A tissue storage and / or transport medium and uses thereof

Abstract

The present invention relates to a tissue storage and / or transport medium and uses thereof. In particular, the present invention relates to a tissue storage and / or transport medium comprising resveratrol and enlicarin, methods of preparation, and uses thereof.

Description

TECHNICAL FIELD

[0001] The present invention relates to the fields of biology and medicine. In particular, the present invention relates to the protective effect of biological small molecules on brain-like organoids in room temperature dormancy. BACKGROUND

[0002] The general human development has always been the central theory in the field of developmental biology. Since the initial discovery of human embryonic stem cells (ESCs) and human induced pluripotent stem cells (HIPSCs), a large number of studies have defined the chemical signals that push human pluripotent stem cells to different states and take advantage of the natural tendency of PSCs to form spontaneous 3D neuroectodermal structures, i.e. brain-like organoids. Brain-like organoids have made significant progress in recent years. They are formed by human pluripotent stem cells under three-dimensional culture conditions and can mimic the cell heterogeneity, structure and function of the human brain. These organoids have important scientific significance and application prospects in the field of regenerative medicine, and can be used to simulate brain injury, repair diseased tissues and construct disease models. Researchers have successfully constructed brain organoid models of different brain regions, such as the cerebral cortex, hippocampus, striatum, etc., and have shown potential in drug screening and personalized medicine.

[0003] However, brain organoid research still faces some challenges. Currently, there is no mature cryopreservation and recovery protocol for brain organoids, and cell survival cannot be maintained during long-distance transportation and room temperature storage. In addition, the lack of vascularization of brain organoids leads to central necrosis of the organoid, interfering with normal development and neuronal migration. Although some studies have provided a solution by co-culturing with vascular stem cells, the problems of nutrient supply, cell survival in the central region, cryopreservation, storage and cell protection during long-distance transfer, and the real blood microenvironment still exist. SUMMARY

[0004] In order to solve the problem of significant reduction in cell survival rate of brain organoids during room temperature storage and transportation, the present invention is proposed.

[0005] In a first aspect, the present invention provides an improved tissue storage and / or transportation medium comprising T- resveratrol and enlicarabine.

[0006] In a specific embodiment, the present invention provides an improved tissue storage and / or transportation medium comprising:

[0007] T-

[0008] 0.1-10 μΜ resveratrol, and

[0009] 0.1-10 μΜ enlicarabine.

[0010] In particular embodiments, the improved tissue storage and / or transport medium provided herein has a final concentration of resveratrol of 0.1 μΜ, 0.2 μΜ, 0.3 μΜ, 0.4 μΜ, 0.5 μΜ, 0.6 μΜ, 0.7 μΜ, 0.8 μΜ, 0.9 μΜ, 1.0 μΜ, 1.1 μΜ, 1.2 μΜ, 1.3 μΜ, 1.4 μΜ, 1.5 μΜ, 1.6 μΜ, 1.7 μΜ, 1.8 μΜ, 1.9 μΜ, 2.0 μΜ, 2.1 μΜ, 2.2 μΜ, 2.3 μΜ, 2.4 μΜ, 2.5 μΜ, 2.6 μΜ, 2.7 μΜ, 2.8 μΜ, 2.9 μΜ, 3.0 μΜ, 3.1 μΜ, 3.2 μΜ, 3.3 μΜ, 3.4 μΜ, 3.5 μΜ, 3.6 μΜ, 3.7 μΜ, 3.8 μΜ, 3.9 μΜ, 4.0 μΜ, 4.1 μΜ, 4.2 μΜ, 4.3 μΜ, 4.4 μΜ, 4.5 μΜ, 4.6 μΜ, 4.7 μΜ, 4.8 μΜ, 4.9 μΜ, 5.0 μΜ, 5.1 μΜ, 5.2 μΜ, 5.3 μΜ, 5.4 μΜ, 5.5 μΜ, 5.6 μΜ, 5.7 μΜ, 5.8 μΜ, 5.9 μΜ, 6.0 μΜ, 6.1 μΜ, 6.2 μΜ, 6.3 μΜ, 6.4 μΜ, 6.5 μΜ, 6.6 μΜ, 6.7 μΜ, 6.8 μΜ, 6.9 μΜ, 7.0 μΜ, 7.1 μΜ, 7.2 μΜ, 7.3 μΜ, 7.4 μΜ, 7.5 μΜ, 7.6 μΜ, 7.7 μΜ, 7.8 μΜ, 7.9 μΜ, 8.0 μΜ, 8.1 μΜ, 8.2 μΜ, 8.3 μΜ, 8.4 μΜ, 8.5 μΜ, 8.6 μΜ, 8.7 μΜ, 8.8 μΜ, 8.9 μΜ, 9.1 μΜ, 9.2 μΜ, 9.3 μΜ, 9.4 μΜ, 9.5 μΜ, 9.6 μΜ, 9.7 μΜ, 9.8 μΜ, 9. μΜ 9, or 10 μΜ; or a range between any two of the above values.

[0011] Resveratrol is a non-flavonoid polyphenolic organic compound that is a phytoalexin produced by several plants when under stress, and has the chemical formula C 14 H 12 O3. It is synthesized in the leaves and skins of grapes, and is a bioactive ingredient in red wine and grape juice. It is readily absorbed when taken orally and is excreted in the urine and feces after metabolism. In vitro and animal studies have shown that resveratrol has antioxidant, anti-inflammatory, anticancer, and cardiovascular protective effects.

[0012] In specific embodiments, the improved tissue storage and / or transportation medium provided herein has a final concentration of Enlicarabine of 0.1 μΜ, 0.2 μΜ, 0.3 μΜ, 0.4 μΜ, 0.5 μΜ, 0.6 μΜ, 0.7 μΜ, 0.8 μΜ, 0.9 μΜ, 1.0 μΜ, 1.1 μΜ, 1.2 μΜ, 1.3 μΜ, 1.4 μΜ, 1.5 μΜ, 1.6 μΜ, 1.7 μΜ, 1.8 μΜ, 1.9 μΜ, 2.0 μΜ, 2.1 μΜ, 2.2 μΜ, 2.3 μΜ, 2.4 μΜ, 2.5 μΜ, 2.6 μΜ, 2.7 μΜ, 2.8 μΜ, 2.9 μΜ, 3.0 μΜ, 3.1 μΜ, 3.2 μΜ, 3.3 μΜ, 3.4 μΜ, 3.5 μΜ, 3.6 μΜ, 3.7 μΜ, 3.8 μΜ, 3.9 μΜ, 4.0 μΜ, 4.1 μΜ, 4.2 μΜ, 4.3 μΜ, 4.4 μΜ, 4.5 μΜ, 4.6 μΜ, 4.7 μΜ, 4.8 μΜ, 4.9 μΜ, 5.0 μΜ, 5.1 μΜ, 5.2 μΜ, 5.3 μΜ, 5.4 μΜ, 5.5 μΜ, 5.6 μΜ, 5.7 μΜ, 5.8 μΜ, 5.9 μΜ, 6.0 μΜ, 6.1 μΜ, 6.2 μΜ, 6.3 μΜ, 6.4 μΜ, 6.5 μΜ, 6.6 μΜ, 6.7 μΜ, 6.8 μΜ, 6.9 μΜ, 7.0 μΜ, 7.1 μΜ, 7.2 μΜ, 7.3 μΜ, 7.4 μΜ, 7.5 μΜ, 7.6 μΜ, 7.7 μΜ, 7.8 μΜ, 7.9 μΜ, 8.0 μΜ, 8.1 μΜ, 8.2 μΜ, 8.3 μΜ, 8.4 μΜ, 8.5 μΜ, 8.6 μΜ, 8.7 μΜ, 8.8 μΜ, 8.9 μΜ, 9.1 μΜ, 9.2 μΜ, 9.3 μΜ, 9.4 μΜ, 9.5 μΜ, 9.6 μΜ, 9.7 μΜ, 9.8 μΜ, 9. μΜ 9, or 10 μΜ; or a range between any two of the above values.

[0013] Enlicarabine is a pan-CASPASE pathway inhibitor that inhibits ZIKV-induced increase in CASPASE-3 activity and protects human cortical neural progenitor cells, with neuroprotective and anti-inflammatory effects.

[0014] The Tissue Storage and Transportation Medium (T-Store) Preservative creates a homeostatic environment that is isotonic, isophasic, and isoelectric to human serum and interstitial fluid, which preserves genetic and histological characteristics of tissue samples for up to 72 hours.

[0015] In specific embodiments, the tissue storage and transportation medium of the present application is a T- It is commercially available from Life Science Production, UK.

[0016] In a particular embodiment of the application, the addition of resveratrol and enlicarbin in appropriate concentrations to T- The addition of resveratrol and enlicarbin in appropriate concentrations to T-

[0017] Thus, the tissue storage and / or transport medium provided by the present application can be used for the ambient temperature storage and / or transport of brain-like organs.

[0018] In a second aspect, the present application provides a method of preparing a tissue storage and / or transport medium, said method comprising the addition of resveratrol and enlicarbin to T- .

[0019] In a particular embodiment, the present application provides a method of preparing a tissue storage and / or transport medium, said method comprising the addition of resveratrol and enlicarbin to T- at a final concentration of 0.1-10 μΜ resveratrol and 0.1-10 μΜ enlicarbin.

[0020] In a third aspect, the present application provides the use of a combination of resveratrol and enlicarbin for improving the performance of a tissue storage and / or transport medium.

[0021] In a particular embodiment, the present application provides the use of a combination of resveratrol and enlicarbin for improving the performance of T- .

[0022] In a fourth aspect, the present application provides a method of ambient temperature storage and / or transport of mammalian tissue samples and cells, said method comprising:

[0023] 1 ) transferring the mammalian tissue samples and cells to be stored and / or transported into an improved tissue storage and / or transport medium according to the present application; and

[0024] 2) optionally, removing air from the improved tissue storage and / or transport medium.

[0025] In a particular embodiment, the present application provides a method of ambient temperature storage and / or transport of brain-like organs, said method comprising:

[0026] 1 ) transferring the brain-like organs to be stored and / or transported into an improved tissue storage and / or transport medium according to the present application; and

[0027] 2) optionally, removing air from the improved tissue storage and / or transport medium.

[0028] In a fifth aspect, the present application provides the use of a combination of resveratrol and enlicarbin for the preparation of a medium for the ambient temperature storage and / or transport of mammalian tissue samples and cells.

[0029] In specific embodiments, the mammalian tissue sample is a brain organoid.

[0030] In specific embodiments, the use of the present application comprises adding to the T- a final concentration of 0.1-10 mM resveratrol, and 0.1-10 mM Enlicarabine. BRIEF DESCRIPTION OF DRAWINGS

[0031] FIG. 1A and FIG. 1B Apoptosis staining results and statistical graphs of brain organoids respectively stored in maturation medium in brain organoid maturation kit, Tstore cryoprotectant and cryoprotectant with small molecules listed in this solution for 5, 7, 10 days at room temperature.

[0032] FIG. 2 Survival status of neurons and astrocytes of brain organoids respectively stored in maturation medium, Tstore cryoprotectant and cryoprotectant with small molecules listed in this solution for 10 days at room temperature. Among them, DAPI (blue) is the cell nucleus staining result, ALDH1L1 (green) is the astrocyte staining result, and NEUN (red) is the mature neuron staining result.

[0033] FIG. 3 Differential gene clustering heat map of brain organoids respectively stored in maturation medium, Tstore cryoprotectant and cryoprotectant with small molecules listed in this solution for 10 days at room temperature.

[0034] FIG. 4 GO and KEGG analysis of differential genes of brain organoids respectively stored in maturation medium, Tstore cryoprotectant and cryoprotectant with small molecules listed in this solution for 10 days at room temperature. DETAILED DESCRIPTION

[0035] In order to more specifically explain the present application, the following specific embodiments are provided in the specification, and these specific embodiments are explained in conjunction with the accompanying drawings, but the solutions of the present disclosure are not limited to only this. Those skilled in the art can make appropriate changes to the methods and uses of the present application in conjunction with the common knowledge in the art, as long as they can achieve the functions described in the present application, which should fall within the scope of the present application. If not specifically indicated, the reagents used in the following examples can be commercially purchased.

[0036] Example 1. Culture of induced pluripotent stem cells (IPSC)

[0037] IPSC culture methods known in the prior art can be used.

[0038] 1. Experimental materials and reagents

[0039] Table 1

[0040]

[0041] According to the manufacturer's instructions, and routine operation in the art, the mTeSR TM Plus medium was used for culture, GCDR was used for passaging, and KOSR+DMSO was used for cryopreservation.

[0042] Example 2. Brain-like organoid culture (i.e. ectoderm-neural differentiation direction)

[0043] The brain-like organoid culture method known in the prior art was used according to the manufacturer's instructions.

[0044] 1. Experimental materials and reagents

[0045] Table 2

[0046]

[0047]

[0048] Example 3. Ambient temperature storage of brain-like organoids

[0049] 1. Experimental materials and reagents

[0050] Table 3

[0051]

[0052] 1) Add small molecules Resveratrol and Emricasan to the T-Store protection solution, so that the final concentration of Resveratrol is 2 μM and the final concentration of Emricasan is 1 μM, and configure it into a brain-like organoid ambient temperature protection solution.

[0053] 2) Try to aspirate the medium around the brain-like organoid, wash once with DPBS, and after trying to aspirate the DPBS, use a 200 μL wide-bore gun head or a Pasteur tube to transfer the brain-like organoid to a 1.5 mL EP tube, fill the EP tube with the brain-like organoid ambient temperature protection solution, and try to leave no air.

[0054] 3) Use sealing film to seal the tube opening, and transfer the EP tube storing the brain-like organoid to a light-proof place for storage.

[0055] 4) After ambient temperature storage or transportation is completed, try to aspirate the medium around the brain-like organoid, wash once with DPBS, and after trying to aspirate the DPBS, use a 200 μL wide-bore gun head or a Pasteur tube to transfer the brain-like organoid to the brain-like organoid maintenance medium for normal culture.

[0056] Test Example 1. Frozen section and tissue immunofluorescence staining

[0057] 1. Experimental materials and reagents

[0058] Table 4

[0059]

[0060] 2. Preparation of experimental reagents

[0061] 1) Preparation of 0.1% PBS-T solution

[0062] Take 500 μL Tween-20 stock solution with 1 mL wide-bore gun head, add to 499.5 mL PBS phosphate buffer, and shake well to prepare 500 mL 0.1% PBS-T solution, store at room temperature.

[0063] 2) Preparation of 0.25% Triton X-100-containing permeation solution

[0064] Take 125 μL Triton X-100 stock solution with 200 μL wide-bore gun head, add to 49.875 mL PBS phosphate buffer, and mix well to prepare 50 mL 0.25% Triton X-100-containing tissue permeation solution, store at room temperature.

[0065] 3) Preparation of 15% / 30% sucrose solution for tissue gradient dehydration

[0066] Accurately weigh 6 g of sucrose on an electronic balance, dissolve it in 30 mL of PBS phosphate buffer, stir well until completely dissolved, make up to 40 mL, prepare 15% sucrose solution, store at 4°C; weigh 12 g of sucrose on an electronic balance, dissolve it in 40 mL of PBS phosphate buffer, stir well until dissolved, make up to 40 mL, prepare 30% sucrose solution, store at 4°C.

[0067] 4) Preparation of 0.5% gelatin solution

[0068] Dilute 1% gelatin solution 1:1 with sterile ultrapure water to prepare 0.5% gelatin solution, aliquot 1 mL per tube using 2 mL EP tubes, and store at -20°C for later use.

[0069] 3. Tissue processing (fixation, gradient dehydration, embedding)

[0070] 1) Using 1 mL wide-bore gun head to gently remove the brain-like organ tissues stored at room temperature and the control group into a 15 mL centrifuge tube, try to avoid mechanical damage; slowly suck up the medium around the organoid, add 4-5 mL of 0.1% PBS-T solution each time, wash 3 times and try to remove the surrounding medium, add 2-3 mL of 4% PFA fixing solution which has been restored to room temperature to the centrifuge tube, and place it at 4°C for 16 hours or overnight.

[0071] 2) Carefully suck up the fixing solution, wash 3 times with 4-5 mL of 0.1% PBS-T solution per tube, then discard the medium, add 10 mL of pre-cooled 15% sucrose solution to the centrifuge tube, and rotate the shaker at 4°C for 16 hours or overnight.

[0072] 3) After the brain-like organ tissues settle at the bottom of the 15% sucrose solution, discard the sucrose solution, add 10 mL of pre-cooled 30% sucrose solution, and place it on the shaker at 4°C for 16 hours or overnight.

[0073] 4) Prepare a tin foil embedding box of appropriate size.

[0074] 5) Discard the 30% sucrose solution, wash once with 4-5 mL of 0.1% PBS-T solution, then add 0.4% trypan blue dye to 0.1% PBS-T solution at a ratio of 1:50, and stain the brain-like tissues for 5 minutes.

[0075] 6) Discard the trypan blue dye, wash twice with 4-5 mL of 0.1% PBS-T solution, and gently suck dry the water around the brain-like tissues.

[0076] 7) Add frozen section embedding agent O.C.T to the embedding box, gently transfer the brain-like tissues to the center of O.C.T, avoid air bubbles, add O.C.T to 3 / 4 of the embedding box, immediately transfer to dry ice for quick freezing, and store in a -80°C refrigerator after the tissue block is completely frozen.

[0077] 8) Place the tissue block in a pre-cooled freezing microtome for 30 minutes to balance the temperature, then remove the tin paper from the embedding box in the low-temperature box, use O.C.T to fix the tissue block to the tray, and after solidification, slice it on the freezing microtome at a thickness of 30 μm, and quickly adsorb the tissue section to a 0.5% gelatin pretreated glass slide, 3-4 brain-like tissues are adhered horizontally on each slide, and after baking at 37°C for 20 minutes, store in a -20°C refrigerator.

[0078] 4. Immunofluorescence staining

[0079] 1) Warm the brain-like organ frozen section to room temperature, fix it with 4% PFA fixing solution at room temperature for 15 minutes, and then wash it with 0.1% PBS-T solution for 2-3 times.

[0080] 2) Gently blot off the surrounding moisture, circle the area of the brain-like tissue with the immunohistochemistry pen, evenly cover the tissue area with tissue permeation solution containing 0.25% Triton X-100, and let it stand in a wet box at room temperature for 30 min for permeation; gently blot off the surrounding moisture, and evenly cover the tissue area with QuickBlock TM Incubate the immunostaining blocking solution at room temperature for 30 min for antigen blocking, and then gently blot off the blocking solution without washing.

[0081] 3) Dilute the primary antibody with the primary antibody diluent, select an appropriate dilution ratio to prepare the primary antibody working solution, evenly cover the tissue area, and incubate it in a wet box at 4°C for 16 h or overnight.

[0082] 4) Gently blot off the primary antibody working solution on the slice, wash it with 0.1% PBS-T solution for 3 times, dilute the fluorescent secondary antibody with the antibody diluent to prepare the working solution at an appropriate ratio, evenly cover the tissue area, and incubate it in a wet box at 37°C for 2 h.

[0083] 5) Carefully remove the secondary antibody working solution, wash it with 0.1% PBS-T solution for 1 time, add an appropriate amount of DAPI staining solution to the tissue area, and incubate it in a wet box at room temperature in the dark for 5-10 min to label the cell nucleus.

[0084] 6) Gently blot off the residual liquid on the slice, wash it with 0.1% PBS-T solution for 3 times, blot off the moisture, seal the slice with an appropriate amount of anti-fluorescence decay mounting medium, try to avoid air bubbles, seal the edges of the glass slide with nail polish, and completely dry it at room temperature in the dark.

[0085] 7) Observe it under a fluorescence microscope or a confocal microscope, or store the sealed tissue glass slide at 4°C in the dark for up to one week to prevent fluorescence quenching.

[0086] 5. Experimental results

[0087] The experimental results are shown in FIG. 1A , FIG. 1B and FIG. 2 to FIG. 4 , wherein the mature culture medium used is the brain-like organ mature culture medium STEMDIFF TM (CEREBRAL ORGANOID MATURATION MEDIUM); the TStore protective solution is a commercially available T- which can be commercially available from Life Science Production, UK.

[0088] 1) Tunel staining to detect apoptosis, this scheme compares the traditional TStore protective solution and the culture medium, and the number of cell apoptosis is significantly reduced after 10 days of storage at room temperature.

[0089] 2) Immunofluorescence detection of the expression of astrocyte marker gene ALDH1L1 and mature neuron marker NeuN, compared with traditional TStore protective solution and culture medium, the number of surviving astrocytes and mature neurons increased significantly after 10 days of storage at room temperature.

[0090] 3) Transcriptome sequencing results show that in gene clusters such as metabolism, environmental information interaction, cell process, human disease, etc., compared with traditional TStore protective solution and mature culture medium, the transcriptomic characteristics are closer to the conventional culture control after 10 days of storage at room temperature.

[0091] Reference:

[0092] Wang, Y., Wei, T., Wang, Q., Zhang, C., Li, K., & Deng, J. (2022). Resveratrol’s neural protective effects for the injured embryoid body and cerebral organoid. BMC Pharmacology & Toxicology, 23, 1-12. doi:https: / / doi.org / 10.1186 / s40360-022-00593-3

[0093] Patent: CN112586495-A

Claims

1. An organization for storing and / or transporting a medium, comprising: 1-5 μM resveratrol, and 0.5-2.5μM Enrica.

2. The tissue storage and / or transport medium according to claim 1, comprising: 2μM resveratrol, and 1μM Enriched.

3. The use of the tissue storage and / or transport medium according to claim 1 or 2 for the storage and / or transport of brain-like organs at room temperature.

4. A method for preparing a tissue storage and / or transport medium, the method comprising: injecting... Resveratrol at a final concentration of 1-5 μM and enricazone at a final concentration of 0.5-2.5 μM were added.

5. The method according to claim 4, the method comprising: ... Resveratrol and enricazone were added to a final concentration of 2 μM and 1 μM, respectively.

6. The use of the combination of resveratrol and enricane in improving the performance of tissue storage and / or transport media; wherein The tissue storage and / or transport medium is and The final concentration of resveratrol is 1-5 μM and the final concentration of enricane is 0.5-2.5 μM.

7. A method for storing and / or transporting mammalian tissue samples and cells at room temperature, the method comprising: 1) Transferring mammalian tissue samples and cells to be stored and / or transported into the tissue storage and / or transport medium according to any one of claims 1 or 2; and 2) Optionally, remove air from the tissue storage and / or transport medium.

8. The method of claim 7, wherein the mammalian tissue sample is a brain-like organ.

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