Figure 1. Human Pancreatic Duct Organoids

PancreaCult™ Organoid Initiation Medium (OIM; Human) and Organoid Growth Medium (OGM; Human) support the initiation and expansion of human pancreatic duct organoids from pancreatic tissue or previously established organoid cultures. Shown are organoids grown using PancreaCult™ OIM and OGM and imaged on day 7 of passage 3.

Figure 2. PancreaCult™ Human Enables Initiation and Expansion of Pancreatic Ductal Organoids

Human pancreatic ductal organoids are initiated in PancreaCult™ OIM for the first 3 days of culture, then switched to PancreaCult™ OGM for the remainder of culture. Alternatively, organoids can be grown in PancreaCult™ OGM for a completely serum-free protocol, however, proliferative pancreatic ductal cells are better supported in PancreaCult™ OIM during the first 3 days of culture. Cultures should be passaged after 7 days with further medium changes every 2-3 days. Pancreatic ductal organoids are suitable for experimentation or banking after one full passage in PancreaCult™ OGM. For full culture instructions please refer to the product manual (Document #10000011617)

Figure 3. PancreaCult™ (Human) Provides Robust Expansion of Pancreatic Duct Organoids

(A) Organoid expansion in PancreaCult™ OGM provides robust expansion of organoids across different donors. (B) Organoids may be initiated in PancreaCult™ OGM for a completely serum-free workflow, however, initiation and maintenance of damaged tissue is better supported by initiating cultures in PancreaCult™ OIM. (C) Comparison of PancreaCult™ Human to two different DIY formulations showed more robust expansion of organoids in PancreaCult™ Human. Shown is the cumulative fold expansion of organoid fragments as counted at the end of each passage.

Figure 4. Pancreatic Duct Organoids Display Features of the Pancreatic Ductal Epithelium

Organoids grown using the PancreaCult™ (Human) display marker expression consistent with the pancreatic ductal epithelium when imaged using immunocytochemistry. Shown are organoids grown in PancreaCult™ OGM and stained for (A) pancreatic ductal marker CK19, (B) pancreatic ductal marker SOX9, (C) epithelial marker EPCAM, (D) proliferation marker KI67, (E) apical pancreatic duct marker MUC1, and (F) pancreatic ductal marker CA2. Organoids were imaged on passage 2 (A), passage 3 (B, C) or passage 10 (D-F).

Figure 5. Pancreatic Duct Organoids Cultured With PancreaCult™ Human Show Pancreatic Marker Expression Levels Similar to Exocrine Tissue

Pancreatic duct organoids grown using the PancreaCult™ Human show marker expression levels similar to those observed in exocrine tissue. Analysis by qPCR showed pancreatic duct organoids were enriched for (A) PDX1 (C) CK19 and (F) LGR5 as compared to total pancreatic tissue, demonstrating enrichment of proliferative duct organoids. Comparable expression of (B) SOX9, (D) cystic fibrosis transmembrane receptor (CFTR), and (E) CA2 was observed in pancreatic duct organoids. Expression levels are normalized to TBP and UBC housekeeping genes (ΔCT) and total pancreas for relative expression levels (ΔΔCT).

Figure 6. PancreaCult™ (Human) Efficiently Maintains the Long-Term Growth and Genetic Profile of PDAC (Pancreatic Ductal Adenocarcinoma) Organoids

(A) Pre-established PDAC organoid lines can be maintained and expanded in PancreaCult™ OGM for at least 10 passages (n = 4) and demonstrate comparable growth with PDAC organoid lines maintained in DIY media. *DIY medium culture for this line was terminated after 10 passages. (B) Pre-established PDAC organoids cultured in PancreaCult™ OGM for 5 passages retain somatic SNPs and indels in oncogenic driver and tumor suppressor genes from the parent PDAC organoid line. Whole-exome sequencing data was collected before (teal) and after 5 passages of PDAC organoid cultures in PancreaCult™ OGM (orange) or the DIY media (maroon). Grey and white boxes indicate the overall presence or absence of SNPs or indels in the indicated genes compared to the parent PDAC organoid line pre-established in DIY media. #TP53 reads were undetectable, indicating gene deletion.

Figure 7. PDAC Organoid Line-Specific Differences in GATA6 Expression, Morphology, and Chemotherapy Drug Response

PDAC organoid lines (PDAC1-3) grown using PancreaCult™ (Human) demonstrate line-specific differences in (A) GATA6 (GATA-binding factor 6) gene expression and (B) morphology. Expression levels are normalized to housekeeping genes (ΔCT) and primary pancreatic tissue for relative expression levels (ΔΔCT). PDAC1 and PDAC2 lines were expanded in PancreaCult™ OGM from pre-established lines while normal organoids and the PDAC3 line were established with PancreaCult™ OIM from dissociated primary tissue and maintained in PancreaCult™ OGM. PDAC3 expansion was facilitated by reduced oxygen culture (5%) and maintained in the absence of EGF. Scale bar = 500 μm. (C) PDAC organoid lines were cultured in 96-well plates within Matrigel® domes or layer cultures and treated with irinotecan (left) or 5-fluorouracil (right) and compared to 0.1% DMSO vehicle control. Full-medium changes with fresh compounds were performed each day and analyses were performed 24 hours after the final fresh compound addition (Day 3). Cell viability for treated, vehicle-treated, and untreated PDAC organoid lines was assessed using the CellTiter-Glo® 3D Cell Viability Assay (Promega Catalog #G9681). Irrespective of layer or dome culture format, PDAC organoid lines demonstrate line-specific differences in chemotherapy drug response. Error bars = SD.

Figure 8. Removing Epidermal Growth Factor (EGF) Abrogates Normal Pancreatic Duct Organoid Growth but Does Not Affect PDAC Organoids Cultured in PancreaCult™ (Human)

(A) EGF removal efficiently suppresses normal pancreatic duct organoid growth within the first passage. (B) PDAC organoids were established in PancreaCult™ OIM and maintained in PancreaCult™ OGM for 8 passages in low-oxygen culture (5%) before removing EGF. EGF-depleted PDAC organoids maintained growth for at least 2 passages, indicating the presence of KRAS-activated tumor cells. Scale bar = 500 μm.

Figure 9. Organoids Can Be Established from Cryopreserved PDAC Cells Using PancreaCult™ (Human)

Mutational profile of organoids established and expanded in PancreaCult™ (Human) (Low O2, no EGF). Orange areas indicate areas of gains (top row) and grey areas indicate areas of losses (middle row) in the indicated chromosomes. There is a lack of high certainty loss of heterozygosity (LOH, bottom row). Microarray analysis was performed with Illumina iScan on the Global Diversity Array with Cytogenetics-8 (v1.0). CNVs (gene copy number variations) were called using the MoChA software tool for mosaic chromosomal alterations detection and analysis, MoChA, with human genome hg38 used as a reference.