CN114470331B - Preparation method of collagen scaffold composite umbilical cord stem cells for endometrial repair - Google Patents
Preparation method of collagen scaffold composite umbilical cord stem cells for endometrial repair Download PDFInfo
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- CN114470331B CN114470331B CN202111672015.9A CN202111672015A CN114470331B CN 114470331 B CN114470331 B CN 114470331B CN 202111672015 A CN202111672015 A CN 202111672015A CN 114470331 B CN114470331 B CN 114470331B
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- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3895—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
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- C12M23/48—Holding appliances; Racks; Supports
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0668—Mesenchymal stem cells from other natural sources
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
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- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/50—Proteins
- C12N2533/54—Collagen; Gelatin
Abstract
The invention discloses a preparation method and application of a collagen scaffold composite umbilical cord stem cell for endometrial repair, wherein human umbilical cord mesenchymal stem cells are obtained firstly, wherein the human umbilical cord mesenchymal stem cells are taken out from a refrigeration tank, thawed and inoculated in a culture bottle, then a single cell suspension is prepared, the human umbilical cord mesenchymal stem cells cultured in the culture bottle are subjected to passage, then UC-MSCs pancreatin is digested to prepare a single cell suspension, then composite culture is carried out, namely the single cell suspension is uniformly dripped onto a collagen scaffold, so that the collagen scaffold composite umbilical cord stem cell for endometrial repair is prepared, and finally, the subsequent culture is carried out. The human umbilical cord mesenchymal stem cells are mixed and cultured on the collagen scaffold, and the collagen scaffold approved by SFDA is used for the endometrial repair technology by a simpler technology, so that the safety is good, and the scientific research of treating serious endometrial injury by the stem cells is facilitated.
Description
Technical Field
The invention relates to the technical field of obstetrics and gynecology, in particular to a preparation method and application of a collagen scaffold composite umbilical cord stem cell for endometrium repair.
Background
When the endometrium is seriously injured, functional repair disorder of the endometrium is replaced by connective tissue hyperplasia repair, fibrosis and scar formation occur on the front wall and the rear wall of the uterus, and clinically, the functional repair disorder of the endometrium mainly comprises intrauterine adhesion, amenorrhea, uterine infertility and the like. How to rebuild the intima of these patients and make them return to normal form and function is one of the problems in regenerative medicine today. In the course of the research, the scholars have proposed a plurality of different treatment modes, such as the use of an intrauterine device, a balloon catheter, the application of estrogen or hyaluronic acid after adhesion separation, and the like, and the various treatment modes have the advantages and disadvantages, and the treatment effects are controversial and disappointing in the results reported in the literature. Therefore, new therapeutic approaches are urgently needed for endometrial injuries.
With the discovery and identification of human stem cells from a variety of different sources, the therapeutic application possibilities of stem cells are continuously being explored, and a new medical branch of regenerative medicine is established, aiming at treating diseases which can not be cured by medicines at present by using stem cells. Collagen is the main component of extracellular matrix, has the advantages of wide source, good biocompatibility, degradability, simple preparation process and the like, and is approved by the national food and drug administration (CFDA) to be applied to clinical skin and oral mucosa repair. Mesenchymal Stem Cells (MSCs) are cells derived from mesenchymal tissues, have biological properties similar to those of bone marrow mesenchymal stem cells (BM-MSCs), and have multipotentiality. MSCs are multipotent adult stromal cells that can differentiate into a variety of tissues of mesenchymal origin, such as osteogenic, cartilage and adipocytes, and under special circumstances, can also differentiate laterally into a variety of other tissue cells, such as muscle cells, nerve cells, vascular endothelial cells, hepatic pancreatic cells, and the like. Human umbilical cord mesenchymal stem cells (UC-MSCs) are one type of MSCs. UC-MSCs had low SH2, CD106 and HLA-1 expression compared to BM-MSCs and positive OCT mRNA expression, indicating that umbilical cord UC-MSCs are a more primitive population of MSCs. A large number of in vitro experiments prove that the UC-MSCs can be differentiated not only to tissue cells of various mesoderm sources, such as osteoblasts, chondrocytes, adipocytes and myoblasts, but also to neuron-like cells and hepatocyte-like cells of ectoderm and endoderm sources under different induction conditions. This suggests that we can use UC-MSCs for endometrial repair.
Due to the particularity of the cavity structure of the uterus of human beings, the cells are difficult to be positioned at the damaged intima by the treatment of stem cells, the cells are easy to diffuse and lose tissues along with the flow of blood, body fluid and the like, the effectiveness and the safety of clinical application of the cells are restricted, and the conditions for the adhesion of the cells to local tissues are required to be provided while the cells are repaired. Therefore, the collagen scaffold composite umbilical cord stem cells suitable for endometrial repair need to be prepared in advance, and the characteristics of simple operation process and high safety are expected.
Disclosure of Invention
The invention aims to provide a preparation method and application of a collagen scaffold composite umbilical cord stem cell for endometrial repair.
In order to achieve the purpose, the invention provides the following technical scheme:
a preparation method of collagen scaffold composite umbilical cord stem cells for endometrial repair comprises the following steps:
s1: obtaining human umbilical cord mesenchymal stem cells, wherein the human umbilical cord mesenchymal stem cells in a frozen state are taken out from a refrigeration tank, unfrozen, added with complete culture solution and inoculated in a culture bottle;
s2: preparing single cell suspension, wherein human umbilical cord mesenchymal stem cells cultured in a culture bottle are subjected to passage, and then the UC-MSCs are subjected to pancreatin digestion to prepare the single cell suspension;
s3: performing composite culture, namely uniformly dropwise adding the single-cell suspension onto a collagen scaffold to prepare collagen scaffold composite umbilical cord stem cells for repairing endometrium;
s4: and (4) performing subsequent culture, namely adding the prepared collagen scaffold composite umbilical cord stem cells into a complete culture medium for continuous culture, and then putting the cells into an incubator for culture.
Preferably, the number of cells in the single cell suspension in S2 is about one hundred thousand per cubic centimeter.
Preferably, the temperature in the incubator in S4 is controlled to 36.2-36.8 deg.C, the content in the incubator is 3-4.5% CO2, and the incubation time in the incubator is controlled to 1-1.5 hours.
Preferably, in S1, the human umbilical cord mesenchymal stem cells in the frozen state after being taken out of the refrigerating tank are immediately placed in a water bath and thawed by being slowly shaken all the time, then the thawed stem cells are transferred to a centrifuge tube, a centrifugation operation is performed after a complete culture solution is added, and the stem cells and the culture solution are inoculated in a culture flask after the centrifugation.
Preferably, during the passage operation in S2, the human umbilical cord mesenchymal stem cell fluid after thawing and centrifugation is subcultured in a culture bottle with a serum-free culture solution at a concentration of 1 × 10/ml, and is subjected to a clone formation experiment while standing culture at a temperature of 37 ℃ and a carbon dioxide concentration of 5% for one week, the clone formation rate of each generation is counted, mechanical separation and passage are performed every 7-9 days, a limited dilution method is adopted for mechanical separation and passage, i.e., a single cell suspension prepared from the original human umbilical cord mesenchymal stem cells is diluted, then the diluted cell suspension is dropped into a 96-well culture plate, a culture well with only one cell is selected as a mark, dynamic observation is performed, after the single cell clone sphere grows, mechanical separation into the single cell suspension is performed, then all cells of one clone are planted in the culture bottle, and after the next generation clone grows, continuous passage is performed, and finally a large amount of subclones from a certain single cell is obtained.
Preferably, in S3, the cell suspension is uniformly dripped on the collagen scaffold, the number of the cells is about 1 × 10/CM, and the 6-well plate is placed in an incubator environment with 37 ℃ and 5% of carbon dioxide for culturing for 15 minutes.
Preferably, the human umbilical cord mesenchymal stem cell in S1 is one of mesenchymal stem cells, the mesenchymal stem cell is a cell derived from mesenchymal tissue, has biological properties similar to those of bone marrow mesenchymal stem cells, and has a multipotential differentiation potential, and the mesenchymal stem cell is a multipotential adult stromal cell, can differentiate into various tissues of mesenchymal origin, such as osteogenic, cartilage and fat cells, and can also differentiate transversely into various other tissue cells, such as muscle cells, nerve cells, vascular endothelial cells, liver pancreatic cells, and the like, under a special environment.
Preferably, the culture bottle used in the step S2 comprises a front frame, a rear frame, a bottle body and a driving rod, the front frame and the rear frame are connected through a plurality of groups of connecting rods, a set of first bearings are arranged on the front frame and the rear frame, a set of rotating shafts are arranged in the inner rings of the two sets of first bearings, a ring body is fixedly connected between the two sets of rotating shafts, a bottom plate is installed at the bottom of the ring body through four transition frames, the bottle body is placed on the bottom plate, a swing arm is fixedly connected to the front end of the rotating shaft located on the front side, a motor is arranged at the top of the front frame, a cross rod is arranged at the output end of the motor, a driving shaft is arranged at the end, far away from the motor, of the cross rod, a connecting shaft is connected to the front end of the swing arm, a first driving hole is arranged at one end, a second driving hole is arranged at one end, close to the driving shaft passes through the first driving hole, and a second driving shaft passes through the second driving hole.
As preferred, the transition frame that the blake bottle is located left side and right side is gone up the symmetry and is provided with clamping mechanism, and clamping mechanism includes H type slider, threaded rod and splint, is provided with rectangular hole on the transition frame, H type slider and rectangular hole sliding connection, threaded connection has the bolt on the H type slider, bolt one end and transition frame contact, threaded rod and H type slider threaded connection, threaded rod one end and splint are connected, and the threaded rod other end is provided with the hand wheel.
More preferably, the bottom plate is hollowed out.
More preferably, a key is provided between the swing arm and the rotating shaft located at the front side.
More preferably, the left side and the right side of the bottom of the front frame and the rear frame are both provided with rubber pads.
More preferably, the first driving hole is provided with a second bearing, the connecting shaft is connected with the inner ring of the second bearing, the second driving hole is provided with a third bearing, and the driving shaft is connected with the inner ring of the third bearing.
More preferably, the clamping plate is made of rubber.
More preferably, two sets of handles are arranged on the top of the front frame and the rear frame.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention provides a preparation method and application of a collagen scaffold composite umbilical cord stem cell for endometrial repair, wherein the human umbilical cord mesenchymal stem cell is obtained firstly, the human umbilical cord mesenchymal stem cell is taken out from a refrigerating tank to be unfrozen and is inoculated in a culture bottle, then a single cell suspension is prepared, the human umbilical cord mesenchymal stem cell cultured in the culture bottle is subjected to passage, then UC-MSCs (umbilical cord mesenchymal stem cells) are subjected to trypsinization to prepare a single cell suspension, then composite culture is carried out, namely the single cell suspension is uniformly dripped on a collagen scaffold, so that the collagen scaffold composite umbilical cord stem cell for endometrial repair is prepared, and finally, the subsequent culture is carried out. The human umbilical cord mesenchymal stem cells are mixed and cultured in the collagen scaffold, and the collagen scaffold approved by SFDA is used for the endometrial repair technology by a simpler technology, so that the safety is good;
2. the culture bottle for preparing the single-cell suspension used in the step S2 is provided with a plurality of groups of connecting rods, the front frame and the rear frame are connected, the swing arms, the ring body, the bottle body, the bottom plate and the like can rotate under the support of the two groups of first bearings, the front frame and the rear frame, when the culture bottle is normally used, the bottle body is placed on the bottom plate, the motor is started to enable the output shaft of the motor to rotate, the cross rod is driven to rotate, the driving shaft is driven to rotate by taking the output shaft of the motor as an axis, and the two ends of the driving rod are respectively and rotatably connected with the driving shaft and the connecting shaft, so that the bottle body and the like are driven to reciprocate by taking the rotating shaft as an axis after the motor is electrified, the bottle body does not need to be manually shaken by operators, the labor intensity is reduced, the use convenience is improved, the working efficiency is improved, the pollution is reduced to the minimum range, the resources can be saved, the cleanness and the sanitation of the experimental environment is ensured, the preparation of the single-cell suspension with high quality is facilitated, and the original characteristics of cells are maintained.
Drawings
FIG. 1 is an overall flow diagram of the present invention;
FIG. 2 is a schematic structural view of the present invention;
FIG. 3 is a top right side front perspective view of the present invention;
in the drawings, the reference numbers: 1. a front frame; 2. a rear frame; 3. a bottle body; 4. a drive rod; 5. a connecting rod; 6. a first bearing; 7. a rotating shaft; 8. a ring body; 9. a transition frame; 10. a base plate; 11. swinging arms; 12. a motor; 13. a cross bar; 14. a drive shaft; 15. a connecting shaft; 16. an H-shaped slider; 17. a threaded rod; 18. a splint; 19. a bolt; 20. a hand wheel; 21. a key; 22. a rubber pad; 23. a second bearing; 24. a third bearing; 25. a handle.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Referring to the figure 1 of the drawings, in which,
a preparation method of collagen scaffold composite umbilical cord stem cells for endometrial repair comprises the following steps:
s1: the method comprises the steps of obtaining human umbilical cord mesenchymal stem cells, wherein the human umbilical cord mesenchymal stem cells are one of mesenchymal stem cells, the mesenchymal stem cells are derived from mesenchymal tissues, have biological properties similar to those of bone marrow mesenchymal stem cells and have multidirectional differentiation potential, are multipotent adult matrix cells, can be differentiated into various tissues of mesenchymal origin, such as osteogenic cells, cartilage cells and fat cells, and can be transversely differentiated into various other tissue cells such as muscle cells, nerve cells, vascular endothelial cells, liver pancreatic cells and the like under special environments, wherein the human umbilical cord mesenchymal stem cells in a frozen state are taken out from a refrigeration tank, are thawed and are added with complete culture solution and inoculated into a culture bottle, the human umbilical cord mesenchymal stem cells in a frozen state taken out from the refrigeration tank are immediately placed into a water bath and are slowly shaken to be thawed, then are transferred into a centrifuge tube, are added with the complete culture solution and are centrifuged, and then the human umbilical cord mesenchymal stem cells and the culture solution are inoculated into the culture bottle.
S2: preparing single cell suspension, wherein human umbilical cord mesenchymal stem cells cultured in a culture bottle are subjected to passage, and then UC-MSCs are subjected to pancreatin digestion to prepare the single cell suspension, wherein the number of the single cell suspension cells is about one hundred thousand per cubic centimeter; during passage operation, firstly, the human umbilical cord mesenchymal stem cell liquid after unfreezing and centrifugal treatment is subcultured in a culture bottle by using a serum-free culture solution according to the concentration of 1 multiplied by 10/ml, the suspension is kept still for one week under the conditions that the temperature is 37 ℃ and the carbon dioxide concentration is 5%, meanwhile, a clone formation experiment is carried out, the clone formation rate of each generation is counted, mechanical separation passage is carried out every 7-9 days, a limited dilution method is adopted for mechanical separation passage, namely, a single cell suspension prepared from the original human umbilical cord mesenchymal stem cells is diluted, then the diluted cell suspension is dripped into a 96-hole culture plate, a culture hole with only one cell is selected as a mark, dynamic observation is carried out, the single cell suspension is mechanically separated after the single cell clone ball grows, all cells of one clone are planted into the culture bottle, and continuous passage is carried out after the next generation clone grows, and finally, a large number of subclones from a certain single cell are obtained.
S3: and (3) performing composite culture, namely uniformly dropwise adding the single-cell suspension onto a collagen scaffold to prepare the collagen scaffold composite umbilical cord stem cells for repairing endometrium, uniformly dropwise adding the cell suspension onto the collagen scaffold, wherein the cell number is about 1 multiplied by 10/CM, and culturing the 6-hole plate for 15 minutes in an incubator environment with the temperature of 37 ℃ and the carbon dioxide of 5%.
S4: and (3) adding the prepared collagen scaffold composite umbilical cord stem cells into a complete culture medium for continuous culture, and then putting the mixture into an incubator for culture, wherein the temperature in the incubator is controlled to be 36.2 ℃, the content in the incubator is also 3% of CO2, and the culture time of the incubator is controlled to be 1.5 hours.
The culture bottle used in the step S2 comprises a front frame 1, a rear frame 2, a bottle body 3 and a driving rod 4, as shown in fig. 2 and 3, wherein the front frame 1 and the rear frame 2 are connected through a plurality of groups of connecting rods 5, a group of first bearings 6 are respectively arranged on the front frame 1 and the rear frame 2, a group of rotating shafts 7 are respectively arranged on inner rings of the two groups of first bearings 6, a ring body 8 is fixedly connected between the two groups of rotating shafts 7, a bottom plate 10 is installed at the bottom of the ring body 8 through four transition frames 9, the bottle body 3 is placed on the bottom plate 10, a swing arm 11 is fixedly connected to the front end of the rotating shaft 7 positioned on the front side, a motor 12 is arranged at the top of the front frame 1, a cross rod 13 is arranged at the output end of the motor 12, a driving shaft 14 is arranged at one end of the cross rod 13, a connecting shaft 15 is connected to the front end of the swing arm 11, a first driving hole is arranged at one end of the driving rod 4, which is close to the connecting shaft 15, a second driving hole is arranged at one end of the driving rod 4, the driving shaft 14, the connecting shaft 15 passes through the first driving hole, and the driving shaft 14 passes through the second driving hole; the multi-group connecting rod 5 connects the front frame 1 and the rear frame 2, the two groups of first bearings 6 are arranged under the support of the front frame 1 and the rear frame 2, the swing arm 11, the ring body 8, the bottle body 3, the bottom plate 10 and the like can rotate, during normal use, the bottle body 3 is placed on the bottom plate 10, the motor 12 is started to rotate the output shaft of the motor 12, the cross rod 13 is driven to rotate, the driving shaft 14 is driven to rotate by taking the output shaft of the motor 12 as an axis, the two ends of the driving rod 4 are respectively connected with the driving shaft 14 and the connecting shaft 15 in a rotating mode, the motor 12 is driven to drive the bottle body 3 and the like to reciprocate and swing by taking the rotating shaft 7 as an axis after being electrified, the bottle body 3 does not need to be manually shaken by operators, labor intensity is reduced, and convenience in use is improved.
A clamping mechanism is symmetrically arranged on transition frames 9 on the left side and the right side of a culture bottle for preparing single cell suspension, the clamping mechanism comprises an H-shaped sliding block 16, a threaded rod 17 and a clamping plate 18, a long hole is formed in the transition frames 9, the H-shaped sliding block 16 is in sliding connection with the long hole, a bolt 19 is in threaded connection with the H-shaped sliding block 16, one end of the bolt 19 is in contact with the transition frames 9, the threaded rod 17 is in threaded connection with the H-shaped sliding block 16, one end of the threaded rod 17 is connected with the clamping plate 18, and a hand wheel 20 is arranged at the other end of the threaded rod 17; the left and right sides of the bottle body 3 are clamped by a clamping mechanism, which has the following principle: firstly, the height of the H-shaped slider 16 is adjusted according to the height of the bottle body 3, after the adjustment is finished, the bolt 19 is screwed to fix the position of the H-shaped slider 16, and then the hand wheel 20 is rotated to enable the clamp plate 18 to be in contact with the bottle body 3, so that the bottle body 3 is fixed, and the shaking during the swinging is prevented.
The bottom plate 10 is hollowed out; the bottom plate 10 is hollowed out, so that the overall weight of the device is reduced, and the load of the motor 12 is reduced. A key 21 is arranged between the swing arm 11 and the rotating shaft 7 positioned at the front side; by providing the key 21, the swing arm 11 and the rotating shaft 7 located on the front side can be prevented from rotating relatively, and the use reliability can be improved. Rubber pads 22 are arranged on the left and right sides of the bottoms of the front frame 1 and the rear frame 2; in normal use, the rubber pad 22 supports the front frame 1 and the rear frame 2, increasing the friction force with the supporting surface and reducing the damage to the supporting surface.
A second bearing 23 is arranged at the first driving hole, the connecting shaft 15 is connected with the inner ring of the second bearing 23, a third bearing 24 is arranged at the second driving hole, and the driving shaft 14 is connected with the inner ring of the third bearing 24; by providing the second bearing 23 and the third bearing 24, the smoothness of the bottle body 3 during reciprocating swing is improved. The clamping plate 18 is made of rubber; the clamping plate 18 is made of rubber, so that the damage to the bottle body 3 is reduced. Two groups of handles 25 are arranged at the top parts of the front frame 1 and the rear frame 2; the two groups of handles 25 are held, so that the whole device can be conveniently moved, and the use convenience is improved.
The invention relates to a culture bottle for preparing single cell suspension, when in work, a plurality of groups of connecting rods 5 connect a front frame 1 and a rear frame 2, under the support of two groups of first bearings 6, the front frame 1 and the rear frame 2, a swing arm 11, a ring body 8, a bottle body 3, a bottom plate 10 and the like can rotate, when in normal use, a rubber pad 22 supports the front frame 1 and the rear frame 2 to increase the friction force with a supporting surface and simultaneously reduce the damage to the supporting surface, the bottle body 3 is placed on the bottom plate 10, the left side and the right side of the bottle body 3 are clamped by a clamping mechanism, a motor 12 is started to enable an output shaft of the motor 12 to rotate, so as to drive a cross rod 13 to rotate, and drive a driving shaft 14 to rotate by taking the output shaft of the motor 12 as a shaft, and two ends of a driving rod 4 are respectively rotatably connected with the driving shaft 14 and a connecting shaft 15, so that the motor 12 drives the bottle body 3 and the like to reciprocate by taking a rotating shaft 7 as a shaft after being electrified, and the principle of the clamping mechanism is as follows: firstly, the height of the H-shaped sliding block 16 is adjusted according to the height of the bottle body 3, after the adjustment is finished, the bolt 19 is screwed to fix the position of the H-shaped sliding block 16, and then the hand wheel 20 is rotated to enable the clamping plate 18 to be in contact with the bottle body 3, so that the bottle body 3 is fixed, and the shaking during the swinging is prevented.
The invention provides a preparation method and application of a collagen scaffold composite umbilical cord stem cell for endometrial repair, wherein the human umbilical cord mesenchymal stem cell is obtained firstly, wherein the human umbilical cord mesenchymal stem cell taken out from a refrigeration tank is unfrozen and inoculated in a culture bottle, then a single cell suspension is prepared, the human umbilical cord mesenchymal stem cell cultured in the culture bottle is subjected to passage, UC-MSCs pancreatin is digested to prepare a single cell suspension, then composite culture is carried out, namely the single cell suspension is uniformly dripped on a collagen scaffold, so that the collagen scaffold composite umbilical cord stem cell for endometrial repair is prepared, and finally, the subsequent culture is carried out. The invention uses human umbilical cord mesenchymal stem cells to perform mixed culture on the collagen scaffold, and uses the collagen scaffold approved by SFDA in the endometrial repair technology through a simpler technology.
Example 2
The same as example 1, except that the temperature in the incubator in the step S4 was controlled to 36.8 ℃, the content in the incubator was 4.5% CO2, and the incubation time of the incubator was controlled to 1 hour.
Example 3
The same as example 1, except that the temperature in the incubator in the step S4 was controlled to 36.5 ℃, the contents of the incubator were also 4% CO2, and the incubation time of the incubator was controlled to 1.3 hours.
Comparative example 1
The same as example 1, except that the flasks used for the single cell suspension preparation were conventional flasks on the market. The temperature in the incubator in the step S4 was controlled to 36.8 ℃, the content in the incubator was 4.5% by volume CO2, and the incubation time in the incubator was controlled to 0.6 hours.
Comparative example 2
The same as example 2, except that the flasks used for the single cell suspension preparation were conventional flasks on the market. In the step S4, the temperature in the incubator was controlled to 35 ℃, the content in the incubator was also 3% CO2, and the incubation time in the incubator was controlled to 1 hour.
Comparative example 3
The same as example 3, except that the flask used for the single cell suspension preparation was a commercial conventional flask. In the above step S4, the temperature in the incubator is controlled to 36.8 deg.C, the content in the incubator is further 2.8%.
TABLE 1 comparison of cell parameters for different examples and comparative examples
Experimental description from examples and comparative examples:
1. the culture bottle for preparing the single cell suspension can prepare the single cell suspension with high quality, and keeps the original characteristics of cells;
2. the preparation method of the collagen scaffold composite umbilical cord stem cell for repairing the endometrium is helpful for cell parameters and can improve cell performance.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.
Claims (3)
1. A preparation method of collagen scaffold composite umbilical cord stem cells for endometrial repair is characterized by comprising the following steps:
s1: obtaining human umbilical cord mesenchymal stem cells, wherein the human umbilical cord mesenchymal stem cells in a frozen state are taken out from a refrigeration tank and are fine
Thawing the cells, adding complete culture solution, and inoculating in a culture bottle;
s2: preparing single cell suspension, wherein human umbilical cord mesenchymal stem cells cultured in a culture bottle are subjected to passage, and then the UC-MSCs are subjected to pancreatin digestion to prepare the single cell suspension;
s3: performing composite culture, namely uniformly dropwise adding the single-cell suspension onto a collagen scaffold to prepare collagen scaffold composite umbilical cord stem cells for repairing endometrium;
s4: subsequent culture, adding the prepared collagen scaffold composite umbilical cord stem cells into a complete culture medium for continuous culture, and then putting the cells into an incubator for culture;
in the S1, the human umbilical cord mesenchymal stem cells in a frozen state are taken out from the refrigerating tank and immediately placed in a water bath, and slowly shaken all the time to be thawed, then the thawed stem cells are transferred into a centrifuge tube, a centrifugal operation is carried out after a complete culture solution is added, and the stem cells and the culture solution are inoculated into a culture bottle after centrifugation;
the culture bottle used in the step S2 comprises a front frame (1), a rear frame (2), bottle bodies (3) and a driving rod (4), wherein the front frame (1) and the rear frame (2) are connected through a plurality of groups of connecting rods (5), the front frame (1) and the rear frame (2) are respectively provided with a group of first bearings (6), the inner rings of the two groups of first bearings (6) are respectively provided with a group of rotating shafts (7), a ring body (8) is fixedly connected between the two groups of rotating shafts (7), the bottom of the ring body (8) is provided with a bottom plate (10) through four transition frames (9), the bottle bodies (3) are placed on the bottom plate (10), the front ends of the rotating shafts (7) positioned on the front side are fixedly connected with swing arms (11), the top of the front frame (1) is provided with a motor (12), the output end of the motor (12) is provided with a transverse rod (13), one ends of the transverse rods (13) far away from the motor (12) are provided with driving shafts (14), the front ends of the swing arms (11) are connected with driving rods (15), one ends of the driving rods (4) close to the connecting shafts (15) are provided with second driving holes (14), and the driving rods (15) pass through the second driving holes (14);
clamping mechanisms are symmetrically arranged on the transition frame (9) on the left side and the right side and comprise an H-shaped sliding block (16), a threaded rod (17) and a clamping plate (18), a long hole is formed in the transition frame (9), the H-shaped sliding block (16) is in sliding connection with the long hole, a bolt (19) is in threaded connection with the H-shaped sliding block (16), one end of the bolt (19) is in contact with the transition frame (9), the threaded rod (17) is in threaded connection with the H-shaped sliding block (16), one end of the threaded rod (17) is connected with the clamping plate (18), and a hand wheel (20) is arranged at the other end of the threaded rod (17);
a second bearing (23) is arranged at the first driving hole, the connecting shaft (15) is connected with the inner ring of the second bearing (23), a third bearing (24) is arranged at the second driving hole, and the driving shaft (14) is connected with the inner ring of the third bearing (24);
during normal use, put the bottle on the bottom plate, open the motor and make the motor output shaft rotatory to it is rotatory to drive the horizontal pole, and drive the drive shaft and use the motor output shaft to rotate as the axle, because the drive lever both ends rotate with drive shaft and connecting axle respectively and be connected, thereby drive the bottle after the motor circular telegram and use the pivot to take place reciprocating swing as the axle.
2. The method for preparing the collagen scaffold composite umbilical cord stem cell for endometrial repair according to claim 1, wherein the collagen scaffold composite umbilical cord stem cell for endometrial repair comprises the following steps: the number of single cell suspension cells in S2 is about one hundred thousand per cubic centimeter.
3. The method for preparing the collagen scaffold composite umbilical cord stem cell for endometrial repair according to claim 1, wherein the collagen scaffold composite umbilical cord stem cell for endometrial repair comprises the following steps: the temperature in the incubator in S4 is controlled to 36.2-36.8 ℃, and the content in the incubator is 3-4.5% 2 The culture time of the incubator is controlled to be 1-1.5 hours.
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| WO2014039429A1 (en) * | 2012-09-04 | 2014-03-13 | Anthrogenesis Corporation | Methods of tissue generation |
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| CN105983133A (en) * | 2015-01-30 | 2016-10-05 | 南京鼓楼医院 | Stem cell composite collagen scaffold kit used for repairing endometrial damage, and preparation method thereof |
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| CN112043727A (en) * | 2020-09-17 | 2020-12-08 | 山西宾大干细胞生物科技有限公司 | Preparation method and application of cell composite endometrium repair gel |
| CN215162812U (en) * | 2021-06-10 | 2021-12-14 | 天津长和生物技术有限公司 | Umbilical cord mesenchymal stem cell culture uses indirect operation case |
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2014039429A1 (en) * | 2012-09-04 | 2014-03-13 | Anthrogenesis Corporation | Methods of tissue generation |
| CN103705984A (en) * | 2013-12-17 | 2014-04-09 | 南京大学医学院附属鼓楼医院 | Preparation method and application of collagen scaffold composite bone marrow-derived mesenchymal stem cells (BMSCs) |
| CN105983133A (en) * | 2015-01-30 | 2016-10-05 | 南京鼓楼医院 | Stem cell composite collagen scaffold kit used for repairing endometrial damage, and preparation method thereof |
| CN106730013A (en) * | 2016-12-06 | 2017-05-31 | 徐妍 | For preventing Asherman's syndrom and the cell preparation of endometrial impairment reparation and preparation method thereof |
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