CN112481191A - Method for extracting CD90 high-expression target cells from adipose tissues - Google Patents
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Abstract
The invention discloses a method for extracting CD90 high-expression target cells from adipose tissues, which comprises the following steps: s1: removing impurity cells in adipose tissues, and performing enzyme digestion and primary target cell separation; s2: performing primary culture on the primarily separated target cells, and separating and removing the hybrid cells; s3: and (4) harvesting the target cells. The method can effectively remove the quantity of red blood cells, platelets, suspension lymphocytes, non-adherent cells and the like in the final product, and the purity of the separated CD90 high-expression target cells can reach more than 98 percent, so that the method has great scientific research and clinical research values.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a method for extracting CD90 high-expression target cells from adipose tissues.
Background
CD90 is a member of the immunoglobulin superfamily, and has a relative molecular mass of (25-37). times.103Attached to the cell membrane, primarily by anchoring of the diglyceride to the carboxy-terminus of the glycosylphosphatidylinositol. CD90 is a glycoprotein molecule on cell surface, is expressed in fibroblast, hematopoietic stem cell, nerve cell and other cell types, and can regulate cell-cell and intercellular, and cell-mesenchymal reactionAnd (4) acting.
At present, the method for extracting the CD90 high-expression target cells mainly comprises the traditional flow cytometry separation method after the dispersion of adipose tissues, but the problem of low extraction purity generally exists.
Disclosure of Invention
The invention aims to provide a method for extracting CD90 high-expression target cells from adipose tissues, so as to solve the problem of low extraction purity of CD90 high-expression target cells in the prior art.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a method for extracting CD90 high-expression target cells from adipose tissues, which comprises the following steps:
step S1, removing impurity cells in adipose tissues, and performing enzyme digestion and primary target cell separation;
step S2, performing primary culture on the primarily separated target cells, and separating and removing the hybrid cells;
and step S3, harvesting the target cells.
Further, in step S1, the adipose tissue is transferred to a sterile 50ml centrifuge tube, a proper amount of physiological saline (preferably, the ratio of the adipose tissue to the physiological saline is 1:1, and the total amount of the liquid in the centrifuge tube is not more than 40 ml) is added thereto, the mixture is blown with a pipette 10 to 20 times, the mixture is left for 1min, then the liquid in the lower layer of the tissue is sucked out with the pipette, and the process is repeated until the sucked liquid is transparent and has no blood cells.
Further, in the step S1, 0.3% collagenase type I is added into the centrifuge tube containing the adipose tissues at a volume ratio of 1:5, and then PBS containing calcium and magnesium ions is added to the centrifuge tube to make the volume of the centrifuge tube to 30ml, the centrifuge tube is sealed and placed in a 37 ℃ constant temperature shaking table, and digestion is performed for 30min with shaking at 180 rpm.
Further, in the step S1, after digestion is completed, the lower layer liquid in the centrifuge tube is sucked out, filtered into a new 50ml centrifuge tube through a 100 μm filter screen, centrifuged at 1500rpm for 10min, the cell sediment is resuspended in physiological saline, centrifuged at 1500rpm for 10min and washed once again, and the supernatant is discarded to obtain the primary separated target cells.
Further, the step S2 includes:
counting the preliminarily separated target cells, adding 10% fetal bovine serum-containing alpha-MEM cell culture medium at a density of 3 x 10^6 cells/ml, suspending the cells 3-6 times, placing the cells in a culture bottle with a vent cover, and placing the culture bottle at 37 ℃ with 5% CO2An incubator; culturing for 3-4 days, and keeping the culture flask physically still.
Further, in step S2, when the target cells are growing adherently, the culture flask is washed 3 times with 10ml of PBS to remove foreign cells such as suspension cells and dead cells.
Further, the step S3 includes:
the culture flask was washed 2 times with 10ml of physiological saline; sucking and discarding the normal saline in the bottle, adding 2ml of pancreatin digestive juice, observing digestion under a 37 ℃ mirror, filtering by using a 0.22 mu m filter after cells begin to retract and become round, stopping digestion, and (softly) blowing and beating the bottom surface for 2-3 times, and then collecting all liquid into a centrifuge tube; adding 10ml of normal saline, blowing the bottom surface for 2-3 times (softly), and transferring the normal saline into the same centrifugal tube; centrifuging at 1200 rpm for 8 minutes, and discarding the supernatant to obtain the target cells.
Preferably, the pancreatin digest, physiological saline and fresh medium are placed in a 37 ℃ water bath for 3 minutes before use.
The invention has the following beneficial effects:
the method for extracting the CD90 high-expression target cells from the adipose tissues, provided by the invention, can effectively remove the number of red blood cells, platelets, suspension lymphocytes, non-adherent cells and the like in a final product, and the purity of the separated CD90 high-expression target cells can reach more than 98%, so that the method has great scientific research and clinical research values.
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FIG. 1 is a flow chart of the method for extracting CD90 high expression target cells from adipose tissues according to the present invention.
FIG. 2 is a diagram showing the flow assay results of example 1 of the present invention.
FIG. 3 is a graph showing the results of flow assay in comparative example 1 of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In addition, unless otherwise specified, each step in the examples can be realized by using materials and conventional technical means in the field.
Example 1
In this example, the method for extracting target cells highly expressing CD90 from adipose tissue employs methods of adipose tissue enzymatic digestion, primary culture, and target cell harvesting. The method specifically comprises the following steps:
and step S1, removing impurity cells from human adipose tissues, and performing enzyme digestion and primary target cell separation.
S1-1, transferring 20g of human adipose tissues into a sterile 50mL centrifuge tube, adding 20mL of normal saline into the centrifuge tube, blowing the saline with a suction tube for 10-20 times, standing for 1min, sucking out the liquid at the lower layer of the tissues with the suction tube, and repeating the steps until the sucked liquid is transparent and does not contain blood cells;
s1-2, adding 0.3% collagenase type I into a centrifugal tube containing adipose tissues according to the volume ratio of 1:5, adding PBS (pH7.4) containing calcium and magnesium ions to a constant volume of 30ml, sealing the centrifugal tube, placing the centrifugal tube in a constant temperature shaking table at 37 ℃, and digesting the centrifugal tube for 30min by oscillating at 180 rpm;
s1-3, sucking out the lower layer liquid in the centrifuge tube, filtering the lower layer liquid into a new 50ml centrifuge tube through a 100 mu m filter screen, centrifuging the centrifuge tube at 1500rpm for 10min, re-suspending the cell sediment with physiological saline, centrifuging the centrifuge tube at 1500rpm for 10min, washing the cell sediment once again, and discarding the supernatant.
And step S2, performing primary culture on the primarily separated target cells, and separating and removing the hybrid cells.
After counting the cells, the cells were cultured in a cell culture medium of alpha-MEM containing 10% fetal bovine serum at a density of 3X 10^6 cells/ml and blownSuspending for 3-6 times, placing in a culture flask with a vent cover, and placing the culture flask in 5% CO at 37 deg.C2An incubator; culturing for 3-4 days, and keeping the culture flask physically still. After 4 days, the target cells are in adherent growth, and the culture bottle is washed 3 times by using 10ml PBS to remove impurity cells such as suspension cells, dead cells and the like.
And step S3, harvesting the target cells.
Placing pancreatin digestive juice, normal saline and fresh culture medium (LONZA 12-725F) in a water bath kettle at 37 ℃ for 3 minutes; the culture flask was washed 2 times with 10ml of physiological saline; removing the normal saline in the bottle, adding 2ml of pancreatin digestive juice (Gibco 25200-072), observing digestion under a 37 ℃ mirror, and filtering by using a 0.22 mu m filter after cells begin to retract and become round; adding a small amount of fresh culture medium into a culture bottle to terminate digestion, gently blowing and beating the bottom surface for 2-3 times, and collecting all liquid into a centrifuge tube; adding 10ml of normal saline, gently blowing and beating the bottom surface for 2-3 times, and transferring the normal saline into the same centrifugal tube; after balancing, the cell suspension is centrifuged at 1200 rpm for 8 minutes, the supernatant is discarded, the cell sediment is resuspended by using a proper amount of normal saline, and a sample is taken for flow detection.
Comparative example 1
Adopts the traditional flow cytometry separation method after the adipose tissues are scattered. The method comprises the following steps:
1. standing 20g of small fat tissue, naturally separating the expansion liquid and the screened fat according to different specific gravities, obtaining the fat tissue through a screening net, and sucking the fat tissue into an injector through the injector to obtain small fat tissue particles;
2. preparing a fat single cell suspension, digesting small fat tissue particles by using a cell separation reagent, adding serum to stop digestion, dispersing, centrifuging and layering, and removing a supernatant; resuspending the cells with a buffer solution, and obtaining a single cell suspension by a filter screen;
3. flow cytometric sorting, enriching CD90+ cells from the obtained cell population;
4. and (3) culturing CD90+ cells, inoculating the obtained CD90+ cells into a culture dish, purifying the cells by an adherence screening method, and performing in vitro culture by adopting a serum-free culture medium to obtain the CD90+ cells.
The test results are detailed in table 1.
TABLE 1 flow assay results of example 1 and comparative example 1
| Group of | Proportion of nucleated cells | CD90 high expression cell ratio | Positive rate of CD73 | Positive rate of CD105 |
| Example 1 | 81.4% | 99.8% | 97.4% | 95.8% |
| Comparative example 1 | 29.9% | 82.2% | 99.4% | 95% |
The experimental results show that: the adipose tissue enzyme digestion, primary culture and target cell harvesting methods are adopted to effectively remove non-target cells such as erythrocytes, platelets and CD90 weakly-expressed lymphocytes, meanwhile, the positive expression rates of CD73 and CD105 are not influenced by the method, and the separation purity of the target cells with high expression of CD90 is greatly improved.
Claims (9)
1. A method for extracting CD90 high-expression target cells from adipose tissues comprises the following steps:
step S1, removing impurity cells in adipose tissues, and performing enzyme digestion and primary target cell separation;
step S2, performing primary culture on the primarily separated target cells, and separating and removing the hybrid cells;
and step S3, harvesting the target cells.
2. The method of claim 1, wherein in step S1, the adipose tissues are transferred to a sterile centrifuge tube, a proper amount of physiological saline is added to the sterile centrifuge tube, the tube is used for blowing 10-20 times, the tube is left for 1min, then the liquid in the lower layer of the tissue is sucked out by the tube, and the steps are repeated until the sucked liquid is transparent and has no blood cells.
3. The method for extracting the cells with the high expression of the CD90 from the adipose tissue as claimed in claim 2, wherein the ratio of adipose tissue: the ratio of the normal saline is 1:1, and the total amount of liquid in the centrifugal tube does not exceed 40 ml.
4. The method for extracting CD 90-highly expressed target cells from adipose tissues according to claim 2 or 3, wherein in step S1, 0.3% collagenase type I is added into the centrifuge tube containing adipose tissues at a volume ratio of 1:5, then PBS containing calcium and magnesium ions is added to the centrifuge tube to make the volume of the centrifuge tube to 30ml, the centrifuge tube is sealed and placed in a 37 ℃ constant temperature shaker, and the centrifuge tube is digested for 30min with 180rpm shaking.
5. The method for extracting CD90 high-expression target cells from adipose tissues according to claim 4, wherein in the step S1, after digestion is completed, lower liquid in a centrifuge tube is sucked out, the lower liquid is filtered into a new 50ml centrifuge tube through a 100 μm filter screen, the centrifuge tube is centrifuged at 1500rpm for 10min, the cell sediment is resuspended in physiological saline, the centrifuge tube is centrifuged at 1500rpm for 10min and then washed once, and the supernatant is discarded, so that the primarily separated target cells are obtained.
6. The method for extracting CD 90-highly expressing target cells from adipose tissue as claimed in claim 1, wherein the step S2 comprises:
counting the preliminarily separated target cells, adding 10% fetal bovine serum-containing alpha-MEM cell culture medium at a density of 3 x 10^6 cells/ml, suspending the cells 3-6 times, placing the cells in a culture bottle with a vent cover, and placing the culture bottle at 37 ℃ with 5% CO2An incubator; culturing for 3-4 days, and keeping the culture flask physically still.
7. The method of claim 6, wherein in step S2, when the target cells are growing adherently, the culture flask is washed 3 times with 10ml PBS to remove contaminating cells including suspension cells and dead cells.
8. The method for extracting CD 90-highly expressing target cells from adipose tissues according to any one of claims 1, 6 and 7, wherein the step S3 comprises:
the culture flask was washed 2 times with 10ml of physiological saline; sucking and discarding the normal saline in the bottle, adding 2ml of pancreatin digestive juice, observing digestion under a 37 ℃ mirror, filtering by using a 0.22 mu m filter when cells begin to retract and become round, stopping digestion, and collecting all liquid into a centrifuge tube after blowing and beating the bottom surface for 2-3 times; adding 10ml of normal saline, blowing and beating the bottom surface for 2-3 times, and transferring the normal saline into the same centrifugal tube; centrifuging at 1200 rpm for 8 minutes, and discarding the supernatant to obtain the target cells.
9. The method for extracting CD 90-highly expressed target cells from adipose tissue as claimed in claim 8, wherein pancreatin digest, physiological saline and fresh medium are placed in a 37 ℃ water bath for 3 minutes before use.
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