  Citation: Bufalo, M.C.; Almeida, M.E.S.d.; Jensen, J.R.; DeOcesano-Pereira, C.; Lichtenstein, F.; Picolo, G.; Chudzinski-Tavassi, A.M.; Sampaio, S.C.; Cury, Y.; Zambelli, V.O. Human Sensory Neuron-like Cells and Glycated Collagen Matrix as a Model for the Screening of Analgesic Compounds. Cells 2022, 11, 247. https://doi.org/ 10.3390/cells11020247 Academic Editor: Albert Quintana Received: 15 December 2021 Accepted: 8 January 2022 Published: 12 January 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). cells Article Human Sensory Neuron-like Cells and Glycated Collagen Matrix as a Model for the Screening of Analgesic Compounds Michelle Cristiane Bufalo 1,2 , Maíra Estanislau Soares de Almeida 2,3 , José Ricardo Jensen 4 , Carlos DeOcesano-Pereira 2 , Flavio Lichtenstein 2 , Gisele Picolo 1 , Ana Marisa Chudzinski-Tavassi 2,5 , Sandra Coccuzzo Sampaio 3,6 , Yara Cury 1, * and Vanessa Olzon Zambelli 1,2, * 1 Laboratory of Pain and Signaling, Butantan Institute, São Paulo 05503-900, Brazil; michelle.bufalo@butantan.gov.br (M.C.B.); gisele.picolo@butantan.gov.br (G.P.) 2 Center of Excellence in New Target Discovery, Butantan Institute, São Paulo 05503-900, Brazil; mesalmeida@gmail.com (M.E.S.d.A.); carlos.ocesano@butantan.gov.br (C.D.-P.); flavio.lichtenstein@butantan.gov.br (F.L.); ana.chudzinski@butantan.gov.br (A.M.C.-T.) 3 Laboratory of Pathophysiology, Butantan Institute, São Paulo 05503-900, Brazil; sandra.coccuzzo@butantan.gov.br 4 Immunogenetics Laboratory, Butantan Institute, São Paulo 05503-900, Brazil; jose.jensen@butantan.gov.br 5 Innovation and Development Laboratory, Innovation and Development Center, Butantan Institute, São Paulo 05503-900, Brazil 6 Department of Pharmacology, Institute of Biomedical Sciences, University of São Paulo, São Paulo 05508-220, Brazil * Correspondence: yarac57@gmail.com (Y.C.); vanessa.zambelli@butantan.gov.br(V.O.Z.); Tel.: +55-11-2627-9765(Y.C. & V.O.Z.) Abstract: Increased collagen-derived advanced glycation end-products (AGEs) are consistently related to painful diseases, including osteoarthritis, diabetic neuropathy, and neurodegenerative disorders. We have recently developed a model combining a two-dimensional glycated extracellular matrix (ECM-GC) and primary dorsal root ganglion (DRG) that mimicked a pro-nociceptive microen- vironment. However, culturing primary cells is still a challenge for large-scale screening studies. Here, we characterized a new model using ECM-GC as a stimulus for human sensory-like neurons differentiated from SH-SY5Y cell lines to screen for analgesic compounds. First, we confirmed that the differentiation process induces the expression of neuron markers (MAP2, RBFOX3 (NeuN), and TUBB3 (β-III tubulin), as well as sensory neuron markers critical for pain sensation (TRPV1, SCN9A (Nav1.7), SCN10A (Nav1.8), and SCN11A (Nav1.9). Next, we showed that ECM-GC increased c-Fos expression in human sensory-like neurons, which is suggestive of neuronal activation. In addition, ECM-GC upregulated the expression of critical genes involved in pain, including SCN9A and TACR1. Of interest, ECM-GC induced substance P release, a neuropeptide widely involved in neuroinflam- mation and pain. Finally, morphine, the prototype opiate, decreased ECM-GC-induced substance P release. Together, our results suggest that we established a functional model that can be useful as a platform for screening candidates for the management of painful conditions. Keywords: two-dimensional culture; nociception; inflammatory disease; in vitro assays; analgesic compounds; high-content screening 1. Introduction Collagen glycation is frequently observed in neuroinflammatory and neurodegen- erative disorders, such as Parkinson’s and Alzheimer’s diseases, osteoarthritis, diabetic neuropathy, amyloid polyneuropathy, and the aging process [1,2]. Glycation is an ir- reversible and spontaneous process in which proteins, such as collagen, bind to sugar molecules, forming advanced glycation end products (AGEs) [3]. AGEs can activate spe- cific cell surface receptors that transduce signals through downstream proteins, leading to the inflammatory process. Cells 2022, 11, 247. https://doi.org/10.3390/cells11020247 https://www.mdpi.com/journal/cells