CN111893087A

CN111893087A - Endometrium collection device and endometrium stem cell harvesting method

Info

Publication number: CN111893087A
Application number: CN202010814320.6A
Authority: CN
Inventors: 曹毓琳; 林俊堂; 滕睿頔; 白志惠; 贺伟
Original assignee: Beijing Zhen Huikang Biological Technology Co ltd
Current assignee: Beijing Zhen Huikang Biological Technology Co ltd
Priority date: 2020-08-13
Filing date: 2020-08-13
Publication date: 2020-11-06
Anticipated expiration: 2040-08-13
Also published as: CN111893087B

Abstract

The invention provides an endometrium collecting device, which comprises a tampon (1) and a microcarrier (2) arranged on the tampon (1), wherein the microcarrier (2) comprises a gel ball core formed by crosslinking degradable microcarrier matrix materials, a gelatin layer coated on the ball core and DEAE coupled on gelatin; the degradable microcarrier matrix material is alginate or pectin, and the alginate is calcium alginate; the endometrium collecting device provided by the invention can solve the problem that the endometrium in the collected menstrual blood is difficult to separate in the prior art, can obviously improve the separation amount of the endometrium in the menstrual blood, and can obtain a large amount of endometrium stem cells after culture.

Description

Endometrium collection device and endometrium stem cell harvesting method

Technical Field

The invention belongs to the technical field of stem cells, and particularly relates to an endometrium collecting device and an endometrium stem cell harvesting method.

Background

The endometrial stem cells are a kind of adult stem cells existing in the endometrial basal layer, have the self-renewal, unlimited proliferation and multidirectional differentiation potential of common adult stem cells, participate in the periodic regeneration of endometrial epithelium and mesenchymal cells, and have a vital role in the dynamic regeneration and repair of endometrium.

Menstrual blood is a mixed liquid of blood and some fallen endometrium, the year and night of cervix, and the like, and in 2008, american scientist Patel and the like found that endometrial stem cells can be separated from menstrual blood of healthy women, and due to the advantages of abundant sources of menstrual blood, non-invasive collection process, non-ethical disputes and the like, more endometrium is hopefully separated from the menstrual blood, but when the menstrual blood is attached to a cotton sliver or other mediums, the endometrium in the menstrual blood is difficult to centrifugally collect.

Disclosure of Invention

In order to solve the technical problems, the invention provides an endometrium collecting device which comprises a tampon and a microcarrier arranged on the tampon, wherein the microcarrier comprises a gel ball core formed by crosslinking degradable microcarrier matrix materials, a gelatin layer coated on the ball core and DEAE coupled on gelatin; the degradable microcarrier matrix material is alginate or pectin, and the alginate is calcium alginate.

DEAE is cellulose, and it is known that when menstrual blood is directly attached to a tampon, the endometrium is difficult to separate from the tampon.

Based on the conventional usage of the tampon, all the endometrial collecting devices of the present invention can further comprise structures such as a catheter, a propulsion tube, etc., and the connection relationship among the structures is the same as that of the existing tampon, and the description is omitted.

Further, the cross section of the tampon is U-shaped, the surface of the tampon is coated with a net structure, and the microcarrier is attached to the net structure.

The microcarrier is arranged on the net-shaped structure in the cotton sliver, the endometrium is adsorbed on the net-shaped structure when the microcarrier is used, and only the net-shaped structure needs to be centrifuged when stem cells are separated, so that the microcarrier is simple and convenient to operate.

Further, the mesh size of the mesh structure is between 0.3cm and 1.5 cm.

According to the invention, by limiting the mesh size of the mesh structure, most endometrium can be successfully intercepted at the mesh structure, and blood or mucus and the like are absorbed by the tampon through the mesh structure, so that more endometrium cells can be harvested after the mesh structure is centrifuged and digested.

Furthermore, the bottom end of the reticular structure is provided with an opening, and the endometrium collecting device also comprises a plurality of pull ropes; all the pull ropes are fixedly connected to the bottom of the tampon, one of the pull ropes passes through the opening at the bottom end of the net structure, and the rest of the pull ropes pass through the grids of the net structure.

Wherein the openings of the net structure are large enough for the tampon to pass through; the plurality of pull ropes can be arranged according to the requirement, and the net-shaped structure can be easily peeled off from the tampon by arranging the plurality of pull ropes.

Furthermore, the mesh structure is provided with a plurality of layers from inside to outside, and the mesh size of the mesh structure is increased from inside to outside in sequence.

By arranging the multilayer mesh structures and limiting the mesh sizes of all the layers of mesh structures, namely, the outer layer mesh structure intercepts a large endometrium, the inner layer mesh structure intercepts a endometrium with a relatively small volume, and all the layers of mesh structures are in work and cooperation, so that the endometrium stem cells can be better adsorbed.

Further, the preparation method of the microcarrier comprises the following steps:

(1) preparing balls: dripping 1-2% nano sodium alginate solution into 0.5-3% calcium chloride solution to react to generate calcium alginate gel beads, wherein the volume ratio of the nano sodium alginate solution to the calcium chloride solution is 1:3-1: 5;

(2) washing: removing the excess calcium chloride solution and washing once with sterile purified water;

(3) coating: uniformly mixing an adsorption solution with the concentration of 10-20% and a glutaraldehyde solution with the concentration of 0.3-1% in equal proportion to prepare a mixed solution, soaking calcium alginate micro-gel beads in the mixed solution for reaction, wherein the volume ratio of the mixed solution to the nano sodium alginate solution is 1-3: 1;

(4) washing: after the coating reaction is finished, removing redundant mixed liquid, and washing with sterile purified water for three times;

(5) neutralizing: adding 0.5-2% glycine solution to react, wherein the volume of the glycine solution is the same as that of the mixed solution in the step (3);

(6) washing: the waste liquid after reaction is discarded and washed with sterile purified water for three times;

(7) coupling DEAE-HCl: taking washed calcium alginate micro-gel beads, adding 1-3mol/L NaOH solution, stirring, adding 0.5-2mol/L DEAE-HCl solution, and stirring; the ratio of the volume of the calcium alginate micro-gel beads to the volume of the NaOH solution and the volume of the DEAE-HCl solution is 1:1-3: 1-3;

(8) washing: removing excess NaOH solution and DEAE-HCl solution, sequentially using sterile purified water, 0.1M hydrochloric acid, 0.1mM hydrochloric acid, and Ca-free²⁺And Mg²⁺Washing with PBS buffer solution (5 rpm), stirring at 300rpm for 8min, discharging the washing solution, and washing with sterile purified water, 0.1M hydrochloric acid, 0.1mM hydrochloric acid, and no Ca²⁺、Mg²⁺Volume ratio of PBS buffer solution to calcium alginate micro-gel beads1-3:1 in all cases, and preparing a wet microcarrier;

(9) freeze-drying: and (4) freeze-drying the microcarrier obtained in the step (8) to obtain the microcarrier.

Further, the adsorption factor comprises chitosan, fibronectin and gelatin in a mass ratio of 1-2:1-2: 5-10; preferably, the adsorption factor comprises chitosan, fibronectin and gelatin in a mass ratio of 1:1: 5. It was found that gelatin has a general effect of adsorbing endometrial stem cells when coated, and microcarriers prepared using an adsorption factor composed of chitosan, fibronectin and gelatin in the above ratio have a significantly improved effect of adsorbing endometrial stem cells.

Further, the specific operation method of washing at least one of the step (2), the step (4) and the step (6) with sterile purified water is as follows: stirring at 50-300rpm for 5-10min, removing the washing solution to remove excessive liquid, and mixing the purified water and calcium alginate microcapsule beads at a volume ratio of 1-3: 1.

Further, the reaction conditions of the step (3) of soaking the calcium alginate micro-gel beads in the mixed solution are as follows: the temperature is 20-60 ℃, the stirring speed is 50-300rpm, and the time is 30min-2 h; the reaction conditions of step (5) are as follows: the neutralization temperature is 40-60 ℃, the stirring speed is 50-300rpm, and the neutralization time is 2-3 h; the specific conditions of the two times of stirring in the step (7) are as follows: the stirring speed is 50-300rpm, the stirring temperature is 60-80 ℃, and the stirring time is 0.5-2 h; the washing method of the step (8) is as follows: stirring at 50-300rpm for 5-10min, and removing the washing solution.

The invention also provides a method for harvesting the endometrium stem cells, which comprises the steps of placing the tampon or the reticular structure (3) adhered with the endometrium in a centrifugal tube, adding the normal saline, centrifuging for 15-25min at the rotating speed of 1500-2500rpm, taking out the centrifuged tampon or the reticular structure (3), adding the centrifuged tampon or the reticular structure into a new centrifugal tube, adding EDTA (ethylene diamine tetraacetic acid) for digesting for 5-10min, immersing the tampon or the reticular structure (3) in the EDTA, washing the tampon or the reticular structure (3) after digestion, collecting washing liquid, centrifuging for 4-6min at the speed of 1500-2500rpm, taking out the sediment to obtain the endometrium cells, and culturing the prepared endometrium cells.

The above washing was carried out using 0.9% sodium chloride solution.

The invention also provides a method for culturing the endometrial stem cells, which comprises the steps of putting the tampon or the reticular structure (3) adhered with the endometrium into a reactor, and culturing at 37 ℃ and 5% CO 2; the first change was performed 24 hours later, every 3 days thereafter, and passage was performed after the cells had grown to confluence. The passage method comprises the following steps: sucking out old culture medium in the culture bottle, adding PBS for washing for 3 times, adding digestive juice for digesting for 5min, wherein the digestive juice comprises 0.25% pancreatin and 0.04% EDTA in a volume ratio of 1: 1; gently blowing and beating the cells to separate the cells from the bottom of the bottle to obtain a single cell suspension; and centrifuging and blowing off the single cell suspension, and carrying out passage according to the ratio of 1: 8.

The endometrium collecting device provided by the invention can solve the problem that the endometrium in the collected menstrual blood is difficult to separate in the prior art, can obviously improve the yield of the endometrium in the menstrual blood, and can collect a large amount of endometrium stem cells after separation, digestion and culture.

Drawings

FIG. 1 is a schematic structural view of the endometrial collection device of example 1;

FIG. 2 is a schematic structural view of the endometrial collection device of example 1;

FIG. 3 is a schematic structural view of the endometrial collection device of example 1;

FIG. 4 is a schematic structural view of the endometrial collection device of example 1;

wherein, 1 is a tampon, 2 is a microcarrier, 3 is a net structure, 4 is an opening, and 5 is a pull rope.

Detailed Description

Example 1

This example provides a microcarrier prepared as follows:

(1) preparing balls: dripping 5L of 1% nanocrystallized sodium alginate solution into 0.5% calcium chloride solution to react to generate calcium alginate gel beads, wherein the volume ratio of the nanocrystallized sodium alginate solution to the calcium chloride solution is 1: 3;

(2) washing: removing redundant calcium chloride solution, washing with sterile purified water once, wherein the washing method comprises adding purified water, stirring at 50rpm for 5min, discarding washing solution to remove redundant liquid, and the volume ratio of the purified water to the calcium alginate micro-gel beads is 1: 1;

(3) coating: uniformly mixing 10L of 10% adsorption solution and 10ml of 0.3% glutaraldehyde solution in equal proportion to obtain a mixed solution, soaking calcium alginate micro-gel beads in the mixed solution, and reacting for 30min at the temperature of 20 ℃ and the stirring speed of 50rpm, wherein the volume ratio of the mixed solution to the nano sodium alginate solution is 1: 1;

(4) washing: after the coating reaction is finished, removing redundant mixed liquid, washing the mixture for three times by using sterile purified water, adding purified water, stirring the mixture at the rotating speed of 50rpm for 5min, and removing the redundant liquid by using the washing liquid, wherein the volume ratio of the purified water to the calcium alginate micro-gel beads is 1: 1;

(5) neutralizing: adding 0.5% glycine solution, neutralizing for 2h at 40 deg.C and stirring speed of 50rpm, wherein the volume of glycine solution is the same as that of the mixed solution in step (3);

(6) washing: discarding the waste liquid after the reaction, washing the waste liquid for three times by using sterile purified water, adding the purified water, stirring the mixture at a rotating speed of 50rpm for 5min, discarding the washing liquid to remove redundant liquid, wherein the volume ratio of the purified water to the calcium alginate micro-gel beads is 1: 1;

(7) coupling DEAE-HCl: adding 1mol/L NaOH solution into the washed calcium alginate micro-gel beads, stirring, and adding 0.5mol/L DEAE-HCl solution, and stirring; the ratio of the volume of the calcium alginate micro-gel beads to the volume of the NaOH solution and the volume of the DEAE-HCl solution is 1:1: 1; the specific conditions of the two times of stirring are as follows: the stirring speed is 50rpm, the stirring temperature is 60 ℃, and the stirring time is 0.5 h;

(8) washing: removing excess NaOH solution and DEAE-HCl solution, sequentially using sterile purified water, 0.1M hydrochloric acid, 0.1mM hydrochloric acid, and Ca-free²⁺And Mg²⁺Washing with PBS buffer solution (5 min) at 50rpm, discharging the washing solution, and washing with sterile purified water, 0.1M hydrochloric acid, 0.1mM hydrochloric acid, and Ca-free solution²⁺、Mg²⁺The volume ratio of the PBS buffer solution to the calcium alginate micro-gel beads is 1:1, and a wet microcarrier is prepared;

(9) freeze-drying: freeze-drying the microcarrier obtained in the step (8), and performing radiation sterilization by using cobalt 60 gamma rays, wherein the radiation dose is 20KGy, so as to prepare the microcarrier;

the adsorption solution is prepared by dissolving adsorption factors with water, wherein the adsorption factors comprise chitosan, fibronectin and gelatin in a mass ratio of 1:2: 8;

the chitosan, fibronectin and gelatin involved in all examples and controls of the present invention were purchased from Sigma company of usa and will not be described in detail below.

Example 2

This example provides a microcarrier prepared as follows:

(1) preparing balls: dripping 5L of 1.5% nanocrystallized sodium alginate solution into 1.5% calcium chloride solution to react to generate calcium alginate gel beads, wherein the volume ratio of the nanocrystallized sodium alginate solution to the calcium chloride solution is 1: 4;

(2) washing: removing redundant calcium chloride solution, washing with sterile purified water once, wherein the washing method comprises adding purified water, stirring at 175rpm for 8min, discarding washing solution to remove redundant liquid, and the volume ratio of purified water to calcium alginate micro-gel beads is 2: 1;

(3) coating: uniformly mixing 10L of 15% adsorption solution and 20mL of 0.6% glutaraldehyde solution to obtain a mixed solution, soaking calcium alginate micro-gel beads in the mixed solution, and reacting for 1.5h at the temperature of 40 ℃ and the stirring speed of 175rpm, wherein the volume ratio of the mixed solution to the nano sodium alginate solution is 2: 1;

(4) washing: after the coating reaction is finished, removing redundant mixed liquid, washing the mixture for three times by using sterile purified water, adding purified water, stirring the mixture for 8min at the rotating speed of 175rpm, and removing the redundant liquid by using the washing liquid, wherein the volume ratio of the purified water to the calcium alginate micro-gel beads is 2: 1;

(5) neutralizing: adding 1.5% glycine solution, and neutralizing at 50 deg.C and stirring speed of 175rpm for 2.5h, wherein the volume of glycine solution is the same as that of the mixed solution in step (3);

(6) washing: discarding the waste liquid after the reaction, washing the waste liquid for three times by using sterile purified water, adding the purified water, stirring the mixture for 8min at the rotating speed of 175rpm, discarding the washing liquid to remove redundant liquid, wherein the volume ratio of the purified water to the calcium alginate micro-gel beads is 2: 1;

(7) coupling DEAE-HCl: adding a NaOH solution with the concentration of 2mol/L into the washed calcium alginate micro-gel beads, stirring, and then adding a DEAE-HCl solution with the concentration of 1.5mol/L, and stirring; the ratio of the volume of the calcium alginate micro-gel beads to the volume of the NaOH solution and the volume of the DEAE-HCl solution is 1:2: 2; the specific conditions of the two stirring steps are as follows: the stirring speed is 150rpm, the stirring temperature is 70 ℃, and the stirring time is 1.5 h;

(8) washing: removing excess NaOH solution and DEAE-HCl solution, sequentially using sterile purified water, 0.1M hydrochloric acid, 0.1mM hydrochloric acid, and Ca-free²⁺And Mg²⁺The PBS buffer solution is washed, each washing is stirred for 8min at the rotating speed of 175rpm, then washing solution is discharged, and the volume ratio of the sterile purified water, 0.1M hydrochloric acid, 0.1mM hydrochloric acid, Ca-free 2+, Mg2+ PBS buffer solution and calcium alginate bead is 2:1, preparing a wet microcarrier;

(9) freeze-drying: freeze-drying the microcarrier obtained in the step (8), and performing radiation sterilization by using cobalt 60 gamma rays, wherein the radiation dose is 19KGy, so as to prepare the microcarrier;

the adsorption solution is prepared by dissolving adsorption factors with water, wherein the adsorption factors comprise chitosan, fibronectin and gelatin in a mass ratio of 1:1: 5.

Example 3

This example provides a microcarrier prepared as follows:

(1) preparing balls: dripping 5L of 2% nanocrystallized sodium alginate solution into 3% calcium chloride solution to react to generate calcium alginate gel beads, wherein the volume ratio of the nanocrystallized sodium alginate solution to the calcium chloride solution is 1: 5;

(2) washing: removing redundant calcium chloride solution, washing with sterile purified water once, wherein the washing method comprises the steps of adding purified water, stirring at the rotating speed of 300rpm for 10min, removing the redundant liquid by discarding the washing solution, and the volume ratio of the purified water to the calcium alginate micro-gel beads is 3: 1;

(3) coating: uniformly mixing 10L of 20% adsorption solution and 30mL of 1% glutaraldehyde solution to obtain a mixed solution, soaking calcium alginate micro-gel beads in the mixed solution, and reacting for 2 hours at the temperature of 60 ℃ and the stirring speed of 300rpm, wherein the volume ratio of the mixed solution to the nano sodium alginate solution is 3: 1;

(4) washing: after the coating reaction is finished, removing redundant mixed liquid, washing the mixture for three times by using sterile purified water, adding purified water, stirring the mixture at the rotating speed of 300rpm for 10min, and removing the redundant liquid by using the washing liquid, wherein the volume ratio of the purified water to the calcium alginate micro-gel beads is 3: 1;

(5) neutralizing: adding 2% glycine solution, neutralizing for 3h at 60 deg.C and stirring speed of 300rpm, wherein the volume of glycine solution is the same as that of the mixed solution in step (3);

(6) washing: discarding the waste liquid after the reaction, washing the waste liquid for three times by using sterile purified water, adding the purified water, stirring the mixture at the rotating speed of 300rpm for 10min, discarding the washing liquid to remove redundant liquid, wherein the volume ratio of the purified water to the calcium alginate micro-gel beads is 3: 1;

(7) coupling DEAE-HCl: adding the washed calcium alginate micro-gel beads into a NaOH solution with the concentration of 3mol/L for stirring, and then adding a DEAE-HCl solution with the concentration of 2mol/L for stirring; the ratio of the volume of the calcium alginate micro-gel beads to the volume of the NaOH solution and the volume of the DEAE-HCl solution is 1:3: 3; the specific conditions of the two stirring steps are as follows: the stirring speed is 300rpm, the stirring temperature is 80 ℃, and the stirring time is 2 hours;

(8) washing: removing excess NaOH solution and DEAE-HCl solution, sequentially using sterile purified water, 0.1M hydrochloric acid, 0.1mM hydrochloric acid, and Ca-free²⁺And Mg²⁺The PBS buffer solution is washed, the washing solution is discharged after stirring for 8min at the rotating speed of 300rpm for each washing, and the volume ratio of the sterile purified water, 0.1M hydrochloric acid, 0.1mM hydrochloric acid, Ca-free 2+, Mg2+ PBS buffer solution and calcium alginate bead is 3:1, preparing a wet microcarrier;

the adsorption solution is prepared by dissolving adsorption factors with water, wherein the adsorption factors comprise chitosan, fibronectin and gelatin in a mass ratio of 2:1: 10.

Example 4

The embodiment provides an endometrium collecting device, as shown in fig. 1, the endometrium collecting device comprises a tampon 1 and a microcarrier 2 arranged on the tampon 1, the preparation method is that the microcarrier and the tampon material fluff fiber are mixed uniformly, the endometrium collecting device also comprises a conduit part which is not shown in the figure, a tampon body is sleeved in one end of the conduit, a propelling tube is sleeved in the other end of the conduit, a pull rope is arranged at one end of the tampon body close to the propelling tube, and the pull rope passes through the propelling tube;

the microcarriers in this example were prepared using the method of example 1;

the tampon of this example is provided with 1g of microcarriers.

Example 5

The embodiment provides an endometrium collecting device, as shown in fig. 2, the endometrium collecting device comprises a tampon 1, the cross section of the tampon 1 is in a U shape, a reticular structure 3 is wrapped on the surface of the tampon, a microcarrier 2 is attached to the reticular structure 3, the reticular structure is sleeved on the outer side wall of the tampon, the reticular structure is made of cotton and artificial fiber, the material of the reticular structure is the same as that of the tampon in the market and is only made into a reticular structure, the connection mode of the microcarrier and the reticular structure is also that the microcarrier and the villus fiber of the reticular structure are uniformly mixed, the endometrium collecting device further comprises a conduit (part of components are not shown in the figure), a tampon body is sleeved inside one end of the conduit, a propelling tube is sleeved inside the other end of the conduit, a pulling rope is arranged at one end of the tampon body close to the propelling tube, and the pulling; the mesh size of the mesh structure is 0.5 cm;

the microcarriers in this example were prepared using the method of example 2;

the tampon of this example is provided with 1g of microcarriers;

in the comparison example, the cross section of the tampon is limited to be U-shaped, the net-shaped structure is arranged, and the microcarrier is arranged on the net-shaped structure, so that the coverage area of the net-shaped structure can be obviously improved, and the adsorption amount of the tampon to endometrial stem cells is increased.

Example 6

The embodiment provides an endometrium collecting device, as shown in fig. 3, the endometrium collecting device comprises a tampon, a reticular structure 3 is wrapped on the surface of the tampon, an opening 4 is arranged at the bottom end of the reticular structure 3, and the endometrium collecting device further comprises 3 pull ropes 5; all the pull ropes 5 are fixedly connected to the bottom of the tampon 1, wherein one pull rope 5 passes through an opening at the bottom end of the net-shaped structure 3, and the rest 2 pull ropes 5 pass through the grids of the net-shaped structure 3; the outer side wall of the tampon is sleeved with the reticular structure, the material of the reticular structure is cotton and artificial fiber, the material of the reticular structure is the same as that of the tampon on the market, only the reticular structure is made, the connection mode of the microcarrier and the reticular structure is also that the microcarrier and the fluff fiber of the reticular structure are uniformly mixed, the endometrium collecting device also comprises a conduit part (not shown in the figure of a component), the tampon is sleeved in one end of the conduit, a propelling pipe is sleeved in the other end of the conduit, and a pull rope at the bottom of the tampon passes through the propelling pipe; the mesh size of the mesh structure is 0.5 cm; the openings of the net structure are large enough for the tampon to pass through;

the microcarriers in this example were prepared using the method of example 3;

the tampon of this example is provided with 1g of microcarriers;

in the embodiment, the net-shaped structure and the tampon can be quickly peeled off by arranging the plurality of pull ropes, and the using method comprises the following steps: during the use, pull 3 stay cords simultaneously and take out the tampon that has absorbed menstrual blood from the chamber way, hold the network structure of aseptic tweezers fixed tampon top (being the open end of U type structure) in one hand, pull the stay cord that stretches out from the network structure open end in the other hand opposite direction, at this moment, the network structure is fixed motionless, tampon downstream, two other stay cords follow tampon downstream, break away from in the network of network structure, until whole tampon breaks away from in network structure, and is convenient quick, easy to operate.

Example 7

The embodiment provides an endometrium collecting device, as shown in fig. 3, the endometrium collecting device comprises a tampon 1, the cross section of the tampon 1 is U-shaped, a reticular structure 3 is wrapped on the surface of the tampon, 2 layers of the reticular structure 3 are arranged from inside to outside, the mesh size of the reticular structure 3 is sequentially increased from inside to outside, a microcarrier 2 is attached to the reticular structure 3, the reticular structure is sleeved on the outer side wall of the tampon, the reticular structure is made of cotton and artificial fiber, is the same as the tampon in the market and is only made into the reticular structure, the connection mode of the microcarrier and the reticular structure is also the mode of uniformly mixing the microcarrier and the villus fiber of the reticular structure, the endometrium collecting device further comprises a conduit (part of which is not shown in the figure), a tampon body is sleeved inside one end of the conduit, a propelling tube is sleeved inside the other end of the conduit, a pull rope is arranged at one end of the tampon body close to the propelling pipe and penetrates through the propelling pipe; the mesh sizes of the mesh structure are respectively 1cm and 0.3cm from outside to inside;

the microcarriers in this example were prepared using the method of example 2;

the tampon of this example is provided with 1g of microcarriers;

this embodiment is through setting up two-layer grid structure, and two-layer grid structure's mesh size is different, can intercept the endometrium that the size is different, and the cooperation of dividing the work of work adsorbs more endometrium stem cells.

Example 8

The embodiment provides an endometrial stem cell harvesting method, which comprises the steps of placing a tampon adhered with an endometrium in a centrifugal tube, adding normal saline, centrifuging for 15min, immersing the tampon in the normal saline at a rotating speed of 1500rpm, taking out the centrifuged tampon, adding the centrifuged tampon into a new centrifugal tube, adding EDTA (ethylene diamine tetraacetic acid) for digestion for 5min, immersing the tampon in the EDTA, washing the tampon with 0.9% sodium chloride solution after digestion, collecting washing liquid, centrifuging at a speed of 1500rpm for 4min, taking sediment to obtain endometrial cells, culturing the prepared endometrial cells, and culturing the obtained endometrial cells in a culture methodThe method comprises the following steps: cell suspension was prepared at 1X 10⁶/cm²Inoculating into culture flask, and adding 5% CO at 37 deg.C₂Culturing in an incubator; the first fluid change was performed after 12 hours, every 3 days thereafter, and passaging was performed after the cells had grown to confluence. The passage method comprises the following steps: sucking out old culture medium in the culture bottle, adding PBS for washing for 2 times, adding digestive juice for digestion for 3min, wherein the digestive juice comprises 0.25% pancreatin and 0.04% EDTA in a volume ratio of 1: 1; gently blowing and beating the cells to separate the cells from the bottom of the bottle to obtain a single cell suspension; and centrifuging and blowing off the single cell suspension, and carrying out passage according to the ratio of 1: 8.

Example 9

The embodiment provides an endometrial stem cell harvesting method, the method is that a reticular structure adhered with an endometrium is placed in a centrifugal tube, normal saline is added into the centrifugal tube, the centrifugation is carried out for 20min, the rotating speed is 2000rpm, the centrifuged reticular structure is taken out, a new centrifugal tube is added, EDTA is added for digestion for 8min, the EDTA submerges the reticular structure, a 0.9% sodium chloride solution is used for washing a tampon after digestion, washing liquid is collected, the washing liquid is centrifuged at the speed of 2000rpm for 5min, sediment is taken to obtain endometrial cells, the prepared endometrial cells are cultured, and the culture method is as follows: the cell suspension was adjusted to 1.5X 10⁶/cm²Inoculating into culture flask, and adding 5% CO at 37 deg.C₂Culturing in an incubator; the first change was performed 18 hours later, every 3 days thereafter, and passage was performed after the cells had grown to confluence. The passage method comprises the following steps: sucking out old culture medium in the culture bottle, adding PBS for washing for 3 times, adding digestive juice for digestion for 4min, wherein the digestive juice comprises 0.25% pancreatin and 0.04% EDTA in a volume ratio of 1: 1; gently blowing and beating the cells to separate the cells from the bottom of the bottle to obtain a single cell suspension; and centrifuging and blowing off the single cell suspension, and carrying out passage according to the ratio of 1: 8.

Example 10

The embodiment provides an endometrial stem cell harvesting method, which comprises the steps of placing a reticular structure adhered with an endometrium in a centrifugal tube, adding normal saline, centrifuging for 25min at the rotating speed of 2500rpm, taking out the centrifuged reticular structure, adding the centrifuged reticular structure into a new centrifugal tube, adding EDTA (ethylene diamine tetraacetic acid) for digestion for 10min, immersing the reticular structure in the EDTA, washing a tampon with 0.9% sodium chloride solution after digestionCollecting washing liquid, centrifuging at 2500rpm for 6min, collecting precipitate to obtain endometrium cell, and culturing the obtained endometrium cell by the culture method: cell suspension was adjusted to 2X 10⁶/cm²Inoculating into culture flask, and adding 5% CO at 37 deg.C₂Culturing in an incubator; the first change was performed 24 hours later, every 3 days thereafter, and passage was performed after the cells had grown to confluence. The passage method comprises the following steps: sucking out old culture medium in the culture bottle, adding PBS for washing for 3 times, adding digestive juice for digesting for 5min, wherein the digestive juice comprises 0.25% pancreatin and 0.04% EDTA in a volume ratio of 1: 1; gently blowing and beating the cells to separate the cells from the bottom of the bottle to obtain a single cell suspension; and centrifuging and blowing off the single cell suspension, and carrying out passage according to the ratio of 1: 8.

Comparative example 1

This comparative example provides a microcarrier which differs from example 2 in that the 15% strength adsorption solution is replaced by a 15% strength gelatin solution prepared by dissolving gelatin in water.

Comparative example 2

This comparative example provides a microcarrier which differs from example 2 in that the 15% strength adsorption solution was replaced with a 15% strength chitosan solution prepared by dissolving chitosan in water.

Comparative example 3

This comparative example provides a microcarrier which differs from example 2 in that the 15% concentrated adsorption solution was replaced with a 15% concentrated fibronectin solution prepared by dissolving fibronectin in water.

Comparative example 4

The control example provides an endometrium collecting device, which comprises a tampon and a microcarrier arranged on the tampon, and the preparation method is that the microcarrier and the tampon material fluff fiber are mixed uniformly, the endometrium collecting device also comprises a guide pipe part which is not shown in the figure, one end of the guide pipe is internally sheathed with a tampon body, the other end of the guide pipe is internally sheathed with a propelling pipe, one end of the tampon body close to the propelling pipe is provided with a pull rope, and the pull rope passes through the propelling pipe;

the microcarrier in the comparative example was prepared by the method of comparative example 1;

the tampon of this comparative example was provided with 1g of microcarriers.

Comparative example 5

the microcarrier in the comparative example was prepared by the method of comparative example 2;

the tampon of this comparative example was provided with 1g of microcarriers.

Comparative example 6

the tampon of this comparative example was provided with 1g of microcarriers.

Test example 1 endometrial Collection Effect

Collecting menstrual blood: collecting 60ml of menstrual blood samples of a healthy female according to the menstrual blood collection package instruction after consultation and consent, diluting the menstrual blood samples to 180ml with 0.9% sodium chloride solution, equally dividing the menstrual blood samples into 12 parts, each of which is 10ml, placing the menstrual blood samples into 10ml test tubes, taking 3 and 18 of the endometrial collection devices of examples 4-6 and comparative examples 4-6, placing the tampons of the endometrial collection devices of examples 4 and comparative examples 2 into the test tubes for adsorption, placing the tampons and the net structures of the endometrial collection devices of examples 5-6 and comparative examples 5-6 into the test tubes for adsorption, taking out the endometrial collection devices of each group after all menstrual blood diluent is adsorbed by the endometrial collection devices of each group, and performing separation culture on the tampons of example 4 and comparative example 4 by the method of example 8, the networks of examples 5 to 6 and comparative examples 5 to 6 were separately cultured by the methods of examples 9 to 10, and the number of harvested cells after separation and the cell growth factor after culture were measured, respectively, and the results are shown in Table 1.

Table 1. number of cells collected and isolated by each set of methods and proliferation fold of cells after culture.

As can be seen from table 1, the number of cells collected by the endometrial collection device of examples 4 to 6 is significantly higher than that of the control examples 4 to 6, which proves that the microcarrier provided by the present invention significantly improves the adsorption of the microcarrier to endometrial stem cells by improving the components of the coating step, while the number of cells collected by examples 5 to 6 is higher than that of example 4, which proves that the harvesting amount of endometrial stem cells can be significantly increased by collecting the endometrium using the mesh structure instead of the whole tampon, and when the whole tampon is used, a small portion of endometrial stem cells are attached to the tampon and cannot be harvested by centrifugation.

Therefore, the invention is not limited to the specific embodiments and examples, but rather, all equivalent variations and modifications are within the scope of the invention as defined in the claims and the specification.

Claims

1. An endometrium collection device, which is characterized by comprising a tampon (1) and a microcarrier (2) arranged on the tampon (1), wherein the microcarrier (2) comprises a gel ball core formed by crosslinking degradable microcarrier matrix materials, a gelatin layer coated on the ball core and DEAE coupled on gelatin; the degradable microcarrier matrix material is alginate or pectin, and the alginate is calcium alginate.

2. The endometrial collection device of claim 1, wherein the cross-section of the tampon (1) is "U" shaped, and wherein a mesh structure (3) is attached to the surface of the tampon, and wherein the microcarriers (2) are attached to the mesh structure (3).

3. An endometrial harvesting device according to claim 2, characterized in that the mesh structure (3) has a mesh size between 0.3cm and 1.5 cm.

4. The endometrial collection device according to claim 2, wherein the bottom end of the mesh structure (3) is provided with an opening (4), and the endometrial collection device further comprises a plurality of pull cords (5); all the pull ropes (5) are fixedly connected to the bottom of the tampon (1), one pull rope (5) passes through an opening at the bottom end of the net-shaped structure (3), and the rest pull ropes (5) pass through the meshes of the net-shaped structure (3).

5. The endometrial collection device of claim 2, wherein the mesh structure (3) comprises a plurality of layers from the inside to the outside, and the mesh size of the mesh structure (3) increases from the inside to the outside.

6. The endometrial collection device of claim 1, wherein the microcarrier is prepared by a method comprising:

(3) coating: uniformly mixing an adsorption solution with the concentration of 10% -20% and a glutaraldehyde solution with the concentration of 0.3% -1% in equal proportion to prepare a mixed solution, soaking the calcium alginate micro-gel beads in the mixed solution for reaction, wherein the volume ratio of the mixed solution to the nano sodium alginate solution is 1-3: 1;

(7) coupling DEAE-HCl: taking washed calcium alginate micro-gel beads, adding 1-3mol/L NaOH solution, stirring, adding 0.5-2mol/L DEAE-HCl solution, and stirring; the ratio of the volume of the calcium alginate micro-gel beads to the volume of the NaOH solution to the volume of the DEAE-HCl solution is 1:1-3: 1-3;

(8) washing: removing excess NaOH solution and DEAE-HCl solution, sequentially using sterile purified water, 0.1M hydrochloric acid, 0.1mM hydrochloric acid, and Ca-free²⁺And Mg²⁺Washing with PBS buffer solution (5 rpm), stirring at 300rpm for 8min, discharging the washing solution, and washing with sterile purified water, 0.1M hydrochloric acid, 0.1mM hydrochloric acid, and no Ca²⁺、Mg²⁺The volume ratio of the PBS buffer solution to the calcium alginate micro-gel beads is 1-3:1, and a wet microcarrier is prepared;

(9) freeze-drying: freeze-drying the microcarrier obtained in the step (8) to obtain a microcarrier;

the adsorption solution is prepared by dissolving adsorption factors in water.

7. The endometrial harvesting device of claim 6, wherein the adsorption factors comprise chitosan, fibronectin, and gelatin in a mass ratio of 1-2:1-2: 5-10; preferably, the adsorption factors include chitosan, fibronectin and gelatin in a mass ratio of 1:1: 5.

8. The endometrial collection device of claim 6, wherein at least one of step (2), step (4) and step (6) is performed by washing with sterile purified water: stirring at the rotating speed of 50-300rpm for 5-10min, and removing the washing solution to remove redundant liquid, wherein the volume ratio of the purified water to the calcium alginate micro-gel beads is 1-3: 1; the reaction conditions of the step (3) of soaking the calcium alginate micro-gel beads in the mixed solution for reaction are as follows: the temperature is 20-60 ℃, the stirring speed is 50-300rpm, and the time is 30min-2 h; the reaction conditions in the step (5) are as follows: the neutralization temperature is 40-60 ℃, the stirring speed is 50-300rpm, and the neutralization time is 2-3 h; the specific conditions of the two times of stirring in the step (7) are as follows: the stirring speed is 50-300rpm, the stirring temperature is 60-80 ℃, and the stirring time is 0.5-2 h; the washing method in the step (8) is as follows: stirring at 50-300rpm for 5-10min, and removing the washing solution.

9. A method for harvesting endometrial stem cells is characterized in that a tampon or a net-shaped structure (3) adhered with an endometrial membrane is placed in a centrifugal tube, normal saline is added into the centrifugal tube, the centrifugal tube is centrifuged for 15-25min at the rotation speed of 1500 + 2500rpm, the centrifuged tampon or net-shaped structure (3) is taken out, the new centrifugal tube is added, EDTA is added for digestion for 5-10min, the EDTA submerges the tampon or net-shaped structure (3), the tampon or net-shaped structure (3) is washed after digestion, a washing solution is collected and centrifuged at the speed of 1500 + 2500rpm for 4-6min, a precipitate is taken, endometrial cells are obtained, and the prepared endometrial cells are cultured.

10. A method for culturing endometrial stem cells is characterized in that a tampon or a reticular structure (3) adhered with an endometrium is placed in a reactor and cultured under the conditions of 37 ℃ and 5% CO 2; the first change was performed 24 hours later, every 3 days thereafter, and passage was performed after the cells had grown to confluence. The passage method comprises the following steps: sucking out old culture medium in a culture bottle, adding PBS for washing for 3 times, adding digestive juice for digestion for 5min, wherein the digestive juice comprises 0.25% of pancreatin and 0.04% of EDTA in a volume ratio of 1: 1; gently blowing and beating the cells to separate the cells from the bottom of the bottle to obtain a single cell suspension; and centrifuging and blowing off the single cell suspension, and carrying out passage according to the ratio of 1: 8.