A use 89 Method for Zr imaging tracing of human mesenchymal stem cells

Abstract

The present invention discloses a method for utilizing 89 A method for Zr imaging tracing of human mesenchymal stem cells, the method is performed as follows: 89 HBSS was added to the Zr-DFO-NCS reaction solution and the pH was adjusted to obtain 89 Zr-DFO-NCS mixed solution; collect human mesenchymal stem cells and use 89 Resuspend the cells in Zr-DFO-NCS mixed solution and incubate; remove the supernatant by centrifugation, then wash the labeled cells several times with PBS, re-plate and continue to culture. 89 Zr-labeled human mesenchymal stem cells. The present invention uses DFO as an intermediate chelating agent to improve 89 The stability of Zr and hMSCs labeling and the accuracy of in vivo tracing results. 89 Zr-hMSCs rather than simply washing, firstly restore cell activity, and secondly remove the markers on cell debris. 89 Zr improves the accuracy of imaging tracing results.

Description

Technical Field

[0001] The present invention relates to the field of nuclear medicine technology, and in particular to a method of using 89 Method for Zr imaging tracing of human mesenchymal stem cells. Background Art

[0002] Mesenchymal stem cells (MSCs) are a type of adult progenitor cells with multidirectional differentiation potential, which can differentiate into various mesenchymal tissues, among which bone, cartilage and fat are more typical. MSCs can self-renew and proliferate in vitro. After infusion into the body, they can regulate the level of inflammation in the body, regulate the immune system, repair damaged tissue structures, and form new tissue cells through differentiation and regeneration through paracrine secretion and (or) release of immune factors. Transplantation of human MSCs (hMSCs) therapy has brought hope for intractable diseases in humans. Before the large-scale clinical application of hMSCs therapy, its effectiveness and safety need to be further verified and its therapeutic mechanism needs to be clarified. The in vivo tracking of the survival number and retention site of exogenous transplanted hMSCs in the host body, and the real-time evaluation of the cell viability and migration path of hMSCs have opened a window for further studying the therapeutic mechanism of hMSCs therapy and evaluating the effectiveness and safety of hMSCs therapy.

[0003] At present, the in vivo imaging tracing technology of hMSCs mainly includes fluorescence imaging tracing technology and magnetic resonance imaging tracing technology, which uses fluorescent proteins, luciferases, and magnetic substances to trace and image hMSCs. Fluorescence imaging tracing technology has a relatively low spatial resolution, and the substrate has certain immunogenicity. In addition, it is limited by the fluorescence intensity of the fluorescent group and cannot be used in large animals. Magnetic resonance imaging tracing technology has a long imaging time and low spatial sensitivity, and cannot be used for whole-body imaging and tracing of large animals.

[0004] Radionuclide labeling technology can image and trace the retention site of hMSCs after transplantation in real time in vivo. The main principle of this technology is to use radioactive isotopes to label hMSCs in vitro, and then transplant and infuse them into experimental animals. By performing single photon emission computed tomography (SPECT) or positron emission tomography (PET) on the experimental animals, the retention site of the transplanted MSCs in the experimental animals can be imaged and traced, and the survival time, migration path, homing ability, etc. of hMSCs can be further analyzed.

[0005] At this stage, 11 carbon( 11 C) 64 copper( 64 Cu)、 18 fluorine( 18 F), iodine ( 124 I. 125 I.131 I)、 89 pick( 89 Zr) and other radionuclides can be used for in vivo imaging and tracing of cells. 89 Zr, with a half-life of 78.4 h, is sufficient to continuously track cells for at least 21 days, and has broad application prospects in imaging and tracking transplanted hMSCs. 89 Zr labeling of hMSCs generally requires an intermediate carrier. Currently, the commonly used carrier is oxygen. For example, the patent application with application number 202111498846.9 discloses a 89 Method for labeling human umbilical cord mesenchymal stem cells with Zr, wherein the hMSCs cells to be labeled are 89 Zr-oxine was mixed and incubated to obtain 89 Zr-hMSCs cells. Oxine is used as an intermediate carrier with poor labeling stability. 89 Zr may be transferred to the outside of hMSCs and absorbed by liver cells. Summary of the invention

[0006] One of the technical problems to be solved by the present invention is that the existing imaging and tracing technology of hMSCs in large animals has limitations in terms of spatial sensitivity, spatial specificity, tissue depth, and the need to sacrifice experimental animals for detecting pathological tissues. 89 The imaging tracing technology of Zr-labeled hMSCs uses PET to image the hMSCs infused into the experimental animals at different time points after transplantation, thereby tracing the retention sites of hMSCs in the experimental animals. The advantages of this method are high spatial sensitivity, strong spatial specificity, rapid imaging, and the ability to achieve in vivo tracing of large animals without sacrificing experimental animals and saving experimental animal resources.

[0007] The second technical problem to be solved by the present invention is that the existing 89 The Zr-labeled hMSCs imaging tracing technology has the disadvantages of single intermediate carrier selection, unstable labeling, and poor result accuracy.

[0008] In view of the above technical problems, the present invention provides a technical solution: a method of using 89 A method for Zr imaging tracing of human mesenchymal stem cells, the method is performed as follows: 89 HBSS was added to the Zr-DFO-NCS reaction solution, and the pH was adjusted with alkaline solution to obtain 89 Zr-DFO-NCS mixed solution; collect human mesenchymal stem cells and use 89Resuspend the cells in the Zr-DFO-NCS mixture and incubate; remove the supernatant by centrifugation, and then wash the labeled cells several times (2-4 times) with PBS to obtain 89 Zr-labeled human mesenchymal stem cells 89 Zr-DFO-hMSCs; 89 Zr-DFO-hMSCs were replated and cultured continuously.

[0009] Furthermore, the 89 The preparation process of Zr-DFO-NCS reaction solution is as follows:

[0010] Step (1) taking 13-15 times the volume of zirconium chloride solution, adding 10-15 times the volume of 0.8-1.2M oxalic acid solution;

[0011] Step (2) adding 20-23 times the volume of 0.8-1.2 M HEPES-NaOH buffer;

[0012] Step (3) gently shake and add 0.8-1.2M Na2CO3 dropwise to adjust the pH value to 6.8-7.2 at room temperature;

[0013] Step (4) add 2-5 times volume of DFO-NCS solution, shake and chelate at room temperature for 0.5-1.5h to obtain 89 Zr-DFO-NCS reaction solution.

[0014] Furthermore, the concentration of zirconium chloride is 0.35-0.45 ng / μL, and the concentration of DOF-NCS is 3-7 μg / μL.

[0015] Furthermore, the 89 The volume ratio of Zr-DFO-NCS reaction solution to HBSS solution is 60-65:500-640; the concentration of HBSS is 1X~1.5X.

[0016] Furthermore, the number of human mesenchymal stem cells is 89 The volume ratio of Zr-DFO-NCS mixed solution is 1-3×10 6 : 1-1.5mL.

[0017] Furthermore, the 89 The culture medium of Zr-DFO-hMSCs is: 85-90% low-glucose DMEM + 10-14% FBS + 0.5-1.5% PS.

[0018] Furthermore, the 89 After resuspending the cells in the Zr-DFO-NCS mixture, incubate at 36-38°C and 200-400 rpm for 20-40 min; 89Zr-DFO-hMSCs were replated and cultured for 18-30h.

[0019] Further, 89 After adding HBSS to the Zr-DFO-NCS mixture, 0.8-1.2 M Na2CO3 was added dropwise to adjust the pH to 7.3-7.7.

[0020] The present invention uses calcium chloride as the starting compound and adds oxalic acid to increase the chelation rate with the intermediate carrier DFO. Using DFO as the intermediate chelating agent instead of oxygen increases 89 The stability of Zr and hMSCs labeling improves the accuracy of in vivo tracing results. 89 Zr-hMSCs were washed for 24 h instead of PBS 89 Zr-hMSCs, firstly, restored the cell activity of hMSCs, and secondly, removed the markers on the cell debris. 89 Zr improves the accuracy of imaging tracing results. The present invention can non-invasively monitor a series of biological changes such as the survival, retention site, and homing ability of transplanted mesenchymal stem cells in vivo, and provide reference data for evaluating the effectiveness and safety of mesenchymal stem cells in treating refractory diseases in humans using primates.

[0021] Compared with the prior art, the present invention has the following beneficial effects: 89 The Zr labeling method for human mesenchymal stem cells was replaced by 89 The starting radioactive compound of Zr improves the intermediate carrier DFO and 89 The speed of Zr binding.

[0022] The present invention 89 The Zr-labeled human mesenchymal stem cell method has better labeling stability, high accuracy of in vivo imaging and tracing results, and reduces the risk of cell fragments or 89 Experimental error caused by competitive uptake of Zr by liver cells.

[0023] The present invention transplants radionuclides by intravenous injection 89 When hMSCs stably labeled with Zr are placed in experimental animals for imaging and tracing, this imaging and tracing method is not limited by tissue depth, has a fast speed, can achieve whole-body imaging and tracing of large animals, has high spatial sensitivity, low detection volume, and can achieve long-term tracing.

[0024] The present invention transplants radionuclides by intravenous injection 89When hMSCs stably labeled with Zr are placed in experimental animals for imaging and tracing, in vivo imaging and tracing of large and small experimental animals can be achieved, which can save primate experimental animal resources and is more in line with the 3R principle of experimental animal ethics requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Single injection into the tail vein of Kunming mice 89 PET / CT images of Zr-DFO-MSCs;

[0026] Figure 2 A single injection into the tail vein of cynomolgus monkeys 89 PET / CT images of Zr-DFO-MSCs;

[0027] Figure 3 Single injection into the tail vein of Kunming mice 89 PET / CT images of Zr-Oxine-MSCs;

[0028] Figure 4 A single injection into the tail vein of cynomolgus monkeys 89 PET / CT images of Zr- Oxine -MSCs. DETAILED DESCRIPTION

[0029] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications. The English abbreviations involved in the following examples are shown in Table 1.

[0030] Table 1

[0031] English abbreviation Chinese meaning English abbreviation Chinese meaning DFO-NCS p-SCN-Bn-Deferoxamine, p-SCN-Bn-Deferoxamine hMSCs Human Mesenchymal Stem Cells HEPES-NaOH 4-Hydroxyethylpiperazineethanesulfonic acid-NaOH PBS Phosphate buffered saline HBSS Hanks' Balanced Salt Solution FBS Fetal bovine serum PS Streptomycin Penicillin

[0032] Example 1 89 Synthesis of Zr-DFO-NCS

[0033] Take 13 μL of zirconium chloride / oxalic acid solution (5 ng zirconium chloride) and add 15 μL of 1 M oxalic acid;

[0034] Add 20 μL 1 M HEPES-NaOH buffer (pH 7.2);

[0035] Shake gently and add 1 M Na2CO3 dropwise to adjust the pH value to 6.8-7.2 (3 min at room temperature).

[0036] Add 3 μL (15 μg) DFO-NCS solution and shake at room temperature for 1 h to obtain 89 Zr-DFO-NCS reaction solution.

[0037] Example 2 89 Zr-DFO Labeling of Human Mesenchymal Stem Cells

[0038] 50~61μL volume 89 500 μL of 28.6 mM HBSS was added to the Zr-DFO-NCS reaction solution, and 1 M Na2CO3 was added dropwise to adjust the pH to 7.5;

[0039] HBSS solution was added dropwise to make the total liquid volume accurately 700 μL.

[0040] Collect 2×10 6 Mesenchymal stem cells (conventional sources) were used 89 The Zr-DFO-NCS mixture was resuspended and incubated at 37°C and 300 rpm for 30 min.

[0041] The supernatant was removed by centrifugation, and the labeled cells were washed 3 times with PBS to obtain 89 Zr-labeled human mesenchymal stem cells 89 Zr-DFO-hMSCs. 89 Zr-DFO-hMSCs were replated and cultured for another 24 h.

[0042] 89 The culture medium of Zr-DFO-hMSCs is: 87% low-glucose DMEM + 12% FBS + 1% PS.

[0043] The cell viability was calculated using a cell counter (JMBIO) before labeling and after continued culture for 24 hours. The cell activity was 97.56% before labeling and 96.05% after continued culture for 24 hours.

[0044] Example 3 Kunming mice / cynomolgus monkeys single tail vein injection 89 PET / CT imaging after Zr-DFO-hMSCs

[0045] Three Kunming mice and one healthy cynomolgus monkey were injected into the tail vein once 89 Zr-labeled human mesenchymal stem cells. The cell numbers were 1×10 6 cells / Kunming mouse, 1.5×10 6 PET / CT imaging was performed at four time points: 4h, 24h, 72h and 168h after cell injection, and the PET / CT scan data were processed using the PHILIPS Nebula workstation. Figure 1 Kunming mice were injected with tail vein 89 PET / CT scan results at different time points after Zr-DFO-hMSCs. Figure 2 Injection of tail vein into cynomolgus monkeys89 PET / CT scan results at different time points after Zr-DFO-hMSCs. (Philips PET / CT scanner model: IngenuityTF; PET / CT scan image processing platform: PHILIPS Nebula workstation).

[0046] Comparative Example 1 Replacement of intermediate carrier DFO / Oxine

[0047] In step 4 of Example 1, DFO-NCS was replaced with Oxine, and the remaining steps were exactly the same as those of Example 1. 89 Zr-Oxine continued to label human mesenchymal stem cells, and the operation steps were exactly the same as those in Example 2. 89 Zr-Oxine-hMSCs were injected into three Kunming mice and one cynomolgus monkey via the tail vein, following the same procedure as in Example 3. Figure 3 Kunming mice were injected with tail vein 89 PET / CT scan results at different time points after Zr-Oxine-hMSCs. Figure 4 Injection of tail vein into cynomolgus monkeys 89 PET / CT scan results at different time points after Zr- Oxine -hMSCs administration.

[0048] When using Oxine as an intermediate carrier, 89 When Zr-Oxine-hMSCs were imaged and traced in Kunming mice, 24 hours after tail vein injection, the signal gradually shifted from the lungs to the liver. When Zr-Oxine-hMSCs were imaged and traced in cynomolgus monkeys, 24 hours after tail vein injection, the signal was enriched in the bones of cynomolgus monkeys and gradually shifted from the lungs to the liver. This experimental result is different from other conventional experimental results in academia because 89 Zr-Oxine-hMSCs labeling method 89 Zr exists in the labeled cells and will be freed to the outside of the cells and competitively absorbed by liver cells and deposited in the bones. Using DFO as an intermediate carrier for labeling is more stable.

[0049] It should be noted that the above embodiments are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Equivalent replacements or substitutions made on the basis of the above embodiments all fall within the protection scope of the present invention.

Claims

1. A kind of utilization 89 The method for Zr imaging tracing of human mesenchymal stem cells is characterized by: The method operates as follows: 89 HBSS was added to the Zr-DFO-NCS reaction solution, and the pH was adjusted with alkaline solution to obtain 89 Zr-DFO-NCS mixed solution; Human mesenchymal stem cells were collected and used 89 Resuspend the cells in the Zr-DFO-NCS mixture and incubate; The supernatant was removed by centrifugation, and the labeled cells were washed several times with PBS to obtain 89 Zr-labeled human mesenchymal stem cells 89 Zr-DFO-hMSCs; 89 Zr-DFO-hMSCs were re-plated and cultured to restore the cell activity of hMSCs and remove the labeled cell debris. 89 Zr, for imaging tracer; Said 89 The preparation process of Zr-DFO-NCS reaction solution is as follows: Step (1) taking 13-15 times the volume of zirconium chloride solution, adding 10-15 times the volume of 0.8-1.2M oxalic acid solution; Step (2) adding 20-23 times the volume of 0.8-1.2 M HEPES-NaOH buffer; Step (3) gently shake and add 0.8-1.2M Na2CO3 dropwise to adjust the pH value to 6.8-7.2 at room temperature; Step (4) add 2-5 times volume of DFO-NCS solution, shake and chelate at room temperature for 0.5-1.5h to obtain 89 Zr-DFO-NCS reaction solution; Said 89 After resuspending the cells in the Zr-DFO-NCS mixture, incubate at 36-38°C and 200-400 rpm for 20-40 min; 89 Zr-DFO-hMSCs were re-plated and cultured for 18-30 h; The concentration of the zirconium chloride solution is 0.35-0.45 ng / μL, and the concentration of the DOF-NCS solution is 3-7 μg / μL.

2. The use according to claim 1 89 The method for Zr imaging tracing of human mesenchymal stem cells is characterized by: Said 89 The volume ratio of Zr-DFO-NCS reaction solution to HBSS solution is 60-65:500-640.

3. The use according to claim 1 89 The method for Zr imaging tracing of human mesenchymal stem cells is characterized by: The number of human mesenchymal stem cells and 89 The volume ratio of Zr-DFO-NCS mixed solution is (1-3)×10 6 : 1-1.5mL.

4. The use according to claim 1 89 The method for Zr imaging tracing of human mesenchymal stem cells is characterized by: Said 89 The culture medium of Zr-DFO-hMSCs is: 85-90% low-glucose DMEM, 10-14% FBS and 0.5-1.5% PS.

5. The use according to claim 1 89 The method for Zr imaging tracing of human mesenchymal stem cells is characterized by: 89 After adding HBSS to the Zr-DFO-NCS reaction solution, 0.8-1.2 M Na2CO3 was added dropwise to adjust the pH to 7.3-7.7.

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