Magnetic nanoparticle-mediated mesenchymal stem cell rapid separation method

Abstract

The invention provides a magnetic nanoparticle mediated mesenchymal stem cell rapid separation method, and belongs to the technical field of cell separation.The magnetic nanoparticle mediated mesenchymal stem cell rapid separation method comprises the following steps that 1, a cell sample is obtained from tissue, and a cell suspension is obtained after digestion; 2) mixing the cell suspension with first magnetic nanoparticles, and performing first incubation; 3) adding second magnetic nanoparticles, and carrying out second incubation; then carrying out magnetic separation and elution to obtain mesenchymal stem cells; the first magnetic nanoparticles are modified with a CD73 antibody and a CD90 antibody; and the second magnetic nanoparticles are modified with a CD105 antibody. According to the method, through two-step magnetic nanoparticle incubation and one-step magnetic separation, the purity of the mesenchymal stem cells obtained through separation is improved on the basis of ensuring sufficient separation of the mesenchymal stem cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell separation, and in particular relates to a method for rapid separation of mesenchymal stem cells mediated by magnetic nanoparticles. Background Art

[0002] Mesenchymal stem cells (MSCs), a type of adult stem cell with self-renewal and multidirectional differentiation potential, have shown tremendous potential for application in regenerative medicine, tissue engineering, immunomodulation, and cell therapy. In regenerative medicine, MSCs can differentiate into bone cells, chondrocytes, adipocytes, and other cells, which are used to repair damaged tissues and organs. In immunomodulation, they can regulate the body's immune system, providing a new approach for the treatment of autoimmune diseases. However, the efficient and precise isolation of MSCs from complex cell populations is a key prerequisite for fully realizing their application value.

[0003] Currently, the main methods for isolating mesenchymal stem cells include traditional adherent selection, density gradient centrifugation, and magnetic-activated cell sorting (MACS). Traditional adherent selection relies on the ability of mesenchymal stem cells to adhere to surfaces and removes non-adherent cells through multiple medium changes, allowing for the isolation of mesenchymal stem cells. However, this method has significant drawbacks: First, the isolation cycle is lengthy, typically requiring 7 to 14 days or even longer, significantly limiting research efficiency and the timeliness of clinical applications. Second, cells undergo multiple passages during the isolation process, which can alter their biological properties, such as reduced differentiation potential and altered gene expression profiles. Third, the lack of specific separation criteria makes it difficult to effectively remove other cells with similar adherent properties to mesenchymal stem cells, resulting in low separation purity, typically only 70% to 80%, which falls short of meeting the requirements of high-quality research and clinical applications. Density gradient centrifugation exploits differences in cell density to enrich mesenchymal stem cells in a specific density gradient medium through centrifugation. However, this method suffers from low separation efficiency, a limited number of cell samples that can be processed at a time, and the significant mechanical forces exerted on cells during centrifugation, which can easily lead to cell damage or even death. Furthermore, because differences in cell density are not absolute, some cells with similar densities to mesenchymal stem cells are difficult to effectively separate, resulting in incomplete separation. The purity of the resulting mesenchymal stem cells typically fluctuates between 75% and 85%, making it difficult to achieve high-purity separation.

[0004] Magnetic-activated cell sorting (MACS) uses magnetic nanoparticles conjugated with specific antibodies to label target cells and separate them under the influence of a magnetic field. While relatively simple to use, existing magnetic nanoparticle-mediated separation technologies still have numerous shortcomings. For one thing, the antibodies used to modify the surface of the magnetic nanoparticles are either of a single type or in an illogical combination, resulting in insufficient recognition of specific markers on the surface of mesenchymal stem cells, an inability to fully capture target cells, and consequently, incomplete separation. Furthermore, due to issues such as nonspecific adsorption, the purity of the isolated mesenchymal stem cells rarely exceeds 90%, making it difficult to meet the stringent requirements for high-purity, high-activity cells required for clinical-grade cell therapy. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a method for rapid separation of mesenchymal stem cells mediated by magnetic nanoparticles. The present invention improves the purity of the separated mesenchymal stem cells while ensuring sufficient separation of the mesenchymal stem cells through two steps of magnetic nanoparticle incubation and one step of magnetic separation.

[0006] The present invention provides a method for rapid separation of mesenchymal stem cells mediated by magnetic nanoparticles, comprising the following steps:

[0007] 1) Obtain cell samples from tissues and obtain cell suspension after digestion;

[0008] 2) mixing the cell suspension with the first magnetic nanoparticles and incubating them for a first time;

[0009] 3) then adding a second magnetic nanoparticle and performing a second incubation; then magnetic separation and elution to obtain mesenchymal stem cells;

[0010] The first magnetic nanoparticles are modified with CD73 antibodies and CD90 antibodies;

[0011] The second magnetic nanoparticles are modified with CD105 antibodies.

[0012] Preferably, in step 2), the ratio of the number of cells to the first magnetic nanoparticles in the cell suspension is 1:(15-35).

[0013] Preferably, in step 3), the ratio of the number of cells to the second magnetic nanoparticles in the cell suspension is 1:(15-35).

[0014] Preferably, the first magnetic nanoparticles are obtained by coupling carboxyl microspheres to CD73 antibodies and CD90 antibodies; the ratio of CD73 antibodies to CD90 antibodies is (2-3): (2-3); the second magnetic nanoparticles are obtained by coupling carboxyl microspheres to CD105 antibodies.

[0015] Preferably, the first incubation time in step 2) is 30 to 50 minutes.

[0016] Preferably, the second incubation time in step 3) is 15 to 35 minutes.

[0017] Preferably, the density of the cell suspension in step 1) is (1-5)×10 6 pieces / mL.

[0018] Compared with existing technologies, the present invention has the following advantages: The present invention provides a method for rapid isolation of mesenchymal stem cells mediated by magnetic nanoparticles, achieved through a two-step incubation and a one-step magnetic separation. The two-step incubation captures as many mesenchymal stem cells as possible from the sample, improving yield. The method also utilizes antibodies corresponding to different specific antigens on the surface of mesenchymal stem cells for specific enrichment, improving extraction efficiency while ensuring the purity of the obtained mesenchymal stem cells. The one-step separation procedure reduces the damage to the activity of the captured mesenchymal stem cells caused by magnetic separation, ensuring that the isolated mesenchymal stem cells maintain good activity. DETAILED DESCRIPTION

[0019] The present invention provides a method for rapid separation of mesenchymal stem cells mediated by magnetic nanoparticles, comprising the following steps: 1) obtaining a cell sample from a tissue, and obtaining a cell suspension after digestion; 2) mixing the cell suspension with a first magnetic nanoparticle and performing a first incubation; 3) then adding a second magnetic nanoparticle, and performing a second incubation; magnetic separation and elution to obtain mesenchymal stem cells; the first magnetic nanoparticles are modified with CD73 antibodies and CD90 antibodies; and the second magnetic nanoparticles are modified with CD105 antibodies.

[0020] In the present invention, a cell sample is first obtained from a tissue, and a cell suspension is obtained after digestion. In the present invention, the tissue is preferably selected from adipose tissue, bone marrow tissue, umbilical cord tissue, etc. After the tissue is obtained in the present invention, it is preferably pretreated, and the pretreatment preferably includes rinsing and mincing; the rinsing is preferably performed using PBS, and the tissue is minced to obtain tissue fragments, which are then digested. In the present invention, the digestion is preferably performed using type I collagenase or trypsin; the digestion time is preferably 20 to 50 minutes, and more preferably 30 to 40 minutes. After the digestion is completed, the present invention adds culture medium to terminate the digestion; then, filtration is performed to obtain a single cell suspension, and the filtration is preferably performed using a cell sieve to filter out incompletely digested tissue fragments to obtain a single cell suspension. The present invention preferably also includes the step of adjusting the concentration of the cell suspension, and the density of the cell suspension is preferably (1 to 5) × 10 6 / mL, more preferably (2 to 4) × 10 6 pieces / mL.

[0021] After obtaining the cell suspension, the present invention mixes the cell suspension with the first magnetic nanoparticles and incubates them for the first time. In the present invention, CD73 antibodies and CD90 antibodies are modified on the first magnetic nanoparticles. In the present invention, the first magnetic nanoparticles are obtained by coupling CD73 antibodies and CD90 antibodies to carboxyl microspheres; the coupling is preferably carried out by the EDC / Sulfo-NHS two-step method. The present invention has no special restrictions on the specific steps of the coupling, and can refer to the instructions. In the present invention, the ratio of the number of cells to the first magnetic nanoparticles in the cell suspension is preferably 1: (15-35), more preferably 1: (20-30), and more preferably 1: 25. The time of the first incubation is preferably 30-50 min, more preferably 35-45 min, and more preferably 40 min; the first incubation is preferably carried out in an incubator at 37°C and 5% CO2.

[0022] After the first incubation, the present invention adds second magnetic nanoparticles for a second incubation, followed by magnetic separation and elution to obtain mesenchymal stem cells. In the present invention, the second magnetic nanoparticles are modified with CD105 antibodies. In the present invention, the second magnetic nanoparticles are obtained by coupling CD105 antibodies to carboxyl microspheres; the coupling is preferably performed using a two-step EDC / Sulfo-NHS method. The specific steps of the coupling are not particularly limited in the present invention and can be performed by referring to the instructions. In the present invention, the ratio of cells to second magnetic nanoparticles in the cell suspension is preferably 1:(15-35), more preferably 1:(20-30), and even more preferably 1:25. In the present invention, the duration of the second incubation is preferably 15-35 minutes, more preferably 20-30 minutes, and even more preferably 25 minutes. The second incubation is preferably performed in an incubator at 37°C and 5% CO2. In the present invention, the magnetic separation is preferably performed using a magnetic separation column, and the elution is performed using a medium containing EDTA.

[0023] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0024] Carboxyl microspheres, CD73 antibody, CD90 antibody, CD105 antibody and corresponding secondary antibodies were all purchased commercially.

[0025] Subcutaneous adipose tissue was obtained from liposuction samples, and the samples were collected with informed consent.

[0026] Example 1

[0027] Preparation of the first magnetic nanoparticles:

[0028] Activation / coupling buffer: 50 mM MES, pH 6.0

[0029] Blocking buffer: 50 mM Tris, pH 8.0, 0.5% (w / v) casein.

[0030] Prepare a 200 mM EDC solution by adding 19.2 mg of room temperature EDC to 500 μL of a clean microcentrifuge tube. In the water.

[0031] To prepare a 200 mM Sulfo-NHS solution, add 21.7 mg of Sulfo-NHS to 500 μL of activation / coupling buffer in a clean microcentrifuge tube.

[0032] To 1 mL of washed microspheres, quickly add 24 μL of 200 mM EDC solution and 240 μL of 200 mM Sulfo-NHS solution. Vortex to mix and incubate on a rotating disk for 30 min at room temperature. Centrifuge to pellet the microspheres and discard the supernatant. Add 1 mL of activation / coupling buffer to wash the microspheres and mix thoroughly.

[0033] Centrifuge again to pellet the microspheres and resuspend the microspheres in 700 μL of activation / coupling buffer to obtain pretreated microspheres.

[0034] Take CD73 antibody and CD90 antibody respectively and use activation / coupling buffer to prepare antibody solutions with a concentration of 2 mg / mL.

[0035] Add 300 μL of antibody (2 mg / mL, 150 μL each of CD73 antibody and CD90 antibody) to 700 μL of microspheres, mix on a rotary mixer for 2.5 h, mix thoroughly, centrifuge and precipitate the microspheres, resuspend in 1 mL of blocking buffer, mix on a rotary mixer for 1 h, precipitate the microspheres again, and repeat blocking twice.

[0036] Preparation of the second magnetic nanoparticles:

[0037] Prepare a 1 mg / mL antibody solution of CD105 antibody using activation / coupling buffer.

[0038] Add 300 μL of antibody (1 mg / mL, STRO-1 antibody) to 700 μL of pretreated microspheres, mix on a rotary mixer for 2.5 h, mix thoroughly, centrifuge and precipitate the microspheres, resuspend in 1 mL of blocking buffer, mix on a rotary mixer for 1 h, precipitate the microspheres again, and repeat blocking twice.

[0039] Qualitative testing:

[0040] Immunofluorescence was used to detect the modified first and second magnetic nanoparticles. The modified magnetic nanoparticles were incubated with fluorescein FITC-labeled anti-mouse IgG antibodies (secondary antibodies against the aforementioned monoclonal antibodies) at room temperature for 1 hour and then washed with PBS. Under fluorescence microscopy, clear fluorescent signals appeared on the surfaces of both the first and second magnetic nanoparticles, indicating that the corresponding antibodies were successfully modified onto the magnetic nanoparticle surfaces.

[0041] Example 2

[0042] 1. Preparation of Single-cell Suspension

[0043] Sample: Human adipose tissue was rinsed three times with PBS buffer (containing 1% penicillin and streptomycin). Between rinses, gently pipette the tissue to thoroughly suspend impurities. The tissue was then allowed to settle for 2 minutes, and the supernatant was discarded. Connective tissue, including blood vessels and fascia, was removed using forceps and scissors, minced, and transferred to a centrifuge tube. 0.5% collagenase type I was added to the centrifuge tube at a 1:1 volume ratio of adipose tissue to 0.5% collagenase type I, mixed thoroughly, and digested at 37°C on a shaker at 50 rpm for 50 minutes. During digestion, the tube was removed and gently inverted every 10 minutes to ensure uniform digestion. An equal volume of complete culture medium was added to the centrifuge tube and gently pipetted to inactivate collagenase activity. The mixture was filtered through a 70 μm cell sieve into a new 50 mL centrifuge tube. The cell sieve was rinsed with an appropriate amount of complete culture medium to ensure complete cell collection. Centrifuge the tube containing the single-cell suspension at 4°C and 300×g for 3 minutes, discard the supernatant, collect the cell pellet, add an appropriate amount of complete culture medium to the cell pellet, and gently pipette to fully resuspend the cells to make a single-cell suspension. Count the cells under a microscope and adjust the cell suspension density to 5×10 using complete culture medium. 6 pieces / mL.

[0044] 2. Two-step incubation

[0045] Transfer 1 mL of the cell suspension with adjusted density to a sterile 15 mL centrifuge tube. Add the first magnetic nanoparticles to each tube of cell suspension at a ratio of 1:25 between the number of cells and the first magnetic nanoparticles, and gently blow to mix. Place the centrifuge tube in an incubator at 37°C and 5% CO2 for the first incubation of 40 minutes. During the incubation process, take out the centrifuge tube every 10 minutes and gently invert and mix to ensure that the cells and the magnetic nanoparticles are fully combined. After the first incubation is completed, add the second magnetic nanoparticles to each tube at a ratio of 1:25 between the number of cells and the second magnetic nanoparticles, and gently blow to mix. Place the centrifuge tube again in an incubator at 37°C and 5% CO2 for the second incubation of 25 minutes, and also invert and mix at regular intervals during the incubation.

[0046] 3. Magnetic Separation and Cell Elution

[0047] Mount the magnetic separation column on the matching magnetic stand, rinse the column with complete culture medium, and discard the flow-through. Slowly add the cell suspension labeled with magnetic nanoparticles to the separation column, allowing the cell suspension to flow naturally and allowing the cell-magnetic nanoparticle complex to be adsorbed within the separation column.

[0048] Rinse the separation column three times with complete culture medium. During each rinse, wait until the liquid has completely flowed out before proceeding to the next rinse to remove unbound impurities.

[0049] The separation column was removed from the magnetic field and placed on a new 15 mL centrifuge tube. EDTA-containing culture medium (1 mM) was added to the separation column, and the separation column was slowly rinsed. The eluate was collected and resuspended in culture medium after centrifugation to obtain the separated and purified mesenchymal stem cells.

[0050] After the obtained mesenchymal stem cells were counted and their purity was identified, they were cultured and the cell viability was measured.

[0051] The entire operation of this embodiment was repeated 5 times.

[0052] Example 3

[0053] 1. Preparation of Single-cell Suspension

[0054] Sample: Human adipose tissue was rinsed three times with PBS buffer (containing 1% penicillin and streptomycin). Between rinses, gently pipette the tissue to thoroughly suspend impurities. The tissue was then allowed to settle for 2 minutes, and the supernatant was discarded. Use forceps and scissors to remove connective tissue, such as blood vessels and fascia. Mince the tissue and transfer it to a centrifuge tube. Add 0.5% type I collagenase to the tube at a 1:1 volume ratio of adipose tissue to 0.5% type I collagenase, mix well, and digest for 50 minutes at 37°C on a shaker at 50 rpm. During digestion, remove the tube and gently invert every 10 minutes to ensure uniform digestion. Add an equal volume of complete culture medium to the tube and gently pipette to inactivate collagenase activity. Filter the mixture through a 70 μm cell sieve into a new 50 mL centrifuge tube. Rinse the cell sieve with an appropriate amount of complete culture medium to ensure complete cell collection. Centrifuge the tube containing the single-cell suspension at 4°C and 300×g for 3 minutes, discard the supernatant, collect the cell pellet, add an appropriate amount of complete culture medium to the cell pellet, and gently pipette to fully resuspend the cells to make a single-cell suspension. Count the cells under a microscope and adjust the cell suspension density to 1×10 6 pieces / mL.

[0055] 2. Two-step incubation

[0056] Transfer 1 mL of the cell suspension with adjusted density to a sterile 15 mL centrifuge tube. Add the first magnetic nanoparticles to each tube of cell suspension at a ratio of 1:20 between the number of cells and the first magnetic nanoparticles, and gently blow to mix. Place the centrifuge tube in an incubator at 37°C and 5% CO2 for the first incubation for 40 minutes. During the incubation process, take out the centrifuge tube every 10 minutes and gently invert and mix to ensure that the cells and the magnetic nanoparticles are fully combined. After the first incubation is completed, add the second magnetic nanoparticles to each tube at a ratio of 1:15 between the number of cells and the second magnetic nanoparticles, and gently blow to mix. Place the centrifuge tube again in an incubator at 37°C and 5% CO2 for the second incubation for 25 minutes, and also invert and mix at regular intervals during the incubation.

[0057] 3. Magnetic Separation and Cell Elution

[0058] Install the magnetic separation column on the matching magnetic rack, rinse the separation column with complete culture medium, and discard the effluent.

[0059] The cell suspension labeled with magnetic nanoparticles is slowly added to the separation column, and the cell suspension is allowed to flow down naturally, so that the cell and magnetic nanoparticle complexes are adsorbed in the separation column.

[0060] Rinse the separation column three times with complete culture medium. During each rinse, wait until the liquid has completely flowed out before proceeding to the next rinse to remove unbound impurities.

[0061] The separation column was removed from the magnetic field and placed on a new 15 mL centrifuge tube. EDTA-containing culture medium (1 mM) was added to the separation column, and the separation column was slowly rinsed. The eluate was collected and centrifuged, and then resuspended in culture medium to obtain the separated and purified mesenchymal stem cells.

[0062] After the obtained mesenchymal stem cells were counted and their purity was identified, they were cultured and the cell viability was measured.

[0063] The entire operation of this embodiment was repeated 5 times.

[0064] Comparative Example 1

[0065] The difference from Example 2 is that the incubation is changed to a one-step process, and the first magnetic nanoparticles are used for incubation, followed by direct magnetic separation.

[0066] The entire operation of this comparative example was repeated 5 times.

[0067] Comparative Example 2

[0068] The difference from Example 2 is that a two-step incubation and two-step magnetic separation is used, i.e., a first magnetic nanoparticle incubation is performed followed by magnetic separation (the magnetic separation and elution methods are the same as in Example 2). The separated cells are collected and incubated with a second magnetic nanoparticle, followed by a second magnetic separation (the magnetic separation and elution methods are the same as in Example 2).

[0069] The entire operation of this comparative example was repeated 5 times.

[0070] Experimental Example 1

[0071] The adipose-derived mesenchymal stem cells isolated in Example 2, Example 3, Comparative Example 1, and Comparative Example 2 were tested for purity by flow cytometry. The positive markers detected were CD73, CD90, and CD105, and the negative markers detected were CD34, CD45, and CD31. The test results are shown in Table 1.

[0072] Table 1 Purity test results of adipose-derived mesenchymal stem cells

[0073] Grouping Total cell number purity(%) Example 2 <![CDATA[8×10 4 ]]> 97.2％ Example 3 <![CDATA[3×10 4 ]]> 95.9％ Comparative Example 1 <![CDATA[5×10 3 ]]> 84.6％ Comparative Example 2 <![CDATA[3×10 3 ]]> 98.2％

[0074] It can be seen that the total number of cells obtained by the two-step incubation and one-step separation method of Examples 2 and 3 is significantly more than that of Comparative Examples 1 and 2, and the cell purity is higher. The number of cells obtained by the one-step incubation in Comparative Example 1 is small and the purity is low; although the two-step incubation and two-step separation method of Comparative Example 2 obtains high cell purity, the total number of cells separated is small.

[0075] The adipose-derived mesenchymal stem cells isolated and obtained in Example 2, Example 3, Comparative Example 1, and Comparative Example 2 were cultured and passaged, and the survival rates and induced differentiation of cells at passages P1 to P3 were detected. The results are shown in Table 2.

[0076] Table 2 Cell survival rate and induced differentiation of P1 to P3 generations

[0077]

[0078] This demonstrates that the adipose-derived mesenchymal stem cells isolated using the separation method of the present invention exhibit high survival rates at passages P1 to P3, excellent inducibility, and retention of the stemness of the original cells. In Comparative Example 2, due to the two magnetic separations, the cells sustained significant damage, leading to a partial loss of stemness, which was partially restored by passage 3.

[0079] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for rapid separation of mesenchymal stem cells mediated by magnetic nanoparticles, characterized in that: The following steps are involved: 1) Obtain cell samples from tissues and obtain cell suspension after digestion; 2) mixing the cell suspension with the first magnetic nanoparticles and incubating them for a first time; 3) then adding a second magnetic nanoparticle and performing a second incubation; then magnetic separation and elution to obtain mesenchymal stem cells; The first magnetic nanoparticles are modified with CD73 antibodies and CD90 antibodies; The second magnetic nanoparticles are modified with CD105 antibodies.

2. The method for rapid isolation of mesenchymal stem cells according to claim 1, characterized in that: Step 2) The ratio of the number of cells to the first magnetic nanoparticles in the cell suspension is 1:(15-35).

3. The method for rapid isolation of mesenchymal stem cells according to claim 1 or 2, characterized in that: Step 3) The ratio of the number of cells to the second magnetic nanoparticles in the cell suspension is 1:(15-35).

4. The method for rapid isolation of mesenchymal stem cells according to claim 1, characterized in that: The first magnetic nanoparticles are obtained by coupling carboxyl microspheres with CD73 antibodies and CD90 antibodies; the ratio of CD73 antibodies to CD90 antibodies is (2-3): (2-3); the second magnetic nanoparticles are obtained by coupling carboxyl microspheres with CD105 antibodies.

5. The method for rapid isolation of mesenchymal stem cells according to claim 1, characterized in that: Step 2) The first incubation time is 30 to 50 minutes.

6. The method for rapid isolation of mesenchymal stem cells according to claim 1, characterized in that: Step 3) The second incubation time is 15 to 35 minutes.

7. The method for rapid isolation of mesenchymal stem cells according to claim 1, characterized in that: The density of the cell suspension in step 1) is (1-5)×10 6 pieces / mL.

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