Application of baricitinib in static cold preservation of isolated heart and / or heart organoid

Abstract

The invention belongs to the technical field of heart transplantation, and particularly relates to application of baricitinib in static cold preservation of an isolated heart and / or heart organoid. The baricitinib can be used as a mitochondrial protective agent to improve the mitochondrial structure and delay mitochondrial damage, so that the static cold preservation time of the isolated heart and / or heart organs is prolonged. The result of the embodiment shows that when 0.1 mu M of baricitinib is used in the freezing model of the heart organoid, the contraction function of the heart organoid can be relieved. Meanwhile, 0.1 mu M of baricitinib is used in human primary heart tissue and mouse heart and is subjected to cold preservation, and it is found that by adding the baricitinib, the heart function can be replenished, and mitochondrial injury can be delayed. Therefore, the baricitinib can prolong the cold preservation time in the heart transplantation process, and a foundation is laid for prolonging the heart cold transfer time in heart transplantation.

Description

Technical Field

[0001] The present invention belongs to the technical field of cardiac transplantation, and specifically relates to the application of baricitinib in static cold storage of isolated hearts and / or cardiac organoids. Background Art

[0002] Heart transplantation is the last surgical treatment for heart failure caused by a variety of diseases. However, insufficient donor hearts and maintenance of function after transplantation have always been bottlenecks restricting the development of the field. At present, static cold storage (SCS) is used for conventional donor heart transportation in China. The cold storage time during the transportation process is directly related to the survival time of the recipient patient after transplantation. However, too long static cold storage time can cause heart damage and greatly reduce the survival rate of heart transplant patients. Therefore, the development of protective agents that prolong the cold storage time during heart transplantation is the key to solving the short transplant window period. At present, there is an urgent need for an agent that can prolong the static cold storage time of the heart. Summary of the invention

[0003] The object of the present invention is to provide the use of baricitinib in static cold storage of isolated heart and / or heart organoids, wherein baricitinib can increase the static cold storage time of isolated heart and / or heart organoids.

[0004] The present invention provides use of baricitinib in static cold storage of isolated hearts and / or cardiac organoids.

[0005] The present invention also provides the use of baricitinib in prolonging the static cold storage time of an isolated heart and / or a heart organoid.

[0006] As a preferred embodiment, the effective concentration of baricitinib is 0.1-10 μM.

[0007] As a preferred embodiment, the application includes at least one of delaying myocardial cell mitochondrial damage, improving myocardial cell calcium conduction function, reducing myocardial cell oxidative stress ability and improving cardiac contractile function.

[0008] As a preferred embodiment, the delaying of myocardial cell mitochondrial damage includes improving the myocardial cell mitochondrial structure.

[0009] As a preferred embodiment, the reducing the oxidative stress capacity of cardiomyocytes includes reducing the level of reactive oxygen species in cardiomyocytes.

[0010] As a preferred embodiment, the construction of the cardiac organoid comprises the following steps: inducing the human pluripotent stem cells or human embryonic stem cells to differentiate into cardiomyocytes; digesting the cardiomyocytes into single cells; mixing the single cells with a hydrogel system to cultivate cardiac organoids;

[0011] The hydrogel system comprises fibrinogen, matrix gel and thrombin; the volume ratio of the fibrinogen, matrix gel and thrombin is 10:5:1.

[0012] As a preferred embodiment, when the human pluripotent stem cells or human embryonic stem cells are induced to differentiate into cardiomyocytes, the method comprises: when the confluence of the human pluripotent stem cells or human embryonic stem cells is 80%, inducing differentiation to obtain cardiomyocytes;

[0013] On the 0th day of differentiation induction, the first differentiation medium was added for culturing; on the 1st day of differentiation induction, the differentiation medium was added for culturing; on the 3rd day of differentiation induction, the second differentiation medium was added for culturing; on the 5th day of differentiation induction, the differentiation medium was added for culturing; after the 5th day of differentiation induction, the differentiation medium was replaced every 2 days;

[0014] The differentiation medium is based on 1640RPMI as the basic medium and also contains 50×B27 -INS and 0.4 mg / mL L-ascorbic acid-2-phosphate magnesium salt hydrate;

[0015] The first differentiation medium is a differentiation medium as a basal medium and further contains a Wnt signaling pathway inhibitor, CHIR99021;

[0016] The second differentiation medium is based on the differentiation medium as a basal medium and further contains IWR-1, an inhibitor of the Wnt signaling pathway.

[0017] As a preferred embodiment, the final concentration of CHIR99021 is 2.5-12.5 μM; the final concentration of IWR-1 is 5-10 μM.

[0018] As a preferred embodiment, after the cardiac organoids are obtained by culturing, the method further comprises: demolding the cardiac organoids and placing them in a 12-well plate; adding a first early culture medium for culturing on the first day after demolding; adding an early culture medium for culturing on the second day after demolding, and replacing the culture medium every 2 days; adding a late culture medium for culturing on the seventh day after demolding, and replacing the culture medium every 2 days;

[0019] The early culture medium is based on 1640RPMI as the basic culture medium and also contains 50×B27 -INS , 0.4 mg / mL L-ascorbic acid-2-phosphate magnesium salt hydrate, 2 mg / mL 6-aminohexanoic acid, 0.45 μM 1-thioglycerol, 1% 100× non-essential amino acids and 1% 100× sodium pyruvate; the first early culture medium is based on the early culture medium and also contains 10 μM Y-27632 and 10 μM 5-bromo-2'-deoxyuridine;

[0020] The late stage culture medium is based on low-glucose DMEM and further contains 2% fetal bovine serum, 0.4 mg / mL L-ascorbic acid-2-phosphate magnesium salt hydrate, 2 mg / mL 6-aminohexanoic acid, 0.45 μM 1-thioglycerol and 1% 100× non-essential amino acids.

[0021] Beneficial effects: The present invention provides the use of baricitinib in static cold storage of isolated hearts and / or heart organoids. Baricitinib can be used as a mitochondrial protectant to improve mitochondrial structure, delay mitochondrial damage, and thus prolong the static cold storage time of isolated hearts and / or heart organoids. The results of the embodiment show that when 0.1 μM Baricitinib is used in the frozen model of heart organoids, the drug-added group can alleviate the contractile function of the heart organoids compared with the untreated group. At the same time, in human primary heart tissue and mouse heart, 0.1 μM Baricitinib was used and cold stored, and it was found that the addition of Baricitinib can restore heart function and delay mitochondrial damage. It can be seen that Baricitinib can prolong the cold storage time during heart transplantation, laying the foundation for prolonging the cold transport time of donor hearts in heart transplantation. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0023] Figure 1 The survival rate and transcriptome analysis diagram under static cold storage in Example 1; A is a static cold storage time and survival rate diagram; B is a biological pathway enrichment diagram of transcriptome biological pathway analysis; C is a heat map of genes related to fatty acid metabolism and NADH metabolism;

[0024] Figure 2 The diagrams of mitochondrial damage of myocardial tissue under static cold storage in Example 1; A is a diagram of mitochondrial structure at different times of static cold storage; B is a diagram of mitochondrial structural damage ratio;

[0025] Figure 3 The electrocardiograms of rat hearts treated differently in Example 2; A is a representative electrocardiogram under different treatments; B is the frequency of heartbeats in the electrocardiogram; C is the PR interval in the electrocardiogram; D is the QRS complex in the electrocardiogram; E is the QT interval in the electrocardiogram; F is the VVD ventricular vector deviation;

[0026] Figure 4 Figures are the hearts of rats treated differently in Example 2; A is a representative pseudo-color image of the left ventricular electrical conduction process in different groups; B is a diagram of left ventricular conduction velocity and conduction dispersion; C is a representative pressure waveform diagram of different groups; D is a diagram of left ventricular pressure signal; E is a diagram of the hearts of mice treated differently;

[0027] Figure 5 Flow chart for static cryopreservation of cardiac organoids;

[0028] Figure 6 : ROS and calcium transient detection diagram in Example 6; A is a representative diagram of cellRox (detecting mitochondrial reactive oxygen levels) and mitotracker (detecting mitochondrial membrane potential levels, Mito) in different groups and their ratio statistics; B is a representative calcium transient time-lapse fluorescence imaging diagram in different groups and its corresponding waveform diagram; C is a calcium transient parameter statistics diagram;

[0029] Figure 7 It is a diagram of the contractile ability of the cardiac organoids in Example 7; wherein A is a representative waveform diagram of the contractile force of different groups under different stretching degrees; and B is a statistical diagram of the active contractile force and passive contractile force of the cardiac organoids. DETAILED DESCRIPTION

[0030] The present invention provides use of baricitinib in static cold storage of isolated hearts and / or cardiac organoids.

[0031] The present invention also provides the use of baricitinib in prolonging the static cold storage time of an isolated heart and / or a heart organoid.

[0032] As an embodiment, the effective concentration of baricitinib in the present invention is 0.1 to 10 μM. As an embodiment, the effective concentration of baricitinib can be any value in the range of 0.1 to 10 μM, such as 0.1 μM, 0.5 μM, 1 μM, 5 μM or 10 μM; in a specific embodiment of the present invention, the effective concentration of baricitinib is 0.1 μM.

[0033] As an embodiment, the application includes at least one of delaying myocardial cell mitochondrial damage, improving myocardial cell calcium conduction function, reducing myocardial cell oxidative stress capacity and improving cardiac contractile function.

[0034] As an embodiment, the delaying of myocardial cell mitochondrial damage includes improving myocardial cell mitochondrial structure. As an embodiment, the reducing myocardial cell oxidative stress capacity includes reducing myocardial cell reactive oxygen levels.

[0035] The cardiac organoids of the present invention include myocardial organoids. As an embodiment, the construction of the cardiac organoids includes the following steps: inducing the differentiation of the human pluripotent stem cells or human embryonic stem cells into myocardial cells; digesting the myocardial cells into single cells; mixing the single cells with a hydrogel system to cultivate cardiac organoids.

[0036] In a specific embodiment of the present invention, the human induced pluripotent stem cell line WTC cells are inoculated in a six-well plate, and on the first day of inoculation, mTeSR1 medium containing 10 μM Y27632 is added for maintenance. As an embodiment, when the confluence of the human pluripotent stem cells or human embryonic stem cells is 80%, differentiation medium is used to induce differentiation to obtain cardiomyocytes. The Y27632 described in the present invention is a Rho-associated protein kinase (ROCK) inhibitor that can inhibit the cell apoptosis pathway, thereby increasing the number of surviving cells and improving the efficiency of clone formation. The present invention does not specifically limit the source of the Y27632, and it can be purchased through conventional purchasing channels in the art. In a specific embodiment of the present invention, the Y27632 is purchased from Sigma (A8960).

[0037] As an embodiment, on the 0th day of induction of differentiation, a first differentiation medium is added for culturing; on the 1st day of induction of differentiation, a differentiation medium is added for culturing; on the 3rd day of induction of differentiation, a second differentiation medium is added for culturing; on the 5th day of induction of differentiation, a differentiation medium is added for culturing; after the 5th day of induction of differentiation, the differentiation medium is replaced every 2 days. As an embodiment, the differentiation medium is based on 1640RPMI as the basic medium, containing 50×B27 -INS and 0.4 mg / mL L-ascorbic acid-2-phosphate magnesium salt hydrate. In the present invention, the B27 -INS It is a serum analogue that can be used to replace serum, supplement the growth factors, hormones, neurotrophic factors, vitamins and minerals required in the differentiation process, and can simulate the in vivo environment. -INS The source of B27 is not particularly limited and can be purchased through conventional purchasing channels in the art. -INSPurchased from Gibco (A1895601). As an embodiment, the first differentiation medium uses differentiation medium as a basal medium, and further contains Wnt signaling pathway inhibitor CHIR99021; the final concentration of the CHIR99021 is 2.5 to 12.5 μM. In a specific embodiment of the present invention, the final concentration of the CHIR99021 can be any value between 2.5 and 12.5 μM, such as 2.5 μM, 5 μM, 7.5 μM, 10 μM or 12.5 μM. As an embodiment, the second differentiation medium uses differentiation medium as a basal medium, and further contains Wnt signaling pathway inhibitor IWR-1; the final concentration of the IWR-1 is 5 to 10 μM. In a specific embodiment of the present invention, the final concentration of the IWR-1 can be any value between 5 and 10 μM, such as 5 μM, 6 μM or 10 μM. The Wnt signaling pathway inhibitor CHIR99021 described in the present invention can promote the differentiation of stem cells into mesodermal cells; the Wnt signaling pathway inhibitor IWR-1 can promote the differentiation of mesodermal cells into cardiac progenitor cells.

[0038] The present invention digests the cardiomyocytes into single cells; as an embodiment, type I collagenase is added to the cardiomyocytes and placed at 37°C, 5% CO 2 The cells were digested in an incubator for 1 hour. The cardiomyocytes after digestion were collected, the upper layer of type I collagenase was removed, and high-glucose EMEM culture medium was added to dilute and terminate the type I collagenase digestion. The high-glucose EMEM culture medium was removed by centrifugation, and trypsin was added. The cells were shaken in a 37°C water bath to obtain single cells.

[0039] The present invention mixes the single cell with a hydrogel system to cultivate a cardiac organoid; as an embodiment, the hydrogel system includes fibrinogen, matrigel and thrombin; the volume ratio of the fibrinogen, matrigel and thrombin is 10:5:1. The fibrinase and matrigel in the hydrogel system of the present invention can be used as a skeleton structure between cardiomyocytes to construct a cardiac organoid.

[0040] After the cardiac organoids are obtained by the cultivation, the present invention further comprises: demolding the cardiac organoids and placing them in a 12-well plate; adding a first early culture medium for culturing on the first day after demolding; adding an early culture medium for culturing on the second day after demolding, and replacing the culture medium every 2 days; adding a late culture medium for culturing on the seventh day after demolding, and replacing the culture medium every 2 days; as an embodiment, the early culture medium is based on 1640RPMI as a basic culture medium and also contains 50×B27 -INS, 0.4 mg / mL L-ascorbic acid-2-phosphate magnesium salt hydrate (Ac-Mg), 2 mg / mL 6-aminocaproic acid, 0.45 μM 1-thioglycerol, 1% 100× non-essential amino acids and 1% 100× sodium pyruvate; the addition of Ac-Mg and 1-thioglycerol to the early culture medium of the present invention has an antioxidant effect, can reduce the degree of cell oxidative damage, and improve cell survival rate; 6-aminocaproic acid can prevent the degradation of fibrin, maintain the stability of the cell culture environment, and help maintain the normal morphology and function of the cells; non-essential amino acids can reduce the metabolic burden of cells, improve cell growth and activity, and maintain the balance of the culture medium. The first early culture medium is based on the early culture medium, and also contains 10μM Y-27632 and 10μM 5-bromo-2'-deoxyuridine (BrdU); the late culture medium is based on low-glucose DMEM, and also contains 2% fetal bovine serum, 0.4mg / mL Ac-Mg, 2mg / mL 6-aminocaproic acid, 0.45μM 1-thioglycerol and 1% 100× non-essential amino acids. The addition of fetal bovine serum to the late culture medium of the present invention can provide rich nutrients and growth factors, thereby promoting the maturation of cardiomyocytes. The 5-bromo-2'-deoxyuridine (BarU) of the present invention is a nucleoside analog that can be incorporated into the newly synthesized DNA chain during DNA synthesis, providing a marker for detecting cell proliferation.

[0041] To further illustrate the present invention, the application of baricitinib provided by the present invention in static cold storage of isolated hearts and / or cardiac organoids is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0042] Example 1

[0043] Statistics on static cold storage time and patient survival rate of heart transplant surgery in the United States, and statistics on static cold storage time and patient survival rate of heart transplant surgery in Wuhan Union Hospital, such as Figure 1 As shown in A. The results showed that the survival rate of patients decreased with the extension of cold storage time. To further explore the biological events that occurred during static cold storage of the heart, positron emission tomography-computed tomography (PET-CT) was performed on the recipient heart to evaluate the cold storage of the recipient heart tissue and the sample processing method.

[0044] 1) Heart scan: A PET-CT scan of the recipient's heart is performed to assess cardiac metabolism.

[0045] 2) Tissue collection: Based on the scan results, myocardial tissue with normal metabolism was collected and immersed in UW organ preservation solution (Bridgetolife, catalog number: CHD051623), and Baricitinib was added at a final concentration of 0.1 μM.

[0046] 3) Cold preservation: The myocardial tissue immersed in UW organ preservation solution in step 2) was cold preserved in a 4°C refrigerator, and the time points were set as 0h, 6h and 8h, which were recorded as SCS-0h, SCS-6h and SCS-8h respectively.

[0047] 4) Sample extraction: After cold storage, myocardial tissue is removed to collect RNA samples and electron microscopy samples.

[0048] 5) RNA sample processing: The myocardial tissue was cut into small pieces, and the surface liquid was absorbed with absorbent paper. After being wiped dry, the pieces were placed in a -80°C refrigerator for freeze-drying, and then sent to Shanghai Ouyi Biomedical Technology Co., Ltd. for RNA extraction and subsequent processing.

[0049] 6) Electron microscopy sample processing: The electron microscopy samples were immersed in glutaraldehyde with a mass volume concentration of 2.5% and stored in a refrigerator at 4°C.

[0050] 7) Subsequent analysis: The RNA samples and electron microscopy samples in steps 5) and 6) were sent to Shanghai Ouyi Biomedical Technology Co., Ltd. for transcriptome analysis and electron microscopy photography. The transcriptome analysis results are as follows: Figure 1 As shown in B and C, the electron microscopy results are as follows Figure 2 As shown, Figure 2 The middle arrow points to the necrotic mitochondria in the cardiomyocytes. According to the transcriptome biological pathway analysis, some biological pathways enriched in static cold storage for 8 hours compared with static cold storage for 0 hours were mainly related to mitochondrial metabolism and temperature homeostasis ( Figure 1 B), and based on the enriched biological pathways, we selected genes related to fatty acid metabolism and NADH metabolism, and found that the expression of related genes decreased after static cold storage for 8 hours ( Figure 1 C). Combined Figure 2 It can be seen that cold storage can cause mitochondrial damage in cardiomyocytes, and the degree of mitochondrial damage in cardiomyocytes increases with the extension of cold storage time.

[0051] Example 2 In vitro perfusion and static cold storage of rat hearts and detection of rat cardiac function

[0052] 1) Heart sampling and connection: The isolated rat heart was obtained, and the aorta was connected to the perfusion needle and fixed with an aortic clamp and surgical thread.

[0053] 2) Perfusion preparation: Use oxygen-enriched (95% O 2 , 5% CO 2) was perfused with KH solution at 37°C constant temperature and flow. After the residual blood was drained, about 10 mL of pre-cooled UW solution was injected. The components of the KH solution are: 119 mMol / L NaCl, 4 mMol / L KCl, 1 mMol / L MgCl 2 6H 2 O, 25 mMol / L NaHCO 3 , 10mMol / L D-glucose, 1.2mMol / LKH 2 PO 4 and 1.8 mMol / L CaCl 2 .

[0054] 3) Sample preservation: The heart and perfusion needle in step 2) were placed in a centrifuge tube filled with UW organ preservation solution (Bridgetolife, catalog number: CHD051623), and randomly divided into 3 groups. One group was cold-preserved in a 4°C refrigerator for 6 h, recorded as SCS-6h; one group was cold-preserved in a 4°C refrigerator for 8 h, recorded as SCS-8h; and one group was added with 0.1 μM final concentration of Baricitinib in UW organ preservation solution and cold-preserved in a 4°C refrigerator for 8 h, recorded as SCS-8h-Bari.

[0055] 4) Flushing step: After the cold storage is completed, use a syringe to slowly push oxygen-rich KH solution at room temperature (25°C) to flush the UW organ preservation solution until the heart begins to peristalsis.

[0056] 5) Low-pressure perfusion: Transfer the heart to the low-pressure perfusion system and continue to perfuse oxygen-rich room-temperature KH solution for 15 minutes.

[0057] 6) Constant pressure perfusion and rewarming: The heart is then transferred to the constant pressure perfusion system for trapezoidal rewarming and perfusion recovery.

[0058] 7) Electrode installation: Place the ECG electrodes in the right atrium and left ventricle of the heart, attach two 64-channel pen electrodes to the left atrium and left ventricle, and place the stimulation electrode at the apex of the heart.

[0059] 8) Signal recording: spontaneous and frequency stimulation, S1S2 field potential signals and ECG signals and electrical mapping detection were recorded. The results are shown in Table 1 and Figure 3 Shown and Figure 4 As shown in A and B. Among them, Figure 3A is a representative electrocardiogram under different treatments; HR is the heart rate; PR is the PR interval, which refers to the time from the start of the P wave to the start of the QRS wave, reflecting the electrical conduction time from the atrium to the ventricle; QRS is the QRS complex, which usually indicates the process of ventricular depolarization; QT is the QT interval, the time interval from the start of the QRS wave to the end of the T wave, reflecting the total time of ventricular depolarization and repolarization; VVD is the ventricular vector deviation, which can be used to describe the vector deviation phenomenon of the ventricular part of the electrocardiogram; Figure 4 A is a representative pseudo-color image of the left ventricular conduction velocity of the rat heart detected by electrical mapping, and B is a statistical graph of the left ventricular conduction velocity and conduction dispersion of the rat heart.

[0060] 9) Pressure signal measurement: After the electrical signal acquisition is completed, the balloon is inserted into the left ventricular cavity, 0.1 mL of liquid is pushed in until the balloon is filled, and the left ventricular pressure signal is recorded; LVSbp is defined as the value of the highest point of the recorded left ventricular pressure waveform, and LVDbp is defined as the value of the lowest point of the recorded left ventricular pressure waveform; the pressure difference is defined as the difference between the highest point and the lowest point of the recorded left ventricular pressure waveform. The results are shown in Table 1 and Figure 4 As shown in C and D; rat hearts before (Control) and after (Baricitinib) addition. Figure 4 As shown in E.

[0061] Table 1 ECG and pressure values ​​of rat hearts treated with different methods

[0062]

[0063] The heart rate of normal rats at rest is 250 to 300 beats per minute. According to the experimental results, the heart rate of rats decreased after cold storage, indicating that their blood supply capacity decreased, which was replenished after drug addition (Table 1 and Figure 3 Middle B); The QRS time of rat hearts stored cold for 8 hours was prolonged, and its prolongation would lead to intraventricular conduction block, which would increase the risk of arrhythmia, while the QRS time was shortened after drug addition (Table 1 and Figure 3 The main function of the left ventricle of the heart is to pump blood from the heart to the whole body to supply oxygen and nutrition. According to the experimental results, the conduction velocity of the left ventricle of the rat heart decreased significantly after cold storage, and its conduction dispersion increased (Table 1 and Figure 4 B) in the figure shows that the left ventricular conduction function of rats decreased after cold storage, and the risk of arrhythmia increased due to increased dispersion, which could be significantly improved after adding the drug. It can be seen that adding baricitinib during cold storage of rat hearts can save the electrophysiological function, conduction function and contraction function of rat hearts.

[0064] Example 3

[0065] A. Human induced pluripotent stem cell line (hi-PSC) WTC differentiation into cardiomyocytes

[0066] 1) Cell seeding: Human induced pluripotent stem cell line (hi-PSC) WTC was seeded in a six-well plate pre-coated with Matrigel, and mTeSR1 culture medium (Stem Cell, catalog number: 85850) was used for cell maintenance.

[0067] 2) Anti-apoptosis treatment: On the first day of inoculation, Y-27632 was added to the culture medium to inhibit cell apoptosis. The final concentration of Y-27632 was 10 μM.

[0068] 3) Cell confluence monitoring: When the cell confluence reaches 80%, the initial differentiation of cells begins.

[0069] 4) Initial differentiation medium: After the initial differentiation, the cells were cultured in a differentiation medium, which used 1640 RPMI as a basal medium and contained 50×B27-INS and 50 mg / mL L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate.

[0070] 5) Directed differentiation day 0: Add differentiation medium containing Wnt signaling pathway inhibitor CHIR99021 (Stem cell, catalog number: 72054) for culture. The CHIR99021 can differentiate stem cells into mesoderm cells. The final concentration is 7.5 μM.

[0071] 6) Directed differentiation day 1: Culture the cells using differentiation medium without other components.

[0072] 7) Directed differentiation day 3: Add differentiation medium containing Wnt signaling pathway inhibitor IWR-1 (Stem cell, catalog number: 72564) for culture. The IWR-1 can differentiate mesoderm cells into cardiac progenitor cells. The final concentration is 5 μM.

[0073] 8) Day 5 of directed differentiation: Culture the cells using differentiation medium without other components.

[0074] 9) Subsequent culture: During the subsequent culture process, the differentiation medium without other components was replaced every 2 days.

[0075] B. Planar cell resurfacing

[0076] 1) Cell detachment: Type I collagenase was added to the cell culture plate of the cardiomyocytes obtained in Example 3 and placed at 37°C and 5% CO 2 The cells were cultured in an incubator for 1 h to allow the cells to detach from the culture plate.

[0077] 2) Cell collection and termination of digestion: The detached cells were collected into a 15 mL centrifuge tube, the type I collagenase on the upper layer was removed, and 10 mL of high-glucose DMEM was added to dilute and terminate the type I collagenase digestion.

[0078] 3) Cell digestion: After centrifugation, add 1 mL of trypsin and place the centrifuge tube in a 37°C water bath and shake for about 4 minutes to digest the cells into single cells.

[0079] 4) Termination of digestion: After the trypsin digestion is completed, a stop solution is added to terminate the digestion. The volume ratio of the stop solution components is high-glucose DMEM: fetal bovine serum (FBS): trypsin = 1:1:1, and 5 μg / mL of DNase I is added.

[0080] 5) Cell counting: Perform cell counting and calculate the total amount of cells needed to prepare cardiac organoids.

[0081] Example 4 Planar cardiomyocyte culture

[0082] 1) Planar cell seeding: After the cardiomyocytes obtained in step B of Example 3 are counted, cells are seeded on the pre-laid confocal dishes for mitochondrial oxidative stress assay, with 0.25 million cells seeded in each dish.

[0083] 2) Medium replacement: On the first day of inoculation, use a 1:1 medium containing Y-27632 (10 μM) and 5-bromo-2'-deoxyuridine (BrdU, 10 μM); on the second day, change to a 1:1 medium, and then change the 1:1 medium every two days.

[0084] The 1:1 culture medium is a mixture of a differentiation culture medium and a screening culture medium in a volume ratio of 1:1. The screening culture medium is based on sugar-free DMEM and further contains 50 mg / mL L-Ascorbic acid 2-phosphatesesquimagnesium salthydrate and 4 μM lactic acid.

[0085] Example 5 Construction of cardiac organoids

[0086] 1) Cell preparation: Use the single cells obtained by digestion in Example 3. The amount of cells required for each cardiac organoid is 0.5 Milligrams.

[0087] 2) Preparation of hydrogel system:

[0088] ①. Prepare a large sample of liquid A: The components of liquid A are 48 μL 1× culture medium and 2 μL thrombin. The components of the 1× culture medium are: Low Glucose DMEM (Gibco, catalog number: 10567014; 8.5 mL) + 10% FBS (volume concentration; 1 mL) + 1% PS (volume concentration; 100 μL) + 2 μg / mL V-B12 vitamin (4 mg / mL; 5 μL) + 1 mg / mL 6AA aminoacetic acid (50 mg / mL; 200 μL).

[0089] ②. Prepare a large sample of liquid B: The components of liquid B are 20 μL 2× culture medium, 10 μL Matrigel and 20 μL fibrinogen. The components of the 2× culture medium are: Low Glucose DMEM (7.5 mL) + 20% FBS (volume concentration; 2 mL) + 1% PS (volume concentration; 100 μL) + 4 μg / mL VB12 (4 mg / mL; 10 uL) + 2 mg / mL 6AA (50 mg / mL; 400 μL).

[0090] ③. Liquid mixing: Take out 51 μL (1 μL more to prevent loss of sample volume) of liquid A in step ① and dispense it into 1.5 mL EP tubes until liquid A is dispensed; then take out 50 μL of liquid B in step ②, mix liquid A and liquid B in a volume ratio of 50 μL:50 μL to obtain a mixed liquid, i.e., the hydrogel system.

[0091] Add to the grooves of the given membrane, add 50 μL of the mixture to each groove.

[0092] 3) Reaction and solidification: Add the hydrogel system in step 2) into the grooves of the given membrane, add 50 μL of the mixed solution to each groove, and place the membrane at 37°C and 5% CO 2 Incubate in an incubator for about 20 minutes to allow thrombin and fibrinogen to react enzymatically and coagulate.

[0093] 4) Demolding the cardiac organoids: Demolding the solidified cardiac organoids and placing them in a 12-well plate.

[0094] 5) Early culture: On the first day after preparation, add early culture medium containing Y-27632 (10 μM) and BrdU (10 μM) and incubate at 37°C, 5% CO 2Incubator. After the second day, early culture medium without other components was used for culture, and the culture medium was replaced every 2 days. The early culture medium is based on 1640 culture medium, containing B27 additive (minus insulin) (B27 (-Ins)), 0.4 mg / mL Ac-Mg, 2 mg / mL 6-aminocaproic acid (6-Aminocaproic Acid), 0.45 μM 1-thioglycerol (1-thioglycerol), 1% (v / v) 100× non-essential amino acids (Non-EssentialAminoAcid) and 1% (v / v) 100X sodium pyruvate (SodiumPyruvate).

[0095] 6) Late culture: Seven days after preparation, add late culture medium for culture, and replace the late culture medium every 2 days.

[0096] The late stage culture medium is based on low-glucose DMEM, containing 2% FBS, 0.4 mg / mL Ac-Mg, 2 mg / mL 6-Aminocaproic Acid, 0.45 μM 1-thioglycerol and 1% 100XNon-EssentialAminoAcid.

[0097] Example 6 Live cell staining of planar cardiomyocytes to detect ROS and calcium transients

[0098] A. Static cryopreservation of planar cardiomyocytes / cardiac organoids

[0099] 1) Culture medium replacement: After the cells in Example 4 were inoculated and cultured for 7 days, they were randomly divided into three groups, one of which was stored at 37°C and recorded as the Control group; one was stored at 4°C and recorded as SCS-8h; and one was added with Baricitinib and stored at 4°C and recorded as SCS-8h-Bari. Before cold storage, UW organ preservation solution was used. Baricitinib was added to the group that needed to be treated at a concentration of 0.1 μM.

[0100] 2) Preparation for hypoxia treatment: Place the cell plate to be stored in cold water in the hypoxia chamber and open the cover of the cell plate.

[0101] 3) Sealing the anoxic chamber: Seal the lid of the anoxic chamber and connect pure nitrogen gas to the specific air inlet of the anoxic chamber for oxygen removal.

[0102] 4) Oxygen removal: Oxygen was removed at a certain flow rate for 5 minutes, while the outlet of the anoxic chamber was kept open to remove air.

[0103] 5) Turn off the gas flow: After the oxygen is exhausted, close the nitrogen and oxygen-deficient chamber inlet and outlet in sequence.

[0104] 6) Static cold storage: Place the hypoxic chamber at 4℃±0.5℃ for static cold storage. The cold storage diagram is as follows Figure 5 shown.

[0105] B. Live cell staining of planar cardiomyocytes to detect ROS and calcium transients

[0106] 1) Washing: After the planar cardiomyocytes have completed static cold storage, the confocal dish is taken out and washed twice with pure 1640 RPMI.

[0107] 2) Dye addition: Add live cell dye Mito-Tracker (addition ratio 1:2000, Thermo Fisher, catalog number: M7512), CellRox (addition ratio 1:1000, Thermo Fisher, catalog number: C10492) and Fluo4 (addition ratio 1:1000, Thermo Fisher, catalog number: F14201), and then place the confocal dish at 37°C, 5% CO 2 Incubate in an incubator for 25 min.

[0108] 3) Wash the plate with pure 1640 RPMI for three times to wash away the dye.

[0109] 4) Medium treatment: Add the 1:1 medium described in Example 4 containing Blebbistatin (10 μM).

[0110] 5) Imaging: Imaging was performed on a Zeiss 980 microscope.

[0111] 6) Data processing: ImageJ software was used to process the imaging data. The results are shown in Table 2 and Figure 6 As shown, in Figure A, CellRox detects the level of mitochondrial reactive oxygen species, and Mito / CellRox / DAPI detects the level of mitochondrial membrane potential.

[0112] Table 2 Data of ROS and calcium transients in cardiomyocytes treated with different methods

[0113]

[0114] The results showed that after static cold storage of planar cardiomyocytes, ROS increased ( Figure 6 However, after adding Baricitinib, the ROS of cardiomyocytes decreased. After static cold storage of planar cardiomyocytes, the maximum amplitude is generally considered to be the most powerful indicator of myocardial calcium conduction capacity. According to the experimental results, the calcium processing capacity is weakened after cold storage ( Figure 6B and C), while the calcium handling capacity was improved after drug addition.

[0115] Example 7

[0116] A. Static cold storage of cardiac organoids: The cardiac organoids were randomly divided into three groups, one of which was stored at 37°C, denoted as the Control group; one was stored at 4°C, denoted as SCS-8h; and one was added with Baricitinib and stored at 4°C, denoted as SCS-8h-Bari. The operation was the same as step A in Application Example 1.

[0117] B. Contractility testing of human cardiac organoids

[0118] 1) Equipment preparation: Start and debug the mechanical testing device in advance, turn on the sensor, mechanical sensor (self-made), stepper motor (Feichuang Yida, model: TSC-1-SR2) and temperature controller (self-made).

[0119] 2) Sensor adjustment: Adjust the direction of the mechanical sensor so that the contraction force wave is facing upward.

[0120] 3) Stretching program setting: Open the stepper motor software and adjust the stretching program to stretch the heart organoid by 2% of its original length each time, for a total of 5 stretches. After each stretch, measure the contraction force data for 30 seconds before the next stretch.

[0121] 4) Temperature control: Turn on the temperature controller to maintain the bath temperature at 37°C. Rinse the bath with pure water and then add 1.8 mM Ca 2+ Tyrode's solution.

[0122] 5) Preparation of cardiac organoids: After the static cold storage time in step A reaches 8 hours, remove the cardiac organoids and use small scissors to cut them into two sections, one of which is connected to the mechanical sensor (free end) and the other is connected to the bath (fixed end).

[0123] 6) Data recording: Start the recording software of the mechanical sensor and record the contraction force waveform. After recording the contraction force plateau for 40 seconds, turn on the electrical stimulator (Chengdu Instrument, item number: YC-2) and perform continuous single stimulation with the parameters of voltage 10V, delay 0ms, wave width 50ms, and stimulation frequency 1.5Hz.

[0124] 7) Perform stretching: After recording for another 40 seconds, start the mechanical stretching program and perform stretching.

[0125] 8) Data storage: After the program measurement is completed, the data is stored.

[0126] 9) Data analysis: The active and passive contractile forces of cardiac organoids were analyzed using the analysis code written in MATLAB. The results are shown in Table 3 and Figure 7 shown.

[0127] Table 3 Contractility data of cardiac organoids

[0128]

[0129] According to Table 3 and Figure 7 It can be seen that after adding Baricitinib, the heart contractility was improved, which can prolong the static cold storage time of heart organoids. Among them, active contractility generally reflects the contractile function of myocardial cells. According to Table 3 and Figure 7 As shown in Figure A, the active contraction force of cardiac organoids decreased significantly after static cold storage, but was restored after the addition of Baricitinib. The passive contraction force generally reflects the hardness of cardiac organoids. According to Table 3 and Figure 7 As shown in Figure B, there was no significant difference in the passive contraction force among the three groups, indicating that the hardness of the three groups of cardiac organoids was the same, thereby eliminating the possibility that differences in active contraction force may be caused by different tissue hardness, and also increasing the credibility of the active contraction force.

[0130] In summary, Baricitinib can improve the mitochondrial function of cardiomyocytes, reduce oxidative stress, enhance cardiac contractility, and prolong the static cold storage time of isolated hearts and cardiac organoids.

[0131] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of baricitinib in static cryopreservation of isolated hearts and / or cardiac organoids.

2. Application of baricitinib in prolonging the static cold storage time of isolated hearts and / or cardiac organoids.

3. The use according to claim 2, characterized in that: The effective concentration of baricitinib is 0.1 to 10 μM.

4. The use according to claim 2 or 3, characterized in that: The application includes at least one of delaying myocardial cell mitochondrial damage, improving myocardial cell calcium conduction function, reducing myocardial cell oxidative stress ability and improving cardiac contractile function.

5. The use according to claim 4, characterized in that: The delaying of cardiomyocyte mitochondrial damage includes improving the cardiomyocyte mitochondrial structure.

6. The use according to claim 4, characterized in that: The reducing the oxidative stress capacity of cardiomyocytes includes reducing the level of active oxygen in cardiomyocytes.

7. The use according to claim 1 or 2, characterized in that: The construction of the cardiac organoid comprises the following steps: inducing the human pluripotent stem cells or human embryonic stem cells to differentiate into cardiomyocytes; digesting the cardiomyocytes into single cells; mixing the single cells with a hydrogel system to cultivate cardiac organoids; The hydrogel system comprises fibrinogen, matrix gel and thrombin; the volume ratio of the fibrinogen, matrix gel and thrombin is 10:5:

1.

8. The use according to claim 7, characterized in that: Inducing the human pluripotent stem cells or human embryonic stem cells to differentiate into cardiomyocytes comprises: when the confluence of the human pluripotent stem cells or human embryonic stem cells is 80%, inducing differentiation to obtain cardiomyocytes; On the 0th day of differentiation induction, the first differentiation medium was added for culturing; on the 1st day of differentiation induction, the differentiation medium was added for culturing; on the 3rd day of differentiation induction, the second differentiation medium was added for culturing; on the 5th day of differentiation induction, the differentiation medium was added for culturing; after the 5th day of differentiation induction, the differentiation medium was replaced every 2 days; The differentiation medium is based on 1640RPMI as the basic medium and also contains 50×B27 -INS and 0.4 mg / mL L-ascorbic acid-2-phosphate magnesium salt hydrate; The first differentiation medium is a differentiation medium as a basal medium and further contains a Wnt signaling pathway inhibitor, CHIR99021; The second differentiation medium is based on the differentiation medium as a basal medium and further contains IWR-1, an inhibitor of the Wnt signaling pathway.

9. The use according to claim 8, characterized in that: The final concentration of CHIR99021 is 2.5 to 12.5 μM; the final concentration of IWR-1 is 5 to 10 μM.

10. The use according to claim 7, characterized in that: After the cardiac organoids are obtained by culturing, the method further includes: demolding the cardiac organoids and placing them in a 12-well plate; adding a first early culture medium for culturing on the first day after demolding; adding an early culture medium for culturing on the second day after demolding, and replacing the culture medium every 2 days; adding a late culture medium for culturing on the seventh day after demolding, and replacing the culture medium every 2 days; The early culture medium is based on 1640RPMI as the basic culture medium and also contains 50×B27 -INS , 0.4 mg / mL L-ascorbic acid-2-phosphate magnesium salt hydrate, 2 mg / mL 6-aminohexanoic acid, 0.45 μM 1-thioglycerol, 1% 100× non-essential amino acids and 1% 100× sodium pyruvate; the first early culture medium is based on the early culture medium and also contains 10 μM Y-27632 and 10 μM 5-bromo-2'-deoxyuridine; The late stage culture medium is based on low-glucose DMEM and further contains 2% fetal bovine serum, 0.4 mg / mL L-ascorbic acid-2-phosphate magnesium salt hydrate, 2 mg / mL 6-aminohexanoic acid, 0.45 μM 1-thioglycerol and 1% 100× non-essential amino acids.

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