A 3D Bio-Printed-Based Model for Pancreatic Ductal Adenocarcinoma

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is a disease with a very poor prognosis, characterized by incidence rates very close to death rates. Despite the efforts of the scientific community, preclinical models that faithfully recreate the PDAC tumor microenvironment remain limited. Currently, the use of 3D bio-printing is an emerging and promising method for the development of cancer tumor models with reproducible heterogeneity and a precisely controlled structure. This study presents the development of a model using the extrusion 3D bio-printing technique. Initially, a model combining pancreatic cancer cells (Panc-1) and cancer-associated fibroblasts (CAFs) encapsulated in a sodium alginate and gelatin-based hydrogel to mimic the metastatic stage of PDAC was developed and comprehensively characterized. Subsequently, efforts were made to vascularize this model. This study demonstrates that the resulting tumors can maintain viability and proliferate, with cells self-organizing into aggregates with a heterogeneous composition. The utilization of 3D bio-printing in creating this tumor model opens avenues for reproducing tumor complexity in the future, offering a versatile platform for improving anti-cancer therapy models.

Keywords: 3D bio-printing; co-culture; pancreatic ductal adenocarcinoma.

Figure 1

The bio-printing process involved trypsinizing cells and embedding them in hydrogel (2% sodium alginate + 15% gelatin) to create bio-ink. Tumor models were printed as cylinders measuring 6 mm in diameter and 1.5 mm thick, with a volume of 62.5 µL of bio-ink for each structure. The printed structures were then cross-linked (using CaCl₂ 100 mM for 7 min), supplemented with fresh culture medium, and placed in an incubator at 37 °C with 5% CO₂ until the experiments were conducted.

Figure 2

Evaluation of the shear-thinning behavior of bio-ink: viscosity (black) and shear stress (red) vs. shear strain rate.

Figure 3

Optimization of co-culture cellular concentration Panc-1/MeWo cells (comparisons on days 1 and day 7 after bio-printing at concentrations of 2, 3, and 4·10⁶ cells·mL⁻¹) by HES staining to evaluate the behavior of cells within the matrix.

Figure 4

Live/Dead staining was conducted followed by confocal microscopy imaging on the bio-printed tumor models. Live cells emit green fluorescence, while dead cells emit red fluorescence. Tile scan images were obtained by combining images from multiple acquisition fields (10× objective) to provide an overall view of the bio-printed structure. NC designates negative control (bio-printed structures exposed to methanol for 30 min). Representative images from three independent experiments are displayed.

Figure 5

WST-1 and AlamarBlue assays performed on bio-printed structures to evaluate cellular activity over time. For both tests, the results are expressed as a percentage of cellular viability after numeric treatment and normalization to the highest value (on day 3), defined as 100% of cellular viability. Three independent experiments were performed. Errors bars correspond to standard deviations.

Figure 6

Histological analysis (HES staining) of bio-printed structures (co-culture condition) on days 1 to day 14 after bio-printing. Cells are individualized and homogenously distributed within the bio-printed structures. Purple stands for nuclear staining, while yellow/brown stains stand for collagen fibers. Representative images are shown.

Figure 7

The evolution of aggregate dimensions over time in the bio-printed structures was analyzed. For each aggregate, a small diameter (SDi) and a large diameter (LDi) were defined. The small diameters (SDis) and large diameters (LDis) were determined using ImageJ image processing software. These measurements represent the diameters of the ellipse passing through the center of the ellipse, with the large axis (large diameter) (a) and small axis (small diameter) (b) intersecting at the central point of the ellipse. The SDi and LDi of 50 different aggregates are measured for each time point, and the average results are presented. Error bars indicate standard deviations. To confirm the statistical significance of the differences in cell diameters between small and large aggregates, we performed a Mann–Whitney U test at each time point. The calculated p-values are as follows: day 1: p = 0.0017, day 3: p = 0.0027, day 7: p = 0.0030, day 10: p = 0.0012, day 14: p = 0.0062. These results indicate statistically significant differences at each time point (p < 0.01).

Figure 8

Immunohistochemistry (IHC) performed on 3D bio-printed structures to assess the expression of different markers. All sections were performed on day 7 after bio-printing. Both monoculture and co-culture conditions were analyzed. Representative images are shown.

Figure 9

Vascularization assay demonstrating the tri-culture Panc-1/MeWo/HUVEC-GFP within the artificial matrix during 7 days of observation. These images represent 10 µm cryosections. The green color is associated with GFP-transfected HUVEC. Three independent experiments were performed. Representative images are shown.