Heart organoid cryopreservation liquid and application thereof

Abstract

The invention relates to a heart organoid cryopreservation solution and application thereof, and belongs to the technical field of biology. The freezing medium capable of maintaining the structure and function stability of the heart organoid is obtained through screening, and the freezing medium is selected from one or more of fetal calf serum, dimethyl sulfoxide, a ROCK inhibitor, methylcellulose, a basic fibroblast growth factor and L-ascorbic acid. The method can effectively protect cells of the heart organoid from being damaged in the cryopreservation process, obviously reduce apoptosis of the cells, maintain the stability of the structure and function of the cryopreserved heart organoid, and provide possibility for large-scale preservation and transportation of heart organoid samples and subsequent use of a large number of heart organoid as experimental models.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biology, and particularly to a heart organoid cryopreservation solution and application thereof. BACKGROUND

[0002] An organoid is a kind of 3D micro-organ structure cultured in vitro, which can simulate the structure, physiological function and development process of a real organ, and plays an important role in revealing the mechanism of diseases, drug development and regenerative medicine. As an important model for simulating cardiovascular diseases, the heart organoid plays a key role in the research of pathogenesis analysis, drug screening and personalized treatment of cardiovascular diseases. However, the heart organoid has a large volume, a dense structure, a complex cell composition, and cell-cell interactions. In addition, the myocardial cells, as the key component of the heart organoid, have weak proliferative capacity. If the cryopreservation method is not appropriate, it may not only cause the fragmentation of the organoid, but also reduce the cell activity and cause cell death, thereby affecting the structure and function of the heart organoid. Therefore, for the cryopreservation of the heart organoid, the above-mentioned situations need to be considered comprehensively, and a new cryopreservation scheme suitable for the heart organoid needs to be developed, so as to provide stable preservation conditions for the construction of heart disease models, drug screening and regenerative medicine, and ensure the stability of batch experiments.

[0003] Traditional cell cryopreservation generally uses dimethyl sulfoxide (DMSO) or glycerol as a cryoprotective agent for cell cryopreservation and long-term preservation. However, traditional cryopreservation methods have certain limitations in organoid cryopreservation due to their strong cytotoxicity, large osmotic pressure difference, and the complex composition of organoid cells, as well as the difficulty in maintaining the three-dimensional structure of organoids. After recovery, the functional recovery of organoids is also a challenge. In recent years, researchers have begun to explore natural sources of cryoprotective agents such as trehalose, sucrose, and proline. These substances have lower cytotoxicity and can better protect the structure and function of organoids. Currently, organoid cryopreservation mainly uses traditional slow cooling methods and vitrification techniques. Researchers are also developing specific cryopreservation solutions for specific types of organoids. Existing organoid cryopreservation solutions, such as Corning KMBanker II, support the cryopreservation of multiple organoids, but require gradient cooling or direct -80°C cryopreservation. Corning KMBanker II contains serum albumin, which can affect the electrophysiological properties of cardiac cells. In addition, the addition of biomaterials during cryopreservation can also protect organoids to some extent. For example, a study has developed a Fe3O4-based nano-warming system for the cryopreservation of cardiac organoids, which effectively avoids cell damage caused by ice crystals and osmotic pressure, achieving efficient protection of organoids. However, the distribution of Fe3O4 nanoparticles in cardiac organoids may not be uniform, leading to rapid heating in some areas and insufficient heating in other areas, which can cause local thermal stress, leading to cell damage or tissue rupture. In summary, existing cardiac organoid cryopreservation solutions can damage the structure and function of recovered organoids, so there is an urgent need for a cardiac organoid cryopreservation solution that does not damage the structure and function of cardiac organoids. SUMMARY

[0004] To this end, the technical problem to be solved by the present application is to overcome the lack of a cryopreservation solution that avoids damage to the structure and function of cardiac organoids after cryopreservation and recovery in the prior art.

[0005] To solve the above technical problems, the present application provides a cardiac organoid cryopreservation solution and its application. The present application has screened a cryopreservation solution that can maintain the structure and function of cardiac organoids. The cryopreservation solution of the present application is selected from one or more of fetal bovine serum, dimethyl sulfoxide, a ROCK inhibitor, methyl cellulose, basic fibroblast growth factor, and L-ascorbic acid, which can effectively protect the cells of cardiac organoids during cryopreservation, significantly reduce cell apoptosis, and maintain the stability of the structure and function of cardiac organoids after cryopreservation.

[0006] A first object of the present application is to provide a heart organoid cryopreservation solution, which comprises fetal bovine serum, dimethyl sulfoxide, a ROCK inhibitor and an auxiliary material selected from one or more of ethylene glycol, methyl cellulose, basic fibroblast growth factor and L-ascorbic acid.

[0007] Further, the synergistic effect of the above-mentioned components is the core of achieving efficient cryopreservation of heart organoids: dimethyl sulfoxide as a classic cryoprotective agent can penetrate the cell membrane to reduce the freezing point and reduce the physical damage of ice crystal formation to the cell structure, laying the foundation for the action of other components; fetal bovine serum is rich in various growth factors, proteins and nutrients, which can not only provide energy support for cells, but also maintain cell membrane stability and alleviate the stress response caused by low temperature; the ROCK inhibitor effectively blocks the apoptosis signaling pathway by inhibiting the activity of Rho-associated kinase, especially reducing low-temperature-induced myocardial cell apoptosis, and synergistically enhances cell survival ability with fetal bovine serum; methyl cellulose as a high molecular substance can increase the viscosity of the cryopreservation solution, slow down the migration speed of water molecules during the cooling process, and further inhibit the growth of ice crystals, forming a "physical + chemical" double anti-ice crystal protection network with dimethyl sulfoxide; basic fibroblast growth factor can activate the myocardial cell proliferation-related pathway to promote the repair and regeneration of cells after cryopreservation, while L-ascorbic acid as an antioxidant can scavenge free radicals generated during the cryopreservation process, reducing the damage of oxidative stress to myocardial cell function, and both of them synergistically improve the functional activity of organoids after resuscitation. Through the synergistic regulation of multiple components, this cryopreservation solution builds a low-temperature protection system for heart organoids from multiple dimensions such as physical protection, cell survival and functional repair, achieving efficient maintenance of the structural integrity and functional stability of organoids.

[0008] Further, the ROCK inhibitor comprises Y-27632, Thiazovivin or GSK429286A. The ROCK inhibitor can reduce the fragmentation phenomenon of heart organoids after cryopreservation and resuscitation, and retain the cell diversity and functional activity of heart organoids, especially the function of myocardial cells.

[0009] Further, basic fibroblast growth factor has both biological effects related to mitosis, such as promoting cell differentiation, proliferation and angiogenesis, and non-mitotic effects, such as inhibiting cell apoptosis, promoting the production of nitric oxide and antagonizing myocardial cell damage.

[0010] Further, the volume ratio of fetal bovine serum to heart organoid cryopreservation solution is (2-7):10.

[0011] When the auxiliary material comprises methyl cellulose, the concentration of the methyl cellulose is 0.02-0.5 mM / L.

[0012] The second object of the present application is to provide an application of the above-mentioned cardiac organoid cryopreservation solution in the preservation of cardiac organoids.

[0013] The third object of the present application is to provide a cardiac organoid cryopreservation method, which comprises freezing a cardiac organoid under the precondition of adding the above-mentioned cardiac organoid cryopreservation solution, and then transferring the cardiac organoid to liquid nitrogen for cryopreservation.

[0014] Further, the cardiac organoid cryopreservation method comprises freezing a cardiac organoid cultured in an induction medium under the precondition of adding the above-mentioned cardiac organoid cryopreservation solution, and then transferring the cardiac organoid to liquid nitrogen for cryopreservation. Since different cardiac organoids use different induction media, the present application does not specifically limit the composition of the induction medium. Since the cardiac organoid is cultured in the induction medium, it is only necessary to add the cardiac organoid cryopreservation solution of the present application to the induction medium containing the cardiac organoid for cryopreservation.

[0015] Further, the freezing comprises primary freezing and secondary freezing, the temperature of the primary freezing is-3~-5℃, the time is 1-3 hours, the temperature of the secondary freezing is-75~-85℃, and the time is 20-30 hours.

[0016] Further, the cardiac organoid is prepared by the following steps:

[0017] S1, inoculating induced pluripotent stem cells into an organoid induction medium added with a Wnt signal activator, bone morphogenetic protein 4, activin A, basic fibroblast growth factor and LY-29004 for first induction culture to obtain a first induction product;

[0018] S2, culturing the first induction product in an organoid induction medium containing a Wnt signal inhibitor, insulin, bFGF and tretinoin for second induction culture to obtain a second induction product;

[0019] S3, culturing the second induction product in an organoid induction medium containing BMP4, Insulin and bFGF for third induction culture to obtain a third induction product;

[0020] S4, culturing the third induction product in an organoid induction medium containing Insulin for fourth induction culture to obtain the cardiac organoid.

[0021] Further, the induced pluripotent stem cell refers to a stem cell with differentiation potential obtained by reprogramming a terminally differentiated somatic cell through introduction of specific transcription factors. The induced pluripotent stem cell is similar to the pluripotent stem cell in morphology, gene and protein expression, and differentiation ability, etc. The induced pluripotent stem cell used in the present application is commercially available or obtained by reprogramming a somatic cell.

[0022] Further, it is particularly pointed out that the pluripotent stem cell used in the present application, whether an embryonic stem cell or an induced pluripotent stem cell, cannot develop into a complete organism, and is a pluripotent stem cell established through ethical review.

[0023] Further, the organoid induction medium comprises modified Dulbecco's medium, F12 medium, bovine serum albumin, transferrin and alpha-thioglycerol.

[0024] The fourth object of the present application is to provide a use of the above-mentioned cardiac organoid cryopreservation solution in myocardial cell cryopreservation.

[0025] The fifth object of the present application is to provide a resuscitation method of a cardiac organoid, which comprises the following steps:

[0026] (1) taking the cryopreserved cardiac organoid out of liquid nitrogen and thawing at a temperature ranging from 25 to 37 DEG C;

[0027] (2) transferring the thawed cardiac organoid to an organoid medium containing fetal bovine serum for washing;

[0028] (3) inoculating the washed cardiac organoid into an organoid medium containing Thiazovivin, basic fibroblast growth factor and fetal bovine serum for recovery culture;

[0029] (4) culturing the recovery-cultured cardiac organoid in an organoid medium;

[0030] The cryopreserved cardiac organoid refers to a cardiac organoid cryopreserved by using the above-mentioned cardiac organoid cryopreservation solution, and the organoid medium comprises CDM, CMD3, BPEL and Claycomb medium. The CDM medium has a product number of D8062, the CMD3 medium has a product number of C11875500BT, the BPEL medium has a product number of FG0465, and the Claycomb medium has a product number of 51800C-500ML.

[0031] The present application has the following beneficial effects:

[0032] The cryopreservation solution of the application can effectively protect the cardiac organoid cells from damage during the freezing process, significantly reduce the apoptosis of cardiomyocytes, and maintain the stability of the structure and function of the cardiac organoid after freezing. It can be used as an effective freezing solution for cardiac organoids. The application of the application to the production and storage of cardiac organoids can solve the problems of difficult storage of cardiac organoids and easy destruction of structure and function after freezing, and provides the possibility for large-scale preservation, transportation and subsequent use of cardiac organoid samples as experimental models. BRIEF DESCRIPTION OF DRAWINGS

[0033] In order to make the content of the application more easily understood, the application will be further described in detail below according to specific embodiments of the application and in conjunction with the drawings.

[0034] Figure 1 is a result graph of the influence of different concentrations of FBS and MC on the freezing and recovery of cardiac organoids;

[0035] Figure 2 is an experimental process of freezing and recovery of cardiac organoids using different freezing solutions and a result graph of function detection of the cardiac organoids after freezing and recovery;

[0036] Figure 3 is a fluorescence staining graph and a cell apoptosis statistical graph of the cardiac organoids after freezing and recovery using different freezing solutions; wherein, cTnT represents cardiac troponin T, Hoechst represents cell nucleus staining, and Merge represents channel merging image;

[0037] Figure 4 is a result graph of the influence of the freezing solution of OCM1 group, OCM2 group and OCM3 group on the freezing and recovery of cardiac organoids;

[0038] Figure 5 is a result graph of the influence of the freezing solution of the application on the freezing and recovery of other cardiac organoids. DETAILED DESCRIPTION

[0039] The application will be further described below in conjunction with the drawings and specific embodiments, so that those skilled in the art can better understand the application and implement it, but the embodiments are not limiting the application.

[0040] Example 1: Construction and freezing of cardiac organoids

[0041] I. Construction of cardiac organoids

[0042] 1. Maintenance and subculture of H1 cells

[0043] Commercial H1 cells were inoculated on Matrigel-coated 6-well plates, and chemically defined feeder-free medium PSCeasy® maintain the stemness and proliferation of H1 cells.

[0044] When the cell density is about 85%, use 0.5 mM EDTA to dissociate at 37°C for 3 min, and after stopping the digestion, inoculate at a ratio of 1:6. To inhibit the programmed death triggered by cell dispersion during the subculture process, supplement 2 μM Thiazovivin (CAS: 1226056-71-8, a ROCK inhibitor) in the culture medium. Place the cells and organoids in a cell incubator at 37°C, 5% CO2.

[0045] 2. Induction and culture of self-organizing heart organoids

[0046] The H1 cell line is cultured with mTeSR medium, and when the density is about 80%, use 0.5 mM EDTA to digest and resuspend as single cells. Count with a cell counting plate, and inoculate about 6500 cells / well of the starting aggregation cells into a Corning 96-well ultra-low adsorption round-bottom culture plate, with a uniform culture solution of 150 μL per well, and add 2 μM of Thiazovivin. After adding, stand still for 3-5 min on the operating table, then balance and centrifuge at 300 g for 3 min in a constant speed centrifuge to make the cells aggregate at the bottom of the well, and then place in a cell incubator for 24 h.

[0047] On days 0-2, add organoid induction medium CDM (50% IMDM + 50% F12 + 10% BSA + Transferrin + α-monothioglycerol) containing Wnt signal activator CHIR99021 (CAS No.: 1797989-42-4, an effective activator of the Wnt / beta-catenin signaling pathway) and bone morphogenetic protein 4 (BMP4) 10 ng / mL, Activin A 50 ng / mL, basic fibroblast growth factor (bFGF) 30 ng / mL, and LY-29004 (CAS No.: 154447-36-6) 5 μM to induce.

[0048] On days 2-6, add Wnt signal inhibitor (IWR-1) 1 μM, and BMP4 10 ng / mL, Insulin 10 μg / mL, bFGF-8 ng / mL, Retinoic acid (RA) 0.5 μM to promote cell proliferation and heart development. Change the liquid every two days to induce the development of heart progenitor cells from mesoderm.

[0049] At day 6-8, remove IWR-1 and RA, continue to use small molecule compounds containing BMP4 10 ng / mL, insulin 10 μg / mL, bFGF 8 ng / mL for cardiac cell differentiation. Finally, after D8, use CDM containing only insulin 5 μg / mL for organoid stabilization and maintenance, and perform functional detection analysis of organoids at D12.

[0050] II. Cryopreservation and recovery of cardiac organoids

[0051] 1. Cryopreservation of cardiac organoids

[0052] (1) Aspirate the target organoid in a centrifuge tube, let it settle naturally, discard the supernatant, and wash it twice with phosphate buffered saline (DPBS).

[0053] (2) Transfer the organoid to a cryovial, remove the remaining liquid, and add the prepared cryopreservation solution.

[0054] (3) Label the cryopreservation information on the cryovial, and transfer it to a gradient cooling cryopreservation box.

[0055] (4) First place it in a 4°C refrigerator for 2h, then transfer it to a -80°C refrigerator for 24h; after cooling is complete, quickly remove the cryovial from the cryopreservation box and transfer it to liquid nitrogen for long-term storage.

[0056] 2. Recovery of cardiac organoids

[0057] (1) From the liquid nitrogen tank, immediately place the target organoid cryovial in a 37°C water bath, gently shake until the ice crystals melt until the size of a green bean, and then remove it (≤2 min).

[0058] (2) Transfer the cryopreserved organoid to a 15 mL centrifuge tube containing 4 mL of preheated 20% FBS organoid medium, dilute the cryopreservation solution, and let the organoid settle naturally before discarding the supernatant. Then gently add 4 mL of preheated 20% FBS organoid medium.

[0059] (3) After washing and natural settling, gently transfer the organoid to a low-absorption well plate, and add 2 μM Thiazovivin, 50 ng / μL bFGF, and 20% fetal bovine serum (FBS) organoid medium for organoid recovery culture.

[0060] (4) After 2 days of culture, replace it with regular organoid medium without serum for long-term culture. Generally, after one week of culture, subsequent experimental detection can be performed.

[0061] Example 2: Effect of different cryopreservation solutions on recovery of cardiac organoids after cryopreservation

[0062] The present embodiment first designs different concentration gradients of fetal bovine serum (20% FBS means that FBS accounts for 20% of the total system volume, 70% FBS means that FBS accounts for 70% of the total system volume, and 90% FBS means that FBS accounts for 90% of the total system volume) and different concentrations of methyl cellulose (0.2% MC means 0.02 mM / L of MC, 1% MC means 0.1 mM / L of MC, and 5% MC means 0.5 mM / L of MC), and the heart organoids are frozen. The organoids are placed in different concentrations of FBS and MC for freezing, and the state of the heart organoids after resuscitation is observed:

[0063] 20% FBS group: CDM + 20% FBS + 1.41 mol / L DMSO + 10 μM Thiazovivin;

[0064] 70% FBS group: CDM + 70% FBS + 1.41 mol / L DMSO + 10 μM Thiazovivin;

[0065] 90% FBS group: 90% FBS + 1.41 mol / L DMSO + 10 μM Thiazovivin;

[0066] 0.2% MC group: CDM + 20% FBS + 0.2% MC + 1.41 mol / L DMSO + 10 μM Thiazovivin;

[0067] 1% MC group: CDM + 20% FBS + 1% MC + 1.41 mol / L DMSO + 10 μM Thiazovivin;

[0068] 5% MC group: 5% MC + 1.41 mol / L DMSO + 10 μM Thiazovivin;

[0069] DMSO group: CDM + 20% FBS + 1.41 mol / L DMSO + 10 μM ROCK inhibitor;

[0070] CTRL group: heart organoids without freezing treatment and treated at the same time.

[0071] The morphology of the organoids after resuscitation at day 0 and day 7 was recorded. The bright field photography of the FBS group showed that the edge structure of the resuscitated organoids was relatively loose, and the morphology structure recovered after conventional organoid culture, indicating that FBS played a protective role in freezing of the heart organoids. Figure 1 A). The diameters of the heart organoids after resuscitation for one week were counted, and the diameters of the 70% FBS organoids were significantly higher than those of the other two groups. Figure 1B), viability testing of cardiac organoids 7 days after resuscitation showed that the organoids exhibited the highest viability under the condition of 70% FBS cryopreservation. Figure 1 (C) indicates that 70% FBS is a suitable concentration for cryopreservation of heart organoids.

[0072] Heart organoids were cryopreserved using different concentrations of methylcellulose (0.2% MC, 1% MC, 5% MC), and the morphology of the organoids was photographed and recorded on day 0 and day 7 after thawing. Figure 1 D). The results showed that the diameter of organoids in the 0.2% MC and 1% MC groups was larger than that in the 0.2% MC group, while the diameter of organoids in the 5% MC group was larger than that in the control group. Figure 1 E). Cell viability was assessed 7 days after thawing. The viability of the 0.2% MC and 1% MC groups was significantly higher than that of the DMSO group, while the viability of the organoids in the 5% MC group was significantly lower than that in the DMSO group, indicating that the organoids in the 5% MC group suffered irreversible damage during cryopreservation. Figure 1 F). In conclusion, 70% FBS and 1% MC are more suitable for the cryopreservation of cardiac organoids.

[0073] In this embodiment, based on the commonly used 10% DMSO cell cryopreservation solution, the following candidate reagents were screened as supplementary components of the cryopreservation solution according to the components required in the construction and culture of cardiac organoids: ethylene glycol (EG), methylcellulose (MC), glucose, basic fibroblast growth factor (bFGF), and L-ascorbic acid (LAA). These were used to screen for components that could stabilize the structural integrity of cardiac organoids, protect cardiomyocytes, and maintain the function of cardiac organoids during cryopreservation. The corresponding cryopreservation solution composition is as follows: CDM consists of 50% IMDM medium + 50% F12 medium + 10% BSA + transferrin + α-monothioglycerol.

[0074] DMSO group: CDM + 20% FBS + 1.41 mol / L DMSO + 10 μM ROCK inhibitor;

[0075] EG group: CDM + 20% FBS + 1.41 mol / L DMSO + 10 μM ROCK inhibitor + 10% EG;

[0076] MC group: CDM + 20% FBS + 1.41 mol / L DMSO + 10 μM ROCK inhibitor + 1% MC;

[0077] Glucose group: CDM + 20% FBS + 1.41 mol / L DMSO + 10 μM ROCK inhibitor + 5% Glucose;

[0078] bFGF group: CDM + 20% FBS + 1.41 mol / L DMSO + 10 μM ROCK inhibitor + 200 ng / uL bFGF;

[0079] LAA group: CDM + 20% FBS + 1.41 mol / L DMSO + 10 μM ROCK inhibitor + 500 μg / mL LAA;

[0080] FBS group: CDM + 70% FBS + 1.41 mol / L DMSO + 10 μM ROCK inhibitor.

[0081] Select heart organoids of similar size for freezing ( Figure 2 A), freeze the heart organoids in the heart organoid culture medium supplemented with 1.41 mol / L DMSO group alone or added with the above candidate reagents (10% ethylene glycol, 1% methyl cellulose, 5% Glucose, 200 ng / mL bFGF, 500 μg / mL LAA) and supplemented with FBS, then thaw and recover, compare the morphology of the organoids before and after freezing and the survival of myocardial cells, to screen out suitable components as supplements.

[0082] Morphological observation: after freezing of the DMSO group and the Glucose group, a small amount of fragments of the organoids were generated after thawing. However, the degree of cell fragmentation in the EG group, the bFGF group, and the LAA group was very small ( Figure 2 B and Figure 2 C). Comparing the diameters of the heart organoids before and after freezing and thawing, the results showed that the diameter of the heart organoids after thawing in the EG group decreased significantly, while the other freezing media had a significant increase compared with the conventional freezing DMSO group ( Figure 2 D).

[0083] Myocardial cell detection: immunofluorescence staining results showed that the myocardial cell proportion in the Glucose group as a supplement component of the freezing solution group was low after thawing, the connection was weak, and the myocardial cells were dispersed ( Figure 3 A and Figure 3 C). The myocardial cell proportion in other groups was significantly improved.

[0084] Apoptosis detection: The apoptosis of the cells in each group after freezing and recovery was detected, and the results showed that the apoptosis of the cells in the other groups was less than that in the DMSO group and the EG group Figure 3 B).

[0085] The above results show that, except for the Glucose group, the rest of the candidate reagents play a certain protective role in the freezing process of the cardiac organoids.

[0086] Example 3: Function detection of cardiac organoids after freezing and recovery of different combinations of freezing solutions

[0087] In order to further improve the freezing efficiency of the cardiac organoids, on the basis of the above research, the present application subsequently tried different combinations of these candidate reagents (referred to as OCM1-OCM3) in the freezing medium of the cardiac organoids, and the corresponding compositions are as follows:

[0088] DMSO group: CDM+20% FBS+1.41mol / L DMSO+10 μM Y27632;

[0089] OCM1 group: CDM+70% FBS+1.41mol / L DMSO+500 μg / mL LAA+200 ng / mL bFGF+10 μM Y27632;

[0090] OCM2 group: CDM+70% FBS+1.41mol / L DMSO+1% MC+200 ng / mL bFGF+10 μM Y27632;

[0091] OCM3 group: CDM+70% FBS+1.41mol / L DMSO+1% MC+500 μg / mL LAA+200 ng / mL bFGF+10 μM Y27632;

[0092] Morphology statistics: The size of the organoids in the OCM1 group and the OCM3 group was significantly restored, while the OCM2 group had no obvious size change Figure 4 A).

[0093] Beating detection of organoids: It can be seen that, except for the DMSO group, the beating of the organoids in the OCM1-OCM3 groups after recovery had no obvious difference with that before freezing, and the cell viability recovery of the OCM3 was better Figure 4 B).

[0094] Myocardial cell detection: The structure of the organoids after freezing was detected by immunofluorescence staining, and the results showed that the cardiac area of the organoids in the OCM3 treatment group was higher than that in the OCM1 and OCM2 groups Figure 4 C and 4D).

[0095] In summary, the present application found that OCM3 group (CDM + 70% FBS + 1.41 mol / L DMSO + 1% MC + 500 μg / mL LAA + 200 ng / mL bFGF + 10 μM Y27632, referred to as FMBL cryopreservation solution) is the best cryopreservation solution for heart organoids.

[0096] Example 4: Universality of heart organoid cryopreservation solution

[0097] Other induction methods for inducing heart organoids:

[0098] (1) Three-cell mixed organoids (CEF): three-cell mixed organoids composed of myocardial cells (CMs), endothelial cells (ECs) and fibroblasts (CFs) induced from commercial human pluripotent stem cells. First, the three types of functional cells induced successfully were dissociated into CMs, ECs and CFs using the corresponding concentration of trypsin. The dissociation effect of each cell type was observed in real time under a microscope to ensure single cell detachment. The three types of cells were resuspended in 1 mL of BPEL medium, and 20 μL of centrifuge tubes were prepared for each. Cell counting was performed using a cell counting plate. Prepare a 15 mL conical tube, dilute ECs, CFs and CMs with BPEL medium containing 50 ng / mL VEGF and 5 ng / mL FGF-2 according to the preset ratio of 70% CMs, 15% ECs and 15% CFs (3,500 CMs, 750 ECs and 750 CFs per microtube). Key steps require 50 μL of cell suspension per well (to be inoculated in a 96-well V-bottom microplate later). Gently resuspend the cell suspension using a pipette and transfer it to a storage tube.

[0099] (2) Vaschamcardioids (vcCOs): Dissociate 80% density commercialized induced pluripotent stem cells (hPSCs) and resuspend in differentiation medium (DMEM:F12 medium, 20% KSR (KnockOut Serum Replacement), L-glutamine solution (Glutamax), NEAA (Non Essential Amino Acid)) and culture in ultra-low attachment 6-well plate overnight. After the formation of aggregates in the presence of Thiazovivin, induce by continuous treatment of CHIR99021 (12 μM) for 2 days, then in differentiation medium containing BMP4 (25 ng / mL), bFGF (25 ng / mL) and VEGF (50 ng / mL) for 6 days, and change the differentiation medium every two days. This process completes the differentiation of hPSCs to vascular lineage cells and the formation of vascular organoid precursors. Each differentiated completed vascular organoid precursor is then mixed with 1 x 10 5 hPSCs-derived purified cardiomyocytes are mixed together in one well of an ultra-low attachment 96-well plate and cultured in suspension in Claycomb medium with the addition of 15% fetal bovine serum (FBS), bFGF (100 ng / mL), vascular endothelial growth factor (VEGF, 100 ng / mL) and Thiazovivin. After the aggregation of CMs around the vascular organoid precursors, the newly formed cardiac organoids, continue to culture to induce the migration of vascular oriented cells to the periphery of the myocardium, and finally form vascularized chamber cardiac organoids with vascularized chamber structure.

[0100] 2. Cryopreservation results:

[0101] To verify the universality of the FMBL cardiac organoid cryopreservation solution, this example constructs three-cell mixed organoids (CEF) and cardiac vascular chamber organoids (vcCOs) according to the above induction method, and cryopreservation in DMSO group and FMBL cryopreservation solution, respectively. Record the morphology of the organoids after recovery in each group, and perform immunofluorescence staining to count the proportion of myocardial cell area at day 7 after recovery, and compare with the same period of culture without cryopreservation CTRL group organoids without cryopreservation.

[0102] Bright-field imaging was used to record CEF and vcCOs cardiac organoids from the DMSO and FMBL groups on day 0 after resuscitation, and compared with CEF and vcCOs cardiac organoids from the CTRL group. The results showed that, compared with the CTRL group, the CEF and vcCOs structure of the DMSO group was loose, while the cardiac organoids cryopreserved using FMBL had a denser structure, indicating that FMBL played a certain protective role in the structure of both types of cardiac organoids. Figure 5 A and Figure 5 D), immunofluorescence staining was performed on cardiac organoids cultured for 7 days after resuscitation and cardiac organoids from the CTRL group. Figure 5 B and Figure 5 E), and the myocardial area of ​​each group was counted separately. Among them, the myocardial area of ​​the DMSO group cryopreserved organoids was much lower than that of the CTRL and FMBL groups. However, the proportion of myocardial cell area in the FMBL cryopreserved and thawed cardiac organoids was similar to that of the CTRL group. This indicates that FMBL effectively protects the survival of myocardial cells in cardiac organoids during cryopreservation. Figure 5 C and Figure 5 The above results indicate that FMBL can effectively protect cardiac organoids induced by different methods during cryopreservation, suggesting that FMBL can be used for cryopreservation of cardiac organoids induced by different methods.

[0103] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A cryopreservation solution for heart organoids, characterized in that, The cryopreservation solution for the heart organoids includes fetal bovine serum, dimethyl sulfoxide, ROCK inhibitors, and excipients, wherein the excipients are selected from one or more of ethylene glycol, methylcellulose, basic fibroblast growth factor, and L-ascorbic acid.

2. The cryopreservation solution for heart organoids according to claim 1, characterized in that, The ROCK inhibitors include Y-27632, Thiazovivin, or GSK429286A.

3. The cryopreservation solution for heart organoids according to claim 1, characterized in that, The volume ratio of fetal bovine serum to cardiac organoid cryopreservation solution is (2-7):10; or, When the excipients include methylcellulose, the concentration of methylcellulose is 0.02-0.5 mM / L.

4. The use of the cardiac organoid cryopreservation solution according to any one of claims 1-3 in the preservation of cardiac organoids.

5. The use of the cardiac organoid cryopreservation solution according to any one of claims 1-3 in the cryopreservation of cardiomyocytes.

6. A method for cryopreserving heart organoids, characterized in that, The method for cryopreserving cardiac organoids is as follows: the cardiac organoids are frozen in the presence of the cryopreservation solution for cardiac organoids as described in any one of claims 1-3, and then transferred to liquid nitrogen for cryopreservation.

7. The method for cryopreserving heart organoids according to claim 6, characterized in that, The freezing process includes primary freezing and secondary freezing. The primary freezing temperature is -3 to -5°C and the time is 1 to 3 hours. The secondary freezing temperature is -75 to -85°C and the time is 20 to 30 hours.

8. The method for cryopreserving cardiac organoids according to claim 6, characterized in that, The heart organoid was prepared through the following steps: S1. Induced pluripotent stem cells were seeded in organoid induction culture medium supplemented with Wnt signaling activator, bone morphogenetic protein 4, activin A, basic fibroblast growth factor and LY-29004 for the first induction culture to obtain the first induction product. S2. The first induction product is subjected to a second induction culture in organoid induction medium containing Wnt signaling inhibitor, insulin, basic fibroblast growth factor and retinoic acid to obtain the second induction product. S3. The second induction product is induced for a third time in organoid induction medium containing bone morphogenetic protein 4, insulin and basic fibroblast growth factor to obtain the third induction product. S4. The third induction product is induced for a fourth time in an organoid induction medium containing insulin to obtain the heart organoid.

9. The method for cryopreserving cardiac organoids according to claim 8, characterized in that, The organoid induction culture medium includes modified Duchenne broth, F12 broth, bovine serum albumin, transferrin, and α-thioglycerol.

10. A method for resuscitating a cardiac organoid, characterized in that, The recovery method includes the following steps: (1) Remove the frozen heart organoids from liquid nitrogen and thaw them in a temperature range of 25-37℃; (2) The thawed heart organoids were transferred to organoid culture medium containing fetal bovine serum and washed; (3) The washed heart organoids were inoculated into organoid culture medium containing Thiazovivin, basic fibroblast growth factor and fetal bovine serum for recovery culture; (4) The restored cardiac organoids were cultured in organoid culture medium; The cryopreserved cardiac organoids refer to cardiac organoids cryopreserved using the cardiac organoid cryopreservation solution described in any one of claims 1-3, and the organoid culture medium is selected from one or more of CDM culture medium, CMD3 culture medium, BPEL culture medium, and Claycomb culture medium.

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