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TE07 Project

English (Engleză)
Project title: Biophotonic micro-opto-fluidic platform for innovative cellular analyses
Periode: May 2018 - December 2020

Contact Person:
Project Director: Dr. Felix SIMA (INFLPR)

National Institute for Laser, Plasma and Radiation Physics - I.N.F.L.P.R.
Atomiștilor 409, Măgurele, Romania

The laser micromachining and micro- and nanostructuring are now approaching the biomedical field by developing new strategies for design and fabrication of microfluidic glass biochips. It is facing now the challenge of showing the exclusive potential of hybrid processing of both inorganic and organic materials. With this project new hybrid laser fabrication methods will be implemented in order to allow 2D and 3D microprocessing and structure functionalization. We propose a combination of laser bioprinting techniques and microfluidic approaches. In particular, a microfluidic device with an open microchannel functionalized surface that simultaneously generate multiple static microenvironments with variable composition (and/or topography) will be assembled. Microtopography, composition and pulsatile fluid flow stimulation will be the means of creating the necessary loading for inducing mesenchymal stem cells (MSCs) differentiation into osteoblasts. Both seeding of cells in the microfluidic device and continuous or intermittent diffusion of soluble growth factors will be introduced by perfusion. We expect that MSCs will modify behavior dependent either on the relative position on the surface gradient or concentration of introduced soluble factor or both. Further, we aim at creating biomimetic 3D polymeric environments by laser bioprinting and implement an optical interrogation method to study 3D MSCs characteristics inside the assembled biochip.

• Subtractive laser assisted fabrication and assembling of glass biochip;
• Laser modification of glass channel topographical features and functionalization;
• Laser printing of biomimetic microenvironments with variable composition;
• Laser printing of 3D polymeric configurations and functionalization;
• Implementation of a new laser interrogation method for 3D cellular studies.

Research Team:
• Dr. Felix Sima.
• Dr. Emanuel Axente
• Dr. Florin Jipa
• Dr. Cristina Pana
• Dr. Cristian Butnaru
• PhD. student Stefana Iosub
• PhD. student Mihai Sopronyi

Română (Romanian)
Titlul proiectului: Platforma biofotonica micro-opto-fluidica pentru analize celulare inovative
Perioada: Mai 2018 - Decembrie 2020

Persoana de contact:
Director Proiect: Dr. Felix SIMA (INFLPR)

Institutul Național pentru Fizica Laserilor, Plasmei și Radiației - I.N.F.L.P.R.
Atomiștilor 409, Măgurele, ROMÂNIA

Micro-procesarea cu ajutorul laserului si structurarea la scala micro- si nanometrica se apropie in prezent de aplicatiile in domeniul biomedical prin dezvoltarea unor noi strategii de configurare si fabricare a unor dispozitive microfluidice de sticla. Recent se abordeaza provocarile de demonstrare a potentialului exclusiv de procesare hibrida atat a materialelor anorganice pe baza de sticla cat si organice. In acest proiect, propunem implementarea unor noi metode hibride de fabricare laser ce permit micro-procesarea bi-dimensionala (2D) si tri-dimensionala (3D) si functionalizarea acestor structuri. Vom combina tehnici de bio-printare laser si abordari microfluidice. In particular, vom asambla un dispozitiv microfluidic cu canal deschis a carei suprafata functionalizata cu laserul va genera simultan multiple micro-medii statice de compozitie (si/sau topografie) variabila. Micro-topografia, compozitia dar si stimularea fluidica prin curgere pulsata vor reprezenta mijloacele de activare pentru inducerea diferentierii unor celule stem mezenchimale (MSC) in celule osteoblaste. Atat cultivarea celulelor in dispozitivul microfluidic cat si introducerea unor factori de crestere prin difuzie continua sau intermitenta vor fi realizate prin perfuzie. Ne asteptam ca MSC sa-si modifice comportamentul in functie de pozitia relativa pe gradientul compozitional (topografic) al suprafetei sau de concentratia factorului de crestere perfuzat sau de amandoua. Mai mult, vom propune dezvoltarea unor medii polimerice biomimetice 3D prin bio-printare laser si implementarea unei metode optice de interogare pentru studiul 3D al caracteristicilor MSC in interiorul dispozitivului biocip asamblat.

• Fabricarea substractiva laser si asamblarea unui dispozitiv microfluidic pe baza de sticla;
• Modificare laser de suprafete ale canalelor microfluidice si functionalizare;
• Printare laser a unor medii biomimetice polimerice cu compozitie variabila;
• Printare laser a unor medii biomimetice 3D polimerice cu diverse configuratii si functionalizari;
• Implementarea unor tehnici de interogare optica cu laser pentru teste celulare 3D.

Echipa de cercetare:
• Dr. Felix Sima.
• Dr. Emanuel Axente
• Dr. Florin Jipa
• Dr. Cristina Pana
• Dr. Cristian Butnaru
• Doctorand Stefana Iosub
• Doctorand Mihai Sopronyi


Scientific Reports 2018
Femtosecond (fs) lasers are nowadays the most promising tools for transparent glass processing. The subtractive fabrication process creating 3D hollow structures in glass, known as fs laser-assisted etching (FLAE), is based on selective removal of the laser-modified regions by successive chemical etching in diluted hydrofluoric acid solutions. In this project we demonstrated the possibility to generate embedded hollow channels in photosensitive Foturan glass volume by high repetition rate picosecond (ps) laser-assisted etching (PLAE). In particular, the influence of the critical irradiation doses and etching rates were evaluated in comparison of two different wavelengths of ultraviolet (355 nm) and visible (532 nm) ranges. Fast and controlled fabrication of a basic structure composed of an embedded micro-channel connected with two open reservoirs, commonly used in the biochip design, were achieved inside glass. Distinct advantages such as good aspect-ratio, reduced processing time for large areas, and lower fabrication cost were evidenced (Nanomaterials 8 (8), 583, 2018).

Scientific Reports 2019
Fast 3D fabrication in Foturan photosensitive glass by picosecond (ps) laser assisted etching assisted etching (PLAE) of a compact microfluidic system (biochip) with scale up-scale-down characteristics consisting of open microreservoirs connected through embedded channels for diffusion based gradient formation of biological relevant molecules over long periods of time to induce controllable cell migration Laser irradiation (Nd:YVO 4 , λ=532 nm, τ~10ps) followed by thermal treatment and wet chemical etching of photosensitive glasses allowed fabricating 3D embedded micro-channels, with high precision, down to micrometric sizes. A second thermal treatment is herein proposed for improvement of optical, topographical and morphological quality of glass surface processed by PLAE. By tailoring the temperature and treatment time interval, very smooth, defect free, with roughness down to few nanometers were achieved for glass processed surfaces. Cell adhesion studies on laser processed Foturan glass surfaces Cell adhesion is a biological process of fundamental importance with implications in the correct development, cell to cell signaling and regulation; moreover, the mechanical interaction between a cell and its extracellular matrix (ECM) can influence and control cell behavior and function. In this study we test the laser processed glass surfaces from the point of view of cell adhesion and proliferation. We have performed immunofluorescence, staining F-actin to evidence the cytoskeleton and the focal adhesion points with vinculin since they play key roles in the cell shape control. Furthermore, to test if the coating or the laser-processed Foturan surface can improve the adherence at early stages, the two different surfaces functionalized with and without collagen coating were used to analyze the cells at two time points, 1h and 24h after cell seeding. For this purpose, we used mesenchymal stem cells (MSC), which were grown on either Foturan or cover glass, coated with and without collagen. Although for this type of cell line, the number of attached cells is higher on un-coated cover glass, the collagen still provides a better adherence as it can be demonstrated by quantification of the degree of actin and vinculin co-localization. Same studies with MSC cells grown on laser processed glass surfaces indicate a good biocompatibility supported by cell attachment after one hour and by cell adhesion after 24 hours. We noticed no difference in the number of attached cells while the increase in cell adherence is observed when collagen is used, similarly to studies carried out on the cover glass samples.

Scientific Reports 2020
It is challenging to create synthetic environments that mimic in vivo characteristics, fabricating relevant biosystems along with imaging techniques for sub-cellular visualization in order to understand mechanism of cell migration. Femtosecond laser assisted chemical etching (FLAE) is a technology performing subtractive processing of glass in order to create 3D microfluidic structures embedded in a microchip with the micrometric feature size. We evaluate herein relevant glass platforms capable to offer both observation of collective cells migration over long periods and individual visualization at unicellular and subcellular levels on the target cell. Glass microfluidic biochips with micrometric characteristics are first fabricated by FLAE, hosting in vivo like microenvironments. Then, by applying two photon polymerization one may generate biomimetic polymeric hierarchical architectures with confining channels inside microchannels. The fabricated 3D glass nanofluidics is applied to observe behavior of cancer cell deformation and migration in narrow spaces, providing new findings. We have then developed a confocal microscopy station by assembling a supercontinuum white laser from NKT Photonics with a NKT variable filter Varia, a 3D translational stage with nanometric resolution on X-Y-Z axes from Aerotech and a CCD camera from Nikon. In this configuration we have tested fluorescence green emission from a photoresist layer to check the best X-Y resolution and applied for imaging cells in different 3D configurations.

[1] F Jipa, S Iosub, B Calin, E Axente, F Sima, K Sugioka “High Repetition Rate UV versus VIS Picosecond Laser Fabrication of 3D Microfluidic Channels Embedded in Photosensitive Glass”, Nanomaterials 8 (8), 583, 2018;
[2] F. Sima, H. Kawano, A.Miyawaki, L.Kelemen, P.Ormos, D. Wu, J. Xu, K.Midorikawa, K. Sugioka,“3D Biomimetic Chips for Cancer Cell Migration in Nanometer-Sized Spaces Using “Ship-in-a-Bottle” Femtosecond Laser Processing, ACS Applied Biomaterials, ACS Applied Bio Materials 1 (5), 1667-1676, 2018.
[3] E. Axente, F. Sima, "Biomimetic Nanostructures with Compositional Gradient Grown by Combinatorial Matrix-Assisted Pulsed Laser Evaporation for Tissue Engineering", Current Medicinal Chemistry (27), 1-16, 2020
[4] F Sima, H Kawano, A Miyawaki, K Obata, D Serien, K Sugioka, “3D glass nanofluidics fabricated by femtosecond laser processing for study of cancer cell metastasis and invasion”, Proceedings SPIE, Volume 11267, Laser Applications in Microelectronic and Optoelectronic Manufacturing (LAMOM) XXV; 1126714, 2020, https://doi.org/10.1117/12.2547222
[5] E Axente, LE Sima, F Sima, “Biomimetic Coatings Obtained by Combinatorial Laser Technologies”, Coatings 10 (5), 463, 2020
[6] LE Sima, G Chiritoiu, I Negut, V Grumezescu, S Orobeti, CVA Munteanu, F Sima, E Axente, “Functionalized graphene oxide thin films for anti-tumor drug delivery to melanoma cells”, Frontiers in Chemistry 8, 184, 2020.

Conference presentations :
[1] F Sima, F Jipa, S Iosub, B Calin, E Axente, K Sugioka, “Biomaterial 2D and 3D processing by innovative laser technologies: application to cancer cell study”, Invited, ISCP-INDLAS, Alba Iulia, Romania, September 3-7, 2018;
[2] F. Sima, Interaction of Ultrashort Laser Pulses with Transparent Materials: Processing and Manufacturing of 3D Lab-on-a-Chip Biosystems, Invited, Ignition Summer School 2018, Sibiu, Romania, 02-06 July 2018;
[3] S. Iosub, “In Vitro Testing of Innovative Glass and PDMS Biosystems Prepared by Laser-Assisted Methods Laser” Poster, Ignition Summer School 2018, Sibiu, Romania, 02-06 July 2018 (Second best poster award)
[4] F. Jipa “Microfluidic device fabrication in photosensitive glasses by high repetition rate picosecond lasers” Poster, Ignition Summer School 2018, Sibiu, Romania, 02-06 July 2018
[5] B Călin, M Zamfirescu, I Păun, C Luculescu, F Jipa, Ş Iosub, E Axente, F. Sima “Multiphoton processing technologies applied in laser-based 3D printing” Frontiers in Optics, JTu3A. 138, poster Laser Science 2018, Washignton, DC United States, 16–20 September 2018
[6] F. Sima, F.Jipa, S.Iosub, C.Butnaru, E.Axente, G.Chiritoiu, L.E. Sima, K.Sugioka, “Picosecond Laser Processing of Photosensitive Glasses for Lab-on-a-Chip Applications”, INVITED, FLAMN Conference, June 30 – July 4, St. Petersburg, Russia, 2019
[7] F. Sima,F. Jipa,S. Iosub,C. Butnaru,E. Axente,G. Ghiritoiu,L. E. Sima, K. Sugioka “Picosecondlaser3D processing of Foturanglass:Towardsfabricationofcomplexlargearea microfluidics”, ORAL, LAMP Conference, May 21-24, Hiroshima, Japan, 2019.
[8] F. Sima, “Laser processing and manufacturing of micro- and nanoscale biosystems”, INVITED, EuroNanoForum Conference, PILLAR 4 – NANO for EU RE-INDUSTRIALISATION, Nano Biomaterials and nanomembranes, June 12-14, Bucharest, Romania,2019
[9] S. Iosub (Orobeti), “Microfluidic glass biochip fabricated by picosecond laser assisted etching for controlled release of biomolecules”, BEST STUDENT POSTER AWARD, EuroNanoForum Conference, June 12-14, Bucharest, Romania, 2019
[10] C. Pana, “Mesoporous carbon thin films synthesized by Matrix-Assisted Pulsed Laser Evaporation”, POSTER, EuroNanoForum Conference, June 12-14, Bucharest, Romania, 2019
[11] F. Jipa, “Precise 3D micro-fabrication of photosensitive glass materials by high repetition rate picosecond laser-assisted etching”, POSTER, EuroNanoForum Conference, June 12-14, Bucharest, Romania, 2019
[12] F. Sima, D. Serien, D. Wu, J. Xu, H. Kawano, K. Midorikawa, K. Sugioka, “Micro and nano- biomimetic structures for cell migration study fabricated by hybrid subtractive and additive 3D femtosecond laser processing”, Invited at Photonics West, San Francisco, USA, 1-6 Feb., 2020 (invited)
[13] F. Sima, F. Jipa, C. Butnaru, S. Orobeti, E. Axente, H. Kawano, A. Miyawaki, K. Obata, D. Serien, Koji Sugioka, “Glass microfluidic lab-on-chip devices by ultrafast laser processing”, International Workshop of Materials Physics 2020 – Materials and structures for bio-applications, Magurele, Romania, 21 septembrie, 2020 (invited)
[14] F. Sima, Hiroyuki Kawano, Masahiko Hirano, Atsushi Miyawaki, Kotaro Obata, Daniela Serien, Koji Sugioka, “3D Nanofluidics Fabricated by Femtosecond Laser Processing in Glass for Cancer Research”, ICALEO Virtual Conference, 19 – 20 Octombrie, 2020 (oral, On-Demand session)

Master thesis:
[1] S. Iosub (Orobeti), Ultrafast laser fabrication of 3D microfluidic structures in photosensitive glasses for dynamic gradient formation of biomolecules,University POLITEHNICA of Bucharest, Faculty of Medical Engineering, Smart Biomaterials and Applications programme (Coordinators Prof. Izabela Stancu and Dr. Felix Sima), 2019

PhD Thesis
[1] M. Sopronyi, Thin films and nanostructures by advanced pulsed laser technologies for biomedicine, sensing and energy storage applications, University of Bucharest, Faculty of Physics, PhD Supervisor: Professor Dr. Ion MIHĂILESCU, PhD Referees: Dr. Felix SIMA, Dr.Camelia MATEI-GHIMEU, Univ. Conf. Dr. Eng. Elena LĂCĂTUŞ /