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International Journal of Bioprinting In vitro 3D pancreatic acinar unit

epithelial cells surrounded by a thin basal lamina, stromal 3D biomimetic platforms, which fail to fully recapitulate
tissue, and pancreatic stellate cells (PSCs). The PSCs the native compartmentalized architecture of tumor
1,2
are stromal cells responsible for the intense desmoplastic microenvironment, which is known to affect cell activity
reaction occurring during the PDAC development. and cancer-cell response to drugs. 5,22,23 Specifically, the
3,4
Indeed, in healthy tissue, PSCs are characterized by high glandular morphology has been mimicked by using different
expression of both ectodermal and mesenchymal markers techniques, 24-27 which however have limitations such as low
and significant amount of retinoids such as vitamin A in reproducibility, throughput, and shape fidelity.
5
lipid droplets. The present work focuses on the development of a
Under the influence of inflammatory cues and cancer 3D in vitro model that resembles the functional unit of
cells-derived factors, PSCs become active and assume exocrine pancreas through melt-electrowriting (MEW).
a myofibroblast-like phenotype capable of deregulating MEW technology combines principles of conventional
extracellular matrix (ECM) homeostasis. Typically, activated electrospinning (solution electrospinning) with melt
6
PSCs surround the cancer cells and communicate with them extrusion-based methods, 28,29 and it can be defined as
via different cytokines and factors (i.e., interleukin [IL]- a contactless 3D printing technique due to the longer
6, IL-1β, and transforming growth factor beta [TGF-β]), distance between needle and print bed. Indeed, like solution
through complex autocrine and paracrine signaling electrospinning, a high voltage (HV) is applied between the
pathways. Therefore, the interplay between cancer and nozzle and the collector to exert an electrostatic attraction
7-9
stromal cells plays a key role in tumor evolution, promoting on the molten material, and a so-called Taylor cone forms
the pancreatic cancer progression by significantly affecting at the nozzle tip. The electrical field forces the formation of
gene expression patterns, metabolic activities, invasion/ a microscale polymer filament from the Taylor cone, which
10
metastasis phenomena, and resistance mechanisms. is deposited into a micrometer fiber on the collector. 28,29 Due
However, the mechanisms implicated in the early stages to the distance between nozzle and platform and the fiber
of PDAC evolution, as well as the complex and dynamic stretching induced by the electric field, the size of the extruded
phenomena involved in the crosstalk between cancer filament in MEW is about ten times lower than the filament
epithelial and stromal cells, still remain unclear. This lack of diameter obtained by fused deposition modeling (FDM)
11
knowledge limits the possibilities of early PDAC diagnosis, where the distance between nozzle and collector is minimal
30
thereby resulting in a poor clinical prognosis. Indeed, the (i.e., a few hundreds of microns), thus resulting in a better
31
number of patients undergoing surgical resection is below resolution of the printed structures. Compared to other
10%, as most of clinical cases have spread metastases extrusion-based techniques, such as 3D bioprinting, MEW
at diagnosis. 12,13 Furthermore, the extremely complex allows to achieve better resolutions, in terms of minimum
bioarchitecture of the pancreatic tumor microenvironment feature width, and higher geometrical accuracy due to the use
(TME) affects the efficacy of drugs and causes the failure of of thermoplastic polymers instead of cell-laden hydrogels. 32,33
current therapeutic strategies that are inadequate to treat this For these reasons, MEW was adopted in this work
extremely aggressive cancer. 12,14 To improve the knowledge to obtain microscale polycaprolactone (PCL) scaffolds
of the disease with the ultimate goal of performing early mimicking the half structure of the pancreatic acinus
diagnoses and designing effective therapies for enhancing (Figure 1a and b) having a resolution and size not
patients’ prognosis, functional and reliable in vitro models achievable with the 3D printing technologies available so
need to be designed and fabricated. 15-20 far. The use of this synthetic thermoplastic polymer (PCL)
For many years, cancer research has been conducted has been widely adopted in the fabrication of scaffolds as
using two-dimensional cell cultures and animal models, it is highly processable by electrospinning 34-36 and additive
which poorly recapitulate the biological complexity of manufacturing, 37-40 and it is recognized to support cell
the disease in humans. Recent efforts in creating more attachment, migration, growth, and long-term cultures.
representative preclinical in vitro models have led to the Then, MEW scaffolds were seeded with stromal cells
development of experimental replica of human tissues in (human fibroblasts) (Figure 1c ) and HPDE (both healthy
i
a 3D environment or inside a microfluidic chip (organ- HPDE and HPDE overexpressing KRAS oncogene) to
on-chip). PDAC-on-chip permits the recapitulation of the develop the 3D models (Figure 1c ) having a biomimetic
ii
physiological crosstalk between different cell phenotypes geometry and cell distribution.
and the high-throughput analysis, but it is unable to mimic
21
the 3D complex gland morphology of the functional unit 2. Materials and methods
of exocrine pancreas. Nevertheless, it remains a challenge 2.1. Cell culture
to reproduce the microanatomy of the exocrine pancreas Human pancreatic ductal epithelial cells (HPDE)—both
(intended as 3D architecture and cellular composition) in wild-type HPDE (HPDE-WT) and HPDE stably expressing

Volume 10 Issue 2 (2024) 414 doi: 10.36922/ijb.1975