Disclosure of Invention
The present invention aims to provide a mesenchymal stem cell preparation prepared from peritock tissue-derived mesenchymal stem cells, particularly umbilical cord-derived mesenchymal stem cells, and has unexpectedly been found to be capable of significantly improving physiological changes in the lung by intravenous injection of the mesenchymal stem cell preparation into a virus-infected viral pneumonia mouse. The present invention has been completed based on such findings.
To this end, the first aspect of the present invention provides a cell preparation comprising umbilical mesenchymal stem cells and a pharmaceutically acceptable vehicle.
The cell preparation according to the first aspect of the present invention, wherein the umbilical cord mesenchymal stem cells are prepared by a method comprising the steps of:
(1) Preparing umbilical cord tissue frozen stock solution: the umbilical cord tissue frozen stock solution comprises human serum albumin and DMSO (dimethyl sulfoxide), and the prepared frozen stock solution is subjected to low-temperature refrigeration at a temperature of 1-7 ℃ (e.g. 4 ℃);
(2) Sterilizing and cleaning: disinfecting the surface of the umbilical cord tissue with a disinfectant (e.g., alcohol), shearing open the umbilical cord, tiling, and washing the umbilical cord tissue with a buffer (e.g., PBS buffer) to reduce red blood cells on the umbilical cord tissue;
(3) Umbilical cord tissue treatment: shearing umbilical cord tissues obtained in the step (2) into tissue blocks;
(4) Umbilical cord tissue cold storage: adding the tissue mass and the frozen stock solution into a frozen stock container under a low temperature environment of 0-15 ℃ (e.g. 1 ℃ to 7 ℃, e.g. 4 ℃), then placing the frozen stock container into a program cooling device, firstly refrigerating at a low temperature of 1 ℃ to 7 ℃ (e.g. 4 ℃) for 0.2-2 hours (e.g. 0.5 hours), then refrigerating at a temperature of-10 ℃ to-150 ℃ (e.g. -80 ℃) for 0.25-3 days (e.g. 1 day), and then refrigerating the frozen stock container in liquid nitrogen for later use;
(5) Cryopreserved umbilical cord tissue resuscitates: taking out the frozen umbilical cord tissue in the step (4) from liquid nitrogen, thawing to 20% -70% (e.g. 50%) of frozen stock solution, starting thawing, cleaning the umbilical cord tissue by using a mesenchymal stem cell culture medium, and filtering to remove waste liquid so as to resuscitate the frozen umbilical cord tissue;
(6) Umbilical cord tissue adherence treatment: taking cell culture plates, spreading the tissue blocks in the plates, and keeping the number of the tissue blocks in each plate at 5-20 blocks (for example, 10-15 blocks), so that the tissue blocks are air-dried for 2-50 minutes until the tissues are attached to the plates;
(7) Umbilical cord tissue culture: slowly adding a mesenchymal stem cell culture medium along the edge of the plate until the tissue is submerged; place the dishes into CO2 Culturing in a 37 deg.C incubator with a concentration of 5%, taking out the plate from the incubator until 3-7 days (e.g. 4-6 days, e.g. 5 days), and supplementing a proper amount of mesenchymal stem cell culture medium (for submerging tissues); removing the culture medium from the dish on days 9-11 (such as day 10), adding appropriate amount of fresh mesenchymal stem cell culture medium (for submerging tissue), and continuing culturing; all umbilical cord tissue blocks were removed and culture continued on days 11-13 (e.g., day 12); after which the whole change is performed every 1-3 days (e.g. every 2 days);
(8) Cell passage: when the fusion rate of the adherent cells in the plate reaches about 50-70%, using digestive enzymes (such as TrypLE Express, invitrogen products) to separate the adherent cells from the bottom of the plate; centrifuging, and removing supernatant to obtain P0 generation umbilical cord mesenchymal stem cells; adding a mesenchymal stem cell culture medium into the P0 generation cells to re-suspend the cells, and then inoculating the cells into a T25 cell culture bottle for passage and amplification culture; changing the liquid every 1-3 days (for example every 2 days) until the fusion rate reaches 70-90%, obtaining the P1 generation umbilical cord mesenchymal stem cells, and completing the passage of the P0 generation to the P1 generation cells; and repeating the passage operation from the generation P0 to the generation P1 in sequence to respectively carry out the passage of the cells from the generation P1 to the generation P2, the generation P2 to the generation P3 to the generation P15, so as to obtain the umbilical cord mesenchymal stem cells from the generation P1 to the generation P15.
The cell preparation according to the first aspect of the present invention, wherein in the process of preparing mesenchymal stem cells, further comprising:
(9) Detecting, for the umbilical cord mesenchymal stem cells obtained in step (8), at least one of the following items: cell activity, cell contamination, genetic disease, HLA-ABC/DR ligand.
The cell preparation according to the first aspect of the present invention, wherein in the process of preparing mesenchymal stem cells, further comprising:
(10) Freezing each passage of umbilical cord mesenchymal stem cells obtained in the step (8) in liquid nitrogen, for example, placing the cells in a cell freezing solution for freezing; and
(11) Establishing a database of umbilical cord stem cells containing the information detected in step (9) above, and correlating the database with the cryopreserved cells of step (10).
The cell preparation according to the first aspect of the present invention, wherein the cell purity of each generation of umbilical cord mesenchymal stem cells obtained in the process of preparing mesenchymal stem cells is greater than 90%.
The cell preparation according to the first aspect of the invention, wherein the umbilical cord is fresh ex vivo tissue of the umbilical cord.
The cell preparation according to the first aspect of the present invention, wherein the umbilical cord tissue-freezing solution comprises human serum albumin and DMSO. In one embodiment, the umbilical cord tissue-freezing stock solution contains 55-95 parts by weight of human serum albumin. In one embodiment, the umbilical cord tissue-freezing solution contains 70-90 parts by weight of human serum albumin. In one embodiment, the umbilical cord tissue-cryopreservation solution contains 80 parts by weight of human albumin. In one embodiment, the umbilical cord tissue cryopreservation solution contains 4-20 parts by weight DMSO. In one embodiment, the umbilical cord tissue cryopreservation solution contains 7-15 parts by weight DMSO. In one embodiment, the umbilical cord tissue cryopreservation solution contains 10 parts by weight DMSO. In one embodiment, the umbilical cord tissue-cryopreservation solution contains about 80 parts by weight human albumin, about 10 parts by weight DMSO. In the above embodiment, the total content of each component in the umbilical cord tissue frozen stock solution is 100%. The present inventors have found that umbilical cord tissue cryopreservation solutions containing about 80 parts by weight of human albumin, about 10 parts by weight of DMSO (i.e., wherein the weight ratio of human albumin to DMSO is 80:10) are particularly preferred, as compared to formulations that vary the content of any component of the umbilical cord tissue cryopreservation solution by more than 10% have significant advantages in protecting umbilical cord tissue from damage by the freezing process, and the like. In the present invention, since other liquid-preparing solvents or solutes commonly used by those skilled in the art may be contained in the frozen stock solution, the amounts of human serum albumin and DMSO expressed in "parts by weight" mentioned above are relative amounts, which may be milligrams, grams, kilograms, etc.
The cell preparation according to the first aspect of the present invention, wherein the refrigeration of step (1) is stored in a refrigerator.
The cell preparation according to the first aspect of the invention, wherein the umbilical cord is fresh ex vivo tissue of the umbilical cord.
The cell preparation according to the first aspect of the invention, wherein the umbilical cord tissue of step (2) is treated in a biosafety cabinet.
The cell preparation according to the first aspect of the invention, wherein the step of plating in step (2) is to plate umbilical cord tissue in a culture plate, the culture plate being a culture plate having a diameter of 5-20cm, preferably a culture plate having a diameter of 10 cm.
The cell preparation according to the first aspect of the invention, wherein the concentration of alcohol is 25% -95%, preferably 75%.
The cell preparation according to the first aspect of the invention, wherein the PBS buffer is formulated as sodium and/or potassium phosphate salt, having a pH of 5.0-8.0, preferably a pH of 5.5-76, preferably a pH of 6.0-7.0. In one embodiment, the phosphate concentration in the PBS buffer is 0.01 to 0.5M, preferably 0.02 to 0.1M. In the experiments described below, the PBS buffer used was sodium phosphate, wherein the phosphate concentration was 0.025M and the pH was 6.5. The inventors found that the concentration of the PBS buffer and the pH value in the above-described range had little effect on the effect of the method of the present invention.
The cell preparation according to the first aspect of the invention, wherein step (3) is transferring the umbilical cord tissue to another cell culture plate, preferably a culture plate having a diameter of 5-20cm, preferably a culture plate having a diameter of 10cm, and then shearing the umbilical cord tissue into tissue pieces.
The cell preparation according to the first aspect of the present invention, wherein the low temperature environment of step (4) is 0-15 ℃, preferably 1 ℃ to 7 ℃, preferably 4 ℃.
The cell preparation according to the first aspect of the present invention, wherein the cryopreservation vessel of step (4) is a cryopreservation tube.
The cell preparation according to the first aspect of the present invention, wherein the cooling device in step (4) is a cooling box.
The cell preparation according to the first aspect of the present invention, wherein the tissue mass of step (4) is in a cryopreservation vessel at a density of 0.1 to 0.8 units of tissue mass per unit space, preferably 0.1 to 0.8 units/cm, to ensure that any tissue mass has sufficient DMSO to provide protection as an upper limit3 Preferably 0.5 pieces/cm3 。
The cell preparation according to the first aspect of the present invention, wherein the low temperature refrigeration and low temperature freezing of step (4) is achieved by placing a temperature reducing device (e.g., a temperature reducing box) in a refrigerator.
The cell preparation according to the first aspect of the present invention, wherein the sample refrigeration process of step (4) is a low temperature refrigeration process at a temperature of 1 ℃ to 7 ℃ for 0.2 to 2 hours. In one embodiment, the low temperature refrigeration is at a temperature of 2 ℃ to 6 ℃. In one embodiment, the low temperature refrigeration is at a temperature of 3 ℃ to 5 ℃. In one embodiment, the low temperature refrigeration is at a temperature of 5 ℃. In one embodiment, the cold storage time is from 0.3 to 1.5 hours. In one embodiment, the cold storage time is from 0.4 to 1 hour. In one embodiment, the cold storage time is 0.5 hours. The inventors found that cryopreservation at a temperature of 4℃for 0.5 hours is particularly preferred, and that sufficient fusion of DMSO and umbilical cord tissue can be ensured. The DMSO and the umbilical cord tissue can be fused under other refrigeration temperature conditions and refrigeration time, the fusion effect can meet the basic requirement of the invention, but the fusion effect of 0.5 hour of low-temperature refrigeration under the temperature condition of 4 ℃ is most sufficient, and the method has obvious advantages.
The cell preparation according to the first aspect of the present invention, wherein the sample freezing procedure of step (4) is freezing at a temperature of-10 ℃ to-150 ℃ for 0.25 to 3 days. In one embodiment, the freezing is at a temperature of-30 ℃ to-120 ℃. In one embodiment, the freezing is at a temperature of-50 ℃ to-100 ℃. In one embodiment, the freezing is at a temperature of-80 ℃. In one embodiment, the freezing time is from 0.4 to 2 days. In one embodiment, the freezing time is from 0.8 to 1.5 days. In one embodiment, the freezing time is 1 day. The inventors found that freezing for 1 day at a temperature of-80 ℃ is particularly preferred, as compared to a freezing procedure that varies by more than 10% in any temperature, time of the freezing procedure, has significant advantages in protecting umbilical cord tissue from damage by the freezing process, etc.
The cell preparation according to the first aspect of the invention, wherein the thawing of step (5) is performed in a thermostatic water bath.
The cell preparation according to the first aspect of the present invention, wherein the mesenchymal stem cell medium comprises FBS, L-Glutamine, gentamicin and DMEM-F12. In one embodiment, the mesenchymal stem cell medium contains 10-20% FBS. In one embodiment, the mesenchymal stem cell medium contains about 15% FBS. In one embodiment, the mesenchymal stem cell medium contains 0.5-2% L-Glutamine. In one embodiment, the mesenchymal stem cell medium contains about 1% L-Glutamine. In one embodiment, the mesenchymal stem cell medium contains 0.02-0.1% Gentamicin (Gentamicin). In one embodiment, the mesenchymal stem cell medium contains about 0.05% Gentamicin. In one embodiment, the mesenchymal stem cell medium contains 80-90% DMEM-F12. In one embodiment, the mesenchymal stem cell medium contains about 84% DMEM-F12. In one embodiment, the mesenchymal stem cell medium contains about 15 parts by weight of FBS, about 1 part by weight of L-Glutamine, about 0.05 part by weight of Gentamicin and about 84 parts by weight of DMEM-F12. The present inventors found that a mesenchymal stem cell medium containing about 15 parts by weight of FBS, about 1 part by weight of L-Glutamine, about 0.05 part by weight of Gentamicin and about 84 parts by weight of DMEM-F12 is particularly preferable, and that a formulation in which the content of any one component of the medium is changed by 10% or more has significant advantages in the effects of increasing the adherence of umbilical cord tissue, reducing the time for adherent cells to climb out of the tissue, and the like.
The cell preparation according to the first aspect of the present invention, wherein the mesenchymal stem cell medium of step (5) is umbilical cord tissue washed by a drip method. The drip method can effectively clean the DMSO in the tissue, thereby avoiding the loss of the cell survival rate in the resuscitation process.
The cell preparation according to the first aspect of the invention, wherein said filtering of the waste liquid of step (5) is achieved by a 50-200 μm filter, preferably a 100 μm filter.
The cell preparation according to the first aspect of the invention, wherein the culture plate is a culture plate having a diameter of 5-20cm, preferably a culture plate having a diameter of about 10 cm.
The cell preparation according to the first aspect of the invention, wherein in step (6) the tissue mass is air dried for a period of 2-50 minutes, such as 5-25 minutes, such as 10-15 minutes. The present inventors found that the effects of increasing the adhesion of umbilical cord tissue, reducing the time for adherent cells to climb out of the tissue, and the like are optimal in the air-drying time of 10 to 15 minutes, and significantly different from those of the air-drying time of less than 5 minutes or the air-drying time of more than 25 minutes.
According to the cell preparation of the first aspect of the present invention, wherein in the step (7), the inventors found that, at the time of culturing to the 5 th day, the plate was taken out of the incubator, an appropriate amount of the mesenchymal stem cell medium was supplemented, the culturing was continued, the medium in the plate was removed at the 10 th day, an appropriate amount of fresh mesenchymal stem cell medium was added, the culturing was continued, all umbilical cord tissue pieces were removed at the 12 th day and the culturing was continued, and thereafter, the whole liquid change was performed every 2 days, which was particularly preferable. The time for the adherent cells to reach the appointed fusion rate is effectively shortened by the liquid exchange and the tissue removal time.
According to the cell preparation of the first aspect of the present invention, in step (9), the cell activity detection is counting the number of living cells before and after freezing by trypan blue staining.
According to the cell preparation of the first aspect of the present invention, in step (9), the cell contamination detection detects whether the cells are contaminated with fungi and bacteria using a small amount of cell culture. In one embodiment, the cell contamination detection is by etiology methods to detect whether the cell is infected with one or more selected from the group consisting of: hepatitis B virus, hepatitis C virus, HIV, cytomegalovirus, EB virus and syphilis, hbsAg, hbsAb, HBcAb, hbeAg, hbeAb, HCVAb, HIV-1/2Ab, CMV-IgM and EBV-IgA, TRUST.
According to the cell preparation of the first aspect of the present invention, in step (9), the genetic disease detection is a method of detecting the presence or absence of a genetic disease in frozen cells using molecular genetics.
According to the cell preparation of the first aspect of the invention, in step (9), the HLA-ABC/DR ligand is a test cell HLA-ABC/DR phenotype.
According to the cell preparation of the first aspect of the present invention, in step (10), the umbilical cord mesenchymal stem cells are frozen in liquid nitrogen through a programmed cooling process.
According to the cell preparation of the first aspect of the present invention, in step (10), the umbilical cord mesenchymal stem cells are present in a cell cryopreservation solution. In one embodiment, the cell cryopreservation solution comprises DMEM-F12, dimethyl sulfoxide, and human serum albumin. In one embodiment, the cell cryopreservation solution comprises about 65 parts DMEM-F12, about 10 parts dimethyl sulfoxide, about 15 parts human serum albumin. In one embodiment, the cell cryopreservation solution comprises 50% low sugar DMEM broth, 40% fbs, 10% dimethyl sulfoxide.
According to the cell preparation of the first aspect of the present invention, the method for preparing the umbilical cord mesenchymal stem cells comprises the steps of:
(1) Preparing umbilical cord tissue frozen stock solution: the umbilical cord tissue frozen stock solution comprises 80 parts by weight of human serum albumin and 10 parts by weight of DMSO (dimethyl sulfoxide ), and the prepared frozen stock solution is stored in a refrigerator at 4 ℃ until being used;
(2) Sterilizing and cleaning: in a biosafety cabinet, disinfecting the surface of umbilical cord tissue by alcohol, cutting the umbilical cord from the middle, spreading the umbilical cord on a sterile 10cm cell culture plate, and cleaning the tissue by PBS to reduce red blood cells on the tissue;
(3) Umbilical cord tissue treatment: transferring the umbilical cord tissue obtained in the step (2) to another 10cm cell culture plate, and shearing the umbilical cord tissue into pieces with a size of 1cm3 Is square in shape;
(4) Umbilical cord tissue cold storage: adding tissue blocks and frozen stock solution into a frozen stock tube in a low-temperature environment of 4 ℃, then placing the frozen stock tube into a program cooling box, firstly refrigerating for 0.5 hour at a low temperature of 4 ℃, then refrigerating for 1 day at a temperature of-80 ℃, and then refrigerating the frozen stock tube in liquid nitrogen for later use;
(5) Cryopreserved umbilical cord tissue resuscitates: taking out the frozen umbilical cord tissue from the step (4) from liquid nitrogen, thawing the umbilical cord tissue in a constant-temperature water bath until a half of frozen stock solution starts to melt, performing a drip method on the umbilical cord tissue by using a mesenchymal stem cell culture medium (comprising 15% FBS+1% L-glutamine+0.05% Gentamicin+84% DMEM-F12, for example), and removing waste liquid by using a 100um filter;
(6) Umbilical cord tissue adherence treatment: spreading the recovered frozen umbilical cord tissue in another 10cm cell culture dish, maintaining the number of tissue blocks in each dish at 10-15, and air-drying the tissue blocks for 10-15 minutes until the tissue is attached to the dish;
(7) Umbilical cord tissue culture: slowly add mesenchymal stem cell culture medium (which contains, for example, 15% fbs+1% l-glutamine+0.05% genomicin+84% dmem-F12) along the plate edge to tissue flooding; place the dishes on CO2 Culturing in a 37 ℃ incubator with the concentration of 5%, taking the plate out of the incubator until the fifth day, and supplementing 5ml of mesenchymal stem cell culture medium; transferring the culture medium in the dish on the tenth day, and adding 15ml of fresh mesenchymal stem cell culture medium; on the twelfth day, all cord tissue pieces were removed and culture continued, every two days thereafterPerforming one-time full liquid exchange;
(8) Cell passage: when the fusion rate of the adherent cells in the plate reaches about 60%, the adherent cells can be separated from the bottom of the plate by using digestive enzyme (TrypLE Express), and supernatant is removed after centrifugation to obtain P0 generation umbilical cord mesenchymal stem cells; adding a mesenchymal stem cell culture medium into the P0 generation cells to re-suspend the cells, inoculating the cells into a T25 cell culture bottle for passage, and performing amplification culture; after the liquid is changed once every two days until the fusion rate reaches 80%, obtaining the umbilical cord mesenchymal stem cells of the generation P1, and completing the passage of the cells from the generation P0 to the generation P1; and repeating the passage operation from the generation P0 to the generation P1 in sequence to respectively carry out the passage of the cells from the generation P1 to the generation P2, the generation P2 to the generation P3 to the generation P15, so as to obtain the umbilical cord mesenchymal stem cells from the generation P1 to the generation P15.
The cell preparation according to the first aspect of the invention, wherein the umbilical cord mesenchymal stem cells have a cell purity of more than 90%, such as more than 95%. In one embodiment, the umbilical cord mesenchymal stem cells are greater than 90%, such as greater than 95%, in cell purity after more than 3 passages.
The cell preparation according to the first aspect of the present invention, wherein the umbilical cord mesenchymal stem cells are prepared according to a method comprising the steps of: (1) preparing umbilical cord tissue frozen stock solution: the umbilical cord tissue frozen stock solution comprises human serum albumin and DMSO, and the prepared frozen stock solution is refrigerated at a low temperature of 1-7 ℃;
(2) Sterilizing and cleaning: sterilizing the surface of the umbilical cord tissue with sterilizing liquid alcohol, shearing and spreading the umbilical cord, and cleaning the umbilical cord tissue with PBS buffer solution to reduce red blood cells on the umbilical cord tissue;
(3) Umbilical cord tissue treatment: shearing umbilical cord tissues obtained in the step (2) into tissue blocks;
(4) Umbilical cord tissue cold storage: adding tissue blocks and frozen stock solution into a frozen stock container in a low-temperature environment of 0-15 ℃, then putting the frozen stock container into a program cooling device, firstly refrigerating at a low temperature of 1-7 ℃ for 0.2-2 hours, then refrigerating at a temperature of-10-150 ℃ for 0.25-3 days, and then refrigerating the frozen stock container in liquid nitrogen for later use;
(5) Cryopreserved umbilical cord tissue resuscitates: taking out the frozen umbilical cord tissue in the step (4) from liquid nitrogen, thawing to 20% -70% of frozen stock solution, cleaning the umbilical cord tissue by using a mesenchymal stem cell culture medium, and filtering to remove waste liquid so as to revive the frozen umbilical cord tissue;
(6) Umbilical cord tissue adherence treatment: spreading the tissue blocks in the plates, and keeping the number of the tissue blocks in each plate at 5-20, so that the tissue blocks are air-dried for 2-50 minutes until the tissues are attached to the plates;
(7) Umbilical cord tissue culture: slowly adding a mesenchymal stem cell culture medium along the edge of the plate until the tissue is submerged; place the dishes into CO2 Culturing in a 37 ℃ incubator with the concentration of 5%, taking the plate out of the incubator when culturing to 3-7 days, and supplementing a proper amount of mesenchymal stem cell culture medium; removing the culture medium in the dish at 9-11 days, adding a proper amount of fresh mesenchymal stem cell culture medium, and continuing culturing; removing all umbilical cord tissue blocks and continuing culturing on days 11-13; after that, the liquid is completely changed every 1-3 days;
(8) Cell passage: when the fusion rate of the adherent cells in the plate reaches 50-70%, the adherent cells are separated from the bottom of the plate by using digestive enzyme TrypLE Express; centrifuging, and removing supernatant to obtain P0 generation umbilical cord mesenchymal stem cells; adding a mesenchymal stem cell culture medium into the P0 generation cells to re-suspend the cells, inoculating the cells into a T25 cell culture bottle for passage, and performing amplification culture; changing the liquid once every 1-3 days until the fusion rate reaches 70-90%, obtaining the P1 generation umbilical cord mesenchymal stem cells, and completing the passage of the P0 generation to the P1 generation cells; and repeating the passage operation from the generation P0 to the generation P1 in sequence to respectively carry out the passage of the cells from the generation P1 to the generation P2, the generation P2 to the generation P3 to the generation P15, so as to obtain the umbilical cord mesenchymal stem cells from the generation P1 to the generation P15.
The cell preparation according to the first aspect of the present invention, wherein the umbilical mesenchymal stem cell preparation process further comprises the following steps:
(9) Detecting, for the umbilical cord mesenchymal stem cells obtained in step (8), at least one of the following items: cell activity, cell contamination, genetic disease, HLA-ABC/DR ligand.
The cell preparation according to the first aspect of the present invention, wherein the umbilical cord mesenchymal stem cells are prepared according to a method comprising the steps of:
(1) Preparing umbilical cord tissue frozen stock solution: the umbilical cord tissue frozen stock solution comprises human serum albumin and DMSO, and the prepared frozen stock solution is refrigerated at a low temperature of 4 ℃;
(2) Sterilizing and cleaning: sterilizing the surface of the umbilical cord tissue with sterilizing liquid alcohol, shearing and spreading the umbilical cord, and cleaning the umbilical cord tissue with PBS buffer solution to reduce red blood cells on the umbilical cord tissue;
(3) Umbilical cord tissue treatment: shearing umbilical cord tissues obtained in the step (2) into tissue blocks;
(4) Umbilical cord tissue cold storage: adding tissue blocks and frozen stock solution into a frozen stock container in a low-temperature environment of 1-7 ℃, then placing the frozen stock container into a program cooling device, firstly refrigerating at a low temperature of 4 ℃ for 0.5 hour, then freezing for 1 day in a temperature of-80 ℃, and then freezing the frozen stock container in liquid nitrogen for later use;
(5) Cryopreserved umbilical cord tissue resuscitates: taking out the frozen umbilical cord tissue in the step (4) from liquid nitrogen, thawing until 50% of frozen stock solution begins to melt, cleaning the umbilical cord tissue by using a mesenchymal stem cell culture medium, and filtering to remove waste liquid so as to revive the frozen umbilical cord tissue;
(6) Umbilical cord tissue adherence treatment: taking cell culture plates, spreading the tissue blocks in the plates, and keeping the number of the tissue blocks in each plate at 10-15 blocks, so that the tissue blocks are air-dried for 2-50 minutes until the tissues are attached to the plates;
(7) Umbilical cord tissue culture: slowly adding a mesenchymal stem cell culture medium along the edge of the plate until the tissue is submerged; place the dishes into CO2 Culturing in a 37 ℃ incubator with the concentration of 5%, taking the plate out of the incubator until the 4 th to the 6 th days, and supplementing a proper amount of mesenchymal stem cell culture medium; removing the culture medium in the plate on the 10 th day, adding a proper amount of fresh mesenchymal stem cell culture medium, and continuing culturing; removing all umbilical cord tissue blocks and continuing culturing on day 12; every 2 days thereafterPerforming primary full liquid exchange;
(8) Cell passage: when the fusion rate of the adherent cells in the plate reaches 50-70%, the adherent cells are separated from the bottom of the plate by using digestive enzyme TrypLE Express; centrifuging, and removing supernatant to obtain P0 generation umbilical cord mesenchymal stem cells; adding a mesenchymal stem cell culture medium into the P0 generation cells to re-suspend the cells, inoculating the cells into a T25 cell culture bottle for passage, and performing amplification culture; after that, changing the liquid once every 2 days until the fusion rate reaches 70-90%, obtaining the umbilical cord mesenchymal stem cells of the generation P1, and completing the passage of the cells from the generation P0 to the generation P1; and repeating the passage operation from the generation P0 to the generation P1 in sequence to respectively carry out the passage of the cells from the generation P1 to the generation P2, the generation P2 to the generation P3 to the generation P15, so as to obtain the umbilical cord mesenchymal stem cells from the generation P1 to the generation P15.
The cell preparation according to the first aspect of the present invention, wherein the umbilical cord mesenchymal stem cells are prepared such that the umbilical cord tissue-freezing solution contains 80 parts by weight of human serum albumin and 10 parts by weight of DMSO.
The cell preparation according to the first aspect of the present invention, wherein in the step (3) of preparing the umbilical cord mesenchymal stem cells, the sheared tissue size is 0.2-2.5 cubic centimeters.
The cell preparation according to the first aspect of the present invention, wherein in the step (5) of preparing the umbilical cord mesenchymal stem cells, the washing of umbilical cord tissue with the mesenchymal stem cell medium is performed by a drip method.
The cell preparation according to the first aspect of the present invention, wherein the mesenchymal stem cells are prepared by comprising FBS, L-glutamine, gentamicin and DMEM-F12 in the mesenchymal stem cell medium.
The cell preparation according to the first aspect of the present invention, wherein the mesenchymal stem cells are prepared by mixing 15 parts by weight of FBS, 1 part by weight of L-glutamine, 0.05 part by weight of gentamicin and 84 parts by weight of DMEM-F12 in the mesenchymal stem cell medium.
The cell preparation according to the first aspect of the present invention, wherein the umbilical cord mesenchymal stem cells are prepared according to a method comprising the steps of:
(1) Preparing umbilical cord tissue frozen stock solution: the umbilical cord tissue frozen stock solution comprises 80 parts by weight of human serum albumin and 10 parts by weight of DMSO, and the prepared frozen stock solution is stored in a refrigerator at 4 ℃ until being used;
(2) Sterilizing and cleaning: in a biosafety cabinet, disinfecting the surface of umbilical cord tissue by alcohol, cutting the umbilical cord from the middle, spreading the umbilical cord on a sterile 10cm cell culture plate, and cleaning the tissue by PBS to reduce red blood cells on the tissue;
(3) Umbilical cord tissue treatment: transferring the umbilical cord tissue obtained in the step (2) to another 10cm cell culture plate, and shearing the umbilical cord tissue into pieces with a size of 1cm3 Is square in shape;
(4) Umbilical cord tissue cold storage: adding tissue blocks and frozen stock solution into a frozen stock tube in a low-temperature environment of 4 ℃, then placing the frozen stock tube into a program cooling box, firstly refrigerating for 0.5 hour at a low temperature of 4 ℃, then freezing for 1 day at a temperature of-80 ℃, and then freezing the frozen stock tube in liquid nitrogen for later use;
(5) Cryopreserved umbilical cord tissue resuscitates: taking out the umbilical cord tissue frozen in the step (4) from liquid nitrogen, thawing in a constant-temperature water bath until a half of frozen stock solution begins to be thawed, performing drip washing on the umbilical cord tissue by using a mesenchymal stem cell culture medium, and removing waste liquid by using a 100um filter;
(6) Umbilical cord tissue adherence treatment: spreading the recovered frozen umbilical cord tissue in another 10cm cell culture dish, maintaining the number of tissue blocks in each dish at 10-15, and air-drying the tissue blocks for 10-15 minutes until the tissue is attached to the dish;
(7) Umbilical cord tissue culture: slowly adding a mesenchymal stem cell culture medium along the edge of the plate until the tissue is submerged; place the dishes on CO2 Culturing in a 37 ℃ incubator with the concentration of 5%, taking the plate out of the incubator until the fifth day, and supplementing 5ml of mesenchymal stem cell culture medium; transferring the culture medium in the dish on the tenth day, and adding 15ml of fresh mesenchymal stem cell culture medium; removing all umbilical cord tissue blocks on the twelfth day and continuing to culture, and performing total liquid exchange every two days thereafter;
(8) Cell passage: when the fusion rate of the adherent cells in the plate reaches 60%, the adherent cells are separated from the bottom of the plate by using digestive enzyme TrypLE Express; centrifuging, and removing supernatant to obtain P0 generation umbilical cord mesenchymal stem cells; adding a mesenchymal stem cell culture medium into the P0 generation cells to re-suspend the cells, inoculating the cells into a T25 cell culture bottle for passage, and performing amplification culture; after that, changing the liquid once every 2 days until the fusion rate reaches 80%, obtaining the umbilical cord mesenchymal stem cells of the generation P1, and completing the passage of the cells from the generation P0 to the generation P1; and repeating the passage operation from the generation P0 to the generation P1 in sequence to respectively carry out the passage of the cells from the generation P1 to the generation P2, the generation P2 to the generation P3 to the generation P15, so as to obtain the umbilical cord mesenchymal stem cells from the generation P1 to the generation P15.
The cell preparation according to the first aspect of the present invention, wherein the mesenchymal stem cells are prepared by mixing 15 parts by weight of FBS, 1 part by weight of L-glutamine, 0.05 part by weight of gentamicin and 84 parts by weight of DMEM-F12 in the mesenchymal stem cell medium.
The cell preparation according to the first aspect of the present invention, wherein the pharmaceutically acceptable vehicle is water.
The cell preparation according to the first aspect of the invention is a preparation for use in an injection mode.
The cell preparation according to the first aspect of the present invention, wherein the umbilical cord mesenchymal stem cells are cells subcultured to 1 to 20 passages, for example, to 2 to 15 passages, for example, to 2 to 12 passages.
The cell preparation according to the first aspect of the present invention, wherein the concentration of the umbilical mesenchymal stem cells is (1 to 10). Times.106 And each ml.
The cell preparation according to the first aspect of the present invention, wherein the concentration of the umbilical cord mesenchymal stem cells is (1.5 to 8). Times.106 And each ml.
The cell preparation according to the first aspect of the present invention, wherein the concentration of the umbilical mesenchymal stem cells is (2 to 5). Times.106 And each ml.
The cell preparation according to the first aspect of the present invention, further comprising sodium chloride.
The cell preparation according to the first aspect of the present invention further comprises sodium chloride at a concentration of 7 to 10mg/ml.
The cell preparation according to the first aspect of the present invention further comprises sodium chloride at a concentration of 7.5 to 9.5mg/ml.
The cell preparation according to the first aspect of the present invention further comprises sodium chloride at a concentration of 8 to 9mg/ml.
The cell preparation according to the first aspect of the present invention, further comprising chlorogenic acid.
The cell preparation according to the first aspect of the present invention further comprises chlorogenic acid at a concentration of 0.25 to 2.5mg/ml.
The cell preparation according to the first aspect of the present invention further comprises chlorogenic acid at a concentration of 0.3 to 2mg/ml.
The cell preparation according to the first aspect of the present invention further comprises chlorogenic acid at a concentration of 0.5 to 1.5mg/ml.
The cell preparation according to the first aspect of the present invention, further comprising sorbitol.
The cell preparation according to the first aspect of the present invention further comprises sorbitol at a concentration of 2 to 10mg/ml.
The cell preparation according to the first aspect of the present invention further comprises sorbitol at a concentration of 2.5 to 7.5mg/ml.
The cell preparation according to the first aspect of the present invention further comprises sorbitol at a concentration of 3 to 5mg/ml.
The cell preparation according to the first aspect of the invention, having a formulation according to any one of the embodiments of the invention.
In the present invention, sodium chloride, chlorogenic acid, sorbitol, etc. may be referred to as pharmaceutical excipients.
The cell preparation according to the first aspect of the invention is prepared according to a method comprising the steps of: dissolving sodium chloride (and pharmaceutical excipients such as chlorogenic acid and sorbitol) with proper amount of water to obtain saline solution; transferring the mesenchymal stem cells obtained by subculture into a centrifuge tube, centrifuging (for example, centrifuging at 2000rpm for 5 min), discarding the supernatant, and adding saline solution to resuspend the cells to a specified cell concentration to obtain a cell preparation.
The cell preparation according to the first aspect of the present invention is a cell preparation for use in the treatment of pneumonia, such as viral pneumonia.
Further, the second aspect of the present invention relates to the use of a cell preparation comprising umbilical mesenchymal stem cells and a pharmaceutically acceptable vehicle for the manufacture of a medicament for the treatment of pneumonia, such as viral pneumonia.
The use according to the second aspect of the invention, wherein the umbilical cord mesenchymal stem cells are prepared by a method comprising the steps of:
(1) Preparing umbilical cord tissue frozen stock solution: the umbilical cord tissue frozen stock solution comprises human serum albumin and DMSO (dimethyl sulfoxide), and the prepared frozen stock solution is subjected to low-temperature refrigeration at a temperature of 1-7 ℃ (e.g. 4 ℃);
(2) Sterilizing and cleaning: disinfecting the surface of the umbilical cord tissue with a disinfectant (e.g., alcohol), shearing open the umbilical cord, tiling, and washing the umbilical cord tissue with a buffer (e.g., PBS buffer) to reduce red blood cells on the umbilical cord tissue;
(3) Umbilical cord tissue treatment: shearing umbilical cord tissues obtained in the step (2) into tissue blocks;
(4) Umbilical cord tissue cold storage: adding the tissue mass and the frozen stock solution into a frozen stock container under a low temperature environment of 0-15 ℃ (e.g. 1 ℃ to 7 ℃, e.g. 4 ℃), then placing the frozen stock container into a program cooling device, firstly refrigerating at a low temperature of 1 ℃ to 7 ℃ (e.g. 4 ℃) for 0.2-2 hours (e.g. 0.5 hours), then refrigerating at a temperature of-10 ℃ to-150 ℃ (e.g. -80 ℃) for 0.25-3 days (e.g. 1 day), and then refrigerating the frozen stock container in liquid nitrogen for later use;
(5) Cryopreserved umbilical cord tissue resuscitates: taking out the frozen umbilical cord tissue in the step (4) from liquid nitrogen, thawing to 20% -70% (e.g. 50%) of frozen stock solution, starting thawing, cleaning the umbilical cord tissue by using a mesenchymal stem cell culture medium, and filtering to remove waste liquid so as to resuscitate the frozen umbilical cord tissue;
(6) Umbilical cord tissue adherence treatment: taking cell culture plates, spreading the tissue blocks in the plates, and keeping the number of the tissue blocks in each plate at 5-20 blocks (for example, 10-15 blocks), so that the tissue blocks are air-dried for 2-50 minutes until the tissues are attached to the plates;
(7) Umbilical cord tissue culture: slowly adding a mesenchymal stem cell culture medium along the edge of the plate until the tissue is submerged; place the dishes into CO2 Culturing in a 37 deg.C incubator with a concentration of 5%, taking out the plate from the incubator until 3-7 days (e.g. 4-6 days, e.g. 5 days), and supplementing a proper amount of mesenchymal stem cell culture medium (for submerging tissues); removing the culture medium from the dish on days 9-11 (such as day 10), adding appropriate amount of fresh mesenchymal stem cell culture medium (for submerging tissue), and continuing culturing; all umbilical cord tissue blocks were removed and culture continued on days 11-13 (e.g., day 12); after which the whole change is performed every 1-3 days (e.g. every 2 days);
(8) Cell passage: when the fusion rate of the adherent cells in the plate reaches about 50-70%, using digestive enzymes (such as TrypLE Express, invitrogen products) to separate the adherent cells from the bottom of the plate; centrifuging, and removing supernatant to obtain P0 generation umbilical cord mesenchymal stem cells; adding a mesenchymal stem cell culture medium into the P0 generation cells to re-suspend the cells, and then inoculating the cells into a T25 cell culture bottle for passage and amplification culture; changing the liquid every 1-3 days (for example every 2 days) until the fusion rate reaches 70-90%, obtaining the P1 generation umbilical cord mesenchymal stem cells, and completing the passage of the P0 generation to the P1 generation cells; and repeating the passage operation from the generation P0 to the generation P1 in sequence to respectively carry out the passage of the cells from the generation P1 to the generation P2, the generation P2 to the generation P3 to the generation P15, so as to obtain the umbilical cord mesenchymal stem cells from the generation P1 to the generation P15.
The use according to the second aspect of the invention, wherein the umbilical cord mesenchymal stem cells are prepared by the method according to any one of the embodiments of the first aspect of the invention.
The use according to the second aspect of the invention, wherein the pharmaceutically acceptable vehicle in the cell preparation is water.
The use according to the second aspect of the invention, wherein the cell preparation is a preparation for use by injection.
The use according to the second aspect of the invention, wherein the umbilical cord mesenchymal stem cells in the cell preparation are cells subcultured to 1-20 passages, for example to 2-15 passages, for example to 2-12 passages.
The use according to the second aspect of the present invention, wherein the concentration of umbilical mesenchymal stem cells in the cell preparation is (1-10). Times.106 And each ml.
The use according to the second aspect of the present invention, wherein the concentration of umbilical mesenchymal stem cells in the cell preparation is (1.5-8). Times.106 And each ml.
The use according to the second aspect of the present invention, wherein the concentration of umbilical mesenchymal stem cells in the cell preparation is (2-5). Times.106 And each ml.
The use according to the second aspect of the invention, wherein the cell preparation further comprises sodium chloride.
The use according to the second aspect of the present invention, wherein the cell preparation further comprises sodium chloride at a concentration of 7 to 10mg/ml.
The use according to the second aspect of the invention, wherein the cell preparation further comprises sodium chloride at a concentration of 7.5 to 9.5mg/ml.
The use according to the second aspect of the present invention, wherein the cell preparation further comprises sodium chloride at a concentration of 8 to 9mg/ml.
The use according to the second aspect of the invention, wherein chlorogenic acid is also comprised in the cell preparation.
The use according to the second aspect of the present invention, wherein the cell preparation further comprises chlorogenic acid in a concentration of 0.25 to 2.5mg/ml.
The use according to the second aspect of the present invention, wherein the cell preparation further comprises chlorogenic acid in a concentration of 0.3 to 2mg/ml.
The use according to the second aspect of the present invention, wherein the cell preparation further comprises chlorogenic acid in a concentration of 0.5 to 1.5mg/ml.
The use according to the second aspect of the invention, wherein sorbitol is also comprised in the cell preparation.
The use according to the second aspect of the present invention, wherein the cell preparation further comprises sorbitol in a concentration of 2 to 10mg/ml.
The use according to the second aspect of the invention, wherein the cell preparation further comprises sorbitol in a concentration of 2.5-7.5 mg/ml.
The use according to the second aspect of the present invention, wherein the cell preparation further comprises sorbitol in a concentration of 3 to 5mg/ml.
The use according to the second aspect of the invention, wherein the cell preparation is prepared according to a method comprising the steps of: dissolving sodium chloride (and pharmaceutical excipients such as chlorogenic acid and sorbitol) with proper amount of water to obtain saline solution; transferring the mesenchymal stem cells obtained by subculture into a centrifuge tube, centrifuging (for example, centrifuging at 2000rpm for 5 min), discarding the supernatant, and adding saline solution to resuspend the cells to a specified cell concentration to obtain a cell preparation.
Of the various operating steps described above, although specific steps are described herein as being distinguished in some details or language description from those described in the preparation examples of the detailed description section below, those skilled in the art can readily generalize the method steps described above based on the detailed disclosure of the invention as a whole.
Any of the embodiments of any of the aspects of the invention may be combined with other embodiments, provided that they do not contradict. Furthermore, in any of the embodiments of any of the aspects of the present invention, any technical feature may be applied to the technical feature in other embodiments as long as they do not contradict. The present invention is further described below.
All documents cited herein are incorporated by reference in their entirety and are incorporated by reference herein to the extent they are not inconsistent with this invention. Furthermore, various terms and phrases used herein have a common meaning known to those skilled in the art, and even though they are still intended to be described and explained in greater detail herein, the terms and phrases used herein should not be construed to be inconsistent with the ordinary meaning in the sense of the present invention.
In the present invention, the term "umbilical cord mesenchymal stem cells" refers to mesenchymal stem cells derived from umbilical cord. Thus, in the context of the present invention, and in particular in relation to the present invention, the term "umbilical cord mesenchymal stem cells" may be used interchangeably with "umbilical cord stem cells", "mesenchymal stem cells", unless explicitly indicated otherwise.
In the present invention, the term "PBS buffer" or "PBS" refers to phosphate buffer. The general formulation and method of formulation of the PBS used in the context of the present invention and their general properties such as pH or pH range are well known to those skilled in the art, and these PBS buffers are typically commercially available pre-formulations (or pre-powders), e.g., PBS used in the field of the present invention is typically a commercial buffer of pH7.4 (±0.1), e.g., hyClone brand PBS buffer; the typical PBS buffer composition used in this field includes 137mM sodium chloride, 2.7nM potassium chloride and 10mM phosphate, and the composition of the PBS used in this invention is that described herein unless otherwise specified.
In the present invention, the term "umbilical cord" refers to neonatal umbilical cord, in particular to umbilical cord within 4 hours after delivery.
The mesenchymal stem cells are adult stem cells with self-replication and multidirectional differentiation potential, have the advantages of easy separation, culture and amplification, low immunogenicity, no expression of a type II main tissue compatibility complex (MHC), can be used in a variant, have strong migration and immunoregulation capacity, promote tissue injury repair and regeneration in a paracrine mode, and are ideal seed cells for regenerative medicine.
The viral pneumonia is described in detail in Han Xudong (Han Xudong, et al, viral pneumonia, journal of doctor's repair (department edition), 2004, 27 (2): 12). Viral pneumonia is often present as an inhalation infection, which is transmitted by droplets and intimate contact, and may be caused by the downward spread of upper respiratory viral infection, and may also be secondary to eruptive viral infection, often accompanied by tracheal-bronchial infection. Influenza virus is the most common pathogen of adult and elderly viral pneumonia, and infant viral pneumonia is often caused by respiratory syncytial virus infection. Other viruses such as parainfluenza virus, cytomegalovirus, coronaviruses, adenoviruses, rhinoviruses and certain enteroviruses such as coxsackie, epstein barr virus and the like can also cause viral pneumonia. In the non-bacterial pneumonia, the viral pneumonia accounts for 25% -50%, is frequently generated in winter and spring, can be sporadic or epidemic, and is frequently seen in infants, the elderly and patients with the original chronic cardiopulmonary diseases. In recent years, because immunosuppressive drugs are widely applied to organ transplantation patients and the number of people suffering from AIDS is increased, the incidence rate of viral pneumonia is gradually increased, and the epidemic of SARS makes the viral pneumonia particularly important. The clinical manifestations of general viral pneumonia are mostly slight, similar to mycoplasma pneumonia symptoms, and the disease course is 1-2 weeks. Severe pneumonia may be associated with persistent hyperpyrexia, palpitations, shortness of breath, dyspnea, cyanosis, and may also be accompanied by shock and respiratory failure. In viral pneumonia, in addition to direct damage to the body caused by viruses, autoimmune damage also plays an important role in viral diseases. The invasion of viruses into bronchiole epithelium can cause bronchiolitis, infection, and pneumonia caused by pulmonary interstitial and alveoli. Congestion and bleeding of lesion parts; a strong inflammatory response involving monocytes occurs. Fibrin-containing monocytes, and occasionally polymorphonuclear leukocytes, may be present in the alveoli, and in severe cases the hyaline membrane may be present, resulting in a severe impairment of alveolar diffuse function. Adenovirus, cytomegalovirus, respiratory syncytial virus or varicella-zoster virus can see characteristic intracellular viral inclusion bodies. After absorption of the lesions, fibrosis or nodular calcification may remain. The lung and immune organs are the main target organs for SARS virus attack. Early changes in the lung are desquamative alveolar inflammatory changes, and convalescence is an organized glomerular pneumonitis change; after absorption of the lesions, lung fibrosis of varying degrees may be left behind.
The present invention uses classical animal tests in the art to verify the biological effects of the cell preparations of the present invention in the use of pneumonia, such as viral pneumonia, and to present satisfactory results, which provide a solid basis for the effectiveness of clinical applications.
Detailed Description
The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples. Those skilled in the art will appreciate that various changes and modifications can be made to the invention without departing from the spirit and scope thereof. The present invention generally and/or specifically describes the materials used in the test as well as the test methods. Although many materials and methods of operation are known in the art for accomplishing the objectives of the present invention, the present invention will be described in as much detail herein.
CN102660497B (chinese patent No. ZL 2012101599162), the entire content of which is incorporated herein by reference, describes a detailed umbilical mesenchymal stem cell acquisition method. The reagents for the chemical reagents such as chlorogenic acid used in the present invention are commercially available.
Example 1 method for cryopreservation, resuscitation of umbilical cord tissue, separation and expansion of Stem cells after resuscitation
The umbilical cord tissue cryopreservation method comprises the following steps:
(1) Preparing umbilical cord tissue frozen stock solution: the umbilical cord tissue frozen stock solution comprises 80 parts by weight of human serum albumin and 10 parts by weight of DMSO (dimethyl sulfoxide ), and the prepared frozen stock solution is stored in a refrigerator at 4 ℃ until being used;
(2) Sterilizing and cleaning: in a biosafety cabinet, disinfecting the surface of umbilical cord tissue by alcohol, cutting the umbilical cord from the middle, spreading the umbilical cord on a sterile 10cm cell culture plate, and cleaning the tissue by PBS to reduce red blood cells on the tissue;
(3) Umbilical cord tissue treatment: transferring the umbilical cord tissue obtained in the step (2) to another 10cm cell culture plate, and shearing the umbilical cord tissue into square shapes with the size of 1cm < 3 >;
(4) Umbilical cord tissue cold storage: adding tissue blocks and frozen stock solution into a frozen stock tube in a low-temperature environment of 4 ℃, then placing the frozen stock tube into a program cooling box, firstly refrigerating for 0.5 hour at a low temperature of 4 ℃, then refrigerating for 1 day at a temperature of-80 ℃, and then refrigerating the frozen stock tube in liquid nitrogen for later use;
the resuscitation method matched with the cryopreservation method comprises the following steps:
(5) Cryopreserved umbilical cord tissue resuscitates: taking out the umbilical cord tissue frozen in the step (4) from liquid nitrogen, thawing in a constant-temperature water bath until a half of frozen stock solution starts to melt, performing infusion method on the umbilical cord tissue by using a mesenchymal stem cell culture medium (comprising 15% FBS+1% L-glutamin+0.05% Gentamicin+84% DMEM-F12), and removing waste liquid by using a 100um filter;
The method for separating and amplifying the mesenchymal stem cells after resuscitation comprises the following steps:
(6) Umbilical cord tissue adherence treatment: spreading the recovered frozen umbilical cord tissue in another 10cm cell culture dish, maintaining the number of tissue blocks in each dish at 10-15, and air-drying the tissue blocks for 10-15 minutes until the tissue is attached to the dish;
(7) Umbilical cord tissue culture: slowly adding a mesenchymal stem cell culture medium (containing 15% FBS+1% L-Glutamine+0.05% Gentamicin+84% DMEM-F12) along the edge of the dish until the tissue is submerged; placing the plate in a 37 ℃ incubator with the CO2 concentration of 5%, culturing, taking the plate out of the incubator until the fifth day, and supplementing 5ml of mesenchymal stem cell culture medium; transferring the culture medium in the dish on the tenth day, and adding 15ml of fresh mesenchymal stem cell culture medium; removing all umbilical cord tissue blocks on the twelfth day and continuing to culture, and performing total liquid exchange every two days thereafter;
(8) Cell passage: when the fusion rate of the adherent cells in the plate reaches about 60%, the adherent cells can be separated from the bottom of the plate by using digestive enzyme (TrypLE Express), supernatant is removed after centrifugation, and P0 generation umbilical cord mesenchymal stem cells are obtained (the resuscitated umbilical cord tissue starts to climb out of the adherent cells on the 10 th day of culture, and the fusion rate of the adherent cells reaches 60% on the 17 th day of culture); adding a mesenchymal stem cell culture medium into the P0 generation cells to re-suspend the cells, inoculating the cells into a T25 cell culture bottle for passage, and performing amplification culture; after the solution is changed once every two days until the fusion rate reaches 80%, obtaining the umbilical cord mesenchymal stem cells of the generation P1, and completing the passage of the cells from the generation P0 to the generation P1 (the fusion rate reaches 90% on the 22 th day); and repeating the passage operation from the generation P0 to the generation P1 in sequence to respectively carry out the passage of the cells from the generation P1 to the generation P2, the generation P2 to the generation P3 to the generation P15, so as to obtain the umbilical cord mesenchymal stem cells from the generation P1 to the generation P15. The purity of the cells obtained after the P3 generation is more than 95 percent.
The method and results for identifying the biological properties of umbilical cord MSC obtained in this example 1 can be described in example 7 of CN102660497B (Chinese patent No. ZL 201210159916.2).
In the case of the cell preparation of the present invention, the amount per 1ml is not less than 50ml per batch in actual preparation, unless otherwise specified. In the examples of the invention for preparing cell preparations, stem cells are obtained by the method of example 1 herein, unless otherwise indicated. In the context of the present invention, the preparation of mesenchymal stem cells (i.e. the cell preparation) is carried out under aseptic conditions.
EXAMPLE 11a preparation of mesenchymal Stem cells
Mesenchymal stem cells (P6 generation): 3.5X106 The number of the two-dimensional space-saving type,
sodium chloride: 8.5mg of the total weight of the composition,
chlorogenic acid: 1mg of the extract of the plant,
sorbitol: 4mg of the total weight of the composition,
water, in an amount of 1ml.
The preparation method comprises the following steps: dissolving sodium chloride, chlorogenic acid and sorbitol with proper amount of water to obtain saline solution; the mesenchymal stem cells obtained by passaging the cells in the step (8) of example 1 were transferred into a centrifuge tube, centrifuged at 2000rpm for 5min, the supernatant was discarded, and the cells were resuspended by adding a saline solution to prepare a cell preparation.
Example 11a1 preparation of mesenchymal Stem cells
The cell preparation of this example 11a1 was obtained with reference to the formulation and preparation of example 11a, except that sorbitol was not added.
Example 11a2, preparationPreparation of mesenchymal stem cells
The cell preparation of example 11a2 was obtained by referring to the formulation and preparation of example 11a, except that chlorogenic acid was not added.
Example 11a3 preparation of mesenchymal Stem cells
The cell preparation of this example 11a3 was obtained with reference to the formulation and preparation of example 11a, except that neither sorbitol nor chlorogenic acid was added.
EXAMPLE 11b preparation of mesenchymal Stem cell preparation
Mesenchymal stem cells (P3 generation): 2X 106 The number of the two-dimensional space-saving type,
sodium chloride: 9mg of the extract of the plant,
chlorogenic acid: 1.5mg of the total amount of the,
sorbitol: 3mg of the total amount of,
water, in an amount of 1ml.
The preparation method comprises the following steps: dissolving sodium chloride, chlorogenic acid and sorbitol with proper amount of water to obtain saline solution; the mesenchymal stem cells obtained by passaging the cells in the step (8) of example 1 were transferred into a centrifuge tube, centrifuged at 2000rpm for 5min, the supernatant was discarded, and the cells were resuspended by adding a saline solution to prepare a cell preparation.
Example 11c preparation of mesenchymal Stem cell preparation
Mesenchymal stem cells (P12 generation): 5X 106 The number of the two-dimensional space-saving type,
sodium chloride: 8.5mg of the total weight of the composition,
chlorogenic acid: 0.5mg of the total amount of the components,
sorbitol: 5mg of the extract of the plant,
water, in an amount of 1ml.
The preparation method comprises the following steps: dissolving sodium chloride, chlorogenic acid and sorbitol with proper amount of water to obtain saline solution; the mesenchymal stem cells obtained by passaging the cells in the step (8) of example 1 were transferred into a centrifuge tube, centrifuged at 2000rpm for 5min, the supernatant was discarded, and the cells were resuspended by adding a saline solution to prepare a cell preparation.
EXAMPLE 11d preparation of mesenchymal Stem cell preparation
Mesenchymal stem cells (P5 generation): 8X 106 The number of the two-dimensional space-saving type,
sodium chloride: 8mg of the extract, which is obtained by mixing,
chlorogenic acid: 0.3mg of the total amount of the,
sorbitol: 2.5mg of the total weight of the composition,
water, in an amount of 1ml.
The preparation method comprises the following steps: dissolving sodium chloride, chlorogenic acid and sorbitol with proper amount of water to obtain saline solution; the mesenchymal stem cells obtained by passaging the cells in the step (8) of example 1 were transferred into a centrifuge tube, centrifuged at 2000rpm for 5min, the supernatant was discarded, and the cells were resuspended by adding a saline solution to prepare a cell preparation.
Example 11e preparation of mesenchymal Stem cell preparation
Mesenchymal stem cells (P9 generation): 1.5X106 The number of the two-dimensional space-saving type,
sodium chloride: 9mg of the extract of the plant,
chlorogenic acid: 2mg of the extract of the plant, wherein,
sorbitol: 7.5mg of the total weight of the powder,
water, in an amount of 1ml.
The preparation method comprises the following steps: dissolving sodium chloride, chlorogenic acid and sorbitol with proper amount of water to obtain saline solution; the mesenchymal stem cells obtained by passaging the cells in the step (8) of example 1 were transferred into a centrifuge tube, centrifuged at 2000rpm for 5min, the supernatant was discarded, and the cells were resuspended by adding a saline solution to prepare a cell preparation.
Test example 1: determination of cell viability:
cell viability assays are widely used in a number of scientific fields, such as drug screening, cell proliferation assays, drug toxicity assays, tumor drug sensitivity assays, and the like. Currently, the most commonly used method for detecting cell viability is MTT colorimetric [ Fischer D, li Y, ahlemeyer B, et al in vitro cytotoxicity testing of polycations: influence of polymer structure on cell viability and hemolysis [ J ]. Biomaterials,2003,24 (7): 1121-1131], CCK-8 colorimetry [ Chen CL, lin CF, chang WT, et al Ceramide products p38 MAPK and JNK activation through a mechanism involving a thioredoxin-interacting protein-mediated path [ J ]. Blood,2008,111 (8): 4365-4374], trypan blue staining exclusion [ Louis KS, siegel ac. Cell viability analysis using trypan blue: manual and automated Methods [ J ]. Methods Mol,2011,740:7-12], etc.
This test example uses MTT colorimetry to determine the cell viability of the various cell preparations prepared herein. Specifically, the cell concentration of each cell preparation at 0 was measured, and then the cells were left in the dark at 2 to 6℃for 48 hours, and the cell concentration at 48 hours was measured, and the average value was obtained by measuring 5 times for each cell preparation in the same manner, and the percentage obtained by dividing the cell concentration at 48 times by the cell concentration at 0 and multiplying the divided value by 100% was used as the cell viability.
Results: example 11a=94.4%, example 1a1=95.2%, example 1a2=93.4%, example 1a3=94.7%, example 11b=95.2%, example 1c=94.3%, example 11d=94.7%, example 1e=96.3%. From these results, cell preparations of different formulations were substantially identical in terms of cell viability after 48 hours, showing no differences.
Test example 2: test of effectiveness of mesenchymal Stem cells in treating viral pneumonia
The lung index is the percentage of the body's lung mass to the body mass, and the magnitude of the lung index value is often used to indicate the severity of pneumonic lesions. Influenza virus infection causes toxic pneumonia in mice, inflammatory infiltration increases lung mass, and the greater the lung index value, the more severe the degree of pneumopathy. The lung index is used as an index for judging the action of the medicine, has a quantitative concept, and can objectively reflect the change degree of the pneumonic lesions.
The experimental example is carried out by the method carried out by Nanjing university of Chinese medicine journal, 2010, 26 (4): 315 referring to Zhou Kunfu (Zhou Kunfu, et al) for the influence of 3 methods on the pulmonary index and the pulmonary index inhibition rate of mice infected with influenza virus.
This test example 2 was conducted using the cell preparations obtained in examples 11a, 11a1, 11a2 and 11a 3.
Influenza virus A mouse lung adapted strain, namely FM1 strain, is provided by the national academy of sciences of Chinese traditional medicine and stored at-80 ℃. Chick embryo at 10 days of age after resuscitation (Boringer's diseasegram-Hun-Witong Co.) allantoic cavity was serially passaged 2 times, the hemagglutination titer was 1:512, and the half-lethal dose LD50 for mice was 10-4.28 . Male ICR mice weighing 18-22 g were purchased from the university of Chinese medicine laboratory animal center in Nanjing and bred on a regular basis. Ribavirin tablets are commercially available (100 mg/tablet, chongqing Kerui H20073882).
Test mice were randomly grouped, 15 per group, and: normal, model, positive (ribavirin), example 11a1, example 11a2, example 11a 3.
And (3) testing: each group of animals was fed in a conventional standard manner under equivalent conditions to the SPF class animal house;
the animals in the normal group are routinely raised during the whole test period, and the animals are not detoxified or dosed;
Each cell preparation was treated with 1X 10 cells daily on day 0 and day 1, respectively5 The dose of individual/animals was injected into the mice of the 4 example groups via the tail vein;
mice in model, positive and 4 example groups were infected nasally with viral allantoic droplets under shallow ether anaesthesia at day 3, 15 times LD50 challenge per mouse;
the model group does not perform any treatment after virus attack;
ribavirin 75mg/kg was given daily for 5 consecutive days following challenge with the positive group virus;
on day 8, 10 mice in each group were weighed, sacrificed by cervical vertebrae removal, dissected to obtain the lungs, weighed accurately, and lung index inhibition were calculated.
And (3) data processing:
the lung index and lung index inhibition were calculated as follows:
lung index = mouse lung mass/mouse mass
Pulmonary index inhibition = (model group pulmonary index mean-treatment group pulmonary index mean)/model group pulmonary index mean x 100%
All data are expressed as mean ± standard deviation, and are compared using t-test.
In addition, in this example, the serum interleukin 4 level of mice was also detected using an ELISA method, as follows: 24h after the last virus excitation, randomly selecting 5 mice from each group, taking 1mL of blood by adopting an eyeball removal method after full anesthesia, performing heparin anticoagulation, centrifuging for 5min at 800r/min, taking upper serum, and detecting the level of interleukin 4 in the serum strictly according to ELISA kit specifications.
The results are shown in the following table:
| group of | Lung index (. Times.0.001) | Lung index inhibition rate | Interleukin 4 levels |
| Normal group | 833±86 | - | 22.84±6.41ng/L |
| Model group | 1673±186△△ | - | 79.36±7.83ng/L△△ |
| Positive group | 1086±169** | 35.09％ | 29.17±7.22ng/L** |
| Example 11a group | 947±153** | 43.40％ | 30.17±7.83ng/L** |
| Example 11a1 group | 1289±211*# | 22.95％ | 43.74±6.87ng/L**# |
| Example 11a2 group | 1337±238*# | 20.08％ | 45.25±7.84ng/L*## |
| Example 11a3 group | 1304±194*# | 22.07％ | 46.32±5.42ng/L**# |
Note that: in comparison with the normal group, deltaDeltaP <0.01; comparing with the model group, P <0.05, P <0.01; compared to example 11a group, #p <0.05, #p <0.01.
The result shows that the lung index of the model group is obviously different from that of the normal group (P < 0.01), the influenza virus can cause the increase of the lung index of the normal mice, and the modeling is successful; the lung index of the 4 cell treatment groups, the positive group and the model group are obviously different (P <0.01, P < 0.05), which indicates that the lung index value of the mice infected by the influenza virus can be reduced by the 4 cells and the positive medicament; in addition, the biological effects of the 4 cell preparations were significantly different from the group of example 11a, the group of example 11a1, the group of example 11a2, and the group of example 11a3 (P < 0.05). In addition, satisfactory differences between groups in terms of changes in interleukin 4 levels were also presented.
Furthermore, in combination with the above measurement results regarding cell viability, it was shown that chlorogenic acid and sorbitol added in the cell preparation do not directly act on stem cells, but act on the animal body together with the stem cells, i.e., it was unexpectedly found that the simultaneous addition of both sorbitol and chlorogenic acid in the cell preparation can significantly improve the biological effects of the cell preparation.
The above-described embodiments are merely preferred embodiments for fully explaining the present application, and the scope of the present application is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present application, and are intended to be within the scope of the present application. The protection scope of the application is subject to the claims.