Title Karakterizacija neurona spinalnih ganglija uzgojenih na mikro-igličastim podlogama Title (english) Characterization of dorsal root ganglion neurons cultured on micro-pillar substrates Author Tihana Marciuš Mentor Damir Sapunar (mentor)Committee member Jasna Marinović Ljubković (predsjednik povjerenstva)Committee member Srećko Gajović (član povjerenstva)Committee member Maja Valić (član povjerenstva)Granter University of Split Defense date and country 2024-07-15, Croatia Scientific / art field, BIOMEDICINE AND HEALTHCARE Universal decimal classificationUDC ) 61 - Medical sciences Abstract Na ponašanje kultiviranih staničnih modela, posebice neurona, značajno utječu kemijska i fizička svojstva njihovog mikro-okoliša. Za razliku od tradicionalnih monoslojnih sustava, trodimenzionalne (3D) platforme za uzgoj stanica, koje integriraju mikro- i nano-strukture, poboljšavaju performanse staničnih modela oponašajući arhitekturu bioloških tkiva. Stoga se 3D uzgojne podloge sve više koriste u staničnoj biologiji kako bi se stvorili nosači (engl. scaffolds) za široki spektar primjena u neurobiologiji, kardiologiji i bioinženjeringu.
Cilj našeg istraživanja i ove doktorske disertacije bio je ispitati učinke 3D uzgojnih podloga na bazi silicija s topografskim strukturama okomitih stupića (iglica) (engl. micro-pillar substrates, MPS) na morfološka i elektrofiziološka svojstva neurona spinalnog ganglija in vitro. Proveli smo temeljitu analizu rasta i raspodjele neurona, te njihove morfologije (usmjerenost, grananje i dužina neurita) na različitim topografijama MPS podloga, kao i karakteristika akcijskih potencijala u usporedbi s onima na standardnim staklenim površinama.
Rezultati pokazuju da su MPS podloge podjednako pogodne za rast i adultnih i neonatalnih neurona spinalnog ganglija kao i kontrolne staklene podloge, pri čemu neonatalni neuroni preferiraju područja s najužim razmakom između struktura (0.6-1.4 μm), dok adultni neuroni favoriziraju nešto širi razmak (1.6-3.2 μm). U ovim MPS područjima (0.6-3.2 μm) neuroni spinalnog ganglija imali su i bolje morfološke karakteristike, pri čemu su i adultni i neonatalni neuroni razvili manji broj dužih neurita te specifičan usmjereni rast neurita u svim podtipovima neurona (N52-, IB4- i CGRP-pozitivni) usmjeravajući se duž tri glavne osi (30°, 90° i 150°), što nije bio slučaj u preostalom području MPS-a i kontrolnim staklenim podlogama gdje je rast neurita nasumično orijentiran. To naglašava utjecaj topografije MPS-a na usmjeravanje rasta neurita i oblikovanje ukupne morfologije neurona. Na dalje, elektrofiziološka analiza putem patch-clamp tehnike pokazuje da neuroni na MPS-u održavaju parametre akcijskih potencijala, te sposobnost okidanja akcijskih potencijala slične onima na staklu, osiguravajući očuvanje ključnih elektrofizioloških svojstava neurona spinalnog ganglija na površinama MPS-a.
Ova otkrića ističu potencijal MPS uzgojnih podloga kao optimalnog 3D sustava za primjene u neuroznanosti, utirući put za razvoj implantata tkivnog inženjerstva i naprednih čipova za snimanje neurona, poput mikro-elektrodnih čipova (MEA), s precizno definiranim fizičkim značajkama koje potiču i strukturni integritet i funkcionalnu održivost kultiviranih neurona.
Abstract (english) Cultured cellular models, especially neurons, are profoundly influenced by their microenvironment’s chemical and physical properties. Unlike traditional monolayer culture systems, three-dimensional (3D) cell culture platforms, integrating micro- and nanostructures, enhance cellular model performance by mimicking biological tissues architecture, leading to their increased use in cell biology to create scaffolds for a broad spectrum of applications across neurobiology, cardiology, and bioengineering.
Our research explores the effects of silicon-based substrates with vertically aligned micro-pillar structures (MPS) on both the morphological and electrophysiological properties of dorsal root ganglion (DRG) neurons in vitro. We conducted a thorough analysis of neuronal growth and distribution, morphology (neurite alignment, branching, and length) across different MPS topographies, as well as action potential characteristics compared to those on control glass.
The results indicate that MPS environments are as conductive to the growth of both adult and neonatal DRG neurons as traditional glass surfaces, with neonatal neurons preferring areas with the narrowest spacing between micro-pillar structures (0.6-1.4 μm), and adult neurons favoring a slightly wider spacing (1.6-3.2 μm). In these MPS areas (0.6-3.2 μm) both neonatal and adult DRG neurons had better morphological characteristics, exhibiting smaller number of longer neurites and directed growth of neurites in all subtypes of DRG neurons (N52-, IB4-, and CGRP-positive) aligning along three major axes (30°, 90°, and 150°), which is not the case in the remaining areas of MPS and control glass where neurite growth is randomly oriented. This highlights the influence of MPS topography on directing neurite outgrowth and shaping the overall morphology of DRG neurons. Moreover, electrophysiological analysis via whole-cell patch-clamp recordings indicates that neurons on MPS maintain action potential parameters and firing patterns similar to those on glass, ensuring the preservation of DRG neurons’ crucial electrophysiological properties on MPS surfaces.
These findings underscore the potential of MPS as an optimal 3D system for neuroscience, facilitating the development of tissue-engineered constructs and advanced neuro-electronic interfaces, including high-throughput micro-electrode arrays (MEA), with precise physical features that foster both the structural integrity and functional viability of cultured neurons.
Keywords
spinalni živčani korijeni
spinalni gangliji
neuroni
tehnike stanične kulture
Keywords (english)
Spinal Nerve Roots
Spinal Ganglia
Neurons
Cell Culture Techniques
Language croatian URN:NBN urn:nbn:hr:171:244036 Project Number: IP-2013-11-4126 Study programme Title: Translational Research in Biomedicine - TRIBE Type of resource Text File origin Born digital Access conditions Open access Terms of use Created on 2024-07-11 11:03:44
