wo-photon polymerization has recently emerged as a promising technique to fabricate scaffolds for three-dimensional (3D) cell culture and tissue engineering. Here, we combined 3D-printed microscale scaffolds fabricated using two-photon polymerization with a bioactive layer-by-layer film coating. This bioactive coating consists of hyaluronic acid and poly(l-lysine) of controlled stiffness, loaded with fibronectin and bone morphogenic proteins 2 and 4 (BMP2 and BMP4) as matrix-bound proteins. Planar films were prepared using a liquid handling robot directly in 96-well plates to perform high-content studies of cellular processes, especially cell adhesion, proliferation, and BMP-induced signaling. The behaviors of two human pancreatic cell lines PANC1 (immortalized) and PAN092 (patient-derived cell line) were systematically compared and revealed important context-specific cell responses, notably in response to film stiffness and matrix-bound BMPs (bBMPs). Fibronectin significantly increased cell adhesion, spreading, and proliferation for both cell types on soft and stiff films; BMP2 increased cell adhesion and inhibited proliferation of PANC1 cells and PAN092 on soft films. BMP4 enhanced cell adhesion and proliferation of PANC1 and showed a bipolar effect on PAN092. Importantly, PANC1 exhibited a strong dose-dependent BMP response, notably for bBMP2, while PAN092 was insensitive to BMPs. Finally, we proved that it is possible to combine a microscale 3D Ormocomp scaffold fabricated using the two-photon polymerization technique with the bioactive film coating to form a microscale tumor tissue and mimic the early stages of metastatic cancer.

1. Rengaraj, Arunkumar, et al. "Engineering of a Microscale Niche for
Pancreatic Tumor Cells Using Bioactive Film Coatings Combined with
3D-Architectured Scaffolds." ACS Applied Materials &
Interfaces (2022).

2. PEM - Biomimetic basement membrane
4/1/2022
2
L Fourel, et al. J. Cell Biol. 2016
STIFF
SOFT
(1) Biomimetic film (2) Crosslinking (EDC) (3) Protein loading
TCPS, Glass
T. Crouzier, et al. Small, 2009
Presentation
mode
Dorsal Ventral
Biomolecule Soluble Matrix-bound
Diffusion Free Restricted
Life Time 13h Months
Stiffness Fixed Tunable
Co-presentation of
ligands
NO YES

3. Objectives
3
4/1/2022
 To prepare a platform for analyzing the cellular dynamics with the stimulated biomimetic
conditions.
Fabricating bioactive niche with a controlled 3D microenvironment for self assembled micro-
tumor model.
Rengaraj, Arunkumar, et al. "Engineering of a Microscale Niche for Pancreatic Tumor Cells Usi
Coatings Combined with 3D-Architectured Scaffolds." ACS Applied Materials & Interfaces (202

4. Cell adhesion, spreading, and proliferation of PANC1 on soft (EDC30)
4
Figure D. Cell adhesion, spreading, and proliferation of PANC1 on soft (EDC30)
and stiff (EDC70) PEM film. Fluorescence imaging on the cells with nucleus
marked with DAPI (blue) and the actin cytoskeleton marked with rhodamine (red)
on (D.1) soft film and (D.2) stiff film with FN, BMP2 or BMP4. The cell adhesion
(D.3, D.6, D.9), cell spreading (D.4, D.7, D.9) and the proliferation (D.5, D.8, D.11)
was studied in presence of fibronectin (D.3-5), BMP2 (D.6-8) or BMP4 (D.9-11). The
mean of 3 independent experiments is plotted with SEM. The star indicates that
the value is significantly different from the control condition.
Brightfield images of PANC1 cells after 120h of culture on EDC30, loaded with increasing
concentrations of either bFN, bBMP2, or bBMP4. The images were taken with a 4X objective
Brightfield images of PANC1 cells after 120h of culture on EDC70, loaded with increasing
concentrations of either bFN, bBMP2, or bBMP4. The images were taken with a 4X
objective.

5. 4/1/2022
5
Fabrication and characterization of 3D Ormocomp scaffold. Setup and steps in 2-photon
polymerization (a), with the computer-associated design (b). Optical imaging in bright field of
the top view (c) and 3D reconstruction (d). Scanning Electronic Microscope images of the
scaffold (e) and its surface (f).
Characterization of the coated 3D PEM-Ormocomp structure and protein loading
on the scaffold using a scanning confocal microscope. PEM coating marked with
FITC (green) on the scaffold (a, b), and BMP2-rhodamine (red) loading on the
hybrid structure (c, d).
Microstructure fabrication (2PP), PEM grafting on Ormocomp
scaffold
Rengaraj, Arunkumar, et al. "Engineering of a Microscale Niche for Pancreatic Tumor Cells Using Bioac
Coatings Combined with 3D-Architectured Scaffolds." ACS Applied Materials & Interfaces (2022).

6. 4/1/2022
6
PANC1 cells on PEM-Ormocomp hybrid structure
Figure G. 3D culture of PANC1 cells on the bioactive scaffold loaded with 5 µg/ml of fibronectin. (G.1) Bright-field images of cells after 3, 4, 10, and 20 days in culture.
Confocal images of PANC1 cells, (G.2) with nucleus stained in blue (DAPI) and cytoskeleton stained in red (Rhodamine) after 11 days of culture and (G.3) Live & Dead
assay after 20 days in culture with living cells marked in green and dead cells marked in red.
Rengaraj, Arunkumar, et al. "Engineering of a Microscale Niche for Pancreatic Tumor Cells Using
Bioactive Film Coatings Combined with 3D-Architectured Scaffolds." ACS Applied Materials & Interfaces

7. NBA Nano/Bio engineering/Analysis Laboratory
L
Conclusion
7
 We provided a bioactive niche with a controlled 3D microenvironment for cells to create on their own a mini-tumor-
like tissue through an almost fully automated process.
 In particular, we successfully created 3D microscale bioactive scaffold with micrometer scale resolution.
 We also showed how PEM (biomimetic coating) could mimic the biophysical condition of tumor
microenvironments, such as the stiffness and delivery of adhesion proteins and growth factors.
 We observed that cell adhesion, spreading, and proliferation processes were influenced by the biomechanical
(stiffness) and biochemical signals provided along by the PEM system.
 We also observed the PAN092 patient-derived cells to be unresponsive to a BMP2 stimulation, suggesting that these
cells might possess the pSMAD mutation reported in previous studies.
 This important information will help screen the optimal drug for the treatment. We are continuously working on
passivating the 2D surface to prevent unwanted cells and protein loading.