Tracing method of myocardial cells for inducing differentiation of pluripotent stem cells in pharmacokinetics

Abstract

The invention relates to the technical field of biological medicines, and relates to a tracing method of myocardial cells for inducing differentiation of pluripotent stem cells in pharmacokinetics, and the method specifically comprises the following steps: providing a test substance comprising a radioactive marker for an organism, and tracing; the in-vivo distribution rule of the i PSC-derived myocardial cells is monitored, and a relationship is established between dynamic distribution of the marker after the cells enter the body and the cell curative effect, so that the clinical curative effect of cell drugs is effectively improved, the risk is reduced, and the interaction of the drugs is reduced.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and relates to a method for tracing cardiomyocytes differentiated from induced pluripotent stem cells in pharmacokinetics. Background Art

[0002] Induced pluripotent stem cells have the potential to differentiate into various functional cells. Similarly, induced pluripotent stem cells can generate cardiomyocytes with contractile function by regulating corresponding signaling pathways. Cardiomyocytes with stable electrophysiological characteristics can not only be used to study the pathogenesis of heart diseases and screen drugs for treating heart diseases, but can even carry out clinical studies on cardiomyocyte treatment of heart diseases, further expanding the contribution of cardiomyocytes in the treatment of diseases such as myocardial infarction and heart failure. The mechanism of cardiomyocytes as a drug for treating heart diseases is not yet clear, and the survival, retention, proliferation, migration and distribution of cardiomyocytes in vivo also need to be further studied. Therefore, it is necessary to trace and label cardiomyocytes.

[0003] Human cardiomyocytes have biological characteristics different from those of traditional drugs such as small molecules and macromolecules. The pharmacokinetic system for evaluating drug absorption, distribution and metabolism of traditional drugs is not suitable for the evaluation of cell drugs. Therefore, it is impossible to monitor the distribution, metabolism and excretion rules of human iPSC-derived cardiomyocytes in vivo, and further, it is impossible to improve the clinical efficacy of cell drugs, reduce risks and reduce drug interactions.

[0004] Existing labeling methods mainly target traditional drugs and cannot effectively label human cardiomyocytes, making it difficult to effectively establish a relationship between the dynamic distribution of cell drugs after entering the body and cell efficacy. To solve the above problems, the present solution proposes a new technical solution to overcome the above problems. Summary of the Invention

[0005] The purpose of the present invention is that through 89 labeling with Zr, cardiomyocytes can be traced or tracked in vivo. When administered into a living organism, through detection, the changes or distribution of cardiomyocytes in vivo can be accurately reflected.

[0006] To achieve the above purpose, the present invention proposes the following technical solution specifically. A method for tracing cardiomyocytes differentiated from induced pluripotent stem cells in pharmacokinetics, characterized in that

[0007] Prepare the test substance: Take 89 Zr to react with oxine to obtain 89 Zr-oxine; and take out 89 Zr-oxine and place them into the resuscitated cardiomyocytes (iPSC-CM) respectively. After incubation for 15 minutes, centrifuge, and then resuspend with 5% human albumin and centrifuge and wash 3 times.

[0008] Administer the test substance to an organism;

[0009] Through ingestion 89 Zr-oxine and 89 The Zr-labeled cardiomyocytes are detected and traced by positron emission tomography.

[0010] As an improved technical solution of this application, preparing the test substance specifically includes:

[0011] S1. Prepare a cell solvent with 0.9% sodium chloride injection and 20% human albumin. Flush the cell cryopreservation tube with the cell solvent, add the cardiomyocyte resuspension and then centrifuge. Subsequently, add the cell solvent to dilute the cell stock solution for standby; the cell stock solution refers to the revived cardiomyocytes;

[0012] S2. Take 89 React Zr with oxine to obtain 89 Zr-oxine; and take out 89 Zr-oxine and place it into the revived cardiomyocytes (iPSC-CM) respectively. After incubating for 15 minutes, centrifuge, and then resuspend with 5% human albumin and centrifuge and wash 3 times.

[0013] As an improved technical solution of this application, the specific ratio of sodium chloride injection to human albumin is 3:1.

[0014] As an improved technical solution of this application, administering the test substance to an organism includes: injecting and detecting: injecting the test substance into the organism respectively; monitoring and delineating the PET imaging and standard uptake value in the body at different time points.

[0015] As an improved technical solution of this application, before preparing 89 Zr-oxine, adjust the pH. Take 89 Zr and add HEPES and Na2CO3 to adjust the pH value to 7.

[0016] As an improved technical solution of this application, the organism is a non-human mammal; the non-human mammal can be a mouse, a monkey, a cow, a sheep or a dog.

[0017] As an improved technical solution of this application, the radiochemical purity (RCP) of the test substance is not less than 90%.

[0018] As an improved technical solution of this application, when administering the drug, the diluent of the test substance is physiological saline containing 5% HSA.

[0019] The organism refers to the subject, which is a non-human mammal.

[0020] It should be noted that the cardiomyocytes described in any implementation manner of the embodiments of the present invention and the tracing method thereof are for non-diagnostic or therapeutic purposes of diseases.

[0021] As can be seen from the above technical solutions, the technical solutions of the present invention provide the following beneficial effects:

[0022] On the one hand, through this method, the stability of the in vivo tracing method can be achieved, and in vivo distribution of cells can be monitored by combining in vivo PET imaging. On the other hand, by monitoring the in vivo distribution law of human iPSC-derived cardiomyocytes, and then establishing a relationship between the dynamic distribution of the marker after the cells enter the body and the cell efficacy, the clinical efficacy of cell drugs can be effectively improved, the risk can be reduced, and the drug interaction can be reduced.

[0023] It should be understood that all combinations of the foregoing concepts and additional concepts described in more detail below can be regarded as part of the inventive subject matter of the present disclosure as long as such concepts do not contradict each other.

[0024] The foregoing and other aspects, embodiments, and features of the teachings of the present invention can be more fully understood from the following description in conjunction with the accompanying drawings. Other additional aspects of the present invention, such as the features and / or beneficial effects of the exemplary embodiments, will be apparent in the following description, or will be learned through the practice of the specific embodiments according to the teachings of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component shown in each figure may be represented by the same reference numeral. For clarity, not every component is labeled in each figure. Now, embodiments of various aspects of the present invention will be described by way of example and with reference to the drawings, wherein:

[0026] Attached Figure 1 PET scan images at different time points after injecting 89 Zr-cardiomyocytes into cynomolgus monkeys G1-M-01 (hereinafter referred to as G1); injecting 89 Zr-Oxine into cynomolgus monkeys G2-M-01 (hereinafter referred to as G2);

[0027] Attached Figure 2 Bar graphs showing the changes in radioactivity uptake values in various tissues over time after injecting 89 Zr-cardiomyocytes and 89 Zr-Oxine into cynomolgus monkeys G1 and G2 respectively;

[0028] Attached Figure 3 Showing the changes in radioactivity uptake values in various tissues over time after injecting 89 Zr-cardiomyocytes into KM mice;

[0029] Appendix Figure 4 PET scan images of KM mice at different time points after injection of 89 Zr-myocardial cells;

[0030] Appendix Figure 5 Radioactivity uptake values of various tissues in NCG mice over time after injection of 89 Zr-myocardial cells;

[0031] Appendix Figure 6 PET scan images of NCG mice at different time points after injection of 89 Zr-myocardial cells;

[0032] Appendix Figure 7 Comparison chart of biodistribution and contour drawing data of KM mice after 192h imaging technology; (where biodistribution: in vitro detection of each organ; contour drawing data: in vivo detection by PET) Detailed implementation manners

[0033] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the described embodiments of the present invention without creative efforts fall within the scope of protection of the present invention. Unless otherwise defined, the technical terms or scientific terms used herein shall have the ordinary meanings understood by those of ordinary skill in the art to which the present invention pertains.

[0034] The terms "first", "second" and similar terms used in the specification and claims of this patent application of the present invention do not denote any order, quantity or importance, but are only used to distinguish different components. Similarly, unless the context clearly indicates otherwise, singular forms such as "a", "an" or "the" and similar terms do not denote a quantity limitation, but mean that there is at least one. The terms "including" or "comprising" and similar terms mean that the elements or objects appearing before "including" or "comprising" cover the features, wholes, steps, operations, elements and / or components listed after "including" or "comprising", and do not exclude the existence or addition of one or more other features, wholes, steps, operations, elements, components and / or their combinations. The terms "upper", "lower", "left", "right", etc. are only used to indicate relative positional relationships, and when the absolute position of the object being described changes, the relative positional relationship may also change accordingly.

[0035] Definition:

[0036] iPSC Induced pluripotent stem cells

[0037] PET Positron Emission Tomography Scanner

[0038] PMOD Medical Analysis Software

[0039] %ID / g Percent Injected Dose per Gram of Tissue

[0040] SUV Standardized Uptake Value

[0041] Activity Meter An instrument for measuring the activity of radionuclides

[0042] μCi Microcurie, the former unit of radioactivity

[0043] ROI Refers to the term "Region of Interest" in image processing

[0044] RCP Radiochemical Purity

[0045] SUV Standardized Uptake Value

[0046] 89 Zr Zirconium-89

[0047] Main Materials and Reagents:

[0048] iPSC-CM: Provided by Nanjing Elpis Regenerative Medicine Technology Co., Ltd.;

[0049] Oxine: Purchased from Sigma Aldrich;

[0050] 89 Zr-oxalate: Purchased from Nanjing Andico;

[0051] 89 Zr-Cardiomyocytes: 89 Zr-labeled cardiomyocytes.

[0052] 2 cynomolgus monkeys, normally raised by Midu (Nanjing) Biotechnology Co., Ltd.; Experimental Animal Use License: SYXK (Su) 2017-0065.

[0053] 1 KM mouse and 1 NCG mouse, provided by Changzhou Cavens Experimental Animal Co., Ltd., and normally raised in the clean-grade environmental IVC system of the Experimental Animal Center of Jiangsu Institute of Atomic Medicine. All animal studies strictly comply with national laws and regulations, and all animal experiments have been approved by the Experimental Animal Management and Ethics Committee of Jiangsu Institute of Atomic Medicine.

[0054] Unless otherwise specified, the practice of the present invention will employ conventional techniques of cell biology, which are within the capabilities of those skilled in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art related to the present invention.

[0055] Example 1, Discrimination of Biological Markers

[0056] 1) Tissue Distribution Experiment on Cynomolgus Monkeys (Non-Human Primates)

[0057] Two normal cynomolgus monkeys, male, each with a unique animal identification number, denoted as G1-M-01 and G2-M-01 respectively. The grouping and dosing regimens of cynomolgus monkeys in this experiment are shown in Table 1 below:

[0058] Table 1

[0059]

[0060] 2) Tissue Distribution Experiment on Kunming (KM) Mice

[0061] One normal Kunming (KM) mouse, male, denoted as KM mouse. The dosing regimen of KM mouse in this experiment is shown in Table 2 below:

[0062] Table 2

[0063]

[0064] 3) Tissue Distribution Experiment on Immunodeficient Mice (NCG Mice)

[0065] One normal NCG mouse, male. The dosing regimen of NCG mouse in this experiment is shown in Table 3 below:

[0066] Table 3

[0067]

[0068] Example 2, Cell Resuscitation

[0069] (1) Take out the original cell solution to be resuscitated from the liquid nitrogen tank, including 3 groups of cardiomyocytes (iPSC-CM), with 8×10 8 cells in each group, and place them in dry ice for temporary storage; take out the 20% human albumin solution from the 4°C refrigerator and equilibrate it in the dark at 15 - 25°C for 10 minutes;

[0070] (2) Add cell solvent to the centrifuge tube. The cell solvent is prepared by mixing 0.9% sodium chloride injection and 20% human albumin in a ratio of 3:1, and mix well for later use;

[0071] (3) Take out the original cell solution from dry ice and put it into cryopreservation tubes respectively. Quickly immerse the lower 1 / 2 of the cryopreservation tube in a 37°C ± 1°C water bath, and quickly shake it for 1 - 2 minutes until there is only a small piece of ice left in the tube, then take out the cryopreservation tube;

[0072] (4) After wiping the surface of the cryopreservation tube with 75% alcohol, gently pipette about 5 times and transfer all the liquid in each cryopreservation tube into a 50 mL centrifuge tube respectively;

[0073] (5) Use a pipette to take 1 ml of cell solvent to rinse the cell cryopreservation tube, and slowly add the rinsing solution into the above-mentioned 50 ml centrifuge tube respectively;

[0074] (6) Use a pipette to slowly add 19 mL of cell resuspension solution, centrifuge at 300 g for 5 min at 15 - 25 °C;

[0075] (7) After centrifugation, aspirate the supernatant, resuspend with cell solvent and then count. Add cell solvent to dilute the cell stock solution to the required concentration, thus completing the administration preparation.

[0076] Example 3: Nuclide Labeling

[0077] Take 50 ul 89 Zr, and measure 633 uCi;

[0078] Adjust the pH = 7, add 200 ul of HEPES and 32.5 ul of Na2CO3,

[0079] The concentration of the HEPES is 0.1 mol / L, and the concentration of Na2CO3 is 1 mol / L;

[0080] Then add 5 uL of Oxine, and the concentration of the Oxine is 5 ug / uL, to obtain 89 Zr - Oxine, and measure 240 uCi.

[0081] Take out the 3 groups of myocardial cell resuscitation liquids in Example 1 respectively, and perform the following labeling on each group of cell resuscitation liquids:

[0082] Take respectively 89 Zr - Oxine into the 3 groups of 8×10 8 myocardial cells in Example 1, incubate at room temperature for 15 min; centrifuge at 300 g for 1 min.

[0083] Then resuspend with 5% human albumin, centrifuge at 300 g for 1 min, and centrifuge and wash 3 times.

[0084] Respectively prepare 89 Zr - oxine; and 3 groups of 89 Zr - myocardial cells.

[0085] The diluent for the test substance during administration is physiological saline containing 5% HSA.

[0086] 89 Zr - oxine is stored in dimethyl sulfoxide (DMSO), and the DMSO content during administration is ≤ 10%.

[0087] Example 4, Injection and Its PET Imaging

[0088] 89 Injection of 8 Zr - cardiomyocytes into the myocardium of cynomolgus monkeys (G1 - M - 01): The administration dose was 1×10

[0089] 89 cells per monkey, the injection volume was 1.25 mL, and the radioactive dose was 1.5 uCi. The first image was formed by PET scanning. 8 Injection of

[0090] 89 Zr - oxine into the myocardium of cynomolgus monkeys (G2 - M - 01): The administration dose was 1×10 6 cells per monkey, the injection volume was 1.25 mL, and the radioactive dose was 1.5 uCi. The second image was formed by PET scanning.

[0091] 89 Injection of 5 Zr - cardiomyocytes into the myocardium of KM mice: The administration dose was 2×10

[0092] cells per mouse, the injection volume was 50 μL, and the radioactive dose was 1 uCi. The third image was formed by PET scanning.

[0093] Injection of

[0094] Zr - cardiomyocytes into the myocardium of NCG mice: The administration dose was 5×10

[0095] cells per mouse, the injection volume was 200 μL, and the radioactive dose was 1 uCi. The fourth image was formed by PET scanning. The first image and the second image were respectively subjected to PET whole - body static scanning at 1.5 h, 2 h, 4 h, 8 h, 24 h, 2 d, 3 d, 8 d, 14 d, and 21 d after injection. The third image and the fourth image were subjected to PET imaging detection at 1 h, 4 h, 6 h, 16 h, 24 h, 40 h, 48 h, 72 h, 96 h, 120 h, 144 h, 168 h, and 192 h after injection. In principle, all the above scanning time points are consistent with the theoretical time. If there are deviations, the allowable deviation ranges are as follows: for the time points of 0 - 4 h (including 0 h and 4 h), the deviation is controlled within ±5 min; for the time points of 4 - 24 h, the deviation is controlled within ±10 min; for the time points of ≥24 h (including 24 h), the deviation is controlled within ±5%. It can be observed in real - time and the error is reduced. The reflection amount was detected using a γ - counter, and the cell tissue distribution was detected. In Example 4, the main tissue organs were outlined and the radioactive uptake values were obtained.After the PET scan is completed, image reconstruction is performed, and the PMOD software is used to process the images and data. Regions of interest (ROIs) are delineated. The ROIs of cynomolgus monkeys include organs such as the heart, liver, spleen, kidneys, whole brain, muscle, knee joint, bone marrow, and gonads; the ROIs of KM mice include the heart, lungs, liver, spleen, and bone marrow; the ROIs of NCG mice include the lungs, liver, bone marrow, and muscle. The radioactivity concentration (i.e., the radioactivity value per unit volume) of the regions of interest (ROIs) is obtained. The percentage injected dose per gram of tissue (abbreviated as %ID / g value) of each organ is calculated based on the administered dose, or the standardized uptake value (abbreviated as SUV value) of each organ is calculated based on the animal's body weight. The formulas are as follows:

[0096]

[0097] For the standardized uptake value (SUV), the method for determining SUV is as follows in detail:

[0098] Determine r, where r is the radioactivity concentration (μCi / g) obtained by performing image reconstruction after the PET scan in the region of interest (ROI) and processing the images and data using the PMOD software;

[0099] Determine s, where s is the administered dose (μCi) of the test substance injected;

[0100] Determine w, the body weight (g) of the organism; that is,

[0101]

[0102] Appendix Figure 2 Describes the standardized uptake value (SUV); Appendix Figure 3 and Appendix Figure 5 and Appendix Figure 7 All describe the percentage injected dose per gram of tissue (abbreviated as %ID / g value), and their theoretical meanings are the same.

[0103] Example 5. PET imaging results and radioactive uptake values

[0104] Verified labeled cardiomyocytes, 89 Zr-oxine was injected into cynomolgus monkeys to trace the in vivo distribution of cells Distribution

[0105] After G1 cynomolgus monkeys were injected with 89 Zr - cardiomyocytes, the radioactive substances were mainly distributed in the heart, liver, and spleen, followed by the lungs, kidneys, bone joints, bone marrow - femur, bone marrow - spine, and bone marrow - tibia, and then the testes and tibial cortical bone, with lower distribution in the brain and muscle.

[0106] After G2 cynomolgus monkeys were injected with 89After Zr-Oxine, the radioactive substances were mainly distributed in the heart, liver, spleen and kidneys, followed by the lungs and bone marrow - spine, and then by the bone joints and testes. The distribution in bone marrow - femur, bone marrow - tibia, tibial cortical bone, brain and muscle was relatively low. See attachment Figure 1

[0107] Judging from the radioactive uptake values and their changing trends, cynomolgus monkeys G1 and G2 were respectively injected with 89 Zr - cardiomyocytes, 89 After Zr-Oxine, there were differences in the radioactive uptake values of the liver, spleen, lungs, kidneys and bone joints: specifically as follows:

[0108] The SUV - mean values of the liver and spleen in cynomolgus monkey G1 were higher than those in G2, and the SUV - mean values of the liver and spleen in cynomolgus monkey G1 generally showed a trend of rising first and then falling, while the change in G2 was not obvious;

[0109] The SUV - mean value of the lungs in cynomolgus monkey G1 was higher than that in G2, and the SUV - mean value of the lungs in cynomolgus monkey G1 showed a gradually decreasing trend, while the change in G2 was not obvious;

[0110] The SUV - mean of the kidneys in cynomolgus monkey G1 was lower than that in G2, and the SUV - mean value of the kidneys in cynomolgus monkey G1 generally showed a trend of rising first and then falling, while that in G2 showed a gradually decreasing trend;

[0111] The SUV - mean of the bone joints of the lungs in cynomolgus monkey G1 was higher than that in G2, and both showed a trend of rising first and then falling. For details, see attachment Figure 2 。

[0112] By labeling the cardiomyocytes differentiated from induced pluripotent stem cells and injecting the labeled cardiomyocytes into cynomolgus monkeys, the distribution of the radioactive labeled signals in the animals after cell injection was detected with an animal in - vivo imager, and based on the PET scan images of the tissues, the in - vivo process of the cells was traced. Compared with single injection of 89 Zr - Oxine, it was shown that after injecting the labeled cardiomyocytes into the animals, the changing trend of the radioactive uptake value was relatively obvious.

[0113] Further verification 89 of the distribution of Zr - labeled cardiomyocytes injected into KM mice

[0114] Through attachment Figure 4 it can be known that KM mice were injected with 89 Zr - cardiomyocytes: Over time 89Zr-myocardial cells are transferred from the heart to parts such as the lungs, liver, and bones; the uptake in the heart gradually decreases and starts to slowly decrease after 40 h. The uptake in the liver first decreases and then the amount of decrease becomes smaller and tends to be stable after 24 h. The uptake in the lungs slowly decreases over time and the amount of decrease becomes smaller and tends to be stable after 48 h. The uptake in the bones gradually increases over time and the change amount becomes smaller after 144 h.

[0115] By attachment Figure 3 It can be seen that after KM mice are injected with 89 Zr-myocardial cells, the uptake in the animal heart is the highest. Over time 89 Zr-myocardial cells are gradually transferred from the heart to parts such as the lungs, liver, and bones. The uptake in the heart gradually decreases and starts to tend to be stable after 48 h. The uptake in the liver first decreases and then the amount of decrease becomes smaller and tends to be stable after 24 h; the uptake in the lungs slowly decreases over time and the amount of decrease becomes smaller and tends to be stable after 48 h. At 192 h, the uptake in the liver is about 5.1 times that in the lungs. The uptake in the bones gradually increases over time and the change amount becomes smaller after 144 h and reaches the peak at 192 h. At 192 h, the uptake in the bones is about 15.4 times that in the lungs and 3.0 times that in the liver.

[0116] The above results show that after these labeled myocardial cells are injected into animals, the changing trend of the radioactive uptake value is also relatively obvious. It can be traced in vivo and has normal biological activity, accurately reflecting the change process or distribution of these cells.

[0117] Further verification 9 Distribution of Zr-labeled cardiomyocytes injected into NCG mice

[0118] By attachment Figure 6 It can be seen that after NCG mice are injected with 89 Zr-myocardial cells: over time, myocardial cells are gradually transferred from the lungs to parts such as the liver and bones; the uptake in the lungs gradually decreases and starts to tend to be stable after 48 h. The uptake in the liver slowly increases over time and tends to be stable after 48 h. The uptake in the bones first increases and then starts to decrease after 72 h.

[0119] By attachment Figure 5 It can be seen that after NCG mice are injected with 89 Zr-myocardial cells for 1 h, the uptake in the animal lungs is the highest. Over time 89The Zr-myocardial cells gradually metastasize from the lungs to the liver and bone regions. The uptake in the lungs gradually decreases and begins to stabilize after 48 h. The liver uptake slowly increases with time, reaches a peak at 40 h, slightly decreases after 72 h, and starts to remain stable at 96 h; the liver uptake at 192 h is about 1.7 times that of the lungs. The uptake in the bone reaches a peak at 72 h and then gradually decreases until 192 h; the uptake in the bone at 192 h is about 3.1 times that of the lungs and 1.8 times that of the liver. Therefore, it can be shown that after injecting such labeled myocardial cells into animals, the changing trend of the radioactive uptake value is also relatively obvious, and it can accurately trace the time-dependent distribution of the labeled cells in vivo.

[0120] Example 6. Cellular tissue distribution (KM mice)

[0121] Further verify the accuracy of tissue radioactivity uptake values in the in-vivo state

[0122] The mice were sacrificed immediately after the 192-h imaging. Hearts, livers, spleens, lungs, and joints of KM mice were taken, and the reflected amounts were detected using a γ counter to detect the cellular tissue distribution. Biological distribution: Detection of each organ in vitro; Contouring data: PET in vivo detection. Refer to the appendix for details. Figure 7 .

[0123] KM mice were injected with 89 The Zr-myocardial cells were sacrificed after the 192-h imaging. Hearts, livers, spleens, lungs, and joints were immediately dissected, and the tissue uptake was detected. During in vivo detection, the uptake value of the joints was the largest, followed by the heart and liver; during the in vitro detection of each organ, the uptake value of the joints was also the largest, followed by the heart and liver. Therefore, there is a match between the in vitro detection and in vivo detection of each organ.

[0124] The PET contouring uptake value is about 2 times that of the in vitro heart uptake value. There is no significant difference between the PET contouring uptake value and the in vitro liver uptake value. The in vitro spleen uptake value is about 5 times that of the PET contouring uptake value. The in vitro lung uptake value is about 2 times that of the PET contouring uptake value. There is no significant difference between the PET contouring uptake value and the in vitro joint uptake value. See the appendix. Figure 7 .

[0125] In summary, the present invention has realized research on the early distribution of cells, efficacy prediction, visual detection, etc. Monitoring the in vivo distribution law of human iPSC-derived myocardial cells and establishing a relationship between the dynamic distribution after the cell drug enters the body and the cell efficacy will effectively improve the clinical efficacy of the cell drug, reduce risks, and reduce drug interactions. Radioisotope labeling is a stable in vivo tracer method, combined with in vivo PET imaging analysis, which provides an effective role for monitoring the in vivo distribution of cells.

[0126] Although the present invention has been disclosed above in preferred embodiments, it is not intended to limit the present invention. Those of ordinary skill in the art to which the present invention pertains can make various modifications and refinements without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the claims.

Claims

1. A method for tracing the pharmacokinetics of cardiomyocytes differentiated from induced pluripotent stem cells, characterized in that: Prepare the test substance: Take 89 Zr reacts with oxine to obtain 89 Zr-oxine; and take out 89 Zr-oxine is respectively placed in the resuscitated cardiomyocytes (iPSC-CM), centrifuged after incubation for 15 min, and then resuspended with 5% human albumin and centrifuged and washed 3 times; Administer a test substance to an organism; By ingestion 89 Zr-oxine and 89 Zr-labeled cardiomyocytes are detected and traced by positron emission tomography.

2. The method for tracing cardiomyocytes differentiated from induced pluripotent stem cells in pharmacokinetics according to claim 1, wherein Preparing the test substance specifically includes: S1. Prepare a cell solvent by mixing 0.9% sodium chloride injection and 20% human albumin. Flush the cell cryopreservation tube with the cell solvent, add the cardiomyocyte resuspension and centrifuge, and then add the cell solvent to dilute the cell stock solution for standby; the cell stock solution refers to the cardiomyocytes after resuscitation; S2. Take 89 React Zr with oxine to obtain 89 Zr-oxine; and take out 89 Place Zr-oxine into the revived cardiomyocytes (iPSC-CM) respectively. After incubation for 15 min, centrifuge, and then resuspend with 5% human albumin, and wash by centrifugation 3 times.

3. The tracing method of cardiomyocytes differentiated from induced pluripotent stem cells in pharmacokinetics according to claim 2, characterized in that, The specific ratio of sodium chloride injection to human albumin is 3:

1.

4. A method for tracing cardiomyocytes differentiated from induced pluripotent stem cells in pharmacokinetics according to claim 1, characterized in that, Administering the test substance to the organism includes: injecting and detecting: injecting the test substance into the organism respectively; monitoring and delineating the PET imaging and standard uptake value in the body at different time points.

5. A method for tracing cardiomyocytes differentiated from induced pluripotent stem cells in pharmacokinetics, as described in claim 1, characterized in that Preparation 89 Before Zr-oxine, pH adjustment is also included. Take 89 Zr, add HEPES and Na2CO3, and adjust the pH value to 7.

6. The tracing method of cardiomyocytes differentiated from induced pluripotent stem cells in pharmacokinetics according to claim 1, wherein The organism is a non-human mammal; the non-human mammal can be a mouse, a monkey, a cow, a sheep or a dog.

7. A method for tracing cardiomyocytes differentiated from induced pluripotent stem cells in pharmacokinetics according to claim 1, characterized in that, The radiochemical purity (RCP) of the test substance is not less than 90%.

8. The tracing method of cardiomyocytes differentiated from induced pluripotent stem cells in pharmacokinetics according to claim 1, characterized in that When administering the drug, the test substance diluent is physiological saline containing 5% HSA.

Images