CN112574948A - Separation culture method of human amniotic mesenchymal stem cells - Google Patents
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Abstract
The invention provides a separation culture method of human amniotic mesenchymal stem cells, which comprises the steps of stripping a amniotic membrane layer from a placenta, cleaning, shearing and digesting, and is characterized in that: removing the stroma tissue under the basement membrane to make the separated amnion as thin as possible and remove blood cells after the amnion layer is stripped from the placenta, and then shearing and digesting; wherein the placenta tissue is fresh placenta tissue of a full-term birth fetus, and the placenta tissue is sterilized and cleaned to separate a amniotic membrane layer from the placenta under the aseptic condition; and the human amniotic membrane does not need to be digested and separated by epithelial cells, but is directly used for digesting and separating the human amniotic mesenchymal stem cells. The human amniotic mesenchymal stem cells obtained by the separation and culture method of the human amniotic mesenchymal stem cells have high purity, strong activity and quick proliferation. And the conclusion establishes a method for separating, culturing and identifying the human amniotic mesenchymal stem cells. hAMSCs have the phenotypic characteristics of bone marrow mesenchymal stem cells.
Description
Technical Field
The invention belongs to the field of cell separation culture, and particularly relates to a separation culture method of human amniotic mesenchymal stem cells.
Background
The human amniotic membrane source stem cell has the multidirectional differentiation potential, and has the characteristics of easiness in obtaining, no ethical dispute, rich stem cell content, low immunogenicity and the like, so that the human amniotic membrane source stem cell has the potential to become an important seed cell source for future regenerative medicine clinical application.
Recent studies prove that human amniotic mesenchymal stem cells (hAMSCs) have a phenotype spectrum similar to bone marrow mesenchymal stem cells (BMSCs), have multi-lineage differentiation potential, can be differentiated into cells of three germ layers and different types, have stronger amplification capability than BMSCs [ 1-8 ], have wide sources, do not involve medical ethical problems and other advantages, and therefore, the hAMSCs are very concerned as cell resources of regenerative medicine (regenerative medicine). However, there is no standardized protocol for isolation, culture and identification of hAMSCs, so it is necessary to establish a method for isolation of hAMSCs with high abundance, high purity and high activity, a simple and clear identification index and a suitable in vitro subculture protocol.
The Chinese patent with publication number CN106244521A discloses a human amniotic epithelial cell, a separation culture method and application. The method comprises the following steps: (1) peeling the amnion from the placenta tissue and cleaning; (2) cutting the cleaned amnion, and digesting with pancreatin; (3) sieving the digestive juice obtained after the digestion treatment, collecting cell filtrate, and centrifuging; (4) suspending the cells obtained by centrifugal treatment with a culture medium, inoculating, and culturing; (5) and when the confluency of the cultured cells reaches 80-90%, adding pancreatin for digestion, then adding a culture medium to stop digestion, and carrying out subculture to obtain the human amniotic epithelial cells. In the method, the amniotic epithelial cells are extracted, separated and purified from the placenta and then expanded, proliferated and cultured in vitro, so that the amniotic epithelial cells can be simply, conveniently and massively obtained. Meanwhile, the human amniotic epithelial cells cultured by the method have high purity, can be used for treating diseases such as diabetes and the like, and can effectively control the blood sugar level of a patient.
The sumo paper of von qian of Beijing institute of medicine, "qualitative research of human amniotic epithelial cells" discloses the isolation of human amniotic mesenchymal stem cells: taking a specimen, fixing the specimen, embedding and slicing paraffin, performing histological staining on the whole layer of the human amniotic membrane, digesting and separating the human amniotic mesenchymal stem cells after digesting and separating the human amniotic epithelial cells.
The above prior art has the following drawbacks: the obtained human amniotic mesenchymal stem cells have more impurities, and have low separation purity, weak activity and slow proliferation.
Disclosure of Invention
In order to solve the technical problem of more impurities in the prior art, the invention provides a separation culture method of human amniotic mesenchymal stem cells, which has the advantages of high purity, strong activity and rapid proliferation of the human amniotic mesenchymal stem cells.
In order to achieve the purpose, the following technical scheme is adopted: a separation culture method of human amniotic mesenchymal stem cells comprises stripping a amniotic membrane layer from a placenta, cleaning, shearing, digesting, further comprises stripping the amniotic membrane layer from the placenta, removing matrix tissue under a basement membrane to make the separated amniotic membrane as thin as possible and remove blood cells, and then shearing and digesting; wherein the placenta tissue is fresh placenta tissue of a full-term birth fetus, and the placenta tissue is sterilized and cleaned to separate a amniotic membrane layer from the placenta under the aseptic condition; and the human amniotic membrane does not need to be digested and separated by epithelial cells, but is directly used for digesting and separating the human amniotic mesenchymal stem cells.
Preferably, the method for separating and culturing the human amniotic mesenchymal stem cells comprises the following steps:
(1) taking placenta tissue, and cleaning;
(2) separating the amniotic membrane layer from the placenta,
(3) removing the stromal tissue under the basement membrane to make the separated amniotic membrane as thin as possible;
(4) removing blood cells;
(5) processing the amnion obtained in the step (4) into small fragments;
(6) digesting the amniotic membrane;
(7) collecting cell filtrate, centrifuging, removing supernatant, and collecting precipitate; resuspending the precipitate, centrifuging again, and discarding the supernatant; adding the precipitate into a complete culture medium for resuspension, counting, inoculating the cells into the culture medium, and culturing;
(8) and (3) changing the liquid after culturing for a certain time, then changing the liquid once at intervals of fixed time, and subculturing according to the proportion of 1:3 after the cell fusion degree reaches 80 percent to obtain the separated and purified human amniotic mesenchymal stem cells.
Preferably, in any of the above protocols, in step (1), the surface of the placenta tissue is sterilized and washed with 75% alcohol or 5% double antibody-added PBS.
Preferably in any of the above embodiments, step (2) is carried out under aseptic conditions.
Preferably, in any of the above embodiments, in step (2), the amniotic membrane layer is blunt-separated from the placenta.
In any of the above embodiments, preferably, in the step (3), the stromal tissue under the basement membrane is removed with a sterile cotton swab.
In any of the above embodiments, preferably, in the step (4), the method for removing blood cells comprises: the amniotic membrane was placed in a petri dish containing 1/2 volumes of PBS containing 1% double antibody, the amniotic membrane was placed in a petri dish and rinsed and the dish was replaced to remove blood cells.
In any of the above embodiments, it is preferred that in step (5), the small pieces have a diameter of 1 to 3 mm.
In any of the above embodiments, preferably, in step (6), the digestion is specifically performed by: transferring the small amnion fragments to a centrifuge tube, uniformly mixing the small amnion fragments with pancreatin digestive juice and collagenase II digestive juice, and placing the mixture in a constant temperature shaking table for oscillation; then stopping digestion, mixing uniformly, sieving by a cell sieve, and collecting filtrate; adding the undigested amnion into the digestive juice for digestion, and repeating the steps until the amnion tissue is basically digested.
In any of the above embodiments, preferably, in the step (6), the pancreatin digestive juice is composed of 0.25% of pancreatin + 0.02% of EDTA by mass percent.
In any of the above embodiments, preferably, in step (6), the mass percentage concentration of the collagenase II digest is 0.1%.
In any of the above embodiments, preferably, in step (6), the small pieces of amnion, the pancreatin digest, and the collagenase II digest are mixed in a volume ratio of 1:1: 1.
In any of the above schemes, preferably, in the step (6), the temperature in the constant temperature shaking table is 37 ℃, the rotating speed is 250r/min, and the oscillation time is 10-20 min.
In any of the above-mentioned protocols, preferably, in step (6), termination of digestion is carried out by adding 10% FBS in low-sugar DMEM/F12 medium.
Preferably, in any of the above embodiments, in step (6), the cell sieve is 200 mesh.
In any of the above schemes, preferably, in the step (7), the first centrifugal rotation speed is 2000r/min, and the subsequent centrifugal rotation speed is 1500 r/min.
In any of the above protocols, it is preferred that in step (7), the complete medium added in the second resuspension is low-sugar DMEM/F12 medium containing 10% FBS.
The method for separating and culturing the human amniotic mesenchymal stem cells adopts a low-speed rotation-trypsin-collagenase digestion method to separate the human amniotic mesenchymal stem cells (hAMSCs), adopts immunocytochemistry and flow cytometry to analyze the phenotypic characteristics of the human amniotic mesenchymal stem cells, and uses a low-sugar DMEM culture medium containing 10% FBS to culture the human amniotic mesenchymal stem cells. The separated human amniotic mesenchymal stem cells have high purity, strong activity and quick proliferation. Establishes the method for separating, culturing and identifying the human amniotic mesenchymal stem cells. hAMSCs have the phenotypic characteristics of bone marrow mesenchymal stem cells.
Drawings
FIG. 1 is a growth curve of human amniotic mesenchymal stem cells of different generations according to a preferred embodiment of the isolated culture method of human amniotic mesenchymal stem cells of the present invention.
Detailed Description
In order that the invention may be more fully and clearly understood, reference will now be made to the following description and accompanying drawings, which are included to provide a further understanding of the invention.
Example 1
The separation and culture method of the human amniotic mesenchymal stem cells comprises the steps of stripping a amniotic membrane layer from a placenta, cleaning, shearing and digesting, and is characterized in that: removing the stroma tissue under the basement membrane to make the separated amnion as thin as possible and remove blood cells after the amnion layer is stripped from the placenta, and then shearing and digesting; wherein the placenta tissue is fresh placenta tissue of a full-term birth fetus, and the placenta tissue is sterilized and cleaned to separate a amniotic membrane layer from the placenta under the aseptic condition; the human amniotic membrane is directly used for digesting and separating the human amniotic mesenchymal stem cells without firstly digesting and separating epithelial cells; the method comprises the following specific steps:
1. fresh placenta tissue of a full-term parturient fetus was taken, sterilized with 75% alcohol or 5% double-antibody-added PBS, and the tissue surface was washed.
2. Under aseptic conditions, a transparent blood vessel-free membrane, the amniotic membrane, was blunt-separated from the placenta using gloved hands.
3. The stromal tissue under the basement membrane was removed with a sterile cotton swab to make the isolated amniotic membrane as thin as possible.
4. The amniotic membrane was placed in a 100mm diameter petri dish containing 1/2 volumes of PBS containing 1% double antibody. The amniotic membrane was rinsed with surgical forceps while the culture dish was changed 3-5 times to remove blood cells.
5. The rinsed amniotic membrane was placed in a dry 100mm petri dish and cut into small pieces of approximately 2mm in diameter by surgical scissors.
6. Transferring the amnion to a 50ml centrifuge tube, uniformly mixing the amnion and (0.25% pancreatin + 0.02% EDTA) pancreatin digestive juice and 0.1% collagenase II digestive juice according to the volume ratio of 1:1:1, and placing the mixture in a constant temperature shaking table at 37 ℃ and 250r/min for 10-20 min by oscillation. Terminating digestion with low-sugar DMEM/F12 medium containing 10% FBS, mixing, sieving with 200 mesh cell sieve, and collecting filtrate. Adding undigested amnion into digestive solution for digestion, repeating for 3-5 times until the amnion tissue is substantially digested.
7. And (3) putting the collected cell filtrate into a 50ml centrifuge tube, centrifuging for 5min at 2000r/min, discarding the supernatant, collecting the precipitate, resuspending the precipitate with PBS, centrifuging for 5min at 1500r/min, and discarding the supernatant. Complete medium (low-sugar DMEM/F12 medium with 10% FBS) was added, resuspended, counted, and plated at 2 × 106 cells in T175 cell culture flasks. The cells were placed in a carbon dioxide incubator at 37 ℃ and 5% CO2 for primary culture.
8. After about 72h, the medium is changed, and after 48h, the medium is changed once, and when the cell fusion degree is 80%, subculture is carried out according to the ratio of 1: 3.
The subculture method comprises removing the culture medium, adding 10ml PBS, cleaning for 2 times, adding 1ml 0.25% pancreatin, spreading uniformly, placing into carbon dioxide incubator for digesting for 30s, and observing cell obviously shrinking and rounding under microscope. The cells were removed from the flask wall by pipetting, adding complete medium to stop digestion and aliquoting.
As a result, the number of hAMSCs isolated from amniotic membrane was (6.72+1.21) × 107/min (n ═ 12), and the isolated hAMSCs significantly expressed the mesenchymal cell marker vimentin, did not express the epithelial cell marker keratin CK19, and highly expressed CD29 and CD 44. The number of hAMSCs was 167-fold increased after 10 days of culture (see FIG. 1). And the conclusion establishes a method for separating, culturing and identifying the human amniotic mesenchymal stem cells. hAMSCs have the phenotypic characteristics of bone marrow mesenchymal stem cells.
Example 2
Unlike example 1, in step (5), the small pieces had a diameter of 1 mm.
Example 3
Unlike example 1, in step (5), the small pieces had a diameter of 1.5 mm.
Example 4
Unlike example 1, in step (5), the small pieces had a diameter of 2.5 mm.
Example 5
Unlike example 1, in step (5), the small pieces had a diameter of 3 mm.
It should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention, and not for limiting the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A separation culture method of human amniotic mesenchymal stem cells comprises the steps of stripping a amniotic membrane layer from a placenta, cleaning, shearing and digesting, and is characterized in that: removing the stroma tissue under the basement membrane to make the separated amnion as thin as possible and remove blood cells after the amnion layer is stripped from the placenta, and then shearing and digesting; wherein the placenta tissue is fresh placenta tissue of a full-term birth fetus, and the placenta tissue is sterilized and cleaned to separate a amniotic membrane layer from the placenta under the aseptic condition; and the human amniotic membrane does not need to be digested and separated by epithelial cells, but is directly used for digesting and separating the human amniotic mesenchymal stem cells.
2. The isolated culture method of human amniotic mesenchymal stem cells according to claim 1, wherein the isolated culture method comprises the following steps: the separation culture method of the human amniotic mesenchymal stem cells comprises the following steps:
(1) taking placenta tissue, and cleaning;
(2) separating the amniotic membrane layer from the placenta,
(3) removing the stromal tissue under the basement membrane to make the separated amniotic membrane as thin as possible;
(4) removing blood cells;
(5) processing the amnion obtained in the step (4) into small fragments;
(6) digesting the amniotic membrane;
(7) collecting cell filtrate, centrifuging, removing supernatant, and collecting precipitate; resuspending the precipitate, centrifuging again, and discarding the supernatant; adding the precipitate into a complete culture medium for resuspension, counting, inoculating the cells into the culture medium, and culturing;
(8) and (3) changing the liquid after culturing for a certain time, then changing the liquid once at intervals of fixed time, and subculturing according to the proportion of 1:3 after the cell fusion degree reaches 80 percent to obtain the separated and purified human amniotic mesenchymal stem cells.
3. The isolated culture method of human amniotic mesenchymal stem cells according to claim 2, wherein the isolated culture method comprises: the placental tissue surface is preferably disinfected and washed with 75% alcohol or 5% double antibody-added PBS.
4. The isolated culture method of human amniotic mesenchymal stem cells according to claim 3, wherein the isolated culture method comprises: the step (2) is carried out under aseptic conditions; preferably, the amniotic membrane layer is separated from the placenta by blunt force; the stromal tissue under the basement membrane is preferably removed with a sterile cotton swab.
5. The isolated culture method of human amniotic mesenchymal stem cells according to claim 4, wherein the isolated culture method comprises: in the step (4), the method for removing blood cells comprises: the amniotic membrane was placed in a petri dish containing 1/2 volumes of PBS containing 1% double antibody, the amniotic membrane was placed in a petri dish and rinsed and the dish was replaced to remove blood cells.
6. The isolated culture method of human amniotic mesenchymal stem cells according to claim 5, wherein the isolated culture method comprises the following steps: in the step (5), the diameter of the small fragments is 1-3 mm.
7. The isolated culture method of human amniotic mesenchymal stem cells according to claim 6, wherein the isolated culture method comprises the following steps: in the step (6), the specific operation of digestion is as follows: transferring the small amnion fragments to a centrifuge tube, uniformly mixing the small amnion fragments with pancreatin digestive juice and collagenase II digestive juice, and placing the mixture in a constant temperature shaking table for oscillation; then stopping digestion, mixing uniformly, sieving by a cell sieve, and collecting filtrate; adding the undigested amnion into the digestive juice for digestion, and repeating the steps until the amnion tissue is basically digested.
8. The isolated culture method of human amniotic mesenchymal stem cells according to claim 7, wherein the isolated culture method comprises: in the step (6), the pancreatin digestive juice consists of 0.25 percent of pancreatin and 0.02 percent of EDTA in percentage by mass; preferably, the mass percentage concentration of the collagenase II digestive juice is 0.1%; preferably, the small pieces of amnion, the pancreatin digestive juice and the collagenase II digestive juice are mixed according to the volume ratio of 1:1: 1.
9. The isolated culture method of human amniotic mesenchymal stem cells according to claim 8, wherein the isolated culture method comprises: in the step (6), the temperature in the constant temperature shaking table is 37 ℃, the rotating speed is 250r/min, and the oscillation time is 10-20 min; preferably, termination of digestion is achieved by low-sugar DMEM/F12 medium with 10% FBS added; preferably, the cell sieve is 200 mesh.
10. The isolated culture method of human amniotic mesenchymal stem cells according to claim 9, wherein the isolated culture method comprises: in the step (7), the first centrifugal rotating speed is 2000r/min, and the subsequent centrifugal rotating speed is 1500 r/min; preferably, in the second resuspension, the complete medium added is low-sugar DMEM/F12 medium containing 10% FBS.
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