Neuroendocrine tissue engineering in rotating wall vessel bioreactors under simulated microgravity conditions

Abstract

interactions that promote and maintain tissue-specific differentiation. Phenotypic diversity can be maintained by growing cells under conditions which provide the necessary environmental cues for organ-like assembly, for example by growing cell aggregates in suspension culture. However, the level of tissue-specific differentiation in these aggregates is limited, in part, because of the detrimental effects of the high fluid shear stress encountered in conventional stirred fermentors. Some of these problems have recently been overcome by the introduction of the Rotating Wall Vessel (RWV) Bioreactor cell culture biotechnology, which achieves spatial co-location of cells cultured in suspension, through "simulated microgravity (µG)", i.e., randomized gravitational vectors (10 -2 g) and very low shear stress, viz. < 0.5 dynes/cm 2 ([1]. Cell culture in the RWV Bioreactors has significantly advanced our capabilities to create macroscopic three-dimensional tissue assemblies. Several groups have shown that the cell culture conditions in the RWV Bioreactors facilitate the assembly of tissue-like 3-D constructs and induce tissue-specific differentiation in a variety of normal and transformed cell types [2,3.Functionally, 3-D constructs obtained from both homotypic and heterotypic cell cultures maintained in the RWV Bioreactors, express tissue-specific markers and synthesize "products", such as soluble differentiation markers, in an organ-specific fashion [4]. Tissue-specific differentiation of the 3-D constructs is enhanced in the presence of diverse cell types in heterotypic co-cultures [2]. Thus, the culture conditions in the RWV provide an excellent in vitro system for studying the effects of environmental cues on tissuespecific cell assembly and differentiation. PC12 cells as model for neuroendocrine differentiation:PC12 pheochromocytoma cells, which are derived from a tumor of rat adrenal chromaffin cells, are an established model system for the bipotential differentiation of neural crest-derived cells of the sympathoadrenal lineage [5]. Depending on microenvironmental cues, cells of this lineage can differentiate in the presence of neurotrophins (such as NGF) into either sympathetic neurons or, in the presence of glucocorticoids (such as dexamethasone) or other differentiative agents into the neuroendocrine, chromaffin cell phenotype. Being bipotential as well as easily available, PC12 cells have become one of the best studied model systems for elucidating signaling pathways associated with neuronal and neuroendocrine differentiation [6]. Report Documentation Page

Proteomics and genomics of microgravity

“…They found enhanced phenylethanolamine-Nmethyl transferase expression, with subsequent catecholamine synthesizing enzyme activity. A downregulation of "neuronal" genes (GAP 43) and an upregulation of "neuroendocrine" genes (chromogranin A) was also observed, suggesting selective activation of signal transduction pathways leading to enhanced neuroendocrine differentiation of PC12 cells (64).…”

Bioreactor Technologies for Enhanced Organoid Culture

“…While commercially sold equipment is often either single-use and sold in sterile packaging or designed to be autoclavable, bioreactors made in the lab must be sterilized via other means. While the glass and PDMS often used for microfluidic bioreactors may be autoclaved, acrylics and 3D-printed materials must be sterilized with protocols utilizing ethanol, sodium hydroxide, or microwaves for sterilization, as has been described elsewhere [68,101,102]. Care must be taken when sterilizing the input/output ports and connectors, as these are often a source of contamination, especially in long-term cultures.…”

“…For example, neuronal/neuroendocrine PC12 cells (derived from a tumor of rat adrenal) have been extensively studied in SMG conditions [66]. In a further study on neuroendocrine interactions [67], upon seeding single cells into the RWV, the cells aggregated, forming a neuroendocrine organoid, which were cultured for up to 30 days. After one day in SMG in the RWV, multiple pathways, including the ERK, p38, and jnk signaling pathways and the adrenergic enzyme PNMT, were upregulated compared to static conditions (VueLife ® bags).…”

“…HARV condition organoids showing better-formed vasculature throughout the organoid from the HARV condition. Reproduced with permission from Lelkes et al [67].…”

The Impact of Simulated and Real Microgravity on Bone Cells and Mesenchymal Stem Cells

“…In addition, the endothelial nitric oxide synthase is phosphorylated by phosphoinositide 3-kinase under weightlessness, simultaneously with Akt [ 96 ]. The organoid formation by PC12 pheochromocytoma cells in a RWV bioreactor is accompanied by prolonged activation of the ERK, p38, and jnk signaling pathways [ 97 ].…”