Gastric cancer tissues were collected from 3 patients with gastric cancer, who underwent primary surgery at The First Hospital of Lanzhou University (Lanzhou, China) from July to August 2021.

Inclusion criteria were preoperative pathologically confirmed gastric cancer in patients over 18 years of age and informed consent. The exclusion criteria were as follows: Patients who received radiotherapy and/or chemotherapy prior to surgery or did not agree to participate in the experiment.

The clinical and pathological data are as follows: Patient 1 was a 75-year-old male, cardia cancer, moderately differentiated; Patient 2 was a 58-year-old male, gastric antrum cancer, poorly differentiated; Patient 3 was a 74-year-old male, gastric antrum cancer, moderately-poorly differentiated. After surgery, this patient received six cycles of systemic chemotherapy (XELOX).

The present study was approved by the Ethics Committee of the First Hospital of Lanzhou University (Lanzhou, China; approval no. LDYYLL2022-292) and written informed consent was obtained from the patients.

The 24-well culture plates were purchased from Corning, Inc. DMEM/F12 medium, fetal bovine serum and cryopreservation solution were purchased from Biological Industries. GlutaMax, TrypLE, collagenase II and dispase II were from Gibco (Thermo Fisher Scientific, Inc.). The light microscope was from Nikon Corporation.

The complete medium consisted of DMEM/F12 supplemented with 1X GlutaMax, 10% fetal bovine serum and 1% penicillin and streptomycin. The tissue transport medium comprised DMEM/F12 with 1% penicillin and streptomycin. The mixed digestive enzymes medium comprised DMEM/F12 with 1 mg/ml collagenase II and 1.5 mg/ml dispase II.

The principle of aseptic operation was strictly followed. First, an appropriate amount of fresh tumor tissue (~5 mm²) was cut according to the size of the tumor and stored in the transport solution. The retrieved cancer tissue samples were placed in a 60-mm petri dish and washed with PBS to remove blood clots, as well as necrotic and fibrous components. A sterile surgical blade was then used to excise a part of the tumor tissue and a portion of the sample was frozen in a −80°C refrigerator for subsequent experiments. The remaining tissue (~0.3 cm³) was minced as much as possible, added to a 15-ml centrifuge tube, mixed with 10 ml digestive enzymes solution and placed in a shaker to be digested for ~40 min at 37°C. During this period, the digestion of the tissue block was closely observed. Once the tissue volume was reduced by half, the digestion solution was filtered through a 100-mesh cell sieve, collected into a 15-ml centrifuge tube and centrifuged at 400 × g for 4 min at 4°C, after which the supernatant was discarded. The centrifugation step was repeated twice, the cell pellet was collected and the cells were resuspended in 300 µl complete culture medium. The cell suspensions of 2 patients were individually mixed with 300 µl Matrigel^® (BD Pharmingen) at a ratio of 1:1 and then inoculated into 24-well culture plates (cat. no. 3527; Corning, Inc.) at 100 µl/well for organoid culture for 3 weeks. For the third case, the cell suspension of 6 ml was directly inoculated into a 24-well ultra-low adhesion plate (cat. no. 3473; Corning, Inc.) at 1 ml/well and organoids were formed by suspension culture at 37°C in a humidified atmosphere containing 5% CO₂.

The organoids were passaged when they reached a size of 100 µm under the microscope. The organoids and complete medium to be passaged were transferred to a 15-ml centrifuge tube, an appropriate amount of PBS was added, and the suspension was gently mixed with a pipette tip and then centrifuged at 400 × g for 4 min at 4°C, before the supernatant was removed and the pellet collected. Subsequently, ~2 ml TrypLE was added to the pellet and the suspension was gently mixed with a pipette to disperse the clumps, followed by digestion at 37°C while maintaining microscopic observation until the organoids were enzymatically digested to a small cell clump state of 3–5 cells. When terminating the digestion, 5 ml complete medium was added to the cell pellet, which was gently mixed with a pipette tip and centrifuged at 400 × g for 4 min at 4°C before removing the supernatant and collecting the pellet. The passage ratio was 1:2-1:3. A part of the organoids was added to the freezing medium and frozen at a density of ~1×10⁶ cells/ml; they were placed in a gradient freezing box at −80°C overnight and then transferred to liquid nitrogen for long-term storage. Prior to subsequent experiments, the cryopreservation tube was placed in a 37°C water bath. After thawing, the sample was quickly transferred to a 15-ml centrifuge tube, 5 ml complete medium was added, and the cell suspension was mixed by passing it through a pipette three times.

The organoids cultured in suspension were separated by centrifugation at 400 × g for 4 min at 4°C, immersed in 4% paraformaldehyde solution at 37°C for 24 h and then dehydrated with an ethanol gradient (50, 70, 80, 95, 100% ×2; 30 min for each gradient step). Subsequently, the dehydrated organoids were placed into 50% xylene and 50% ethanol for 30 min and immersed in pure xylene for 1 h twice at 37°C. A total of 200 organoids were agar-embedded, cut into thin slices with a thickness of ~5 µm, placed onto glass slides and dried.

The slices of the aforementioned steps were placed in xylene I for 10 min, xylene II for 10 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 95% ethanol for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min, 70% ethanol for 5 min and then distilled water for washing at 37°C. The sections were then stained with Harris hematoxylin for 3–8 min at 37°C, washed with tap water, differentiated with 1% hydrochloric acid ethanol for several seconds, rinsed with tap water, incubated in 0.6% ammonia water for nuclear staining, and rinsed with running water. The sections were then stained in eosin staining solution at 37°C for 1–3 min. Thereafter, the slices were placed for 5 min each into 95% ethanol I, 95% ethanol II, absolute ethanol I, absolute ethanol II, xylene I and xylene II to dehydrate and generate transparent slices at 37°C. After the last step, the slices were removed from xylene and dried before being sealed with neutral gum. Finally, the slides were examined under a light microscope (S40-Slider; Leica Microsystems) and images were acquired.

A total of 500-1,000 PDO 3D spheres were centrifuged at 500 × g for 5 min at 4°C, the supernatant was discarded, 4% paraformaldehyde was added, and spheres were fixed overnight at 25°C. Subsequently, they were dehydrated with a graded ethanol series, cleared with xylene for 1 h and soaked in paraffin (temperature, <50°C) twice, each for 1 h. After embedding, serial sections were made with a thickness of 3 µm. Another part of the fresh gastric cancer tissue removed by surgery was immersed in formaldehyde solution and then routinely fixed, dehydrated, embedded and sliced. The PDO sections and primary tumor sections were placed in a 65°C oven for 30 min, dewaxed with xylene, hydrated with a gradient of ethanol, processed with 0.5 mol/l sodium citrate antigen retrieval solution under high pressure at 98°C for 5 min and naturally cooled to 25°C. The sections were then blocked with 3% bovine serum albumin (Wuhan Servicebio Technology Co., Ltd.) at 25°C for 15 min and then incubated with primary antibodies against human P53, Ki-67, cytokeratin low molecular weight (CKL) and cytokeratin (CK)18 (Table I) at 4°C overnight. Subsequently, the sections were incubated with 3% hydrogen peroxide at 25°C for 15 min, horseradish peroxidase-conjugated goat anti-mouse IgG (cat. no. GB23303; 1:200 dilution; Wuhan Servicebio Technology Co., Ltd.) at 25°C for 15 min and horseradish peroxidase-labeled streptavidin (cat. no. A0303; 1:500 dilution; Beyotime Institute of Biotechnology) at 25°C for 15 min, and the samples were then washed three times with PBS for 3 min. After color development with 3,3′-diaminobenzidine, the nuclei were stained with hematoxylin at 37°C for 150 sec, then dehydrated with a gradient concentration of ethanol, made transparent with xylene and observed under a light microscope (IX73+DP74; Olympus Corporation).

The organoids cultured for 1 week were centrifuged and the pellet was obtained and resuspended in complete medium as previously mentioned. Subsequently, the cell suspension was seeded into 24-well ultra-low adhesion plate (cat. no. 3473; Corning, Inc.) at 0.5 ml/well (~500 organoids), and 0.5 ml complete medium was added to top up the total liquid volume to 1 ml per well. The experiment consisted of one control group and three experimental groups. In the control group, the same concentration of solvent was used. In the experimental groups, the commonly used chemotherapy drugs for gastric cancer, paclitaxel (Jiangsu Osaikang Pharmaceutical Co., Ltd.), oxaliplatin (Jiangsu Hengrui Pharmaceutical Co., Ltd.) and fluorouracil (Tianjin Jinyao Pharmaceutical Co., Ltd.), were used. At 24 h after inoculation, chemotherapy drugs were added to each experimental group. According to the relevant literature data (20,21), the final concentrations were 20 ng/ml for paclitaxel, 20 µM for oxaliplatin and 384 µM for fluorouracil. The morphological changes of the organoids (morphology, structure, size and quantity) were then observed under a light microscope (S40-Slider; Leica Microsystems) and compared every 24 h for 96 h.

After the gastric cancer tissues from 3 patients were individually digested with mixed enzymes, the organoids were successfully established by gradually increasing from a small granular type (Fig. 1Aa) to single spherical or multiple spore-like structures of different sizes (Fig. 1B). This culturing process took between 7 and 21 days when organoids reached a size of 100 µm under the microscope, depending on the characteristics of the tumor itself.

Under 2D culture conditions, gastric cancer cells exhibited a typical epithelioid cell morphology and cobblestone-like adherent growth, whereas the cell morphology was mainly of the short-spindle and round type (Fig. 2A and B). Under suspension culture conditions, early tumor cells first formed spherical sac-like structures (Fig. 2C), and with time, the spherical sac-like structures continued to develop into irregular clumps, and finally, gastric cancer organoids formed typical irregular lobulated lobes (Fig. 2D).

After 2 weeks of culture, gastric cancer organoids were embedded, sliced and analyzed by H&E staining. It was observed that the gastric cancer organoids had cystic or irregular glandular duct-like structures, an irregular cell arrangement, large and hyperchromatic nuclei and irregular ring-shaped glandular structures (Fig. 3A). This was highly similar to the biological characteristics of the original gastric cancer tissues (Fig. 3B).

Immunohistochemistry was used to detect the protein levels of P53, Ki-67, cytokeratin low molecular weight (CKL) and cytokeratin (CK)18 in gastric cancer organoids and original tumor tissues (Fig. 4). The results indicated that P53 (Fig. 4Aa), Ki-67 (Fig. 4Ba) and CKL (Fig. 4Ca) were all expressed in the cultured gastric cancer organoids, while staining for CK18 was negative (Fig. 4Da). The results were consistent with those for the original gastric cancer tissue (Fig. 4Ab, Bb, Cb and Db).

Paclitaxel, oxaliplatin and fluorouracil were added to the gastric cancer organoids cultured to the 8th day. The morphological changes of the organoids were observed every 24 h. Using light microscopy, it was indicated that, compared with that in the control group (Fig. 5A), the morphology of the experimental group was more disordered than that of the control group with certain organoids exhibiting cell death, among which the paclitaxel and oxaliplatin groups exhibited the most obvious changes. Organoid death gradually increased as the culture time progressed. After 96 h, it was observed that most of the organoids in the paclitaxel and oxaliplatin groups had disintegrated appearance and lost their morphological structure, while only a few organoids in the fluorouracil group had disintegrated (Fig. 5B-D). Patient 3 was treated with oxaliplatin and capecitabine combination chemotherapy for six cycles after surgery; carcinoembryonic antigen (Fig. 6A) and carbohydrate antigen 19-9 (Fig. 6B) have been stable, and no signs of tumor recurrence were found for 7 months after the operation. This verifies the feasibility and effectiveness of organoid drug screening to a certain extent.