Hypoxia preconditioned serum (HPS) and platelet-rich plasma (PRP) promote proliferation, neurite network formation, and neurite elaboration in N2a cells

Abstract

The use of autologous growth factors, such as those contained in Hypoxia-Preconditioned Serum (HPS) and Platelet-Rich Plasma (PRP), represents a promising strategy to enhance regenerative processes in injured nerves. As an initial in vitro study, we investigated the effects of these secretomes on the neuroblastoma cell line N2a. HPS and PRP were first compared with normal serum (NS) using a protein microarray to detect neurologically related growth factors. Subsequently, N2a cells were cultured with three different secretome concentrations (0.1%, 1%, and 10%) for up to 96 h and analyzed for proliferation (cell counting), viability (Alamar Blue), cytotoxicity (LDH assay), combined proliferation and migration (scratch assay), and neurite outgrowth. Microarray analysis identified 30 neurotrophic, regulatory, and inflammatory factors in both HPS and PRP. Compared with PRP, HPS contained significantly higher levels of BDNF, CNTF, and VEGF-A, as well as the regulatory factors IL-6, TGF-β, and LIF. Several neurodegeneration-associated markers, including FAS, TNF-α, and IL-8, were also elevated in HPS. Proliferation and migration were most strongly stimulated by the 1% concentration of HPS and PRP, which also maintained high viability and low cytotoxicity. After 96 h, both PRP and HPS markedly enhanced neurite outgrowth by increasing total neurite count, neurite length, neurite-covered area, and branching points. PRP-0.1% and -1% produced the strongest effects, with up to 1.7-fold higher values across assessed parameters than the negative control, whereas HPS-0.1% increased these parameters by up to 1.4-fold. Further analyses demonstrated that these effects occurred alongside marked cell proliferation. Although the proportion of neurite-bearing cells (NBCs) declined over time because total cell expansion outpaced the increase in NBCs, normalization of neurite parameters to NBCs revealed up to 1.7-fold increases in neurite count, neurite length, neurite-covered area, and branching points per NBC, with the strongest effects observed in HPS-0.1% and PRP-0.1%. HPS and PRP promoted expansion of the neurite network while enhancing neurite elaboration of NBCs despite pronounced proliferative effects. These findings identify HPS and PRP as biologically active secretomes with potential to support neuronal structural remodeling, warranting investigation in primary neuronal cultures and in vivo models of peripheral nerve regeneration.