CN110484491B

CN110484491B - Method for obtaining amniotic membrane and amniotic fluid derived endothelial progenitor cells and purification culture method thereof

Info

Publication number: CN110484491B
Application number: CN201910770838.1A
Authority: CN
Inventors: 赵亚; 李志刚; 杜颖; 王煜骏; 丁炜; 阮志; 王颖颖
Original assignee: BEIJING JING-MENG STEM CELL TECHNOLOGY CO LTD
Current assignee: BEIJING JING-MENG STEM CELL TECHNOLOGY CO LTD
Priority date: 2019-08-20
Filing date: 2019-08-20
Publication date: 2022-03-18
Anticipated expiration: 2039-08-20
Also published as: CN110484491A

Abstract

The invention discloses a method for obtaining endothelial progenitor cells from human and mammal amniotic membrane and amniotic fluid, which comprises the following steps: pretreating the amnion stripped from the placenta; mechanically removing the pretreated sponge layer and epithelial layer of the amniotic membrane to obtain an amniotic membrane tissue; cutting the amniotic tissue into a preset size, mixing the cut amniotic tissue with a collagenase IV solution, digesting, and discarding the supernatant obtained by digestion; and mixing the amniotic membrane tissue after the first digestion step with a fresh collagenase IV solution again, digesting, centrifuging, and discarding the supernatant to obtain the endothelial progenitor cells. The amniotic membrane tissue block does not need to be ground and filtered, epithelial cells and mesenchymal stem cells in the amniotic membrane are fully removed by mechanical scraping and a gradient stepwise digestion method, and the technical problem that high-purity endothelial progenitor cells cannot be obtained from the amniotic membrane tissue in the prior art is solved.

Description

Method for obtaining amniotic membrane and amniotic fluid derived endothelial progenitor cells and purification culture method thereof

Technical Field

The invention belongs to the cell separation and amplification technology in the technical field of bioengineering, and particularly relates to an acquisition method and a purification culture method of amniotic membrane and amniotic fluid derived endothelial progenitor cells.

Background

Endothelial Progenitor Cells (EPC) are precursor cells capable of circulating, proliferating and differentiating into vascular endothelial cells, and are differentiated into endothelial cells under the stimulation of various physiological and pathological factors to participate in the repair and angiogenesis of damaged endothelium, thereby playing an important role in the development of cardiovascular diseases, especially atherosclerotic lesions. In 1997, Asahara et al demonstrated the presence of EPC in adult peripheral blood, and introduced the concept of stem cells for the first time in the area of angiogenesis. Subsequent studies found the presence of EPC in bone marrow, cord blood, adipose tissue.

Traditional EPC is mainly collected from bone marrow and cord blood, but the bone marrow is inconvenient to take and is limited by personal physical conditions; umbilical cord blood is not only inconvenient to take and easy to pollute, but also limited in amount, and the EPC from the umbilical cord blood needs HLA matching, and meanwhile, allogeneic input may have side effects such as immunological rejection. Because EPC has limited sources and extremely rare quantity, on one hand, the research difficulty of researchers on endothelial cells is increased, and on the other hand, the clinical application of the endothelial cells in diseases such as cardiovascular system diseases and various tissue injuries is limited.

The amnion is the innermost membrane of the fetal membrane, is connected with the amniotic membrane layer covering the placenta and the umbilical cord, and has the function of secreting amniotic fluid. It has been shown that amnion contains abundant trophoblasts and a large amount of different collagens, including fibronectin, laminin, etc., because these cells and components make amnion not only prevent leukocyte infiltration, but also produce some growth factors into amniotic fluid, such as: basic Fibroblast Growth Factor (BFGF), Hepatocyte Growth Factor (HGF), transforming growth factor-beta (TGF-beta), etc., Vascular Endothelial Growth Factor (VEGF), epithelial cell growth factor (EGF). These factors can promote proliferation, migration and differentiation of epithelial, mesenchymal and endothelial cells. In addition, as the amniotic membrane tissue is a product derived from a fetus, the human leukocyte antigen DR on the surface of the amniotic membrane tissue is low in expression and does not express HLA-A, B and C antigens when exposed to the monitoring of a maternal immune system, and the characteristic that the human amniotic membrane cell is reported to have no immunogenicity is firstly reported in 1982 by Andiolfi. In addition, the amniotic membrane tissue and amniotic fluid series postpartum wastes have no ethical problems; and because the sources of the amniotic membrane tissue cells are sufficient, the amniotic membrane has wide application prospect as a novel and reliable donor material.

At present, researchers at home and abroad separate cell groups capable of adhering to the wall from amnion, however, because the amnion tissue structure is complex, on one hand, the digestion mode is complicated, the pollution source is easy to introduce, the operation process is difficult to control and repeat, on the other hand, the obtained cells are complex in variety, and the cell differentiation potential and the cell differentiation trend are diversified. Although researchers have been able to isolate a single cell type with high purity from amnion in recent years, mainly mesenchymal stem cells of mesenchymal type have been used, and there has been no case of obtaining endothelial progenitor cells with high purity from amnion and amniotic fluid.

For example, patent applications entitled "treatment of radiation injury using amnion-derived adherent cells" and "amnion-derived adherent cells" under application numbers 201280040374.5 (published as 2015.03.25) and 201710437293.3 (published as 2017.9.26) disclose methods for obtaining a cell composition with adherent properties from amnion, wherein the cell population comprises hematopoietic stem cells, somatic stem cells, chondrocytes, fibroblasts, myocytes, endothelial cells, hemangioblasts, endothelial progenitor cells, pericytes, cardiomyocytes, myocytes, cardiomyocytes, myoblasts, embryonic stem cells, or a combination of membrane stem cells treated similarly to embryonic stem cells. On the one hand, the patent provides a method for obtaining cells and cell groups with differentiation and angiogenesis from amnion, but it is difficult to separate endothelial progenitor cells with high purity from a complex cell mixture; and the steps of the digestion process of the amnion are complicated, the processing container which is independently constructed by utilizing the glass processing container and the circulating water area pipeline needs to be used for multiple times, and the amnion fragments need to be intermittently stirred and filtered for multiple times, so that the steps are complicated, the pollution is easy, the operation process is difficult to control, a stable standard operation program SOP is difficult to form, and the industrialization is difficult to realize.

For example, the invention patent of application No. 201811259326.0 (published as 2019.02.12), entitled "efficient separation and amplification of human amniotic mesenchymal stem cells" discloses a method for separating mesenchymal stem cells from amniotic membrane by using two-step enzyme digestion method, which comprises the steps of firstly cutting the amniotic membrane into tissue blocks with uniform size by using a scissors, mixing trypsin 0.05% and EDTA0.02%, shaking table shaking to digest and remove amniotic epithelial stem cells (37 ℃, 145 rpm/min), co-digesting for two times, each time for 15min, washing with physiological saline to remove epithelial cells, and then adding trypsin and EDTA digestive enzyme again; then, 0.1% of collagenase I and 0.02% of DNase I are used for digesting for 1.0h, and the amniotic mesenchymal stem cells are obtained through separation. The method has the advantages of complex operation steps, multiple mixed digestive enzymes which are required to be added for multiple times in the digestion process, complex digestive enzyme configuration, easy pollution, higher cost and no contribution to industrial preparation, and in addition, the cells obtained by digestion are stem cell populations mainly comprising mesenchyme.

The invention aims to provide a method for collecting a large number of vascular endothelial progenitor cells with high purity from amnion and amniotic fluid.

Disclosure of Invention

The invention provides a separation method and a culture method of amniotic membrane and amniotic fluid derived endothelial progenitor cells and prepared endothelial progenitor cells, which can obtain the endothelial progenitor cells with higher purity from the amniotic membrane, have simpler and more convenient separation method and easily controlled operation process, and can effectively reduce the possibility of pollution in the separation process, thereby realizing the industrial preparation of the amniotic membrane and amniotic fluid derived endothelial progenitor cells.

The invention provides amniotic membrane and amniotic fluid derived endothelial progenitor cells obtained from human or mammalian amniotic membrane.

The invention also provides an amniotic membrane derived endothelial progenitor cell acquisition method, which comprises the following steps:

a pretreatment step of pretreating an amnion detached from a placenta;

removing the sponge layer and the epithelial cell layer, namely removing the sponge layer and the epithelial cell layer on the pretreated amnion by adopting a mechanical separation method to obtain an amnion tissue;

a first digestion step, cutting the amniotic membrane tissue into a preset size, and mixing the cut amniotic membrane tissue with a collagenase solution according to a volume ratio of 1: (1-5), placing the mixture in an incubator at 37 ℃ for digestion for 1.0-8.0 h, and washing the digested amniotic tissue with physiological saline;

a second digestion step, namely mixing the amniotic membrane tissue subjected to the first digestion step with a collagenase solution according to a volume ratio of 1: (1-2), placing the mixture in an incubator at 37 ℃ for digestion for 1.0-2.0 h, mixing the digestive juice with normal saline, centrifuging, and removing supernatant to obtain endothelial progenitor cells.

In one embodiment, the collagenase is any one of collagenase type I, collagenase type II, collagenase type III, and collagenase type IV; the concentration of the collagenase solution is 0.05% -0.25%.

In one embodiment, in the pretreatment step, the pretreatment includes a normal saline washing treatment containing penicillin and streptomycin, a chorionic membrane removal treatment, and a decontamination treatment.

In one embodiment, before the step of removing the sponge layer and the epithelial cell layer, the method further comprises the following steps:

cutting the pretreated amnion into a second preset size, and soaking and washing the cut amnion with physiological saline containing penicillin and streptomycin.

In one embodiment, after the step of removing the sponge layer and the epithelial cell layer, the method further comprises the following steps:

the amniotic tissue is soaked and washed with normal saline containing penicillin and streptomycin.

In one embodiment, the acquiring method further includes the following steps:

mixing the collected amniotic fluid and/or the amnion preserved preservation solution with normal saline containing penicillin and streptomycin according to the volume ratio of 1: (0.5-1.5), centrifuging, discarding the supernatant, mixing the obtained cell precipitate with physiological saline, centrifuging, and discarding the supernatant to obtain the endothelial progenitor cells.

The invention also provides a method for obtaining the amniotic fluid-derived endothelial progenitor cells, which comprises the following steps:

The invention also provides an endothelial progenitor cell, which is obtained by the method.

The invention also provides a method for purifying and culturing the endothelial progenitor cells, which comprises the following steps:

and (3) inoculating the endothelial progenitor cells into a culture solution, naturally settling for 6.0-24.0 h, adhering to the wall, removing residual interstitial cells to obtain an endothelial progenitor cell suspension, and performing secondary adherence to obtain endothelial progenitor cell primary seed cells for subculture.

The amniotic membrane and amniotic fluid derived endothelial progenitor cells have vigorous growth capacity, stable multiplication capacity and karyotype as shown by the flow cell cycle, and typical angioplastic capacity, low-density lipoprotein uptake capacity and lectin binding capacity.

The method for obtaining the endothelial progenitor cells from the amniotic membrane adopts a method combining mechanical separation and enzyme digestion, firstly utilizes blunt separation of amniotic membrane tissues to remove chorions, utilizes a mechanical method to scrape epithelial cell layers, then using enzyme gradient to digest step by step, removing residual epithelial cells and easily digestible interstitial cells in the amniotic membrane tissue in the first step of digestion, and then the seed cells of the endothelial progenitor cells are obtained by using the second step of digestion, the technical scheme of combining the step-by-step mechanical separation to remove different types of cells and the digestion treatment method to screen cell types is adopted, the technical problem that high-purity endothelial progenitor cells cannot be obtained in the prior art is solved, the technical defect that the digestion treatment effect is single in the traditional separation method is overcome, and the digestion treatment is used for screening cell types to achieve the purpose of obtaining the high-purity endothelial progenitor cells. The digestion mode is simple and easy to implement, other auxiliary digestive enzymes are not required to be added, the reagent cost is reduced, the steps of tissue grinding, filtering and the like are not required, the pollution caused by manual operation is reduced, the whole digestion process is easy to form a complete standard operation procedure, and the industrial operation is facilitated.

Drawings

FIG. 1 is a diagram of clonal colony of endothelial progenitor cells 4X in example 1 of the present invention;

FIG. 2 is a 10X diagram of clonal colonies of endothelial progenitor cells according to example 1 of the present invention;

FIG. 3 is a 20X map of clonal endothelial progenitor cell colonies according to example 1 of the present invention;

FIG. 4 is a graph showing the results of the flow cytometric phenotypic assay of endothelial progenitor cells in example 1 of the present invention;

FIG. 5 is a graph showing the confirmation of the vascularization ability of endothelial progenitor cells in example 1 of the present invention;

FIG. 6 is a graph showing the results of the determination of phagocytic activity and binding activity of endothelial progenitor cells in example 1 of the present invention;

FIG. 7 is a graph showing the results of the evaluation of the continuous expansion ability of endothelial progenitor cells according to example 1 of the present invention;

FIG. 8 is a graph showing the result of karyotyping the endothelial progenitor cells in example 1 of the present invention;

FIG. 9 is a graph showing the results of cell cycle identification of endothelial progenitor cells in example 1 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the detailed description and specific examples, while indicating the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.

The invention provides an amniotic membrane and amniotic fluid derived endothelial progenitor cell, which is obtained from a human or mammal peripheral product amniotic membrane and has extremely strong angiogenesis capacity after subculture. Further experimental verification shows that the amnion and amniotic fluid derived EPC have vigorous growth capacity, and cell cycle detection shows that cells are in an exponential growth phase, have stable cell multiplication capacity and karyotype, and have typical angioplastic capacity, low-density lipoprotein uptake capacity and lectin binding capacity.

Amnion-derived stem cells are more nutritious, and studies have shown that amnion-derived stem cells express stem cell factors at higher concentrations than other tissue-derived stem cells, as well as hematopoietic stem cell factors. The amniotic membrane tissue is a product from a fetus, is exposed to the monitoring of a maternal immune system, not only provides nutrition for the development of the fetus, but also is a wonderful barrier for preventing two variant tissues of the fetus and the maternal from mutually rejecting, and the specificity of the structure and the development ensures that the amniotic membrane tissue lacks immunogenicity and variant immune rejection, and in addition, the amniotic membrane cell does not express telomerase and has no tumorigenicity, so that the amniotic membrane-derived stem cell organism has higher nourishment and the biological safety is ensured. Currently, amniotic stem cells have been used in a variety of clinical programs, such as ophthalmic diseases and endometrial diseases, and in recent years, research on the tissue engineering skin, blood vessel construction, biological scaffold materials, biological slow release materials and the like of amniotic stem cells and amniotic cells has been advanced greatly. Therefore, the amniotic cells have higher scientific research value and wide application prospect advantage than other tissue cells. The amniotic fluid derived endothelial progenitor cells have extremely strong angiogenesis capacity after subculture.

The invention provides an embodiment of the method for obtaining the amniotic membrane derived endothelial progenitor cells, which comprises the following steps:

a pretreatment step of pretreating an amnion detached from a placenta;

removing a sponge layer, namely removing the sponge layer and an epithelial cell layer on the pretreated amnion by adopting a mechanical separation method to obtain an amnion tissue;

The method for obtaining the endothelial progenitor cells from the amniotic membrane adopts a method combining mechanical separation and enzyme digestion, firstly utilizes blunt separation of amniotic membrane tissues to remove chorions, utilizes a mechanical method to scrape epithelial cell layers, then using enzyme gradient to digest step by step, digesting and removing residual epithelial cells and easily digestible interstitial cells in the amniotic tissue in the first step, and then the seed cells of the endothelial progenitor cells are obtained by using the second step of digestion, the technical scheme of combining the step-by-step mechanical separation to remove different types of cells and the digestion treatment method to screen cell types is adopted, the technical problem that high-purity endothelial progenitor cells cannot be obtained in the prior art is overcome, the technical defect that the digestion treatment effect is single in the traditional separation method is overcome, and the digestion treatment is used for screening cell types to achieve the purpose of obtaining the high-purity endothelial progenitor cells. The digestion mode is simple and easy to implement, other auxiliary digestive enzymes are not required to be added, the reagent cost is reduced, the steps of tissue grinding, filtering and the like are not required, the pollution caused by manual operation is reduced, the whole digestion process is easy to form a complete standard operation procedure, and the industrial operation is facilitated.

In the prior art of cell isolation and expansion in bioengineering, there is no case of isolating high purity EPC from amniotic membrane and amniotic fluid. Firstly, because the amnion structure is complicated, the cell composition is various, and the amnion comprises 3 layers of single-layer epithelial layer, basal lamina and intermediate lamina from inside to outside, and wherein the intermediate lamina can be divided into compact layer, fiber cell layer and sponge layer again. The monolayer epithelial cells mainly secrete amniotic fluid and trophic factors; the amnion basal membrane layer contains unique intercellular substance and a large number of different glue elements, mainly including type I, III, IV, V and VII collagens, fibronectin, laminin and other components; the stratum interna is thick, and adherent cells containing more than dozens of components in the stratum interna are known at present, and various progenitor cells including neural stem cells, fibroblasts, mesenchymal cells, hematopoietic cells, hemangioblasts and the like; secondly, because the material of the amniotic membrane tissue is inconvenient to obtain, although the amniotic membrane is very easy to perform blunt separation on a spongy layer from a chorion layer, the epithelial layer and a mesenchymal layer of the amniotic membrane tissue cannot be completely separated, so that the adherent cell group can be obtained from the amniotic membrane in the prior report, but the components in the cell group cannot be purified; thirdly, the cells separated from the amniotic membrane mainly comprise mesenchymal stem cells and multipotential stromal cells, and hematopoietic stem cells and vascular stromal cells are few, so reports on obtaining epithelial cells and mesenchymal stem cells with higher purity from the amniotic membrane have been made so far, however, the successful case of obtaining endothelial progenitor cells with higher purity by separating from the amniotic membrane is not seen. The adherent cell population obtained by the traditional amnion separation technology has various types and complex components, and although the adherent cell population can be obtained from the amnion, only a few vascular cells exist, and the quantity and the proportion are low. In recent years, it has been reported that high-purity stem cells are obtained from amnion, but mesenchymal stem cells are mainly used as mesenchymal stem cells. For example, chinese patent 201811259326.0 discloses a high efficiency separation and amplification technique of human amniotic mesenchymal stem cells, a method for separating mesenchymal stem cells from amniotic membrane, on one hand, because digestive enzymes are various in kind during digestion, not only is the configuration cumbersome and easy to bring into pollution source, but also the reagent cost is increased; because the structure of the amniotic membrane is complex, the inventor verifies that the obtained cells are mesenchymal stem cells by repeating the digestion of the amniotic membrane tissue by using the two-step enzyme digestion method, and tests show that the method is not suitable for obtaining the amniotic membrane derived endothelial progenitor cells. On the other hand, the trypsin has extremely strong digestion capability and can digest amniotic tissues into cell suspensions in a short time, so adherent cell groups obtained by the traditional amniotic membrane separation technology are various in types and complex in components, and although adherent cell groups can be obtained from the amniotic membrane, only a small part of the adherent cell groups are vascular cells, but the proportion of epithelial cells and interstitial cells is high; in addition, due to the fact that the target cell membrane protein is easily damaged to different degrees due to the fact that the protein dissolving and digesting capacity of trypsin is too strong, and the individual difference of the amniotic membrane tissue is large, the digestion time is difficult to control and form stable SOP, and the cell seed digestion method utilized in tissue engineering rarely uses trypsin. However, the research of the invention finds that the collagenase belongs to protease, can specifically recognize a protein sequence and cut peptide bonds in the protein sequence, is suitable for separating epithelial cells, mesenchymal stem cells and angioblast stem cells in connective tissues and tissue blocks, particularly collagenase IV has low pancreatic enzyme activity, has mild performance, and has unique advantages of digesting cells and maintaining the integrity of cell membrane protein. The invention utilizes low-concentration collagenase IV to slowly digest and separate epithelial layer cells and interstitial cells from amniotic membrane tissues, and because vascular stromal cells are difficult to digest, adherent cells collected at last by controlling the digestion time are high-purity hemangioblasts. And then removing residual interstitial cells easy to adhere to the wall by a differential adherence method to obtain primary seed cells of endothelial progenitor cells with higher purity.

In the method for obtaining the amniotic membrane derived endothelial progenitor cells, firstly, amniotic membrane tissues are subjected to blunt separation to remove chorions, epithelial layer cells are scraped by a mechanical method, then enzyme gradient stepwise digestion is utilized, firstly, residual epithelial layer cells and interstitial cells in the amniotic membrane are fully removed by first digestion, and then, a vascular stroma angioblast stem cell group is obtained by second digestion, so that the existing technical bias is broken through and overcome. The cells obtained by the method have the advantages of high EPC quantity, high purity and endothelial-like cell characteristics, obvious EPC clone presentation is observed when the cells are cultured for 5-7 days, cobblestone-like cell shapes are presented, the cell phenotype of the EPC is verified by flow verification, the cells have typical EPC cell phenotypes, high-expression endothelial cell specific markers CD31, CD29, CD44, CD73 and CD105, the positive rate is up to 90-99%, hematopoietic stem markers are not expressed, the positive rate of CD45 is lower than 1%, human leukocyte antigens are not expressed, the immunogenicity is extremely low, and the HLA-DR positive rate is lower than 1%. Functional verification experiments of EPC prove that the EPC cells obtained by the method have the function of differentiating into vascular cells in vitro. The cell long-term amplification culture result proves that the EPC obtained by the method has vigorous growth capacity, and the flow cell cycle detection shows that the cells are in an exponential growth phase, have stable cell multiplication capacity and karyotype, and have typical angioplastic capacity, low-density lipoprotein phagocytic capacity and lectin binding capacity.

The invention discloses an amniotic membrane derived endothelial progenitor cell acquisition method and an amniotic fluid derived endothelial progenitor cell acquisition method. Optionally, the amniotic membrane and amniotic fluid may be collected human amniotic membrane and amniotic fluid, or amniotic membrane and amniotic fluid of other mammals.

Optionally, the amniotic fluid is collected during the childbirth of the pregnant woman, placenta and amniotic tissue are obtained after the childbirth, the amniotic membrane is peeled from the placenta by surgical scissors and placed in a storage box containing preservation solution, and after informed consent is signed by family members, the amniotic fluid is timely delivered to a laboratory in a cold chain under the preservation condition of 2-8 ℃; numbering the amniotic fluid to avoid pollution.

As an alternative embodiment, in the pretreatment step, the pretreatment includes a normal saline immersion washing treatment containing penicillin and streptomycin, a chorionic membrane removal treatment, and a decontamination treatment. The specific operation method can be as follows:

wiping the surface of the sample bottle containing the amnion with 75% alcohol, and then placing the sample bottle into a II-level biological safety cabinet; carefully opening the bottle cap of the sample bottle, transferring the amniotic membrane into a plastic basin filled with 100-200 mL of physiological saline (containing 100U/mL of penicillin and 100 microgram/mL of streptomycin) by using large sterile forceps, performing immersion washing for 2-3 min, and then performing blunt separation on the amniotic membrane and the chorion by using elbow forceps to remove the chorion. Lifting one end of the amnion by using a pair of tweezers, using the other tweezers to strip out residual blood solution in the amnion as much as possible for decontamination treatment, then transferring the amnion into a new plastic basin filled with 100-200 mL of physiological saline (containing 100U/mL of penicillin and 100 microgram/mL of streptomycin), carrying out immersion cleaning for 2-3 min, and repeating the above operations until no blood pollution exists in the physiological saline, wherein the number of times is about 2-3.

Further optionally, in order to increase the number of the obtained endothelial progenitor cells and fully utilize the raw materials, adding an equal volume of physiological saline (containing penicillin 100U/mL and streptomycin 100 mug/mL) into the amniotic fluid and amniotic membrane preservation solution, transferring into a 50mL sterile centrifuge tube, centrifuging at 1200 rpm-1800 rpm for 8 min-12 min, and discarding the supernatant. Washing the cell precipitate with normal saline, mixing, centrifuging again, discarding supernatant, resuspending the precipitate in normal saline, and storing at 4 deg.C for use.

As an alternative embodiment, before the step of removing the sponge layer and the epithelial cell layer, the method further comprises cutting the pretreated amnion into a second predetermined size, for example, the second predetermined size may be 100 cm²A block of 200cm²The cut amnion is soaked and washed by physiological saline containing penicillin and streptomycin.

The step of removing the sponge layer and the epithelial cell layer comprises the operation of sequentially removing the sponge layer and the epithelial cell layer, and specifically can be:

taking an amnion, fixing the edge of the amnion by the elbow part of the elbow forceps, and then separating the sponge layer at the end from the amnion by the elbow part of the elbow forceps lightly.

The amnion of getting rid of the sponge layer is arranged in disposable plastic ware, uses the little tweezers of operation to tile amnion epithelial layer up, uses the fixed amnion one end of surgical tweezers on one hand, and the other end adopts the cell scraper from inside to outside, and the epithelial layer is scraped to the horizontal direction, and 5~ 10 times are scraped to same direction probably, can get rid of most epithelial cells through mechanical scraping.

Further optionally, the method further comprises the step of immersing the amniotic membrane tissue in a physiological saline containing penicillin and streptomycin in the middle of the step of removing the sponge layer and the epithelial cell layer and after the step of removing the sponge layer and the epithelial cell layer.

The specific operation steps can be as follows:

the amniotic membrane was spread on a disposable plastic dish and divided into pieces (200 cm) with a scalpel²Block), then transferring all amnions to 120-150 mL physiological saline (containing penicillin 100U/mL, streptomycin 100 mug/mL) for immersion washing for 1 time; one of the amnion is taken out,fixing the edge of an amniotic membrane by using an elbow part of an elbow forceps, slightly separating a sponge layer at the end from the amniotic membrane by using the elbow part of another elbow forceps, finally, washing all the amniotic membranes with the sponge layer removed in normal saline (containing 100U/mL of penicillin and 100 mug/mL of streptomycin) for 1 time to obtain amniotic membrane tissues, then spreading the obtained amniotic membranes in a plastic dish with the epithelial layer facing upwards, exposing the epithelial layer facing upwards and outwards, fixing one section of the amniotic membrane by using the elbow forceps, slightly scraping endothelial cells on the surface of the inner membrane from inside to outside by using a cell scraper at the other end for 8 times, and when scraping, the actions are gentle and uniform, the angle between the scraper and the surface is about 60 ℃, and the scraping direction of each part is the same. Transferring the amnion into a new plastic basin filled with antibiotics by using another pair of tweezers, and performing immersion washing for 2-3 times, which is the same as the above operation.

As an alternative, the specific operation method of the first digestion step and the second digestion step may be:

the amniotic tissue is transferred to a disposable plastic dish and spread, one end of the amniotic tissue is fixed with forceps, and the de-epithelialized amniotic tissue is cut into a predetermined size, for example, into 2cm × 2cm tissue pieces with a scalpel. Loading the cut amniotic membrane tissue fragments into a 50mL centrifuge tube, and mixing the cut amniotic membrane tissue with a collagenase solution according to a volume ratio of 1: (1-5) adding a collagenase solution, placing the mixture in an incubator at 37 ℃ for digestion for 1.0-8.0 h, discarding a supernatant of a digestion product (the supernatant of the digestion product contains epithelial cells and interstitial cells which are left after the step of mechanically removing the sponge layer and the epithelial layer cells of the amniotic membrane), transferring the digested amniotic tissue from the mucus of the digestion enzyme to a culture dish by using forceps, and washing the amniotic tissue twice by using normal saline to remove the epithelial cells and the interstitial cells attached to the amniotic membrane as much as possible.

Placing the digested amniotic membrane tissue into a new 50mL centrifuge tube, and mixing the digested amniotic membrane tissue with a collagenase solution according to a volume ratio of 1: (1-2) adding a collagenase solution, placing the mixture in an incubator at 37 ℃ for digestion for 1.0-2.0 h, adding a proper amount of normal saline into the digestion solution, mixing, centrifuging at 1100-1500 rpm for 3-7 min, and removing the supernatant of the digestion product to obtain the endothelial progenitor cell single cell suspension. Alternatively, an appropriate amount of physiological saline is added to the endothelial progenitor cells and stored at 4 ℃ for future use.

In an alternative embodiment, the collagenase is any one of collagenase type I, collagenase type II, collagenase type III, and collagenase type IV; the concentration of the collagenase solution is 0.05% -0.25%. Preferably, the collagenase is a more milder performing collagenase type IV. The concentration of the collagenase solution is 0.05% to 0.20%, and may be, for example, 0.05%, 0.07%, 0.09%, 0.10%, 0.11%, 0.13%, 0.15%, 0.16%, 0.19%, 0.20%. Further preferably, the concentration of the collagenase solution is 0.1%. Optionally, the collagenase is prepared into a collagenase solution with the concentration of 0.05% -0.25% by any one of basic culture media 1640, DMEM, MEM and DMEM/F12. The gradient stepwise digestion according to the invention is preferably a two-step digestion, wherein the addition volume, concentration and digestion time of the collagenase in the first and second digestion steps have an important influence on the treatment effect. In the first digestion step, if the concentration of collagenase is too high, the amount is too large or the digestion time is too long, the number of endothelial progenitor cells finally obtained is reduced; however, if the concentration of collagenase is too low, the amount of collagenase is too small, or the digestion time is too short, epithelial and mesenchymal cells remain too much, which affects the purity of the obtained cells; in contrast, in the second digestion step, if the collagenase is too high in concentration, too large in amount, or too long in digestion time, it will result in increased cell death rate, limited cell activity, and decreased purity of the cells obtained; if the collagenase concentration is too low, too small an amount or too short a digestion time, the number of endothelial progenitor cells finally obtained will be reduced.

Compared with the existing amnion stem cell separation method, the invention obtains high-purity endothelial progenitor cells by using a mild collagenase IV stepwise digestion method, only one digestive enzyme is needed in the digestion process, other exogenous enzymes are not needed to be introduced, the separation process is simplified, the risk of the exogenous substances influencing the quality of cell preparations is reduced, the operation is simple, the existing material-taking part is changed, the tissues and cells are not needed to be filtered, in addition, the cells obtained by separation are convenient to purify, only residual mesenchymal stem cells easy to adhere to the wall are needed to be removed through differential adherence, the prepared stem cells are more in quantity, the cell activity is good, the purity is high, and the proliferation capacity is stronger.

As an optional implementation, the obtaining method further includes the following steps: mixing the collected amniotic fluid and/or the amnion preserved preservation solution with normal saline containing penicillin and streptomycin according to the volume ratio of 1: (0.5-1.5), centrifuging, discarding the supernatant, mixing the obtained cell precipitate with physiological saline, centrifuging, and discarding the supernatant to obtain the endothelial progenitor cells. The obtained endothelial progenitor cells can be mixed with amnion-derived endothelial progenitor cells and cultured. The specific operation method can be as follows:

adding equal volume of physiological saline (containing penicillin 100U/mL and streptomycin 100 microgram/mL) into the amniotic fluid and amniotic membrane preservation solution, transferring into a 50mL sterile centrifuge tube, centrifuging at 1200-1800 rpm for 8-12 min, and discarding the supernatant. Washing the cell precipitate with normal saline, mixing, centrifuging again, discarding supernatant, resuspending the precipitate in normal saline, and storing at 4 deg.C for use.

Mixing the amnion-derived endothelial progenitor cell suspension and the amniotic fluid-derived endothelial progenitor cell suspension, adding equal volume of normal saline to prepare a mixed solution, subpackaging the mixed solution into 50mL centrifuge tubes, adding normal saline to 45mL, centrifuging at 1800-2000 rpm for 10 min. Discarding the supernatant, re-suspending the centrifugal precipitate with normal saline, synthesizing into 1 tube, centrifuging at 1800-2000 rpm for 10min, and discarding the supernatant for later use.

Amniotic fluid is convenient to collect with amnion at the same time, and considerable endothelial progenitor cells can be separated from the amniotic fluid to obtain endothelial progenitor cells; in addition, the amniotic membrane preserved preserving fluid may also contain appreciable amounts of endothelial progenitor cells. The endothelial progenitor cells in the amniotic fluid or the amnion-preserved preserving fluid are obtained by separation, so that the number of the obtained endothelial progenitor cells can be further increased, the utilization rate of raw materials is increased, and the preparation cost of the endothelial progenitor cells is reduced.

The invention also provides a method for obtaining the amniotic fluid-derived endothelial progenitor cells, which comprises the following steps: mixing the collected amniotic fluid and/or the amnion preserved preservation solution with normal saline containing penicillin and streptomycin according to the volume ratio of 1: (0.5-1.5), centrifuging, discarding the supernatant, mixing the obtained cell precipitate with physiological saline, centrifuging, and discarding the supernatant to obtain the endothelial progenitor cells. Considerable endothelial progenitor cells can be separated from the amniotic fluid to obtain the endothelial progenitor cells; in addition, the amniotic membrane preserved preserving fluid may also contain appreciable amounts of endothelial progenitor cells. The endothelial progenitor cells in the amniotic fluid or the amnion-preserved preserving fluid are obtained by separation, so that the number of the obtained endothelial progenitor cells can be further increased, the utilization rate of raw materials is increased, and the preparation cost of the endothelial progenitor cells is reduced. The harvesting method can be used alone, or can be applied to the method for harvesting the amniotic membrane derived endothelial progenitor cells as described above.

In a second broad aspect of the invention, there is also provided an endothelial progenitor cell obtained by the method described above.

The third aspect of the present invention also provides a method for purifying and culturing the endothelial progenitor cells, which comprises the following steps:

Optionally, the endothelial progenitor cells are mixed with a culture medium to prepare a cell suspension, and the cell suspension is subjected to a single inoculation culture at a volume fraction of 5% CO₂Culturing for 6.0-24.0 h, preferably 8.0-18.0 h, and more preferably 11.0-15.0 h in an incubator at the temperature of 37 ℃; after the natural sedimentation adherence in the period of time, the residual interstitial stem cells with fast adherence can be removed through the first adherence, the non-adherence cells and the cell suspension after the first inoculation culture are subjected to the second inoculation culture, and the volume fraction of CO is 5 percent₂Culturing for 24.0-55.0 h in an incubator at 37 ℃, performing full liquid exchange after the secondary wall adhesion to obtain endothelial progenitor cells with high purity, and performing full liquid exchange or half liquid exchange at regular time according to the culture condition until the cell fusion rate reaches 80-90%.

The primary progenitor cells obtained by the gradient stepwise digestion method are further subjected to differential adherent culture by using the purification culture method, residual interstitial cells can be removed, endothelial progenitor cell seed cells with higher purity are obtained by secondary adherence, the obtained endothelial progenitor cells have a typical clonal growth state in the culture process by a factor optimization method of a culture solution, and the cells are in a cobblestone or short fusiform typical endothelial progenitor cell shape.

The method has the advantages of simple operation, easy repetition, low requirement of the obtained cell culture, strong in-vitro amplification capacity and short proliferation time, and overcomes the difficulties of low quantity of self-derived EPC, slow in-vitro amplification and the like. The purification and culture steps are simple, convenient and easy to implement, economical, strong in practicability and low in operation requirement.

Optionally, before preparing the cell suspension, the endothelial progenitor cells to be used are washed with physiological saline, and the specific operation method can be as follows:

adding equal volume of normal saline into amnion-derived endothelial progenitor cell suspension and/or amniotic fluid-derived endothelial progenitor cell suspension to prepare mixed solution, subpackaging the mixed solution into 50mL centrifuge tubes, supplementing normal saline to 45mL, centrifuging at 1800-2000 rpm for 8-12 min. Discarding the supernatant, re-suspending the centrifugal precipitate with normal saline to synthesize 1 tube at 1800-2000 rpm for 8-12 min, and discarding the supernatant for later use.

Optionally, the culture medium used in the method for purifying and culturing endothelial progenitor cells is any one of IMDM, α -MEM, DMEM, EGM, CGM and DMEM/F12.

Optionally, the culture solution is supplemented with any one of platelet lysate, endothelial growth factor, epithelial growth factor, and basic fibroblast growth factor.

Further optionally, the content of the endothelial cell growth factor in the culture solution is 8 ng/mL-12 ng/mL, preferably 10 ng/mL; the content of the epidermal growth factor is 8 ng/mL-12 ng/mL, preferably 10 ng/mL; the content of the basic fibroblast growth factor is 8 ng/mL-12 ng/mL, and preferably 10 ng/mL. The content of the platelet lysate in the culture solution is 3-7 per mill (V/V), preferably 5 per mill (V/V).

Further optionally, the method for purifying and culturing endothelial progenitor cells further comprises the following steps: and digesting adherent cells with the cell fusion rate of 80-90% by using trypsin, and then carrying out subculture. Alternatively, the concentration of trypsin is 0.05% -0.25% (w/v); preferably, the concentration of trypsin is 0.1% (w/v). The specific operation method can be as follows:

directly digesting adherent cells with the cell fusion rate of 80% -90% for 2 min by using 0.1% trypsin for passage, and carrying out subsequent amplification culture. Meanwhile, a small number of passage cells are taken for flow cytometry detection and karyotype detection so as to analyze the dry maintenance capacity and stability of the in vitro long-term culture of the cells.

The invention adopts collagenase IV gradient stepwise digestion method to remove epithelial cells and interstitial cells in amnion, so as to obtain stem cell group of hemangioblast, and residual interstitial cells are removed by differential adherence in the adherence process, so as to obtain endothelial progenitor cell seed cells with higher purity. The single digestive enzyme is used in the digestion process, the method is simple and easy to implement, the operations such as tissue grinding, filtering and the like are not needed, the operation steps are simple and easy to implement, the complete SOP is easy to form, and the industrial transformation and industrialization are easy to realize.

Example 1

Preparation before experiment:

1) after ultraviolet sterilization of the level II biological safety cabinet for 20 min, blowing the table top for 15min by a fan;

2) personal protective articles necessary for wearing include work clothes, work shoes, hats, masks, double-layer sterile gloves and protective glasses;

3) the required sterile consumables, instruments, reagents, etc. are in place and the wash vessel is ready for use.

A pretreatment step:

1) collecting amniotic fluid in the process of childbirth of a pregnant woman, obtaining placenta and amniotic tissue after childbirth, peeling the amniotic tissue from the placenta by using surgical scissors, placing the amniotic tissue in a storage box containing preservation solution, signing informed consent by family members, and then carrying the amniotic tissue back to a laboratory at 2-8 ℃ in a timely cold chain manner; in order to avoid pollution, the numbers are numbered.

2) Wiping the surface of a sample bottle of the human amniotic membrane with 75% alcohol, and then placing the sample bottle into a II-level biological safety cabinet; carefully opening the bottle cap of the sample bottle, transferring the human amniotic membrane into a plastic basin filled with 150mL of physiological saline (penicillin 100U/mL, streptomycin 100 mug/mL) by using a large sterile forceps, performing immersion washing for 2-3 min, and then performing blunt separation on the amniotic membrane and the chorion by using an elbow forceps.

3) One end of the amnion is lifted by a pair of tweezers, the residual blood solution in the amnion is stroked out as much as possible by the other tweezers, then the amnion is transferred to a new plastic basin filled with 150mL of physiological saline (penicillin 100U/mL and streptomycin 100 mug/mL), the amnion is soaked and washed for 2-3 min, and the operations are repeated until the physiological saline is free of blood contamination (2-3 times).

4) Adding equal volume of physiological saline (penicillin 100U/mL, streptomycin 100 mug/mL) into the amniotic fluid and amniotic fluid preservation solution, transferring into a 50mL sterile centrifuge tube, centrifuging at 1500rpm for 10min, and discarding the supernatant. Washing the cell precipitate with normal saline, mixing, centrifuging again, discarding the supernatant, resuspending the precipitate in normal saline, and storing at 4 deg.C.

Removing cells of the spongy layer and the epithelial layer:

1) the amniotic membrane was spread on a disposable plastic dish and divided into pieces (200 cm) with a scalpel²And/piece), then transferring all amnions to 120-150 mL physiological saline (penicillin 100U/mL, streptomycin 100 mug/mL) for immersion washing for 1 time.

2) Taking one of the amniotic membranes, fixing the edge of the amniotic membrane at the elbow part of the elbow forceps, then separating the sponge layer at the end from the amniotic membrane in a blunt manner by using the other elbow part of the elbow forceps, and finally, soaking and washing all the amniotic membrane tissues with the sponge layer removed in normal saline (penicillin 100U/mL, streptomycin 100 mug/mL) for 1 time.

3) The amnion is placed in a disposable plastic dish, the epithelial layer of the amnion is laid upwards by using small surgical forceps, one end of the amnion is fixed by using the surgical forceps on one hand, and the epithelial layer is scraped from inside to outside in the horizontal direction by using a cell scraper on the other end.

A first digestion step:

1) the amniotic tissue is transferred to a disposable plastic dish and spread, one end of the amniotic tissue is fixed by a pair of tweezers, and the de-epithelialized amniotic tissue is cut into 2 x 2cm tissue pieces by a scalpel.

2) The cut amniotic membrane tissue fragments are put into a 50mL centrifuge tube, 0.1% collagenase IV is added according to the volume ratio of 1 (1-5), and the cover is screwed tightly; digesting the amniotic tissue in an incubator at 37 ℃ for 1.0-3.0 h.

3) And (5) after the digestion is finished. Transferring the amniotic membrane tissue from the digestive enzyme mucus to a culture dish by using forceps, and washing twice by using normal saline; epithelial cells and mesenchymal cells attached to the amniotic membrane are removed as much as possible.

A second digestion step:

placing the amniotic membrane tissue into a new 50mL centrifuge tube, adding 0.1% collagenase IV into collagenase (1-3) according to the volume ratio of 1, and screwing down a cover; continuously digesting the amniotic tissue in the incubator at 37 ℃ for 0.5-2.0 h. After digestion, adding a proper amount of normal saline, carrying out centrifugation at 1300 rpm/min for 5 min; discarding the supernatant and the tissue; 10mL of physiological saline was added to each tube to prepare a cell suspension for use.

Collecting endothelial progenitor cells:

mixing the cell suspension obtained by digesting the amniotic membrane with the cell suspension obtained from the amniotic fluid source, adding physiological saline with the same volume, subpackaging the solution into 50mL centrifuge tubes, adding the physiological saline to 45mL, performing centrifugation at 1800-2000 rpm for 10 min. Abandoning the supernatant, resuspending the centrifugal precipitate with normal saline, combining 1 tube, centrifuging at 1800-2000 rpm for 10min, abandoning the supernatant, and collecting endothelial progenitor cells for later use.

Purified culture of endothelial progenitor cells:

adding a certain amount of EPC culture solution into a centrifuge tube filled with endothelial progenitor cells, suspending the cells, counting, and performing 1 × 10⁴/cm²Density cells were seeded in sterile culture flasks and placed at a volume fraction of 5% CO₂An incubator at 37 ℃ and saturated humidity, and sampling for microorganism detection.

After 12 h of cell culture under the above culture conditions, the nonadherent cells and cell suspension were re-inoculated into a new culture plate and placed in a volume fraction of 5% CO₂Culturing for 48 h in an incubator at 37 ℃ under saturated humidity conditions, carrying out first total liquid change, removing redundant tissue blocks and non-adherent cells, adding fresh culture solution, changing the total liquid for 1 time every other day, and digesting and passaging by 0.05% (w/v) pancreatin (purchased from sigma) when the cell fusion degree reaches 90%, so as to obtain the amniotic endothelial cells. At the same time, a small number of passage cells are taken forFlow cytometry, karyotype detection, to analyze the dry maintenance capacity and stability of long-term cell culture in vitro.

Wherein the culture medium is IMDM medium (purchased from gibco) containing 5% (V/V) platelet lysate (purchased from UltraGRO), 10ng/mL VEGF, 10ng/mL EGF and 10ng/mL bFGF (purchased from peprotech), and in other embodiments, DMEM/F12, alpha-MEM, ECM, EGM and other cell culture media can be selected for amplification culture.

Identification of human amniotic endothelial cells

Firstly, the growth characteristics and morphological characteristics of human amniotic endothelial cells

Observing the purified culture of the endothelial progenitor cells in example 1 by using a microscope, inducing human amniotic endothelial cells adherent for 2 days by using a total solution change for the first time (expanding the cells after differential adherent culture of the endothelial progenitor cells), as shown in fig. 1 and 2, the adherent cells with a scattered paving stone shape can be seen under the microscope, the monoclonal cells can be seen to form after culturing for 3-4 days after the total solution change (fig. 1), and when the cells are cultured until the cell fusion degree reaches about 90% (fig. 2), the cells are in a typical paving stone shape, have a faster proliferation speed and relatively uniform shape, and can be used for identifying the biological characteristics of the cells. The morphology of the cells after further culturing is shown in FIG. 3. According to the identification, the purification culture method is simple, EPC seed cells with high purity are obtained by a simple differential adherence method and primary cell digestion, the obtained EPC has a clonal growth state in the culture process by a factor optimization method of a culture medium, and the cells are in a cobblestone or short spindle-shaped typical EPC form.

Flow cytometry for identifying human amniotic endothelial cell surface marker

Taking 3 rd generation human amniotic endothelial cells from the amniotic source, and detecting a cell surface marker by using flow cytometry. The specific method comprises the following steps: digesting and collecting cells, counting, and taking 1 × 10⁶Washing with PBS for 1 time, centrifuging at 1500rpm for 10min, discarding supernatant, leaving 200uL, blowing to mix well, adding FITC labeled CD31 (CD 45) and PERCP labeled CD105, PE labeled CD44 (CD 73) and APC labeled CD29 (HLA-DR) 10uL each in two groups, respectively, purchasing antibody from BD, and setting 1 tube as emptyWhite control, reaction at 4 ℃ in dark for 30min, washing with PBS for 1 time, centrifuging at 1500rpm for 10min, discarding supernatant, leaving 200uL, and detecting on machine.

The flow cytometry detection results of the human amniotic endothelial cell surface markers are shown in table 1 and fig. 4, and the EPC obtained by the method for obtaining the endothelial progenitor cells of the invention from amniotic membrane and amniotic fluid is high in purity, and the flow cytometry phenotype proves that the EPC obtained by the method not only highly expresses important phenotype stem cell markers CD29, CD44, CD73 and CD105 of stem cells, but also highly expresses endothelial progenitor cell specific antigen CD31 (platelet-endothelial cell adhesion molecules), the positive rate of the EPC is as high as more than 90 percent, the EPC obtained by the method has no expression or low expression of a hematopoietic stem cell marker CD45 and cellular immune characteristic HLA-DR (< 5%), which shows that the cellular immunogenicity is low, immune rejection after transplantation does not exist, and the method is suitable for seed sources applied to tissue engineering and regenerative medicine.

TABLE 1 flow cytometry assay results for human amniotic endothelial progenitor cells

Biomarker compounds	Test results (%)
		CD29	99.48
CD31	94.64
		CD44	97.45
CD73	99.02
		CD105	99.54
CD45	0.26
		HLA-DR	0.49

Application of human amniotic endothelial cells

The human amniotic endothelial cells isolated in example 1 of the present invention can be used for differentiation into blood vessels after in vitro amplification. The method specifically comprises the following steps of preparing a 24-hole plate, and paving a layer of Matrigel matrix of 200 mu L/hole under an ice bath condition. The mixture was incubated in an incubator at 37 ℃ for 1 hour to give a gummy solid. The exponential growth phase of EPC from example 2 was then trypsinized into single cells and plated onto Matrigel gel at 10 ten thousand/well. After overnight incubation, the ability of the EPCs to vascularize in vitro in three-dimensional culture environment was observed (figure 5, see, when the EPC was seeded on the matrix layer colloid, the EPC was able to migrate into the colloid like other endothelial cells and form a capillary-like structure, in addition, in order to verify the in vitro angiogenic ability of EPC isolated and cultured by this method, we tested in vitro amplification culture until the third generation EPC cells were trypsinized into single cells, then seeded into Matrigel gel to be freely differentiated, after overnight incubation, the angiogenic ability of EPC in vitro in three-dimensional culture environment was observed, as can be seen in FIG. 5, when the EPC is inoculated on matrigel colloid, the EPC can migrate into the colloid like other endothelial cells, and forming a capillary-like structure, the lumen structure of the amnion-derived EPC of figure 5 further verifies the angiogenic function of the EPC obtained by this method.

Therefore, the human amniotic endothelial cells can be used for establishing in vitro blood vessel formation and repairing in vivo damaged blood vessels, and have wide application prospect.

Fourth, the functional identification of human amniotic endothelial progenitor cells

The amniotic endothelial cells obtained by separation and culture of the invention also have the characteristics of endothelial cell adhesion, uptake of acetylated low-density lipoprotein (ac-LDL) and combination of vitex bean phytohemagglutinin (UEA-I), and the verification experiment is as follows:

after the primary endothelial progenitor cells are cultured in vitro to the third generation, 4 mu g of acetylated low-density lipoprotein Dil-ac-LDL is added into the adherent cells in exponential growth phase, the adherent cells are placed in an incubator for incubation for 4 h, washed by PBS for 3 times, fixed by 20 g/L paraformaldehyde for 20 min, rinsed by PBS once, and 10mg/L of green fluorescence labeled FITC-UEA-I4 mu g is added into the specimen for incubation for 1 h at 37 ℃, and the cells are observed under a fluorescence microscope after DAPI staining, and the result is shown in figure 6. The distribution of cells under white light is shown in figure 6A (white light), DAPI staining shows that cell nuclei are shown in figure 6B (purple, DAPI), the staining is green to show that amniotic endothelial progenitor cells are combined with UEA-I to show that figure 6C (green light, FITC-UEA-I), the staining is red to show that cells take ac-LDL to show that figure 6D (red light, DiI-ac-LDL), the images are combined and then are shown in figure 6E (combined), the diagram shows that the amniotic endothelial progenitor cells can not only take low-density lipoprotein ac-LDL and can be combined with phytohemagglutinin UEA-I, the rate of double-staining positive cells reaches more than 95%, and the obtained cells have high purity.

Continuous amplification culture of human amniotic membrane derived EPC

In order to evaluate the nuclear stability of the continuous proliferation capability of the human amniotic endothelial progenitor cells, the endothelial progenitor cells obtained by separation and culture in the embodiment 1 of the invention are cultured in a full culture medium EGM and continuously expanded to the 10 th generation, and the result shows that the cells grow rapidly in an oval shape after passage, the cells proliferate rapidly, and can be passaged for 1 time every 2-4 d, and the passage ratio can reach 1: 5-6, the cell shape is stable after passage, and the cell grows in accordance with the exponential phase. After culturing to 10 generations, the adherent cells are taken and sent to carry out chromosome G band analysis and flow cycle analysis (cell and molecular clinical laboratory institute of Beijing Mongolian stem cell technology, Inc.), thereby carrying out karyotype and cell cycle detection. As shown in FIG. 8, the G-band analysis showed that the cells had a normal karyotype (46 XY), indicating that the cells maintained genetic stability during the continuous amplification culture; as shown in fig. 9, the cell cycle showed cell ratios of 94.63%, 0.63% and 5.36% in the pre-DNA synthesis phase (G1 phase), the DNA synthesis phase (S phase) and the post-DNA synthesis phase (G2 phase), respectively. The growth ability of the cells is vigorous, most cell substances are actively metabolized, RNA and protein are rapidly synthesized, and rapid mitosis can be performed.

Example 2

Preparation before experiment:

A pretreatment step:

1) collecting amniotic fluid in the process of childbirth of a pregnant woman, obtaining placenta and amniotic tissue after childbirth, peeling the amniotic tissue from the placenta by using surgical scissors, placing the amniotic tissue in a storage box containing preservation solution, signing informed consent by family members, and timely conveying the amniotic tissue back to a laboratory at the temperature of 2-8 ℃; in order to avoid pollution, the numbers are numbered.

Removing the sponge layer and the epithelial cell layer:

1) the amnion is laid on a disposable plastic dish, the amnion is divided into blocks (200 cm 2/block) by a scalpel, and then all the amnion is transferred to 120-150 mL of physiological saline (penicillin 100U/mL, streptomycin 100 mug/mL) to be soaked and washed for 1 time.

A first digestion step:

2) Loading the cut amniotic membrane tissue fragments into a 50mL centrifuge tube, adding 0.2% collagenase IV according to the volume ratio of 1:1, and screwing down a cover; the amniotic tissue was digested in a 37 ℃ incubator for 1.0 h.

A second digestion step:

placing the amniotic membrane tissue into a new 50mL centrifuge tube, adding 0.2% collagenase IV into collagenase according to the volume ratio of 1:1, and screwing down a cover; the incubator at 37 ℃ continues to digest the amniotic tissue for 0.5 h. After digestion, adding a proper amount of normal saline, carrying out centrifugation at 1300 rpm/min for 5 min; discarding the supernatant and the tissue; 10mL of physiological saline was added to each tube to prepare a cell suspension for use.

Collecting endothelial progenitor cells:

Purified culture of endothelial progenitor cells:

adding a certain amount of EPC culture solution into a centrifuge tube filled with endothelial progenitor cells, suspending the cells, counting, and performing 5 × 10⁴/cm²Density cells were seeded in sterile culture flasks and placed at a volume fraction of 5% CO₂An incubator at 37 ℃ and saturated humidity, and sampling for microorganism detection.

After 6.0 h of cell culture under the above culture conditions, the nonadherent cells and cell suspension were re-inoculated into a new culture plate and placed in a volume fraction of 5% CO₂Culturing for 45 h in an incubator at 37 ℃ under saturated humidity conditions, performing first total liquid exchange, removing redundant tissue blocks and non-adherent cells, adding fresh culture solution, performing total liquid exchange for 1 time every other day, and digesting and passaging with 0.05% (w/v) pancreatin (purchased from sigma) when the cell fusion degree reaches 90%, so as to obtain the amniotic endothelial cells. Meanwhile, a small number of passage cells are taken for flow cytometry detection and karyotype detection so as to analyze the dry maintenance capacity and stability of the in vitro long-term culture of the cells.

Example 3

Preparation before experiment:

A pretreatment step:

Removing the sponge layer and the epithelial cell layer:

A first digestion step:

2) Loading the cut amniotic membrane tissue fragments into a 50mL centrifuge tube, adding 0.2% collagenase IV according to the volume ratio of 1:5, and screwing down a cover; the amniotic tissue was digested in a 37 ℃ incubator for 3.0 h.

A second digestion step:

placing the amniotic membrane tissue into a new 50mL centrifuge tube, adding 0.2% collagenase IV into collagenase according to the volume ratio of 1:5, and screwing down a cover; the incubator at 37 ℃ continues to digest the amniotic tissue for 2.0 h. After digestion, adding a proper amount of normal saline, carrying out centrifugation at 1300 rpm/min for 5 min; discarding the supernatant and the tissue; 10mL of physiological saline was added to each tube to prepare a cell suspension for use.

Collecting endothelial progenitor cells:

Purified culture of endothelial progenitor cells:

adding a certain amount of EPC culture solution into a centrifuge tube filled with endothelial progenitor cells, suspending the cells, counting, and performing 5 × 10⁴/cm²Density cells were seeded in sterile culture flasks and placed at a volume fraction of 5% CO₂、37℃、Incubators saturated with humidity and sampling for microorganism detection.

After 18 h of cell culture under the above culture conditions, the nonadherent cells and cell suspension were re-inoculated into a new culture plate and placed in a volume fraction of 5% CO₂Culturing at 37 deg.C for 55 hr in incubator with saturated humidity, performing first total liquid exchange, removing excessive tissue blocks and non-adherent cells, adding fresh culture solution, changing total liquid for 1 time every other day, and digesting with 0.05% (w/v) pancreatin (purchased from sigma) for passage when cell fusion degree reaches 90% to obtain amnion endothelial cell. Meanwhile, a small number of passage cells are taken for flow cytometry detection and karyotype detection so as to analyze the dry maintenance capacity and stability of the in vitro long-term culture of the cells.

Example 4

Preparation before experiment:

A pretreatment step:

Removing the sponge layer and the epithelial cell layer:

A first digestion step:

2) Loading the cut amniotic membrane tissue fragments into a 50mL centrifuge tube, adding 0.05% collagenase IV according to the volume ratio of 1:2, and screwing down a cover; the amniotic tissue was digested at 37 ℃ in an incubator 1.0.

A second digestion step:

placing the amniotic membrane tissue into a new 50mL centrifuge tube, adding 0.05% collagenase IV into collagenase according to the volume ratio of 1:2, and screwing down a cover; the incubator at 37 ℃ continues to digest the amniotic tissue for 0.5 h. After digestion, adding a proper amount of normal saline, carrying out centrifugation at 1300 rpm/min for 5 min; discarding the supernatant and the tissue; 10mL of physiological saline was added to each tube to prepare a cell suspension for use.

Collecting endothelial progenitor cells:

Purified culture of endothelial progenitor cells:

After 14 h of cell culture under the above culture conditions, the nonadherent cells and cell suspension were re-inoculated into a new culture plate and placed in a volume fraction of 5% CO₂Culturing for 50 h in an incubator at 37 ℃ under saturated humidity conditions, carrying out first total liquid change, removing redundant tissue blocks and non-adherent cells, adding fresh culture solution, changing the total liquid for 1 time every other day, and digesting and passaging by 0.05% (w/v) pancreatin (purchased from sigma) when the cell fusion degree reaches 90%, so as to obtain the amniotic endothelial cells. Meanwhile, a small number of passage cells are taken for flow cytometry detection and karyotype detection so as to analyze the dry maintenance capacity and stability of the in vitro long-term culture of the cells.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Claims

1. An amniotic membrane derived endothelial progenitor cell harvesting method, comprising the steps of:

a pretreatment step of pretreating an amnion detached from a placenta; in the pretreatment step, the pretreatment comprises a normal saline immersion washing treatment containing penicillin and streptomycin, a chorion removal treatment and a decontamination treatment;

removing a sponge layer and an epithelial cell layer, namely removing the pretreated amniotic sponge layer and the pretreated epithelial cell layer by adopting a mechanical separation method to obtain an amniotic tissue;

a first digestion step, cutting the amniotic tissue into a preset size, and mixing the cut amniotic tissue with an IV type collagenase solution with the concentration of 0.05% -0.25% according to the volume ratio of 1: (1-5), placing the mixture in an incubator at 37 ℃ for digestion for 1.0-8.0 h, and washing the digested amniotic tissue with physiological saline;

a second digestion step, namely mixing the amniotic membrane tissue subjected to the first digestion step with an IV type collagenase solution with the concentration of 0.05-0.25% according to the volume ratio of 1: (1-2), placing the mixture in an incubator at 37 ℃ for digestion for 1.0-2.0 h, mixing the digestion solution with physiological saline, centrifuging, removing supernatant, and carrying out cell resuspension to obtain cell suspension;

mixing the cell suspension obtained by digesting the amniotic membrane with the cell suspension obtained from the amniotic fluid source, adding the same volume of physiological saline, mixing, centrifuging at 1800-2000 rpm, discarding the supernatant, mixing the obtained cell precipitate with the physiological saline, centrifuging, discarding the supernatant, and obtaining the endothelial progenitor cell.

2. The method of claim 1, wherein prior to the step of removing the sponge layer and epithelial cell layer, further comprising the steps of:

cutting the pretreated amnion into a second preset size, and soaking and washing the cut amnion by using physiological saline containing penicillin and streptomycin.

3. The method of claim 2, further comprising, after said step of removing the sponge layer and epithelial cell layer, the steps of:

the amniotic tissue is treated by immersion in a physiological saline solution containing penicillin and streptomycin.

4. The obtaining method of claim 3, further comprising a purification culture method of the endothelial progenitor cells, i.e. inoculating the endothelial progenitor cells into the culture solution, naturally settling and attaching the cells for 6.0-24.0 h, removing the residual mesenchymal cells to obtain a suspension of the endothelial progenitor cells, and obtaining the primary seed cells of the endothelial progenitor cells for subculture after secondary attachment.