.
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