A tissue preservation solution for fresh tumor tissue preservation and transportation and application thereof

Abstract

The application relates to the field of biological culture medium, in particular to a tissue preservation solution for fresh tumor tissue preservation and transportation and application; the tissue preservation solution comprises a culture medium, an HEPES buffer, penicillin-streptomycin-amphotericin B, N-acetylcysteine, adenine, trimethyl-glycine, taurine, a ROCK inhibitor Y27632 and fetal bovine serum; long-distance transportation of tumor tissues for a long time is carried out by using the tissue preservation solution, high activity of tumor cells can be maintained, the risk of contamination of tumor organoid culture is reduced, and the success rate of tumor organoid culture is improved; the preparation of the tissue preservation solution reduces the use of growth factors such as EFG, FGF and HGF, and reduces the cost of the tissue preservation solution.

Description

Technical Field

[0001] This invention relates to the field of biological culture medium technology, specifically to a tissue preservation solution for the preservation and transportation of fresh tumor tissue and its application. Background Technology

[0002] Patient-derived tumor organoids are produced by mimicking the in vivo microenvironment, enabling the self-renewal of adult stem cells, and allowing tissue to grow in vitro. These organoids possess similar spatial organization to their corresponding organs, replicating their functions and providing a highly physiologically relevant "in vitro surrogate." Patient-derived tumor organoids can be used for in vitro drug screening research and can reveal clinical patient responses to drugs. This is a clinically relevant and timely in vitro drug sensitivity testing method, used to guide precise medication for patients and achieve personalized medicine. Therefore, to serve clinical needs, tumor organoid culture requires shorter culture times and higher success rates. The viability of tumor cells in the sample is one of the most critical factors affecting organoid culture success. Differences in the source location of the tissue sample, required transportation time and distance, etc., can significantly affect the viability of the tissue sample and reduce the success rate of organoid culture.

[0003] Meanwhile, tissues have a complex cellular composition, consisting of multiple cell types. Tissue preservation solutions need to be effective at preserving various primary cell types. Furthermore, due to the dense structure of tissues, preservation media do not easily penetrate the tissue cells; therefore, the preservation solution needs a certain degree of tissue permeability. However, while improving the tissue permeability of the preservation medium, it is crucial not to damage the spatial structure and morphology of the tissue. Therefore, compared to cell preservation, achieving tissue preservation is far more challenging.

[0004] Current methods for preserving and transporting fresh tumor tissue:

[0005] The first method involves transporting tissues via cryopreservation in liquid nitrogen or dry ice.

[0006] The second method involves placing the tissue in a tissue preservation solution for low-temperature (2-8°C) transport.

[0007] The first method, cryopreservation followed by transportation, is technically demanding and costly. Furthermore, the subsequent thawing process can severely damage cells, reducing cell viability and the success rate of organoid culture. The second method causes varying degrees of frostbite to tissue cells during cryopreservation, impairing some cell viability. Therefore, these types of preservation solutions are only suitable for short-term preservation of cell viability and are not suitable for long-distance transport. Existing sample preservation solutions also suffer from significant osmotic pressure changes, leading to reduced cell viability. Additionally, some developed sample preservation solutions contain multiple recombinant proteins, increasing costs. Current tissue preservation solutions are not suitable for long-distance transport and can damage tumor cell viability, limiting the clinical and research applications of organoids.

[0008] For example, Chinese invention patent (CN112586495B) discloses a tumor tissue preservation solution, which is a serum-free basal culture medium containing serum albumin, recombinant human insulin, transferrin, linoleic acid, Trace Elements A, free radical scavengers, and cell protectants; the serum-free basal culture medium is Leibovitz's L-15; the serum albumin is bovine serum albumin at a concentration of 2% w / v; the recombinant human insulin in the tumor tissue preservation solution is 10 μg / ml; the transferrin in the tumor tissue preservation solution is 5 μg / ml; the linoleic acid in the tumor tissue preservation solution is 5 μg / ml; the cell protectants are trehalose and γ-polyglutamic acid, wherein the trehalose concentration is 2 mg / ml and the 500 kDa γ-polyglutamic acid concentration is 0.075% w / v; the Trace Elements A is a serum-free basal culture medium containing serum albumin, recombinant human insulin, transferrin, linoleic acid, Trace Elements A, free radical scavengers, and cell protectants; the serum-free basal culture medium is Leibovitz's L-15; the serum albumin is bovine serum albumin at a concentration of 2% w / v; the recombinant human insulin in the tumor tissue preservation solution is recombinant human insulin at a concentration of 10 μg / ml; the transferrin in the tumor tissue preservation solution is recombinant human insulin at a concentration of 5 μg / ml; the linoleic acid in the tumor tissue preservation solution is linoleic acid; the cell protectants are trehalose and γ-polyglutamic acid, wherein the trehalose concentration is 2 mg / ml and the 500 kDa γ-polyglutamic acid concentration is 0.075% w / v; the Trace Elements A is a serum-free basal culture medium containing serum albumin, recombinant human insulin, transferrin, linoleic acid, T Elements A is 1000×, with a content of 0.1% v / v; the free radical scavenger is 100× Glutamax, with a content of 1% v / v; the tumor tissue preservation solution also contains Primocin, with a content of 100 μg / ml; the tumor tissue is gastric cancer, colorectal cancer, and / or breast cancer tissue. This patent develops a serum-free tumor tissue preservation solution suitable for long-term sealed preservation and transportation of ex vivo tumor tissue under low-temperature conditions; however, this preservation solution uses multiple recombinant proteins, resulting in high cost, and the solution contains many components, making its preparation relatively complex. Summary of the Invention

[0009] To address the shortcomings of existing technologies, the viability of tumor tissue samples is a key factor affecting the success of tumor organoid culture. Therefore, this invention develops a novel tissue preservation solution formulation that reduces the use of various recombinant proteins and growth factors. By appropriately selecting and adjusting the components in the preservation solution, it can be used for long-term, long-distance transport of tumor tissue at low temperatures, while maintaining high cell viability and reducing the risk of contamination in organoid culture, which is crucial for tumor organoid culture.

[0010] The objective of this invention can be achieved through the following technical solutions:

[0011] The first aspect of this invention discloses a tissue preservation solution for the preservation and transportation of fresh tumor tissue, comprising culture medium, HEPES buffer, penicillin-streptomycin-amphoteric B, N-acetylcysteine, taurine, adenine, trimethylglycine, ROCK inhibitor Y27632, and fetal bovine serum.

[0012] The culture medium was AdvancedDMEM / F12;

[0013] The concentration of the HEPES buffer is 8-12 mM;

[0014] The concentration of penicillin in the penicillin-streptomycin-amphotericidal B mixture is 100-800 U / mL, the concentration of streptomycin is 0.1-0.8 mg / mL, and the concentration of amphotericin B is 0.25-2 μg / mL.

[0015] The concentration of the N-acetylcysteine ​​is 4-6 mM;

[0016] The concentration of taurine is 0.4-0.6 mM;

[0017] The dosage of adenine is 0.7-1.5 g / L;

[0018] The amount of trimethyl-glycine used is 2-6 g / L;

[0019] The concentration of Y27632 is 8-12 μM;

[0020] The fetal bovine serum content is 1%-5% of the tissue preservation solution.

[0021] N-acetyl-L-cysteine ​​(NAC) is an aminothiol antioxidant and a precursor to intracellular cysteine ​​and reduced glutathione (GSH). It can either diffuse directly into cells to resynthesize GSH or be converted into metabolites that stimulate glutathione synthesis through first-pass metabolism in the liver, thereby promoting detoxification and scavenging free radicals.

[0022] Taurine (T), also known as 2-aminoethanesulfonic acid, can reduce the level of oxidative stress induced during cryopreservation, thus preserving the activity and integrity of tumor tissue.

[0023] Preferably, the concentration of the HEPES buffer is 10 mM;

[0024] Preferably, the concentration of penicillin in the penicillin-streptomycin-amphotericidal B is 500 U / mL, the concentration of streptomycin is 0.5 mg / mL, and the concentration of amphotericin B is 1.25 μg / mL.

[0025] Preferably, the concentration of N-acetylcysteine ​​is 5 mM;

[0026] The concentration of taurine is 0.5 mM;

[0027] Preferably, the amount of adenine used is 1 g / L;

[0028] Preferably, the amount of trimethyl-glycine used is 4 g / L;

[0029] Preferably, the concentration of Y27632 is 10 μM;

[0030] Preferably, the fetal bovine serum content accounts for 2.5% of the tissue preservation solution.

[0031] The second aspect of this invention discloses a method for preparing the above-mentioned tissue preservation solution, comprising the following steps:

[0032] S1: Dissolve adenine and trimethylglycine in AdvancedDMEM / F12 solution and then filter.

[0033] S2: Add HEPES buffer, penicillin-streptomycin-amphoteric B, N-acetylcysteine, taurine and fetal bovine serum to AdvancedDMEM / F12 to obtain tissue preservation solution;

[0034] S3: Y27632 is added to the tissue preservation solution obtained in S2 after the tissue sample is placed in it.

[0035] The dissolution method in S1 is to dissolve in a 37°C water bath, during which the AdvancedDMEM / F12 is shaken.

[0036] The third aspect of the present invention discloses the use of the above-mentioned tissue preservation solution in the preservation and transportation of fresh tumor tissue, preferably for colorectal cancer and lung cancer.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] This invention adds adenine to the tissue preservation solution to maintain the energy metabolism of cells in a low-temperature environment and prolong the survival time of cells after they are ex vivo.

[0039] The tissue preservation solution of the present invention adds trimethyl-glycine as a cell osmotic pressure protectant and a cryo-damage protectant to reduce cell damage in the preservation solution and maintain high activity for a longer period of time;

[0040] The N-acetylcysteine ​​and taurine added in this invention have antioxidant activity. The combination of the two significantly improves the antioxidant performance of the preservation solution, enhances the removal of free radicals, and inhibits the induction of tumor tissue differentiation by serum-containing preservation solution.

[0041] The addition of penicillin-streptomycin-amphotericidal B in this invention can effectively inhibit the proliferation of microorganisms;

[0042] This invention reduces the use of growth factors such as EFG, FGF, and HGF, thereby lowering the cost of tissue preservation solutions.

[0043] The tissue preservation solution prepared in this invention significantly reduces the need for animal-derived serum or exogenous added growth factors, and can maintain more than 90% cell viability of tumor tissue for a preservation time of up to 72 hours. Attached Figure Description

[0044] Figure 1 The proportion of viable cells relative to 0h after 48h and 72h of preservation of colorectal cancer samples.

[0045] Figure 2 The proportion of viable cells relative to 0h after 48h and 72h of storage for gastric cancer samples.

[0046] Figure 3 The culture status of colorectal cancer organoids in the tissue preservation solution of Example 1.

[0047] Figure 4 This shows the culture status of gastric cancer organoids in the tissue preservation solution of Example 1.

[0048] Figure 5 The culture status of lung cancer organoids in the tissue preservation solution of Example 1. Detailed Implementation

[0049] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0050] Comparative Example 1

[0051] Prepare a standard sample preservation solution: AdvancedDMEM / F12, 10% FBS, 1X penicillin-streptomycin.

[0052] Comparative Example 2

[0053] Preparation of tissue preservation solution:

[0054] The tissue preservation solution was formulated as follows: AdvancedDMEM / F12, 10mM HEPES, 500U / mL penicillin-0.5mg / mL streptomycin-1.25μg / mL amphotericin B, 5mM N-acetylcysteine, 1g / L adenine, 4g / L trimethylglycine, 10μMY27632, and FBS (2.5%).

[0055] Comparative Example 3

[0056] Preparation of tissue preservation solution:

[0057] The tissue preservation solution was formulated as follows: AdvancedDMEM / F12, 10mM HEPES, 500U / mL penicillin-0.5mg / mL streptomycin-1.25μg / mL amphotericin B, 5mM N-acetylcysteine, 0.5mM taurine, 1g / L adenine, 50g / L dextran, 10μMY27632, and FBS (2.5%).

[0058] Example 1

[0059] Preparation of tissue preservation solution:

[0060] The tissue preservation solution was formulated as follows: AdvancedDMEM / F12, 10mM HEPES, 500U / mL penicillin-0.5mg / mL streptomycin-1.25μg / mL amphotericin B, 5mM N-acetylcysteine, 0.5mM taurine, 1g / L adenine, 4g / L trimethylglycine, 10μMY27632, and FBS (2.5%).

[0061] In Example 1, adenine and trimethylglycine were dissolved in Advanced DMEM / F12 and then filtered (dissolved in a 37°C water bath with shaking). This sample preservation solution can be stored at 4°C, and Y27632 was added after the tissue sample was placed in it.

[0062] Example 2

[0063] Cell isolation and cell viability assay in tumor tissue:

[0064] 1. Collect surgical samples of colorectal or gastric cancer from the hospital and wash the samples multiple times with saline solution. Use sterile surgical scissors to cut the tumor tissue into 1-2mm pieces. 3 The tumor mass was divided into 5 equal parts. Two parts were placed in 10 mL of sample preservation solution, and two parts were placed in 10 mL of ordinary sample preservation solution and stored at 4°C for 48 h and 72 h, respectively. The remaining tissue mass was processed as follows.

[0065] 2. Transfer all tissue blocks and fluids to 15mL centrifuge tubes to prepare approximately 10mL of sample enzymatic hydrolysate. Place the centrifuge tubes in a shaker at 37℃ and 300rpm for 60-120min. Every 30min, take a small amount of the hydrolysate and observe the cell hydrolysis under a microscope. Stop the hydrolysis when a large number of cells have dissociated and are mostly in cell clusters. Then filter the solution through a 100μm filter to obtain the filtrate.

[0066] 3. Transfer the tissue block from the upper part of the filter to a new centrifuge tube, add 10 mL of sample enzymatic digestion solution, and enzymatically digest at 37℃ and 300 r / min for 60-120 min. After enzymatic digestion, filter the solution through a 100 μm filter to obtain the filtrate.

[0067] 4. Centrifuge the filtrates obtained from the two enzymatic digestions at 1000 rpm for 5 min. After centrifugation, discard the supernatant, resuspend the precipitate in 1×HBSS buffer, wash once, and centrifuge again to obtain the cell pellet.

[0068] 5. Resuspend the cell pellet in sterile water, mix by pipetting for about 30 seconds, and immediately add the same volume of 1.8% sodium chloride aqueous solution to make the final concentration reach 0.9% physiological saline. Then centrifuge to obtain the cell pellet from which red blood cells have been removed.

[0069] 6. At 0h (after the tumor tissue was minced, before it was preserved with preservation solution, this time is recorded as 0h), 48h and 72h respectively using the tissue preservation solutions in Comparative Examples 1-3 and Examples 1-3, live and dead cells were stained with Trypan Blue and the proportion of live cells was calculated.

[0070] Experimental results are as follows Figure 1 , 2 As shown, after 72 hours of preservation in the sample preservation solution, the proportion of viable cells in colorectal cancer tissue remained above 85%, and the proportion of viable cells in gastric cancer tissue remained above 90%. These cell viability data are higher than those of ordinary sample preservation solutions.

[0071] The results of comparing Comparative Example 2 and Example 1 show that the complex antioxidant component composed of taurine can enhance cell viability in the tissue preservation solution, indicating that it can improve the preservation effect of the preservation solution on tumor tissue compared with N-acetylcysteine ​​alone.

[0072] As can be seen from the comparison between Comparative Example 3 and Example 1, the present invention uses trimethyl-glycine as an osmotic regulator. Compared with dextran, its combination with N-acetylcysteine ​​and taurine can improve the preservation effect of the tissue preservation solution. Trimethyl-glycine also has antioxidant activity, which can enhance the antioxidant and free radical scavenging effects of N-acetylcysteine ​​and taurine in the preservation solution.

[0073] Example 3

[0074] Isolation of cells from tumor tissue and culture of organoids:

[0075] 1. Collect tumor surgical samples of lung cancer, gastric cancer and colorectal cancer from the hospital, place them in the tissue sample preservation solution in Example 1 of the present invention, and transfer them to the laboratory by low temperature transportation.

[0076] 2. Washing. Transfer the tumor sample to 10 mL of sample washing solution, place it on a rotary mixer, and wash 3 times, 5 minutes each time. Replace with fresh washing solution after each wash.

[0077] 3. Mince the tissue. Place the cleaned tumor tissue into a culture dish, add 5-6 mL of HBSS, and use ophthalmic scissors and forceps to mince the tissue into pieces smaller than 1-2 mm. 3 Small pieces.

[0078] 4. Enzymatic hydrolysis of cells. Transfer all liquid to a 15mL centrifuge tube to prepare approximately 10mL of sample hydrolysate. Place the centrifuge tube in a shaker at 37℃ and 300 rpm for 60-120 minutes. Every 30 minutes, take a small amount of the hydrolysate and observe the cell hydrolysis under a microscope. Stop the hydrolysis when a large number of cells have dissociated and are mostly in cell clusters. Then filter the solution through a 100μm filter to obtain the filtrate.

[0079] 5. Secondary enzymatic hydrolysis. The tissue block on the upper part of the filter screen is transferred back to a new centrifuge tube, 10 mL of sample enzymatic hydrolysis solution is added, and the mixture is incubated at 37℃ and 300 r / min for 60-120 min. After enzymatic hydrolysis, the solution is filtered through a 100 μm filter screen to obtain the filtrate.

[0080] 6. Washing the enzymatic hydrolysate: Centrifuge the filtrate obtained from the two enzymatic hydrolysates at 1000 rpm for 5 min. After centrifugation, discard the supernatant, resuspend the precipitate in 1×HBSS buffer, wash once, and centrifuge again to obtain the cell pellet.

[0081] 7. Rupture red blood cells. Resuspend the cell pellet in sterile water, mix by pipetting for about 30 seconds, and immediately add an equal volume of 1.8% sodium chloride aqueous solution to bring the final concentration to 0.9% physiological saline. Then centrifuge to obtain a cell pellet free of red blood cells.

[0082] 8. Seeding the Cell Specimen. Remove as much supernatant as possible from the obtained cell pellet. Depending on the amount of cell pellet, resuspend the cell pellet with 40-200 μL of matrix gel (be careful not to generate air bubbles). After resuspending and mixing, seed the matrix gel mixture into 24-well plates (the plates should be preheated overnight in a cell culture incubator to facilitate matrix gel formation), 1 drop per well, approximately 50 μL / well.

[0083] 9. Gel preparation. Place the plate flat in a cell culture incubator and allow it to solidify for approximately 30 minutes. Remove the plate, add 350 μL of preheated (37°C) culture medium to each well, and incubate in an incubator.

[0084] 10. Change the medium. Monitor cell growth. Replace with fresh medium after 2-3 days of culture.

[0085] 11. Passaging: After 7 days of culture, individual organoids reached approximately 300 μm in diameter and were passaged. The culture medium was removed from the wells, and 400 μm / well of cell recovery buffer was added. The wells were incubated at 4°C for 3 hours. The organoids were observed scattering from the matrix gel under a microscope. The organoids were transferred to 15 mL centrifuge tubes, repeatedly pipetted 20 times with a 1 mL pipette, centrifuged, and washed twice with 10 mL of pre-chilled HBSS. The supernatant was removed by centrifugation, and 5 mL of pre-warmed 37°C LTrypLE solution was added. The tubes were incubated at 37°C for 5 min, washed with 5 mL of HBSS buffer, centrifuged to collect the organoid pellet, counted, and resuspended in the appropriate amount of matrix gel at a concentration of 500-1500 cells / 10 μL. Following the culture procedure, the tubes were seeded into plates, incubated at 37°C until the matrix gel solidified, and then culture medium was added. The culture medium was changed every 3 days. The growth status of the organoids was observed and photographed. The culture results for colorectal cancer, gastric cancer, and lung cancer were as follows. Figure 3 , 4 As shown in Figures 5 and 6, a large number of organoids can be formed in a relatively short period of time.

[0086] The success rate of organoid culture from colorectal cancer was statistically analyzed. A total of 33 samples were preserved using the sample preservation solution developed in this invention, with 30 successfully cultured, resulting in a success rate of 90.9%. Three samples failed to culture. Two of these failed due to lack of cell activity after enzymatic digestion during room temperature transport, leading to contamination during later culture. The third failed sample was from a neoadjuvant chemotherapy patient, whose cells showed low activity after enzymatic digestion. These results demonstrate that the sample preservation solution of this invention can effectively improve the success rate of organoid culture.

Claims

1. A tissue preservation solution for the preservation and transportation of fresh tumor tissue, characterized in that, Includes culture medium, HEPES buffer, penicillin-streptomycin-amphoteric B, N-acetylcysteine, taurine, adenine, trimethylglycine, ROCK inhibitor Y27632, and fetal bovine serum; The culture medium was Advanced DMEM / F12; The concentration of the HEPES buffer is 10 mM; The concentration of penicillin in the penicillin-streptomycin-amphotericidal B mixture is 500 U / mL, the concentration of streptomycin is 0.5 mg / mL, and the concentration of amphotericin B is 1.25 μg / mL. The concentration of the N-acetylcysteine ​​was 5 mM; The concentration of taurine is 0.5 mM; The dosage of adenine is 1 g / L; The amount of trimethyl-glycine used is 4 g / L; The concentration of the ROCK inhibitor Y27632 was 10 μM; The fetal bovine serum content accounts for 2.5% of the tissue preservation solution.

2. The method for preparing a tissue preservation solution for preserving and transporting fresh tumor tissue as described in claim 1, characterized in that, Includes the following steps: S1: Dissolve adenine and trimethylglycine in Advanced DMEM / F12 until fully dissolved, then filter. S2: Add HEPES buffer, penicillin-streptomycin-amphoteric B, N-acetylcysteine, taurine and fetal bovine serum to Advanced DMEM / F12 to obtain tissue preservation solution; S3: The tissue preservation solution obtained in S2 is then supplemented with the ROCK inhibitor Y27632 after the tissue sample is placed in it.

3. The method for preparing a tissue preservation solution for preserving and transporting fresh tumor tissue according to claim 2, characterized in that, The dissolution method in S1 is to dissolve in a 37°C water bath, during which the Advanced DMEM / F12 is shaken.

4. The use of the tissue preservation solution as described in claim 1 for the preservation and transportation of fresh tumor tissue.

5. The use as described in claim 4, characterized in that, The fresh tumor tissue includes colorectal cancer and lung cancer.

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