CN114796275A - Stem cell gel preparation and preparation method and application thereof - Google Patents
Stem cell gel preparation and preparation method and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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Abstract
The invention relates to the technical field of biomedicine, and particularly discloses a stem cell gel preparation as well as a preparation method and application thereof. The stem cell gel preparation comprises mesenchymal stem cells of menstrual blood source, autologous platelet-rich plasma and chitosan. The stem cell gel preparation provided by the invention can promote the repair of endometrial tissue through immune regulation, improve the inflammatory reaction of intrauterine adhesion, obviously increase the thickness of an inner membrane of a patient with serious intrauterine adhesion, improve the fertility possibility of a patient with moderate and severe intrauterine adhesion, and has good prevention and treatment effects on intrauterine adhesion.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a stem cell gel preparation and a preparation method and application thereof.
Background
Endometrial damage is mainly characterized by intrauterine adhesions (IUA). At present, the incidence of uterine cavity adhesion is high, and besides abnormal menstruation and pelvic pain, the uterine cavity adhesion patients can also prevent blastocyst implantation, damage uterus and embryo blood supply, and finally lead to repeated abortion or infertility of the patients. Hysteroscope is a common surgical auxiliary treatment means in the field of female reproductive diseases, and mainly aims to relieve the adhesion of uterine cavities and restore normal uterine cavity anatomy and reproductive function. Hysteroscopic treatment of intrauterine adhesions, however, is potentially dangerous for subsequent pregnancies and for endometrium-harming people. Repeated hysteroscopic separation surgery can cause severe trauma to the basal layer of the endometrium, which in turn damages basal layer stem cells and may result in endometrial damage that is difficult to repair. Later, the technology of injecting medicines into the uterine cavity for treating and preventing the uterine cavity adhesion appears, the technology is simple to operate and has small invasiveness to patients, and the situation of serious wound in the treatment process is avoided to a certain extent. But the existing clinical treatment means has no obvious prevention and treatment effect on the repair of the repeatedly adhered endometrium.
Disclosure of Invention
Aiming at the problems of the existing clinical treatment means of the intrauterine adhesion, the invention provides a stem cell gel preparation, a preparation method and an application thereof.
In order to achieve the purpose of the invention, the embodiment of the invention adopts the following technical scheme:
a stem cell gel preparation comprises mesenchymal stem cells of menstrual blood source, autologous platelet-rich plasma and chitosan.
Compared with the prior art, the stem cell gel preparation provided by the invention combines blood-derived mesenchymal stem cells, autologous platelet-rich plasma and chitosan, can regulate the expression quantity of inflammation-inhibiting factors and proinflammatory factors of endometrial cells, increase the quantity of inflammation-inhibiting cells, improve the inflammatory reaction of intrauterine adhesion, promote the repair of endometrial injury, inhibit the formation of intrauterine tissue adhesion and scars, is beneficial to maintaining normal pregnancy, particularly has an obvious treatment effect on patients with moderate and severe intrauterine adhesion, can increase the thickness of the endometrium of the patients with moderate and severe intrauterine adhesion, and increase the possibility of fertility of the patients with moderate and severe intrauterine adhesion in the treatment of diseases such as endometrial repair.
Preferably, the cell concentration of the mesenchymal stem cells of blood source in the stem cell gel preparation is (0.5-1). times.10 7 one/mL.
Preferably, the concentration of platelets in the stem cell gel preparation is (600- 9 /L。
Preferably, the concentration of said chitosan in said stem cell gel formulation is between 0.4 wt% and 0.6 wt%.
Preferably, the preparation method of the autologous mesenchymal stem cells of the blood source comprises the following steps:
a. mixing menstrual blood with preservation solution, adding into Ficoll separating medium, centrifuging, removing supernatant to obtain menstrual blood mononuclear cells, and re-suspending with mesenchymal stem cell culture medium to obtain menstrual blood-derived mesenchymal stem cell suspension;
b. adding the inactivated peripheral blood plasma of the patient and the cefoperazone sodium sulbactam sodium into the menstrual blood source mesenchymal stem cell suspension to obtain a menstrual blood source mesenchymal stem cell culture solution, and placing the menstrual blood source mesenchymal stem cell culture solution at the temperature of 35-37 ℃ and CO 2 Culturing under the condition that the volume concentration is 4% -6%, and performing digestion passage when the cell fusion degree reaches 50% -70% to obtain P1 generation cells; continuously subculturing, and performing second digestion and subculture when the cell fusion degree reaches 80-90% to obtain P2 generation cells; continuously subculturing until the cells of P3 generation are obtained, and sucking out culture solution except the cells to obtain menstrual blood-derived mesenchymal stem cells growing adherently;
c. adding pancreatin into the blood-derived mesenchymal stem cells which grow adherently for digestion, stopping digestion after the blood-derived mesenchymal stem cells become round, centrifuging, and removing supernatant to obtain the blood-derived mesenchymal stem cells.
The autologous blood-derived mesenchymal stem cells prepared by the preferable preparation method of the autologous blood-derived mesenchymal stem cells do not contain xenogenic serum components, the initial amount of culturable cells is low, the cell culture period is shorter, the purity of the obtained cells is higher, and the autologous blood-derived mesenchymal stem cells have better activity when being combined with other components of the gel preparation and applied to endometrial repair, so that the speed of endometrial repair is further increased. Meanwhile, the application of the autologous menstrual blood-derived mesenchymal stem cells is not restricted by ethics, and can meet the clinical use standard.
The pancreatin used is under the brand name gibco and under the trade name CTS TM TrypLE TM Select, cat # A1285901-01.
Preferably, in the step a, the preservation solution consists of normal saline, cefoperazone sodium and sulbactam sodium and amphotericin B; the concentration of the cefoperazone sodium and sulbactam sodium in the preservation solution is 3.8-4.2 mg/mL; the volume content of the amphotericin B is 1.8-2.2% of the volume of the preservation solution.
Preferably, in the step a, the mixing volume of the menstrual blood and the preservation solution is 1: 0.8-1.2.
Preferably, in the step a, the density of the Ficoll separating medium is 1.07-1.08g/mL, and the volume of the Ficoll separating medium is 1.8-2.2 times of the volume of the menstrual blood.
Preferably, in step a, the preparation method of the mesenchymal stem cell culture medium comprises: collecting umbilical cord of healthy full-term newborn, peeling amnion and arteriovenous blood vessel, and tearing umbilical cord Wharton jelly; carrying out primary cell culture on the Wharton jelly by adopting a tissue block adherence method, and when the cell fusion degree reaches 30-50%, carrying out digestion passage and removing tissue blocks to obtain P1 generation cells; continuously subculturing, and performing second digestion and passage when the cell fusion degree reaches 80-90% to obtain P2 generation cells; and (3) continuing subculturing, when the cell fusion degree reaches 80-90%, collecting culture supernatant, and mixing the collected supernatant with a mesenchymal stem cell serum-free culture medium produced by Beijing Mongolia stem cell technology Limited according to the ratio of 1: mixing the mixture in a volume ratio of 0.8-1.2 to obtain the mesenchymal stem cell culture medium.
The menstrual blood-derived mesenchymal stem cells are prepared by using the preferable mesenchymal stem cell culture medium, so that the purity, increment and cell adherence rate of the prepared menstrual blood-derived mesenchymal stem cells can be further improved. The success rate of the culture of the mesenchymal stem cells of the menstrual blood source under the condition of extremely small menstrual blood volume is increased, and the possibility of promoting the autograft of a patient with the clinical repeated moderate and severe intrauterine adhesion menstrual blood volume which is obviously less (1-2mL) is met.
Preferably, in step a, the concentration of the mesenchymal stem cells of blood source in the mesenchymal stem cell suspension of blood source is (1-3) × 10 6 one/mL.
Preferably, in step b, the patient peripheral blood plasma is added in an amount of 8-12% of the volume of the mesenchymal stem cell suspension of the menstrual blood source.
Preferably, in step b, the concentration of the cefoperazone sodium and sulbactam sodium in the culture solution of the mesenchymal stem cells of the menstrual blood source is 0.8mg/mL-1.2 mg/mL.
The addition of the cefoperazone sodium and sulbactam sodium with the concentration has no influence on cell proliferation while achieving the bacteriostatic action, and has no statistical difference.
Preferably, in step c, the digestion is terminated by adding physiological saline to the rounded mesenchymal stem cells of blood source.
Preferably, the preparation method of the autologous platelet-rich plasma comprises the following steps:
collecting peripheral blood of a patient, performing anticoagulation treatment, centrifuging to separate the peripheral blood into an upper layer, a middle layer and a lower layer, sucking the upper layer and the middle layer, performing secondary centrifugation, removing an upper layer separation solution, adding thrombin and 8-10 wt.% calcium chloride solution into the rest part, and activating to obtain the autologous platelet-rich plasma; the volume percentage content of the calcium chloride solution in the autologous platelet-rich plasma is 8-12%, and the content of thrombin is 15-25U/mL.
The invention also provides a preparation method of the stem cell gel preparation, which comprises the following steps: and (3) resuspending the mesenchymal stem cells from blood source by using the autologous platelet-rich plasma to obtain a cell suspension, adding chitosan into the cell suspension, and uniformly mixing to obtain the stem cell gel preparation.
Compared with the prior art, the stem cell gel preparation provided by the invention has the advantages of simple preparation method, easily obtained raw material components, no ethical limitation and capability of meeting the clinical use requirement in a short time.
The invention also provides application of the stem cell gel preparation in preparing a medicament for preventing and treating endometrial injury.
The invention also provides application of the stem cell gel preparation in preparing a medicament for treating severe intrauterine adhesion.
Drawings
Fig. 1 is a micrograph of P0 generation menstrual blood-derived mesenchymal stem cells in the test group and the control group in example 1 of the present invention;
FIG. 2 is a micrograph of P3 generation mesenchymal stem cells of menstrual blood origin in the test group and the control group in example 1 of the present invention;
FIG. 3 is a graph showing the increase in HUVECs in the test group and the control group in example 1 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
Preparation of Stem cell gel preparation
1. Environmental requirements
1) Isolation, culture and formulation preparation of blood-derived mesenchymal stem cell (MenSCs) seed cells were performed in class B clean rooms, and open operations were performed in class a environments (biosafety cabinets).
2) The preparation personnel carry out ultraviolet sterilization on the cell operation room and the biological safety cabinet in advance, the ultraviolet irradiation time is 30min, the ultraviolet lamps of the operation units related to the biological safety cabinet in the B-level clean room are started to irradiate for 30min, the ultraviolet lamps of the operation units are closed, then fresh air is introduced for 10min, and the operation units are kept in the running state.
3) All reagents, consumables, samples and the like which enter the laboratory must be transmitted after being subjected to disinfection and sterilization operations such as ultraviolet irradiation, alcohol wiping and disinfection and the like.
4) The preparation personnel need to carry out the dressing change according to the SOP standard of the personnel dressing change standard and then can enter the clothes.
2. Preparing mesenchymal stem cell culture medium
Collecting umbilical cord of healthy full-term newborn, peeling amnion and arteriovenous blood vessel, and tearing umbilical cord Wharton jelly; carrying out primary cell culture on the Wharton jelly by adopting a tissue block adherence method, and when the cell fusion degree reaches 40%, carrying out digestion and passage, and removing tissue blocks to obtain P1 generation cells; continuously carrying out subculture, and carrying out second digestion subculture when the cell fusion degree reaches 85% to obtain P2 generation cells; and (3) continuing subculturing, when the cell fusion degree reaches 85%, collecting culture supernatant, and mixing the collected supernatant with a mesenchymal stem cell serum-free culture medium produced by Beijing Mongolia stem cell technology Limited according to the ratio of 1: mixing the mixture in a volume ratio of 0.8-1.2 to obtain the mesenchymal stem cell culture medium. The formula of a culture medium used for primary cell culture and subculture is as follows: the shelf number of the serum-free culture medium for the mesenchymal stem cells produced by Beijing Mongolian Gaokou stem cell technology Limited is (MSC1201B + MSC 1201S)/set.
3. Preparation of mesenchymal stem cells of menstrual blood source
1) Menstrual blood sample treatment
Take out the menstrual blood preservative fluid and place room temperature balance 1h in, open biological safety cabinet ultraviolet irradiation 30min, will this experiment required consumptive material also ultraviolet irradiation disinfection simultaneously, open fan and lighting system after the completion, it is steady to wait for the air current, spreads into the menstrual blood sample into biological safety cabinet in, according to 1: adding the prepared preservation solution in a proportion of 1, fully and uniformly mixing, and preserving at 4 ℃ to obtain a treated sample (treated in 48 h). Wherein the preservation solution consists of normal saline, cefoperazone sodium and sulbactam sodium and amphotericin B; the concentration of the cefoperazone sodium and sulbactam sodium in the preservation solution is 4 mg/mL; the content of amphotericin B by volume was 2% of the volume of the preservation solution.
2) Preparation of patient peripheral blood plasma
Carrying out centrifugal treatment on the peripheral blood of the patient at 3500rpm for 10min, wherein the set parameters of the centrifugal machine are as follows: and (3) rising the temperature to 9 ℃ and reducing the temperature to 20 ℃, transferring the separated plasma into a new centrifugal tube, sealing the centrifugal tube for inactivation, and inactivating the plasma at 56 ℃ for 30 min. After inactivation, the plasma is centrifuged for 10min at 2000rpm, and the centrifuge setting parameters are as follows: the temperature is increased by 9 ℃ and decreased by 9 ℃ and 20 ℃, and the supernatant (the blood plasma of the peripheral blood of the patient) is taken for standby.
3) MenSCs separation
Taking out the reagents required by the experiment in advance, balancing the reagents at room temperature for 1h, opening the biological safety cabinet for ultraviolet irradiation for 30min, simultaneously performing ultraviolet irradiation disinfection on the consumables required by the experiment, opening a fan and an illumination system after the completion of the ultraviolet irradiation disinfection, waiting for the stable airflow, transferring the processed menstrual blood treatment sample into the biological safety cabinet, and transferring the menstrual blood treatment sample into a new centrifugal tube.
Secondly, according to the following steps of 1: 2 ratio the blood-treated sample was added to a Ficoll separation medium having a density of 1.077. Centrifuging at 2000rpm for 25min, and setting parameters of the centrifuge as follows: the temperature is increased by 9, decreased by 2 and decreased by 20 ℃. After the centrifugation is finished, the middle PBMC layer is sucked by a suction pipe, the sucked cells are transferred into a new centrifugal pipe, and the suspension is the menstrual blood mononuclear cells. Centrifuging the cell suspension at 1500rpm for 5min at room temperature, wherein the centrifuge setting parameters are as follows: 9 and 9 are increased and decreased, and 20 ℃ is increased and decreased. After centrifugation, the supernatant was discarded, and the cell pellet was washed and repeated three times.
Thirdly, after the washing is finished, the cell sediment is re-suspended by the prepared mesenchymal stem cell culture medium, 10 percent of inactivated patient plasma and cefoperazone sodium and sulbactam sodium (1:1) are added according to the volume ratio to obtain cell suspension, wherein the final concentration of the cefoperazone sodium and sulbactam sodium is 1 mg/mL.
4. Culture of MenSCs
Adding the uniformly mixed cell suspension into a cell culture bottle, and marking related information on the culture bottle. Observing cell inoculation density under a mirror, and then placing the culture flask at 37 ℃ and CO 2 Culturing in an incubator with 5% volume concentration. After 48h, the solution is changed for the first time, the new mesenchymal stem cell culture medium is changed (the cells are P0 generation), and cefoperazone sodium and sulbactam sodium (1:1) are added, the final concentration of the cefoperazone sodium and sulbactam sodium is 1mg/mL, and then the solution is changed every 3 d. Observing the cell fusion degree under a microscope, and when the cell fusion degree reaches 60%, carrying out digestion passage to obtain P1 generation cells; continuously subculturing, and performing second digestion passage when the cell fusion degree reaches 85% to obtain P2 generation cells; continuously subculturing until the cells of P3 generation, and sucking out the culture solution except the cells to obtain the menstrual blood-derived mesenchymal stem cells growing adherently.
The growth of the P0 generation cells and the P3 generation cells was recorded under a microscope, and the culture medium of mesenchymal stem cells was replaced with fetal bovine serum + DMEM medium as a control. The micrograph of the P0 generation cells is shown in FIG. 1. The micrograph of the P3 passage cells is shown in FIG. 2. From the cell growth states in fig. 1 and 2, it can be seen that the test groups obtained significantly higher numbers of cell proliferations after the same density of inoculation.
The flow identification is carried out on the P3 generation menstrual blood source mesenchymal stem cells, and the identification result is shown in the table 1:
TABLE 1 flow-type identification of mesenchymal stem cells from blood sources
Note: p < 0.05 > and P < 0.01
As can be seen from table 1, the surface labeling index of the passage P3 mesenchymal stem cells obtained from the test group showed significantly higher purity and very significant difference (P < 0.01) compared to the control group.
Statistics are carried out on the culture periods of the menstrual blood-derived mesenchymal stem cells of the test group and the control group, wherein the culture periods of the menstrual blood-derived mesenchymal stem cells of the P3 generation are obtained, and the statistical results are shown in the table 2:
TABLE 2 statistical results of the incubation periods
Note: p < 0.05, P < 0.01;
as can be seen from table 2, the test group menstrual blood-derived mesenchymal stem cells have a short culture cycle in each initial blood volume collection group, and have a very significant difference (P < 0.01), and the difference is the largest in the case of a very small amount of menstrual blood collection (1-2 mL).
Statistics is carried out on the growth and proliferation conditions of the menstrual blood-derived mesenchymal stem cells of the P3 generation obtained in the test group and the control group, and the statistical results are shown in Table 3:
TABLE 3 statistics of cell growth proliferation
Note: < 0.05 > and < 0.01;
as can be seen from table 3, the difference between the amount of proliferated menstrual blood-derived mesenchymal stem cells and the control group by the fold is larger in the case of a smaller amount of menstrual blood, and the difference is very significant (P < 0.01), and in the case of an extremely small amount of menstrual blood (1-2mL), the cell order of magnitude of clinical transplantation can be satisfied when the generation number of cells reaches 3 generations, and in the case of a single moderate collection amount of menstrual blood (> 4mL), the amount of cell expansion is also very significant (P < 0.01) compared to the control group.
In the in vitro cell culture, the inoculation number of cells and the components of a culture medium have important influence on the cell growth, the cell growth can be inhibited when the cell adherence number is too small, at the moment, the growth substance in the cells is too much dispersed outside the cells, the cells can not be proliferated again to cause culture failure, and if the pH of the culture medium is continuously changed to alkali, the cells become round and can not adhere to the walls until the cells die. The difference of different culture systems on the adherent density and the cell proliferation condition of primary cells is obvious, statistics is carried out on the culture success rate of menstrual blood source mesenchymal stem cells, and the statistical results are shown in table 4:
TABLE 4 success rate of culturing mesenchymal stem cells from blood source
The results in Table 4 show that the higher the amount of menstrual blood, the higher the success rate of culture in each group. In the case of very small (1-2mL) menstrual blood volume, the experimental composition power was very significantly different from the control group (P < 0.01).
Therefore, the specific mesenchymal stem cell culture medium is added, so that the number of initial cells of the mesenchymal stem cells from menstrual blood sources can be reduced, the culture period is shortened, more cells can be obtained, the culture success rate under the condition of extremely small menstrual blood volume is increased, and the possibility of autologous transplantation is improved for patients with clinical repeated moderate-severe intrauterine adhesion menstrual blood volume which is obviously less (1-2 mL).
② observing the growth condition of the menstrual blood-derived mesenchymal stem cells growing adherently under an inverted microscope, adding new culture medium on the non-cell culture surface of the culture bottle, then placing the culture bottle at 37 ℃ and CO 2 Culturing in an incubator with the volume concentration of 5%, and subculturing according to the adherent number and the fusion degree of the primary cells and the fusion degree to be achieved of about 60% -80% of the cell cloning condition.
And thirdly, sucking the culture solution out by using a pipette as much as possible until the culture solution is completely sucked, adding physiological saline into a non-cell culture surface, slightly shaking to wash the cells to be attached to the wall surface, discarding and repeating for 2 times. Pancreatin (the pancreatin brand is gibco, under the trade name CTS) was added TM TrypLE TM Select, cat # A1285901-01), allowing pancreatin to uniformly infiltrate the cells and attach to the wall surface, incubating at 37 deg.C for 2min, adding normal saline to stop the digestion process after the cells become round, blowing the cells and attaching to the wall surface with a pipette, and suckingThe cell suspension is transferred to a centrifuge tube. Centrifuging the cell suspension at 1500rpm for 5min at room temperature, wherein the centrifuge setting parameters are as follows: 9 and 9 are increased and decreased, and 20 ℃ is increased and decreased. And after the centrifugation is finished, discarding the supernatant to obtain the mesenchymal stem cells of the menstrual blood source.
5. Preparation of autologous platelet-rich plasma
Collecting peripheral blood of a patient, carrying out anticoagulation treatment, and carrying out centrifugal treatment at 2500rpm for 10min, wherein the centrifuge setting parameters are as follows: 9 and 9 are increased and decreased, and 20 ℃ is increased and decreased. After centrifugation, the blood was divided into 3 layers: the upper layer is Platelet Poor Plasma (PPP), the middle layer is buffy coat layer, which is the concentrated platelet, and the lower layer is red blood cells. Sucking the upper layer, the middle layer and part of the red blood cells (the proportion of the red blood cells to the whole red blood cells is not higher than 25%) adjacent to the middle layer by using a pipette, transferring the red blood cells to a new centrifugal tube, and performing secondary centrifugation. The rotation speed of the second centrifugal treatment is 4100rpm, the time is 20min, and the setting parameters of the centrifugal machine are as follows: 9 and 9 are increased and decreased, and 20 ℃ is increased and decreased. After the centrifugation is finished, dividing the liquid in the centrifuge tube into 3 layers, removing PPP on the upper layer part by using a straw, retaining the middle and lower layers, removing erythrocyte sediment, fully and uniformly mixing, adding thrombin and 10 wt.% calcium chloride solution for activation, and obtaining autologous platelet-rich plasma; the volume percentage content of the calcium chloride solution in the autologous platelet-rich plasma is 10 percent, and the content of the thrombin is 20U/mL.
6. Preparation of stem cell gel preparation
Mixing the obtained mesenchymal stem cells with autologous platelet-rich plasma and medical chitosan, mixing well, and making into stem cell gel preparation with the concentration of the mesenchymal stem cells of blood source of 1 × 10 7 Per mL, platelet concentration 1000X 10 9 The concentration of chitosan was 0.5 wt%. Sealing and packaging the prepared stem cell gel preparation in a medical disposable syringe.
7. Method for detecting endothelial cell growth promoting capability of stem cell gel preparation
Endometrial tissue from patients with cervical adhesions exhibits vascular occlusion, i.e., revascularization in the damaged endometrium facilitates endometrial repair. The experiment judges the repair capability of the stem cell gel preparation on endometrium by detecting the increment influence of the stem cell gel preparation on Human Umbilical Vein Endothelial Cells (HUVECs).
Isolation and culture of HUVECs: taking part of newborn umbilical cords, timely putting sterile normal saline into the umbilical cords, clamping one end of each hemostatic forceps to be cut off, searching the thickest venous blood vessel with thin wall, washing the venous blood vessels to be colorless by normal saline, injecting pancreatin for digestion, collecting digestive juice into a centrifugal tube of a complete culture medium, centrifuging, discarding supernatant, suspending and precipitating by using the complete culture medium, inoculating the venous blood vessels into a culture bottle, culturing in an incubator with the temperature of 37 ℃ and the volume concentration of CO2 of 5%, and carrying out passage for subsequent experiments when the cell fusion degree reaches 80-90%. The complete medium was DMEM/F12+ 10% fetal bovine serum.
Detecting the influence of different groups on the HUVECs proliferation by a CCK8 method: HUVECs cell suspension was counted at 5X 10 per well 3 Inoculating each cell to a 96-well plate, culturing by using a complete culture medium until the cell fusion degree reaches more than 90 percent, changing into serum-free DMEM/F12 starvation treatment for 12 hours, adding 200uL of intervention liquid (the experimental group is a stem cell gel preparation, the contrast group 1 is 0.5 wt% of chitosan, the contrast group 2 is 0.5 wt% of chitosan + the blood source mesenchymal stem cells) into each well according to the experimental group, continuously culturing for 24 hours, changing the complete culture medium, adding 10uL of CCK8, incubating for 2 hours, detecting the absorbance value of each well at the wavelength of 490nm by using an enzyme labeling instrument, measuring once every 24 hours, continuously detecting for 6 days, and analyzing the proliferation capacity of the cells.
The effect of the test group on the proliferation of HUVECs compared with the control group 1 and the control group 2 counted by the above test is shown in Table 5, and the proliferation curve of HUVECs is shown in FIG. 3.
TABLE 5 Effect of the various formulations on the proliferation of HUVECs
Note: p < 0.05 > and < 0.01.
As can be seen from the results in table 5 and fig. 3, the combination of chitosan with autologous PRP and menstrual-derived mesenchymal stem cells has a significant promoting effect on the proliferation of HUVECs.
8. Stem cell gel formulations promote endometrial tissue repair through immunomodulation
The damaged endometrium is highly expressed with proinflammatory factors INF-gamma and IL-2, and shows an inflammation reaction mainly based on Th1 cells, while Th2 cells mainly show an inflammation inhibition reaction, and when Th2 cells are mainly used, the endometrium repair is facilitated, and the normal pregnancy is maintained.
When possible effects and effects of clinical application of the stem cell gel preparation are researched, most of intimal injuries of patients with moderate and severe intrauterine adhesion are considered to be serious, the intimal thickness is extremely thin, objective indexes can be obtained for avoiding further injury to the patients, besides the observation of the intimal thickness, a patient menstrual blood detection immune cytokine (flow cytometry) is selected creatively to replace intimal pathological biopsy to reflect the condition of endometrial repair. The results are shown in Table 6.
TABLE 6 endometrial cell proliferation and expression of related factors
Note: p < 0.05 > and < 0.01.
The results show that the gel formulation stayed in the uterine cavity for about 4-7 days with varying degrees of increase in endometrial thickness; meanwhile, compared with the detection results of menstrual blood immune cells Th2/Th1 before and after treatment, the significant difference (P < 0.05) is found after 6 cases of treatment.
Example 2
Preparation of Stem cell gel preparation
1. Environmental requirements
1) Isolation, culture and formulation preparation of blood-derived mesenchymal stem cell (MenSCs) seed cells were performed in class B clean rooms, and open operations were performed in class a environments (biosafety cabinets).
2) The preparation personnel carry out ultraviolet sterilization on the cell operation room and the biological safety cabinet in advance, the ultraviolet irradiation time is 30min, the ultraviolet lamps of the operation units related to the biological safety cabinet in the B-level clean room are started to irradiate for 30min, the ultraviolet lamps of the operation units are closed, then fresh air is introduced for 10min, and the operation units are kept in the running state.
3) All reagents, consumables, samples and the like which enter the laboratory must be transmitted after being subjected to disinfection and sterilization operations such as ultraviolet irradiation, alcohol wiping and disinfection and the like.
4) The preparation personnel need to carry out the dressing change according to the SOP standard of the personnel dressing change standard and then can enter the clothes.
2. Configuring mesenchymal stem cell culture medium
Collecting umbilical cord of healthy full-term newborn, peeling amnion and arteriovenous blood vessel, and tearing umbilical cord Wharton jelly; carrying out primary cell culture on the Wharton jelly by adopting a tissue block adherence method, and when the cell fusion degree reaches 50%, carrying out digestion and passage, and removing tissue blocks to obtain P1 generation cells; continuously subculturing, and performing second digestion and subculture when the cell fusion degree reaches 90% to obtain P2 generation cells; and (4) continuing subculturing, when the cell fusion degree reaches 90%, collecting culture supernatant, and mixing the collected supernatant with the mesenchymal stem cell serum-free culture medium according to the ratio of 1: mixing the mixture in a volume ratio of 0.8-1.2 to obtain the mesenchymal stem cell culture medium. The formula of a culture medium used for primary cell culture and subculture is as follows: the serum-free culture medium is produced by the Beijing Mongolian stem cell technology limited company.
3. Preparation of mesenchymal stem cells of menstrual blood source
1) Menstrual blood sample treatment
Take out the menstrual blood preservative fluid and place room temperature balance 1h in, open biological safety cabinet ultraviolet irradiation 30min, will this experiment required consumptive material also ultraviolet irradiation disinfection simultaneously, open fan and lighting system after the completion, it is steady to wait for the air current, spreads into the menstrual blood sample into biological safety cabinet in, according to 1: adding the prepared preservation solution in a proportion of 1.2, fully and uniformly mixing, and preserving at 4 ℃ to obtain a treated sample (treated in 48 h). Wherein the preservation solution consists of normal saline, cefoperazone sodium and sulbactam sodium and amphotericin B; the concentration of the cefoperazone sodium and sulbactam sodium in the preservation solution is 4.2 mg/mL; the content of amphotericin B by volume was 2.2% of the volume of the preservation solution.
2) Preparation of patient peripheral blood plasma
Carrying out centrifugal treatment on the peripheral blood of the patient at 3500rpm for 10min, wherein the set parameters of the centrifugal machine are as follows: and (3) rising the temperature to 9 ℃ and reducing the temperature to 20 ℃, transferring the separated plasma into a new centrifugal tube, sealing the centrifugal tube for inactivation, and inactivating the plasma at 56 ℃ for 30 min. After inactivation, the plasma is centrifuged for 10min at 2000rpm, and the centrifuge setting parameters are as follows: the temperature is increased by 9 ℃ and decreased by 9 ℃ and 20 ℃, and the supernatant (the blood plasma of the peripheral blood of the patient) is taken for standby.
3) MenSCs separation
Taking out the reagents required by the experiment in advance, balancing the reagents at room temperature for 1h, opening the biological safety cabinet for ultraviolet irradiation for 30min, simultaneously performing ultraviolet irradiation disinfection on the consumables required by the experiment, opening a fan and an illumination system after the completion of the ultraviolet irradiation disinfection, waiting for the stable airflow, transferring the processed menstrual blood treatment sample into the biological safety cabinet, and transferring the menstrual blood treatment sample into a new centrifugal tube.
Secondly, according to the following steps of 1:1 ratio the blood-treated sample was added to a Ficoll separation medium having a density of 1.077. Centrifuging at 2000rpm for 25min, and setting parameters of the centrifuge as follows: the temperature is increased by 9, decreased by 2 and decreased by 20 ℃. After the centrifugation is finished, the middle PBMC layer is sucked by a suction pipe, the sucked cells are transferred into a new centrifugal pipe, and the suspension is the menstrual blood mononuclear cells. Centrifuging the cell suspension at 1500rpm for 5min at room temperature, wherein the centrifuge setting parameters are as follows: 9 and 9 are increased and decreased, and 20 ℃ is increased and decreased. After centrifugation, the supernatant was discarded, and the cell pellet was washed and repeated three times.
Thirdly, after the washing is finished, the cell sediment is re-suspended by the prepared mesenchymal stem cell culture medium, 10 percent of inactivated patient plasma and cefoperazone sodium and sulbactam sodium (1:1) are added according to the volume ratio to obtain cell suspension, wherein the final concentration of the cefoperazone sodium and sulbactam sodium is 1.2 mg/mL.
4. Culture of MenSCs
Adding the uniformly mixed cell suspension into a cell culture bottle, and marking related information on the culture bottle. Observing cell inoculation density under a mirror, and then placing the culture flask at 37 ℃ and CO 2 Culturing in an incubator with 6% volume concentration. After 48h, the solution is changed for the first time, the new mesenchymal stem cell culture medium is changed (the cells are P0 generation at the moment), and the solution is addedCefoperazone sodium and sulbactam sodium (1:1), the final concentration of cefoperazone sodium and sulbactam sodium is 1.2mg/mL, and then the liquid is changed every 3 d. Observing the cell fusion degree under a microscope, and performing digestion passage when the cell fusion degree reaches 70% to obtain P1 generation cells; continuously subculturing, and performing second digestion passage when the cell fusion degree reaches 90% to obtain P2 generation cells; continuously subculturing until the cells of P3 generation, and sucking out the culture solution except the cells to obtain the menstrual blood-derived mesenchymal stem cells growing adherently.
② observing the growth condition of the menstrual blood-derived mesenchymal stem cells growing adherently under an inverted microscope, adding new culture medium on the non-cell culture surface of the culture bottle, then placing the culture bottle at 37 ℃ and CO 2 Culturing in an incubator with the volume concentration of 6 percent, and subculturing according to the adherent number and the fusion degree of the primary cells and the fusion degree to be achieved of about 60-80 percent of the cell cloning condition.
And thirdly, sucking out the culture solution by using a pipette as much as possible until the culture solution is completely sucked, adding physiological saline into the non-cell culture surface, slightly shaking to wash the cells and pasting the cells on the wall surface, discarding and repeating for 2 times. Adding pancreatin, allowing pancreatin to uniformly infiltrate the cell and stick to the wall surface, incubating at 37 deg.C for 2min, adding normal saline to terminate the digestion process after the cell becomes round, blowing the cell and stick to the wall surface with a pipette, sucking out the cell suspension, and transferring to a centrifuge tube. Centrifuging the cell suspension at 1500rpm for 5min at room temperature, wherein the centrifuge setting parameters are as follows: 9 and 9 are increased and decreased, and 20 ℃ is increased and decreased. And after the centrifugation is finished, discarding the supernatant to obtain the mesenchymal stem cells of the menstrual blood source.
5. Preparation of autologous platelet-rich plasma
Collecting peripheral blood of a patient, carrying out anticoagulation treatment, and carrying out centrifugal treatment at 2500rpm for 10min, wherein the centrifuge setting parameters are as follows: 9 and 9 are increased and decreased, and 20 ℃ is increased and decreased. After centrifugation, the blood was divided into 3 layers: the upper layer is Platelet Poor Plasma (PPP), the middle layer is buffy coat layer, which is the concentrated platelet, and the lower layer is red blood cells. Sucking the upper layer, the middle layer and part of the red blood cells (the proportion of the red blood cells to the whole red blood cells is not higher than 25%) adjacent to the middle layer by using a pipette, transferring the red blood cells to a new centrifugal tube, and performing secondary centrifugation. The rotation speed of the second centrifugal treatment is 4100rpm, the time is 20min, and the setting parameters of the centrifugal machine are as follows: 9 and 9 are increased and decreased, and 20 ℃ is increased and decreased. And after the centrifugation is finished, dividing the liquid in the centrifuge tube into 3 layers, removing PPP on the upper layer part by using a straw, retaining the middle and lower layers, removing erythrocyte sediment, fully and uniformly mixing, and activating by adding thrombin and calcium chloride (10:1) to obtain the autologous platelet-rich plasma.
6. Preparation of stem cell gel preparation
Mixing the obtained mesenchymal stem cells with autologous platelet-rich plasma and medical chitosan, mixing well, and making into stem cell gel preparation, wherein the concentration of the mesenchymal stem cells is 0.8 × 10 7 Per mL, platelet concentration 1200X 10 9 The concentration of chitosan was 0.6 wt%. Sealing and packaging the prepared stem cell gel preparation in a medical disposable syringe.
Example 3
Preparation of Stem cell gel preparation
1. Environmental requirements
1) Isolation, culture and formulation preparation of blood-derived mesenchymal stem cell (MenSCs) seed cells were performed in class B clean rooms, and open operations were performed in class a environments (biosafety cabinets).
2) The preparation personnel carry out ultraviolet sterilization on the cell operation room and the biological safety cabinet in advance, the ultraviolet irradiation time is 30min, the ultraviolet lamps of the operation units related to the biological safety cabinet in the B-level clean room are started to irradiate for 30min, the ultraviolet lamps of the operation units are closed, then fresh air is introduced for 10min, and the operation units are kept in the running state.
3) All reagents, consumables, samples and the like which enter the laboratory must be transmitted after being subjected to disinfection and sterilization operations such as ultraviolet irradiation, alcohol wiping and disinfection and the like.
4) The preparation personnel need to carry out the dressing change according to the SOP standard of the personnel dressing change standard and then can enter the clothes.
2. Configuring mesenchymal stem cell culture medium
Collecting umbilical cord of healthy full-term newborn, peeling amnion and arteriovenous blood vessel, and tearing umbilical cord Wharton jelly; carrying out primary cell culture on the Wharton jelly by adopting a tissue block adherence method, and when the cell fusion degree reaches 30%, carrying out digestion and passage, and removing tissue blocks to obtain P1 generation cells; continuously subculturing, and performing second digestion and subculture when the cell fusion degree reaches 80% to obtain P2 generation cells; and (3) continuing subculturing, when the cell fusion degree reaches 80%, collecting culture supernatant, and mixing the collected supernatant with a mesenchymal stem cell serum-free culture medium produced by Beijing Mongolia stem cell technology Limited according to the ratio of 1: mixing the mixture in a volume ratio of 0.8-1.2 to obtain the mesenchymal stem cell culture medium. The formula of a culture medium used for primary cell culture and subculture is as follows: the serum-free culture medium is produced by the Beijing Mongolian stem cell technology limited company.
3. Preparation of mesenchymal stem cells of menstrual blood source
1) Menstrual blood sample treatment
Take out the menstrual blood preservative fluid and place room temperature balance 1h in, open biological safety cabinet ultraviolet irradiation 30min, will this experiment required consumptive material also ultraviolet irradiation disinfection simultaneously, open fan and lighting system after the completion, it is steady to wait for the air current, spreads into the menstrual blood sample into biological safety cabinet in, according to 1: adding the prepared preservation solution in a proportion of 0.8, fully and uniformly mixing, and preserving at 4 ℃ to obtain a treated sample (treated in 48 h). Wherein the preservation solution consists of normal saline, cefoperazone sodium and sulbactam sodium and amphotericin B; the concentration of the cefoperazone sodium and sulbactam sodium in the preservation solution is 3.8 mg/mL; the content of amphotericin B by volume was 1.8% of the volume of the preservation solution.
2) Preparation of patient peripheral blood plasma
Carrying out centrifugal treatment on the peripheral blood of the patient at 3500rpm for 10min, wherein the set parameters of the centrifugal machine are as follows: and (3) rising the temperature to 9 ℃ and reducing the temperature to 20 ℃, transferring the separated plasma into a new centrifugal tube, sealing the centrifugal tube for inactivation, and inactivating the plasma at 56 ℃ for 30 min. After inactivation, the plasma is centrifuged for 10min at 2000rpm, and the centrifuge setting parameters are as follows: the temperature is increased by 9 ℃ and decreased by 9 ℃ and 20 ℃, and the supernatant (the blood plasma of the peripheral blood of the patient) is taken for standby.
3) MenSCs separation
Taking out the reagents required by the experiment in advance, balancing the reagents at room temperature for 1h, opening the biological safety cabinet for ultraviolet irradiation for 30min, simultaneously performing ultraviolet irradiation disinfection on the consumables required by the experiment, opening a fan and an illumination system after the completion of the ultraviolet irradiation disinfection, waiting for the stable airflow, transferring the processed menstrual blood treatment sample into the biological safety cabinet, and transferring the menstrual blood treatment sample into a new centrifugal tube.
Secondly, according to the following steps of 1: 1.8 ratio the blood-treated sample was added to a Ficoll separation medium having a density of 1.077. Centrifuging at 2000rpm for 25min, and setting parameters of the centrifuge as follows: the temperature is increased by 9, decreased by 2 and decreased by 20 ℃. After the centrifugation is finished, the middle PBMC layer is sucked by a suction pipe, the sucked cells are transferred into a new centrifugal pipe, and the suspension is the menstrual blood mononuclear cells. Centrifuging the cell suspension at 1500rpm for 5min at room temperature, wherein the centrifuge setting parameters are as follows: 9 and 9 are increased and decreased, and 20 ℃ is increased and decreased. After centrifugation, the supernatant was discarded, and the cell pellet was washed and repeated three times.
Thirdly, after the washing is finished, resuspending the cell sediment by using the prepared mesenchymal stem cell culture medium, and adding 8 percent of inactivated patient peripheral blood plasma and cefoperazone sodium and sulbactam sodium (1:1) according to the volume ratio to obtain cell suspension, wherein the final concentration of the cefoperazone sodium and sulbactam sodium is 0.8 mg/mL.
4. Culture of MenSCs
Adding the uniformly mixed cell suspension into a cell culture bottle, and marking related information on the culture bottle. Observing cell inoculation density under the mirror, and then placing the culture bottle at 35 deg.C and CO 2 Culturing in an incubator with the volume concentration of 4 percent. After 48h, the solution is changed for the first time, the new mesenchymal stem cell culture medium is changed (the cells are P0 generation), and cefoperazone sodium and sulbactam sodium (1:1) are added, the final concentration of the cefoperazone sodium and sulbactam sodium is 0.8mg/mL, and then the solution is changed every 3 d. Observing the cell fusion degree under a microscope, and performing digestion passage when the cell fusion degree reaches 50% to obtain P1 generation cells; continuously subculturing, and performing second digestion passage when the cell fusion degree reaches 80% to obtain P2 generation cells; continuously subculturing until the cells of P3 generation, and sucking out the culture solution except the cells to obtain the menstrual blood-derived mesenchymal stem cells growing adherently.
② observing the growth condition of the menstrual blood-derived mesenchymal stem cells growing adherently under an inverted microscope, adding new culture medium on the non-cell culture surface of the culture bottle, then placing the culture bottle at the temperature of 35 ℃ and CO 2 Culturing in an incubator with a volume concentration of 4%Subculture can be carried out according to the adherent number and the fusion degree of primary cells and the fusion degree of cell cloning condition of about 60% -80%.
And thirdly, sucking the culture solution out by using a pipette as much as possible until the culture solution is completely sucked, adding physiological saline into a non-cell culture surface, slightly shaking to wash the cells to be attached to the wall surface, discarding and repeating for 2 times. Adding pancreatin, allowing pancreatin to uniformly infiltrate the cell and stick to the wall surface, incubating at 37 deg.C for 2min, adding normal saline to terminate the digestion process after the cell becomes round, blowing the cell and stick to the wall surface with a pipette, sucking out the cell suspension, and transferring to a centrifuge tube. Centrifuging the cell suspension at 1500rpm for 5min at room temperature, wherein the centrifuge setting parameters are as follows: 9 and 9 are increased and decreased, and 20 ℃ is increased and decreased. And after the centrifugation is finished, discarding the supernatant to obtain the mesenchymal stem cells of the menstrual blood source.
5. Preparation of autologous platelet-rich plasma
Collecting peripheral blood of a patient, carrying out anticoagulation treatment, and carrying out centrifugal treatment at 2500rpm for 10min, wherein the centrifuge setting parameters are as follows: 9 and 9 are increased and decreased, and 20 ℃ is increased and decreased. After centrifugation, the blood was divided into 3 layers: the upper layer is Platelet Poor Plasma (PPP), the middle layer is buffy coat layer, which is the concentrated platelet, and the lower layer is red blood cells. Sucking the upper layer, the middle layer and part of the red blood cells (the proportion of the red blood cells to the whole red blood cells is not higher than 25%) adjacent to the middle layer by using a pipette, transferring the red blood cells to a new centrifugal tube, and performing secondary centrifugation. The rotation speed of the second centrifugal treatment is 4100rpm, the time is 20min, and the setting parameters of the centrifugal machine are as follows: 9 and 9 are increased and decreased, and 20 ℃ is increased and decreased. And after the centrifugation is finished, dividing the liquid in the centrifuge tube into 3 layers, removing PPP on the upper layer part by using a straw, retaining the middle and lower layers, removing erythrocyte sediment, fully and uniformly mixing, and activating by adding thrombin and calcium chloride (10:1) to obtain the autologous platelet-rich plasma.
6. Preparation of stem cell gel preparation
Mixing the obtained mesenchymal stem cells with autologous platelet-rich plasma and medical chitosan, mixing well, and making into stem cell gel preparation, wherein the concentration of the mesenchymal stem cells is 0.5 × 10 7 Per mL, platelet concentration of 600X 10 9 The concentration of chitosan was 0.4 wt%. Sealing and packaging the prepared stem cell gel preparation in medical useA disposable syringe.
The stem cell gel preparations prepared in examples 2 and 3 were tested to have substantially the same effects and effects in promoting endometrial repair as the stem cell gel preparation prepared in example 1.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. A stem cell gel formulation, comprising: comprises mesenchymal stem cells of blood source, autologous platelet-rich plasma and chitosan.
2. The stem cell gel formulation of claim 1, wherein: the cell concentration of the mesenchymal stem cells of menstrual blood source in the stem cell gel preparation is (0.5-1) x 10 7 Per mL;
and/or the concentration of platelets in the stem cell gel preparation is (600- 9 /L；
And/or the concentration of the chitosan in the stem cell gel preparation is 0.4 wt% to 0.6 wt%.
3. The stem cell gel formulation of claim 1, wherein: the preparation method of the autologous menstrual blood-derived mesenchymal stem cells comprises the following steps:
a. mixing menstrual blood with preservation solution, adding into Ficoll separating medium, centrifuging, removing supernatant to obtain menstrual blood mononuclear cells, and re-suspending with mesenchymal stem cell culture medium to obtain cell suspension;
b. adding the inactivated peripheral blood plasma of the patient and the cefoperazone sodium and sulbactam sodium into the cell suspension to obtain a menses source mesenchymal stem cell culture solution, and placing the culture solution at the temperature of 35-37 ℃ and CO 2 Culturing under the condition that the volume concentration is 4% -6%, and performing digestion passage when the cell fusion degree reaches 50% -70% to obtain P1 generation cells; continuously subculturing, and fusing cellsCarrying out second digestion passage when the degree of contact reaches 80-90% to obtain P2 generation cells; continuously subculturing until the cells of P3 generation are obtained, and sucking out culture solution except the cells to obtain menstrual blood-derived mesenchymal stem cells growing adherently;
c. adding pancreatin into the blood-derived mesenchymal stem cells which grow adherently for digestion, stopping digestion after the blood-derived mesenchymal stem cells become round, centrifuging, and removing supernatant to obtain the blood-derived mesenchymal stem cells.
4. A stem cell gel formulation according to claim 3, wherein: in the step a, the preservation solution consists of normal saline, cefoperazone sodium and sulbactam sodium and amphotericin B; the concentration of the cefoperazone sodium and sulbactam sodium in the preservation solution is 3.8-4.2 mg/mL; the volume content of the amphotericin B is 1.8-2.2% of the volume of the preservation solution;
and/or in the step a, the mixing volume of the menstrual blood and the preservation solution is 1: 0.8-1.2;
and/or in the step a, the density of the Ficoll separating medium is 1.07-1.08g/mL, and the volume of the Ficoll separating medium is 1.8-2.2 times of the menstrual blood volume;
and/or in the step a, the preparation method of the mesenchymal stem cell culture medium comprises the following steps: collecting umbilical cord of healthy full-term newborn, peeling amnion and arteriovenous blood vessel, and tearing umbilical cord Wharton jelly; carrying out primary cell culture on the Wharton jelly by adopting a tissue block adherence method, and when the cell fusion degree reaches 30-50%, carrying out digestion passage and removing tissue blocks to obtain P1 generation cells; continuously subculturing, and performing second digestion and passage when the cell fusion degree reaches 80-90% to obtain P2 generation cells; and (3) continuing subculturing, when the cell fusion degree reaches 80-90%, collecting culture supernatant, and mixing the collected supernatant with the mesenchymal stem cell serum-free culture medium according to the ratio of 1: mixing the mixture in a volume ratio of 0.8-1.2 to obtain the mesenchymal stem cell culture medium;
and/or in step a, the concentration of the mesenchymal stem cells of the blood source in the mesenchymal stem cell suspension of the blood source is (1-3) multiplied by 10 6 one/mL.
5. A stem cell gel formulation according to claim 3, wherein: in the step b, the addition amount of the peripheral blood plasma of the patient is 8-12% of the volume of the menstrual blood-derived mesenchymal stem cell suspension;
and/or in the step b, the concentration of the cefoperazone sodium and sulbactam sodium in the menstruum-derived mesenchymal stem cell culture solution is 0.8mg/mL-1.2 mg/mL.
6. A stem cell gel formulation according to claim 3, wherein: in the step c, the digestion is terminated by adding physiological saline to the rounded mesenchymal stem cells.
7. The stem cell gel formulation of claim 1, wherein: the preparation method of the autologous platelet-rich plasma comprises the following steps:
collecting peripheral blood of a patient, performing anticoagulation treatment, centrifuging to separate the peripheral blood into an upper layer, a middle layer and a lower layer, sucking the upper layer and the middle layer, performing secondary centrifugation, removing an upper layer separation solution, adding thrombin and 8-10 wt.% calcium chloride solution into the rest part, and activating to obtain the autologous platelet-rich plasma; the volume percentage content of the calcium chloride solution in the autologous platelet-rich plasma is 8-12%, and the content of thrombin is 15-25U/mL.
8. The method for producing a stem cell gel preparation according to any one of claims 1 to 7, characterized in that: the method comprises the following steps: and (3) resuspending the mesenchymal stem cells from blood source by using the autologous platelet-rich plasma to obtain a cell suspension, adding chitosan into the cell suspension, and uniformly mixing to obtain the stem cell gel preparation.
9. Use of a stem cell gel formulation according to any one of claims 1 to 7 as a medicament for the prevention and treatment of endometrial damage.
10. Use of a stem cell gel formulation according to any one of claims 1 to 7 as a medicament for the treatment of severe intrauterine adhesions.
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| JP2021107385A (en) * | 2019-12-27 | 2021-07-29 | 医療法人社団暁慶会 | Pharmaceutical composition containing culture supernatant of menstrual blood-derived stem cell |
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| CN110693911A (en) * | 2019-10-30 | 2020-01-17 | 南通大学 | Menstrual blood-derived endometrial stem cell preparation and preparation method and application thereof |
| JP2021107385A (en) * | 2019-12-27 | 2021-07-29 | 医療法人社団暁慶会 | Pharmaceutical composition containing culture supernatant of menstrual blood-derived stem cell |
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