Table 2.

Studies reporting GI cancers PDOs and their main culture characteristics

GI cancer type	Sample obtention	Method	Main outcomes	References
Colon adenocarcinoma	Surgery resected tissues from 20 patients with colon cancer	Digestion: collagenase type IX and dispase type II for 30 min at 37 ℃ Culture: 50% Matrigel in basal culture medium (advanced Dulbecco’s modified Eagle medium/ F12 supplemented with penicillin/streptomycin, Glutamax, B27, n-acetylcysteine) supplemented with EGF, gastrin, A83-01, SB202190 without growth factors	Optimization of long-term expansion protocol to colon tumor epithelial PDOs	[60]
Colon adenocarcinoma	22 surgery resected tissues and 19 normal-adjacent were derived from 20 patients with colon tumors	Digestion: Collagenase II, hyaluronidase and Ly27632 for 30 min at 37 °C Culture: BME with Human Stem cell Medium (HICS) minus Wnt (basal culture medium with 20% Rspo-1 conditioned medium, 10% Noggin conditioned medium, B27, n-acetylcysteine, nicotinamide, hEGF, Gastrin, A83-01, SB202190 and Primocin Drug screening: 15–20,000 organoids/ml seeded in 2% BME/growth media and 7-point of 83 compound library drug were tested. At 6 days, the cell viability was analyzed using CellTiter-Glo	CC-PDOs culture was successful in 81,5% (22/27) and closely recapitulate histological and genetic properties of the original tumor PDOs are amenable to high throughput drug screens allowing detection of gene-drug association	[40]
Metastatic colorectal cancers	14 patients’ biopsies from accessible metastatic lesions Clinical trial: NCT01855477	Digestion: was performed as described Sato et al. [60] Culture: Matrigel and basal culture medium supplemented with 20% Rspo-1 conditioned medium and 10% Noggin conditioned medium, nicotinamide, human EGF, gastrin, A83-01, SB202190, PGE2 and Primocin	CRCPDOs culture was successful in 71% (10 of 14 cases) and 90% of somatic mutations were shared and DNA copy number profiles were correlated between PDOs and biopsies from the same patients	[22]
Colorectal tumors	52 tumor samples, 23 surgery resected tissues and 29 endoscopic biopsies from 43 patients	Digestion: was performed as described Sato et al. [60] with several modifications. Liberase digestion for 60 min at 37 °C Culture: Matrigel and advanced DMEN/F12 supplemented with penicillin/streptomycin, HEPES, Glutamax, B27, gastrin I, n-acetylcysteine. Niche factors: EGF, Noggin, 10% Rspo-1 conditioned medium and 50% Wnt-3A, A83-01 and SB202190	55 CRCPDOs was successful in 100% of tissues. The PDOs often displayed Wnt or hypoxia dependency or SB sensitivity. The niche factors dependency decreases in adenoma-carcinoma transition	[62]
Metastatic gastrointestinal cancers	Ultrasound, CT-guide and endoscopic biopsies of 16 mCRC; 4 mGOC and 1 mCC from cancer patient. Clinical trials: NCT02994888 and NCT03010722	Digestion: PBS/EDTA containing 2 × TrypLe for 1 h at 37 °C Culture: Matrigel and organoid medium containing DMEN/F12 medium supplemented with EGF, Noggin, Rspo-1, gastrin I, FGF-10, Wnt-3A, PGE2, Y-27632, nicotinamide, A83-01 and SB202190. HGF only in cholangiocarcinoma organoids Drug screening: 4500–6000 cells were seeded in 30% of Matrigel and were treated with 50 custom-made library compounds in technical triplicate at 1 mM concentration. At 6–8 days, the cell viability assayed using CellTiter-Blue	GIs-PDOs culture was successful in 70% of biopsies. 96% overlap in mutational spectrum was observed between PDOs and their parental biopsies 100% sensitivity, 93% specificity, 88% positive predictive value, and 100% negative predictive value in forecasting response to targeted agents or chemotherapy in patients	[52]
Colorectal cancers and paired liver metastasis	72 surgery resected tissues, 36 primary CRC and their liver metastasis matched	Digestion: DMEM medium containing collagenase IV, collagenase II, hyaluronidase, dispase type II and YM-27632 for 30 min at 37 °C Culture: Medium and Matrigel 1:2 ratio and organoid medium containing DMEN/F12 medium supplemented with Rspo-1, Noggin, EGF, HEPES, Glutamax, Normocin, Gentamicin/amphotericin B, N2, B27, n-acetylcysteine, nicotinamide, A83-01, SB202190, gastrin and PGE2 Drug screening: 200 ±50 PDOs cells were seeded in Matrigel and were treated at 7-point concentration of 5-FU, CPT11, oxaliplatin and FOLFOX and FOLFIRI. At 3 days, the cell viability assayed using CellTiter-Glo 3D	PDOs culture was successful in 86,1% of CRC and 75.0% of liver metastasis and capture intra and interpatient heterogeneity at multiomic level PDOs in vitro response correlated with RECISTs and prognosis. PDOs may have a predictive value to determinate the risk of disease progression with FOLFOX and FOLFIRI	[37]
Metastatic colorectal cancers	67 tumor biopsies from 61 patients before start of treatment from TUMOROID clinical trial study (NL49002.031.14)	Digestion: Biopsy was dissociated with sharp needles Culture: The PDOs were cultured as a previously described in Sato et al. [60] and Dijkstra et al. [103] Drug screening: PDOs previously dissociated using dispase II. 100 PDOs per well were resuspended at 1:2 concentration of medium: geltrex. PDOs were incubated in four-fold drug matrices of 5-FU + oxaliplatin or 5-FU + irinotecan and two-fold single-drug doses response curves in technical triplicate. At 72 h of incubation the cell viability was measure using CellTiter-Glo 3D	PDOs culture was successful in 63% DOs predicted response for Irinotecan in more of 80% of patients. The PDOs do not predict response to 5-FU/capecitabine plus oxaliplatin combined therapy	[38]
Rectal cancer	65 rectal cancer PDOs from 58 individual patients collected by surgery and biopsy	Digestion: collagenase Type XI and 125 mg/mL dispase type II for 40 min at 37 °C Culture: Matrigel and organoid medium containing advanced DMEM/ F12 supplemented with antibiotic–antimycotic, B27, N2, Glutamax, HEPES, N-acetylcysteine, nicotinamide, 50% Wnt3a, 20% Rspo-1 conditioned medium, Noggin, EGF, A83-01, Y-27632 and SB202190 Drug screening: 50.000 cells were coated with 50% Matrigel. The PDOs were cultures with different doses to 5-FU, FOLFOX. For radiation experiment, 10.000 cells embedded 30 mL Matrigel and irradiated at 250 kVp and 12 mA. At 6 or 10–13 days, the cell viability assayed using CellTiter-Glo	PDOs overall success rate of 77% (65/84) PDOs retained molecular features of the derived tumors, and their ex vivo responses to clinically relevant chemotherapy and radiation treatment correlated with the clinical responses	[63]
Locally Advanced Rectal Cancer	112 Biopsy tumors of primary rectal cancer tumor, Clinical Trial: NCT02605265	Digestion: collagenase IV, collagenase II, hyaluronidase and dispase II (0.1 mg/mL) for 30–60 min at 37 °C Culture: Matrigel and organoid medium containing advanced DMEM/ F12 supplemented with Rspo-1, Noggin, EGF, HEPES, Glutamax, Normocin, Gentamicin/amphoteritin B, N2, B27, n-Acetylcysteine, Niacinamide, Alk 4/5/7 inhibitor, p38 inhibitor, Gastrin and PGE2 Drug screening: 200 ± 50 PDOs in 15 ml Matrigel with 300 mL medium. PDOs with or without chemotherapy treatment (10 mM 5-FU or 10 mM irinotecan (CPT-11) were exposed to X-rays (246 cGy/min, 250.0 kV, 12.00 mA, SSD = 50 cm). The cell viability was evaluated each three days using CellTiter-Glo 3.0	PDOs culture was successful in 85.7% (96/112) PDOs closely recapitulate the pathophysiology and genetic changes of corresponding tumors Chemoradiation responses in patients are highly matched to RCO responses, with 84.43% accuracy, 78.01% sensitivity, and 91.97% specificity	[48]
Ductal pancreatic cancer	20 primary tumor samples	Digestion: collagenase (Roche) Culture: Matrigel and tumor organoid medium containing DMEN with B27, ascorbic acid, insulin hydrocortisone, FGF-2, all-trans retinoic acid and Y267632 Drug screening: 2,500 cells seeded in Matrigel. At day 1 and 4 inhibitors and gemcitabine were added. At day 8, cell growth was analyzed using CellTiter 96 nonradioactive cell proliferation assay	PDOs culture was successful in 85% (17/20) samples and maintain phenotypic heterogeneity of the primary tumor PDOs retain patient-specific physiologic changes including hypoxia, oxygen consumption, epigenetic marks, and differential sensitivity to EZH2 inhibition	[42]
Liver tumors	Resected HCC (n = 3), CC (n = 3), combined CHC tumors (n = 2) and one healthy-liver derived donor (as a control)	Digestion: collagenase D, DNase I for 20–40 at 37 °C Culture: BME plus advanced DMEM/F12 supplemented with Penicillin/Streptomycin, Glutamax, 10 mM HEPES, B27 (without Vitamin A), N2, n-acetylcysteine, 10% (vol/vol) Rspo-1 conditioned medium, 30% (vol/vol) Wnt3a conditioned medium, nicotinamide, gastrin I, EGF, FGF-10, HGF, Forskolin, A83-01, Noggin and Y27632 Drug screening: 15,000–20,000 organoids seeded with 2% Matrigel. At following day were treated 29 anticancer compound, 4 point for 6 days and cell viability was evaluated using CellTiter-Glo	Establishment of PDOs was 100% and were expanding long-term (~ 1 year) Primary PDOs recapitulate the histological, > 80% of the cancer-related variant and transcriptomic profiles present in the original tumor In vivo xenograft of PDOs showed tumorigenic and metastatic potential PDOs facilitate the prediction of drug sensitivity and/ resistance in patient-specific manner	[41]
Liver tumors	Needle biopsies with ultrasound guidance	Digestion: collagenase IV and DNase I at 37 °C Culture: BME-2 plus advanced DMEM/F12 supplemented B27, N2, nicotinamide, n-acetylcysteine, gastrin, forskolin, A83-01, EGF, FGF-10, 10% Rspo-1 conditioned medium and 30% (vol/vol) Wnt3a conditioned medium Drug screening: 5,000 cells in BME2. At 6-day, sorafenib was added and cell viability was evaluated using CellTiter-Glo	HCC-PDOs with efficiency of generation of 33% (8/24) and retain the morphology and expression of HCC markers and preserve the genetic heterogeneity of the original tumors. HCC-PDOs display variable sensitivity to sorafenib Xenograph implantation in 80% of HCC-PDOs (16/20)	[64]
Ductal pancreatic cancer	20 primary tumor samples	Digestion: collagenase (Roche) Culture: Matrigel and tumor organoid medium (DMEM with B27, ascorbic acid, insulin hydrocortisone, FGF-2, all-trans retinoic acid and Y267632) Drug screening: 2500 cells seeded in Matrigel. At day 1 and 4 inhibitors and gemcitabine were added. At day 8, cell growth was analyzed using CellTiter-Glo 96 nonradioactive cell proliferation assay	PDOs culture was successful in 85% (17/20) samples PDOs maintain phenotypic heterogeneity of the primary tumor and retain patient-specific physiologic changes including hypoxia, oxygen consumption, epigenetic marks, and differential sensitivity to EZH2 inhibition	[42]
Ductal pancreatic cancer	10 human samples from surgical resection	Digestion: Collagenase II in human complete medium at 37 ºC for 16 h and treatment with TrypLe for 15 min at 37 ºC Culture: Matrigel and DMEM/F12 medium supplemented with HEPES, Glutamax, penicillin/streptomycin, B27, Primocin, n-acetylcysteine, Wnt3a-conditioned medium (50% v/v), Rspo-1conditioned medium (10% v/v), Noggin conditioned medium (10% v/v) or recombinant protein, EGF, gastrin, FGF-10, nicotinamide and A83-01	PDOs culture was successful in 75% (3/4) and 83% (5/6) samples in Netherlands and USA respectively Human and murine pancreatic PDOs with molecular and cellular properties to evaluate neoplastic progression	[66]
Pancreatic cancer	159 samples from primary tumors and 138 metastases from surgical resections, fine-needle biopsies or rapid rapids autopsies	Digestion: Collagenase XI, DNAse and Y-27632 for 1 h at 37 °C Culture: Matrigel plus DMEM/F12 supplemented with HEPES, Glutamax, A83-01, hEGF, mNoggin, hFGF-10, hGastrin I, n-acetylcysteine, nicotinamide, PGE2 1, B27, Rspo-1 conditioned media 10% and afamin/Wnt3A conditioned media 50% Drug screening: 500 cells seeded in 10% of Matrigel. At following day were treated with 5 chemotherapeutics agents. At 6 days were measure viability using CellTiter-Glo	PDOs (n = 66) with efficiency of generation of 75% and 78% harbored genetic alterations and transcriptomic analysis revealed unique cluster consistent with PDAC PDOs exhibited heterogenous responses to chemotherapy and other target agents. In a single-cell case study, the retrospective clinical data was equal with the PDO chemosensitivity profile	[44]
Pancreatic cancer	30 patient-derived organoids from pancreas and distal bile duct tumor	Digestion: Collagenase incubate at 37 ºC Culture: Cultrex growth factor reduced BME type 2 plus medium supplemented with Wnt3a-conditioned medium (50% v/v), B27, n-acetylcysteine, nicotinamide, A83-01, FGF-10 and Noggin Drug Screening: PDOs were dissociated in single cells and cultured in 384-well, 24 h later drug was added. After 72 h was evaluated the viability of the cells using CellTiter-Glo	PDOs Biobank with 30 samples and retain histological features from tissue which derived and present the genetic alterations common in this type of tumor. In drug assay identify 76 compounds could be target of therapies in pancreatic tumor. 1 PDOs present similar resistance to gemcitabine compare with the clinical response	[67]
Pancreatic cancer	Biopsied tissue from patient enrolled in Clinical Trial: NCT03563248	Digestion: mechanical digestion and enzymatic digestion Culture medium: Matrigel plus Human complete feeding medium: DMEM/F12, HEPES Glutamax, A83-01, hEGF, mNoggin, hFGF-10, hGastrin I, n-acetylcysteine, nicotinamide, PGE2 1 μM, B27, Rspo-1 conditioned media 10%, Afamin/Wnt3A conditions media 50% Drug assay: Gemcitabine, paclitaxel, 5FU and oxaliplatin were tested in logarithmically curve. At 5 days, the viability was evaluated using CellTiter Glo	PDOs culture was successful in 77% (59/77). Tissue derived from biopsy the establishment was 78% (34/45) and samples from resection was 75% (24/32). The results demonstrate that the protocol reported could facility precision medicine in the treatment of pancreatic cancer	[43]
Pancreatic cancer	Biopsy tissue	Digestion: STEMxyme for 30–40 min. Then, Accutase for 30 min Culture: 5% Matrigel plus Y-27632 plus DMEM supplemented with growth factors, insulin and FGF-2 Drug screening: PDOs were dissociated to single cells and diluted to 25.000 cells/mL and cultured for 4 days. Then the drug was added. At days, cell growth was measured using Cyto Tox-Glo	PDOs culture was successful in 41% (31 of 75). In 12 PDOs were tested drug, showed sensitives to Gemcitabine, 5FU, oxaliplatin, SN-38 and paclitaxel Reported a strong indication that drug sensitives in PDO models have the capacity to predict clinical response	[51]
Pancreatic cancer	94 patients with PDAC, 43 from biopsy and 73 from surgical specimen	Digestion: Collagenase XI, DNAse I and Y-27632 in Human complete medium Culture: Matrigel plus Human Complete Medium: advanced DMEM/F12, HEPES, Glutamax, Primocin, A83-01, hEGF, mNoggin, hFGF-10, hGastrin I, n-acetylcysteine, nicotinamide, B27, 10% Rspon-1 conditioned media, 50% Afamin/Wnt3A conditional media Drug Screening: PDOS were dissociated in single cells, the drug evaluated were Gemcitabine, placitaxel, SN-38, and 5-FU and oxaplatin, cell viability was measure with CellTiter Glo	Generation a biobank with a total of 117 patients with different ethnicity. PDOs culture was successful > 70% and feasible from tissue originating from biopsy or surgical resection	[54]
Pancreatic cancer	Tissue from surgical resected PDAC Clinical trial (NCT03563248)	Digestion: culture medium and collagenase XI Culture: 5% Matrigel plus Y-27632 plus DMEM supplemented with growth factors, insulin and FGF-2 Drug Screening: PDOs were dissociated in single cells and plated on 384-well with 10% of Matrigel, Gemcitabine, paclitaxel, irinotecan, 5FU and oxaliplatin were tested in logarithmically curve, cell viability was measured 5 days after drug treatment using CellTiter Glo	Demonstrate the capacity of rapidly establishment to PDOs from PDAC, in according to the clinical time predictive biomarkers of chemotherapeutic response. In addition, was the first prospective experience using PDOs	[68]
Gastric cancer	37 GC, 2 adjacent, 7 nontumor and 9 normal like specimens were collected by surgical resection, endoscopic biopsy and ascites puncture	PDOs were established as reported Bartfeld et al., 2015 [80] Digestion: PBS/EDTA containing 2 × TrypLe for 1 h at 37 °C Culture: Matrigel and DMEN/F12 medium supplemented with HEPES, Glutamax, B27, Gastrin I, n-acetylcysteine, EGF, FGF-10, Noggin, R-spo1, Afamin-Wnt-3A serum-free conditioned medium and A83-01	PDO culture was successful in 74.6% (44/49) PDOs maintained histopathological and molecular subtypes in comparison with their parental tissues	[70]
Gastric cancer	20 tissue samples were obtained from surgical resection	Digestion: Collagenase IX and Dispase II- Culture: Matrigel and DMEN/F12 medium supplemented with Wnt3A 50%, Rspo-1 10%, Noggin 10% (both as conditioned medium), B27, Nicotinamide, N2, n-acetylcysteine, hFGF-10; mEGF, Gastrin and A83-01 Drug screening: PDOs were seeded in Matrigel. At following day were treated with 5 chemotherapy drugs at 3-point and selected PDOs were treated with trastuzumab, Palbociclib and imatinib at 2-point. At 24-72 h the cell viability was assayed using Presto Blue Cell viability reagent	20 PDOs were established and shown to represent characteristics and altered pathway corresponding to primary tissue A differential response to chemotherapy was observed	[46]
Gastric cancer	10 human cancer fundus tissues and normal controls were collected from surgical resection	Digestion: Collagenase for Clostridium histolyticum for 30 min at 37 °C Culture: Matrigel and DMEN/F12 medium supplemented with HEPES, L-glutamine, Pen/Strep, N2, B27, n-acetylcysteine, nicotinamide, EGF, Noggin, R-spo1 conditioned media, Wnt conditioned media, FGF-10, Gastrin I, Y-27632, amphotericin B/gemtamicin and kanamycin Drug screening: organoids were treated with 3 chemotherapy drugs at 8 different concentrations (0–200 mmol/L) for 48 h. The organoids proliferation was measured by MTS assay	PDOs closely resembled the patient´s native tumor tissues Tumor PDOs exhibited differences in the response to drug treatment 1 tumor PDOs were highly responsive to drug treatment and were derived from patient with a near complete tumor response Orthotopic transplantation of PDOs resulted un engraftment and development of human adenocarcinoma	[72]
Gastric cancer	42 PDOs derived from 34 patient tissues obtained from surgical resection	PDOs were established as reported Barker et al., 2010 [116] and Bartfeld et al., 2015 [80] Digestion: collagenases, hyaluronidase and Y-27632 for 1 h at 37 °C Culture: Matrigel and standard gastric organoid medium containing advanced DMEM/F12 supplemented with GlutaMax, HEPES, P/S, 50% Wnt3a, 10% Rspo-1 conditioned medium, 10% Noggin conditioned medium, B27, EGF, FGF-10, n-acetylcysteine, Gastrin, A83-01, Y-27632 and Primocin Drug screening: 15–20.000 PDOs per mL were coated with 50% Matrigel. At following day were treated with 37 compounds, 7 point, three technical replicated. At 6 day the cell viability assayed using CellTiter-Glo 2.0	PDOs success rate > 50% 2–3 weeks and maintained molecular subtypes, capturing regional heterogeneity and subclonal architecture to the original tumors Morphology, transcriptome and genomic profiles remain long-term similarity than in vivo tumors	[47]
Gastric signet ring cell cancer	Tissue from 26 patients who underwent surgery	Digestion: advanced DMEM/F12 containing collagenases, hyaluronidase and Y-27632 Culture: advanced DMEM/F12, HEPES, Glutamax, Pen/Strep, B27 supplement, n-acetylcysteine, nicotinamide, Noggin, Wnt3a, Rspon-1, EGF, EGF2, FGF-10, A83-01, Y-27632 and Gastrin Drug screening: Organoids were dissociated, 5000 cells were evaluated a 6 concentration of 5-FU, oxaliplatin, irinotecan and docetaxel. Viability was measured with CellTiter Blue 72 h post treatment	Generation to biobank of GC organoids, evaluation to respond a therapy was completed in 4 weeks, indicating that the model could be treatment recommendations	[33]
Biliary tract carcinomas (BTC)	18 surgically resected tissue specimens obtained from BTC patients	Digestion: collagenase type XI and dispase type II for 1 h at 37 °C Culture: Matrigel and organoid medium containing advanced DMEM/ F12 supplemented with Glutamax, HEPES, Pen/Strep, N2, B27, EGF, n-Acetylcysteine, gastrin, nicotinamide, 10% Rspo1 conditioned medium, A83-01, Forskolin and Y-27632 Drug screening: 12,000 cells were plated and cultures for 4 days, and compounds were added al final concentration 0.1 mm. After 6 days, the cell viability was evaluated using water-soluble tetra- zolium salts assay	PDOs culture was successful in 50% (3/6) of IHCC and 20% (1/5) of GBC BTC-PDOs recapitulated the histopathology, gene expression and genetic alterations evident in the primary tumors SOX2 could be a potential prognosis biomarker for patients with BTC The antifungal drugs amorolfine and fenticonazole suppressed the growth of PDOs with minimal toxicity to normal epithelial cells	[39]
Gallbladder cancer	Surgically resected tumor tissues from 41 untreated GBC patients	Digestion: collagenase IV for 30–60 min at 37 °C Culture: Matrigel and organoid medium containing advanced DMEM/ F12 supplemented with B27, N2, n-acetylcysteine, Noggin, EGF, FGF-10, IGF, HGF, Gastrin, Y27632, nicotinamide, A83-01, Forskolin, dexamethasome, primocin, Pen/strep, Glutamax and HEPES Drug screening: 5000 cells were plated and the drug were added at final concentration of 10 µM then 24 h after cell seeding. At 4 days, the cell viability was evaluated each three days using CellTiter-Glo	PDOs culture was successful in 12.2% of GBC (5/41) PDOs recapitulates the histopathology, genetic, transcriptomic and intratumoral heterogeneity of the primary tissues PDO are a potentially a useful platform to explore molecular pathogenesis and discover personalized drugs	[49]

B27 B-27 supplement (the standard for neuronal cell culture), N2 N-2 supplement CTS (cell therapy systems), EGF epidermal growth factor, Rspo-1 R-spondin 1, PGE2 prostaglandin E2, CT-guide computed tomography-guide, mCRC metastatic colorectal cancer, mGOC metastatic gastroesophageal cancer, mCC metastatic cholangiocarcinoma, PBS/EDTA phosphate buffered saline (PBS) with EDTA, TrypLe DMEN/F12 Dulbecco’s Modified Eagle Medium/Ham’s F-12 basal media, FGF-10 fibroblast growth factor 10, Wnt-3a recombinant human Wnt-3a protein, HGF human hepatocyte growth factor, HEPES (4-2-hydroxyethyl)-1-piperazineethanesulfonic acid, FOLFOX 5-FU, leucovorin, oxaliplatin, FOLFIRI folinic acid, fluorouracil and irinotecan, RECIST response evaluation criteria in solid tumors, BME basement membrane extract, FGF-2 fibroblast growth factor 2, EZH2 enhancer of zeste 2 polycomb repressive complex 2 subunit, HCC hepatocellular carcinoma; combined, HCC/CC hepatocellular carcinoma plus cholangiocarcinoma, IHCC intrahepatic cholangiocarcinoma, GBC gallbladder cancer, IGF insulin growth factor