Three-dimensional (3D) bioprinting is already being used to fabricate functional living tissues. Although still in its infancy, this technology shows promise for manufacturing transplantable tissues or testing personalized treatments. Remarkable advances in this technology are also appearing in fundamental research to treat cancer. For example, within the DeLIMIT project, researchers from the "Victor Babeș" University of Medicine and Pharmacy and the OncoGen Institute in Timișoara successfully manufactured 3D tissue models of the tumor microenvironment and studied their evolution. However, 3D bioprinting involves numerous difficulties, especially related to the programming of the bioprinters. Writing in G-code, traditionally used to transmit print commands, is difficult and does not allow for easy adjustment or scaling of construct geometry. Therefore, this project aims to develop Python programs capable of generating G-code instructions for printing tissue tumor models with adjustable geometry. In addition, these programs are experimentally validated to optimize the printing process of toroidal or triple-layered tumor models containing SK-BR-3 cells (breast cancer cell line) and peri-tumoral fibroblasts.

The general objective of this project aims at the development and experimental validation of some software packages written in the Python language for optimizing the geometry and bioprinting accuracy of three-dimensional tumor tissue models.

Specific objectives:

1. Adaptation of the G-codes used for bioprinting with the Cellink Inkredible bioprinter (used in the previous DeLIMIT project) for the Cellink Bio X model. 
2. Development of Python programs for generating flexible geometries of toroidal and triple-layered tumor model structures.
3. Optimizing the 3D bioprinting process with the Bio X bioprinter.
4. Optimization of open-source programs intended for classic 3D printers to be used in 3D bioprinting. Comparative analysis of the results obtained with these versus those generated from the programs developed within this project.
5. Biotyping and characterization of tumor models.

Within this project, software packages were developed capable of generating G-code instructions for the bioprinting of toroidal and triple-layered structures with the Cellink BIO X bioprinter (Figure 1). The constructs were analyzed for size and shape reproducibility (Figure 3) and if they agree with the proposed digital models (Figure 2). The developed programs were adapted to allow sequential bioprinting of structures on multiwell plates (Figure 4A). To validate the fabrication methodology of the toroidal and triple-layered tissue models, we printed the two types of constructs from hydrogels mixed with tumor and peri-tumor cells and followed their evolution in vitro (Figure 4B).

1. Bojin F, Robu A, Bejenariu MI, Ordodi V, Olteanu E, Cean A, Popescu R, Neagu M, Gavriliuc O, Neagu A, Arjoca S, Paunescu V. "3D bioprinting of model tissues that mimic the tumor microenvironment", poster , 13th European Biophysics Conference (EBSA 2021), 24-28 July 2021, Vienna, Austria
2. Bojin F, Robu A, Bejenariu MI, Ordodi V, Olteanu E, Cean A, Popescu R, Neagu M, Gavriliuc O, Neagu A, Arjoca S, Paunescu V. "3D bioprinting of model tissues that mimic the tumor microenvironment", poster , International Conference on Biofabrication, September 27-29, 2021, online event, Wollongong, Australia
3. Bojin F, Robu A, Bejenariu MI, Ordodi V, Olteanu E, Cean A, Popescu R, Neagu M, Gavriliuc O, Neagu A, Arjoca S, Paunescu V. "3D bioprinting of model tissues that mimic the tumor microenvironment", poster , 1st Oncohub Conference, October 13-15, 2021, online event, Bucharest, Romania
4. Bojin F, Arjoca S, Bejenariu MI, Ordodi V, Olteanu E, Cean A, Popescu R, Neagu M, Gavriliuc O, Neagu A, Paunescu V. "Biofabtication of tumor models that mimic the tumor microenvironment using extrusion bioprinting" - oral presentation , Tissue Engineering and Regenerative Medicine International Society (TERMIS) European Conference 2022, June 26 – July 1, 2022, Krakow, Poland
5. Arjoca S, Robu A, Bojin F, Ordodi V, Olteanu E, Cean A, Neagu M, Gavriliuc O, Neagu A, Paunescu V. "Post-printing structure formation in bioprinted tissue constructs that mimic the tumor microenvironment" - oral presentation, Tissue Engineering and Regenerative Medicine International Society (TERMIS) European Conference 2022, June 26 – July 1, 2022, Krakow, Poland
6. Bojin F, Arjoca S, Bejenariu MI, Ordodi V, Olteanu E, Cean A, Popescu R, Neagu M, Gavriliuc O, Neagu A, Paunescu V. "Biofabrication of breast cancer models using extrusion bioprinting" - oral presentation, 17th National Conference of Biophysics with International Participation (CNB2022), September 23-25, 2022, Târgu Mureș, Romania
7. Arjoca S, Robu A, Bojin F, Ordodi V, Olteanu E, Cean A, Neagu M, Gavriliuc O, Neagu A, Paunescu V. "Multicellular self-organization in bioprinted models of tumor microenvironment" - oral presentation, 17th National Conference of Biophysics with International Participation (CNB2022), September 23-25, 2022, Târgu Mureș, Romania

1. Bojin F, Robu A, Bejenariu MI, Ordodi V, Olteanu E, Cean A, Popescu R, Neagu M, Gavriliuc O, Neagu A, Paunescu V, 3D Bioprinting of Model Tissues That Mimic the Tumor Microenvironment. Micromachines 2021, 12, 535. https://doi.org/10.3390/mi12050535
2. Arjoca S, Robu A, Neagu M, Neagu A, Mathematical and computational models in spheroid-based biofabrication. Acta Biomaterialia 2023, 165, 125-139. https://doi.org/10.1016/j.actbio.2022.07.024

1. The "Research Project of the Year" award in the Romanian Healthcare Awards 2021 competition, December 10, 2021, Bucharest, Romania
2. Gold medal at InventCor 2021 Invention Fair, December 16-18, 2021, Deva, Romania
3. Participation in the Pro Invent Invention Fair, October 20-22, 2021, Cluj-Napoca, Romania