A tissue preservation and transportation device and a tissue preservation reagent

Abstract

The present application belongs to the technical field of tissue transportation, and particularly relates to a tissue preservation and transportation device and a tissue preservation reagent, which aims to solve the technical problem that the activity of living tissue is reduced or the living tissue is dead after transportation in the prior art, and it is difficult to retain the original tissue characteristics. The tissue preservation and transportation device comprises a tissue preservation reagent and a preservation tube. The tissue preservation reagent comprises a temperature-sensitive gel, an antioxidant stress component and a tissue preservation solution. The preservation tube is used for containing the tissue preservation reagent. The tissue preservation and transportation device and the tissue preservation reagent utilize the tissue tube and the tissue preservation reagent to effectively buffer the jolt suffered by the tissue during transportation. The characteristics of the temperature-sensitive gel facilitate the transfer of the tissue block and the removal of the tissue block. The antioxidant stress component can keep the tissue characteristics unchanged. The tissue preservation solution can keep the tissue cell activity, and plays a good protection role on the tissue during transportation. The tissue preservation and transportation device provides a simple and effective method for tissue preservation and transportation.

Description

Technical Field

[0001] This invention belongs to the field of tissue transport technology, specifically relating to a tissue preservation and transport device and a tissue preservation reagent. Background Technology

[0002] When performing multidimensional analysis on tissue samples, it is often necessary to send them to specialized external institutions for testing, such as gene sequencing, proteomics, transcriptomics, metabolomics, epigenome analysis, single-cell sequencing, organoid creation, and tissue microarray creation. Among these, single-cell sequencing, organoid creation, and tissue microarray creation are particularly critical in ensuring the freshness and stable viability of the tissues. Therefore, maintaining the tissue's viability and its in vivo condition during transport is of paramount importance.

[0003] Some methods involve preservation using gradient cryopreservation or liquid nitrogen flash freezing, followed by dry ice transport. The advantage is long preservation time; a suitable cryopreservation environment can sustain preservation for several years. However, cryopreservation may cause some cell death, leading to reduced activity after dissociation or thawing. The cryopreservation and thawing process can alter the transcriptional levels of tissue cells and may even cause an increase in background RNA in the suspension after dissociation, affecting the smooth progress of subsequent experiments and the validity and accuracy of the detection data. Some methods utilize live tissue transport. The advantage is that it can maintain cell viability as much as possible while removing the rapidly metabolic environment in vivo, allowing the tissue to retain its original tissue characteristics (including the genome and proteome) and avoiding cell loss caused by cryopreservation and thawing. However, the simple in vitro preservation solutions cannot fully match the complexity of in vivo conditions, and mechanical damage is also a significant cause of cell loss. Summary of the Invention

[0004] This invention provides a tissue preservation and transportation device and a tissue preservation reagent, aiming to solve the technical problem in the prior art where the activity of living tissue decreases or dies after transportation, making it difficult to preserve the original tissue characteristics.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides a tissue preservation and transportation device, comprising a tissue preservation reagent and a preservation tube;

[0007] The tissue preservation reagent includes a thermosensitive gel, an antioxidant stress component, and a tissue preservation solution;

[0008] The preservation tube is used to hold tissue preservation reagents.

[0009] Preferred option: The temperature-sensitive gel is type B gelatin derived from pigskin with a gel strength of 180-250.

[0010] A preferred embodiment: the concentration of the thermosensitive gel in the tissue preservation reagent is 2.5-4%.

[0011] Preferred formulation: The antioxidant stress-reducing components include vitamin C, flavonoids, and polyphenols.

[0012] Preferred embodiment: In the tissue preservation reagent, the concentration of vitamin C is 3-8 μg / mL, the concentration of flavonoids is 200-300 μg / mL, and the concentration of polyphenols is 8-15 μg / mL.

[0013] Based on the above scheme, the thermosensitive gel can protect the integrity of transport tissues when it is in a solidified gel state; the vitamin C, flavonoids and polyphenols can reduce the concentration of reactive oxygen species, resist oxidative stress, and maintain the vitality of tissue cells within 48 hours; the tissue preservation solution can maintain the activity of tissue cells and reduce cell metabolism, thus maintaining the stability of gene expression.

[0014] Preferred option: The tissue preservation reagent contains antibiotics.

[0015] Based on the above approach, antibiotics are used to protect tissue cells from microbial contamination.

[0016] A preferred embodiment is provided with a threaded opening at the upper end of the storage tube, a storage tube cap on the threaded opening, and a protective sleeve on the outside of the storage tube.

[0017] Based on the above solution, the preservation tube seals and preserves the tissue, and the outer protective sleeve of the preservation tube can effectively buffer the bumps and collisions that the tissue tube is subjected to during transportation, ensuring the integrity of the transported tissue.

[0018] Preferred embodiment: The temperature-sensitive gel is in a gel state when refrigerated at a temperature of 2-8℃, and in a liquid state when the room temperature is above 25℃.

[0019] Based on the above scheme, the gel is solidified at a refrigeration temperature of 2-8℃. The solidified gel temperature-sensitive gel can effectively protect the tissue and avoid mechanical damage caused by bumps and impacts during transportation. At a room temperature of above 25℃, it is liquid, which helps the tissue to move into the tissue tube. After transportation, the tissue can be removed at room temperature.

[0020] Secondly, the present invention provides a tissue preservation reagent, comprising a thermosensitive gel, an antioxidant stress component, and a tissue preservation solution.

[0021] Preferred method: The preparation steps of the tissue preservation reagent are as follows:

[0022] S1. Place the temperature-sensitive biohydrogel lyophilized powder and PBS balanced salt buffer into a centrifuge tube, and dissolve the temperature-sensitive biohydrogel lyophilized powder completely by heating in a water bath to obtain the temperature-sensitive gel.

[0023] S2. Before the temperature drops, filter and sterilize to obtain a sterile temperature-sensitive gel solution;

[0024] S3. Once the temperature of the thermosensitive gel solution drops below 37°C, add antibiotics, tissue preservation solution, and antioxidant stress-reducing components and mix thoroughly to obtain the tissue preservation reagent.

[0025] Based on the above scheme, in step S1 of the preparation of the tissue preservation reagent, the use of PBS balanced salt buffer can provide a relatively stable ionic environment and maintain a suitable pH. The temperature-sensitive biogel lyophilized powder is rapidly dissolved by water bath heating.

[0026] The beneficial effects of this invention are as follows: This invention provides a tissue preservation and transportation device and a tissue preservation reagent. Utilizing tissue tubes and the tissue preservation reagent, it protects the integrity of the tissue, effectively buffers the bumps and impacts experienced during transportation, and avoids mechanical damage to the tissue. The tissue preservation reagent includes a temperature-sensitive gel, an antioxidant component, and a tissue preservation solution. The temperature-sensitive gel exhibits different states at different temperatures, facilitating the transfer of tissue blocks. The antioxidant component can reduce the concentration of reactive oxygen species, maintain cell viability within 48 hours, and prevent changes in tissue characteristics. The tissue preservation solution can maintain tissue cell activity, reduce cell metabolism, and maintain stable gene expression. This invention solves the technical problem in existing technologies where the activity of living tissue decreases or dies after transportation, making it difficult to preserve the original tissue characteristics. It provides excellent protection for the tissue during transportation and offers a simple and effective method for tissue preservation and transportation. Attached Figure Description

[0027] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is a schematic diagram of an organization-preserved transportation device.

[0029] Figure 2 This is a diagram showing the state of tissue preserved at 4°C in a tissue preservation reagent in a gel state.

[0030] Figure 3 This is a diagram showing the state of tissue preserved at 37°C in a liquid tissue preservation reagent.

[0031] Figure 4 This is a diagram of the tissue after it has been preserved in a tissue preservation reagent for 24 hours and subjected to simulated shock treatment.

[0032] Figure 5 This is a diagram of the tissue after it has been preserved in a standard tissue preservation solution for 24 hours and then subjected to simulated turbulence treatment.

[0033] Figure 6 This is a comparison chart showing the viability of cells harvested after tissue preservation and processing using tissue preserved in tissue preservation reagents and ordinary tissue preservation solutions.

[0034] Figure 7 This is a comparison chart showing the total number of live cells harvested after 24 hours of tissue preservation using tissue preservation reagents and ordinary tissue preservation solutions.

[0035] Figure 8 These are morphological images of the cells harvested from each group and cultured as organoids under a light microscope.

[0036] Figure 9 This is a comparison chart of organoid activity detection results after organoid culture of cells harvested from each group.

[0037] Figure 10 This is a comparison chart showing the proliferation of tissue cells in tissue preservation reagents and tissue cells in ordinary tissue preservation solutions.

[0038] Figure 11 This is a bar chart comparing the intracellular ROS content detection results after different treatment groups.

[0039] Explanation of the labels in the diagram:

[0040] 1-Storage tube cap; 2-Protective sleeve; 3-Storage tube. Detailed Implementation

[0041] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the protection scope of the present invention.

[0042] Example 1:

[0043] This embodiment provides a tissue preservation and transportation device, including a tissue preservation reagent and a preservation tube;

[0044] The tissue preservation reagent includes a thermosensitive gel, an antioxidant stress component, and a tissue preservation solution;

[0045] The preservation tube is used to hold tissue preservation reagents.

[0046] The thermosensitive gel is type B gelatin derived from pigskin with a gel strength of 180-250.

[0047] In the tissue preservation reagent, the concentration of the thermosensitive gel is 2.5%-4%.

[0048] Specifically, the thermosensitive gel, a type B gelatin derived from pigskin with a gel strength of 180-250, has high transparency, making it easy to observe, and can effectively protect tissues when it is in a gel-like state.

[0049] The antioxidant stress-reducing components include vitamin C, flavonoids, and polyphenols.

[0050] In the tissue preservation reagent, the concentration of vitamin C is 3-8 μg / mL, the concentration of flavonoids is 200-300 μg / mL, and the concentration of polyphenols is 8-15 μg / mL.

[0051] Specifically, free radicals are constantly generated during tissue cell metabolism. The harmful substances produced by free radicals can cause irreversible oxidative damage to the structure and function of tissue cells. Therefore, the aforementioned antioxidant stress components, including vitamin C, flavonoids, and polyphenols, are added to the tissue preservation reagent. Vitamin C, as a natural and powerful antioxidant, has reducing properties and eliminates the toxicity of harmful oxygen free radicals through reduction. Flavonoids exert their antioxidant effects by inhibiting the production of free radicals, directly eliminating free radicals, and increasing antioxidant factors in tissues. Polyphenols exert their antioxidant effects by scavenging free radicals, chelating metal ions, and inhibiting oxidase activity. This ensures that tissue cell viability is maintained within 48 hours of transport, and the expression of tissue surface antigens and stress-related genes remains largely unchanged.

[0052] To ensure that the tissue preservation reagent is not contaminated with microorganisms in the above scheme, the tissue preservation reagent contains antibiotics.

[0053] Specifically, the antibiotic can be 100 mg / ml Primocin. TM Antibiotic, Primocin TM Antibiotics can protect primary tissue cells from microbial contamination and have a killing effect on Gram-positive bacteria, Gram-negative bacteria, mycoplasma, and fungi.

[0054] The tissue preservation reagent is loaded into a preservation tube, the upper end of which is provided with a threaded opening, a preservation tube cap is provided on the threaded opening, and a protective sleeve is provided on the outside of the preservation tube.

[0055] For details, please refer to Figure 1 The protective cap 1 in the preservation tube seals and preserves the tissue. The protective sleeve 2 has a hollowed-out mesh structure. The preservation tube 3 is sleeved inside the protective sleeve 2, which can effectively buffer the bumps and collisions that the preservation tube 3 is subjected to during transportation and ensure the integrity of the tissue.

[0056] The temperature-sensitive gel is in a gel state when refrigerated at a temperature of 2-8°C, and in a liquid state when the room temperature is above 25°C.

[0057] Specifically, before transportation, the thermosensitive gel is in a liquid state at room temperature above 25°C. It is then combined with antioxidant stress-relieving components and tissue preservation solution to prepare a tissue preservation reagent, which can be directly injected into the tissue preservation tube. The tissue is placed inside, ensuring the tissue preservation reagent completely submerges the tissue. After sealing the tube, transportation is carried out at 2-8°C. At this temperature, the thermosensitive gel solidifies into a gel-like state, effectively protecting the tissue and preventing mechanical damage caused by bumps and impacts during transportation. After transportation, the tissue can be easily removed at room temperature above 25°C.

[0058] Example 2:

[0059] This embodiment provides a tissue preservation reagent, including a thermosensitive gel, an antioxidant stress component, and a tissue preservation solution.

[0060] The preparation steps of the tissue preservation reagent are as follows:

[0061] S1. Place the temperature-sensitive biohydrogel lyophilized powder and PBS balanced salt buffer into a centrifuge tube, and dissolve the temperature-sensitive biohydrogel lyophilized powder completely by heating in a water bath to obtain the temperature-sensitive gel.

[0062] S2. Before the temperature drops, filter and sterilize to obtain a sterile temperature-sensitive gel solution;

[0063] S3. Once the temperature of the thermosensitive gel solution drops below 37°C, add antibiotics, tissue preservation solution, and antioxidant stress-reducing components and mix thoroughly to obtain the tissue preservation reagent.

[0064] Specifically, in step S1, the high-concentration thermosensitive gel solution is 10-15%. In step S3, the obtained tissue preservation reagent contains: a thermosensitive gel concentration of 2.5%, and the antibiotic can be 100 mg / ml Primocin. TM The concentration of vitamin C added is 3-8 μg / mL, the concentration of flavonoids added is 200-300 μg / mL, and the concentration of polyphenols added is 8-15 μg / mL.

[0065] To more clearly describe the present invention, further description is provided below in conjunction with specific experiments.

[0066] 1. To verify that tissues can be easily transferred in and out of the preservation reagent in this invention, and to reduce mechanical damage during tissue transport, the following experiments were conducted:

[0067] 1.1 Subcutaneous xenograft tumor tissue sampling

[0068] Preparation: Ophthalmic scissors and forceps are sterilized under high temperature and high pressure beforehand; the ultra-clean workbench is pre-cooled and exposed to UV light for 30 minutes before use; ice packs, ice, a micro electronic scale, and a container of 100mg / ml antibiotic (Primocin) are prepared. TM Prepare sterile PBS buffer (4°C) and sterile 60mm culture dishes for later use.

[0069] The specific steps for extracting xenograft tumor tissue are as follows:

[0070] S1. Nude mice with subcutaneous xenografted tumor tissue were euthanized by cervical dislocation, immersed in alcohol for 3-5 seconds, and then placed in a clean bench.

[0071] S2. Use ophthalmic forceps to grasp the lower edge of the subcutaneous tumor tissue about 0.5cm and cut a small opening with ophthalmic scissors. Then bluntly separate the nude mouse skin and subcutaneous tissue or tumor tissue.

[0072] S3. Gently lift the blunt-dissected skin upwards and cut it open with ophthalmic scissors, taking care to avoid damaging the tumor tissue. After fully exposing the tumor tissue, completely dissect the entire tumor tissue with ophthalmic scissors and then place it in a 60mm culture dish for later use.

[0073] 1.2 Preparation of tissue preservation reagents

[0074] Preparation: Analytical balance and clean bench with UV light pre-activated for 30 minutes; antibiotics containing 100 mg / ml (Primocin) are prepared. TM The tissue preservation solution was refrigerated at 4°C for later use, along with antibiotics (Primocin). TM ), PBS balanced salt buffer, temperature-sensitive bio-hydrogel lyophilized powder, 0.22μm filter, pre-opened water bath with temperature set to 50℃, vitamin C additive, flavonoids and polyphenols.

[0075] The specific steps for preparing tissue preservation reagents are as follows:

[0076] S1. Weigh 1g of thermosensitive biohydrogel lyophilized powder and transfer it to a 50mL centrifuge tube. Add 10mL of PBS solution to the tube, tighten the cap, and place the tube in a 50℃ water bath to heat and dissolve. Shake the tube every 2-3 minutes until all the thermosensitive biohydrogel lyophilized powder is completely dissolved to obtain a 10% hydrogel solution.

[0077] S2. Before the temperature drops, quickly filter the 10% photocurable biohydrogel solution obtained in step S1 using a 0.22μm filter to sterilize it, and collect the sterile 10% thermosensitive gel solution.

[0078] S3. In a clean bench, wait for the temperature of the sterile 10% thermosensitive gel solution to drop below 37°C to obtain a thermosensitive gel solution. Add 20 mL of tissue preservation solution to the thermosensitive gel solution, then add antibiotics, vitamin C, flavonoids and polyphenols. Finally, add tissue preservation solution to make up to 40 mL. The final tissue preservation reagent has the following concentrations: thermosensitive gel solution concentration 2.5%, antibiotic concentration 100 mg / mL, vitamin C concentration 3-8 μg / mL, flavonoid concentration 200-300 μg / mL, and polyphenol concentration 8-15 μg / mL.

[0079] 1.3 The specific steps of the organization preservation and transportation process simulation are as follows:

[0080] Preparation: Preheat the biosafety cabinet to UV light for 30 minutes; preheat the water bath to 37°C; prepare tissue preservation solution; 5mL preservation tubes; pipettes; pipette tips; and a 4°C refrigerator.

[0081] The specific steps for simulating the tissue preservation and transportation process are as follows:

[0082] S1. Add the tissue preservation reagent to the preservation tube in the biosafety cabinet, adding 3 ml to each tube, and place the preservation tube in a 37°C water bath for insulation.

[0083] S2. In a biosafety cabinet, divide the tumor tissue in a 60mm dish into equal portions, weigh each tissue portion, place them in a storage tube, label them, and store them in a 4°C refrigerator.

[0084] S3. Set up a control group. In the control group, only tissue preservation solution is added to the preservation tubes, and all other operating conditions are the same.

[0085] S4. Shake and vibrate the storage tube once every 1 hour to simulate the bumps during transportation.

[0086] Experimental results:

[0087] Figure 2 and Figure 3 Tissues preserved in a gel state at 4°C and in a liquid state at 37°C are shown, respectively. (See also...) Figure 2 After immersing the tissue block in the tissue preservation reagent, it sinks to the bottom and, when placed at 4°C, transforms from a solution to a gel state within 2-5 minutes. (See also...) Figure 3 Tissue preservation reagents in gel state can be restored to solution state after being heated in a 37°C water bath for 2-5 minutes.

[0088] Figure 4 and Figure 5 The tissue samples were prepared in tissue preservation reagent and in ordinary tissue preservation solution, respectively. Both samples were preserved for 24 hours and subjected to the same simulated turbulence treatment. Figure 2 After being heated in a 37°C water bath for 24 hours, the tissue preservation reagent was clear, with no obvious turbidity or tissue fragments, and the tissue fragments had clear outlines. Figure 3 Compared to tissues preserved in ordinary tissue preservation solution under the same treatment conditions, tissue preservation solution was significantly cloudy and contained numerous tissue fragments. This demonstrates that tissue preservation reagents can effectively buffer the impact of bumps and knocks during transportation, reducing mechanical damage to the tissues.

[0089] 2. To determine the activity of tissue cells after tissue transport, the changes in cell activity, and the total number of viable cells, the following experiment was conducted:

[0090] Preparation: Preheat the biosafety cabinet to UV light for 30 minutes; preheat the water bath to 37°C; prepare a container containing 100 mg / ml of antibiotic (Primocin). TM Sterile PBS buffer, centrifuge tubes, tissue processing solution, AO / PI dual-fluorescence cell live / dead staining reagent, disposable cell counting chamber, automated cell counter, 100μm cell sieve, EP tubes, pipettes, and pipette tips.

[0091] The specific steps for organizational processing are as follows:

[0092] S1. After transportation, remove the tissue tubes and heat them in a 37°C water bath. Once the tissue preservation reagent changes from a gel to a solution, open it in the biosafety cabinet of the cell culture room. Then, immerse the tissue block in the tissue preservation reagent with a solution containing 100 mg / ml of primordial antibiotics. TM Rinse 5-10 times with sterile PBS buffer, then trim and remove excess fat and connective tissue with ophthalmic scissors, and finally mince the tissue into tissue clumps with a single volume not exceeding 0.5 mm.

[0093] S2. Transfer the shredded tissue mass to a sterile tube, add tissue processing solution, tighten the sterile tube cap, and place it at a constant temperature of 37°C for 60 minutes to mechanically pulverize and obtain tissue fluid.

[0094] S3. Place the 100μm cell sieve in a 60mm culture dish, pour the obtained tissue fluid into the cell sieve, add culture medium, and gently blow and agitate the tissue fluid in the cell sieve until the tissue fluid is basically filtered into the culture dish.

[0095] S4. Transfer the tissue fluid from the culture dish to a 15mL centrifuge tube, centrifuge at 1500rpm for 5 minutes, discard the supernatant, and retain the tissue pellet. Add 5-7mL of a 100mg / ml antibiotic (Primocin) to the tissue pellet. TMResuspend the cells in sterile PBS buffer and mix well to obtain a cell suspension. Take 10 μL of the cell suspension and mix it thoroughly with AO / PI dual-fluorescence cell live / dead staining reagent. Take 10 μL of the mixture and add it to a disposable cell counting chamber. Count the cells using an automated cell counter. Record the total number of live cells and cell viability.

[0096] Experimental results:

[0097] Figure 6 The cell viability is shown for tissues preserved in the tissue preservation reagent and for tissues preserved in ordinary tissue preservation solution, followed by tissue treatment and cell viability. The cell viability after 24 hours of preservation with the ordinary tissue preservation reagent was approximately 58.35%, while the cell viability after 24 hours of preservation with the tissue preservation reagent was approximately 68.71%. This demonstrates that the tissue preservation reagent has a superior ability to protect tissues compared to the ordinary tissue preservation reagent (n=3).

[0098] Figure 7 The total number of viable cells harvested after 24 hours of tissue preservation using tissue preservation reagent and tissue preserved in ordinary tissue preservation solution. The effect of removal quality on the total number is clearly shown; after 24 hours of preservation, the viable cell density of tissue preserved in ordinary tissue preservation solution is 2.20*102. 7 g / cell, the tissue preservation reagent preserves tissue with a viable cell density of 3.88*10⁻⁶ cells / cell. 7 g / cell, which can yield more live cells compared to ordinary tissue preservation solution (n=3).

[0099] 3. To determine the cell morphology, viability, and proliferation rate of the tissue after transport and culture, the following experiments were conducted:

[0100] 3.1 Organoid tissue cell culture

[0101] Preparation: Preheat the biosafety cabinet to UV light for 30 minutes, preheat the water bath to 37°C, prepare centrifuge tubes and substrate gel to thaw at 4°C, prepare ice packs, ice, tissue processing solution, EP tubes, pipettes, pipette tips, and organoid culture medium.

[0102] The specific steps for organoid tissue culture are as follows:

[0103] S1. Centrifuge the cell suspension obtained from tissue processing at 1500 rpm for 5 minutes to finally harvest the cell pellet. Remove the matrix gel from 4°C and place it on ice for later use.

[0104] S2. Take an appropriate amount of organoid culture medium to resuspend the cells according to the number of live cells, then take an appropriate amount of cell suspension and add it to the EP tube, place it on ice for later use. Calculate the amount of matrix gel according to the number of wells of the plate, add the matrix gel to the EP tube, and finally add an appropriate amount of organoid culture medium and mix the cell-gel suspension evenly.

[0105] S3. Add the well-mixed cell-gel suspension to a 96-well plate. After the droplets solidify under temperature-sensitive conditions, add an appropriate amount of organoid culture medium and place the plate in an incubator for incubation. Observe and record the results regularly by taking photos.

[0106] 3.2 Detection of organoid tissue cell viability

[0107] Preparation: Preheat the clean bench to UV light for 30 minutes before use; prepare an ELISA reader (chemiluminescence); prepare cell viability assay reagents; prepare pipettes, pipette tips; prepare a horizontal shaker and an inverted microscope.

[0108] The specific steps for organoid activity testing are as follows:

[0109] S1. Turn on the ultraviolet light in the clean bench for 30 minutes beforehand. Take the organoid culture plate out of the incubator and place it on the clean bench at room temperature for 10 minutes. Observe the organoid culture under an inverted microscope to check for contamination.

[0110] S2. Add an appropriate amount of cell viability assay reagent to the organoid culture plate in a biosafety cabinet and react at room temperature in a horizontal shaker in the dark for 30 minutes.

[0111] S3. Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the organoid culture plates after the reaction and calculate the results.

[0112] Experimental results:

[0113] Figure 8 The results of organoid culture were observed under a light microscope for the harvested cells from each group. On day 2 of culture, typical organoid morphology was observed in both the ordinary tissue preservation solution group and the tissue preservation reagent group, which were round or nearly round structures with a diameter of 30-100 μm. The organoids in the tissue preservation reagent group were larger.

[0114] Figure 9 The results of organoid viability assays were performed on the harvested cells from each group after organoid culture. Cells were plated and cultured at the same density, and cell viability was assessed at 3 and 7 days of culture. Figure 7 It can be seen that the cell viability of cells obtained after 24 hours of preservation with tissue preservation reagent is significantly higher than that of cells obtained after organoid culture.

[0115] like Figure 10As shown, after 3 days of culture, the cell proliferation rate of cells treated with the tissue preservation reagent for 24 hours was 200.69% when used for organoid culture, while the cell proliferation rate of cells treated with ordinary tissue preservation solution for 24 hours was 155.33%. The organoid proliferation rate of the tissue preservation reagent group was 1.34 times that of the ordinary tissue preservation solution group. It can be seen that the cell viability and proliferation rate of cells treated with the tissue preservation reagent for 24 hours were superior to those of cells treated with ordinary tissue preservation solution in organoid culture. The results showed a statistically significant difference (P = 0.049, n = 3).

[0116] 4. In order to more clearly illustrate the antioxidant capacity of the tissue preservation reagent in this invention, the following description is based on specific experiments.

[0117] 4.1 In vitro resuscitation and culture of human umbilical vein endothelial cells (hUVECs)

[0118] Preparation: Preheat the biosafety cabinet to UV light for 30 minutes before use; prepare a container containing 100mg / ml of antibiotics (Primocin). TM Sterile PBS balanced salt buffer, 60mm sterile culture dishes, preheated water bath (37°C), centrifuge tubes, and high-glucose DMEM medium (Gibco) containing 10% FBS. TM ), AO / PI dual-fluorescence cell live / dead staining reagent, disposable cell counting chamber, automated cell counter, pipette and pipette tip.

[0119] The specific steps for the resuscitation and cultivation of hUVECs are as follows:

[0120] S1. Remove the frozen hUVECs from the liquid nitrogen container and place them in a 37°C water bath for rapid thawing. Once the solution in the cryovial is completely thawed, quickly place it in a biosafety cabinet for further processing.

[0121] S2. Transfer the cell suspension to a 15mL centrifuge tube and centrifuge at 1500rpm for 5 minutes. Discard the supernatant, resuspend the cell pellet in PBS balanced salt buffer, mix by pipetting, and centrifuge for 5 minutes. Repeat this step twice.

[0122] S3. After centrifugation, discard the supernatant, leaving the cell pellet. Add 2-4 mL of PBS balanced salt buffer to the cell pellet, resuspend and mix well to obtain a cell suspension. Take 10 μL of the cell suspension and mix it with 10 μL of AO / PI dual-fluorescence cell live / dead staining reagent to obtain a mixed solution. Take 10 μL of the mixed solution and add it to a disposable cell counting chamber. Use an automated cell counter to count the cells. Record the total number of live cells harvested and the cell viability.

[0123] S4. Centrifuge the cell suspension for 5 minutes, discard the supernatant, resuspend the cells in high-glucose DMEM medium (Gibco) containing 10% FBS, and seed the cells into sterile 60mm culture dishes at a density of 1–2 x 10⁻⁶ cells / mL. 5 / mL, with an inoculation volume of 3-4mL;

[0124] S5. Place the cell culture dish in a cell culture incubator for culture under the following conditions: 95% air, 5% carbon dioxide, 37°C, and 70%-80% humidity. Change the medium once on the second day after inoculation, and then change the medium every 2-3 days thereafter.

[0125] 4.2 Passaging of human umbilical vein endothelial cells (hUVECs)

[0126] Preparation: Preheat the biosafety cabinet to UV light for 30 minutes before use; prepare a container containing 100mg / ml of antibiotics (Primocin). TM Sterile PBS balanced salt buffer, 60mm sterile culture dishes, centrifuge tubes, and high-glucose DMEM medium containing 10% FBS (Gibco) were prepared. TM Cell digestion solution (0.25% Trypsin-0.53mM EDTA), pipettes and pipette tips.

[0127] The specific steps for hUVECs passaging are as follows:

[0128] S1. When the cell density in the cell culture dish reaches 80%-90%, discard the supernatant of the culture dish and wash with PBS balanced salt buffer containing 100mg / ml Primocin antibiotic 1-2 times to remove residual serum.

[0129] S2. Add 1 ml of digestion solution to a cell culture dish and incubate at 37°C for 1-2 minutes. Observe the cell digestion under a microscope. When most of the cells become round and detach, add a small amount of culture medium to stop the digestion and gently blow the cells from the bottom of the dish to detach them.

[0130] S3. Centrifuge the harvested cells at 1500 rpm for 5 minutes after digestion; discard the supernatant, resuspend the cells in Gibco high-glucose DMEM medium containing 10% FBS, and re-seed them into new culture dishes for further culture at a passage ratio of 1:2-1:3.

[0131] 4.3 Validation of the antioxidant capacity of the optimized tissue preservation reagent

[0132] Preparation: Preheat biosafety cabinet to UV light for 30 minutes; PBS balanced salt buffer containing 100 mg / ml Primocin antibiotic; 60 mm sterile culture dishes; centrifuge tubes; high-glucose DMEM medium (Gibco) containing 10% FBS; hUVECs; pipettes; pipette tips; tissue preservation reagents; thermosensitive gel; 96-well cell culture plates; hydrogen peroxide (H2O2); incubator; serum-free medium containing DCFH-DA; fluorescence microscope; and digestion solution.

[0133] The specific steps for verifying the antioxidant capacity of the optimized tissue preservation reagent are as follows:

[0134] S1. Resuspend hUVECs in tissue preservation reagent and mix well. Seed the cells into 96-well cell culture plates, with 1*10 cells per well. 4 Add high-sugar DMEM medium (Gibco) containing 10% FBS and incubate overnight in an incubator to allow it to adhere to the wall.

[0135] S2. After overnight incubation, discard the DMEM medium and add the following to the wells according to the group: H2O2 (final concentration 100 μM) + ordinary thermosensitive gel, H2O2 (final concentration 100 μM) + tissue preservation reagent, H2O2 (final concentration 100 μM) + ordinary tissue preservation solution, H2O2 (final concentration 100 μM) and PBS balanced salt buffer (same volume as H2O2). Set up 3 replicates for each group and incubate the samples at 4°C overnight.

[0136] S3. Detect cell ROS levels in samples that have been stored at 4°C overnight. Transfer the samples to a 37°C incubator and aspirate the liquid after 15 minutes. Wash the cells in the cell culture plate once with PBS balanced salt buffer, add serum-free medium containing DCFH-DA, and incubate at room temperature in the dark for 20 minutes.

[0137] S4. Add digestion solution (0.25% trypsin digestion) to the cell culture plate, then add culture medium to stop digestion and make cell suspension. Centrifuge at 1500 rpm for 5 minutes to collect cells. Wash 3 times with PBS balanced salt buffer to fully remove DCFH-DA that has not entered the cells. Centrifuge to collect cell pellet for fluorescence detection.

[0138] S5. Resuspend the collected cells in PBS balanced salt buffer, and then use a fluorescence microplate reader to detect the fluorescence intensity of 2',7'-dichlorofluorescein (DCF) (FITC channel).

[0139] Grouping:

[0140] Control group: PBS balanced salt buffer (same volume as H2O2);

[0141] B: H2O2 (final concentration 100μM);

[0142] C: H2O2 (final concentration 100μM) + ordinary tissue preservation reagent;

[0143] D: H2O2 (final concentration 100μM) + tissue preservation reagent;

[0144] E: H2O2 (final concentration 100μM) + ordinary thermosensitive gel.

[0145] Experimental results:

[0146] Depend on Figure 11 It can be seen that the cells in groups B, C and E maintained a high ROS content, while the ROS content in hUVECs in group D was significantly reduced after treatment with H2O2 following transport and preservation of the tissue preservation reagent.

[0147] The above experiments show that the tissue preservation and transportation device provided by the present invention can reduce the mechanical damage to tissues during transportation and maintain the stability of tissue viability within 48 hours during transportation, thus preserving the characteristics of the transported tissues and providing a simple and effective method for tissue preservation and transportation.

[0148] This invention is not limited to the above-described optional embodiments. Anyone can derive other various forms of products under the guidance of this invention. However, regardless of any changes made in their shape or structure, any technical solution that falls within the scope of the claims of this invention shall be protected by this invention.

Claims

1. A tissue preservation and transportation device, characterized in that, Includes tissue preservation reagents and preservation tubes; The tissue preservation reagent includes a thermosensitive gel, an antioxidant stress component, and a tissue preservation solution; The thermosensitive gel is type B gelatin derived from pigskin with a gelatin strength of 180-250; the concentration of the thermosensitive gel is 2.5%-4%; the antioxidant stress components are vitamin C (3-8 μg / mL), flavonoids (200-300 μg / mL) and polyphenols (8-15 μg / mL); The preservation tube is used to hold tissue preservation reagents; the upper end of the preservation tube is provided with a threaded tube opening, the threaded tube opening is provided with a preservation tube cap, and the preservation tube is provided with a protective sleeve.

2. The tissue preservation and transportation device according to claim 1, characterized in that, The temperature-sensitive gel is in a solidified gel state when refrigerated at a temperature of 2-8℃, and in a liquid state when the room temperature is above 25℃.

3. A tissue preservation reagent, characterized in that, Includes thermosensitive gel, antioxidant stress ingredients, and tissue preservation solution; The thermosensitive gel is a type B gelatin derived from pigskin with a gel strength of 180-250 and a concentration of 2.5%-4%. The antioxidant stress-reducing components include vitamin C, flavonoids, and polyphenols, wherein the concentration of vitamin C is 3-8 μg / mL, the concentration of flavonoids is 200-300 μg / mL, and the concentration of polyphenols is 8-15 μg / mL.

4. The tissue preservation reagent according to claim 3, characterized in that, The preparation steps of the tissue preservation reagent are as follows: S1. Place the temperature-sensitive biohydrogel lyophilized powder and PBS balanced salt buffer into a centrifuge tube, and dissolve the temperature-sensitive biohydrogel lyophilized powder completely by heating in a water bath to obtain the temperature-sensitive gel. S2. Before the temperature drops, filter and sterilize to obtain a sterile temperature-sensitive gel solution; S3. Once the temperature of the thermosensitive gel solution drops below 37°C, add antibiotics, tissue preservation solution, and antioxidant stress-reducing components and mix thoroughly to obtain the tissue preservation reagent.

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