Browsing by Subject "Neuronal culture"

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Defined hydrogel microenvironments for optimized neuronal culture
(2010-05) Seidlits, Stephanie Kristin; Schmidt, Christine E.; Shear, Jason B.
Three-dimensional (3D) in vitro culture systems that provide controlled, biomimetic microenvironments would be a significant technological advance for both basic cell biology research and the development of clinical therapeutics (e.g., as in vivo cell delivery constructs). A variety of signals determine cell phenotype, including those from soluble factors, immobilized biomolecules, mechanical substrates, and culture geometry. My research seeks to create hydrogel culture systems that incorporate these signals in a defined, controllable manner. Specifically, I have focused on developing hydrogels based on the extracellular matrix (ECM) component hyaluronic acid (HA) with precisely specified mechanical, chemical and geometrical microenvironments. For example, the mechanical environment presented by HA hydrogels was tuned to span the threefold range measured for neonatal brain and adult spinal cord by modifying HA with varying numbers of photocrosslinkable methacrylate groups. These hydrogels were used to evaluate the effects of mechanical properties of a 3D culture paradigm on the differentiation of ventral midbrain-derived neural progenitor cells (NPCs) and results demonstrated that the mechanical properties of these scaffolds can assert a defining influence on differentiation. In addition, whole fibronectin was incorporated into HA hydrogels as an adhesive factor to encourage angiogenesis in 3D cultures, as interplay between endothelial cells and neurons is an important determining factor during NPC development and axonal regeneration after injury. To create spatially defined neuronal cultures in three-dimensions, multiphoton excitation (MPE) was used to photocrosslink protein microstructures within HA hydrogels. This method can be used to create complex, 3D architectures that provide both chemical and topographical cues to direct cell adhesion and guidance on size scales relevant to in vivo environments. Using this approach, both dorsal root ganglion cells (DRGs) and hippocampal NPCs could be guided along user-defined, 3D paths. In future studies, these strategies can be combined into a single hydrogel to create a culture microenvironment with multiple types of highly specified cues (i.e., chemical, topographical, and mechanical).
Neuronal activity in motor neuron and V2a interneuron cultures
(2022-08-03) Somavarapu, Rachel; Sakiyama-Elbert, Shelly E.; Wang, Huiliang (Evan)
The consequences of a spinal cord injury can include paralysis, and loss of motor function, sensory function, and proprioception. Furthermore, spinal cord injuries are often associated with potentially fatal complications, such as pneumonia or sepsis. Spinal cord injuries can take a negative toll on a patient’s life by causing discomfort or by hindering them from living a normal life. Current treatments, such as rehabilitative therapy, electrical spinal cord stimulation, and spinal cord surgery are not effective in treating this condition. However, studying the behavior of cultured neurons in-vitro can help determine what combination of cells are most beneficial for spinal cord transplantation or targeting regeneration of endogenous neural populations. The goal of this project is to study two types of neurons that are found in the spinal cord, motor neurons and V2a interneurons, and examine how these cells behave when they are cultured separately versus cocultured together. Selectable cell lines containing puromycin N-acetyl-transferase (PAC) under the control of either the Hb9 or Chx10 gene regulatory element were induced and selected to form neurons over a 6 day period. The neurons were plated in multielectrode array (MEA) plates or 24 well plates. Collected data regarding the neural firing rate, burst frequency, number of spikes observed, network inter-spike interval coefficient of variation, and synchrony were analyzed to determine differences in activity between mono- and co-cultures.