Tissue storage and / or transport medium and use 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 # imgabs0 # resveratrol and enlicasin, methods of preparation thereof, and uses thereof.

Description

Technical Field

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

[0002] Summarizing human development has always been a central doctrine 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 used the natural tendency of PSCs to aggregate into spontaneous 3D neuroectodermal structures, namely brain organoids. Brain organoid research has made significant progress in recent years. They are formed by the differentiation of human pluripotent stem cells under three-dimensional culture conditions and can simulate the cellular heterogeneity, structure and function of the human brain. These organoids have important scientific significance and application prospects in the field of regenerative medicine. They can be used to simulate brain damage, 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 freezing and resuscitation scheme for brain organoids, and it is impossible to keep cells alive during long-distance transportation and room temperature storage. In addition, the lack of vascularization in brain organoids leads to central necrosis of organoids, interfering with normal development and neuronal migration. Although some studies have provided certain solutions through co-culture with vascular stem cells, problems such as nutrient supply, cell survival in the central region, freezing, storage and cell protection during long-distance transfer, and the real blood microenvironment still exist. Summary of the invention

[0004] The present invention is proposed to solve the problem that the cell survival rate of brain organoids is significantly reduced during storage and transportation at room temperature.

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

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

[0007] T-

[0008] 0.1-10 μM resveratrol, and

[0009] 0.1-10 μM Enricain.

[0010] In a specific embodiment, the final concentration of resveratrol in the improved tissue storage and / or transport medium provided by the present invention is 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1.0 μM, 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, 1.5 μM, 1.6 μM, 1.7 μM, 1.8 μM, 1.9 μM, 2. 0μM, 2.1μM, 2.2μM, 2.3μM, 2.4μM, 2.5μM, 2.6μM, 2.7μM, 2.8μM, 2.9μM, 3.0μM, 3.1μM, 3.2μM, 3. 3μM, 3.4μM, 3.5μM, 3.6μM, 3.7μM, 3.8μM, 3.9μM, 4.0μM, 4.1μM, 4.2μM, 4.3μM, 4.4μM, 4.5μM, 4.6 μM, 4.7μM, 4.8μM, 4.9μM, 5.0μM, 5.1μM, 5.2μM, 5.3μM, 5.4μM, 5.5μM, 5.6μM, 5.7μM, 5.8μM, 5.9 μM, 6.0μM, 6.1μM, 6.2μM, 6.3μM, 6.4μM, 6.5μM, 6.6μM, 6.7μM, 6.8μM, 6.9μM, 7.0μM, 7.1μM, 7.2 μM, 7.3μM, 7.4μM, 7.5μM, 7.6μM, 7.7μM, 7.8μM, 7.9μM, 8.0μM, 8.1μM, 8.2μM, 8.3μM, 8.4μM, 8.5 μM, 8.6μM, 8.7μM, 8.8μM, 8.9μM, 9.1μM, 9.2μM, 9.3μM, 9.4μM, 9.5μM, 9.6μM, 9.7μM, 9.8μM, 9.μM 9 or 10μM; or the range between any two values ​​above.

[0011] Resveratrol is a non-flavonoid polyphenol organic compound. It is an antitoxin produced by many plants when stimulated. Its chemical formula is C 14 H 12 O3. It can be synthesized in grape leaves and grape skins and is a biologically active ingredient in wine and grape juice. It is easily absorbed orally and excreted through urine and feces after metabolism. In vitro and animal experiments have shown that resveratrol has antioxidant, anti-inflammatory, anti-cancer and cardiovascular protective effects.

[0012] In a specific embodiment, the final concentration of enlicasin in the improved tissue storage and / or transport medium provided by the present invention is 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1.0 μM, 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, 1.5 μM, 1.6 μM, 1.7 μM, 1.8 μM, 1.9 μM, 2. 0μM, 2.1μM, 2.2μM, 2.3μM, 2.4μM, 2.5μM, 2.6μM, 2.7μM, 2.8μM, 2.9μM, 3.0μM, 3.1μM, 3.2μM, 3. 3μM, 3.4μM, 3.5μM, 3.6μM, 3.7μM, 3.8μM, 3.9μM, 4.0μM, 4.1μM, 4.2μM, 4.3μM, 4.4μM, 4.5μM, 4.6 μM, 4.7μM, 4.8μM, 4.9μM, 5.0μM, 5.1μM, 5.2μM, 5.3μM, 5.4μM, 5.5μM, 5.6μM, 5.7μM, 5.8μM, 5.9 μM, 6.0μM, 6.1μM, 6.2μM, 6.3μM, 6.4μM, 6.5μM, 6.6μM, 6.7μM, 6.8μM, 6.9μM, 7.0μM, 7.1μM, 7.2 μM, 7.3μM, 7.4μM, 7.5μM, 7.6μM, 7.7μM, 7.8μM, 7.9μM, 8.0μM, 8.1μM, 8.2μM, 8.3μM, 8.4μM, 8.5 μM, 8.6μM, 8.7μM, 8.8μM, 8.9μM, 9.1μM, 9.2μM, 9.3μM, 9.4μM, 9.5μM, 9.6μM, 9.7μM, 9.8μM, 9.μM 9 or 10μM; or the range between any two values ​​above.

[0013] Enricasin is a pan-CASPASE pathway inhibitor that can inhibit Zika virus (ZIKV)-induced increase in CASPASE-3 activity and protect human cortical neural progenitor cells, with neuroprotective and anti-inflammatory effects.

[0014] Tissue Storage and Transportation Medium (T-Store) Protective Fluid is osmotic, isotonic, and plasma-buffered with human serum and interstitial fluid to create a steady-state environment that allows tissue samples to retain genetic and histological characteristics for up to 72 hours.

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

[0016] In a specific embodiment of the present invention, in T- Adding appropriate concentrations of resveratrol and enricasin to tissue storage and transport media can significantly alleviate cell apoptosis in brain-like organs at room temperature and protect neurons and astrocytes.

[0017] Therefore, the tissue storage and / or transportation medium provided by the present invention can be used to store and / or transport brain organs at room temperature.

[0018] In a second aspect, the present invention provides a method for preparing a tissue storage and / or transport medium, the method comprising: Resveratrol and enricasin are added.

[0019] In a specific embodiment, the present invention provides a method for preparing a tissue storage and / or transport medium, the method comprising: Resveratrol and enlicasin were added to a final concentration of 0.1-10 μM.

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

[0021] In a specific embodiment, the present invention provides a combination of resveratrol and enlicasin for improving T- performance of the application.

[0022] In a fourth aspect, the present invention provides a method for storing and / or transporting mammalian tissue samples and cells at room temperature, the method comprising:

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

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

[0025] In a specific embodiment, the present invention provides a method for storing and / or transporting a brain organoid at room temperature, the method comprising:

[0026] 1) transferring the brain organoid to be stored and / or transported into the improved tissue storage and / or transport medium according to the present invention; and

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

[0028] In a fifth aspect, the present invention provides a use of a combination of resveratrol and enlicasin in the preparation of a medium for storage and / or transportation of mammalian tissue samples and cells at room temperature.

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

[0030] In a specific embodiment, the use of the present invention comprises providing T- Resveratrol and enlicasin were added to a final concentration of 0.1-10 μM. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1A and Figure 1B These are the apoptosis staining results and statistical graphs of brain organoids after storage in the maturation culture medium in the brain organoid maturation kit, Tstore protective solution, and protective solution with the small molecules listed in this protocol at room temperature for 5, 7, and 10 days, respectively.

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

[0033] Figure 3 This is a heat map of differential gene clustering after brain organoids were stored at room temperature for 10 days in maturation culture medium, Tstore protective solution, and protective solution with small molecules listed in this protocol.

[0034] Figure 4 GO and KEGG analysis of differentially expressed genes after brain organoids were stored in maturation medium, Tstore protective solution, and protective solution with the small molecules listed in this protocol at room temperature for 10 days. DETAILED DESCRIPTION

[0035] In order to explain the present invention in more detail, this specification provides the following specific embodiments, and illustrates these specific embodiments in conjunction with the drawings, but the scheme of the present disclosure is not limited thereto. Those skilled in the art can make appropriate changes to the method and use of the present invention in conjunction with the common knowledge in the art, as long as it can achieve the functions described in the present invention, that is, it should fall within the scope of the present invention. If not specifically specified, the reagents used in the following examples can be purchased commercially.

[0036] Example 1. Cultivation of induced pluripotent stem cells (IPSCs)

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

[0038] 1. Experimental Materials and Reagents

[0039] Table 1

[0040]

[0041] mTeSR was used according to the manufacturer's instructions and conventional procedures in the art. TM Plus medium, passaged in GCDR, and cryopreserved in KOSR+DMSO.

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

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

[0044] 1. Experimental Materials and Reagents

[0045] Table 2

[0046]

[0047]

[0048] Example 3. Storage of brain organoids at room temperature

[0049] 1. Experimental Materials and Reagents

[0050] Table 3

[0051]

[0052] 1) Add small molecule resveratrol and emricasan to the T-Store protective solution, so that the final concentration of resveratrol is 2 μM and the final concentration of emricasan is 1 μM, to prepare a room temperature protective solution for brain organs.

[0053] 2) Aspirate and discard the culture medium around the spheroids as much as possible, wash once with DPBS, aspirate and discard the DPBS as much as possible, and use a 200μL wide-mouth pipette or Pasteur tube to transfer the spheroids to a 1.5mL EP tube. Fill the EP tube with room temperature protective solution for brain organs, and try not to retain air.

[0054] 3) Seal the tube mouth with sealing film and transfer the EP tube containing the spheroids to a dark place for storage.

[0055] 4) After storage or transportation at room temperature, discard the surrounding medium of the brain organoids as much as possible, wash once with DPBS, discard the DPBS as much as possible, and use a 200μL wide-mouth pipette tip or Pasteur tube to transfer the brain organoids to the brain 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) Prepare 0.1% PBS-T solution

[0062] Slowly draw 500 μL of Tween-20 stock solution with a 1 mL wide-mouth pipette tip, add it to 499.5 mL of PBS phosphate buffer, shake well to prepare 500 mL of 0.1% PBS-T solution, and store at room temperature.

[0063] 2) Prepare permeabilization solution containing 0.25% TritonX-100

[0064] Use a 200 μL wide-mouth pipette tip to slowly draw 125 μL of Triton X-100 stock solution, add it to 49.875 mL of PBS phosphate buffer, mix thoroughly to prepare 50 mL of tissue permeabilization solution containing 0.25% Triton X-100, and store it at room temperature.

[0065] 3) Prepare 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 thoroughly until completely dissolved, then dilute to 40 mL to prepare a 15% sucrose solution, and store it at 4°C for later use; weigh 12 g of sucrose on an electronic balance, dissolve it in 40 mL of PBS phosphate buffer, stir thoroughly until dissolved, then dilute to 40 mL to prepare a 30% sucrose solution, and store it at 4°C for later use.

[0067] 4) Prepare 0.5% gelatin solution

[0068] Use sterile ultrapure water to dilute the 1% gelatin solution 1:1 to 0.5% gelatin solution, use 2mL EP tubes to dispense 1mL / tube, and store at -20℃ for use.

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

[0070] 1) Use a 1 mL wide-mouth pipette tip to gently remove the brain organoid tissues stored at room temperature and the control group into a 15 mL centrifuge tube, trying to avoid mechanical damage; slowly aspirate the medium surrounding the organoids, 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 fixative that has been restored to room temperature to the centrifuge tube, and fix at 4°C for 16 hours or overnight.

[0071] 2) Carefully aspirate the fixative, add 4-5 mL of 0.1% PBS-T solution to each tube and wash three times, then discard the medium, add 10 mL of pre-cooled 15% sucrose solution to the centrifuge tube, and dehydrate on a rotary shaker at 4°C for 16 hours or overnight.

[0072] 3) After the brain organoid tissue settles to the bottom of the 15% sucrose solution, the sucrose solution is discarded, and 10 mL of pre-cooled 30% sucrose solution is added, and the tissue is placed on a shaker at 4°C for dehydration for 16 hours or overnight.

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

[0074] 5) The 30% sucrose solution was discarded, and the tissue was washed once with 4-5 mL of 0.1% PBS-T solution. Then, 0.4% trypan blue dye was added to 0.1% PBS-T solution at a ratio of 1:50, and the brain tissue was stained for 5 minutes.

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

[0076] 7) Add the frozen section embedding agent OCT into the embedding box, gently transfer the brain-like tissue to the center of the OCT to avoid bubbles, add OCT to 3 / 4 of the embedding box, and immediately transfer to dry ice for quick freezing. After the tissue block is completely frozen, store it in a -80℃ refrigerator for later use.

[0077] 8) Place the tissue block in a pre-cooled cryostat for 30 minutes to balance the temperature of the two. Then remove the tin foil of the embedding box in the low-temperature box, use OCT to fix the tissue block to the tray, and slice it on the cryostat at a thickness of 30 μm after solidification. The tissue slices are quickly adsorbed onto a 0.5% gelatin pretreated slide. 3-4 brain-like tissues are horizontally adhered to each slide. After baking at 37°C for 20 minutes, store in a -20°C refrigerator.

[0078] 4. Immunofluorescence Staining

[0079] 1) The frozen sections of brain organoids were warmed to room temperature, fixed with 4% PFA fixative for 15 min at room temperature, and then washed 2-3 times with 0.1% PBS-T solution.

[0080] 2) Gently absorb the surrounding water, circle the brain-like tissue area with an immunohistochemical pen, evenly cover the tissue area with a tissue permeabilization solution containing 0.25% TritonX-100, and place it in a wet box at room temperature for 30 minutes for permeabilization; gently wipe the surrounding water, and use QuickBlock TM Incubate the sections with immunostaining blocking solution at room temperature for 30 min to block the antigen, then gently wipe off the blocking solution without washing.

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

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

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

[0084] 6) Gently wipe off the residual liquid on the slices, wash with 0.1% PBS-T solution for 3 times, wipe off the moisture, use an appropriate amount of anti-fluorescence attenuation mounting medium to seal the slices, try to avoid bubbles, seal the edges of the coverslip with nail polish, and dry completely at room temperature away from light.

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

[0086] 5. Experimental Results

[0087] The experimental results are as follows Figure 1A , Figure 1B and Figures 2 to 4 As shown, the maturation medium used is the brain organoid maturation medium STEMDIFF TM (CEREBRAL ORGANOID MATURATION MEDIUM); TStore protective liquid is commercially available T- Commercially available from Life Science Production, UK.

[0088] 1) Tunel staining was used to detect cell apoptosis. Compared with the traditional TStore protective solution and culture medium, the number of apoptotic cells in this protocol was significantly reduced after 10 days of storage at room temperature.

[0089] 2) Tissue immunofluorescence was used to detect the expression of astrocyte marker gene ALDH1L1 and mature neuron marker NeuN. Compared with the traditional TStore protective solution and culture medium, the survival number of astrocytes and mature neurons in this protocol increased significantly after 10 days of storage at room temperature.

[0090] 3) The transcriptome sequencing results showed that in gene clustering such as metabolism, environmental information interaction, cellular process, and human disease, the transcriptomic characteristics of this protocol were closer to the conventional culture control after 10 days of storage at room temperature compared with the traditional TStore protective solution and mature culture medium.

[0091] References:

[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 cerebralorganoid.BMC Pharmacology&Toxicology,23,1-12.doi:https: / / doi.org / 10.1186 / s40360-022-00593-3

[0093] Patent: CN112586495-A

Claims

1. A tissue storage and / or transport medium comprising T- Resveratrol and enricasin.

2. The tissue storage and / or transport medium according to claim 1, comprising: T- 0.1-10 μM resveratrol, and 0.1-10 μM Enricasen; Preferably, it comprises: T- 1-5 μM resveratrol, and 0.5-2.5 μM Enricasen; More preferably, it comprises: T- 2 μM resveratrol, and 1 μM Enricain.

3. The tissue storage and / or transport medium according to claim 1 or 2, which is used for storing and / or transporting brain organs at room temperature.

4. The tissue storage and / or transport medium according to claim 1 or 2, wherein the tissue storage and / or transport medium is combined with T- In comparison, it significantly alleviates cell apoptosis in brain-like organs at room temperature and protects neurons and astrocytes.

5. A method for preparing a tissue storage and / or transport medium, the method comprising: Resveratrol and enricasin are added.

6. The method according to claim 5, comprising providing T- Add resveratrol and enlicasin to a final concentration of 0.1-10 μM; Preferably, resveratrol and enricasin are added at a final concentration of 1-5 μM and 0.5-2.5 μM, respectively; More preferably, resveratrol is added at a final concentration of 2 μM and enricasin is added at a final concentration of 1 μM.

7. Use of a combination of resveratrol and enlicasin for improving the performance of tissue storage and / or transport media; Preferably, the tissue storage and / or transport medium is T- 8. 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 improved tissue storage and / or transport medium according to the present invention; and 2) Optionally, removing air from the improved tissue storage and / or transport medium.

9. The method of claim 8, wherein the mammalian tissue sample is a brain organoid.

10. Use of a combination of resveratrol and enlicasin in the preparation of a medium for storage and / or transportation of mammalian tissue samples and cells at room temperature, preferably, the mammalian tissue sample is a brain organoid.

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