CN112043727A

CN112043727A - Preparation method and application of cell composite endometrium repair gel

Info

Publication number: CN112043727A
Application number: CN202010979123.XA
Authority: CN
Inventors: 傅松涛
Original assignee: Shanxi Bdsc Biology Technology Co ltd
Current assignee: Shanxi Bdsc Biology Technology Co ltd
Priority date: 2020-09-17
Filing date: 2020-09-17
Publication date: 2020-12-08

Abstract

The invention discloses a preparation method of a cell composite endometrium repair gel, which comprises the following steps: step one, extracting and culturing endometrial stem cells to obtain endometrial stem cells; step two, extracting and culturing the mesenchymal stem cells to obtain the mesenchymal stem cells; step three, preparing temperature-sensitive chitosan hydrogel; step four, preparing the cell composite endometrium repair gel. The invention also discloses application of the cell composite endometrium repair gel in treating intrauterine adhesion. The invention has the following beneficial effects: 1. autologous stem cell transplantation, with substantially no rejection risk; 2. the autologous endometrial stem cells and the mesenchymal stem cells are compositely transplanted, so that the regeneration of endometrium, small blood vessels and mesenchyme is accelerated, and the repair time of endometrium is obviously shortened; 3. the temperature-sensitive gel can be formed after local injection, not only can provide a three-dimensional retention space for stem cells, but also can prevent the adhesion of regenerated mucosa.

Description

Preparation method and application of cell composite endometrium repair gel

Technical Field

The invention relates to the field of biomedicine, in particular to a preparation method and application of a cell composite endometrium repair gel.

Background

According to the report of the literature, the incidence rate of intrauterine adhesion caused by artificial abortion and uterine curettage is as high as 25% -30%, and the incidence rate of postoperative re-adhesion is very high, especially severe intrauterine adhesion, and the postoperative re-adhesion rate is as high as 62.5%.

How to completely cure the intrauterine adhesion is a clinical troublesome problem. In recent years, scholars at home and abroad propose that the occurrence of intrauterine adhesion is related to endometrial stem cell injury, endometrial stem cell reduction or function damage are caused by endometrial injury, endometrium cannot be completely regenerated, endometrial scar repair is caused, and finally intrauterine adhesion occurs.

The endometrial stem cells as adult stem cells have the characteristics of easy separation and expansion, stronger colony forming capability and fewer technical problems of extraction, and have huge autologous treatment potential. The umbilical cord is medical waste, the mesenchymal stem cells of the umbilical cord have strong proliferation and differentiation capacity, the immunogenicity is low, and the umbilical cord has the effects of improving and conditioning local microenvironment.

The existing treatment means for intrauterine adhesion generally has the problems of poor clinical treatment effect, rejection in the existing cell transplantation, insufficient cell repair time, postoperative re-adhesion and the like. Therefore, the invention discloses a cell composite endometrium repair gel which can well solve the problems.

Disclosure of Invention

The purpose of the invention is as follows: the invention is improved aiming at the defects of the prior art, namely, the first purpose of the invention is to disclose a preparation method of a cell composite type endometrium repair gel. The second purpose of the invention is to disclose the application of the cell composite type endometrium repair gel. The invention uses the chitosan hydrogel compounded with the endometrial stem cells and the umbilical cord mesenchymal stem cells to carry out the local transplantation of the uterus, thereby increasing the retention time of the cells in the uterine cavity and promoting the adherence of the cells to create conditions for promoting the repair of the endometrium.

The technical scheme is as follows: a preparation method of a cell composite endometrium repair gel comprises the following steps:

step one, extracting and culturing endometrial stem cells to obtain endometrial stem cells;

step two, extracting and culturing the mesenchymal stem cells to obtain the mesenchymal stem cells;

step three, preparing temperature-sensitive chitosan hydrogel;

step four, preparing the cell composite endometrium repair gel.

Further, the first step comprises the following steps:

(1) taking endometrium tissue under aseptic condition, and using Ca-free²⁺And Mg²⁺Is washed at least three times by PBS buffer solution, then transferred into a sterile culture dish, and is cut to the volume of less than 1mm³Small pieces of (2);

(2) adding 0.1 v/v% collagenase aqueous solution with the volume of 1.5-3 times that of the sterile culture dish in the step (1), carrying out water bath oscillation digestion for 30-80 min at 37 ℃, collecting digestion liquid in a centrifuge tube, adding DMEM/F12 complete culture medium into the centrifuge tube to stop digestion, then centrifuging for 10min at 1000rpm, discarding supernatant, and keeping cell precipitation;

(3) adding 4-8 times of volume of erythrocyte lysate into the centrifugal tube, continuously oscillating until the liquid in the centrifugal tube gradually turns red, adding 10ml of PBS buffer solution into the centrifugal tube, centrifuging at 1000rpm for 5min again, removing supernatant, and keeping cell precipitate;

(4) washing the cell precipitate with PBS buffer solution for at least 2 times, discarding the supernatant, and retaining the cell precipitate;

(5) suspending the cell pellet remaining in step (4) with 4 times volume of DMEM/F12 complete medium to prepare a cell suspension;

(6) sequentially sieving the cell suspension through 200-mesh and 400-mesh screens, flushing the cell screens by using a DMEM/F12 complete culture medium, collecting the cell suspension below 400 meshes, centrifuging at 1000rpm for 5min, discarding supernatant, and retaining cell precipitates;

(7) the step of(6) The remaining cell pellet was suspended in DMEM/F12 complete medium and transferred to a cell culture flask at 37 ℃ with 5 v/v% CO₂Culturing in an incubator, performing half-amount liquid change by using a DMEM/F12 complete culture medium for the first time after culturing for 24-48h, performing full-amount liquid change by using a DMEM/F12 complete culture medium every 2-3 days later, discarding non-adherent cells, adding a 0.25% pancreatin aqueous solution containing 0.02% of EDTA for passage when primary cells grow and fuse to 80% -90%, and performing passage by using 6 x 10 EDTA³Individual cell/cm²Subculturing the endometrium to the density of more than three generations to obtain the endometrium stem cells, wherein:

the DMEM/F12 complete medium in the steps (2), (5), (6) and (7) contains 10 v/v% FBS and 1 v/v% streptomycin, and the balance is DMEM/F12.

Furthermore, the endometrial stem cells obtained in the first step are identified and qualified to be used as a raw material for preparing the cell composite type endometrial repair gel, and the identification of the endometrial stem cells comprises identification of a surface marker of the endometrial stem cells, identification of osteogenic differentiation and identification of adipogenic differentiation, wherein:

(a) the identification of the endometrial stem cell surface marker comprises the following steps:

(a1) taking the 3 rd generation endometrial stem cells with the fusion degree of 90% -95% obtained in the step (7), discarding the culture medium, adding PBS buffer solution for washing twice, adding 0.25% pancreatin containing 0.02% EDTA for digestion, and adding an isovolumetric DMEM/F12 complete culture medium to stop digestion when cells mostly become round and float, so as to obtain cell suspension;

(a2) transferring the cell suspension obtained in the step (a1) into a centrifuge tube, centrifuging for 5min at 1000rpm, discarding the supernatant, and keeping the cell precipitate;

(a3) washing the cell sediment obtained in the step (a2) for 2 times by using a PBS buffer solution, centrifuging for 5min at 1000rpm, removing supernatant, and keeping the cell sediment;

(a4) resuspending the cell pellet obtained in step (a3) with PBS buffer to obtain cell suspension, and counting;

(a5)、adjusting the cell concentration of the cell suspension obtained in step (a5) to 2X 10⁶Taking 5 flow tubes, adding 100ul of cell suspension into each tube, blowing, beating and uniformly mixing;

(a6) respectively adding 10ul primary antibodies with FITC or PE fluorescent labels into the 5 flow tubes, wherein the primary antibodies are respectively FITC isotype antibodies, PE isotype antibodies, CD29 antibodies, CD73 antibodies, CD90 antibodies, CD34 antibodies, CD45 antibodies and CD133 antibodies, and incubating for 20-30min at room temperature in a dark condition;

(a7) after incubation, adding 100ul PBS buffer solution into each tube, gently blowing and beating the buffer solution to obtain a single cell suspension, detecting the fluorescence intensity of a surface marker of the single cell suspension by using a flow cytometer, wherein endometrial stem cells show positive CD29 antibody, CD73 antibody and CD90 antibody, and negative CD34 antibody, CD45 antibody and CD133 antibody;

(b) identification of differentiation ability of endometrium stem cell induced osteogenesis

(b1) Taking the 3 rd generation endometrial stem cells with the fusion degree of 90% -95% obtained in the step (7), discarding the culture medium, adding PBS buffer solution for washing twice, adding 0.25% pancreatin containing 0.02% EDTA for digestion, and adding an isovolumetric DMEM/F12 complete culture medium to stop digestion when cells mostly become round and float, so as to obtain cell suspension;

(b2) transferring the cell suspension obtained in the step (b1) into a centrifuge tube, centrifuging for 5min at 1000rpm, discarding the supernatant, and keeping the cell precipitate;

(b3) washing the cell sediment obtained in the step (b2) for 2 times by using a PBS buffer solution, centrifuging for 5min at 1000rpm, removing supernatant, and keeping the cell sediment;

(b4) resuspending the cell pellet obtained in step (b3) with PBS buffer to obtain a cell suspension and counting,

(b5) adjusting the cell concentration of the cell suspension obtained in the step (b4) to 2X 10⁶Adding 100ul of cell suspension into each 6-well plate paved with gelatin, and supplementing 2ml of DMEM/F12 complete culture medium for cell culture;

(b6) when the cell fusion degree is 60-70%, replacing the medium with an adult adipose-derived mesenchymal stem cell osteogenic induction differentiation medium containing 10% FBS and preheated to 37 ℃, replacing the medium every 3 days, and inducing for 2-4 weeks;

(b7) after induction, removing the culture medium, washing for 2 times by using PBS buffer solution, fixing for 30min by using 4% paraformaldehyde, absorbing the paraformaldehyde solution, and eluting the fixing solution for 5min for 2 times by using the PBS buffer solution;

(b8) adding 1ml alizarin red working solution into each hole, dyeing for 5min at room temperature, eluting the dyeing solution by using PBS buffer solution, and observing osteogenic dyeing conditions under a microscope, wherein the osteogenic differentiated endometrial stem cells present red mineralized nodules with different depths; wherein:

the adult adipose-derived mesenchymal stem cell osteogenic induced differentiation culture medium comprises: 175ml of adult adipose-derived stromal cell osteogenesis induced differentiation basal medium, 20ml of adult adipose-derived stromal cell osteogenesis induced differentiation medium special serum, 2ml of diabase, 2ml of glutamine, 400ul of ascorbic acid, 2ml of beta-sodium glycerophosphate and 20ul of dexamethasone;

(c) identification of endometrial stem cell adipogenic differentiation

(c1) Taking 3 rd generation endometrial stem cells with the fusion degree of 90% -95% obtained in the step (7), removing the culture medium, adding PBS buffer solution for washing twice, adding 0.25% pancreatin containing 0.02% EDTA for digestion, and adding an isovolumetric DMEM/F12 complete culture medium to stop digestion when cells mostly become round and float, so as to obtain cell suspension;

(c2) transferring the cell suspension obtained in the step (c1) into a centrifuge tube, centrifuging for 5min at 1000rpm, discarding the supernatant, and keeping the cell precipitate;

(c3) washing the cell sediment obtained in the step (c2) for 2 times by using a PBS buffer solution, centrifuging for 5min at 1000rpm, removing supernatant, and keeping the cell sediment;

(c4) resuspending the cell pellet obtained in step (c3) with PBS buffer to obtain cell suspension and counting;

(c5) adjusting the cell concentration of the cell suspension obtained in the step (c4) to 2X 10⁶Adding 100ul of cell suspension into each 6-well plate paved with gelatin, and supplementing 2ml of DMEM/F12 complete culture medium for cell culture;

(c6) when the cells grow to be completely fused or supersaturated, replacing the culture medium with an adult adipogenic mesenchymal stem cell adipogenic induced differentiation culture medium A containing 10% of FBS, changing the culture medium into an adipogenic induced differentiation culture medium B after three days, sucking the adipogenic induced differentiation culture medium B after 24 hours, and replacing the adipogenic mesenchymal stem cell adipogenic induced differentiation culture medium A containing 10% of FBS with the adipogenic induced differentiation culture medium B for induction;

(c7) after the adult adipose-derived mesenchymal stem cell adipogenic induced differentiation medium A containing 10% of FBS and the adipogenic induced differentiation medium B are alternately used for 5 times, the adult adipose-derived mesenchymal stem cell adipogenic induced differentiation medium A and the adipogenic induced differentiation medium B are cultured for 7 days by using the simple liquid B, and half of the liquid is changed every 2 to 3 days;

(c8) removing the culture medium after induction, washing with PBS buffer solution, washing for 2 times, fixing with 4% paraformaldehyde for 30min, removing paraformaldehyde solution, and eluting the fixing solution with PBS buffer solution for 2 times, each time for 5 min;

(c9) oil red O solution: distilled water 3: 2 diluting the mixed solution into a working solution, adding 1ml of oil red O working solution into each hole, dyeing for 30min at room temperature, eluting the dyeing solution by using PBS (phosphate buffer solution), observing the condition of lipogenic dyeing under a microscope, and enabling most orange red fat drops to appear in lipogenic differentiated endometrial stem cells; wherein:

adult adipose-derived mesenchymal stem cell adipogenic induction differentiation medium containing 10% FBS a: 175ml of A liquid basal medium of adult adipose-derived stromal cell adipogenic induction differentiation medium, 20ml of fetal bovine serum for adult adipose-derived stromal cell adipogenic induction differentiation, 2ml of double antibody, 2ml of glutamine, 400ul of insulin, 200ul of 3-isobutyl-1-methylxanthine, 200ul of rosiglitazone and 200ul of dexamethasone; adipogenic induction differentiation medium B: adult adipose-derived stromal cell adipogenic induction differentiation medium B liquid basic medium 175ml, adult adipose-derived stromal cell adipogenic induction differentiation special fetal bovine serum 20ml, double antibody 2ml, glutamine 2ml, insulin 400 ul.

Further, the mesenchymal stem cells in the second step are umbilical cord mesenchymal stem cells, and the second step comprises the following steps:

(1) taking a healthy umbilical cord specimen by 10-20cm under a sterile condition, and placing the umbilical cord specimen in a PBS buffer solution containing 1 v/v% of double antibody;

(2) transferring the umbilical cord specimen obtained in the step (1) to a super clean bench, and repeatedly washing the umbilical cord specimen with PBS (phosphate buffer solution) to remove residual blood;

(3) after the adventitia and the umbilical cord arteriovenous vessels of the umbilical cord specimen are stripped in a culture dish, the residual tissue blocks are sheared into pieces with the volume of less than 1mm³；

(4) Adding DMEM/F12 complete medium into the culture dish, and placing the culture dish at 37 ℃ and 5 v/v% CO₂Culturing in a carbon dioxide incubator with saturated humidity, performing half-amount liquid change after 4 days, performing liquid change once every 3 days after cell climbing out, transferring to the 3 rd generation, digesting cells by using 0.25 wt% of pancreatin aqueous solution when the umbilical cord mesenchymal stem cells of the 3 rd generation are 80-90% fused, stopping digestion by using EMEM/F12 complete culture medium, slightly blowing the bottom of the culture dish and collecting the cells, centrifuging at 1000rpm for 5 minutes, discarding supernatant, and keeping cell precipitation;

(5) and adding 1ml of DMEM/F12 complete culture medium into the cell sediment, re-suspending the cell sediment, and counting the cells to obtain the umbilical cord mesenchymal stem cells.

Further, the passage method of umbilical cord mesenchymal stem cells in the step (4) comprises the following steps: when the umbilical cord mesenchymal stem cells reach 80-90% fusion, digesting the cells by using a 0.25% pancreatin water solution containing 0.02% EDTA, stopping digestion by using a DMEM/F12 complete culture medium, slightly blowing the bottom of a culture dish, collecting the cells, centrifuging at 1000rpm for 5min, and performing the following steps of 1: passage at a ratio of 2-3.

Further, the mesenchymal stem cells in the second step are adipose mesenchymal stem cells, and the second step comprises the following steps:

s21, under aseptic condition, taking adipose tissue, removing vascular fascia, washing with PBS, removing erythrocytes, shearing into pieces with volume less than 1mm³Then placing the mixture into a 50ml centrifuge tube, adding 0.1 wt% type I collagenase with the volume 5-10 times of that of the mixture, and oscillating the mixture for 40-80min in a water bath kettle with the temperature of 37 ℃ and the rpm of 100;

s22, adding an equal volume of HG-DMEM complete culture medium into the centrifuge tube to terminate digestion, centrifuging for 10min at 4 ℃ and 1500rpm, removing supernatant and upper fat, and leaving cell sediment, wherein:

the HG-DMEM complete culture medium comprises 15% v/vFBS and 1 v/v% streptomycin, and the balance is HG-DMEM;

s23, adding 10-15ml HG-DMEM complete culture medium into the centrifuge tube, fully suspending, filtering with a 200-mesh filter screen, reserving filtrate, centrifuging at 4 ℃ and 1500rpm for 10min, and removing supernatant to obtain cell sediment;

s24, adding 5ml of HG-DMEM complete culture medium into the centrifugal tube, and fully suspending to obtain a cell suspension;

s25, inoculating the cell suspension into a T25 cell culture bottle, placing the bottle at 37 ℃ and 5 v/v% CO₂And after culturing for 48-72h in a saturated humidity incubator, carrying out half-amount liquid exchange for the first time, removing non-adherent cells, then carrying out liquid exchange once every 2-3d, transferring to the 3 rd generation, digesting the cells by using 0.25% pancreatin water solution containing 0.02% EDTA when the 3 rd generation adipose-derived mesenchymal stem cells reach 80% -90%, stopping digestion by using an HG-DMEM complete culture medium, slightly blowing to beat the bottom of a bottle and collecting the cells, centrifuging at 1000rpm for 5min, discarding supernatant, adding 1ml of HG-DMEM complete culture medium into cell precipitates, re-suspending the cell precipitates, counting the cell precipitates, and obtaining the adipose-derived mesenchymal stem cells of the 3 rd generation after the completion.

Further, the passage method of the adipose-derived mesenchymal stem cells comprises the following steps: when the adipose-derived mesenchymal stem cells reach 80% -90% fusion, digesting the cells by using a 0.25% pancreatin water solution containing 0.02% EDTA, stopping digestion by using an HG-DMEM complete culture medium, slightly blowing the bottom of a bottle and collecting the cells, centrifuging at 1000rpm for 5min, and mixing the cells according to the ratio of 1: passage at a ratio of 2-3.

Further, the mesenchymal stem cells in the second step are bone marrow mesenchymal stem cells, and the second step comprises the following steps:

step21, taking out the femur and the tibia under the aseptic condition, cutting from the middle of the femoral shaft and the tibia shaft, and repeatedly flushing the marrow cavity by using DMEM/F12 complete culture medium to obtain a flushing liquid, wherein:

DMEM/F12 complete medium comprising 10 v/v% FBS and 1 v/v% streptomycin with the balance being DMEM/F12;

step22, filtering the flushing liquid by using a 200-mesh filter screen, taking a filtrate, then placing the filtrate in a centrifuge tube, centrifuging for 5min at 1000rpm, and removing a supernatant to obtain a cell precipitate;

step23, adding 5ml DMEM/F12 into the centrifuge tube completelyCulturing, suspending to obtain cell suspension, inoculating into T25 cell culture flask, and placing at 37 deg.C and 5 v/v% CO₂Culturing in a saturated humidity incubator, changing liquid for the first time after 24-48h, removing non-adherent cells, changing liquid once every 2-3d, transferring to the 3 rd generation, digesting the cells by using 0.25% pancreatin solution cells containing 0.02% EDTA when the 3 rd generation mesenchymal stem cells are fused to 80% -90%, stopping digestion by using a DMEM/F12 complete culture medium, slightly blowing to beat the bottom of a bottle, collecting a wash bar, and centrifuging at 1000rpm for 5 min; and (3) removing the supernatant, adding 1ml of DMEM/F12 complete culture medium into the cell sediment, fully suspending, counting, and obtaining the 3 rd generation mesenchymal stem cells.

Further, the passage method of the bone marrow mesenchymal stem cells comprises the following steps: when the bone marrow mesenchymal stem cells reach 80% -90% fusion, digesting the cells by using a 0.25% pancreatin water solution containing 0.02% EDTA, stopping digestion by using an HG-DMEM complete culture medium, slightly blowing to beat the bottom of a bottle and collecting the cells, centrifuging at 1000rpm for 5min, and mixing the cells according to the ratio of 1: passage at a ratio of 2-3.

Further, the mesenchymal stem cells in the second step are placenta mesenchymal stem cells, and the second step comprises the following steps:

step21, taking placenta lobules close to the umbilical cord part under an aseptic condition, stripping amnion and decidua basalis parts, taking placenta fetal surface tissues, repeatedly washing with PBS until the liquid is clear, and shearing with ophthalmic scissors until the volume is less than 1mm³Then placing the mixture into a 50ml centrifuge tube;

step22, adding 0.1 wt% collagenase aqueous solution with the volume 2-4 times that of the centrifuge tube, oscillating at 37 ℃ and 100rpm for 20-50min, adding DMEM/F12 complete culture medium with the same volume, and sieving with a 200-mesh filter screen to obtain cell sediment;

step23, adding 5ml of DMEM/F12 complete culture medium into the centrifuge tube for fully suspending to obtain a cell suspension, inoculating the cell suspension into a T25 cell culture bottle, placing the cell suspension in a5 v/v% CO culture bottle at 37 DEG C₂Culturing in a saturated humidity incubator, changing liquid for the first time after 24-48h, removing non-adherent cells, changing liquid every 2-3d, transferring to 3 rd generation, and adding 0.25% pancreatin water containing 0.02% EDTA when the 3 rd generation placenta mesenchymal stem cells reach 80-90% fusionDigesting cells by using a solution, stopping digestion by using a DMEM/F12 complete culture medium, slightly blowing the bottom of a bottle and collecting the cells, centrifuging for 5min at 1000rpm, discarding the supernatant, adding 1ml of DMEM/F12 complete culture medium into cell precipitates, fully suspending to obtain cell suspension, counting the cell suspension, and obtaining 3 rd generation of placenta mesenchymal stem cells after the cell suspension is completed, wherein:

DMEM/F12 complete medium included 10 v/v% FBS and 1 v/v% streptomycin, with the balance being DMEM/F12.

Further, the passage method of the placenta mesenchymal stem cells comprises the following steps: when the placenta mesenchymal stem cells reach 80-90% fusion, digesting the cells by using a 0.25% pancreatin water solution containing 0.02% EDTA, stopping digestion by using an HG-DMEM complete culture medium, slightly blowing the bottom of a bottle and collecting the cells, centrifuging at 1000rpm for 5min, and mixing the cells according to the weight ratio of 1: passage at a ratio of 2-3.

Further, the third step comprises the following steps:

(31) preparation of chitosan solution

Weighing 0.2-0.3g of chitosan powder with deacetylation degree greater than 90%, adding into a sterile beaker containing 10ml of dilute acetic acid aqueous solution with concentration of 0.1M, and stirring with a stirrer to make the chitosan solution clear and transparent for later use;

(32) preparation of type I collagen solution

Weighing 25-50mg of type I collagen powder, adding the type I collagen powder into a sterile beaker filled with 10ml of 0.02M dilute acetic acid aqueous solution, and stirring the type I collagen powder by using a clean glass rod until the type I collagen powder is completely dissolved to obtain a type I collagen solution;

(33) preparation of beta-sodium glycerophosphate solution

Weighing 0.4-0.6g of beta-sodium glycerophosphate powder, adding the beta-sodium glycerophosphate powder into a 5ml sterile beaker filled with 1ml of distilled water, and stirring the mixture by using a clean glass rod until the beta-sodium glycerophosphate powder is completely dissolved to obtain a beta-sodium glycerophosphate solution;

(34) preparation of sodium alginate solution

Weighing 0.15-0.25g of sodium alginate powder, then adding the sodium alginate powder into a sterile beaker filled with 10ml of distilled water, and stirring the mixture in a stirrer to ensure that the sodium alginate solution becomes clear and transparent for later use;

(35) preparing temperature-sensitive chitosan hydrogel

(351) Respectively weighing 4ml of the chitosan solution obtained in the step (31) and 2ml of the type I collagen solution obtained in the step (32), adding the chitosan solution and the type I collagen solution into a sterile beaker, uniformly stirring the mixture on ice to obtain a mixed solution, and then performing the step (352);

(352) slowly adding the beta-sodium glycerophosphate solution obtained in the step (33) into the mixed solution obtained in the step (351) on ice to form a mixed solution, and then entering the step (353);

(353) adding 3ml of the sodium alginate solution obtained in the step (34) into the mixed solution obtained in the step (352), uniformly stirring to form a mixed solution, and entering the step (354);

(354) and (3) adding the mixed solution obtained in the step (353) into an EP (EP) tube, and immediately putting the mixed solution into a constant-temperature incubator at 37 ℃ for culturing for 8-10min to obtain the temperature-sensitive chitosan hydrogel.

Further, the fourth step includes the steps of:

(41) further diluting the endometrial stem cells prepared in the step one by using a DMEM/F12 complete culture medium, wherein the cell concentration of the diluted endometrial stem cells is 2 x 10⁴～2×10⁶Per ml;

(42) further diluting the mesenchymal stem cells prepared in the step two by using a DMEM/F12 complete culture medium, wherein the cell concentration of the diluted mesenchymal stem cell suspension is 1 multiplied by 10⁵～1×10⁷Per ml;

(43) and (3) mixing the diluted endometrial stem cell suspension obtained in the step (41) with the diluted mesenchymal stem cell suspension obtained in the step (42) according to the volume ratio of 2: 1 to form endometrium stem cell and mesenchymal stem cell suspension, then cooling to 18-20 ℃, and fully mixing the endometrium stem cell and mesenchymal stem cell suspension and the temperature-sensitive chitosan hydrogel prepared in the third step in an ice bath container to obtain the cell composite endometrium repair gel.

The cell composite type endometrium repair gel is applied to treating intrauterine adhesion.

Furthermore, after the cell composite type endometrium repair gel is transplanted locally in the uterine cavity, the cell composite type endometrium repair gel is rapidly aggregated in the body temperature environment of 37 ℃, so that the cell can stay in the uterine cavity and be attached to the endometrium, and the endometrium is repaired.

Chitosan is a natural cationic polysaccharide with good biocompatibility and is a biological material with wide application. The chitosan solution is liquid at room temperature, can wrap active cells and proteins, is mixed with beta-sodium glycerophosphate, is injected into a body, and can form biodegradable gel in situ at the injection site under the condition of body temperature. Type I collagen is an extracellular matrix component that allows cells to better survive. The strength of the whole hydrogel system is improved by adding the sodium alginate. The system is used as a carrier for wrapping active cells, and is successfully applied to the delivery of bioactive growth factors and tissue engineering in a living body. Therefore, the invention uses the endometrial stem cells and umbilical cord mesenchymal stem cells as cell sources, uses the chitosan hydrogel as a cell wrapping material and a 3D bracket, and provides possibility for repairing endometrium by cells through artificial compounding and temperature-sensitive gel polymerization.

The endometrial stem cells as adult stem cells have the advantages of easy separation and expansion, stronger colony forming capability and fewer technical problems of extraction, are taken from the endometrium of a patient, have extremely low immune rejection response, and can be better planted on the surface of the endometrium, thereby playing the function of repairing damaged endometrium. Therefore, the present invention adopts endometrial stem cells as seed cells for endometrial repair.

The mesenchymal stem cells are adult stem cells with multidirectional differentiation potential, and can be induced to differentiate into adipogenic cells, osteoblastic cells, cardiac muscle cells, islet-like cells and the like. The umbilical cord mesenchymal stem cells, the adipose mesenchymal stem cells, the bone marrow mesenchymal stem cells and the placenta mesenchymal stem cells can be used as seed cells due to the advantages of easy acquisition, strong multiplication capacity, no ethical relation and the like. Research proves that the umbilical cord mesenchymal stem cells, the bone marrow mesenchymal stem cells and the placenta mesenchymal stem cells do not express cell surface markers CD80, CD86, CD40 and CD40L related to transplant rejection, and the umbilical cord mesenchymal stem cells are low in immunogenicity and do not generate immune rejection reaction after cell transplantation. Moreover, umbilical cord mesenchymal stem cells can regulate microenvironment, and contribute to the colonization and play of endometrial stem cells. The source of the placenta mesenchymal stem cells is placenta which is medical waste after delivery, the placenta is large in size and rich in stem cells, the placenta stem cells extracted from the placenta are waste utilization, immunogenicity is low, immune rejection is small, and the placenta mesenchymal stem cells are suitable for transplantation. The adipose-derived stem cells can be derived from adipose tissues of a patient, fat is obtained through operations such as liposuction and the like, the adipose-derived stem cells belong to autologous cell in-vitro amplification culture, and the immune rejection reaction is extremely low. The bone marrow mesenchymal stem cells can be derived from the blood of a patient or umbilical cord blood, and have low immunogenicity, thereby being beneficial to the field planting of the transplanted cells.

In order to solve the problems of immunological rejection reaction, too short cell retention time, poor microenvironment and the like in cell transplantation in the endometrial repair process, the chitosan temperature-sensitive hydrogel compounded with the umbilical cord mesenchymal stem cells and the endometrial stem cells is selected, and the cell compound type hydrogel is agglutinated in the body temperature environment and is used for repairing the damaged endometrium.

Has the advantages that: the preparation method and the application of the cell composite endometrium repair gel disclosed by the invention have the following beneficial effects:

1. autologous stem cell transplantation, with substantially no rejection risk;

2. the autologous endometrial stem cells and the mesenchymal stem cells are compositely transplanted, so that the regeneration of endometrium, small blood vessels and mesenchyme is accelerated, and the repair time of endometrium is obviously shortened;

3. the temperature-sensitive gel can be formed after local injection, not only can provide a three-dimensional retention space for stem cells, but also can prevent the adhesion of regenerated mucosa.

The specific implementation mode is as follows:

the following describes in detail specific embodiments of the present invention.

Detailed description of the preferred embodiment 1

A preparation method of a cell composite endometrium repair gel comprises the following steps:

step three, preparing temperature-sensitive chitosan hydrogel;

step four, preparing the cell composite endometrium repair gel.

Further, the first step comprises the following steps:

(2) adding a 2-time volume of 0.1 v/v% collagenase aqueous solution into the sterile culture dish in the step (1), carrying out water bath shaking digestion for 50min at 37 ℃, collecting a digestion solution into a centrifuge tube, adding a DMEM/F12 complete culture medium into the centrifuge tube to stop digestion, then centrifuging for 10min at 1000rpm, discarding a supernatant, and keeping a cell precipitate;

(3) adding 6 times volume of erythrocyte lysate into the centrifuge tube, continuously oscillating until the liquid in the centrifuge tube gradually becomes red, adding 10ml of PBS buffer solution into the centrifuge tube, centrifuging at 1000rpm for 5min again, discarding the supernatant, and keeping cell precipitate;

(7) adding DMEM/F12 to the cell pellet retained in step (6), suspending the cell pellet in a complete culture medium, transferring the cell pellet to a cell culture flask, and culturing the cell pellet at 37 ℃ and 5 v/v% CO₂CulturingCulturing in a box, performing half-amount liquid change with DMEM/F12 complete culture medium for the first time after culturing for 36h, performing full-amount liquid change with DMEM/F12 complete culture medium every 2.5 days, discarding non-adherent cells, adding 0.25% pancreatin water solution containing 0.02% EDTA for passage when primary cells grow and fuse to 85%, and performing passage with 6 × 10³Individual cell/cm²Subculturing the endometrium to the density of more than three generations to obtain the endometrium stem cells, wherein:

(a1) taking the 3 rd generation endometrial stem cells with the fusion degree of 92 percent obtained in the step (7), removing the culture medium, adding PBS buffer solution for washing twice, adding 0.25 percent pancreatin containing 0.02 percent EDTA for digestion, and adding an isovolumetric DMEM/F12 complete culture medium to stop digestion when cells mostly become round and float, so as to obtain cell suspension;

(a5) adjusting the cell concentration of the cell suspension obtained in the step (a5) to 2X 10⁶Taking 5 flow tubes, adding 100ul of cell suspension into each tube, blowing, beating and uniformly mixing;

(a6) respectively adding 10ul primary antibodies with FITC or PE fluorescent labels into the 5 flow tubes, wherein the primary antibodies are respectively FITC isotype antibodies, PE isotype antibodies, CD29 antibodies, CD73 antibodies, CD90 antibodies, CD34 antibodies, CD45 antibodies and CD133 antibodies, and incubating for 25min at room temperature in a dark condition;

(b1) Taking the 3 rd generation endometrial stem cells with the fusion degree of 92 percent obtained in the step (7), removing the culture medium, adding PBS buffer solution for washing twice, adding 0.25 percent pancreatin containing 0.02 percent EDTA for digestion, and adding an isovolumetric DMEM/F12 complete culture medium to stop digestion when cells mostly become round and float, so as to obtain cell suspension;

(b6) when the cell fusion degree is 65%, replacing the culture medium with an adult adipose-derived mesenchymal stem cell osteogenesis induction differentiation culture medium containing 10% FBS and preheated to 37 ℃, replacing the medium once every 3 days, and inducing for 3 weeks;

(c) identification of endometrial stem cell adipogenic differentiation

(c1) Taking the 3 rd generation endometrial stem cells with the fusion degree of 92% obtained in the step (7), removing the culture medium, adding PBS buffer solution for washing twice, adding 0.25% pancreatin containing 0.02% EDTA for digestion, and adding an isovolumetric DMEM/F12 complete culture medium to stop digestion when cells mostly become round and float, so as to obtain cell suspension;

(c7) after the adult adipose-derived mesenchymal stem cell adipogenic induced differentiation medium A containing 10% of FBS and the adipogenic induced differentiation medium B are alternately used for 5 times, the adult adipose-derived mesenchymal stem cell adipogenic induced differentiation medium A and the adipogenic induced differentiation medium B are cultured for 7 days by using the simple liquid B, and half of the liquid is changed every 2.5 days;

(1) taking a healthy umbilical cord specimen by 15cm under a sterile condition, and placing the umbilical cord specimen in PBS buffer solution containing 1 v/v% double antibody;

(4) Adding DMEM/F12 complete medium into the culture dish, and placing the culture dish at 37 ℃ and 5 v/v% CO₂Culturing in a carbon dioxide incubator with saturated humidity, performing half-amount liquid change after 4 days, performing liquid change once every 3 days after cell climbing out, transferring to the 3 rd generation, digesting cells by using 0.25 wt% pancreatin aqueous solution when umbilical cord mesenchymal stem cells of the 3 rd generation are fused to 85%, stopping digestion by using EMEM/F12 complete culture medium, slightly blowing the bottom of the culture dish and collecting cells, centrifuging at 1000rpm for 5 minutes, discarding supernatant, and keeping cell precipitation;

Further, the passage method of umbilical cord mesenchymal stem cells in the step (4) comprises the following steps: when the umbilical cord mesenchymal stem cells reach 85% fusion, digesting the cells by using a 0.25% pancreatin water solution containing 0.02% EDTA, stopping digestion by using a DMEM/F12 complete culture medium, slightly blowing the bottom of a culture dish and collecting the cells, centrifuging at 1000rpm for 5min, and performing centrifugation according to the following steps of 1: passage at a ratio of 2.5.

Further, the third step comprises the following steps:

(31) preparation of chitosan solution

Weighing 0.25g of chitosan powder with deacetylation degree of more than 90%, adding the chitosan powder into a sterile beaker filled with 10ml of dilute acetic acid aqueous solution with concentration of 0.1M, and stirring by a stirrer to ensure that the chitosan solution becomes clear and transparent for later use;

(32) preparation of type I collagen solution

Weighing 40mg of type I collagen powder, adding the type I collagen powder into a sterile beaker filled with 10ml of 0.02M dilute acetic acid aqueous solution, and stirring the type I collagen powder by using a clean glass rod until the type I collagen powder is completely dissolved to obtain a type I collagen solution;

(33) preparation of beta-sodium glycerophosphate solution

Weighing 0.5g of beta-sodium glycerophosphate powder, adding the powder into a 5ml sterile beaker filled with 1ml of distilled water, and stirring the powder by using a clean glass rod until the powder is completely dissolved to obtain a beta-sodium glycerophosphate solution;

(34) preparation of sodium alginate solution

Weighing 0.2g of sodium alginate powder, then adding the sodium alginate powder into a sterile beaker filled with 10ml of distilled water, and stirring the mixture in a stirrer to ensure that the sodium alginate solution becomes clear and transparent for later use;

(35) preparing temperature-sensitive chitosan hydrogel

(354) and (3) adding the mixed solution obtained in the step (353) into an EP (EP) tube, and immediately putting the mixed solution into a constant-temperature incubator at 37 ℃ for culturing for 9min to obtain the temperature-sensitive chitosan hydrogel.

Further, the fourth step includes the steps of:

(41) further diluting the endometrial stem cells prepared in the step one by using a DMEM/F12 complete culture medium, wherein the cell concentration of the diluted endometrial stem cells is 2 x 10⁵Per ml;

(42) further diluting the mesenchymal stem cells prepared in the step two by using a DMEM/F12 complete culture medium, wherein the cell concentration of the diluted mesenchymal stem cell suspension is 1 multiplied by 10⁶Per ml;

(43) and (3) mixing the diluted endometrial stem cell suspension obtained in the step (41) with the diluted mesenchymal stem cell suspension obtained in the step (42) according to the volume ratio of 2: 1 to form endometrium stem cell and mesenchymal stem cell suspension, then cooling to 19 ℃, and fully mixing the endometrium stem cell and mesenchymal stem cell suspension and the temperature-sensitive chitosan hydrogel prepared in the third step in an ice bath container to obtain the cell composite endometrium repair gel.

Specific example 2

The method is substantially the same as the embodiment 1, and only differs from the industrial control conditions in the step two:

the mesenchymal stem cells in the second step are umbilical cord mesenchymal stem cells, and the second step comprises the following steps:

(1) taking a healthy umbilical cord specimen by 10cm under a sterile condition, and placing the umbilical cord specimen in PBS buffer solution containing 1 v/v% double antibody;

(4) Adding DMEM/F12 complete medium into the culture dish, and placing the culture dish at 37 ℃ and 5 v/v% CO₂Culturing in a carbon dioxide incubator with saturated humidity, performing half-amount liquid change after 4 days, performing liquid change once every 3 days after cell climbing out, transferring to the 3 rd generation, digesting cells by using 0.25 wt% pancreatin aqueous solution when the umbilical cord mesenchymal stem cells of the 3 rd generation are fused to 80%, stopping digestion by using EMEM/F12 complete culture medium, slightly blowing the bottom of the culture dish and collecting the cells, centrifuging at 1000rpm for 5 minutes, discarding the supernatant, and keeping cell precipitation;

Further, the passage method of umbilical cord mesenchymal stem cells in the step (4) comprises the following steps: when the umbilical cord mesenchymal stem cells reach 80% fusion, digesting the cells by using a 0.25% pancreatin water solution containing 0.02% EDTA, stopping digestion by using a DMEM/F12 complete culture medium, slightly blowing the bottom of a culture dish and collecting the cells, centrifuging at 1000rpm for 5min, and performing separation according to the weight ratio of 1: 2.

Specific example 3

(1) taking a healthy umbilical cord specimen by 20cm under a sterile condition, and placing the umbilical cord specimen in PBS buffer solution containing 1 v/v% double antibody;

(4) Adding DMEM/F12 complete medium into the culture dish, and placing the culture dish at 37 ℃ and 5 v/v% CO₂Culturing in a carbon dioxide incubator with saturated humidity, performing half-amount liquid change after 4 days, performing liquid change once every 3 days after cell climbing out, transferring to the 3 rd generation, digesting cells by using 0.25 wt% pancreatin aqueous solution when the umbilical cord mesenchymal stem cells of the 3 rd generation are 90% fused, stopping digestion by using EMEM/F12 complete culture medium, slightly blowing the bottom of the culture dish and collecting the cells, centrifuging at 1000rpm for 5 minutes, discarding the supernatant, and keeping cell precipitation;

Further, the passage method of umbilical cord mesenchymal stem cells in the step (4) comprises the following steps: when the umbilical cord mesenchymal stem cells reach 90% fusion, digesting the cells by using a 0.25% pancreatin water solution containing 0.02% EDTA, stopping digestion by using a DMEM/F12 complete culture medium, slightly blowing the bottom of a culture dish and collecting the cells, centrifuging at 1000rpm for 5min, and performing centrifugation according to the following steps of 1: 3, passage.

Specific example 4

step three, preparing temperature-sensitive chitosan hydrogel;

step four, preparing the cell composite endometrium repair gel.

Further, the first step comprises the following steps:

(2) adding a 0.1 v/v% collagenase aqueous solution with the volume being 1.5 times of that of the sterile culture dish in the step (1), carrying out water bath shaking digestion for 30min at 37 ℃, collecting a digestion solution in a centrifuge tube, adding a DMEM/F12 complete culture medium into the centrifuge tube to stop digestion, then centrifuging for 10min at 1000rpm, discarding a supernatant, and keeping a cell precipitate;

(3) adding 4 times volume of erythrocyte lysate into the centrifuge tube, continuously oscillating until the liquid in the centrifuge tube gradually becomes red, adding 10ml of PBS buffer solution into the centrifuge tube, centrifuging at 1000rpm for 5min again, discarding the supernatant, and keeping cell precipitate;

(7) adding DMEM/F12 to the cell pellet retained in step (6), suspending the cell pellet in a complete culture medium, transferring the cell pellet to a cell culture flask, and culturing the cell pellet at 37 ℃ and 5 v/v% CO₂Culturing in incubator, performing half-amount liquid change with DMEM/F12 complete culture medium for the first time after culturing for 24h, performing full-amount liquid change with DMEM/F12 complete culture medium every 2 days, discarding non-adherent cells, adding 0.25% pancreatin aqueous solution containing 0.02% EDTA for passage when primary cells grow and fuse to 80%, and performing passage with 6 × 10³Individual cell/cm²Subculturing the endometrium to the density of more than three generations to obtain the endometrium stem cells, wherein:

(a1) taking the 3 rd generation endometrial stem cells with the fusion degree of 90% obtained in the step (7), removing the culture medium, adding PBS buffer solution for washing twice, adding 0.25% pancreatin containing 0.02% EDTA for digestion, and adding an isovolumetric DMEM/F12 complete culture medium to stop digestion when cells mostly become round and float, so as to obtain cell suspension;

(a5) and the toneThe cell concentration of the cell suspension obtained in the step (a5) was 2X 10⁶Taking 5 flow tubes, adding 100ul of cell suspension into each tube, blowing, beating and uniformly mixing;

(a6) respectively adding 10ul primary antibodies with FITC or PE fluorescent labels into the 5 flow tubes, wherein the primary antibodies are respectively FITC isotype antibodies, PE isotype antibodies, CD29 antibodies, CD73 antibodies, CD90 antibodies, CD34 antibodies, CD45 antibodies and CD133 antibodies, and incubating for 20min at room temperature in a dark condition;

(b1) Taking the 3 rd generation endometrial stem cells with the fusion degree of 90% obtained in the step (7), removing the culture medium, adding PBS buffer solution for washing twice, adding 0.25% pancreatin containing 0.02% EDTA for digestion, and adding an isovolumetric DMEM/F12 complete culture medium to stop digestion when cells mostly become round and float, so as to obtain cell suspension;

(b6) when the cell fusion degree is 60%, replacing the culture medium with an adult adipose-derived mesenchymal stem cell osteogenesis induction differentiation culture medium containing 10% FBS and preheated to 37 ℃, replacing the medium once every 3 days, and inducing for 2 weeks;

(c) identification of endometrial stem cell adipogenic differentiation

(c1) Taking 3 rd generation endometrial stem cells with the fusion degree of 90% obtained in the step (7), removing a culture medium, adding PBS buffer solution for washing twice, adding 0.25% pancreatin containing 0.02% EDTA for digestion, and adding an isovolumetric DMEM/F12 complete culture medium to stop digestion when cells mostly become round and float, so as to obtain a cell suspension;

(c7) after the adult adipose-derived mesenchymal stem cell adipogenic induced differentiation medium A containing 10% of FBS and the adipogenic induced differentiation medium B are alternately used for 5 times, the adult adipose-derived mesenchymal stem cell adipogenic induced differentiation medium A and the adipogenic induced differentiation medium B are cultured for 7 days by using the simple liquid B, and half of liquid is changed every 2 days;

s21, under aseptic condition, taking adipose tissue, removing vascular fascia, washing with PBS, removing erythrocytes, shearing into pieces with volume less than 1mm³Then placing the mixture into a 50ml centrifuge tube, adding 0.1 wt% type I collagenase with the volume 5 times that of the mixture, and oscillating the mixture for 40min in a water bath kettle with the temperature of 37 ℃ and the rpm of 100;

s23, adding 10ml HG-DMEM complete culture medium into the centrifuge tube, fully suspending, filtering through a 200-mesh filter screen, reserving filtrate, centrifuging for 10min at 4 ℃ and 1500rpm, and removing supernatant to obtain cell sediment;

s25, inoculating the cell suspension into a T25 cell culture bottle, placing the bottle at 37 ℃ and 5 v/v% CO₂And after culturing for 48 hours in a saturated humidity incubator, carrying out half-amount liquid exchange for the first time, removing non-adherent cells, carrying out liquid exchange once every 2d, transferring to the 3 rd generation, digesting the cells by using a 0.25% pancreatin water solution containing 0.02% EDTA when the 3 rd generation adipose-derived mesenchymal stem cells reach 80%, stopping digestion by using an HG-DMEM complete culture medium, slightly blowing the bottom of a bottle and collecting the cells, centrifuging at 1000rpm for 5min, discarding the supernatant, adding 1ml of HG-DMEM complete culture medium into cell sediment, resuspending the cell sediment, counting the cell sediment, and obtaining the adipose-derived mesenchymal stem cells of the 3 rd generation after the completion.

Further, the passage method of the adipose-derived mesenchymal stem cells comprises the following steps: when the adipose-derived mesenchymal stem cells reach 80% fusion, digesting the cells by using a 0.25% pancreatin water solution containing 0.02% EDTA, stopping digestion by using an HG-DMEM complete culture medium, slightly blowing the bottom of a bottle and collecting the cells, centrifuging at 1000rpm for 5min, and performing centrifugation according to the weight ratio of 1: 2.

Further, the third step comprises the following steps:

(31) preparation of chitosan solution

Weighing 0.2g of chitosan powder with deacetylation degree of more than 90%, adding the chitosan powder into a sterile beaker filled with 10ml of dilute acetic acid aqueous solution with concentration of 0.1M, and stirring by a stirrer to ensure that the chitosan solution becomes clear and transparent for later use;

(32) preparation of type I collagen solution

Weighing 25mg of type I collagen powder, adding the type I collagen powder into a sterile beaker filled with 10ml of 0.02M dilute acetic acid aqueous solution, and stirring the type I collagen powder by using a clean glass rod until the type I collagen powder is completely dissolved to obtain a type I collagen solution;

(33) preparation of beta-sodium glycerophosphate solution

Weighing 0.4g of beta-sodium glycerophosphate powder, adding the powder into a 5ml sterile beaker containing 1ml of distilled water, and stirring the powder by using a clean glass rod until the powder is completely dissolved to obtain a beta-sodium glycerophosphate solution;

(34) preparation of sodium alginate solution

Weighing 0.15g of sodium alginate powder, then adding the sodium alginate powder into a sterile beaker filled with 10ml of distilled water, and stirring the mixture in a stirrer to ensure that the sodium alginate solution becomes clear and transparent for later use;

(35) preparing temperature-sensitive chitosan hydrogel

(354) and (3) adding the mixed solution obtained in the step (353) into an EP (EP) tube, and immediately putting the mixed solution into a constant-temperature incubator at 37 ℃ for culturing for 8min to obtain the temperature-sensitive chitosan hydrogel.

Further, the fourth step includes the steps of:

(41) further diluting the endometrial stem cells prepared in the step one by using a DMEM/F12 complete culture medium, wherein the cell concentration of the diluted endometrial stem cells is 2 x 10⁴Per ml;

(42) and the mesenchyme prepared in the second stepThe stem cells are further diluted by DMEM/F12 complete medium, and the cell concentration of the diluted mesenchymal stem cell suspension is 1 x 10⁵Per ml;

(43) and (3) mixing the diluted endometrial stem cell suspension obtained in the step (41) with the diluted mesenchymal stem cell suspension obtained in the step (42) according to the volume ratio of 2: 1 to form endometrium stem cell and mesenchymal stem cell suspension, then cooling to 18 ℃, and fully mixing the endometrium stem cell and mesenchymal stem cell suspension and the temperature-sensitive chitosan hydrogel prepared in the third step in an ice bath container to obtain the cell composite endometrium repair gel.

Specific example 5

The method is substantially the same as the specific example 4, and only differs from the industrial control conditions of the step two:

the mesenchymal stem cells in the second step are adipose mesenchymal stem cells, and the second step comprises the following steps:

s21, under aseptic condition, taking adipose tissue, removing vascular fascia, washing with PBS, removing erythrocytes, shearing into pieces with volume less than 1mm³Then placing the mixture into a 50ml centrifuge tube, adding 0.1 wt% collagenase type I with the volume 10 times that of the mixture, and oscillating the mixture for 80min in a water bath kettle with the temperature of 37 ℃ and the rpm of 100;

s23, adding 15ml HG-DMEM complete culture medium into the centrifuge tube, fully suspending, filtering through a 200-mesh filter screen, reserving filtrate, centrifuging for 10min at 4 ℃ and 1500rpm, and removing supernatant to obtain cell sediment;

s25, inoculating the cell suspension into a T25 cell culture bottle, placing the bottle at 37 ℃ and 5 v/v% CO₂After the adipose-derived mesenchymal stem cells are cultured in a saturated humidity incubator for 36 hours, half of the liquid is changed for the first time, the cells which are not attached to the wall are removed, then the liquid is changed once every 3d and is transferred to the 3 rd generation, when the adipose-derived mesenchymal stem cells of the 3 rd generation reach 90%, 0.25% pancreatin water solution containing 0.02% EDTA is used for digesting the cells, the HG-DMEM complete culture medium stops digestion, the bottom of a bottle is lightly blown and the cells are collected, the centrifugation is carried out at 1000rpm for 5min, the supernatant is discarded, 1ml of the HG-DMEM complete culture medium is added into the cell sediment, the cell sediment is resuspended and counted, and the adipose-derived mesenchymal stem cells of the 3 rd generation are obtained after the.

Further, the passage method of the adipose-derived mesenchymal stem cells comprises the following steps: when the adipose-derived mesenchymal stem cells reach 90% fusion, digesting the cells by using a 0.25% pancreatin water solution containing 0.02% EDTA, stopping digestion by using an HG-DMEM complete culture medium, slightly blowing the bottom of a bottle and collecting the cells, centrifuging at 1000rpm for 5min, and performing centrifugation according to the weight ratio of 1: 3, passage.

Specific example 6

s21, under aseptic condition, taking adipose tissue, removing vascular fascia, washing with PBS, removing erythrocytes, shearing into pieces with volume less than 1mm³Then placing the mixture into a 50ml centrifuge tube, adding 0.1 wt% collagenase type I with the volume 8 times of that of the mixture, and oscillating the mixture for 80min in a water bath kettle with the temperature of 37 ℃ and the rpm of 100;

s23, adding 112ml HG-DMEM complete culture medium into the centrifuge tube, fully suspending, filtering with a 200-mesh filter screen, reserving filtrate, centrifuging at 4 ℃ and 1500rpm for 10min, and removing supernatant to obtain cell precipitate;

s25, inoculating the cell suspension into a T25 cell culture bottle, placing the bottle at 37 ℃ and 5 v/v% CO₂And after the cells are cultured in a saturated humidity incubator for 60 hours, half of the cells are firstly changed, the cells which are not attached to the wall are removed, the cells are digested by pancreatin water solution which contains 0.25% of EDTA and contains 0.02% of EDTA every 2.5 days, the digestion of HG-DMEM complete culture medium is stopped, the bottom of a bottle is lightly blown and the cells are collected, the cells are centrifuged at 1000rpm for 5min, the supernatant is discarded, 1ml of HG-DMEM complete culture medium is added into the cell sediment, the cell sediment is resuspended and counted, and the adipose-derived mesenchymal stem cells of the 3 rd generation are obtained after the cell sediment is completely centrifuged for 5 min.

Further, the passage method of the adipose-derived mesenchymal stem cells comprises the following steps: when the adipose-derived mesenchymal stem cells reach 85% fusion, digesting the cells by using a 0.25% pancreatin water solution containing 0.02% EDTA, stopping digestion by using an HG-DMEM complete culture medium, slightly blowing the bottom of a bottle and collecting the cells, centrifuging at 1000rpm for 5min, and performing centrifugation according to the weight ratio of 1: passage at a ratio of 2.5.

Specific example 7

step three, preparing temperature-sensitive chitosan hydrogel;

step four, preparing the cell composite endometrium repair gel.

Further, the first step comprises the following steps:

(2) adding a 0.1 v/v% collagenase aqueous solution with the volume being 3 times that of the sterile culture dish in the step (1), carrying out water bath shaking digestion for 80min at 37 ℃, collecting a digestion solution in a centrifuge tube, adding a DMEM/F12 complete culture medium into the centrifuge tube to stop digestion, then centrifuging for 10min at 1000rpm, discarding a supernatant, and keeping a cell precipitate;

(3) adding 8 times volume of erythrocyte lysate into the centrifuge tube, continuously oscillating until the liquid in the centrifuge tube gradually becomes red, adding 10ml of PBS buffer solution into the centrifuge tube, centrifuging at 1000rpm for 5min again, discarding the supernatant, and keeping cell precipitate;

(7) adding DMEM/F12 to the cell pellet retained in step (6), suspending the cell pellet in a complete culture medium, transferring the cell pellet to a cell culture flask, and culturing the cell pellet at 37 ℃ and 5 v/v% CO₂Culturing in incubator, performing half-amount liquid change with DMEM/F12 complete culture medium for the first time after culturing for 48h, performing full-amount liquid change with DMEM/F12 complete culture medium every 3 days, discarding non-adherent cells, adding 0.25% pancreatin aqueous solution containing 0.02% EDTA for passage when primary cells grow and fuse to 90%, and performing passage with 6 × 10³Individual cell/cm²Subculturing the endometrium to the density of more than three generations to obtain the endometrium stem cells, wherein:

(a1) taking the 3 rd generation endometrial stem cells with the fusion degree of 95% obtained in the step (7), removing the culture medium, adding PBS buffer solution for washing twice, adding 0.25% pancreatin containing 0.02% EDTA for digestion, and adding an isovolumetric DMEM/F12 complete culture medium to stop digestion when cells mostly become round and float, so as to obtain cell suspension;

(a6) respectively adding 10ul primary antibodies with FITC or PE fluorescent labels into the 5 flow tubes, wherein the primary antibodies are respectively FITC isotype antibodies, PE isotype antibodies, CD29 antibodies, CD73 antibodies, CD90 antibodies, CD34 antibodies, CD45 antibodies and CD133 antibodies, and incubating for 30min at room temperature in a dark condition;

(b1) Taking the 3 rd generation endometrial stem cells with the fusion degree of 95% obtained in the step (7), removing the culture medium, adding PBS buffer solution for washing twice, adding 0.25% pancreatin containing 0.02% EDTA for digestion, and adding an isovolumetric DMEM/F12 complete culture medium to stop digestion when cells mostly become round and float, so as to obtain cell suspension;

(b6) when the cell fusion degree is 70%, replacing the culture medium with an adult adipose-derived mesenchymal stem cell osteogenesis induction differentiation culture medium containing 10% FBS and preheated to 37 ℃, replacing the medium once every 3 days, and inducing for 4 weeks;

(c) identification of endometrial stem cell adipogenic differentiation

(c1) Taking the 3 rd generation endometrial stem cells with the fusion degree of 95% obtained in the step (7), removing the culture medium, adding PBS buffer solution for washing twice, adding 0.25% pancreatin containing 0.02% EDTA for digestion, and adding an isovolumetric DMEM/F12 complete culture medium to stop digestion when cells mostly become round and float, so as to obtain cell suspension;

(c7) after the adult adipose-derived mesenchymal stem cell adipogenic induced differentiation medium A containing 10% of FBS and the adipogenic induced differentiation medium B are alternately used for 5 times, the adult adipose-derived mesenchymal stem cell adipogenic induced differentiation medium A and the adipogenic induced differentiation medium B are cultured for 7 days by using the simple liquid B, and half of liquid is changed every 3 days;

step23, adding 5ml DMEM/F12 into a centrifuge tube, completely culturing and fully suspending to obtain a cell suspension, inoculating into a T25 cell culture bottle, placing at 37 ℃ and 5 v/v% CO₂Culturing in a saturated humidity incubator, changing liquid for the first time after 48 hours, removing non-adherent cells, changing liquid every 3d, transferring to the 3 rd generation, digesting cells by using 0.25% pancreatin solution cells containing 0.02% EDTA when the 3 rd generation mesenchymal stem cells reach 90% fusion, stopping digestion by using DMEM/F12 complete culture medium, lightly blowing the bottom of a bottle and collecting a wash barCentrifuging at 1000rpm for 5 min; and (3) removing the supernatant, adding 1ml of DMEM/F12 complete culture medium into the cell sediment, fully suspending, counting, and obtaining the 3 rd generation mesenchymal stem cells.

Further, the passage method of the bone marrow mesenchymal stem cells comprises the following steps: when the bone marrow mesenchymal stem cells reach 90% fusion, digesting the cells by using a 0.25% pancreatin water solution containing 0.02% EDTA, stopping digestion by using an HG-DMEM complete culture medium, slightly blowing the bottom of a bottle and collecting the cells, centrifuging at 1000rpm for 5min, and mixing the cells according to the weight ratio of 1: 3, passage.

Further, the third step comprises the following steps:

(31) preparation of chitosan solution

Weighing 0.3g of chitosan powder with deacetylation degree of more than 90%, adding the chitosan powder into a sterile beaker filled with 10ml of dilute acetic acid aqueous solution with concentration of 0.1M, and stirring by a stirrer to ensure that the chitosan solution becomes clear and transparent for later use;

(32) preparation of type I collagen solution

Weighing 50mg of type I collagen powder, adding the type I collagen powder into a sterile beaker filled with 10ml of 0.02M dilute acetic acid aqueous solution, and stirring the type I collagen powder by using a clean glass rod until the type I collagen powder is completely dissolved to obtain a type I collagen solution;

(33) preparation of beta-sodium glycerophosphate solution

Weighing 0.6g of beta-sodium glycerophosphate powder, adding the powder into a 5ml sterile beaker filled with 1ml of distilled water, and stirring the powder by using a clean glass rod until the powder is completely dissolved to obtain a beta-sodium glycerophosphate solution;

(34) preparation of sodium alginate solution

Weighing 0.25g of sodium alginate powder, then adding the sodium alginate powder into a sterile beaker filled with 10ml of distilled water, and stirring the mixture in a stirrer to ensure that the sodium alginate solution becomes clear and transparent for later use;

(35) preparing temperature-sensitive chitosan hydrogel

(354) and (3) adding the mixed solution obtained in the step (353) into an EP (EP) tube, and immediately putting the mixed solution into a constant-temperature incubator at 37 ℃ for culturing for 10min to obtain the temperature-sensitive chitosan hydrogel.

Further, the fourth step includes the steps of:

(41) further diluting the endometrial stem cells prepared in the step one by using a DMEM/F12 complete culture medium, wherein the cell concentration of the diluted endometrial stem cells is 2 x 10⁶Per ml;

(42) further diluting the mesenchymal stem cells prepared in the step two by using a DMEM/F12 complete culture medium, wherein the cell concentration of the diluted mesenchymal stem cell suspension is 1 multiplied by 10⁷Per ml;

(43) and (3) mixing the diluted endometrial stem cell suspension obtained in the step (41) with the diluted mesenchymal stem cell suspension obtained in the step (42) according to the volume ratio of 2: 1 to form endometrium stem cell and mesenchymal stem cell suspension, then cooling to 20 ℃, and fully mixing the endometrium stem cell and mesenchymal stem cell suspension and the temperature-sensitive chitosan hydrogel prepared in the third step in an ice bath container to obtain the cell composite endometrium repair gel.

Specific example 8

The method is substantially the same as the specific example 7, and only differs from the industrial control conditions of the step two:

step23, adding 5ml DMEM/F12 into a centrifuge tube, completely culturing and fully suspending to obtain a cell suspension, inoculating into a T25 cell culture bottle, placing at 37 ℃ and 5 v/v% CO₂Culturing in a saturated humidity incubator, changing the liquid for the first time after 36 hours, removing non-adherent cells, changing the liquid every 2.5 days, transferring to the 3 rd generation, digesting the cells by using 0.25% pancreatin solution cells containing 0.02% EDTA when the 3 rd generation mesenchymal stem cells reach 85% fusion, stopping digestion by using a DMEM/F12 complete culture medium, slightly blowing the bottom of a bottle, collecting a wash bar, and centrifuging at 1000rpm for 5 min; and (3) removing the supernatant, adding 1ml of DMEM/F12 complete culture medium into the cell sediment, fully suspending, counting, and obtaining the 3 rd generation mesenchymal stem cells.

Further, the passage method of the bone marrow mesenchymal stem cells comprises the following steps: when the bone marrow mesenchymal stem cells reach 85% fusion, digesting the cells by using a 0.25% pancreatin water solution containing 0.02% EDTA, stopping digestion by using an HG-DMEM complete culture medium, slightly blowing the bottom of a bottle and collecting the cells, centrifuging at 1000rpm for 5min, and mixing the cells according to the ratio of 1: passage at a ratio of 2.5.

Specific example 9

step23, adding 5ml DMEM/F12 into a centrifuge tube, completely culturing and fully suspending to obtain a cell suspension, inoculating into a T25 cell culture bottle, placing at 37 ℃ and 5 v/v% CO₂Culturing in a saturated humidity incubator, changing the liquid for the first time after 24 hours, removing non-adherent cells, changing the liquid every 2 days, transferring to the 3 rd generation, digesting the cells by using 0.25% pancreatin solution cells containing 0.02% EDTA when the 3 rd generation mesenchymal stem cells are fused to 80%, stopping digestion by using a DMEM/F12 complete culture medium, slightly blowing the bottom of a bottle, collecting a washing bar, and centrifuging for 5min at 1000 rpm; and (3) removing the supernatant, adding 1ml of DMEM/F12 complete culture medium into the cell sediment, fully suspending, counting, and obtaining the 3 rd generation mesenchymal stem cells.

Further, the passage method of the bone marrow mesenchymal stem cells comprises the following steps: when the mesenchymal stem cells of the bone marrow reach 80 percent fusion, digesting the cells by using a 0.25 percent pancreatin water solution containing 0.02 percent EDTA, stopping digestion by using an HG-DMEM complete culture medium, slightly blowing the bottom of a bottle and collecting the cells, centrifuging at 1000rpm for 5min, and mixing the cells according to the ratio of 1: 2.

Detailed description of example 10

Substantially the same as in example 7, except that the mesenchymal stem cells in step two were different:

the mesenchymal stem cells in the second step are placenta mesenchymal stem cells, and the second step comprises the following steps:

step21, taking placenta lobules close to the umbilical cord part under the aseptic condition, stripping amnion and decidua basalis parts, taking fetal disc and fetal surface tissues, repeatedly flushing the fetal surface tissues with PBS (phosphate buffer solution) until the liquid is clear, and cutting the fetal surface tissues with ophthalmic scissorsShearing into pieces with a volume of less than 1mm³Then placing the mixture into a 50ml centrifuge tube;

step22, adding 0.1 wt% collagenase aqueous solution with the volume 2 times that of the centrifuge tube, oscillating at 37 ℃ and 100rpm for 20min, adding DMEM/F12 complete culture medium with the same volume, and sieving with a 200-mesh filter screen to obtain cell sediment;

step23, adding 5ml of DMEM/F12 complete culture medium into the centrifuge tube for fully suspending to obtain a cell suspension, inoculating the cell suspension into a T25 cell culture bottle, placing the cell suspension in a5 v/v% CO culture bottle at 37 DEG C₂Culturing in a saturated humidity incubator, firstly replacing liquid after 24 hours, removing nonadherent cells, then replacing liquid once every 2d, transferring to the 3 rd generation, digesting the cells by using a 0.25% pancreatin water solution containing 0.02% EDTA when the 3 rd generation of placenta mesenchymal stem cells reach 80% fusion, stopping digestion by using a DMEM/F12 complete culture medium, slightly blowing the bottom of a bottle and collecting the cells, centrifuging at 1000rpm for 5min, discarding the supernatant, adding 1ml of DMEM/F12 complete culture medium into cell sediment, fully suspending to obtain a cell suspension, counting the cell suspension, and obtaining the 3 rd generation of placenta mesenchymal stem cells after completion, wherein:

Further, the passage method of the placenta mesenchymal stem cells comprises the following steps: when the placenta mesenchymal stem cells reach 80% fusion, digesting the cells by using a 0.25% pancreatin water solution containing 0.02% EDTA, stopping digestion by using an HG-DMEM complete culture medium, slightly blowing the bottom of a bottle and collecting the cells, centrifuging at 1000rpm for 5min, and mixing the cells according to the weight ratio of 1: 2.

Specific example 11

step21, taking placenta lobules close to the umbilical cord part under an aseptic condition, stripping amnion and decidua basalis parts, taking placenta fetal surface tissues, repeatedly washing with PBS until the liquid is clear, and shearing with ophthalmic scissors until the volume is less than 1mm³Then, thenPlacing in a 50ml centrifuge tube;

step22, adding 0.1 wt% collagenase aqueous solution with the volume 4 times that of the centrifuge tube, oscillating at 37 ℃ and 100rpm for 50min, adding DMEM/F12 complete culture medium with the same volume, and sieving with a 200-mesh filter screen to obtain cell sediment;

step23, adding 5ml of DMEM/F12 complete culture medium into the centrifuge tube for fully suspending to obtain a cell suspension, inoculating the cell suspension into a T25 cell culture bottle, placing the cell suspension in a5 v/v% CO culture bottle at 37 DEG C₂Culturing in a saturated humidity incubator, firstly replacing liquid after 48 hours, removing nonadherent cells, then replacing liquid once every 3d, transferring to the 3 rd generation, digesting the cells by using a 0.25% pancreatin water solution containing 0.02% EDTA when the 3 rd generation of placenta mesenchymal stem cells reach 90% fusion, stopping digestion by using a DMEM/F12 complete culture medium, slightly blowing the bottom of a bottle and collecting the cells, centrifuging at 1000rpm for 5min, discarding the supernatant, adding 1ml of DMEM/F12 complete culture medium into cell sediment, fully suspending to obtain a cell suspension, counting the cell suspension, and obtaining the 3 rd generation of placenta mesenchymal stem cells after completion, wherein:

Further, the passage method of the placenta mesenchymal stem cells comprises the following steps: when the placenta mesenchymal stem cells reach 90% fusion, digesting the cells by using a 0.25% pancreatin water solution containing 0.02% EDTA, stopping digestion by using an HG-DMEM complete culture medium, slightly blowing the bottom of a bottle and collecting the cells, centrifuging at 1000rpm for 5min, and mixing the cells according to the weight ratio of 1: 3, passage.

Detailed description of example 12

step22, adding 0.1 wt% collagenase aqueous solution with the volume 3 times that of the centrifuge tube, oscillating at 37 ℃ and 100rpm for 30min, adding DMEM/F12 complete culture medium with the same volume, and sieving with a 200-mesh filter screen to obtain cell sediment;

step23, adding 5ml of DMEM/F12 complete culture medium into the centrifuge tube for fully suspending to obtain a cell suspension, inoculating the cell suspension into a T25 cell culture bottle, placing the cell suspension in a5 v/v% CO culture bottle at 37 DEG C₂Culturing in a saturated humidity incubator, firstly replacing liquid after 36 hours, removing nonadherent cells, then replacing liquid once every 2.5 days, transferring to the 3 rd generation, digesting the cells by using 0.25% pancreatin water solution containing 0.02% EDTA when the 3 rd generation of placenta mesenchymal stem cells reach 85% fusion, stopping digestion by using a DMEM/F12 complete culture medium, slightly blowing the bottom of a bottle and collecting the cells, centrifuging at 1000rpm for 5min, discarding the supernatant, adding 1ml of DMEM/F12 complete culture medium into cell sediment, fully suspending to obtain a cell suspension, counting the cell suspension, and obtaining the 3 rd generation of placenta mesenchymal stem cells after completion, wherein:

Further, the passage method of the placenta mesenchymal stem cells comprises the following steps: when the placenta mesenchymal stem cells reach 85% fusion, digesting the cells by using a 0.25% pancreatin water solution containing 0.02% EDTA, stopping digestion by using an HG-DMEM complete culture medium, slightly blowing the bottom of a bottle and collecting the cells, centrifuging at 1000rpm for 5min, and mixing the cells according to the weight ratio of 1: passage at a ratio of 2.5.

And (3) verification test:

firstly, constructing rat intrauterine adhesion model

The female SD rat without mating is raised in an SPF level barrier system, the temperature is 20-26 ℃, the humidity is 50-60 percent, and the female SD rat can freely eat and drink water in day and night for 12 hours.

4 female mice (8 uteruses) with normal estrus cycles are selected, and an intrauterine adhesion model is built by adopting an absolute ethyl alcohol intrauterine injection method.

Firstly, fasting the rat before an operation for 12 hours, carrying out intraperitoneal injection of 10% chloral hydrate according to the dose of 0.3mL/100g, and carrying out the operation after corneal reflex and righting reflex disappear;

② the disposable operation sheet is spread on the operation board, the rat is fixed on the operation board in supine position, the lower abdomen is preserved, and 75% alcohol is disinfected. Making a longitudinal incision at 1.5cm above the pubic symphysis and in the middle of lower abdomen, and sequentially cutting skin and abdominal wall;

thirdly, separating the uterus, slightly clamping the near end and the far end of the uterus at one side by 2 hemostatic forceps respectively, and slightly straightening the uterus by toothless forceps;

fourthly, the needle head of the injector enters the uterine cavity, absolute ethyl alcohol is slowly injected into one end of the uterus, the uterine cavity is kept in a full state, and the needle head stays for 4-5 min. Sucking out residual anhydrous ethanol, washing with normal saline for 2 times, slowly taking out the needle, and loosening the hemostatic forceps;

treating the uterus on the other side by the same method;

sixthly, after the uterus is reset to prevent adhesion, the abdominal cavity is flushed by normal saline, and the abdominal cavity is closed layer by layer after being sucked dry by sterile gauze.

Observed in operation, the rat uterus is pink before operation and has elasticity; the uterus becomes pale and the texture becomes hard after the injection of the absolute ethyl alcohol.

The rats were kept warm before awakening and were protected from infection by intraperitoneal injection of penicillin (10 ten thousand units, once a day) three consecutive days after surgery. After the estrus period of three estrus cycles, the abdomen is opened, the uterus is taken out, 4% paraformaldehyde is fixed, and the gross specimen is observed.

Second, detection of rat intrauterine adhesion model establishment

(1) HE staining

Preparation of a section: after paraformaldehyde fixation, the tissues were placed in groups vertically in an embedding cassette. Making the embedded paraffin into 4um slices, gently placing the slices on a preheated constant-temperature water surface, gradually flattening the slices, gently fishing out the paraffin slices by using a glass slide, and placing the slices in an oven at 60 ℃ overnight;

② dewaxing and hydrating: sequentially placing paraffin sections in xylene I, xylene II, xylene III and xylene IV for 10min respectively; then sequentially placing in gradient alcohol, namely absolute ethyl alcohol I, absolute ethyl alcohol II, 95% alcohol I and 95% alcohol II, each for 30 s; washing with tap water, and soaking in PBS for 5 min;

③ HE dyeing: staining with hematoxylin for 10min, returning blue with running water for 30min, staining with eosin for 1min, and observing the staining condition of the section under a microscope;

dehydrating, transparent and sealing: placing the slices in gradient alcohol, namely 95% alcohol I, 95% alcohol II, absolute ethanol I and absolute ethanol II, respectively for 30s, xylene I for 1min and xylene II for 2min, sealing with neutral resin, observing under an optical microscope, and shooting.

(2) Endometrial thickness measurement

The slices are produced by the HE staining method, observed under an optical microscope and photographed. Measuring the thickness of the endometrium by adopting Image-Pro Plus 6.0 Image processing software, namely the vertical distance from the joint of the endometrium muscular layer to the uterine cavity, selecting 4 parts of each section, namely the widest, narrowest and 2 equal points or symmetrical points of the endometrium for measurement, and taking the average value as the thickness of the endometrium of the section.

Thirdly, the method comprises the following steps: control test:

rat intrauterine adhesion treatment

4 SD rats were randomly divided into the following groups, 1 in each group.

gel/EDSCs panel: after the animal model is established, 10-25ul of the product prepared in specific example 1 is injected locally into uterine cavity and contains 1 × 10%⁶Gel mixtures of individual EDSCs;

② EDSCs group: after animal model is established, 10-25ul of the injection containing 1X 10 is locally injected into uterine cavity⁶PBS of EDSCs;

③ gel group: after an animal model is established, 10-25ul of gel is locally injected into the uterine cavity;

fourthly, negative control group: after an animal model is established, 10-25ul PBS is locally injected into the uterine cavity;

normal control group: normal female rats were taken without drug and gel/eds.

The following operations are then carried out:

opening the abdominal cavity along the original incision, separating out the left endometrium injury side uterus, slowly injecting the four transplantation components into the uterine cavities of four groups of SD rats respectively by using an injector, reserving the needle for 20s, resetting the uterus, flushing the abdominal cavity by using normal saline, sucking the uterus by using sterile gauze, and closing the abdomen layer by layer. After each group of rats was labeled, they were transferred to a 37 ℃ thermostatic plate and returned to the animal house after anaesthesia and awakening.

2. Evaluation of therapeutic Effect of rat intrauterine adhesion

After gel/EDSCs or PBS transplantation, rats were sacrificed by anesthesia overdose during estrus of three estrus cycles, separated by laparotomy and uterus removed, and fixed with 4% paraformaldehyde. After HE staining and endometrial measurements, the increase in endometrial thickness was significantly higher in the gel/endometrial group than in the other groups, essentially consistent with the normal group thickness.

(1) The autologous stem cell transplantation, the gel/EDSCs group and the EDSCs group do not have inflammatory reaction of endometrium;

(2) the autologous endometrial stem cells and the mesenchymal stem cells are compositely transplanted, the regeneration of endometrium, tiny blood vessels and mesenchyme is accelerated, the endometrium of the gel/EDSCs group and the endometrium of the EDSCs group are obviously repaired in a 3-month period, and the thickness change of the endometrium of other groups is not obvious;

(3) the temperature-sensitive gel can be formed after local injection, provides a three-dimensional retention and proliferation space for stem cells, prevents stem cell suspension from flowing out through a cervical orifice and an oviduct, and can prevent adhesion of regenerated mucosa.

The embodiments of the present invention have been described in detail. However, the present invention is not limited to the above-described embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present invention.

Claims

1. A preparation method of a cell composite endometrium repair gel is characterized by comprising the following steps:

step three, preparing temperature-sensitive chitosan hydrogel;

step four, preparing the cell composite endometrium repair gel.

2. The method for preparing the cell composite type endometrium repair gel according to claim 1, wherein the first step comprises the following steps:

(7) adding DMEM/F12 to the cell pellet retained in step (6), suspending the cell pellet in a complete culture medium, transferring the cell pellet to a cell culture flask, and culturing the cell pellet at 37 ℃ and 5 v/v% CO₂Culturing in an incubator, performing half-amount liquid change by using a DMEM/F12 complete culture medium for the first time after culturing for 24-48h, performing full-amount liquid change by using a DMEM/F12 complete culture medium every 2-3 days later, discarding non-adherent cells, adding a 0.25% pancreatin aqueous solution containing 0.02% of EDTA for passage when primary cells grow and fuse to 80% -90%, and performing passage by using 6 x 10 EDTA³Individual cell/cm²Subculturing at the density of (D) to third generation or moreObtaining endometrial stem cells, wherein:

3. The method for preparing a cell composite type endometrium repair gel according to claim 2, wherein the endometrium stem cell obtained in the first step is identified, and can be used as a raw material for preparing the cell composite type endometrium repair gel after qualified identification, and the identification of the endometrium stem cell comprises identification of a surface marker of the endometrium stem cell, identification of osteogenic differentiation and identification of adipogenic differentiation, wherein:

(c) identification of endometrial stem cell adipogenic differentiation

4. The method for preparing the cell composite type endometrium repair gel according to claim 1, wherein the mesenchymal stem cell in the second step is an umbilical cord mesenchymal stem cell, and the second step comprises the following steps:

(4) Adding DMEM/F12 complete medium into the culture dish, and placing the culture dish at 37 ℃ and 5 v/v% CO₂Well being full ofCulturing in a carbon dioxide incubator with humidity, performing half-amount liquid change after 4 days, performing liquid change once every 3 days after cell climbing out, transferring to the 3 rd generation, digesting the cells by using 0.25 wt% of pancreatin aqueous solution when the umbilical cord mesenchymal stem cells of the 3 rd generation are 80-90% fused, stopping digestion by using EMEM/F12 complete culture medium, slightly blowing the bottom of the culture dish and collecting the cells, centrifuging at 1000rpm for 5 minutes, discarding the supernatant, and keeping cell precipitation;

5. The preparation method of the cell composite type endometrium repair gel according to claim 4, wherein the passage method of the umbilical cord mesenchymal stem cells in the step (4) comprises the following steps: when the umbilical cord mesenchymal stem cells reach 80-90% fusion, digesting the cells by using a 0.25% pancreatin water solution containing 0.02% EDTA, stopping digestion by using a DMEM/F12 complete culture medium, slightly blowing the bottom of a culture dish, collecting the cells, centrifuging at 1000rpm for 5min, and performing the following steps of 1: passage at a ratio of 2-3.

6. The method for preparing the cell composite type endometrium repair gel of claim 1, wherein the mesenchymal stem cell in the second step is an adipose mesenchymal stem cell, and the second step comprises the following steps:

7. The preparation method of the cell composite type endometrium repair gel of claim 6, wherein the passage method of the adipose derived mesenchymal stem cells comprises the following steps: when the adipose-derived mesenchymal stem cells reach 80% -90% fusion, digesting the cells by using a 0.25% pancreatin water solution containing 0.02% EDTA, stopping digestion by using an HG-DMEM complete culture medium, slightly blowing the bottom of a bottle and collecting the cells, centrifuging at 1000rpm for 5min, and mixing the cells according to the ratio of 1: passage at a ratio of 2-3.

8. The method for preparing the cell composite type endometrium repair gel of claim 1, wherein the mesenchymal stem cell in the second step is a bone marrow mesenchymal stem cell, and the second step comprises the following steps:

step23, adding 5ml DMEM/F12 into a centrifuge tube, completely culturing and fully suspending to obtain a cell suspension, inoculating into a T25 cell culture bottle, placing at 37 ℃ and 5 v/v% CO₂Culturing in a saturated humidity incubator, changing liquid for the first time after 24-48h, removing non-adherent cells, changing liquid once every 2-3d, transferring to the 3 rd generation, digesting the cells by using 0.25% pancreatin solution cells containing 0.02% EDTA when the 3 rd generation mesenchymal stem cells are fused to 80% -90%, stopping digestion by using a DMEM/F12 complete culture medium, slightly blowing to beat the bottom of a bottle, collecting a wash bar, and centrifuging at 1000rpm for 5 min; and (3) removing the supernatant, adding 1ml of DMEM/F12 complete culture medium into the cell sediment, fully suspending, counting, and obtaining the 3 rd generation mesenchymal stem cells.

9. The method for preparing the cell composite endometrium repair gel according to claim 8, wherein the passage method of the bone marrow mesenchymal stem cells comprises the following steps: when the bone marrow mesenchymal stem cells reach 80% -90% fusion, digesting the cells by using a 0.25% pancreatin water solution containing 0.02% EDTA, stopping digestion by using an HG-DMEM complete culture medium, slightly blowing to beat the bottom of a bottle and collecting the cells, centrifuging at 1000rpm for 5min, and mixing the cells according to the ratio of 1: passage at a ratio of 2-3.

10. The method for preparing the cell composite type endometrium repair gel of claim 1, wherein the mesenchymal stem cell in the second step is a placental mesenchymal stem cell, and the second step comprises the following steps:

step23, adding 5ml of DMEM/F12 complete culture medium into the centrifuge tube for fully suspending to obtain a cell suspension, inoculating the cell suspension into a T25 cell culture bottle, placing the cell suspension in a5 v/v% CO culture bottle at 37 DEG C₂Culturing in a saturated humidity incubator, carrying out liquid exchange for the first time after 24-48h, removing non-adherent cells, then carrying out liquid exchange once every 2-3d, transferring to the 3 rd generation, digesting the cells by using a 0.25% pancreatin water solution containing 0.02% EDTA when the 3 rd generation placental mesenchymal stem cells reach 80-90% fusion, stopping digestion by using a DMEM/F12 complete culture medium, gently blowing to blow the bottom of a bottle and collecting the cells, centrifuging at 1000rpm for 5min, discarding supernatant, adding 1ml of DMEM/F12 complete culture medium into cell sediment, fully suspending to obtain cell suspension, counting the cell suspension, and obtaining the 3 rd generation placental mesenchymal stem cells after completion, wherein:

11. The method for preparing the cell composite endometrium repair gel of claim 10, wherein the passage method of the placenta mesenchymal stem cells comprises the following steps: when the placenta mesenchymal stem cells reach 80-90% fusion, digesting the cells by using a 0.25% pancreatin water solution containing 0.02% EDTA, stopping digestion by using an HG-DMEM complete culture medium, slightly blowing the bottom of a bottle and collecting the cells, centrifuging at 1000rpm for 5min, and mixing the cells according to the weight ratio of 1: passage at a ratio of 2-3.

12. The preparation method of the cell composite type endometrium repair gel of claim 1, wherein the third step comprises the following steps:

(31) preparation of chitosan solution

(32) preparation of type I collagen solution

(33) preparation of beta-sodium glycerophosphate solution

(34) preparation of sodium alginate solution

(35) preparing temperature-sensitive chitosan hydrogel

13. The preparation method of the cell composite type endometrium repair gel of claim 1, wherein the fourth step comprises the following steps:

14. Use of a cell complex endometrial repair gel according to any one of claims 1 to 13 for the treatment of intrauterine adhesions.

15. The use of claim 14, wherein after the cell composite endometrium repair gel is implanted locally in the uterine cavity, the cell composite endometrium repair gel is rapidly aggregated at the body temperature of 37 ℃, so that the cell stays in the uterine cavity and is attached to the endometrium to repair the endometrium.