Preserving fluid for preserving pancreatic cancer tissue and application thereof

Abstract

The invention discloses a preserving fluid for preserving pancreatic cancer tissues and application of the preserving fluid. The preserving fluid is prepared from mannitol, reduced glutathione, allopurinol, magnesium sulfate, gabexate mesylate, orlistat, pyruvic acid, Primocin, dexamethasone, heparin, bovine serum albumin, HEPES and the balance of an advance DMEM / F12 culture medium. The preserving fluid has a good effect in maintaining the pancreatic cancer tissue activity and inhibiting self-digestion and oxidative damage, is beneficial to maintaining the cell activity, and is beneficial to the preservation of pancreatic ductal adenocarcinoma in-vitro tissues and the construction of subsequent organoid.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a preservation solution for preserving pancreatic cancer tissue, and further to the application of said preservation solution in maintaining the activity of pancreatic cancer tissue and inhibiting autodigestion and oxidative damage. Background Technology

[0002] Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant solid tumors, with an extremely low five-year survival rate, difficult early diagnosis, and limited treatment options. The tumor microenvironment of PDAC is highly heterogeneous, fibrotic, and immunosuppressive, making it difficult for traditional two-dimensional cell line models to accurately simulate the biological behavior, drug response, and drug resistance mechanisms of patient tumors. In recent years, patient-derived organoids (PDOs) have become important tools in oncology research because they can preserve the tissue structure, gene expression profile, tumor stem cell characteristics, and drug sensitivity of the primary tumor in vitro, and are widely used in personalized drug screening, disease mechanism research, and the development of novel treatment strategies.

[0003] However, the success of organoid culture is highly dependent on the quality of ex vivo tissue samples. PDAC tissues obtained through clinical surgical resection or biopsy are extremely susceptible to decreased activity and structural damage after ex vivo due to factors such as ischemia, hypoxia, temperature changes, enzymatic autodigestion, and microbial contamination, severely affecting the formation rate and representativeness of organoids. Specifically, ex vivo PDAC tissues face the following main challenges: First, there is ischemic and hypoxic injury. PDAC itself has a poor blood supply. After being removed from the body, the oxygen and nutrient supply is interrupted, the intracellular ATP is rapidly depleted, and the enhanced anaerobic glycolysis leads to lactic acid accumulation and acidosis, which in turn triggers cellular ion homeostasis disorder and activation of apoptosis signaling pathways; Second, there is autodigestion of pancreatic tissue. Pancreatic tissue is rich in various digestive enzymes (such as trypsin and elastase). These enzymes may still be activated after the tissue is removed from the body, triggering autodigestion, damaging cell-cell connections and extracellular matrix structure, and leading to irreversible cell accumulation. Third, the organoid construction process exacerbates damage. In the application of organoid construction, in order to preserve tissue viability and heterogeneous cell subpopulations, tumor tissue needs to be transported out of the body for as short a time as possible. This means that after transport under low temperature and hypoxia conditions, destructive shearing and digestion processes will begin immediately, causing existing damage to be unable to be compensated for and to accumulate further, affecting cell fate.

[0004] Currently, commonly used preservation solutions (such as UW solution) are mainly designed for organ transplantation. They have significant limitations when used for preserving PDAC organoid samples: UW solution has a high viscosity and usually needs to be used after vascular perfusion. Direct use can easily affect the uniformity of preservation results and further affect tissue digestion, reducing cell clump yield and viability; its antioxidant components are relatively simple and have limited concentrations, which is insufficient to protect against the explosive oxidative stress faced by PDAC cells with abnormal metabolism and high levels of oxidative stress during preservation and subsequent processing; it lacks specific enzyme inhibition. UW solution is usually used for the preservation of intact organs with perfusion and washing procedures, but for cancer tissue samples, which must be obtained through surgical resection or puncture, there will be self-digestion effects.

[0005] Therefore, developing a specialized tissue preservation solution for PDAC organoid construction that can effectively maintain tissue viability, inhibit autodigestion and oxidative damage, and is cost-effective is of great clinical significance and market application value. Summary of the Invention

[0006] In view of this, one objective of the present invention is to provide a preservation solution for preserving pancreatic cancer tissue; a second objective of the present invention is to provide a method for preparing the preservation solution; a third objective of the present invention is to provide a method for preserving pancreatic cancer tissue using the preservation solution; a fourth objective of the present invention is to provide the application of the preservation solution in preparing sample preservation reagents for the preparation of pancreatic cancer organoids; and a fifth objective of the present invention is to provide the application of the preservation solution in maintaining the activity of pancreatic cancer tissue and inhibiting autodigestion and oxidative damage.

[0007] To achieve the above objectives, the present invention provides the following technical solution: 1. A preservation solution for preserving pancreatic cancer tissue, the preservation solution comprising the following components: mannitol 1.821~5.465 g / L, reduced glutathione 0.921~1.536 g / L, allopurinol 6.8~27.2 mg / L, magnesium sulfate 0.120~0.601 g / L, gabexate mesylate 4.2~20.8 mg / L, orlistat 2.5~9.9 mg / L, pyruvate 0.440~1.761 g / L, primocin 50~100 mg / L, dexamethasone 0.4 mg / L, heparin 1000 U / L, bovine serum albumin 5 g / L, HEPES 3.574~5.957 g / L, the balance being advanced DMEM / F12 medium, the pH of the preservation solution being 7.2~7.4.

[0008] In some embodiments of the present invention, the preferred amounts of each component in the preservation solution are: mannitol 3.642 g / L, reduced glutathione 1.536 g / L, allopurinol 13.6 mg / L, magnesium sulfate 0.12 g / L, gabexate mesylate 4.2 mg / L, orlistat 2.5 mg / L, pyruvate 0.440 g / L, Primocin 50 mg / L, dexamethasone 0.4 mg / L, heparin 1000 U / L, bovine serum albumin 5 g / L, HEPES 3.574 g / L, with the remainder being advance DMEM / F12 culture medium.

[0009] In some embodiments of the present invention, the preservation solution is sterilized by filtration using a 0.22 μm PES material filter.

[0010] 2. A method for preparing the preservation solution, comprising the following steps: (1) Weigh each component according to the proportion; (2) Dissolve and bring to volume using advanced DMEM / F12; (3) Adjust the pH to 7.2–7.4; (4) Use a 0.22 μm PES filter for sterilization; (5) After repackaging, store at 2-8℃. The shelf life is 1 month.

[0011] 3. A method for preserving pancreatic cancer tissue using the preservation solution, comprising: (1) Cut the pancreatic cancer tissue into pieces no larger than 2 cm. 3 ; (2) Completely immerse the pancreatic cancer tissue block in the preservation solution, with the tissue volume not exceeding 1 / 5 of the preservation solution volume; (3) Store at 2-8℃ for no more than 24 hours.

[0012] In some embodiments of the present invention, the pancreatic cancer tissue is derived from surgical resection or puncture biopsy samples.

[0013] 4. The application of the preservation solution in the preparation of sample preservation reagents for the preparation of pancreatic cancer organoids.

[0014] 5. The application of the preservation solution in maintaining the activity of pancreatic cancer tissue and inhibiting autodigestion and oxidative damage.

[0015] The beneficial effects of this invention are as follows: Compared with existing preservation solutions, the formulation of the tissue preservation solution of this invention has been specifically optimized for organoid construction applications: 1. Using advance 1. DMEM / F12 is used as the preservation substrate to maintain similarity to the components used in organoid culture applications, reducing metabolic adaptation stress and providing sufficient energy and basic substances such as inorganic salts; 2. Mannitol, magnesium sulfate, and bovine serum albumin are used as structural protectants to provide suitable osmotic pressure and cell membrane protection while maintaining low viscosity, which is beneficial for maintaining cell viability and tissue integrity and preserving intercellular communication; 3. Mannitol, reduced glutathione, allopurinol, and pyruvate are used as antioxidants to maintain intracellular redox balance and neutralize reactive oxygen species, minimizing the explosive free radical damage and oxidative stress that may occur during tissue transport and subsequent organoid construction; 4. Gabexate mesylate and orlistat are used to inhibit the autodigestion process caused by the still secretory part of pancreatic cancer tissue, avoiding unnecessary enzymatic damage; 5. Pyruvate and magnesium sulfate are used as energy substrates, which are taken up by cells as pre-reserves, enabling rapid restoration of cell viability and inhibition of apoptosis signaling after rewarming. 6. Using Primocin in combination with dexamethasone can combat contamination while reducing aseptic inflammation caused by cell / tissue debris, thus preventing abnormal cellular responses. Attached Figure Description

[0016] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration: Figure 1 The results of organoid culture using control group 1 are shown in the figure (D0 is the cell morphology at the initial stage of culture, and D2 is the cell morphology after 48 hours of culture). Figure 2 This is a diagram showing the organoid culture results using control group 2. Figure 3 This is a diagram showing the results of organoid culture using control group 3. Figure 4 This is a diagram showing the organoid culture results of the experimental group; Figure 5 This is a statistical chart showing organoid formation. Detailed Implementation

[0017] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0018] Example 1 Pancreatic cancer tumor tissue samples were extracted from a mouse PDX model and trimmed to approximately 1 cubic centimeter using sterile ophthalmic scissors.

[0019] The prepared tissue blocks were grouped and preserved under different conditions as described in Table 2. The tissues were then minced and digested with collagenase I at 37°C until small cell clusters formed. After centrifugation, washing, and counting, the cell suspension was mixed with matrix gel for organoid construction. The pancreatic cancer organoid culture medium disclosed in Chinese Invention Patent CN114426949A was used for 48 hours at 37°C and 5% CO2, and the organoid formation of each group was compared.

[0020] Table 1 Experimental Grouping Control group 1 Control group 2 Control group 3 Experimental group Storage time - 24 hours 24 hours 24 hours Preservation solution - Control preservation solution UW solution The present patent preservation solution The formulations of each preservation solution are shown in Table 2-4 below: Table 2. Control Preservative Formulation Control preservation solution formula (1L) Raw material Dosage FBS 100mL Primosin 50mg Advance DMEM / F12 Dilute to 1mL Table 3 Main Components of UW Fluid UW solution main component (1L) Raw material Dosage Lactose potassium salt 10mmol KH2PO4 25mmol MgSO4 5mmol Insulin 100U Dexamethasone 8mg Sulfo-methisoxazole 5mmol Methoxybenzyl aminopyrimidine 40mg Glutathione 8mg Hydroxyethyl starch 1mmol Raffinose 17.8g Table 4. Preservative formulation of the present invention The present patent preservation solution formula (1L) Raw material Dosage Mannitol 3.642g Reduced glutathione 1.536g Allopurinol 13.6mg Magnesium sulfate 0.12g Gavacide mesylate 4.2mg Orlistat 2.5mg Pyruvic acid 0.440g Primocin 50mg Dexamethasone 0.4mg Heparin 1000U Bovine serum albumin 5g HEPES 3.574g Advance DMEM / F12 Dilute to 1L The preservation solution formulation of this invention has a storage temperature of 2~8℃ and an effective storage period of 1 month. It can be used at a temperature of 2~8℃, supporting cryopreservation of pancreatic cancer tissue samples for 24 hours without significantly affecting organoid construction.

[0021] When using this storage solution, the pancreatic cancer tissue to be preserved must be cut into small pieces no larger than 2 cubic centimeters, fully immersed, and the tissue volume should not exceed 1 / 5 of the storage solution volume.

[0022] Comparison of culture results as follows Figure 1-Figure 4 As shown, the statistical results of organoid formation are as follows: Figure 5 As shown, tissues preserved in the preservation solution of this invention for 24 hours can still form organoids well. Compared to tissues preserved in the preservation solution of control group 3 for 24 hours, tissues preserved in the preservation solution of this invention for 24 hours form more organoids with more consistent overall morphology. However, tissues preserved in the preservation solution of control group 2 for 48 hours fail to form good organoids, and cell apoptosis occurs. There is no significant difference in organoid formation between samples preserved using this patent and fresh samples from control group 1.

[0023] In summary, this invention provides a tissue preservation solution for pancreatic cancer organoid culture, which effectively avoids damage to PDAC ex vivo tissue caused by ischemia-hypoxia, tissue autodigestion, and organoid construction, helps maintain its cell viability, and is beneficial for PDAC ex vivo tissue preservation and subsequent organoid construction.

[0024] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. A preservation solution for preserving pancreatic cancer tissue, characterized in that, The preservation solution contains the following components: mannitol 1.821~5.465 g / L, reduced glutathione 0.921~1.536 g / L, allopurinol 6.8~27.2 mg / L, magnesium sulfate 0.120~0.601 g / L, gabexate mesylate 4.2~20.8 mg / L, orlistat 2.5~9.9 mg / L, pyruvate 0.440~1.761 g / L, primocin 50~100 mg / L, dexamethasone 0.4 mg / L, heparin 1000 U / L, bovine serum albumin 5 g / L, HEPES 3.574~5.957 g / L, with the balance being advance DMEM / F12 culture medium. The pH of the preservation solution is 7.2~7.

4.

2. The preservation solution according to claim 1, characterized in that, The preferred amounts of each component in the preservation solution are: mannitol 3.642 g / L, reduced glutathione 1.536 g / L, allopurinol 13.6 mg / L, magnesium sulfate 0.12 g / L, gabexate mesylate 4.2 mg / L, orlistat 2.5 mg / L, pyruvate 0.440 g / L, Primocin 50 mg / L, dexamethasone 0.4 mg / L, heparin 1000 U / L, bovine serum albumin 5 g / L, HEPES 3.574 g / L, with the remainder being advanceDMEM / F12 culture medium.

3. The preservation solution according to claim 1 or 2, characterized in that, The preservation solution was sterilized by filtration using a 0.22 μm PES material filter.

4. The method for preparing the preservation solution according to any one of claims 1 to 3, characterized in that, Includes the following steps: (1) Weigh each component according to the proportion; (2) Dissolve and bring the volume to 1L using advanced DMEM / F12; (3) Adjust the pH to 7.2–7.4; (4) Use a 0.22 μm PES filter for sterilization; (5) After repackaging, store at 2-8℃. The shelf life is 1 month.

5. A method for preserving pancreatic cancer tissue using the preservation solution according to any one of claims 1 to 3, characterized in that, include: (1) Cut the pancreatic cancer tissue into pieces no larger than 2 cm. 3 ; (2) Completely immerse the pancreatic cancer tissue block in the preservation solution, with the tissue volume not exceeding 1 / 5 of the preservation solution volume; (3) Store at 2-8℃ for no more than 24 hours.

6. The method according to claim 5, characterized in that, The pancreatic cancer tissue was derived from surgical resection or biopsy samples.

7. The use of the preservation solution according to any one of claims 1 to 3 in the preparation of a sample preservation reagent for the preparation of pancreatic cancer organoids.

8. The use of the preservation solution according to any one of claims 1 to 3 in maintaining the activity of pancreatic cancer tissue and inhibiting autodigestion and oxidative damage.

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