CN114129597A - Preparation method of stem cell active factor preparation for treating endometrial injury - Google Patents
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Abstract
The invention discloses a preparation method of a stem cell active factor preparation for treating endometrial injury, which belongs to the technical field of biological pharmacy, wherein the cultured umbilical cord mesenchymal stem cells and umbilical cord blood stem cells have high cell yield, good cell morphology, stable state, simpler culture operation, huge cell number and sufficient source; the dual stem cell active factor combination preparation has high safety performance, uses nontoxic and pollution-free reagents and materials, has low immunogenicity, no toxic or side effect and is safer to use; compared with single stem cell active factor, the dual stem cell active factor combined preparation has more active factors in the synergistic effect of the dual stem cells, and the stem cell active factors mainly comprise fibroblast growth factors, epidermal growth factors, keratinocyte growth factors, vascular endothelial growth factors, transforming growth factors beta and the like. The stem cell active factor combined preparation can stimulate endometrium and promote endometrial growth.
Description
Technical Field
The invention belongs to the technical field of biological pharmacy, and particularly relates to a preparation method of a stem cell active factor preparation for treating endometrial injury.
Background
Endometrial damage is a broad concept that includes various endometrial disorders such as thin endometrium, intrauterine adhesion. In normal menstrual cycle, the endometrium can realize periodic complete regeneration, the endometrium is divided into a functional layer and a basal layer, and the functional layer is continuously and circularly regenerated through a series of processes of growth transformation, stripping, regeneration repair and the like in each cycle. However, with the increasing number of uterine cavity operations such as induced abortion and hysteroscopy, and the effects of various factors such as endometrial infection and drugs, endometrial injuries become more and more common. After damage to the endometrial lining, proliferating inflammatory cells such as monocytes, neutrophils, lymphocytes cause an inflammatory response, secrete various inflammatory mediators, and cause endometrial cell regeneration disorders. Endometrial damage from these infections can result in loss of endometrial stromal cells that proliferate the growing endometrium, causing the endometrium to thin, resulting in infertility.
Currently, the treatment methods for treating thin uterus are divided into drug treatment, physical treatment and hysteroscopic surgery treatment. The medicine treatment is common to oral administration or vaginal administration of large dose of estrogen, subcutaneous injection of growth hormone, oral administration of aspirin or sildenafil to improve endometrial blood supply. Long-term administration of drugs such as sex hormones not only affects the endocrine of patients, but also easily causes drug resistance, and the drug therapy is effective only for a few patients. Common physical treatments include scraping of the endometrium with a curette to cause minor intimal damage, stimulation of intimal growth, and neuromuscular electrical stimulation. When the intimal fibrosis is serious, the patient urgently hopes to treat the infertility caused by intimal injury and recover normal menstruation, and hysteroscopy is the current preferred treatment method. Physical therapy and hysteroscopic therapy are not only inefficient but may cause secondary damage to the endometrium. The existing medicinal preparation has poor curative effect on endometrium, and a safe and efficient therapeutic preparation is urgently needed.
Disclosure of Invention
The invention aims to provide a preparation method of a stem cell active factor preparation for treating endometrial injury, and aims to solve the problems in the background art.
The purpose of the invention can be realized by the following technical scheme: a preparation method of a stem cell active factor preparation for treating endometrial injury comprises the following steps:
step one, collecting raw materials:
placing the collected fresh umbilical cord into a culture dish filled with 100-200mL of collection liquid for later use; after the placenta is dropped after production, inserting a blood collection bag needle containing an anticoagulant into an umbilical vein, collecting while shaking a blood bag so as to fully mix blood and the anticoagulant, and storing at low temperature for later use;
step two, inoculating the umbilical cord tissue blocks:
intercepting an umbilical cord section of 5-10cm by using an operating scissors, transferring the umbilical cord section to a sterilized culture dish, adding an ethanol solution with the mass fraction of 75%, soaking and turning over the umbilical cord in the ethanol solution, sterilizing for 45-60s, removing arteries and veins on the sterilized umbilical cord section, and washing with a buffer solution A until no blood clot exists; draining off water, putting the treated umbilical cord section into a flask, and cutting to pieces to obtain tissue blocks with the diameter of 1-3 mm; inoculating the tissue blocks into a culture dish with a DMEM culture medium, enabling the distance between the tissue blocks to be less than 1cm, and after inoculation is finished, placing the culture dish filled with the tissue blocks in a carbon dioxide incubator in an inverted mode for processing for 1-3 hours; stimulating cells by carbon dioxide to promote cell growth and division;
step three, umbilical cord mesenchymal stem cell primary culture:
adding an MSC culture solution with the volume of 1/5 culture dishes into the culture dishes inoculated with the tissue blocks treated by the carbon dioxide, and placing the culture dishes into a constant-temperature incubator for primary culture; controlling the temperature in the constant-temperature incubator to be 37 ℃ and the carbon dioxide concentration to be 5 percent; after obvious adherence of cells is observed in the culture process, the MSC culture solution is replaced once at intervals of 3 days for 21-27 days;
step four, subculturing umbilical cord mesenchymal stem cells:
collecting cultured primary cells, removing supernatant from the culture dish, washing with 2-3mL buffer solution A, removing waste liquid, addingAdding 1-2mL of digestive juice, and performing digestion culture in an incubator for 5-10 min; after digestion culture, adding 1-2mL of stop solution into a culture dish, blowing and beating the culture dish by using a pipette to completely drop attached cells to obtain cell suspension, transferring the cell suspension into a 50mL centrifuge tube, centrifuging for 5-10min under the condition of 1000-1500r/min, removing supernatant, adding 1mL of MSC culture solution to resuspend the cells, adding 2-10mL of DMEM culture medium to mix uniformly, counting, inoculating into culture bottles, placing in a carbon dioxide incubator for culture, and controlling the number of the cells in each culture bottle to be 1.3-1.6 multiplied by 106A plurality of;
subculturing when the cells in the culture bottle grow to 80-90% of the bottle wall area of the culture bottle, wherein the subculturing comprises the following specific steps:
firstly, taking an original DMEM culture medium, and washing for 1-2 times in a biological safety cabinet by using a DPBS buffer solution or physiological saline; removing waste liquid, adding appropriate amount of digestive juice, and digesting for 3-5min until cells are separated from the culture bottle; adding stop solution to stop digestion, sucking cell suspension, transferring to 50mL centrifuge tube, centrifuging for 5-10min under the condition of 1000-1500r/min, discarding supernatant, blowing uniformly with pipette gun, and selecting 1.3-1.6 × 10 cell counting plate6Adding MSC culture solution into each cell, and carrying out passage under the conditions that the concentration of carbon dioxide is 5% and the saturation humidity is 37 ℃; digesting and collecting cells by using digestive juice after the cells are passaged to 3 rd generation, carrying out sterile detection, endotoxin detection, loss phenotype, cell counting and activity detection, collecting all culture supernatants generated in the culture process, and refrigerating for later use;
step five, carrying out primary separation on cord blood stem cells:
separating umbilical cord blood by adopting a density gradient method, mixing the umbilical cord blood with equivalent physiological saline to obtain blood diluent, mixing the blood diluent with the separation solution according to the volume ratio of 2-3:1, centrifuging for 15-25min under the condition that the centrifugal force is 600-1000g, dividing the mixture into a plasma layer, a white membrane layer and a red blood cell layer from top to bottom after centrifugation, sucking the white membrane layer, and carrying out heavy suspension by using the physiological saline to obtain a first heavy suspension; centrifuging the first heavy suspension for 5min under the condition that the centrifugal force is 300-800g, re-suspending the first heavy suspension by using MSC culture solution after the centrifugation is finished to obtain a second heavy suspension, and inoculating the second heavy suspension into a culture bottle, wherein the inoculation amount is 1/5 of the volume of the culture bottle;
step six, replacing liquid of cord blood stem cells and subculturing:
changing the liquid after carrying out primary culture on umbilical cord blood stem cells in a culture bottle for 48h, carrying out subculture when the cells in the culture bottle grow to 80-90% of the bottle wall area of the culture bottle, adding a digestive juice after the cells are subcultured to 3 rd generation, digesting and collecting the cells, and sequentially carrying out sterile detection, endotoxin detection, loss phenotype, cell counting and vitality detection; collecting all culture supernatants generated in the culture process, and performing aseptic refrigeration for later use;
step seven, preparing an active factor concentrated solution:
transferring culture supernatants collected in the culture process of the umbilical cord mesenchymal stem cells and the umbilical cord blood stem cells to different centrifuge tubes, concentrating for two times to obtain an umbilical cord mesenchymal stem cell active factor concentrated solution and an umbilical cord blood stem cell active factor concentrated solution respectively, and storing at-20 ℃ for later use;
the specific steps of the two-time concentration are as follows:
centrifuging at 500-1500r/min for 5-10min, respectively collecting cell supernatant, and ultrafiltering and dialyzing the supernatant with 30KD ultrafiltration membrane under 1.0bar pressure to obtain dialysate; passing the dialysate through a 3KD ultrafiltration membrane, collecting concentrated solution containing a large amount of cell factors, and completing the first concentration; performing second concentration after the first concentration, wherein the step of the second concentration is the same as that of the first concentration;
step eight: preparing a dual stem cell active factor combined preparation:
1-3mL of the umbilical cord mesenchymal stem cell active factor concentrated solution and the umbilical cord blood stem cell active factor concentrated solution: mixing the components in a dosage ratio of 1-3mL to prepare a dual stem cell active factor combined concentrated solution, and sucking and injecting the dual stem cell active factor combined concentrated solution into sterile physiological saline by using a sterile injector to prepare a stem cell active factor preparation for treating endometrial injury; the dosage ratio of the dual stem cell active factor combined concentrated solution to the sterile normal saline is 1-3mL to 100 mL;
further, the PBS buffer solution comprises NaCl, KCl and Na2HPO4、KH2PO4And distilled water; NaCl, KCl, Na2HPO4、KH2PO4And the amount ratio of distilled water is 4 g: 0.1 g: 0.72 g: 0.12 g: 400 mL;
further, the DPBS buffer solution comprises NaCl, KCl and Na2HPO4、KH2PO4、CaCl, MgCl and distilled water; NaCl, KCl, Na2HPO4、KH2PO4、The dosages of CaCl, MgCl and distilled water are respectively 8 g: 0.2 g: 1.15 g: 0.2 g: 0.1 g: 0.1 g: 1L;
further, buffer a was prepared by the following steps: adjusting the pH value of the PBS buffer solution to 7.2 by using hydrochloric acid with the mass fraction of 10%, adding a double antibody to obtain a buffer solution A, and enabling the content of the double antibody to be 1%;
further, the collection liquid is prepared by the following steps: DMSO, fetal bovine serum, PBS buffer were mixed in 10 mL: 20mL of: mixing 70mL of the mixture according to the dosage ratio, then adjusting the pH to 7.2-7.5 by using 10% hydrochloric acid by mass fraction, adding a double antibody to obtain a mixed solution, and enabling the content of the double antibody to be 2% of the mixed solution; the double antibody is penicillin-streptomycin mixed liquor, wherein the content of penicillin is 10000U/mL, and the content of streptomycin is 10 mg/mL; the anticoagulant is a heparin sodium solution with the mass fraction of 1% or a sodium citrate solution with the mass fraction of 3.8%;
further, the DMEM medium is DMEM/F12DMEM medium purchased from seimer fisher technologies (china) ltd; the MSC culture solution is prepared by mixing a DMEM culture medium, fetal calf serum, a double antibody and an anti-B, wherein the proportion of the fetal calf serum is 10-20%, the content of the double antibody is 100-300 mu L/mL, the content of the anti-B is 100-300 mu L/mL, and the anti-B is amphotericin B; the digestive juice is EDTA/trypsin solution purchased from Saimer Feishale science and technology (China); the stop solution is fetal bovine serum and PBS buffer solution, and the volume ratio of the stop solution is 1 mL: 9mL of the mixture is prepared by mixing; the separation liquid is a lymphocyte separation liquid purchased from sigma aldrich (shanghai) trade company ltd.
The invention has the beneficial effects that:
in the preparation method, the cultured umbilical cord mesenchymal stem cells and umbilical cord blood stem cells have high cell output rate, good cell morphology, stable state, simpler culture operation, huge cell number and sufficient sources; the dual stem cell active factor combination preparation has high safety performance, uses nontoxic and pollution-free reagents and materials, has low immunogenicity, no toxic or side effect and is safer to use; compared with single stem cell active factor, the dual stem cell active factor combined preparation has more active factors in the synergistic effect of the dual stem cells, and the stem cell active factors mainly comprise fibroblast growth factors, epidermal growth factors, keratinocyte growth factors, vascular endothelial growth factors, transforming growth factors beta and the like. The stem cell active factor combined preparation can stimulate endometrium and promote the growth of endometrium, has stronger pertinence and has obvious curative effect on endometrium injury.
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The invention will be further described with reference to the accompanying drawings.
FIG. 1 is a flow chart of the preparation method of the stem cell active factor preparation for treating endometrial injury.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to 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 of the 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.
Example 1
The preparation method of the umbilical cord mesenchymal stem cell active factor concentrated solution comprises the following steps:
placing the collected fresh umbilical cord into a culture dish filled with 100mL of collection liquid for later use;
cutting an umbilical cord section of 5cm by using an operating scissors, transferring the umbilical cord section to a sterilized culture dish, adding an ethanol solution with the mass fraction of 75%, soaking and turning over an umbilical cord in the ethanol solution, sterilizing for 45s, removing arteries and veins on the sterilized umbilical cord section, and cleaning with a buffer solution A until no blood clot exists; draining off water, putting the treated umbilical cord section into a flask, and cutting to pieces to obtain tissue blocks with the diameter of 1 mm; inoculating the tissue blocks into a culture dish with a DMEM culture medium, enabling the distance between the tissue blocks to be less than 1cm, and after inoculation is finished, placing the culture dish filled with the tissue blocks in a carbon dioxide incubator in an inverted mode for treatment for 1 hour; stimulating cells by carbon dioxide to promote cell growth and division;
adding an MSC culture solution with the volume of 1/5 culture dishes into the culture dishes inoculated with the tissue blocks treated by the carbon dioxide, and placing the culture dishes into a constant-temperature incubator for primary culture; controlling the temperature in the constant-temperature incubator to be 37 ℃ and the carbon dioxide concentration to be 5 percent; after obvious adherence of cells is observed in the culture process, the MSC culture solution is replaced once at intervals of 3 days for 21 days;
collecting the cultured primary cells, removing the supernatant in the culture dish, washing with a buffer solution A, removing waste liquid, adding 1mL of digestive juice, and performing digestion culture in an incubator for 5 min; after digestion culture, adding 1mL of stop solution into a culture dish, blowing and beating the culture dish by using a pipette gun to completely drop attached cells to obtain cell suspension, transferring the cell suspension into a 50mL centrifuge tube, centrifuging for 5min under the condition of 1000r/min, removing supernatant, adding 1mL of MSC culture solution to resuspend the cells, adding 2mL of DMEM culture medium to mix uniformly, counting, inoculating into a culture bottle, placing into a carbon dioxide incubator to culture, and controlling the number of the cells in each culture bottle to be 1.3 multiplied by 106A plurality of;
firstly, taking an original DMEM culture medium, and washing for 1 time in a biological safety cabinet by using a DPBS buffer solution or normal saline; removing waste liquid and adding appropriate amount of digestive juice for digestion for 3min until cells are separated from the culture flask; adding stop solution to stop digestion, sucking cell suspension, transferring into 50mL centrifuge tube, centrifuging at 1000r/min for 5min, discarding supernatant, blowing uniformly with pipette gun, and selecting 1.3 × 10 cell counting plate6Adding MSC culture solution into each cell, and carrying out passage under the conditions that the concentration of carbon dioxide is 5% and the saturation humidity is 37 ℃; digesting with digestive juice and collecting cells after passage of cells to 3 rd generation, performing aseptic detection, endotoxin detection, and loss tableDetecting type, cell count and activity, collecting all culture supernatants generated in the culture process, and refrigerating for later use;
transferring culture supernatant collected in the umbilical cord mesenchymal stem cell culture process to a centrifuge tube, centrifuging for 5min under the condition of 500r/min, respectively collecting centrifuged cell supernatant, performing ultrafiltration dialysis on the supernatant by using an ultrafiltration membrane package of 30KD under the pressure of 1.0bar, and taking dialysate; passing the dialysate through a 3KD ultrafiltration membrane, collecting concentrated solution containing a large amount of cell factors, and completing the first concentration; after the first concentration, carrying out second concentration according to phase synchronization steps to obtain concentrated solution of the active factors of the umbilical cord mesenchymal stem cells, and storing the concentrated solution at the temperature of-20 ℃ for later use.
Example 2
The preparation method of the umbilical cord mesenchymal stem cell active factor concentrated solution comprises the following steps:
placing the collected fresh umbilical cord into a culture dish filled with 200mL of collection liquid for later use;
intercepting an umbilical cord section of 10cm by using an operating scissors, transferring the umbilical cord section to a sterilized culture dish, adding an ethanol solution with the mass fraction of 75%, soaking and overturning the umbilical cord in the ethanol solution, sterilizing for 60s, removing arteries and veins on the sterilized umbilical cord section, and cleaning with a buffer solution A until no blood clot exists; draining off water, putting the treated umbilical cord section into a flask, and cutting to pieces to obtain tissue blocks with the diameter of 3 mm; inoculating the tissue blocks into a culture dish with a DMEM culture medium, enabling the distance between the tissue blocks to be less than 1cm, and after inoculation is finished, placing the culture dish filled with the tissue blocks in a carbon dioxide incubator in an inverted mode for treatment for 3 hours; stimulating cells by carbon dioxide to promote cell growth and division;
adding an MSC culture solution with the volume of 1/5 culture dishes into the culture dishes inoculated with the tissue blocks treated by the carbon dioxide, and placing the culture dishes into a constant-temperature incubator for primary culture; controlling the temperature in the constant-temperature incubator to be 37 ℃ and the carbon dioxide concentration to be 5 percent; after obvious adherence of cells is observed in the culture process, the MSC culture solution is replaced once at intervals of 3 days for 27 days;
collecting the cultured primary cells, removing the supernatant from the culture dish, and washing with buffer solution ARemoving waste liquid, adding 2mL of digestive juice, and performing digestion culture in an incubator for 10 min; after digestion culture, adding 2mL of stop solution into a culture dish, blowing and beating the culture dish by using a pipette gun to completely drop attached cells to obtain cell suspension, transferring the cell suspension into a 50mL centrifuge tube, centrifuging for 10min under the condition of 1500r/min, removing supernatant, adding 1mL of MSC culture solution to resuspend the cells, adding 10mL of DMEM culture medium to mix uniformly, after counting, inoculating into a culture bottle and placing into a carbon dioxide incubator to culture, and controlling the number of the cells in each culture bottle to be 1.6 multiplied by 106A plurality of;
firstly, taking an original DMEM culture medium, and washing for 2 times by using a DPBS buffer solution or normal saline in a biological safety cabinet; removing waste liquid and adding appropriate amount of digestive juice for digestion for 5min until cells are separated from the culture flask; adding stop solution to stop digestion, sucking cell suspension, transferring into 50mL centrifuge tube, centrifuging at 1500r/min for 10min, discarding supernatant, blowing uniformly with pipette gun, and selecting 1.6 × 10 cell counting plate6Adding MSC culture solution into each cell, and carrying out passage under the conditions that the concentration of carbon dioxide is 5% and the saturation humidity is 37 ℃; digesting and collecting cells by using digestive juice after the cells are passaged to 3 rd generation, carrying out sterile detection, endotoxin detection, loss phenotype, cell counting and activity detection, collecting all culture supernatants generated in the culture process, and refrigerating for later use;
transferring culture supernatant collected in the umbilical cord mesenchymal stem cell culture process to a centrifuge tube, centrifuging for 10min under the condition of 1500r/min, respectively collecting centrifuged cell supernatant, performing ultrafiltration dialysis on the supernatant by using an ultrafiltration membrane package of 30KD under the pressure of 1.0bar, and taking dialysate; passing the dialysate through a 3KD ultrafiltration membrane, collecting concentrated solution containing a large amount of cell factors, and completing the first concentration; after the first concentration, carrying out second concentration according to phase synchronization steps to obtain concentrated solution of the active factors of the umbilical cord mesenchymal stem cells, and storing the concentrated solution at the temperature of-20 ℃ for later use.
Example 3
The preparation method of the cord blood stem cell active factor concentrated solution comprises the following steps:
after the placenta is dropped after production, inserting a blood collection bag needle containing an anticoagulant into an umbilical vein, collecting while shaking a blood bag so as to fully mix blood and the anticoagulant, and storing at low temperature for later use;
separating umbilical cord blood by adopting a density gradient method, mixing the umbilical cord blood with equivalent physiological saline to obtain blood diluent, mixing the blood diluent with the separation solution according to a volume ratio of 2:1, centrifuging for 15min under the condition that the centrifugal force is 600g, dividing the blood diluent into a plasma layer, a white membrane layer and a red blood cell layer from top to bottom after centrifugation, sucking the white membrane layer, and carrying out heavy suspension by using the physiological saline to obtain a first heavy suspension; centrifuging the first resuspension solution for 5min under the condition that the centrifugal force is 300g, resuspending the first resuspension solution by using MSC culture solution after centrifugation is finished to obtain a second resuspension solution, and inoculating the second resuspension solution into a culture bottle, wherein the inoculation amount is 1/5 of the volume of the culture bottle;
changing the liquid after the umbilical cord blood stem cells in the culture bottle are subjected to primary culture for 48h, carrying out subculture when the cells in the culture bottle grow to 80% of the bottle wall area of the culture bottle, adding a digestive juice after the cells are subjected to passage to the 3 rd generation, digesting and collecting the cells, and sequentially carrying out sterile detection, endotoxin detection, loss phenotype, cell counting and activity detection; collecting all culture supernatants generated in the culture process, and performing aseptic refrigeration for later use;
transferring culture supernatant collected in the process of culturing umbilical cord blood stem cells into a centrifuge tube, centrifuging for 5min under the condition of 500r/min, respectively collecting centrifuged cell supernatant, performing ultrafiltration dialysis on the supernatant by using an ultrafiltration membrane package of 30KD under the pressure of 1.0bar, and taking dialysate; passing the dialysate through a 3KD ultrafiltration membrane, collecting concentrated solution containing a large amount of cell factors, and completing the first concentration; after the first concentration, carrying out second concentration according to phase synchronization steps to obtain a concentrated solution of the cord blood stem cell active factor, and storing at the temperature of minus 20 ℃ for later use.
Example 4
The preparation method of the cord blood stem cell active factor concentrated solution comprises the following steps:
the preparation method of the cord blood stem cell active factor concentrated solution comprises the following steps:
after the placenta is dropped after production, inserting a blood collection bag needle containing an anticoagulant into an umbilical vein, collecting while shaking a blood bag so as to fully mix blood and the anticoagulant, and storing at low temperature for later use;
separating umbilical cord blood by adopting a density gradient method, mixing the umbilical cord blood with equivalent physiological saline to obtain blood diluent, mixing the blood diluent with the separation solution according to a volume ratio of 3:1, centrifuging for 25min under the condition that the centrifugal force is 1000g, dividing the blood diluent into a plasma layer, a white membrane layer and a red blood cell layer from top to bottom after centrifugation, sucking the white membrane layer, and carrying out heavy suspension by using the physiological saline to obtain a first heavy suspension; centrifuging the first resuspension solution for 5min under the condition that the centrifugal force is 800g, resuspending the first resuspension solution by using MSC culture solution after centrifugation is finished to obtain a second resuspension solution, and inoculating the second resuspension solution into a culture bottle, wherein the inoculation amount is 1/5 of the volume of the culture bottle;
changing the liquid after the umbilical cord blood stem cells in the culture bottle are subjected to primary culture for 48h, carrying out subculture when the cells in the culture bottle grow to 90% of the bottle wall area of the culture bottle, adding a digestive juice after the cells are subjected to passage to the 3 rd generation, digesting and collecting the cells, and sequentially carrying out sterile detection, endotoxin detection, loss phenotype, cell counting and activity detection; collecting all culture supernatants generated in the culture process, and performing aseptic refrigeration for later use;
transferring culture supernatant collected in the process of culturing umbilical cord blood stem cells into a centrifuge tube, centrifuging for 10min under the condition of 1500r/min, respectively collecting centrifuged cell supernatant, performing ultrafiltration dialysis on the supernatant by using an ultrafiltration membrane package of 30KD under the pressure of 1.0bar, and taking dialysate; passing the dialysate through a 3KD ultrafiltration membrane, collecting concentrated solution containing a large amount of cell factors, and completing the first concentration; after the first concentration, carrying out second concentration according to phase synchronization steps to obtain a concentrated solution of the cord blood stem cell active factor, and storing at the temperature of minus 20 ℃ for later use.
Example 5
Referring to fig. 1, the preparation of stem cell active factor preparation for treating endometrial injury comprises the following steps:
step one, collecting raw materials:
placing the collected fresh umbilical cord into a culture dish filled with 150mL of collection liquid for later use; after the placenta is dropped after production, inserting a blood collection bag needle containing an anticoagulant into an umbilical vein, collecting while shaking a blood bag so as to fully mix blood and the anticoagulant, and storing at low temperature for later use;
step two, inoculating the umbilical cord tissue blocks:
cutting an umbilical cord section of 8cm by using an operating scissors, transferring the umbilical cord section to a sterilized culture dish, adding an ethanol solution with the mass fraction of 75%, soaking and turning over an umbilical cord in the ethanol solution, sterilizing for 50s, removing arteries and veins on the sterilized umbilical cord section, and cleaning with a buffer solution A until no blood clot exists; draining off water, putting the treated umbilical cord section into a flask, and cutting to pieces to obtain tissue blocks with the diameter of 2 mm; inoculating the tissue blocks into a culture dish with a DMEM culture medium, enabling the distance between the tissue blocks to be less than 1cm, and after inoculation is finished, placing the culture dish filled with the tissue blocks in a carbon dioxide incubator in an inverted mode for treatment for 2 hours;
step three, umbilical cord mesenchymal stem cell primary culture:
adding an MSC culture solution with the volume of 1/5 culture dishes into the culture dishes inoculated with the tissue blocks treated by the carbon dioxide, and placing the culture dishes into a constant-temperature incubator for primary culture; controlling the temperature in the constant-temperature incubator to be 37 ℃ and the carbon dioxide concentration to be 5 percent; after obvious adherence of cells is observed in the culture process, the MSC culture solution is replaced once at intervals of 3 days for 24 days;
step four, subculturing umbilical cord mesenchymal stem cells:
collecting the cultured primary cells, removing the supernatant in the culture dish, washing with 2.5mL of buffer solution A, removing the waste liquid, adding 1.5mL of digestive juice, and performing digestion culture in an incubator for 8 min; after digestion culture, adding 1.5mL of stop solution into a culture dish, blowing and beating the culture dish by using a pipette gun to completely drop attached cells to obtain cell suspension, transferring the cell suspension into a 50mL centrifuge tube, centrifuging for 8min under the condition of 1200r/min, removing supernatant, adding 1mL of MSC culture solution to resuspend the cells, adding 6mL of DMEM culture medium to mix uniformly, after counting, inoculating into a culture bottle and placing into a carbon dioxide incubator to culture, and controlling the number of the cells in each culture bottle to be 1.5 multiplied by 106A plurality of;
subculture is carried out when the cells in the culture bottle grow to 85% of the bottle wall area of the culture bottle, and the specific steps of subculture are as follows:
firstly, taking an original DMEM culture medium, and washing for 2 times by using a DPBS buffer solution or normal saline in a biological safety cabinet; removing waste liquid and adding appropriate amount of digestive juice for digestion for 4min until cells are separated from the culture flask; adding stop solution to stop digestion, sucking cell suspension, transferring into 50mL centrifuge tube, centrifuging at 1300r/min for 8min, discarding supernatant, blowing uniformly with pipette gun, and selecting 1.5 × 10 cell counting plate6Adding MSC culture solution into each cell, and carrying out passage under the conditions that the concentration of carbon dioxide is 5% and the saturation humidity is 37 ℃; digesting and collecting cells by using digestive juice after the cells are passaged to 3 rd generation, carrying out sterile detection, endotoxin detection, loss phenotype, cell counting and activity detection, collecting all culture supernatants generated in the culture process, and refrigerating for later use;
step five, carrying out primary separation on cord blood stem cells:
separating umbilical cord blood by adopting a density gradient method, mixing the umbilical cord blood with equivalent physiological saline to obtain blood diluent, mixing the blood diluent with the separation solution according to the volume ratio of 2.5:1, centrifuging for 20min under the condition that the centrifugal force is 800g, dividing the mixture into a plasma layer, a white membrane layer and a red blood cell layer from top to bottom after centrifugation, sucking the white membrane layer, and re-suspending the mixture by using the physiological saline to obtain a first resuspension solution; centrifuging the first resuspension solution for 5min under the condition that the centrifugal force is 500g, resuspending the first resuspension solution by using MSC culture solution after centrifugation is finished to obtain a second resuspension solution, and inoculating the second resuspension solution into a culture bottle, wherein the inoculation amount is 1/5 of the volume of the culture bottle;
step six, replacing liquid of cord blood stem cells and subculturing:
changing the liquid after the umbilical cord blood stem cells in the culture bottle are subjected to primary culture for 48h, carrying out subculture when the cells in the culture bottle grow to 85% of the bottle wall area of the culture bottle, adding a digestive juice after the cells are subjected to passage to the 3 rd generation, digesting and collecting the cells, and sequentially carrying out sterile detection, endotoxin detection, loss phenotype, cell counting and activity detection; collecting all culture supernatants generated in the culture process, and performing aseptic refrigeration for later use;
step seven, preparing an active factor concentrated solution:
transferring culture supernatants collected in the culture process of the umbilical cord mesenchymal stem cells and the umbilical cord blood stem cells to different centrifuge tubes, concentrating for two times to obtain an umbilical cord mesenchymal stem cell active factor concentrated solution and an umbilical cord blood stem cell active factor concentrated solution respectively, and storing at-20 ℃ for later use;
step eight, preparing a dual stem cell active factor combined preparation:
the concentrated solution of the umbilical cord mesenchymal stem cell active factor and the concentrated solution of the umbilical cord blood stem cell active factor are mixed according to the ratio of 1 mL: 3mL of the double stem cell active factor combination concentrated solution is prepared by mixing, and 1mL of the double stem cell active factor combination concentrated solution is injected into 100mL of sterile physiological saline by a sterile syringe to prepare the stem cell active factor preparation for treating endometrial injury.
Example 6
Referring to fig. 1, the preparation of stem cell active factor preparation for treating endometrial injury comprises the following steps:
step one, collecting raw materials:
placing the collected fresh umbilical cord into a culture dish filled with 150mL of collection liquid for later use; after the placenta is dropped after production, inserting a blood collection bag needle containing an anticoagulant into an umbilical vein, collecting while shaking a blood bag so as to fully mix blood and the anticoagulant, and storing at low temperature for later use;
step two, inoculating the umbilical cord tissue blocks:
cutting an umbilical cord section of 8cm by using an operating scissors, transferring the umbilical cord section to a sterilized culture dish, adding an ethanol solution with the mass fraction of 75%, soaking and turning over an umbilical cord in the ethanol solution, sterilizing for 50s, removing arteries and veins on the sterilized umbilical cord section, and cleaning with a buffer solution A until no blood clot exists; draining off water, putting the treated umbilical cord section into a flask, and cutting to pieces to obtain tissue blocks with the diameter of 2 mm; inoculating the tissue blocks into a culture dish with a DMEM culture medium, enabling the distance between the tissue blocks to be less than 1cm, and after inoculation is finished, placing the culture dish filled with the tissue blocks in a carbon dioxide incubator in an inverted mode for treatment for 2 hours;
step three, umbilical cord mesenchymal stem cell primary culture:
adding an MSC culture solution with the volume of 1/5 culture dishes into the culture dishes inoculated with the tissue blocks treated by the carbon dioxide, and placing the culture dishes into a constant-temperature incubator for primary culture; controlling the temperature in the constant-temperature incubator to be 37 ℃ and the carbon dioxide concentration to be 5 percent; after obvious adherence of cells is observed in the culture process, the MSC culture solution is replaced once at intervals of 3 days for 24 days;
step four, subculturing umbilical cord mesenchymal stem cells:
collecting the cultured primary cells, removing the supernatant in the culture dish, washing with 2.5mL of buffer solution A, removing the waste liquid, adding 1.5mL of digestive juice, and performing digestion culture in an incubator for 8 min; after digestion culture, adding 1.5mL of stop solution into a culture dish, blowing and beating the culture dish by using a pipette gun to completely drop attached cells to obtain cell suspension, transferring the cell suspension into a 50mL centrifuge tube, centrifuging for 8min under the condition of 1200r/min, removing supernatant, adding 1mL of MSC culture solution to resuspend the cells, adding 6mL of DMEM culture medium to mix uniformly, after counting, inoculating into a culture bottle and placing into a carbon dioxide incubator to culture, and controlling the number of the cells in each culture bottle to be 1.5 multiplied by 106A plurality of;
subculture is carried out when the cells in the culture bottle grow to 85% of the bottle wall area of the culture bottle, and the specific steps of subculture are as follows:
firstly, taking an original DMEM culture medium, and washing for 2 times by using a DPBS buffer solution or normal saline in a biological safety cabinet; removing waste liquid and adding appropriate amount of digestive juice for digestion for 4min until cells are separated from the culture flask; adding stop solution to stop digestion, sucking cell suspension, transferring into 50mL centrifuge tube, centrifuging at 1300r/min for 8min, discarding supernatant, blowing uniformly with pipette gun, and selecting 1.5 × 10 cell counting plate6Adding MSC culture solution into each cell, and carrying out passage under the conditions that the concentration of carbon dioxide is 5% and the saturation humidity is 37 ℃; digesting and collecting cells by using digestive juice after the cells are passaged to 3 rd generation, carrying out sterile detection, endotoxin detection, loss phenotype, cell counting and activity detection, collecting all culture supernatants generated in the culture process, and refrigerating for later use;
step five, carrying out primary separation on cord blood stem cells:
separating umbilical cord blood by adopting a density gradient method, mixing the umbilical cord blood with equivalent physiological saline to obtain blood diluent, mixing the blood diluent with the separation solution according to the volume ratio of 2.5:1, centrifuging for 20min under the condition that the centrifugal force is 800g, dividing the mixture into a plasma layer, a white membrane layer and a red blood cell layer from top to bottom after centrifugation, sucking the white membrane layer, and re-suspending the mixture by using the physiological saline to obtain a first resuspension solution; centrifuging the first resuspension solution for 5min under the condition that the centrifugal force is 500g, resuspending the first resuspension solution by using MSC culture solution after centrifugation is finished to obtain a second resuspension solution, and inoculating the second resuspension solution into a culture bottle, wherein the inoculation amount is 1/5 of the volume of the culture bottle;
step six, replacing liquid of cord blood stem cells and subculturing:
changing the liquid after the umbilical cord blood stem cells in the culture bottle are subjected to primary culture for 48h, carrying out subculture when the cells in the culture bottle grow to 85% of the bottle wall area of the culture bottle, adding a digestive juice after the cells are subjected to passage to the 3 rd generation, digesting and collecting the cells, and sequentially carrying out sterile detection, endotoxin detection, loss phenotype, cell counting and activity detection; collecting all culture supernatants generated in the culture process, and performing aseptic refrigeration for later use;
step seven, preparing an active factor concentrated solution:
transferring culture supernatants collected in the culture process of the umbilical cord mesenchymal stem cells and the umbilical cord blood stem cells to different centrifuge tubes, concentrating for two times to obtain an umbilical cord mesenchymal stem cell active factor concentrated solution and an umbilical cord blood stem cell active factor concentrated solution respectively, and storing at-20 ℃ for later use;
step eight, preparing a dual stem cell active factor combined preparation:
concentrating the umbilical cord mesenchymal stem cell active factor concentrated solution and the umbilical cord blood stem cell active factor concentrated solution according to the ratio of 3mL: 1mL of the active factor combination concentrate is prepared, and 2mL of the active factor combination concentrate is injected into 100mL of sterile physiological saline by a sterile syringe to prepare the active factor preparation of the stem cells for treating the endometrial injury.
Example 7:
referring to fig. 1, the preparation of stem cell active factor preparation for treating endometrial injury comprises the following steps:
step one, collecting raw materials:
placing the collected fresh umbilical cord into a culture dish filled with 150mL of collection liquid for later use; after the placenta is dropped after production, inserting a blood collection bag needle containing an anticoagulant into an umbilical vein, collecting while shaking a blood bag so as to fully mix blood and the anticoagulant, and storing at low temperature for later use;
step two, inoculating the umbilical cord tissue blocks:
cutting an umbilical cord section of 8cm by using an operating scissors, transferring the umbilical cord section to a sterilized culture dish, adding an ethanol solution with the mass fraction of 75%, soaking and turning over an umbilical cord in the ethanol solution, sterilizing for 50s, removing arteries and veins on the sterilized umbilical cord section, and cleaning with a buffer solution A until no blood clot exists; draining off water, putting the treated umbilical cord section into a flask, and cutting to pieces to obtain tissue blocks with the diameter of 2 mm; inoculating the tissue blocks into a culture dish with a DMEM culture medium, enabling the distance between the tissue blocks to be less than 1cm, and after inoculation is finished, placing the culture dish filled with the tissue blocks in a carbon dioxide incubator in an inverted mode for treatment for 2 hours;
step three, umbilical cord mesenchymal stem cell primary culture:
adding an MSC culture solution with the volume of 1/5 culture dishes into the culture dishes inoculated with the tissue blocks treated by the carbon dioxide, and placing the culture dishes into a constant-temperature incubator for primary culture; controlling the temperature in the constant-temperature incubator to be 37 ℃ and the carbon dioxide concentration to be 5 percent; after obvious adherence of cells is observed in the culture process, the MSC culture solution is replaced once at intervals of 3 days for 24 days;
step four, subculturing umbilical cord mesenchymal stem cells:
collecting the cultured primary cells, removing the supernatant in the culture dish, washing with 2.5mL of buffer solution A, removing the waste liquid, adding 1.5mL of digestive juice, and performing digestion culture in an incubator for 8 min; after digestion culture, 1.5mL of stop solution was added to the petri dish, and the petri dish was blown with a pipette to completely detach the attached cells to obtainTransferring the cell suspension to a 50mL centrifuge tube, centrifuging for 8min at 1200r/min, removing supernatant, adding 1mL MSC culture solution to resuspend cells, adding 6mL DMEM culture medium, mixing, counting, inoculating to a culture flask, culturing in a carbon dioxide incubator, and controlling the number of cells in each culture flask to be 1.5 × 106A plurality of;
subculture is carried out when the cells in the culture bottle grow to 85% of the bottle wall area of the culture bottle, and the specific steps of subculture are as follows:
firstly, taking an original DMEM culture medium, and washing for 2 times by using a DPBS buffer solution or normal saline in a biological safety cabinet; removing waste liquid and adding appropriate amount of digestive juice for digestion for 4min until cells are separated from the culture flask; adding stop solution to stop digestion, sucking cell suspension, transferring into 50mL centrifuge tube, centrifuging at 1300r/min for 8min, discarding supernatant, blowing uniformly with pipette gun, and selecting 1.5 × 10 cell counting plate6Adding MSC culture solution into each cell, and carrying out passage under the conditions that the concentration of carbon dioxide is 5% and the saturation humidity is 37 ℃; digesting and collecting cells by using digestive juice after the cells are passaged to 3 rd generation, carrying out sterile detection, endotoxin detection, loss phenotype, cell counting and activity detection, collecting all culture supernatants generated in the culture process, and refrigerating for later use;
step five, carrying out primary separation on cord blood stem cells:
separating umbilical cord blood by adopting a density gradient method, mixing the umbilical cord blood with equivalent physiological saline to obtain blood diluent, mixing the blood diluent with the separation solution according to the volume ratio of 2.5:1, centrifuging for 20min under the condition that the centrifugal force is 800g, dividing the mixture into a plasma layer, a white membrane layer and a red blood cell layer from top to bottom after centrifugation, sucking the white membrane layer, and re-suspending the mixture by using the physiological saline to obtain a first resuspension solution; centrifuging the first resuspension solution for 5min under the condition that the centrifugal force is 500g, resuspending the first resuspension solution by using MSC culture solution after centrifugation is finished to obtain a second resuspension solution, and inoculating the second resuspension solution into a culture bottle, wherein the inoculation amount is 1/5 of the volume of the culture bottle;
step six, replacing liquid of cord blood stem cells and subculturing:
changing the liquid after the umbilical cord blood stem cells in the culture bottle are subjected to primary culture for 48h, carrying out subculture when the cells in the culture bottle grow to 85% of the bottle wall area of the culture bottle, adding a digestive juice after the cells are subjected to passage to the 3 rd generation, digesting and collecting the cells, and sequentially carrying out sterile detection, endotoxin detection, loss phenotype, cell counting and activity detection; collecting all culture supernatants generated in the culture process, and performing aseptic refrigeration for later use;
step seven, preparing an active factor concentrated solution:
transferring culture supernatants collected in the culture process of the umbilical cord mesenchymal stem cells and the umbilical cord blood stem cells to different centrifuge tubes, concentrating for two times to obtain an umbilical cord mesenchymal stem cell active factor concentrated solution and an umbilical cord blood stem cell active factor concentrated solution respectively, and storing at-20 ℃ for later use;
step eight, preparing a dual stem cell active factor combined preparation:
concentrating the umbilical cord mesenchymal stem cell active factor concentrated solution and the umbilical cord blood stem cell active factor concentrated solution according to the ratio of 2 mL: 2mL of the double stem cell active factor combination concentrated solution is prepared by mixing, and 3mL of the double stem cell active factor combination concentrated solution is injected into 100mL of sterile physiological saline by a sterile syringe to prepare the stem cell active factor preparation for treating endometrial injury.
It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
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 (8)
1. A preparation method of a stem cell active factor preparation for treating endometrial injury is characterized by comprising the following steps:
step one, inoculating an umbilical cord tissue block; step two, carrying out primary culture on umbilical cord mesenchymal stem cells; thirdly, subculturing umbilical cord mesenchymal stem cells; step four, carrying out primary separation on umbilical cord blood stem cells; step five, replacing liquid of cord blood stem cells and subculturing; step six, preparing a concentrated solution of the umbilical cord mesenchymal stem cell active factor and a concentrated solution of the umbilical cord blood stem cell blood activating factor; step seven: preparing a dual stem cell active factor combined preparation, namely mixing the concentrated solution of the umbilical cord mesenchymal stem cell active factor with the concentrated solution of the umbilical cord blood stem cell active factor according to the proportion of 1-3mL: mixing the components in a dosage ratio of 1-3mL to prepare a double stem cell active factor combined concentrated solution; and mixing the dual stem cell active factor combined concentrated solution and sterile physiological saline according to the dosage ratio of 1-3mL to 100mL to prepare the stem cell active factor preparation for treating endometrial injury.
2. The preparation method of the active factor preparation for the stem cells for treating the endometrial injury according to claim 1, wherein the specific inoculation process in the step one is as follows: taking out the umbilical cord from the collection liquid, intercepting umbilical cord sections of 5-10cm, sterilizing with ethanol solution for 45-60s, washing with buffer solution A, shearing to obtain tissue blocks with diameter of 1-3mm, and inoculating to make the distance between tissue blocks less than 1 cm.
3. The method for preparing the stem cell active factor preparation for treating endometrial injury according to claim 1, wherein the primary culture in step two is as follows: adding MSC culture solution into the tissue block, and culturing at 37 deg.C and 5% carbon dioxide concentration for 21-27 days to obtain primary cells.
4. The preparation method of the stem cell active factor preparation for treating the endometrial injury according to claim 1, wherein the subculture process in the third step is as follows: preparing cell suspension, centrifuging for 5-10min under the conditions of 1000-1500r/min, and separatingThe bottom cells are blown and beaten uniformly and are selected to be 1.3-1.6 multiplied by 106After each cell, MSC culture medium was added and passaged 3 times under the conditions of carbon dioxide concentration of 5% and saturated humidity at 37 ℃.
5. The preparation method of the active factor preparation of stem cells for treating endometrial injury according to claim 2, wherein the buffer A is prepared by the following steps: the pH of the PBS buffer solution is adjusted to 7.2 by hydrochloric acid with the mass fraction of 10%, and the double antibody is added to obtain a buffer solution A, so that the content of the double antibody is 1%.
6. The preparation method of the stem cell active factor preparation for treating the endometrial injury according to claim 2, wherein the collection solution is prepared by the following steps: DMSO, fetal bovine serum, PBS buffer were mixed in 10 mL: 20mL of: mixing 70mL of the mixture according to the dosage ratio, adjusting the pH to 7.2-7.5 by using 10% hydrochloric acid by mass fraction, adding a double antibody to obtain a mixed solution, and enabling the content of the double antibody to be 2% of the mixed solution.
7. The method for preparing the stem cell active factor preparation for treating the endometrial injury according to claim 3, wherein the umbilical cord mesenchymal stem cell subculture process in the three steps is as follows: collecting primary cells in the second step, and taking 1.3-1.6X 10 after resuspension6Passage of each cell 3 times; taking DMEM culture medium of the previous culture for subculture each time, washing for 1-2 times, digesting for 3-5min, stopping digestion, centrifuging, and taking cells for subculture under the conditions of carbon dioxide concentration of 5% and saturated humidity of 37 ℃.
8. The method for preparing the stem cell active factor preparation for treating the endometrial injury according to claim 1, wherein the specific process of primary separation of umbilical cord blood in the fourth step is as follows: separating umbilical cord blood by adopting a density gradient method, mixing the umbilical cord blood with equivalent amount of physiological saline to obtain blood diluent, mixing the blood diluent with the separation solution according to the volume ratio of 2-3:1, centrifuging for 15-25min under the condition that the centrifugal force is 600-800 g, sucking a white membrane layer, re-suspending with the physiological saline, centrifuging for 5min under the condition that the centrifugal force is 300-800g, and re-suspending with an MSC culture solution.
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