CN114457014B - Combined reagent for separating callus and macrophage and application thereof - Google Patents
Combined reagent for separating callus and macrophage and application thereof Download PDFInfo
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Abstract
The invention discloses a combined reagent for separating poroma macrophages and application thereof, wherein the combined reagent mainly comprises a liquid A and a liquid B, and the combined reagent comprises the following components in percentage by weight: the A solution mainly comprises 2wt.% of type II collagenase solution, 2.5wt.% of trypsin-EDTA solution and RPMI 1640 culture medium, wherein the volume ratio of the 2wt.% of type II collagenase solution to the 2.5wt.% of trypsin-EDTA solution is 1-3: 1-3, wherein the used solvents are calcium-magnesium-free PBS buffer solution, and the volume ratio of the total solution amount of the 2wt.% type II collagenase solution and the 2.5wt.% trypsin-EDTA solution to the RPMI 1640 culture medium is 1:3, a step of; and (2) liquid B: 2wt% serum was added to calcium-magnesium free PBS buffer. The combined reagent can efficiently and thoroughly digest poroma tissues, prepare high-activity cell suspension, well reserve surface markers in macrophages and avoid losing key surface markers F4/80; the materials used are easy to obtain, the toxic and side effects are small, the digestion operation method for separating macrophages in poroma is simple and easy, and the experimental burden is reduced.
Description
Technical Field
The invention belongs to the technical field of cell separation, and particularly relates to a combined reagent for separating callus macrophages and application thereof.
Background
Macrophages, originally discovered and named by the teachings of Metchnikoff, are extremely plastic innate immune cells in humans, present in all tissues and exhibit a diversity in function. Macrophages of different tissues, such as microglia, osteoclasts, coulombs and bronchoalveolar macrophages, all have respective gene transcription profiles and biological functions. Macrophages play an important role in body development, homeostasis, tissue repair and immunomodulation. Macrophages are the first line of defense of the body against infection, are an important component of innate immunity, and act in concert with other innate immune cells, such as neutrophils, innate lymphocytes, natural killer cells, and the like. In addition, macrophages are key cells in the coordination of chronic inflammation and related pathological processes, with the ability to adapt to the microenvironment of different tissues and respond to different pathogenic lesions. Understanding the biological functions and specific mechanisms of macrophages more clearly would help to resolve pathophysiological mechanisms of various human diseases and to develop potential therapeutic targets.
In recent years, more and more studies have found that macrophages are critical for bone healing, and the lack of macrophages will severely inhibit intramembranous and endochondral bone formation. In early stage after fracture, macrophages mainly take effect of clearing tissue fragments and releasing inflammatory factors; and in the middle and late stages of the fracture healing process, the phenotype of macrophages is gradually changed, mainly tissue repair, so that the anti-inflammatory effect is generated, the growth of blood vessels is promoted, and the osteogenic differentiation of stem cells is regulated. Therefore, the macrophage plays an important role in the bone healing process, is an important target for treating delayed healing and non-healing of fracture, and has potential application value. However, the prior researchers have limited research means for poroma macrophages after fracture, and cannot directly separate macrophages in poroma tissues. At the in vitro level, the in vivo microenvironment can only be simulated by using macrophage cell lines or extracting primary macrophages, with some stimulation; or by immunohistochemistry and immunofluorescence, the function is estimated from the level in vivo by roughly observing the surface markers.
Previous studies reported that mature macrophage isolation systems have been established in many tissues, such as heart, liver, intestinal tract, tumor, etc., but still lack isolation techniques for callus macrophages. Porosities are heterogeneous in tissue composition, including bone tissue, cartilage tissue, fibrous tissue, etc., which also presents a challenge for efficient separation of porosities. Nowadays, with the continuous and deep research, important roles of macrophages in bone healing are focused, and more students focus on macrophages. However, there is currently no effective technique for isolating macrophages from callus. Therefore, the invention discloses an effective and simple method for separating macrophages in callus, which is important for elucidating the physiological process of bone healing and exploring the pathophysiological mechanism of delayed union and non-union of fracture.
Disclosure of Invention
In order to solve the technical problems that macrophages in callus are difficult to extract and how to effectively dissociate callus and ensure the activity of the macrophages and the stability of surface markers, the application provides a combined reagent for separating the callus macrophages and application thereof, which not only can effectively and rapidly dissociate the callus, but also can reduce the injury to the macrophages, retain the activity of the callus macrophages and maintain the stability of the surface markers. The method for separating macrophages in the callus by using the combined reagent is effective, quick and simple, is beneficial to researchers to separate and obtain the high-activity macrophages in the callus, and is convenient for carrying out related researches on fracture and macrophages.
The technical scheme adopted for solving the technical problems is as follows:
A combined reagent for separating macrophages mainly comprises a poroma digestive juice A (A liquid) and a buffer solution B (B liquid), wherein: the A solution mainly comprises 2wt.% of type II collagenase solution, 2.5wt.% of trypsin-EDTA solution and RPMI 1640 culture medium, wherein the volume ratio of the 2wt.% of type II collagenase solution to the 2.5wt.% of trypsin-EDTA solution is 1-3: 1-3, wherein the solvent is calcium-magnesium-free PBS buffer solution, and the volume ratio of the total solution amount of the 2wt.% type II collagenase solution and the 2.5wt.% trypsin-EDTA solution to the RPMI 1640 culture medium is 1:3, a step of; and (2) liquid B: 2wt.% serum was added to calcium-magnesium free PBS buffer.
As a preferred technical scheme of the application, the solution a further comprises 2wt.% BSA solution, wherein the volume ratio of the 2wt.% collagenase solution type ii, the 2.5wt.% trypsin-EDTA solution and the 2wt.% BSA solution is 100-300: 100-300: 50 to 200.
As a preferred embodiment of the present application, the solution a further comprises 2wt.% BSA solution, wherein the volume ratio of the 2wt.% collagenase solution type ii, the 2.5wt.% trypsin-EDTA solution to the 2wt.% BSA solution is 200:200:50 to 200.
As a preferred embodiment of the present application, the solution a further comprises 2wt.% BSA solution, wherein the volume ratio of the 2wt.% collagenase solution type ii, the 2.5wt.% trypsin-EDTA solution to the 2wt.% BSA solution is 200:200:100.
A combination reagent for macrophage separation, the combination reagent being formulated in proportions comprising: 100-300 mu L of 2wt.% type II collagenase solution, 100-300 mu L of 2.5wt.% trypsin-EDTA solution and 50-200 mu L of 2wt.% BSA solution, wherein the total amount of the solutions is 500 mu L, the solvents are calcium-free magnesium PBS buffer solution, and 1500 mu L of RPMI 1640 culture medium contains double antiserum; buffer B (B) 2wt.% serum was added to calcium magnesium free PBS buffer.
As a preferred technical scheme of the application, the combined preparation comprises the following specific components: 200. Mu.L of 2wt.% type II collagenase solution, 200. Mu.L of 2.5wt.% trypsin-EDTA solution and 100. Mu.L of 2wt.% BSA solution, the solvents of which are calcium-magnesium-free PBS buffer, 1500. Mu.L of RPMI 1640 medium containing double antisera; buffer B (B) 2wt.% serum was added to calcium magnesium free PBS buffer.
The invention also provides the use of any of the aforementioned combination agents for the preparation of an isolated macrophage product.
As a preferred technical scheme of the application, the application comprises the following specific steps: taking a femur of a mouse, removing muscles and other soft tissues, carefully separating femur callus, assembling the callus into an EP tube, and adding A solution for digestion; centrifuging callus digestion liquid after digestion is finished, discarding supernatant after centrifugation, adding red blood cell lysate with 10 times of cell volume to resuspend cell sediment, centrifuging cell suspension after 5min of lysis, discarding supernatant, using solution B to resuspend sediment, centrifuging, discarding supernatant, adding solution B to resuspend sediment, and adding the sediment into an EP tube protected from light; adding CD45, CD11B, F4/80 and flow antibody of dead living cell dye into the cell suspension, rotating on a rotary table at 4 ℃ to incubate the antibody for 30 minutes, adding liquid B after the incubation of the antibody is finished, centrifuging after the cell is resuspended by using the liquid B, cleaning, centrifuging, discarding the liquid B after the centrifugation is finished, and adding the cell to a flow tube for flow cytometry to sort macrophages after the cell is resuspended by using the liquid B.
As a preferred embodiment of the present application, the centrifugation is performed for 5 minutes with 300 g.
As a preferred technical scheme of the application, the application comprises the following specific steps: taking a femur of a mouse, removing muscles and other soft tissues, carefully separating femur callus, assembling the callus into a 5mL EP tube, and adding A solution for digestion; centrifuging 300g of callus digestion solution for 5 minutes after digestion is finished, discarding the supernatant after centrifugation, adding 10 times of cell volume of erythrocyte lysate to resuspend cell sediment, centrifuging 300g of cell suspension for 5 minutes after 5 minutes of lysis, discarding the supernatant, using 5mL of B solution to resuspend sediment, centrifuging 300g for 5 minutes, discarding the supernatant, adding 500 mu L B solution to resuspend sediment, and adding the sediment into a light-resistant 1.5mL EP tube; adding CD45, CD11B, F4/80 and flow antibody of dead living cell dye into the cell suspension, rotating on a rotary table at 4 ℃ for incubation of the antibody for 30 minutes, adding 1mL of B solution after the incubation of the antibody is finished, centrifuging for 5 minutes, using 300g of B solution to resuspend the cell, centrifuging for 5 minutes, discarding the B solution after the centrifugation is finished, using 300 mu L B solution to resuspend the cell, and adding the cell into a flow tube for flow cytometry to sort macrophages.
As a preferable technical scheme of the application, the digestion method is that after the solution A is added into an EP tube, a tissue shear after high-temperature and high-pressure sterilization is used for trimming callus into a tissue block; callus digestion was performed in a constant temperature shaker at 37℃and samples were taken to observe the trend of cell count increase, after which no significant increase in cell number was observed, the digestate was aspirated, filtered through a 70 μm sieve and added to a centrifuge tube placed on ice.
Preferably, the digestion method is to trim callus into 1mm 3 tissue blocks by using tissue scissors after high-temperature and high-pressure sterilization after adding 2mL of A solution into a 5mL EP tube; callus digestion was performed in a constant temperature shaker at 37℃using 150 rpm, cell counts were performed every 5min starting 20min to observe the trend of growth, after no significant increase in cell number was observed, the digests were aspirated, filtered through a 70 μm screen and added to a 15mL centrifuge tube placed on ice.
The combined reagent for separating the poroma macrophages and the application thereof provided by the invention can effectively and rapidly dissociate poroma tissues with mixed components, can reduce the damage to the macrophages, can reduce the cell death rate, and can well retain the activity, the function and the stability of surface markers of the macrophages. The reagent and the material used in the invention are easy to obtain, the toxic and side effects are small, and the operation method for separating and extracting the callus macrophages is simple and easy to implement. The invention is helpful for promoting the related research in the field of macrophage and fracture healing, and provides a brand-new technical method for researching the functions of macrophages in callus and analyzing pathophysiological mechanisms of fracture nonunion.
Drawings
FIG. 1 shows the steps and cell numbers of flow cytometry to sort macrophages in callus cell suspensions.
FIG. 2 is a statistical plot of the number of macrophages isolated from 20g callus tissue in digests containing different proportions of type II collagenase solution and trypsin solution.
FIG. 3 is a graph showing the survival rate of macrophages isolated from 20g callus tissue in a digestive fluid containing different proportions of type II collagenase solution and trypsin solution.
FIG. 4 is a statistical plot of the percentage of macrophages isolated from 20g callus tissue in the presence of digests containing different proportions of type II collagenase solution and trypsin solution.
FIG. 5 is a statistical plot of the survival rate of macrophages isolated in 20g callus tissue at varying concentrations of BSA.
Detailed Description
The following examples will provide those skilled in the art with a more complete understanding of the invention, but are not intended to limit the invention in any way.
Example 1
2 Male C57BL/6 mice with the age of 8-10 weeks and the weight of 25+/-1.5 g are selected for femur fracture modeling. After 14 days, the mice callus macrophages were extracted. A callus digestion solution A (A solution) was prepared, 200. Mu.L of a type II collagenase solution, 200. Mu.L of a 2.5wt.% trypsin-EDTA solution (trypsin-EDTA solution having a trypsin concentration of 25g/L and EDTA concentration of 3.8 g/L) and 100. Mu.L of a 2wt.% BSA solution (both dissolved in calcium-magnesium-free PBS buffer), 1500. Mu.L of RPMI 1640 medium (containing double antiserum); buffer B (B) 2wt.% serum was added to calcium magnesium free PBS buffer.
The mice were anesthetized, sacrificed after anesthesia, the femur removed, muscle and other soft tissues removed, the femur callus carefully isolated, the callus filled into a 5mL EP tube, and the a solution added for digestion.
The callus was digested by adding 2mL of solution A to a 5mL EP tube, and trimming the callus to 1mm 3 pieces with a high-temperature and high-pressure sterilized tissue shears. Callus digestion was performed in a constant temperature shaker at 37℃using 150 rpm, cell counts were performed every 5min starting 20min to observe the trend of growth, after no significant increase in cell number was observed (at about 40-50 min), the digestate was aspirated, filtered through a 70 μm filter and added to a 15mL centrifuge tube placed on ice.
Centrifuging 300g of callus digestion solution for 5 minutes after digestion is finished, discarding the supernatant after centrifugation, adding 10 times of cell volume of erythrocyte lysate to resuspend cell sediment, centrifuging 300g of cell suspension for 5 minutes after 5 minutes of lysis, discarding the supernatant, using 5mLB solution to resuspend sediment, centrifuging 300g for 5 minutes, discarding the supernatant, adding 500 mu L B solution to resuspend sediment, and adding the sediment into a photophobic 1.5mL EP tube; adding CD45, CD11B, F4/80 and flow antibody of dead living cell dye into the cell suspension, rotating on a rotary table at 4 ℃ for incubation of the antibody for 30 minutes, adding 1mL of B solution after the incubation of the antibody is finished, centrifuging for 5 minutes, using 300g of B solution to resuspend the cell, centrifuging for 5 minutes, discarding the B solution after the centrifugation is finished, using 300 mu L B solution to resuspend the cell, and adding the cell into a flow tube for flow cytometry to sort macrophages.
The flow type sorting tube filled with cell suspension is assembled into an 18-color flow type cell sorter for macrophage sorting, living cells are marked in a cell community through a dead living cell antibody of an APC-Cy TM channel, then the macrophages are marked in the living cells through a CD45 antibody of a PerCP-Cy5.5 channel, a CD11b antibody of a BV421 channel and an F4/80 antibody of a PE channel, the macrophages are sorted through the marks, and the macrophages are separated from the digested cell suspension, and the sorting result is shown in figure 1.
In order to find out the optimal ratio of the type II collagenase solution to the trypsin solution in the digestive juice, the mixed solution containing the type II collagenase solution and the trypsin solution in different ratios is used for treating the callus, and the optimal ratio of the type II collagenase solution to the trypsin solution for effectively separating macrophages in the callus is found when the ratio of the type II collagenase solution to the trypsin solution is 1:1. Under the condition that the ratios of other reagents are the same in the formula developed by us, the mixed solution containing the type II collagenase solution and the trypsin solution in different ratios is used for treating the callus, and the 1:1 type II collagenase solution and the trypsin solution are added into the callus digestion solution, so that the recovery efficiency, the survival rate and the recovery quantity of macrophages are all optimal, as shown in fig. 2, fig. 3 and fig. 4.
To increase the survival rate of macrophages in the digestion and separation of the heart, we added BSA to the digestive juice to provide nutrients to the cells and set a concentration gradient to find the optimal concentration to maintain the activity of the cells. We added 50. Mu.L, 100. Mu.L, 150. Mu.L and 200. Mu.L of 2wt.% BSA solution to 2ml of solution A containing 1:1 type II collagenase and trypsin respectively, found that the addition of 100. Mu.L of 2wt.% BSA solution can significantly increase the survival rate of macrophages in callus tissue and save the cost of reagents after digesting the callus tissue of mice, as shown in FIG. 5.
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should make equivalent substitutions or modifications according to the technical scheme of the present invention and the inventive concept thereof, and should be covered by the scope of the present invention.
Claims (5)
1. Use of a combined reagent for macrophage separation in the preparation of a separated macrophage product, the combined reagent for macrophage separation consisting of a solution A and a solution B, wherein the solution A comprises 200 mu L of 2wt.% type II collagenase solution and 200 mu L of 2.5wt.% trypsin
EDTA solution and 100. Mu.L of 2wt.% BSA solution, the solvents of the above solutions being calcium-magnesium-free PBS buffer, 1500. Mu.L of RPMI 1640 medium containing double antisera; adding 2wt.% of serum into a calcium-magnesium-free PBS buffer solution;
The application comprises the following specific steps: taking a femur of a mouse, removing muscles and other soft tissues, carefully separating femur callus, assembling the callus into an EP tube, and adding A solution for digestion; centrifuging callus digestion liquid after digestion is finished, discarding supernatant after centrifugation, adding red blood cell lysate with 10 times of cell volume to resuspend cell sediment, centrifuging cell suspension after 5min of lysis, discarding supernatant, using solution B to resuspend sediment, centrifuging, discarding supernatant, adding solution B to resuspend sediment, and adding the sediment into an EP tube protected from light; adding CD45, CD11B, F4/80 and flow antibody of dead living cell dye into the cell suspension, rotating on a rotary table at 4 ℃ to incubate the antibody for 30 minutes, adding liquid B after the incubation of the antibody is finished, centrifuging after the cell is resuspended by using the liquid B, cleaning, centrifuging, discarding the liquid B after the centrifugation is finished, and adding the cell to a flow tube for flow cytometry to sort macrophages after the cell is resuspended by using the liquid B.
2. Use according to claim 1, characterized in that the centrifugation takes 5 minutes with 300 g.
3. The use according to claim 1, characterized in that it comprises the following specific steps: taking a femur of a mouse, removing muscles and other soft tissues, carefully separating femur callus, assembling the callus into a 5mL EP tube, and adding A solution for digestion; centrifuging 300g of callus digestion solution for 5 minutes after digestion is finished, discarding the supernatant after centrifugation, adding 10 times of cell volume of erythrocyte lysate to resuspend cell sediment, centrifuging 300g of cell suspension for 5 minutes after 5 minutes of lysis, discarding the supernatant, using 5mL of B solution to resuspend sediment, centrifuging 300g for 5 minutes, discarding the supernatant, adding 500 mu L B solution to resuspend sediment, and adding the sediment into a light-resistant 1.5mL EP tube; adding CD45, CD11B, F4/80 and flow antibody of dead living cell dye into the cell suspension, rotating on a rotary table at 4 ℃ for incubation of the antibody for 30 minutes, adding 1mL of B solution after the incubation of the antibody is finished, centrifuging for 5 minutes, using 300g of B solution to resuspend the cell, centrifuging for 5 minutes, discarding the B solution after the centrifugation is finished, using 300 mu L B solution to resuspend the cell, and adding the cell into a flow tube for flow cytometry to sort macrophages.
4. The use according to claim 3, wherein the digestion is performed by adding solution a to the EP tube and then trimming the callus into pieces using high temperature and high pressure sterilized tissue shears; callus digestion was performed in a constant temperature shaker at 37℃and samples were taken to observe the trend of cell count increase, after which no significant increase in cell number was observed, the digestate was aspirated, filtered through a 70 μm sieve and added to a centrifuge tube placed on ice.
5. The use according to claim 4, wherein the digestion method is to trim callus tissue into 1mm 3 tissue pieces with high temperature and high pressure sterilized tissue shears after adding 2mL of a solution to 5mL EP tube; callus digestion was performed in a constant temperature shaker at 37℃using 150 rpm, cell counts were performed every 5min starting 20min to observe the trend of growth, after no significant increase in cell number was observed, the digests were aspirated, filtered through a 70 μm screen and added to a 15mL centrifuge tube placed on ice.
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