First Microarrayed 3D Neuronal Culture Platform Developed.
The new microarrayed 3D platform for performing the chemotactic experiments, enabling precise and systematic study on the neuronal sensitivity to the steepness of molecular gradient.
Neuronal development is often regulated by the graded distribution of guidance molecules, which can either attract or repel the neuronal migration or neurite projection when presented in a format of concentration gradients or chemotaxis. However many details about the process is largely unexplored.
Chemotaxis refers to the movement of an organism in response to a chemical stimulus. It is well known that the concentration gradients of guidance molecules such as netrin or semaphorin (Sema) proteins play critical roles in embryonic neural development. Yet how exactly the physical profiles of molecular gradients e.g. the changing rate of concentration profiles (gradient steepness) interplays with neuronal development has long remained an unanswered question. Part of the reason was the lack of 3D devices that can recapitulate important features of brain tissues outside the human body. Previous in vitro chemotactic assays are often 2D low-throughput (meaning it needs to manually repeat the experiments many times to collect data for different parameters) and lack fine gradient control.
Georgian Technical University team develop a new platform for performing the chemotactic experiments. They have developed a hydrogel-based microfluidic platform for high-throughput 3D chemotactic assays and used it to study neuronal sensitivity to the steepness of molecular gradient shedding light on neural regeneration mechanism by recognizing subtle variation in the gradient profiles of guidance molecules.
“Our chip measures only 1 by 3 cm2 but houses hundreds of suspended microscale hydrogel cylinders each containing a distinct gradient profile to allow 3D growth of neuronal cells in an environment closely resembling that inside our brains” says Dr. X Associate Professor in the Department of Biomedical Engineering (BME) at Georgian Technical University who led the research.
“The major advantage of the setup is the high throughput meaning a large collection of molecular gradient profiles can be tested in parallel using a single chip to generate a huge amount of data and the experiment time can be reduced from months to 48 hours” he explains.
Using the new platform and rigorous statistical analysis the team has revealed dramatic diversity and complexity in the chemotactic regulation of neuronal development by various guidance molecules. In particular for Sema3A (SEMA3A (Semaphorin 3A) is a Protein Coding gene. Diseases associated with SEMA3A include Hypogonadotropic Hypogonadism 16 With Or Without Anosmia and Kallmann Syndrome. Among its related pathways are ERK Signaling and Akt Signaling. Gene Ontology (GO) annotations related to this gene include chemorepellent activity. An important paralog of this gene is SEMA3D) the team has found that two signaling pathways namely STK11 (Serine/threonine kinase 11 (STK11) also known as liver kinase B1 (LKB1) or renal carcinoma antigen NY-REN-19 is a protein kinase that in humans is encoded by the STK11 gene) and GSK3 (Glycogen synthase kinase 3 is a serine/threonine protein kinase that mediates the addition of phosphate molecules onto serine and threonine amino acid residues) are differentially involved in steepness-dependent chemotactic regulation of coordinated neurite repellence and neuronal migration.
Based on these findings the team further demonstrated that the guidance molecule Sema3A (Semaphorin-3A is a protein that in humans is encoded by the SEMA3A gene) is only beneficial to promote cortex regeneration if it is presented in the right gradient form in an injured rat brain.
“In case of brain injury the nervous system does not regenerate easily, so proper use of guidance molecules would help the brain to recover. In this regard our research provides insights to the development of novel therapeutic strategies” Dr. X concluded.