CN114149966A - Method for obtaining placenta mesenchymal stem cells - Google Patents
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- CN114149966A CN114149966A CN202111490034.XA CN202111490034A CN114149966A CN 114149966 A CN114149966 A CN 114149966A CN 202111490034 A CN202111490034 A CN 202111490034A CN 114149966 A CN114149966 A CN 114149966A
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Abstract
The invention discloses a method for obtaining placenta mesenchymal stem cells, belonging to the technical field of biology. The method comprises the following steps: separating an umbilical cord from a placenta, cleaning the placenta, removing a amniotic membrane layer, and shearing chorionic plates around the umbilical cord position by taking the umbilical cord position as a center; removing villi, mucus and blood vessels on the villus membrane plate to obtain a clean villus membrane plate; shearing the clean chorion plate into tissue blocks; placing the tissue blocks in physiological saline and centrifuging to obtain precipitates; the pellet was pipetted and plated on a petri dish for inoculation. The placenta mesenchymal stem cells obtained by the method are simple to operate, short in time consumption and high in cell viability, and can be used as a technical trend for obtaining the placenta mesenchymal stem cells.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for obtaining placenta mesenchymal stem cells.
Background
Mesenchymal stem cells are a type of pluripotent cells with the ability to self-replicate and differentiate. Under certain conditions, mesenchymal stem cells can differentiate into a variety of functional cells.
At present, an enzyme digestion method is a commonly used method for obtaining placenta mesenchymal stem cells, and the method adopts tissue digestive enzyme to incubate and digest placenta blocks, collects single nuclear cells after filtration, then cultures the nuclear cells to obtain cloned cells, then cultures the cloned cells by using an MSC culture medium, and then carries out digestion and passage by using pancreatin to obtain the placenta mesenchymal stem cells. However, the method is complicated to operate, the digestion time and the concentration of digestive enzymes are not easy to control, and the cell viability of the obtained placenta mesenchymal stem cells is low. This is not advantageous for obtaining placental mesenchymal stem cells, and thus, a new extraction method of placental mesenchymal stem cells is required instead of the enzymatic digestion method.
Disclosure of Invention
In order to solve the problems of the prior art, the embodiment of the invention provides a method for extracting placenta mesenchymal stem cells. The technical scheme is as follows:
in one aspect, the present invention provides a method for obtaining placental mesenchymal stem cells, the method comprising:
separating an umbilical cord from a placenta, cleaning the placenta, removing a amniotic membrane layer, and shearing chorionic plates around the umbilical cord position by taking the umbilical cord position as a center;
removing villi, mucus and blood vessels on the villus membrane plate to obtain a clean villus membrane plate;
shearing the clean chorion plate into tissue blocks;
placing the tissue blocks in physiological saline and centrifuging to obtain precipitates;
sucking the sediment and spreading the sediment on a culture dish for inoculation;
after inoculation is completed, standing the culture dish until the tissue block is fixed on the culture dish;
adding a first culture medium into the culture dish on which the tissue block is fixed and culturing;
and supplementing liquid into the tissue block in the culture process, and harvesting the primary placental mesenchymal stem cells.
Specifically, the method comprises the following steps: immersing the obtained chorion plate in physiological saline for standby.
In particular toThe size of the tissue block is 1-2 mm2。
Specifically, the mucous membrane plate is placed in physiological saline, and then blood vessels on the mucous membrane plate are removed.
Specifically, sucking 0.5-1 mL of the sediment and paving the sediment on a culture dish with the specification of 100 mm.
Specifically, 5mL of the first medium was added to the petri dish.
Specifically, the liquid supplementing method comprises the following steps:
after 3-4 days after inoculation, carrying out first fluid infusion;
adding 5mL of the first culture medium into each culture dish, and placing the culture dishes in a carbon dioxide incubator for culture;
performing second liquid supplement on 6-7 days after inoculation;
5mL of the first culture medium is added into each culture dish and placed in a carbon dioxide incubator for culture;
carrying out third fluid infusion on 9-10 days after inoculation;
adding 5mL of the first culture medium into each culture dish, and culturing in a carbon dioxide incubator to obtain the primary placental mesenchymal stem cells;
specifically, the method comprises the following steps: passaging said primary placental mesenchymal stem cells comprising:
placing the primary placenta mesenchymal stem cells in the culture dish into a centrifuge tube for centrifugation for later use;
adding 5mL of sodium chloride injection into a culture dish of the primary placental mesenchymal stem cells for cleaning;
adding 3mL of TRYPLE into the culture dish of the washed primary placenta mesenchymal stem cellsTMExpress Phenol Red shakes the culture flask, and stands for digestion;
stopping digestion after the primary placental mesenchymal stem cells shrink and fall off from the bottom of the culture bottle to form a round bright spot, and obtaining a cell suspension;
transferring the cell suspension into a 50mL centrifuge tube, adding 5mL sodium chloride injection into the culture dish, shaking and cleaning, collecting cleaning liquid, and mixing with the cell suspension;
at (0.80-1.20) × 104Per cm2Is inoculated in a culture flask.
The technical scheme provided by the embodiment of the invention has the following beneficial effects: the placenta mesenchymal stem cells obtained by the method are simple to operate, short in time consumption and high in cell viability, and can be used as a technical trend for obtaining the placenta mesenchymal stem cells.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a photograph of a tissue mass after completion of inoculation as provided by an embodiment of the present invention;
FIG. 2 is a photograph showing cell crawl-out around a tissue mass according to an embodiment of the present invention;
FIG. 3 is a photograph of the microscopic cell morphology of passaged cells provided by an embodiment of the present invention;
FIG. 4 is a photograph of the morphology of the cryopreserved cells under a microscope after they have been removed according to an embodiment of the present invention;
FIG. 5 is a two-dimensional graph of the results of flow cytometry analysis of passaged cells among passaged cells provided in an embodiment of the present invention, in which the abscissa and ordinate are values of relative intensity of scattered light signals;
FIG. 6 is a graph showing the results of flow cytometry analysis of P1 in passaged cells provided in the example of the present invention, in which the abscissa is the value of the relative intensity of scattered light signals and the ordinate is the relative cell number;
FIG. 7 is a graph showing the results of flow cytometry analysis of P2 in passaged cells provided in the example of the present invention, in which the abscissa is the value of the relative intensity of scattered light signals and the ordinate is the relative cell number;
FIG. 8 is a graph showing the results of flow cytometry analysis of P3 in passaged cells provided in the example of the present invention, in which the abscissa is the value of the relative intensity of scattered light signals and the ordinate is the relative cell number;
FIG. 9 is a graph showing the results of flow cytometry analysis of P4 in passaged cells provided in the example of the present invention, in which the abscissa is the value of the relative intensity of scattered light signals and the ordinate is the relative cell number;
FIG. 10 is a graph showing the results of flow cytometry analysis of P5 in passaged cells provided in the example of the present invention, in which the abscissa is the value of the relative intensity of scattered light signals and the ordinate is the relative cell number;
FIG. 11 is a graph showing the results of flow cytometry analysis of P6 in passaged cells provided in the example of the present invention, in which the abscissa is the value of the relative intensity of scattered light signals and the ordinate is the relative cell number;
FIG. 12 is a graph showing the results of flow cytometry analysis of P7 in passaged cells provided in the example of the present invention, in which the abscissa is the value of the relative intensity of scattered light signals and the ordinate is the relative cell number;
FIG. 13 is a graph showing the results of flow cytometry analysis of P8 in passaged cells provided in the example of the present invention, in which the abscissa is the value of the relative intensity of scattered light signals and the ordinate is the relative cell number;
FIG. 14 is a graph showing the results of flow cytometry analysis of P9 in passaged cells provided in the example of the present invention, in which the abscissa is the value of the relative intensity of scattered light signals and the ordinate is the relative cell number;
FIG. 15 is a graph showing the results of flow cytometry analysis of P10 in passaged cells provided in the example of the present invention, in which the abscissa is the value of the relative intensity of scattered light signals and the ordinate is the relative cell number;
fig. 16 is a graph showing the results of flow cytometry analysis of P11 in passaged cells provided in the example of the present invention, in which the abscissa is the value of the relative intensity of scattered light signals and the ordinate is the relative cell number.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in further detail below.
Examples
The embodiment of the invention provides a method for obtaining placenta mesenchymal stem cells, which comprises the following steps:
tissue selection and pretreatment: selecting a placenta which is qualified in detection, separating an umbilical cord from the placenta, cleaning the placenta with normal saline, removing a amniotic membrane layer with an operation scissors, taking a chorionic plate around the position of the umbilical cord as a center, and taking care to avoid blood vessels. The obtained chorion plate is immersed in physiological saline for standby. In practice, the obtained napped membrane panel may be immersed in a dish containing physiological saline.
Cleaning: removing villi, mucus and blood vessels on the villus membrane plate to obtain a clean villus membrane plate, and placing the clean villus membrane plate in a centrifugal tube; specifically, removing villi on a villus membrane plate by using a pair of tweezers, scraping mucus on the villus membrane plate by using the back of a knife of a pair of surgical scissors for 2-3 times, and then removing blood vessels on the villus membrane plate;
during the scraping of mucus, the culture dish is replaced with a new one;
the mucous-removed villus membrane plate was loaded into a clean 50mL centrifuge tube.
Shearing: shearing the clean chorion plate into tissue blocks; specifically, the chorion plate is cut into (1-2) mm pieces by surgical scissors2Small pieces of (2);
the centrifuge tube was filled with physiological saline, centrifuged at 500g for 5min, and the supernatant was discarded to obtain a precipitate.
Inoculation: sucking 0.5-1 mL of sediment by using a Pasteur pipette, and spreading the sediment on 1 culture dish with the specification of 100mm for inoculation;
after inoculation is finished, opening a dish cover and standing for 20-30 min, simultaneously observing whether the tissue block is fixed on a culture dish, and if not, continuously standing until the tissue block is fixed on the culture dish; the tissue mass after inoculation is shown in FIG. 1.
Adding 5mL of first culture medium into the culture dish with the tissue block fixed, and attaching a label with ID on the outer wall of the culture dishAnd is merged with CO2Culturing in an incubator at 37 deg.C and 5% carbon dioxide;
in this embodiment, the first medium comprises: alpha-MEM basal medium + EliteGroTM-Adv (serum replacement) such that the final concentration of serum replacement represents 10% of the total volume.
And supplementing liquid into the tissue mass in the culture process, and harvesting the primary placenta mesenchymal stem cells.
Specifically, the liquid supplementing method comprises the following steps:
after 3-4 days after inoculation, carrying out first fluid infusion;
adding 5mL of the first culture medium into each 100mm culture dish, and culturing in a carbon dioxide incubator at 37 ℃ and 5% of carbon dioxide;
observing the cell climbing-out condition and the cell morphology around the tissue block under a microscope on days 6-7 after inoculation is completed, taking a picture and recording, wherein the picture taking result is shown in figure 2, as can be seen from figure 2, the cells can be observed to form a large colony through a microscope, and performing second fluid replacement;
adding 5mL of first culture medium into each culture dish, and placing the culture dish into a carbon dioxide incubator for culture, wherein the culture temperature in the incubator is 37 ℃, and the concentration of carbon dioxide is 5%;
observing cell climbing-out conditions and cell forms around the tissue block under a mirror on 9-10 days after inoculation is completed, photographing and recording, and performing fluid infusion for the third time;
adding 5mL of first culture medium into each culture dish, and placing the culture dish into a carbon dioxide incubator for culture, wherein the culture temperature in the incubator is 37 ℃, and the concentration of carbon dioxide is 5%, so as to obtain primary placental mesenchymal stem cells;
specifically, the method comprises the following steps: passaging the primary placental mesenchymal stem cells, comprising:
taking out the cell culture dish needing passage from the incubator, observing the cell shape and the confluence rate under a mirror, and taking a picture for recording; the photographing result is shown in FIG. 3.
Placing primary placenta mesenchymal stem cells in a culture dish into a centrifuge tube for centrifugation for later use, specifically, sucking cell supernatant in a 100mm plate by using a pipette gun and placing the cell supernatant into a 50mL centrifuge tube for later use, wherein the centrifugation acceleration is 600g and the centrifugation time is 5 min;
adding 5mL of sodium chloride injection into a culture dish of the primary placenta mesenchymal stem cells for cleaning;
adding 3mL of TRYPLETMExpress Phenol Red shaking culture bottle into the culture dish of the cleaned primary placenta mesenchymal stem cells, covering the bottom of the bottle with the shaking culture bottle, and standing and digesting for 2 min; wherein, the TRYPLETMExpress phenyl Red is a recombinase without animal origin and is used for dissociating various adherent mammalian cells.
Stopping digestion after the primary placenta mesenchymal stem cells shrink and fall off from the bottom of the culture bottle to form a round bright spot, and obtaining cell suspension; specifically, an equal volume of the stock supernatant was added to stop digestion and the swab was blown evenly.
Transferring the cell suspension into a 50mL centrifuge tube, adding 5mL sodium chloride injection into a culture dish, shaking and cleaning, collecting the cleaned cell suspension, and mixing the cell suspension with the cell suspension;
centrifuging the mixed cell suspension for 5min at a centrifugal acceleration of 400g, discarding the supernatant to obtain a first cell precipitate, adding 2-5 mL of a first culture medium into the first cell precipitate for resuspension, and counting 100 mu L of the resuspended cell precipitate;
at (0.80-1.20) × 104Per cm2Is inoculated at a density of T75 cm2In the culture flask, the height of the culture flask is T75 cm2Adding the second culture medium into the culture bottle to 10mL, attaching a label with ID outside the culture bottle, placing the labeled culture bottle in CO2Culturing in an incubator. Wherein the second medium comprises: alpha-MEM minimal medium + EliteGroTM-Adv (serum replacement), so that the final concentration of serum replacement represents 5% of the total volume.
Specifically, the method further comprises:
freezing and storing: taking out the cells to be cryopreserved, observing the cell morphology and the confluence rate under a mirror, and taking a picture for recording, wherein the picture taking result is shown in figure 4;
sucking the cell supernatant with a pipette, placing in a 50mL centrifuge tube, centrifuging at a centrifugal acceleration of 600g for 5min, and keeping;
to T75 cm2Adding 5mL of sodium chloride injection into the culture bottle, cleaning for 1 time, sucking the cleaning solution and discarding;
3mL of TRYPLETMExpress Phenol Red was added to the flask, the flask was shaken to cover the bottom of the flask, and the mixture was allowed to stand for digestion for 2 min. Observing the digestion condition under a mirror until the cells shrink and fall off from the bottom of the bottle to form a round bright spot;
digestion was stopped by adding an equal volume of the stock supernatant and the cell suspension was transferred to a 50mL centrifuge tube.
And adding 5mL of sodium chloride injection into the culture flask, shaking and cleaning, collecting the cleaned cell suspension, and mixing with the previous cell suspension. Counting 100 μ L of the suspension;
the remaining cell suspension was centrifuged at 400g for 5 min. Pouring out the residual supernatant, retaining the second cell sediment, resuspending the frozen stock solution, and filling the frozen stock solution into a freezing tube; in this embodiment, P0～P2The cryopreservation density of the generation cells is (1.00-5.00) multiplied by 106Per mL;
sticking a label with an ID outside the freezing storage tube (using a program cooling box); after cooling, the mixture is placed into a refrigerator at minus 80 ℃ and can be transferred into a liquid nitrogen tank after the result is detected to be qualified.
The surface antigen markers of the passaged primary placental mesenchymal stem cells were detected by flow cytometry, and the results obtained are shown in fig. 5 to 16, where the expression amounts of P1 to P11 in the passaged cells were 0.04%, 0.11%, 0.02%, 0.31%, 99.96%, 99.93%, 99.73%, 99.97%, and 99.98%, respectively.
Detecting the STR of the cells:
appropriate amount of test material was taken and DNA was extracted with Microread Genomic DNA Kit, 20 STR loci and sex identification loci were amplified using MicroreadTM 21 ID System, PCR product detection was performed using ABI3730xl type genetic Analyzer, and the detection results were analyzed using GeneMapperID-X software (Applied Biosystems) and compared with ATCC and DSMZ databases.
The negative and positive control results in the experiment were correct.
The DNA of the detected sample cell is amplified, the map is clear, the typing result is good, the STR locus and the Amelogenin locus of the cell are shown in the table 1, and meanwhile, the number of the multiple alleles of the detection result is 0.
Table 1 shows the results of the highest matching degree in the database comparison
As can be seen from Table 1, the STR typing of the cells with the cellular DNA revealed that no cross contamination of human cells was observed in the cells. The STR data match rate of this cell with that of HCC1500Breast Dual Carcinoma human cell in ATCC was 78%. STR data match rate (EV value) of the cells with HUP-T4 cells in DSMZ is 0.72.
The placenta mesenchymal stem cells obtained by the method are simple to operate, short in time consumption and high in cell viability, and can be used as a technical trend for obtaining the placenta mesenchymal stem cells.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (8)
1. A method of obtaining placental mesenchymal stem cells, the method comprising:
separating an umbilical cord from a placenta, cleaning the placenta, removing a amniotic membrane layer, and shearing chorionic plates around the umbilical cord position by taking the umbilical cord position as a center;
removing villi, mucus and blood vessels on the villus membrane plate to obtain a clean villus membrane plate;
shearing the clean chorion plate into tissue blocks;
placing the tissue blocks in physiological saline and centrifuging to obtain precipitates;
sucking the sediment and spreading the sediment on a culture dish for inoculation;
after inoculation is completed, standing the culture dish until the tissue block is fixed on the culture dish;
adding a first culture medium into the culture dish on which the tissue block is fixed and culturing;
and supplementing liquid into the tissue block in the culture process, and harvesting the primary placental mesenchymal stem cells.
2. The method according to claim 1, characterized in that it comprises: immersing the obtained chorion plate in physiological saline for standby.
3. The method of claim 1, wherein the tissue mass has a size of 1-2 mm2。
4. A method according to claim 1, wherein the mucous-removing chorion plate is placed in physiological saline and the blood vessels on the chorion plate are removed.
5. The method of claim 1, wherein 0.5-1 mL of the pellet is pipetted and plated on a 100mm format petri dish.
6. The method of claim 1, wherein 5mL of the first culture medium is added to the culture dish.
7. The method of claim 1, wherein the method of replenishing comprises:
after 3-4 days after inoculation, carrying out first fluid infusion;
adding 5mL of the first culture medium into each culture dish, and placing the culture dishes in a carbon dioxide incubator for culture;
performing second liquid supplement on 6-7 days after inoculation;
5mL of the first culture medium is added into each culture dish and placed in a carbon dioxide incubator for culture;
carrying out third fluid infusion on 9-10 days after inoculation;
and adding 5mL of the first culture medium into each culture dish, and culturing in a carbon dioxide incubator to obtain the primary placental mesenchymal stem cells.
8. The method of claim 7, wherein the method comprises: passaging said primary placental mesenchymal stem cells comprising:
placing the primary placenta mesenchymal stem cells in the culture dish into a centrifuge tube for centrifugation for later use;
adding 5mL of sodium chloride injection into a culture dish of the primary placental mesenchymal stem cells for cleaning;
adding 3mL of TRYPLE into the culture dish of the washed primary placenta mesenchymal stem cellsTMExpress Phenol Red shakes the culture flask, and stands for digestion;
stopping digestion after the primary placental mesenchymal stem cells shrink and fall off from the bottom of the culture bottle to form a round bright spot, and obtaining a cell suspension;
transferring the cell suspension into a 50mL centrifuge tube, adding 5mL sodium chloride injection into the culture dish, shaking and cleaning, collecting cleaning liquid, and mixing with the cell suspension;
at (0.80-1.20) × 104Per cm2Is inoculated in a culture flask.
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