Application of puromycin in culture of gastrointestinal tumor operation specimen organoids

Abstract

The invention discloses an application of puromycin in culture of gastrointestinal tumor surgery specimen organs. According to the method, in the tissue cleaning process before organoid culture, puromycin with the concentration of 5 [mu] g / mL is added into a cleaning solution to replace Primocin with the concentration of 0.1 mg / mL to clean primary tumor tissue. Results show that in culture of organs derived from 46 patients, 1 sample, 2 samples and 11 samples cleaned and pretreated by Primocin, puromycin and HBSS are polluted respectively. The result shows that the culture success rate of the organoid by adopting puromycin pretreatment can be increased to 95% or above from 76.1%, which is basically equal to that of Primocin pretreatment, but the use concentration of puromycin is only 1 / 20 of that of Primocin, and the cost of a single experiment is reduced to 14% of the original cost. Besides, puromycin used in the invention has no obvious influence on proliferation and passage capacities of tumor organoids, can well retain histological characteristics of primary lesions of tumor patients, and can effectively eliminate microbial contamination in tissue samples, thereby significantly reducing culture failure cases caused by contamination.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a new use of puromycin, specifically the application of puromycin in organoid culture of surgical specimens from gastrointestinal tumors. Background Technology

[0002] Tumor organoids, which retain the intratumoral heterogeneity and subclonal structure of the primary tumor well, are important in vitro models for studying tumor biological behavior, drug resistance mechanisms, and personalized treatment. However, gastrointestinal tumor organoids are susceptible to microbial contamination during culture, especially in patient-derived samples, where microbial contamination significantly exacerbates the failure rate in the early stages of culture. Currently, researchers typically inhibit microbial contamination by adding 0.1 mg / mL of Primocin to the washing solution before tissue processing. While this method reduces the risk of contamination to some extent, using high concentrations of Primocin (0.1 mg / mL) significantly increases the cost per experiment, thereby increasing the cost of establishing an organoid biobank. Summary of the Invention

[0003] Based on the above, this invention provides the application of puromycin in the culture of organoids from gastrointestinal tumor surgical specimens. By adding a low concentration of puromycin to the cleaning solution instead of puromycin during the tissue cleaning process before culturing organoids from gastrointestinal tumor surgical specimens, the problem of high cost of pretreatment cleaning solutions in existing organoid culture of gastrointestinal tumor surgical specimens is effectively solved. This achieves a cleaning effect comparable to traditional methods while significantly reducing costs, thereby promoting the popularization and application of gastrointestinal tumor organoid culture technology.

[0004] To achieve its purpose, the present invention adopts the following technical solution:

[0005] This invention provides the application of puromycin in the culture of organoids from surgical specimens of gastrointestinal tumors, specifically: adding the puromycin during the cleaning process of the surgical specimens of gastrointestinal tumors before culture; wherein the gastrointestinal tumor is gastric cancer or colorectal cancer.

[0006] As a further preferred embodiment of the technical solution of the present invention, the application method includes the following steps:

[0007] (1) Add the antibiotic mixture and puromycin to HBSS buffer to obtain tissue washing solution; the antibiotic mixture is a mixture of penicillin, streptomycin and amphotericin B;

[0008] (2) The tissue was cleaned with a tissue cleaning solution;

[0009] (3) After cleaning, the tissue was immersed in the tissue cleaning solution and incubated at 37°C for 15 min.

[0010] (4) After the culture is completed, the tissue is removed, mechanically cut into pieces, and then enzymatically digested.

[0011] Further, in step (1), the concentration of penicillin in the tissue washing solution is 20 U / mL, the concentration of streptomycin is 20 mg / mL, the concentration of amphotericin B is 25 μg / mL, and the concentration of puromycin is 5 μg / mL.

[0012] Furthermore, in step (3), the culture time is 5-15 min. A culture time of less than 5 min will result in the contaminants not being inhibited, while a culture time of more than 15 min will result in a decrease in the number of viable cells in the gastrointestinal tumor tissue. Within the above time range, 15 min is more preferred.

[0013] By adopting the above technical solution, the beneficial effects of the present invention are as follows:

[0014] This invention utilizes a washing solution containing 5 μg / mL puromycin (Solepro, #P8230, 10 RMB / mg) instead of a washing solution containing 0.1 mg / mL Primocin (InvivoGen, #ant-pm-1, 3.6 RMB / mg) to wash primary tumor tissue before organoid culture of surgical specimens from gastrointestinal tumors. The cost of the puromycin washing solution is 0.05 RMB / mL, while the cost of the Primocin washing solution is 0.36 RMB / mL. Puromycin is a broad-spectrum antibiotic that kills contaminating microorganisms by inhibiting protein synthesis. Experimental results show that in organoid culture of samples from 46 patients with gastrointestinal tumors, one sample washed with Primocin was contaminated, two samples washed with puromycin were contaminated, and 11 samples in the control group washed with HBSS were contaminated, resulting in a contamination rate as high as 23.9%. These results demonstrate that the cleaning method of this invention can increase the success rate of gastrointestinal tumor organoid culture (gastric cancer, GC; colorectal cancer, CRC) from 76.1% to over 95%, which is essentially on par with the results of Primocin cleaning. However, this method significantly reduces experimental costs: the concentration of puromycin used is only 1 / 20 of that of Primocin (5 μg / mL vs. 0.1 mg / mL), reducing the cost per experiment to 14% of the original cost, making it widely applicable in establishing organoid biobanks. Further experiments have shown that when culturing organoids from gastrointestinal tumor tissues, using 5 μg / mL puromycin to clean patient-derived tissues effectively reduces microbial contamination, while having no significant impact on the proliferation and passage capacity of tumor organoids, and effectively preserving the histological characteristics of the primary tumor lesions. By further optimizing the action time of puromycin, microbial contamination in tissue samples can be efficiently removed, thereby significantly reducing culture failures due to contamination. Attached Figure Description

[0015] Figure 1 A flowchart illustrating the process of establishing patient-derived organoids after treatment with different washing solutions is provided; ① HBSS containing antibiotic mixture: HBSS (VivaCellBioscience, #C3571) + antibiotic mixture (P / S / A); ② HBSS containing Puromycin: HBSS + 5 μg / mL Puromycin + P / S / A; ③ HBSS containing Primocin: HBSS + 0.1 mg / mL Primocin + P / S / A;

[0016] Figure 2Results of bacterial contamination prevention during organoid growth in gastrointestinal cancer patients using washing solutions containing puromycin or Primocin; (A) Representative microscopic images of CRC PDO#17 organoid cultures on days 1 and 3. The first microscopic image on the left on day 3 shows organoid cultures contaminated with microorganisms. ① indicates HBSS containing P / S / A; ② indicates ① with the addition of Puromycin; ③ indicates ① with the addition of Primocin; (B) In CRC PDO#17 organoid cultures, after tissue washing with HBSS washing solution containing P / S / A and organoids being seeded onto culture plates, bacterial contamination led to degradation of the Matrigel matrix. ① The red arrow indicates an area of ​​high bacterial concentration, and the culture medium is yellow, indicating active bacterial metabolism. ② and ③ show uncontaminated cultures with normal culture medium color; (C) Total number of contamination events observed under different washing conditions. The bar chart shows the number of contamination events (n=46) under different washing conditions.

[0017] Figure 3 Histological and immunophenotypic features of colorectal cancer organoids (representative micrographs of paired tissues and organoids); both tissue and organoid sections were stained with hematoxylin-eosin (H&E) and CD31 antibody. Scale bar for HE-stained images: 10 μm. Scale bar for immunohistochemical images: 50 μm.

[0018] Figure 4 Figures showing the results of organoid proliferation without the influence of different washing solutions on tissue pretreatment; (AB) shows that the presence of Puromycin or Primocin in the washing solution does not affect organoid proliferation, (A) is PDO of CRC-colorectal cancer, and (B) is PDO of GC-gastric cancer. Organoids marked with yellow circles in each figure are used for area calculation. Each figure contains three organoids, and the scale bar is 200 μm.

[0019] Figure 5 Figures showing that pretreatment of tissues with different washing solutions does not affect organoid passage results. (AB) Characteristics of long-term culture of organoids constructed with puromycin or primomycin in the washing solution. (A) represents CRC-colorectal cancer, and (B) represents GC-gastric cancer. Scale bar is 100 μm. Detailed Implementation

[0020] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0021] Example 1

[0022] Experimental procedures for establishing patient-derived organoids after treatment with different washing solutions were developed. (See results below) Figure 1 )

[0023] 1. Tissue Collection. Two tissue samples from different sites are selected from fresh gastric cancer (GC) or colorectal cancer (CRC) tissue. The tissue surface is gently wiped with sterile gauze to remove contaminants, and the tissue blocks are immediately placed in 15 mL centrifuge tubes containing 7-8 mL of cell culture medium to maintain tissue hydration. The culture medium consists of DMEM / F12 (VivaCell Bioscience, #C3132), supplemented with 10% fetal bovine serum (FBS, VivaCell Bioscience, #C04002) and a 3% antibiotic mixture (containing penicillin 20 U / mL, streptomycin 20 mg / mL, and amphotericin B 25 μg / mL, VivaCell Bioscience, #C3423). This medium is called complete culture medium. One tissue sample is used to establish patient-derived organoids (PDOs), and the other tissue sample is immediately cryopreserved in liquid nitrogen or at -80°C for later use.

[0024] 2. In a sterile laminar flow hood, place the tissue block in a sterile petri dish with a diameter of 10cm, and carefully remove non-target tissues such as fibrous connective tissue and fat. Use Figure 1 The tissue was washed 8 to 10 times with the three different cleaning solutions shown. If there was still obvious blood or contaminants on the tissue surface, the cleaning steps were repeated until the tissue surface was clean. After cleaning, the tissue block was immersed in the specified solution and incubated in a 37°C incubator for 15 minutes, followed by mechanical shearing and enzymatic hydrolysis. Figure 1 ① HBSS containing antibiotic mixture: HBSS (VivaCell Bioscience, #C3571) + antibiotic mixture (P / S / A, containing penicillin 20U / mL, streptomycin 20mg / mL and amphotericin B 25μg / mL, VivaCellBioscience, #C3423); ② HBSS containing Puromycin: HBSS + 5μg / mL Puromycin (Solepro, #P8230) + P / S / A; ③ HBSS containing Primocin: HBSS + 0.1mg / mL Primocin (InvivoGen, #ant-pm-1) + P / S / A.

[0025] 3. Transfer the tissue to a sterile culture dish and finely chop it using a scalpel, making the tissue block approximately 1 mm in volume. 3The chopped tissue blocks were then transferred to a mixture containing type IV collagenase (Gibico, #17104019) and dispersin II (Gibico, #17105041) for enzymatic digestion until the tissue blocks dissociated into homogeneous cell clusters (approximately 100 μm in diameter). The cell clusters were immediately washed twice with pre-cooled sterile phosphate-buffered saline (PBS) to terminate the enzymatic digestion. The cell clusters were collected by centrifugation at 300 g (4 °C, 5 min), and the supernatant was carefully discarded.

[0026] 4. Resuspend the cell pellet in complete culture medium and filter it through a 100 μm cell sieve to remove any remaining large cell clumps or debris. Centrifuge the filtered cell suspension, discard the supernatant completely, and resuspend it in organoid culture medium.

[0027] 5. Thaw Matrigel (ABW, #082703) slowly on ice, then mix it with organoid culture medium at a volume ratio of 2:1.

[0028] 6. Seeding and Culture: Cells were seeded and cultured in 12-well plates. The pellet containing cell clusters was resuspended in a matrix gel-medium mixture, adjusting the total volume to 600 μL. 30 μL of the mixture was seeded into each well, ensuring the droplets were evenly distributed at the bottom. The plate was inverted and incubated at 37°C for 30 minutes to allow the matrix gel to solidify completely. Then, 1 mL of Primocin-free organoid culture medium was added, and the plate was placed in a 37°C, 5% CO2 cell culture incubator for upright culture. The culture medium was replaced with fresh medium every 2-3 days to maintain cell growth.

[0029] Example 2

[0030] use Figure 1 The tissues were washed 8 to 10 times with the three different washing solutions shown, and then the organoids were cultured according to the method described above. Results are shown below. Figure 2 . Figure 2 (A) are representative microscopic images of the #17 colorectal cancer organoid culture on days 1 and 3. The first microscopic image on the left on day 3 shows the organoid culture contaminated with microorganisms. Figure 2(B) is a schematic diagram showing bacterial contamination leading to Matrigel matrix degradation in PDO#17 organoid cultures of colorectal cancer. The red arrow in ① indicates areas of high bacterial concentration, with the culture medium appearing yellow, indicating active bacterial metabolism. ② and ③ show uncontaminated cultures with normal culture medium color. (C) represents the total number of contamination events observed under different washing conditions. This invention performed organoid cultures on samples from 46 patients with gastrointestinal tumors. One sample pretreated with Primocin experienced contamination, two samples pretreated with Puromycin experienced contamination, and 11 samples in the control group washed with HBSS experienced contamination. The results are shown in Table 1.

[0031] Table 1 Summary of contamination events detected in various PDO cultures under different cleaning solution treatment conditions

[0032]

[0033]

[0034] Example 3

[0035] This embodiment utilizes hematoxylin-eosin (H&E) and immunohistochemical analysis to determine whether successfully cultured colorectal cancer organoids can exhibit the histological characteristics of the patient's primary lesion. The results are shown in […]. Figure 3 . Figure 3 Representative micrographs of paired tissue-organoid pairs are shown. Both tissue and organoid sections were stained with hematoxylin and eosin (H&E) and CD31 antibody. Scale bar is 10 μm for HE-stained images and 50 μm for immunohistochemical images. The images demonstrate that colorectal cancer organoids reproduce the characteristics of the primary lesion tissue under both H&E and immunohistochemical staining.

[0036] Example 4

[0037] This example investigated the effects of different washing solutions pretreatment of tissues on organoid proliferation. Results are shown below. Figure 4 . Figure 4 (A) is colorectal cancer #18PDO, (B) is gastric cancer #1PDO. Organoids marked with yellow circles in each figure are used for area calculation. Each figure contains three organoids, and the scale bar is 200μm. Figure 4 The results showed that gastrointestinal cancer organoids are spheroids composed of many cells. After establishing organoids with different washing solutions, continuous observation revealed that after about 5 days of culture, different washing solutions did not affect the proliferation of organoids.

[0038] Example 5

[0039] This study investigated the effects of different washing solutions pretreatment on organoid proliferation. Using three-dimensional (3D) organoid culture technology, gastric and colorectal cancer organoids (PDOs) from 45 patients (patient #44 could not be established due to contamination from all three washing methods) were successfully established. Results are shown below. Figure 5 . Figure 5 Typical morphologies of organoids derived from gastric cancer (#1GC) and colorectal cancer (#18CRC) patients at different passage numbers are shown. (AB) represent the characteristics of organoids constructed with Puromycin or Primocin in the washing solution after long-term culture; (A) represents CRC-colorectal cancer, and (B) represents GC-gastric cancer. Scale bar is 100 μm. Figure 5 The results showed that using different washing solutions for tissue pretreatment had no significant effect on organoid passage.

Claims

1. Application of puromycin in organoid culture of surgical specimens from gastrointestinal tumors.

2. The application of puromycin as described in claim 1 in the culture of organoids from gastrointestinal tumor surgical specimens, characterized in that, The puromycin is added during the tissue cleaning process of gastrointestinal tumor surgical specimens before organoid culture.

3. The application of puromycin as described in claim 1 in the culture of organoids from gastrointestinal tumor surgical specimens, characterized in that, The gastrointestinal tumor is either gastric cancer or colorectal cancer.

4. The application of puromycin as described in any one of claims 1-3 in the culture of organoids from gastrointestinal tumor surgical specimens, characterized in that, The application method includes the following steps: (1) Add the antibiotic mixture and puromycin to HBSS buffer to obtain tissue washing solution; the antibiotic mixture is a mixture of penicillin, streptomycin and amphotericin B; (2) The surgical tissue was cleaned with a tissue cleaning solution; (3) After cleaning, the tissue is immersed in the tissue cleaning solution and incubated at a constant temperature of 37°C in an incubator; (4) After the culture is completed, the tissue is removed, mechanically cut into pieces, and then enzymatically digested.

5. The application of puromycin as described in claim 4 in the culture of organoids from gastrointestinal tumor surgical specimens, characterized in that, In step (1), the concentration of penicillin in the tissue washing solution is 20 U / mL, the concentration of streptomycin is 20 mg / mL, the concentration of amphotericin B is 25 μg / mL, and the concentration of puromycin is 5 μg / mL.

6. The application of puromycin as described in claim 3 or 4 in the culture of organoids from gastrointestinal tumor surgical specimens, characterized in that, In step (3), the culture time is 5-15 min.

7. The application of puromycin as described in claim 5 in organoid culture of surgical specimens from gastrointestinal tumors, characterized in that, In step (3), the culture time is 15 min.

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