Development of a Disease Modeling Framework for Glutamatergic Neurons Derived from Neuroblastoma Cells in 3D Microarrays

Development of a Disease Modeling Framework for Glutamatergic Neurons Derived from Neuroblastoma Cells in 3D Microarrays | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Development of a Disease Modeling Framework for Glutamatergic Neurons Derived from Neuroblastoma Cells in 3D Microarrays Duc Long Nguyen, My Phuong Thi Le, Kyung Won Lee, Jae-Ho Kim, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4934775/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Nov, 2024 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract Neurodegenerative diseases (NDDs) present significant challenges due to limited treatment options and the ethical concerns of traditional animal models and iPSC-derived neurons. We addressed these issues by developing a 3D culture protocol for differentiating SH-SY5Y cells into glutamatergic neurons, enhancing physiological relevance with a 3D microarray culture plate. Our protocol optimized serum concentration and incorporated retinoic acid (RA) to improve differentiation. We analyzed the proportions of N-type and S-type cells, observing that RA in the maturation stage not only reduced cell proliferation but also enhanced the expression of MAP2 and VGLUT1, indicating effective neuronal differentiation. Our approach demonstrates the strong expression of glutamatergic neuron phenotypes in 3D SH-SY5Y neural spheroids, offering a promising tool for high-throughput NDD modeling and advancing drug discovery and therapeutic development. This method overcomes limitations associated with conventional 2D cultures and animal models, providing a more effective platform for NDD research. Biological sciences/Biotechnology/Biomaterials Biological sciences/Biotechnology/Tissue engineering SH-SY5Y cells 3D microarrays glutamatergic neuron disease modeling Retinoic acid Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Neurodegenerative diseases (NDDs) caused by uncontrolled neuronal death and the loss of structure and function of neural networks have detrimentally influenced the lives of more than 50 million people worldwide 1 . Dementia, which is a syndrome associated with various NDDs such as Alzheimer's disease, Tauopathies, Huntington's disease, and Parkinson's disease, is expected to cost the world $ 2.8 trillion in 2030, including direct medical, indirect medical, and informal care costs 1 . The complex pathogenesis and unmanageable environmental and genetic factors hinder the efficacy of treatment attempts. Rodents have long served as models for studying NDDs. However, alongside ethical concerns of animal models, this method is costly, time-consuming, and unable to fully mimic human diseases 2 . Thus, the development of a human-based in vitro model with close physiological resemblance may shed new light for NDDs modeling and drug discovery. Human induced pluripotent stem cells-derived neurons (iPSC-derived neurons) are the most prominent tools for disease modeling due to their specificity. However, it takes six to nine months and costs $ 10,000 to $ 25,000 to develop a research-grade iPSC line from patient samples 3 . Therefore, until further innovations reduce the cost and time of iPSC reprogramming, alternative in vitro disease modeling approach is still necessary. The SH-SY5Y, originating from the bone marrow of a 4-year-old female with metastatic neuroblastoma, has been widely applied in NDD research 4 , 5 . These cells, derived from a human source, express many human-specific proteins and isoforms not present in rodent models 6 . As an immortalized cell line, SH-SY5Y cells can proliferate indefinitely, allowing for large-scale production and serving as a cost-effective model. The cell line has been reported to differentiate into neurons and express neuronal markers such as synapsin 1 (SYN1), microtubule-associated protein 2 (MAP2) or tubulin beta class III (TUJ1) and facilitate several types of neurotransmitter communications 7 – 9 . Easier to handle than animal models and primary neurons SH-SY5Y can be genetically modified to introduce disease-related mutations or gene expression constructs 10 – 12 . Furthermore, as a cell line, SH-SY5Y bypasses the ethical issues associated with animal models and human stem cells. While 2D culture of SH-SY5Y cells has been instrumental in elucidating various aspects of neuronal biology and disease mechanisms 5 , 8 , 11 , 13 – 18 , it has several limitations. These include the inability to mimic the complex 3D microenvironment of the brain, limited cell-cell and cell-matrix interactions, and altered cell morphology and behavior compared to in vivo conditions 19 . Lack of the complexity of the human brain's cellular environment, traditional 2D cultures may not fully recapitulate certain aspects of neuronal physiology and pathology 20 . However, research on differentiation using 3D methods is limited because these processes are time-consuming, labor-intensive, difficult to control, and challenging to mass-produce and apply for downstream applications. While differentiation and maturation protocols towards dopaminergic, cholinergic and adrenergic neurons are established 5 , 18 , 19 , the approach for glutamatergic and GABA neurons are very limited 7 . Additionally, most studies focus on qualitative analysis, reporting the existence of targeted neurons without quantifying the amount of N-type and S-type cells or proportions of neuron types after differentiation and maturation. Thus, to address the limitations of traditional 2D cultures and the challenges associated with 3D differentiation methods, our research focuses on developing a large-scale 3D culture protocol for differentiating and maturing SH-SY5Y cells into glutamatergic neurons using the PAMCELL™ microarray plate. By optimizing serum concentration and incorporating retinoic acid (RA) in the 3D culture environment, we aim to enhance the differentiation process and create a more physiologically relevant model for studying NDDs. This approach not only facilitates high-throughput NDD modeling but also paves the way for more effective drug discovery and therapeutic development. Results Morphological analysis of differentiated SH-SY5Y cells in 2D and 3D culture systems To address the impact of RA in maturation stage (stage II), 2D and 3D SH-SY5Y cells were cultured and differentiated in 4 different kinds of media: growth media (EMEM, 15% hiFBS, 1% PS), differentiation media (EMEM, 2.5% hiFBS, 10 µM RA, 1% PS) and 2 variants of maturation media (Neurobasal, 1% B27, 20 mM KCl, 2 mM Glutamax, 50 mM BDNF, 1% PS): RA free and RA treated (10 µM). Additionally, in differentiation stage (stage I) 3D culture cells were examnined with 2 different serum concentrations (2.5% and 5%) due to the different in metabolic demands. Cellular morphology was observed everyday under inverted microscope. On the final day of Stage I, 2D SH-SY5Y cells exhibited significant morphological changes indicative of differentiation into N-type cells, characterized by the outgrowth of neurites (Fig. 1 A). Despite this differentiation, some S-type and undifferentiated cells were still observed. After being fully matured in media containing RA, the cells adopted a triangular shape and developed dense neurite networks. Although some undifferentiated cells persisted, the majority had acquired a neuron-like phenotype. Conversely, in RA-free media, there was an increase in the population of undifferentiated cells and S-type SH-SY5Y cells, which retained their flat, large, and polygonal shape. Before being treated with differentiation media, the cells in 3D PAMCELL™ plates were nurtured in growth media for 2–3 days to form spheroids (Fig. 1 B). After the 12-day differentiation process, the size of the spheroids was measured. Spheroids differentiated in 2.5% hiFBS had an average diameter of 111.92 ± 33.19 µm (n = 50) in the presence of RA in maturation media and 110.11 ± 31.88 µm in RA-free media, whereas those in 5% hiFBS were larger and exhibited less size variation, with an average diameter of 125.72 ± 19.50 µm in RA-treated media and 131.38 ± 27.08 µm in RA-free media (Fig. 1 C). This demonstrates the impact of serum on spheroid proliferation. Additionally, while RA affected the morphology of 2D cells, it did not significantly impact spheroid size. Flow Cytometry Analysis of serum concentration and the effect of RA in maturation media The observation on cellular morphology were confirmation of the presence of matured SH-SY5Y neurons. Subsequently, we further quantified the amount of differentiation cells via flow cytometry analysis (FACS). The expression of neurogenic markers MAP2, vesicular glutamate transporter 1 (VGLUT1), and Tyrosine Hydroxylase (TH) in SH-SY5Y cells were measured under different conditions. 2D cells were cultured with 2.5% hiFBS, 3D samples were nurtured with 2.5% hiFBS and 5% hiFBS. The analysis was conducted on day 6, following the completion of the differentiation stage, and on day 12, after the maturation stage had concluded. Figure 2 A illustrates the FACS analysis from day 6. While over 70% of events obtained from 2D cultures with 2.5% hiFBS and 3D cultures with 5% hiFBS were cells, only 30% of events in 3D spheroids differentiated in 2.5% hiFBS were considered similar, and these cells did not express any neurogenic markers. By day 12, 3D cultures in 2.5% hiFBS showed a healthier profile, with over 80% of the total 30,000 events being cells, 69.27% of which expressed the MAP2 signal and 90.06% expressed VGLUT1 (Fig. 2 B). This suggests that 2.5% hiFBS requires additional time for differentiation and maturation, and the serum concentration may not be sufficient for the spheroids to maintain both their metabolic status and differentiation. Conversely, the analysis of 2D cultures in 2.5% hiFBS and 3D cultures in 5% hiFBS on day 6 showed that over 84% of single cells were positive for the MAP2 signal, indicating successful differentiation (Fig. 2 A). Interestingly, while the TH signal was expressed in only 10.77% of 2D cultures in 2.5% hiFBS and 12.09% in 3D cultures in 5% hiFBS, 93.8% of 2D cells and 81.14% of 3D cells expressed the VGLUT1 signal, suggesting that the cells were differentiating towards a glutamatergic pathway. Figure 2 B highlights the importance of RA in the maturation media when 38.67% of the 2D cell population in 2.5% hiFBS cultured in RA-free media were negative for the MAP2 signal. The results suggest that the cells continued proliferating in RA-free media, leading to the emergence of unwanted undifferentiated cells. The 3D cultures in 5% hiFBS negated the impact of the absence of RA, maintaining 88.81% of cells expressing the MAP2 signal. However, their mean signal intensity reduced from 312,048 to 159,333. To verify the effect of RA, we compared the FACS results of 2D cells and 3D 5% hiFBS after 12 day maturation with and without RA (Fig. 2 C,D). The amount of MAP2 positive cells were increased from 61.33–93.25% when RA was added into maturation media. VGLUT1 also expressed the same result from 79.43% cell population to 93.25%. Did not heavily impact from lack of RA, 3D 5% hiFBS cells were still showed their improve in signal, with the mean MAP2 signal increasing nearly fourfold to 594,139. VGLUT1 signals also surged sixfold compared to RA-free cells from 41,075 to 252,011. These results suggest that the presence of RA not only inhibited unwanted proliferation but also promoted the maturation process of SH-SY5Y cells in both 2D and 3D culture conditions. The contrast in signals between TH and VGLUT further supports the differentiation of neurons towards a glutamatergic pathway. Additionally, the 3D PAMCELL™ culture with 5% hiFBS demonstrated superior expression of the neurogenic markers MAP2 and VGLUT1 on both day 6 and day 12. This suggests that the 3D PAMCELL™ in 5% hiFBS condition is optimal for the differentiation and maturation of SH-SY5Y cells. Therefore, we recommend using 3D 5% hiFBS for further studies on SH-SY5Y cell differentiation and maturation. Immunocytochemical and mRNA expression analysis of neuronal differentiation and maturation Five mature neuronal markers were analyzed by immunocytochemistry (ICC), as shown in Fig. 3 . Figure 3 A illustrates the expression of VGLUT1, a key indicator for glutamatergic neurons 21 . VGLUT1 was strongly expressed across all conditions, confirming the differentiation protocol's effectiveness towards a glutamatergic pathway. Notably, in the 3D 5% hiFBS condition, VGLUT1 was prominently expressed on the outer layer of the spheroid, suggesting the communication between adjacent spheroids. MAP2, essential for dendritic elongation and structural integrity of neurons 22 , was observed differently across conditions. In the 3D 2.5% hiFBS spheroids, MAP2 was expressed as short but thick lines, indicating the early stage of neurite extension (Fig. 3 C). Conversely, in the 3D 5% hiFBS spheroids, MAP2 appeared as thin, long, and branching lines around the spheroid, suggesting that these spheroids are at a more mature stage of neuronal development. The expression of TUJ1 reinforcing similar observations (Fig. 3 D). 3D 2.5% hiFBS spheroid only expressed the TUJ1 in outer layer cells while 3D 5% hiFBS spheroid displayed a more complex axonal matrix at the center, indicating a higher degree of maturation. mRNA Expression Analysis (Fig. 4 A) further supports that culturing cells in 3D PAMCELL™ with 5% hiFBS creates an optimal environment for SH-SY5Y differentiation and maturation. Figure 4 B, C, D explore the impact of RA on mRNA expression level all 3 culture platform and condition. The expression level of MAP2 , ENO2 , SYN1 and TUBB3 were diverse among platforms. In 2D cultures with RA, there was a decrease in ENO2 levels and no significant change in MAP2 and TUBB3 levels. Conversely, in 3D 5% hiFBS cultures, the levels of these markers increased, with SYN1 and MAP2 showing significant upregulation (9.47-fold and 3.87-fold, respectively). EN1 and GLUL displayed consistent trends across all conditions, with EN1 increasing 3.64-fold in 2D and significantly more in 3D conditions (over 710-fold in 3D 2.5% hiFBS and over 880-fold in 3D 5% hiFBS). GLUL levels were 1.6 times higher in RA-treated 2D cultures compared to RA-free conditions. In 3D cultures, RA-treated spheroids showed 6.6-fold and 32.59-fold increases in GLUL expression in 2.5% hiFBS and 5% hiFBS, respectively. The result suggests that RA plays a crucial role in the maturation stage of SH-SY5Y cells and significantly influencing the expression levels of EN1 and GLUL in the 3D culture platform. Additionally, the overexpression level of GLUL and VGLUT1 (Fig. 3 A) dedicate that the cells were matured into glutamatergic neurons and ready for further application. Glutamate detection in maturated neural spheroid using cyclic voltammetry After confirming the maturation protocol for glutamatergic neurons, we proceeded to measure the levels of glutamate released as a neurotransmitter. Figure 5 A illustrates the design of the electrochemical GO-modified glutamate sensor. The enzyme immobilized on the surface of a platinum (Pt) electrode catalyzes the conversion of glutamate released from the spheroid into α-ketoglutarate, producing hydrogen peroxide (H 2 O 2 ) as a byproduct. The H 2 O 2 then undergoes elecrochemical oxidation at the electrode, each molecule donating two electrons. This electron flow generates an electrical current that is proportional to the glutamate concentration in the sample, allowing for quantitative analysis. Figure 5 B showed cyclic voltammograms of PBS, maturation media, healthy neural spheroids, 2-day starving spheroids and 4-day staring spheroids. The voltammogram of the healthy neural spheroids shows a peak at 1091 µA and is higher than the peak of maturation media (633 µA). The increasing electrochemical activity likely cause by the glutamate communication of spheroids. The current continued increasing in 2-day starving spheroids and 4-day starving spheroids suggest the loss in glutamate homeostasis in spheroid led to glutamatergic hyperactivity 23 . Figure 5 C further illustrates the differences in current among three types of spheroids across three independent experiments. The average peak current for healthy spheroids was 1151 ± 55 µA, while the starving spheroids showed increased average peaks of 1277 ± 71 µA for 2-day starvation and 1594 ± 152 µA for 4-day starvation. These results suggest that the system has significant potential for application in neurodegenerative disease (NDD) modeling and drug discovery. Discussion In this study, we explored the differentiation and maturation of SH-SY5Y cells into glutamatergic neurons within 3D PAMCELL™ microarrays, with a focus on optimizing serum concentrations and incorporating RA. The results provide valuable insights into the critical role of RA in the maturation process and the benefits of using 3D cultures for NDDs modeling. The differentiation and maturation of SH-SY5Y cells involve a two-stage strategy. In Stage I, RA treatment induces the expression of the tropomyosin-related kinase B (TrkB) receptor 18 . When BDNF is introduced in Stage II, the binding of BDNF to the TrkB receptor triggers several downstream pathways, such as the phosphoinositide 3-kinases (PI3K)-AKT signaling pathway, the phospholipase Cγ1 (PLC-γ1) pathway, and the Ras-mitogen-activated protein kinase (MAPK) pathway. These pathways induce differentiation, maturation, and the survival of neurons 24 , 25 . The importance of RA in stage I have been widely accepted, while the presence of RA in the maturation stage remains controversial 14 , 26 – 28 . Shipley et al. 27 argued that the presence of RA during maturation is necessary, whereas Dravids et al. 28 suggested RA was unnecessary in stage II of differentiation. Our study indicated the presence of RA during the maturation stage significantly influenced the differentiation and maturation of SH-SY5Y cells. The FACS analysis (Fig. 2 ) revealed that the cell would continue proliferating even in maturation stage if RA is absent, leading to an unwanted undifferentiated cell population. Conversely, the presence of RA reduces proliferative capacity and promotes the expression of MAP2, VGLUT1 aligning with previous reports 29 – 31 . While SH-SY5Y cells were established as Parkinson’s disease modeling 5 , 13 , our protocol directs the cell into different pathway. By combining effect of B27 and RA, our cells strongly expressed glutamatergic neuron features as presented in FACS analysis (Fig. 2 ), ICC analysis (Fig. 3 A), mRNA expression levels of GLUL (Fig. 4 ). VGLUT1 plays a crucial role in the central nervous system by facilitating the uptake of glutamate into synaptic vesicles, essential for maintaining synaptic efficacy and controlling neuronal activity 21 , 32 . GLUL gene encodes for Glutamate-Ammonia Ligase, which synthesize glutamine from glutamate and ammonia in an ATP-dependent reaction 7 . Additionally, B27 was found to reduce TH expression 7 , which aligns with our FACS results. The cyclic voltammetry results further demonstrated the maturation of glutamatergic neurons by detecting glutamate signals in the spheroids. The 3D spheroid culture system on PAMCELL ™ plates were introduced on our previous report 33 . Each well of the 96-well R100 plate contains over 350 micropads, each with a diameter of 100 µm, allowing for cell migration and the formation of uniformly sized spheroids. These features are suitable not only for large-scale spheroid production but also for high-throughput screening. Additionally, other 3D culture platforms such as hanging drops, bioreactors, and ultra-low attachment plates require careful handling and extensive techniques for downstream applications such as ICC or SEM. These methods often necessitate transferring spheroids to other platforms like cover glass, leading to potential spheroid loss. In contrast, the thin film flat bottom of 3D microarray plate allows users to perform downstream experiments directly on-site, minimizing the risk of cell loss. Leveraging the advantages of the plate, in this study, we verified the differentiation and maturation process of SH-SY5Y cells in 3D environment and optimized the serum condition specifically for 3D culture. Many reports recommended low serum concentration (2.5–1%) when differentiated SH-SY5Y to avoid cell proliferation 7 , 13 , 14 , 16 , 27 – 29 . However, 3D culture with complex cell-cell interaction and microenvironment require different serum concentration to differentiate and mature the neural spheroid. We found that even after 12-day process, spheroids nurtured with 2.5% hiFBS were only in early stage of differentiation with short and thick neurite extension (Fig. 3 ). Conversely, 5% hiFBS spheroids provided complex matrix neurite around and inside the spheroid. Additionally, FACS analysis (Fig. 2 ) mRNA expression levels of 3D 5% hiFBS spheroids also demonstrated that 5% hiFBS is optimal for the differentiation and maturation of SH-SY5Y spheroids. To further demonstrate the potential of our system, we use a GO enzymatic sensor to test the glutamate neurotransmitter release while cell inter-spheroid and intra-spheroid communicate to each other. Glutamate excitotoxicity is one of the important signal of neurodegenerative diseases. Increased extracellular glutamate levels have been observed in the brains of patients with Alzheimer's disease, contributing to synaptic dysfunction and neuronal loss 34 , 35 . Thus, the increasing signals of between healthy, 2-day starving and 4-day starving, respectively showed potential for NNDs research of the platform. In conclusion, we developed a refined protocol for differentiating SH-SY5Y cells into glutamatergic neurons using 3D PAMCELL™ microarrays, optimizing serum concentrations and incorporating retinoic acid (RA). Our results highlight RA's crucial role in both differentiation and maturation, significantly enhancing neuronal development and reducing cell proliferation. The expression of glutamatergic markers like VGLUT1 and GLUL confirmed the shift towards a glutamatergic phenotype. The 3D culture system fostered more physiologically relevant cell interactions, essential for proper neuronal development and function, demonstrated by improved glutamate handling. This protocol offers a valuable tool for neurodegenerative disease research and drug development, setting the stage for future studies to refine this approach and explore its broader applications in neurological disorders. Materials and Methods Cell culture and differentiation Human neuroblastoma cell lines SH-SY5Y (CRL-2266) obtained from American Type Culture Collection (ATCC; Manassas, VA, USA) were proliferation in Eagle's Minimum Essential Medium (EMEM; M4655, Sigma-Aldrich, Burlington, MA, USA) with penicillin and streptomycin and 15% heat-inactivated fetal bovine serum (hiFBS; Thermo Fisher, Waltham, MA, USA). The cells were incubated at 37°C, 5% CO2, and 95% humidity. When the cells reached 80–90% confluence, cells were passaged with 4 mL of TrypLE™ Express Enzyme (Thermo Fisher) for 5 minutes, then the enzyme was inhibited via addition of proliferation media. SH-SY5Y cells were differentiated and matured in 96-well PAMCELL™ R100 plates (ANK, Suwon, Korea) and 12-well culture plates. For neuron spheroid cultures (3D culture), onto 96-well PAMCELL™ R100 plates, which were pre-coated with iMatrix-511 (1:1000 in PBS) for 1 hour. The cells were cultured in EMEM supplemented with various range of hiFBS (2.5%, 5%, 10%, 15%), 1× penicillin/streptomycin, and 10 µM retinoic acid (RA; 223018, Sigma-Aldrich). On day 6, the culture medium was replaced with 2 types of maturation media (Neurobasal (21103049, Thermo Fisher), 1% B27 (Thermo Fisher), 20 mM KCl (Sigma-Aldrich), 1% Penicillin, 2mM Glutamax (Thermo Fisher), 50 mM Brain-derived neurotrophic factor (BDNF; Sigma-Aldrich)): RA-free and RA treated (10 µM) media, and half the medium was changed every day until day 12. To obtain conventional two-dimensional monolayer (2D) cultures, cells were cultured on 12-well plates coated with Matrigel Matrix (Corning Inc., NY, USA) at 1: 1000 ratio in ice-cold DMEM/F12 for 1 hour. Cells were seeded at 30,000 cells per well, and cultured in EMEM supplemented with 2.5% hiFBS, one penicillin/streptomycin, and 10 µM RA for a period of 6 days, similar to the 3D culture method during the maturation phase. Maturation media was replaced every 48 hours during this period (see Scheme 1 for further details). Immunocytochemistry Once the differentiation medium has been removed from the cell culture, the cells were washed with PBS before and after the fixation step the differentiation process was completed for immunocytochemistry procedures as described in the protocol 33 . Briefly, the spheroids were fixed with 4% paraformaldehyde(PFA) for one hour and then permeabilized with 0.3% Triton-X 100 for 30 minutes. Samples were then washed three times in PBS and then soaked in a blocking solution for one hour (50 mM phosphate-buffered saline pH 7.4 (PBS; Thermo Fisher) with 1% bovine serum albumin (BSA; BioWorld, OH, USA). The permeable cells were incubated with primary antibodies against VGLUT, MAP2, TUJ1, EN1, Synapsin for an overnight period at 4°C (Supplementary table S1 ). After washing with blocking buffer, secondary antibodies were treated for 30 min at 4 ° C. Dihydrochloride 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI; Thermo Fisher) was incubated for 20 minutes. The stain must be removed by washing thoroughly three times or more with PBS. To avoid being dried and detached from plates, spheroids are not exposed to air for every single step. The following above protocol was applied for 2D SHSY5 cells with time adjustment: 15 minutes for fixation, 10 minutes for permeability, and 30 minutes for blocking. Florescent images were taken using a confocal microscope (Leica Stellaris 5, Leica Camera AG, Germany). Flow cytometry analysis (FACS) In 3D PAMCELL ™ culture, cells were pipetted to detached from the well and subsequently centrifuged to remove media. Accutase (200 µL for 3D and 1mL for 2D per well; Thermo Fisher) was used to dissociate the spheroids for ten minutes. Observe under microscope frequently to confirm the separation. Then, add media to collect the cell and centrifuge to remove the media. Followed by 1 hour of fixation in 4% PFA. The cells were rinsed three times with PBS, then permeabilized with 0.3% Triton-X 100 for 20 minutes. After centrifugation to remove the media, the cells were incubated in blocking buffer for one hour and then treated with first antibody for one hour (Supplementary table S1 ). Cells were washed 3 times with blocking buffer, followed by a second antibody and incubated for 30 min. FACS solution (50 mM PBS containing 1% BSA, 10% fetal bovine serum (FBS) for 30 min at 4°C after washing. RNA extraction, cDNA conversion and qRT-PCR The mRNA content of differentiated SHSY5 cells was extracted on day 12 using the following method 33 . After removing media from the cells, total RNA was extracted using the Total RNA Extraction Kit (iNtRON Biotechnology, Inc., South Korea), then quantified with NanoDrop One (Thermo Fisher). RNA was converted to cDNA using RT-PCR machine was performed following the manufacturer's instructions using Maxime ™ RT PreMix (Random Primer) (iNtRON Biotechnology, Inc.). In all qRT-PCR reactions, the AriaM x Real-Time PCR system was used with 1 µL of cDNA template, 7 µL of ultra-distilled water, 10 µL of RealMOD green W2 qPCR mix (iNtRON Biotechnology, Inc.), and 1 µL of forward and reverse primers. Supplementary information regarding primers can be found in the following section. Information of primers were used was described in the Supplementary Table S2 . The PCR settings included an initial denaturation at 95°C for 10 minutes, followed by 40 cycles of denaturation at 95°C for 20 seconds, annealing at 55°C for 40 seconds, and a melt curve analysis with one cycle each at 95°C for 10 seconds, 65°C for 10 seconds, and 95°C for 10 seconds. The qPCR results were visualized using Agilent Aria 1.8, and relative expression levels were normalized to GAPDH. Ct values obtained through RT-PCR can be found in Supplementary Table S4 . Glutamate Oxidase (GO) electrodes preparation for glutamate level detection Glutamate oxidase (GO) solution was prepared before enzyme immobilization. First, 1 mL of sodium periodate (1.5 mg/mL in PBS; Sigma-Aldrich) was added to a vial of GO (25 units/vial; Yamasa Corp., Japan). The solution was then stirred at 4°C for 1 hour. Excess chemicals were washed away with PBS. The periodate-oxidized enzyme was collected using a 30 kDa MWCO Amicon® Ultra Centrifugal Filter (Millipore Corp., USA) and resuspended in 50 mM PBS pH 7.4. The solution was stored at 4°C. A platinum electrode was polished with alumina slurry (0.05 um, BASi Corp., USA) and incubated with 20 mM cystamine solution (Sigma-Aldrich) for 12 hours at room temperature after being washed with double-distilled water (DDW). The cystamine-functionalized electrode was then treated in GO solution for 1 hour at room temperature, resulting in immobilization through Schiff-base formation. The GO-immobilized electrode was washed and stored in PBS prior to use. The maturation media were exchanged 6 hours before testing. Electrochemical characterization was conducted using cyclic voltammetry. With the selected anodic oxidation potential of E app (0.65 V vs. Ag/AgCl reference electrode), glutamate concentration in testing sample was evaluated. Statistical analysis All statistical analyses were performed using GraphPad Prism version 9.0.0 (GraphPad, CA, USA). The size of the spheroid were measured by Fiji. Data are shown as the mean ± standard error of the mean. Student’s t-test and one-way analysis of variance (ANOVA) were applied and a p -value or p ≤ 0.05, 0.01, and 0.001 were considered significant. Declarations Authors’ contributions HTMP, DLN, and MPTL conceived and designed the project. HTMP, DLN, MPTL, and KWL performed research, conducted data analyses, and created figures. HTMP, DLN wrote the manuscript. JHK and HCY discussed the results, consulted, and gave critical feedback. All the authors read and approved the manuscript. Data Availability The data sets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. Results of the size of spheroids can be found in the Supplementary Data 3. mRNA expression level can be found in the Supplementary Data 4. Voltammogram raw data can be found in the Supplementary Data 5. Funding This study was supported by the Creative Materials Discovery Program (NRF-2019M3D1A1078943), the Priority Research Centers (NRF-2019R1A6A1A11051471), and the Commercialization Promotion Agency for R&D Outcomes (COMPA) grant funded by the Korean government (MSIT) (no. 2021N100). Ethics declarations Competing interests The authors declare the following competing interests: S.A. Sieber is co-founder of smartbax limited. All other authors declare no competing interests. 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N-acetylaspartate (NAA) induces neuronal differentiation of SH-SY5Y neuroblastoma cell line and sensitizes it to chemotherapeutic agents. Oncotarget 7, 26235 (2016). Ioghen, O. C., Ceafalan, L. C. & Popescu, B. O. SH-SY5Y Cell Line In Vitro Models for Parkinson Disease Research-Old Practice for New Trends. J. Integr. Neurosci. 22, (2023). Kaya, Z. B. et al. Optimizing SH-SY5Y cell culture: exploring the beneficial effects of an alternative media supplement on cell proliferation and viability. Sci. Reports 2024 141 14, 1–11 (2024). Strother, L., Miles, G. B., Holiday, A. R., Cheng, Y. & Doherty, G. H. Long-term culture of SH-SY5Y neuroblastoma cells in the absence of neurotrophins: A novel model of neuronal ageing. J. Neurosci. Methods 362, 109301 (2021). Lopez-Suarez, L., Awabdh, S. Al, Coumoul, X. & Chauvet, C. The SH-SY5Y human neuroblastoma cell line, a relevant in vitro cell model for investigating neurotoxicology in human: Focus on organic pollutants. Neurotoxicology 92, 131–155 (2022). Palanivel, V. et al. Neuroprotective Effects of Neuropeptide Y on Human Neuroblastoma SH-SY5Y Cells in Glutamate Excitotoxicity and ER Stress Conditions. Cells 11, 3665 (2022). de Medeiros, L. M. et al. Cholinergic Differentiation of Human Neuroblastoma SH-SY5Y Cell Line and Its Potential Use as an In vitro Model for Alzheimer’s Disease Studies. Mol. Neurobiol. 56, 7355–7367 (2019). Agholme, L., Lindström, T., Kgedal, K., Marcusson, J. & Hallbeck, M. An in vitro model for neuroscience: differentiation of SH-SY5Y cells into cells with morphological and biochemical characteristics of mature neurons. J. Alzheimers. Dis. 20, 1069–1082 (2010). Falkenburger, B. H. & Schulz, J. B. Limitations of cellular models in Parkinson’s disease research. J. Neural Transm. Suppl. 261–268 (2006) doi: 10.1007/978-3-211-45295-0_40 . Martineau, M., Guzman, R. E., Fahlke, C. & Klingauf, J. VGLUT1 functions as a glutamate/proton exchanger with chloride channel activity in hippocampal glutamatergic synapses. Nat. Commun. 2017 81 8, 1–13 (2017). Harada, A., Teng, J., Takei, Y., Oguchi, K. & Hirokawa, N. MAP2 is required for dendrite elongation, PKA anchoring in dendrites, and proper PKA signal transduction. J. Cell Biol. 158, 541 (2002). Nakamura, T., Gu, Z. & Lipton, S. A. Contribution of glutamatergic signaling to nitrosative stress-induced protein misfolding in normal brain aging and neurodegenerative diseases. Aging Cell 6, 351–359 (2007). Jin, W. Regulation of BDNF-TrkB Signaling and Potential Therapeutic Strategies for Parkinson’s Disease. J. Clin. Med. 9, (2020). Huang, E. J. & Reichardt, L. F. Trk receptors: roles in neuronal signal transduction. Annu. Rev. Biochem. 72, 609–642 (2003). Encinas, M. et al. Sequential Treatment of SH-SY5Y Cells with Retinoic Acid and Brain-Derived Neurotrophic Factor Gives Rise to Fully Differentiated, Neurotrophic Factor-Dependent, Human Neuron-Like Cells. J. Neurochem. 75, 991–1003 (2000). Shipley, M. M., Mangold, C. A. & Szpara, M. L. Differentiation of the SH-SY5Y human neuroblastoma cell line. J. Vis. Exp. 2016, e53193 (2016). Dravid, A., Raos, B., Svirskis, D. & O’Carroll, S. J. Optimised techniques for high-throughput screening of differentiated SH-SY5Y cells and application for neurite outgrowth assays. Sci. Reports 2021 111 11, 1–15 (2021). Kunzler, A. et al. Changes in Cell Cycle and Up-Regulation of Neuronal Markers During SH-SY5Y Neurodifferentiation by Retinoic Acid are Mediated by Reactive Species Production and Oxidative Stress. Mol. Neurobiol. 54, 6903–6916 (2017). Horvat, L., Grubar, M., Madunic, J., Antica, M. & Matulic, M. INHIBITION OF PARP ACTIVITY DOES NOT AFFECT THE DIFFERENTIATION PROCESSES CAUSED BY RETINOIC ACID IN SH-SY5Y CELLS. Hv. L Mol. Exp. Biol. Med. 1, 38–43 (2019). Waetzig, V. et al. Retinoic acid–induced survival effects in SH-SY5Y neuroblastoma cells. J. Cell. Biochem. 120, 5974–5986 (2019). Benarroch, E. E. Glutamate transporters: Diversity, function, and involvement in neurologic disease. Neurology 74, 259–264 (2010). Pham, H. T. M. et al. A novel and cost-effective method for high-throughput 3D culturing and rhythmic assessment of hiPSC-derived cardiomyocytes using retroreflective Janus microparticles. Biomater. Res. 27, 1–18 (2023). Danysz, W. & Parsons, C. G. Alzheimer’s disease, β-amyloid, glutamate, NMDA receptors and memantine–searching for the connections. Br. J. Pharmacol. 167, 324–352 (2012). Lewerenz, J. & Maher, P. Chronic Glutamate Toxicity in Neurodegenerative Diseases-What is the Evidence? Front. Neurosci. 9, (2015). Schemes Scheme 1 is available in the Supplementary Files section Additional Declarations No competing interests reported. Supplementary Files TableS1antibody.docx TableS2Primers.docx TableS4.docx TableS5.docx tables3spheroidsize.docx Scheme1.png Scheme 1: Timeline for differentiation protocol. Cite Share Download PDF Status: Published Journal Publication published 25 Nov, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 23 Sep, 2024 Reviews received at journal 20 Sep, 2024 Reviews received at journal 16 Sep, 2024 Reviews received at journal 15 Sep, 2024 Reviewers agreed at journal 07 Sep, 2024 Reviewers agreed at journal 07 Sep, 2024 Reviewers agreed at journal 06 Sep, 2024 Reviewers invited by journal 06 Sep, 2024 Editor assigned by journal 06 Sep, 2024 Editor invited by journal 29 Aug, 2024 Submission checks completed at journal 28 Aug, 2024 First submitted to journal 18 Aug, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4934775","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":357698471,"identity":"9f312f5a-d078-4ece-975e-c7c032164e7b","order_by":0,"name":"Duc Long Nguyen","email":"","orcid":"","institution":"Ajou University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Duc","middleName":"Long","lastName":"Nguyen","suffix":""},{"id":357698472,"identity":"f657a231-0e08-4c4b-9e7f-b876e10425bd","order_by":1,"name":"My Phuong Thi Le","email":"","orcid":"","institution":"Ajou University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"My","middleName":"Phuong Thi","lastName":"Le","suffix":""},{"id":357698475,"identity":"8e90aaea-9ead-4f71-be95-f21929bef2d6","order_by":2,"name":"Kyung Won Lee","email":"","orcid":"","institution":"Ajou University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kyung","middleName":"Won","lastName":"Lee","suffix":""},{"id":357698478,"identity":"9346776f-412b-4214-b057-cdb3cc9f1bd0","order_by":3,"name":"Jae-Ho Kim","email":"","orcid":"","institution":"Ajou University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jae-Ho","middleName":"","lastName":"Kim","suffix":""},{"id":357698479,"identity":"6e792c28-40c8-4178-a60e-95138cf1909c","order_by":4,"name":"Hyun C. Yoon","email":"","orcid":"","institution":"Ajou University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hyun","middleName":"C.","lastName":"Yoon","suffix":""},{"id":357698481,"identity":"5abcceef-a377-4ab6-9adc-00e76ee2c1de","order_by":5,"name":"Huyen T. M. Pham","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9klEQVRIiWNgGAWjYDACZgY2ZgiLDUQckAOTD0jRYgwmE/Dbg6olsQFE4dOi28577HFh22E5cwa25A+MO+6kzw87/BBoi52cbgN2LWaH+dKNZ7YdNrZsYDsmwXjmWe7G22kGQC3JxmYHcGnhMZPmbTucuOEAexsDY9vh3I2zE0BaDiRuI6ClHqil+QNQS7rh7PQPRGkBmsx2QAKoJUFeOocIW3jOpRtuOMyWJpHY9sxwg3ROwYEEAzx+OX8GqKXMWt7geJvxh49td+TlZ6dv/vChwk4OlxYoaAbFKSQ6DMAqDfAqB4E6BFO+gaDqUTAKRsEoGGEAACZrYI06dbSgAAAAAElFTkSuQmCC","orcid":"","institution":"Ajou University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Huyen","middleName":"T. M.","lastName":"Pham","suffix":""}],"badges":[],"createdAt":"2024-08-18 21:03:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4934775/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4934775/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-80369-3","type":"published","date":"2024-11-25T15:58:03+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":65451576,"identity":"39091e27-5b15-428b-ae22-e80a04c11f8f","added_by":"auto","created_at":"2024-09-27 15:09:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":178948,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphology of differentiated SH-SY5Y cells in 2D and 3D PAMCELL cultures with/without RA in maturation media and different serum condition.\u003c/strong\u003e (A) Bright field images of SH-SY5Y in 2D culture on day 1, day 6, and day 12. Arrow (black) indicated the S-type cells. Images were captured using an inverted epifluorescence microscope at 60X magnification in phase contrast. (B) Bright field images of SH-SY5Y spheroids on day 1 and day 12. (C) Violin plots of the size of spheroids cultured in different serum concentration. Diameters of 50 random spheroids were measured. Significant differences between different cultured conditions were evaluated. (ns = not significant, *p\u0026lt;0.05, ***p\u0026lt;0.001, unpaired Student’s t-test)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4934775/v1/cef5a44ea87957acbb355e7e.png"},{"id":65451994,"identity":"ce73ad1c-7d04-4e8c-9bd6-db476fcd6c85","added_by":"auto","created_at":"2024-09-27 15:17:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":274329,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferentiation and maturation of SH-SY5Y Cells in 2D and 3D Cultures Assessed by Flow Cytometry Analysis. \u003c/strong\u003e(A) FACS analysis on day 6 of differentiation. Black arrow indicated the population of undifferentiated cells (B) FACS analysis of cells cultured in maturation media without RA. Black arrow indicated the population of undifferentiated cells (C) Comparison of neuronal marker expression in 2D cell cultures with and without RA treatment. (D) Comparison of neuronal marker expression in 3D spheroids cultured in 5% hiFBS with and without RA treatment.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4934775/v1/82b09cdb69aec007bc8d3bc3.png"},{"id":65451578,"identity":"c5450c13-9cf3-4c01-a142-ee50a85adb1f","added_by":"auto","created_at":"2024-09-27 15:09:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":246290,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of mature neuronal markers by 2D, 3D 2.5% hiFBS and 3D 5% hiFBS after 12-day maturation in presence of RA. \u003c/strong\u003e(A)Expression of VGLUT1. (B)Expression of EN1. (C)Expression of MAP2. (D)Expression of TUJ1. (E)Expression of Synapsin. (F)Depth code view of 3D spheroid and spheroid formation confirmation. 3D 5% hiFBS were chosen for depth code image.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4934775/v1/070ed52ea576c7fcb93db28a.png"},{"id":65452857,"identity":"15069f59-58a5-40bd-91f3-e009232f7802","added_by":"auto","created_at":"2024-09-27 15:25:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":81379,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRelative expression of different mature neuronal markers in 2D, 3D 2.5% hiFBS and, 3D 5% hiFBS after 12-day maturation process with and without RA. \u003c/strong\u003e(A) Relative expression of 3 culture platforms and conditions with presence of RA in maturation media. Significant differences between different cultured conditions were evaluated. (ns = not significant, *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001, unpaired Student’s t-test, n = 3) (B) The effect of RA on level of neuronal markers in 2D culture. (C) The effect of RA on expression level of neuronal markers in 3D 2.5% hiFBS. (D) The effect of RA on expression level of neuronal markers in 3D 5% hiFBS. The vertical axis shows the relative gene expression levels as the means ± SE (n = 03). Bars represent the means of three replicates ± SD.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4934775/v1/da042675754e2d1796ae28ae.png"},{"id":65451577,"identity":"84d725eb-0fb3-40bf-ab22-5b47bc409bde","added_by":"auto","created_at":"2024-09-27 15:09:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":115699,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConcept and result of cyclic voltammetry of enzymatic glutamate sensor. \u003c/strong\u003e(A) Scheme of GO enzymatic sensor. (B)Detection of glutamate in 3D 5% hiFBS in different condition. (C) Violin plot of current density of spheroid in different condition ((ns = not significant, *p\u0026lt;0.05, ***p\u0026lt;0.001, unpaired Student’s t-test, n = 3).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4934775/v1/024a1b5b3634f848ae1157e4.png"},{"id":70382806,"identity":"f992614f-0d51-47e1-9fc8-dea1337db76c","added_by":"auto","created_at":"2024-12-02 16:31:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1458894,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4934775/v1/66684e02-a348-4e9c-a716-1457f06d3a1a.pdf"},{"id":65451575,"identity":"59237a5a-cd00-44f0-89af-54ca000f124d","added_by":"auto","created_at":"2024-09-27 15:09:03","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16674,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1antibody.docx","url":"https://assets-eu.researchsquare.com/files/rs-4934775/v1/67e58e607b9e5dd066b28181.docx"},{"id":65451997,"identity":"74aaba66-47b7-43d6-a082-fb30f0c1af6f","added_by":"auto","created_at":"2024-09-27 15:17:03","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":16244,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2Primers.docx","url":"https://assets-eu.researchsquare.com/files/rs-4934775/v1/6a17ab896340b5e2c254b5f2.docx"},{"id":65452856,"identity":"9efb22b2-b095-4427-bf58-3d800a0dee8a","added_by":"auto","created_at":"2024-09-27 15:25:03","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":27466,"visible":true,"origin":"","legend":"","description":"","filename":"TableS4.docx","url":"https://assets-eu.researchsquare.com/files/rs-4934775/v1/5768e231b2676257537459f6.docx"},{"id":65451585,"identity":"a6e626f3-f877-43d1-b92b-90fc585d294f","added_by":"auto","created_at":"2024-09-27 15:09:03","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":149482,"visible":true,"origin":"","legend":"","description":"","filename":"TableS5.docx","url":"https://assets-eu.researchsquare.com/files/rs-4934775/v1/c12b9325726865009321026e.docx"},{"id":65452855,"identity":"26f441fe-2a40-4b41-a48c-59c91e2c1f6b","added_by":"auto","created_at":"2024-09-27 15:25:03","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":15816,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"tables3spheroidsize.docx","url":"https://assets-eu.researchsquare.com/files/rs-4934775/v1/90109d084384ef6a1685661b.docx"},{"id":65451582,"identity":"327ed688-7a4e-49f1-8be9-bd6bf4bf9c8e","added_by":"auto","created_at":"2024-09-27 15:09:03","extension":"png","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":126788,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1: Timeline for differentiation protocol.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Scheme1.png","url":"https://assets-eu.researchsquare.com/files/rs-4934775/v1/9d3bc4ae76fa9ff6068a283e.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Development of a Disease Modeling Framework for Glutamatergic Neurons Derived from Neuroblastoma Cells in 3D Microarrays","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNeurodegenerative diseases (NDDs) caused by uncontrolled neuronal death and the loss of structure and function of neural networks have detrimentally influenced the lives of more than 50\u0026nbsp;million people worldwide\u003csup\u003e \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e \u003c/sup\u003e. Dementia, which is a syndrome associated with various NDDs such as Alzheimer's disease, Tauopathies, Huntington's disease, and Parkinson's disease, is expected to cost the world \u003cspan\u003e$\u003c/span\u003e2.8 trillion in 2030, including direct medical, indirect medical, and informal care costs\u003csup\u003e \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e \u003c/sup\u003e. The complex pathogenesis and unmanageable environmental and genetic factors hinder the efficacy of treatment attempts. Rodents have long served as models for studying NDDs. However, alongside ethical concerns of animal models, this method is costly, time-consuming, and unable to fully mimic human diseases\u003csup\u003e \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e \u003c/sup\u003e. Thus, the development of a human-based \u003cem\u003ein vitro\u003c/em\u003e model with close physiological resemblance may shed new light for NDDs modeling and drug discovery. Human induced pluripotent stem cells-derived neurons (iPSC-derived neurons) are the most prominent tools for disease modeling due to their specificity. However, it takes six to nine months and costs \u003cspan\u003e$\u003c/span\u003e10,000 to \u003cspan\u003e$\u003c/span\u003e25,000 to develop a research-grade iPSC line from patient samples\u003csup\u003e \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e \u003c/sup\u003e. Therefore, until further innovations reduce the cost and time of iPSC reprogramming, alternative \u003cem\u003ein vitro\u003c/em\u003e disease modeling approach is still necessary.\u003c/p\u003e \u003cp\u003eThe SH-SY5Y, originating from the bone marrow of a 4-year-old female with metastatic neuroblastoma, has been widely applied in NDD research\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. These cells, derived from a human source, express many human-specific proteins and isoforms not present in rodent models\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. As an immortalized cell line, SH-SY5Y cells can proliferate indefinitely, allowing for large-scale production and serving as a cost-effective model. The cell line has been reported to differentiate into neurons and express neuronal markers such as synapsin 1 (SYN1), microtubule-associated protein 2 (MAP2) or tubulin beta class III (TUJ1) and facilitate several types of neurotransmitter communications\u003csup\u003e\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Easier to handle than animal models and primary neurons SH-SY5Y can be genetically modified to introduce disease-related mutations or gene expression constructs\u003csup\u003e\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Furthermore, as a cell line, SH-SY5Y bypasses the ethical issues associated with animal models and human stem cells.\u003c/p\u003e \u003cp\u003eWhile 2D culture of SH-SY5Y cells has been instrumental in elucidating various aspects of neuronal biology and disease mechanisms\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan additionalcitationids=\"CR14 CR15 CR16 CR17\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, it has several limitations. These include the inability to mimic the complex 3D microenvironment of the brain, limited cell-cell and cell-matrix interactions, and altered cell morphology and behavior compared to in vivo conditions\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Lack of the complexity of the human brain's cellular environment, traditional 2D cultures may not fully recapitulate certain aspects of neuronal physiology and pathology\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. However, research on differentiation using 3D methods is limited because these processes are time-consuming, labor-intensive, difficult to control, and challenging to mass-produce and apply for downstream applications.\u003c/p\u003e \u003cp\u003eWhile differentiation and maturation protocols towards dopaminergic, cholinergic and adrenergic neurons are established\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, the approach for glutamatergic and GABA neurons are very limited\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Additionally, most studies focus on qualitative analysis, reporting the existence of targeted neurons without quantifying the amount of N-type and S-type cells or proportions of neuron types after differentiation and maturation.\u003c/p\u003e \u003cp\u003eThus, to address the limitations of traditional 2D cultures and the challenges associated with 3D differentiation methods, our research focuses on developing a large-scale 3D culture protocol for differentiating and maturing SH-SY5Y cells into glutamatergic neurons using the PAMCELL\u0026trade; microarray plate. By optimizing serum concentration and incorporating retinoic acid (RA) in the 3D culture environment, we aim to enhance the differentiation process and create a more physiologically relevant model for studying NDDs. This approach not only facilitates high-throughput NDD modeling but also paves the way for more effective drug discovery and therapeutic development.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMorphological analysis of differentiated SH-SY5Y cells in 2D and 3D culture systems\u003c/h2\u003e \u003cp\u003eTo address the impact of RA in maturation stage (stage II), 2D and 3D SH-SY5Y cells were cultured and differentiated in 4 different kinds of media: growth media (EMEM, 15% hiFBS, 1% PS), differentiation media (EMEM, 2.5% hiFBS, 10 \u0026micro;M RA, 1% PS) and 2 variants of maturation media (Neurobasal, 1% B27, 20 mM KCl, 2 mM Glutamax, 50 mM BDNF, 1% PS): RA free and RA treated (10 \u0026micro;M). Additionally, in differentiation stage (stage I) 3D culture cells were examnined with 2 different serum concentrations (2.5% and 5%) due to the different in metabolic demands. Cellular morphology was observed everyday under inverted microscope.\u003c/p\u003e \u003cp\u003eOn the final day of Stage I, 2D SH-SY5Y cells exhibited significant morphological changes indicative of differentiation into N-type cells, characterized by the outgrowth of neurites (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Despite this differentiation, some S-type and undifferentiated cells were still observed. After being fully matured in media containing RA, the cells adopted a triangular shape and developed dense neurite networks. Although some undifferentiated cells persisted, the majority had acquired a neuron-like phenotype. Conversely, in RA-free media, there was an increase in the population of undifferentiated cells and S-type SH-SY5Y cells, which retained their flat, large, and polygonal shape.\u003c/p\u003e \u003cp\u003eBefore being treated with differentiation media, the cells in 3D PAMCELL\u0026trade; plates were nurtured in growth media for 2\u0026ndash;3 days to form spheroids (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). After the 12-day differentiation process, the size of the spheroids was measured. Spheroids differentiated in 2.5% hiFBS had an average diameter of 111.92\u0026thinsp;\u0026plusmn;\u0026thinsp;33.19 \u0026micro;m (n\u0026thinsp;=\u0026thinsp;50) in the presence of RA in maturation media and 110.11\u0026thinsp;\u0026plusmn;\u0026thinsp;31.88 \u0026micro;m in RA-free media, whereas those in 5% hiFBS were larger and exhibited less size variation, with an average diameter of 125.72\u0026thinsp;\u0026plusmn;\u0026thinsp;19.50 \u0026micro;m in RA-treated media and 131.38\u0026thinsp;\u0026plusmn;\u0026thinsp;27.08 \u0026micro;m in RA-free media (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). This demonstrates the impact of serum on spheroid proliferation. Additionally, while RA affected the morphology of 2D cells, it did not significantly impact spheroid size.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eFlow Cytometry Analysis of serum concentration and the effect of RA in maturation media\u003c/h2\u003e \u003cp\u003eThe observation on cellular morphology were confirmation of the presence of matured SH-SY5Y neurons. Subsequently, we further quantified the amount of differentiation cells via flow cytometry analysis (FACS). The expression of neurogenic markers MAP2, vesicular glutamate transporter 1 (VGLUT1), and Tyrosine Hydroxylase (TH) in SH-SY5Y cells were measured under different conditions. 2D cells were cultured with 2.5% hiFBS, 3D samples were nurtured with 2.5% hiFBS and 5% hiFBS. The analysis was conducted on day 6, following the completion of the differentiation stage, and on day 12, after the maturation stage had concluded.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA illustrates the FACS analysis from day 6. While over 70% of events obtained from 2D cultures with 2.5% hiFBS and 3D cultures with 5% hiFBS were cells, only 30% of events in 3D spheroids differentiated in 2.5% hiFBS were considered similar, and these cells did not express any neurogenic markers. By day 12, 3D cultures in 2.5% hiFBS showed a healthier profile, with over 80% of the total 30,000 events being cells, 69.27% of which expressed the MAP2 signal and 90.06% expressed VGLUT1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). This suggests that 2.5% hiFBS requires additional time for differentiation and maturation, and the serum concentration may not be sufficient for the spheroids to maintain both their metabolic status and differentiation.\u003c/p\u003e \u003cp\u003eConversely, the analysis of 2D cultures in 2.5% hiFBS and 3D cultures in 5% hiFBS on day 6 showed that over 84% of single cells were positive for the MAP2 signal, indicating successful differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Interestingly, while the TH signal was expressed in only 10.77% of 2D cultures in 2.5% hiFBS and 12.09% in 3D cultures in 5% hiFBS, 93.8% of 2D cells and 81.14% of 3D cells expressed the VGLUT1 signal, suggesting that the cells were differentiating towards a glutamatergic pathway.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB highlights the importance of RA in the maturation media when 38.67% of the 2D cell population in 2.5% hiFBS cultured in RA-free media were negative for the MAP2 signal. The results suggest that the cells continued proliferating in RA-free media, leading to the emergence of unwanted undifferentiated cells. The 3D cultures in 5% hiFBS negated the impact of the absence of RA, maintaining 88.81% of cells expressing the MAP2 signal. However, their mean signal intensity reduced from 312,048 to 159,333.\u003c/p\u003e \u003cp\u003eTo verify the effect of RA, we compared the FACS results of 2D cells and 3D 5% hiFBS after 12 day maturation with and without RA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC,D). The amount of MAP2 positive cells were increased from 61.33\u0026ndash;93.25% when RA was added into maturation media. VGLUT1 also expressed the same result from 79.43% cell population to 93.25%. Did not heavily impact from lack of RA, 3D 5% hiFBS cells were still showed their improve in signal, with the mean MAP2 signal increasing nearly fourfold to 594,139. VGLUT1 signals also surged sixfold compared to RA-free cells from 41,075 to 252,011. These results suggest that the presence of RA not only inhibited unwanted proliferation but also promoted the maturation process of SH-SY5Y cells in both 2D and 3D culture conditions. The contrast in signals between TH and VGLUT further supports the differentiation of neurons towards a glutamatergic pathway.\u003c/p\u003e \u003cp\u003eAdditionally, the 3D PAMCELL\u0026trade; culture with 5% hiFBS demonstrated superior expression of the neurogenic markers MAP2 and VGLUT1 on both day 6 and day 12. This suggests that the 3D PAMCELL\u0026trade; in 5% hiFBS condition is optimal for the differentiation and maturation of SH-SY5Y cells. Therefore, we recommend using 3D 5% hiFBS for further studies on SH-SY5Y cell differentiation and maturation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eImmunocytochemical and mRNA expression analysis of neuronal differentiation and maturation\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFive mature neuronal markers were analyzed by immunocytochemistry (ICC), as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA illustrates the expression of VGLUT1, a key indicator for glutamatergic neurons\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. VGLUT1 was strongly expressed across all conditions, confirming the differentiation protocol's effectiveness towards a glutamatergic pathway. Notably, in the 3D 5% hiFBS condition, VGLUT1 was prominently expressed on the outer layer of the spheroid, suggesting the communication between adjacent spheroids.\u003c/p\u003e \u003cp\u003eMAP2, essential for dendritic elongation and structural integrity of neurons\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, was observed differently across conditions. In the 3D 2.5% hiFBS spheroids, MAP2 was expressed as short but thick lines, indicating the early stage of neurite extension (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Conversely, in the 3D 5% hiFBS spheroids, MAP2 appeared as thin, long, and branching lines around the spheroid, suggesting that these spheroids are at a more mature stage of neuronal development.\u003c/p\u003e \u003cp\u003eThe expression of TUJ1 reinforcing similar observations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). 3D 2.5% hiFBS spheroid only expressed the TUJ1 in outer layer cells while 3D 5% hiFBS spheroid displayed a more complex axonal matrix at the center, indicating a higher degree of maturation.\u003c/p\u003e \u003cp\u003emRNA Expression Analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) further supports that culturing cells in 3D PAMCELL\u0026trade; with 5% hiFBS creates an optimal environment for SH-SY5Y differentiation and maturation.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, C, D explore the impact of RA on mRNA expression level all 3 culture platform and condition. The expression level of \u003cem\u003eMAP2\u003c/em\u003e, \u003cem\u003eENO2\u003c/em\u003e, \u003cem\u003eSYN1\u003c/em\u003e and \u003cem\u003eTUBB3\u003c/em\u003e were diverse among platforms. In 2D cultures with RA, there was a decrease in \u003cem\u003eENO2\u003c/em\u003e levels and no significant change in \u003cem\u003eMAP2\u003c/em\u003e and \u003cem\u003eTUBB3\u003c/em\u003e levels. Conversely, in 3D 5% hiFBS cultures, the levels of these markers increased, with \u003cem\u003eSYN1\u003c/em\u003e and \u003cem\u003eMAP2\u003c/em\u003e showing significant upregulation (9.47-fold and 3.87-fold, respectively). \u003cem\u003eEN1\u003c/em\u003e and \u003cem\u003eGLUL\u003c/em\u003e displayed consistent trends across all conditions, with EN1 increasing 3.64-fold in 2D and significantly more in 3D conditions (over 710-fold in 3D 2.5% hiFBS and over 880-fold in 3D 5% hiFBS). \u003cem\u003eGLUL\u003c/em\u003e levels were 1.6 times higher in RA-treated 2D cultures compared to RA-free conditions. In 3D cultures, RA-treated spheroids showed 6.6-fold and 32.59-fold increases in \u003cem\u003eGLUL\u003c/em\u003e expression in 2.5% hiFBS and 5% hiFBS, respectively. The result suggests that RA plays a crucial role in the maturation stage of SH-SY5Y cells and significantly influencing the expression levels of \u003cem\u003eEN1\u003c/em\u003e and \u003cem\u003eGLUL\u003c/em\u003e in the 3D culture platform. Additionally, the overexpression level of \u003cem\u003eGLUL\u003c/em\u003e and VGLUT1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) dedicate that the cells were matured into glutamatergic neurons and ready for further application.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eGlutamate detection in maturated neural spheroid using cyclic voltammetry\u003c/h2\u003e \u003cp\u003eAfter confirming the maturation protocol for glutamatergic neurons, we proceeded to measure the levels of glutamate released as a neurotransmitter. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA illustrates the design of the electrochemical GO-modified glutamate sensor. The enzyme immobilized on the surface of a platinum (Pt) electrode catalyzes the conversion of glutamate released from the spheroid into α-ketoglutarate, producing hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) as a byproduct. The H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e then undergoes elecrochemical oxidation at the electrode, each molecule donating two electrons. This electron flow generates an electrical current that is proportional to the glutamate concentration in the sample, allowing for quantitative analysis.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB showed cyclic voltammograms of PBS, maturation media, healthy neural spheroids, 2-day starving spheroids and 4-day staring spheroids. The voltammogram of the healthy neural spheroids shows a peak at 1091 \u0026micro;A and is higher than the peak of maturation media (633 \u0026micro;A). The increasing electrochemical activity likely cause by the glutamate communication of spheroids. The current continued increasing in 2-day starving spheroids and 4-day starving spheroids suggest the loss in glutamate homeostasis in spheroid led to glutamatergic hyperactivity\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC further illustrates the differences in current among three types of spheroids across three independent experiments. The average peak current for healthy spheroids was 1151\u0026thinsp;\u0026plusmn;\u0026thinsp;55 \u0026micro;A, while the starving spheroids showed increased average peaks of 1277\u0026thinsp;\u0026plusmn;\u0026thinsp;71 \u0026micro;A for 2-day starvation and 1594\u0026thinsp;\u0026plusmn;\u0026thinsp;152 \u0026micro;A for 4-day starvation. These results suggest that the system has significant potential for application in neurodegenerative disease (NDD) modeling and drug discovery.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we explored the differentiation and maturation of SH-SY5Y cells into glutamatergic neurons within 3D PAMCELL\u0026trade; microarrays, with a focus on optimizing serum concentrations and incorporating RA. The results provide valuable insights into the critical role of RA in the maturation process and the benefits of using 3D cultures for NDDs modeling.\u003c/p\u003e \u003cp\u003eThe differentiation and maturation of SH-SY5Y cells involve a two-stage strategy. In Stage I, RA treatment induces the expression of the tropomyosin-related kinase B (TrkB) receptor \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. When BDNF is introduced in Stage II, the binding of BDNF to the TrkB receptor triggers several downstream pathways, such as the phosphoinositide 3-kinases (PI3K)-AKT signaling pathway, the phospholipase Cγ1 (PLC-γ1) pathway, and the Ras-mitogen-activated protein kinase (MAPK) pathway. These pathways induce differentiation, maturation, and the survival of neurons\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe importance of RA in stage I have been widely accepted, while the presence of RA in the maturation stage remains controversial\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Shipley et al.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e argued that the presence of RA during maturation is necessary, whereas Dravids et al.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e suggested RA was unnecessary in stage II of differentiation. Our study indicated the presence of RA during the maturation stage significantly influenced the differentiation and maturation of SH-SY5Y cells. The FACS analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) revealed that the cell would continue proliferating even in maturation stage if RA is absent, leading to an unwanted undifferentiated cell population. Conversely, the presence of RA reduces proliferative capacity and promotes the expression of MAP2, VGLUT1 aligning with previous reports\u003csup\u003e\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWhile SH-SY5Y cells were established as Parkinson\u0026rsquo;s disease modeling\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, our protocol directs the cell into different pathway. By combining effect of B27 and RA, our cells strongly expressed glutamatergic neuron features as presented in FACS analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), ICC analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), mRNA expression levels of \u003cem\u003eGLUL\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). VGLUT1 plays a crucial role in the central nervous system by facilitating the uptake of glutamate into synaptic vesicles, essential for maintaining synaptic efficacy and controlling neuronal activity\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eGLUL\u003c/em\u003e gene encodes for Glutamate-Ammonia Ligase, which synthesize glutamine from glutamate and ammonia in an ATP-dependent reaction\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Additionally, B27 was found to reduce TH expression\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, which aligns with our FACS results. The cyclic voltammetry results further demonstrated the maturation of glutamatergic neurons by detecting glutamate signals in the spheroids.\u003c/p\u003e \u003cp\u003eThe 3D spheroid culture system on PAMCELL\u003csup\u003e\u0026trade;\u003c/sup\u003e plates were introduced on our previous report\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Each well of the 96-well R100 plate contains over 350 micropads, each with a diameter of 100 \u0026micro;m, allowing for cell migration and the formation of uniformly sized spheroids. These features are suitable not only for large-scale spheroid production but also for high-throughput screening. Additionally, other 3D culture platforms such as hanging drops, bioreactors, and ultra-low attachment plates require careful handling and extensive techniques for downstream applications such as ICC or SEM. These methods often necessitate transferring spheroids to other platforms like cover glass, leading to potential spheroid loss. In contrast, the thin film flat bottom of 3D microarray plate allows users to perform downstream experiments directly on-site, minimizing the risk of cell loss.\u003c/p\u003e \u003cp\u003eLeveraging the advantages of the plate, in this study, we verified the differentiation and maturation process of SH-SY5Y cells in 3D environment and optimized the serum condition specifically for 3D culture. Many reports recommended low serum concentration (2.5\u0026ndash;1%) when differentiated SH-SY5Y to avoid cell proliferation\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. However, 3D culture with complex cell-cell interaction and microenvironment require different serum concentration to differentiate and mature the neural spheroid. We found that even after 12-day process, spheroids nurtured with 2.5% hiFBS were only in early stage of differentiation with short and thick neurite extension (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Conversely, 5% hiFBS spheroids provided complex matrix neurite around and inside the spheroid. Additionally, FACS analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) mRNA expression levels of 3D 5% hiFBS spheroids also demonstrated that 5% hiFBS is optimal for the differentiation and maturation of SH-SY5Y spheroids.\u003c/p\u003e \u003cp\u003eTo further demonstrate the potential of our system, we use a GO enzymatic sensor to test the glutamate neurotransmitter release while cell inter-spheroid and intra-spheroid communicate to each other. Glutamate excitotoxicity is one of the important signal of neurodegenerative diseases. Increased extracellular glutamate levels have been observed in the brains of patients with Alzheimer's disease, contributing to synaptic dysfunction and neuronal loss\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Thus, the increasing signals of between healthy, 2-day starving and 4-day starving, respectively showed potential for NNDs research of the platform.\u003c/p\u003e \u003cp\u003eIn conclusion, we developed a refined protocol for differentiating SH-SY5Y cells into glutamatergic neurons using 3D PAMCELL\u0026trade; microarrays, optimizing serum concentrations and incorporating retinoic acid (RA). Our results highlight RA's crucial role in both differentiation and maturation, significantly enhancing neuronal development and reducing cell proliferation. The expression of glutamatergic markers like VGLUT1 and GLUL confirmed the shift towards a glutamatergic phenotype. The 3D culture system fostered more physiologically relevant cell interactions, essential for proper neuronal development and function, demonstrated by improved glutamate handling. This protocol offers a valuable tool for neurodegenerative disease research and drug development, setting the stage for future studies to refine this approach and explore its broader applications in neurological disorders.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and differentiation\u003c/h2\u003e \u003cp\u003eHuman neuroblastoma cell lines SH-SY5Y (CRL-2266) obtained from American Type Culture Collection (ATCC; Manassas, VA, USA) were proliferation in Eagle's Minimum Essential Medium (EMEM; M4655, Sigma-Aldrich, Burlington, MA, USA) with penicillin and streptomycin and 15% heat-inactivated fetal bovine serum (hiFBS; Thermo Fisher, Waltham, MA, USA). The cells were incubated at 37\u0026deg;C, 5% CO2, and 95% humidity. When the cells reached 80\u0026ndash;90% confluence, cells were passaged with 4 mL of TrypLE\u0026trade; Express Enzyme (Thermo Fisher) for 5 minutes, then the enzyme was inhibited via addition of proliferation media.\u003c/p\u003e \u003cp\u003eSH-SY5Y cells were differentiated and matured in 96-well PAMCELL\u0026trade; R100 plates (ANK, Suwon, Korea) and 12-well culture plates. For neuron spheroid cultures (3D culture), onto 96-well PAMCELL\u0026trade; R100 plates, which were pre-coated with iMatrix-511 (1:1000 in PBS) for 1 hour. The cells were cultured in EMEM supplemented with various range of hiFBS (2.5%, 5%, 10%, 15%), 1\u0026times; penicillin/streptomycin, and 10 \u0026micro;M retinoic acid (RA; 223018, Sigma-Aldrich). On day 6, the culture medium was replaced with 2 types of maturation media (Neurobasal (21103049, Thermo Fisher), 1% B27 (Thermo Fisher), 20 mM KCl (Sigma-Aldrich), 1% Penicillin, 2mM Glutamax (Thermo Fisher), 50 mM Brain-derived neurotrophic factor (BDNF; Sigma-Aldrich)): RA-free and RA treated (10 \u0026micro;M) media, and half the medium was changed every day until day 12. To obtain conventional two-dimensional monolayer (2D) cultures, cells were cultured on 12-well plates coated with Matrigel Matrix (Corning Inc., NY, USA) at 1: 1000 ratio in ice-cold DMEM/F12 for 1 hour. Cells were seeded at 30,000 cells per well, and cultured in EMEM supplemented with 2.5% hiFBS, one penicillin/streptomycin, and 10 \u0026micro;M RA for a period of 6 days, similar to the 3D culture method during the maturation phase. Maturation media was replaced every 48 hours during this period (see Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e for further details).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eImmunocytochemistry\u003c/h2\u003e \u003cp\u003eOnce the differentiation medium has been removed from the cell culture, the cells were washed with PBS before and after the fixation step the differentiation process was completed for immunocytochemistry procedures as described in the protocol \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Briefly, the spheroids were fixed with 4% paraformaldehyde(PFA) for one hour and then permeabilized with 0.3% Triton-X 100 for 30 minutes. Samples were then washed three times in PBS and then soaked in a blocking solution for one hour (50 mM phosphate-buffered saline pH 7.4 (PBS; Thermo Fisher) with 1% bovine serum albumin (BSA; BioWorld, OH, USA). The permeable cells were incubated with primary antibodies against VGLUT, MAP2, TUJ1, EN1, Synapsin for an overnight period at 4\u0026deg;C (Supplementary table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). After washing with blocking buffer, secondary antibodies were treated for 30 min at 4\u003csup\u003e\u0026deg;\u003c/sup\u003eC. Dihydrochloride 4\u0026prime;,6-Diamidino-2-phenylindole dihydrochloride (DAPI; Thermo Fisher) was incubated for 20 minutes. The stain must be removed by washing thoroughly three times or more with PBS. To avoid being dried and detached from plates, spheroids are not exposed to air for every single step.\u003c/p\u003e \u003cp\u003eThe following above protocol was applied for 2D SHSY5 cells with time adjustment: 15 minutes for fixation, 10 minutes for permeability, and 30 minutes for blocking.\u003c/p\u003e \u003cp\u003eFlorescent images were taken using a confocal microscope (Leica Stellaris 5, Leica Camera AG, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry analysis (FACS)\u003c/h2\u003e \u003cp\u003eIn 3D PAMCELL\u003csup\u003e\u0026trade;\u003c/sup\u003e culture, cells were pipetted to detached from the well and subsequently centrifuged to remove media. Accutase (200 \u0026micro;L for 3D and 1mL for 2D per well; Thermo Fisher) was used to dissociate the spheroids for ten minutes. Observe under microscope frequently to confirm the separation. Then, add media to collect the cell and centrifuge to remove the media. Followed by 1 hour of fixation in 4% PFA. The cells were rinsed three times with PBS, then permeabilized with 0.3% Triton-X 100 for 20 minutes. After centrifugation to remove the media, the cells were incubated in blocking buffer for one hour and then treated with first antibody for one hour (Supplementary table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Cells were washed 3 times with blocking buffer, followed by a second antibody and incubated for 30 min. FACS solution (50 mM PBS containing 1% BSA, 10% fetal bovine serum (FBS) for 30 min at 4\u0026deg;C after washing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction, cDNA conversion and qRT-PCR\u003c/h2\u003e \u003cp\u003eThe mRNA content of differentiated SHSY5 cells was extracted on day 12 using the following method \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. After removing media from the cells, total RNA was extracted using the Total RNA Extraction Kit (iNtRON Biotechnology, Inc., South Korea), then quantified with NanoDrop One (Thermo Fisher). RNA was converted to cDNA using RT-PCR machine was performed following the manufacturer's instructions using Maxime\u003csup\u003e\u0026trade;\u003c/sup\u003e RT PreMix (Random Primer) (iNtRON Biotechnology, Inc.). In all qRT-PCR reactions, the AriaM x Real-Time PCR system was used with 1 \u0026micro;L of cDNA template, 7 \u0026micro;L of ultra-distilled water, 10 \u0026micro;L of RealMOD green W2 qPCR mix (iNtRON Biotechnology, Inc.), and 1 \u0026micro;L of forward and reverse primers. Supplementary information regarding primers can be found in the following section. Information of primers were used was described in the Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e. The PCR settings included an initial denaturation at 95\u0026deg;C for 10 minutes, followed by 40 cycles of denaturation at 95\u0026deg;C for 20 seconds, annealing at 55\u0026deg;C for 40 seconds, and a melt curve analysis with one cycle each at 95\u0026deg;C for 10 seconds, 65\u0026deg;C for 10 seconds, and 95\u0026deg;C for 10 seconds. The qPCR results were visualized using Agilent Aria 1.8, and relative expression levels were normalized to GAPDH. Ct values obtained through RT-PCR can be found in Supplementary Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eGlutamate Oxidase (GO) electrodes preparation for glutamate level detection\u003c/h2\u003e \u003cp\u003eGlutamate oxidase (GO) solution was prepared before enzyme immobilization. First, 1 mL of sodium periodate (1.5 mg/mL in PBS; Sigma-Aldrich) was added to a vial of GO (25 units/vial; Yamasa Corp., Japan). The solution was then stirred at 4\u0026deg;C for 1 hour. Excess chemicals were washed away with PBS. The periodate-oxidized enzyme was collected using a 30 kDa MWCO Amicon\u0026reg; Ultra Centrifugal Filter (Millipore Corp., USA) and resuspended in 50 mM PBS pH 7.4. The solution was stored at 4\u0026deg;C.\u003c/p\u003e \u003cp\u003eA platinum electrode was polished with alumina slurry (0.05 um, BASi Corp., USA) and incubated with 20 mM cystamine solution (Sigma-Aldrich) for 12 hours at room temperature after being washed with double-distilled water (DDW). The cystamine-functionalized electrode was then treated in GO solution for 1 hour at room temperature, resulting in immobilization through Schiff-base formation. The GO-immobilized electrode was washed and stored in PBS prior to use.\u003c/p\u003e \u003cp\u003eThe maturation media were exchanged 6 hours before testing. Electrochemical characterization was conducted using cyclic voltammetry. With the selected anodic oxidation potential of \u003cem\u003eE\u003c/em\u003eapp (0.65 V vs. Ag/AgCl reference electrode), glutamate concentration in testing sample was evaluated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll statistical analyses were performed using GraphPad Prism version 9.0.0 (GraphPad, CA, USA). The size of the spheroid were measured by Fiji. Data are shown as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean. Student\u0026rsquo;s t-test and one-way analysis of variance (ANOVA) were applied and a \u003cem\u003ep\u003c/em\u003e-value or \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05, 0.01, and 0.001 were considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHTMP, DLN, and MPTL conceived and designed the project. HTMP, DLN, MPTL, and KWL performed research, conducted data analyses, and created figures. HTMP, DLN wrote the manuscript. JHK and HCY discussed the results, consulted, and gave critical feedback. All the authors read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data sets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. Results of the size of spheroids can be found in the Supplementary Data 3. mRNA expression level can be found in the Supplementary Data 4. Voltammogram raw data can be found in the Supplementary Data 5.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Creative Materials Discovery Program (NRF-2019M3D1A1078943), the Priority Research Centers (NRF-2019R1A6A1A11051471), and the Commercialization Promotion Agency for R\u0026amp;D Outcomes (COMPA) grant funded by the Korean government (MSIT) (no. 2021N100).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare the following competing interests: S.A. Sieber is co-founder of smartbax limited. All other authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003e\u003cem\u003eGlobal Status Report on the Public Health Response to Dementia Methodology for Producing Global Dementia Cost Estimates\u003c/em\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://apps.who.int/bookorders\u003c/span\u003e\u003cspan address=\"http://apps.who.int/bookorders\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDawson, T. M., Golde, T. E. \u0026amp; Lagier-Tourenne, C. Animal models of neurodegenerative diseases. Nat. Neurosci. \u003cem\u003e2018 2110\u003c/em\u003e 21, 1370\u0026ndash;1379 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNeofytou, E., O\u0026rsquo;Brien, C. G., Couture, L. A. \u0026amp; Wu, J. C. Stem cells. J. Clin. 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Chronic Glutamate Toxicity in Neurodegenerative Diseases-What is the Evidence? Front. Neurosci. 9, (2015).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Schemes","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"SH-SY5Y cells, 3D microarrays, glutamatergic neuron, disease modeling, Retinoic acid","lastPublishedDoi":"10.21203/rs.3.rs-4934775/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4934775/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNeurodegenerative diseases (NDDs) present significant challenges due to limited treatment options and the ethical concerns of traditional animal models and iPSC-derived neurons. We addressed these issues by developing a 3D culture protocol for differentiating SH-SY5Y cells into glutamatergic neurons, enhancing physiological relevance with a 3D microarray culture plate. Our protocol optimized serum concentration and incorporated retinoic acid (RA) to improve differentiation. We analyzed the proportions of N-type and S-type cells, observing that RA in the maturation stage not only reduced cell proliferation but also enhanced the expression of MAP2 and VGLUT1, indicating effective neuronal differentiation. Our approach demonstrates the strong expression of glutamatergic neuron phenotypes in 3D SH-SY5Y neural spheroids, offering a promising tool for high-throughput NDD modeling and advancing drug discovery and therapeutic development. This method overcomes limitations associated with conventional 2D cultures and animal models, providing a more effective platform for NDD research.\u003c/p\u003e","manuscriptTitle":"Development of a Disease Modeling Framework for Glutamatergic Neurons Derived from Neuroblastoma Cells in 3D Microarrays","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-27 15:08:58","doi":"10.21203/rs.3.rs-4934775/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-09-23T06:22:16+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-20T12:28:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-16T06:40:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-16T03:35:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"251239051261942733790021814606301998092","date":"2024-09-07T15:15:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"564791649127394180082107124260408304","date":"2024-09-07T12:48:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"70578587784302408236583833184699371512","date":"2024-09-07T00:09:35+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-09-06T23:49:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-09-06T23:39:15+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-08-29T19:11:19+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-08-28T11:57:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-08-18T21:01:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7aeb37cf-df0a-49c5-8636-e39befca8cf3","owner":[],"postedDate":"September 27th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":38048911,"name":"Biological sciences/Biotechnology/Biomaterials"},{"id":38048912,"name":"Biological sciences/Biotechnology/Tissue engineering"}],"tags":[],"updatedAt":"2024-12-02T16:04:23+00:00","versionOfRecord":{"articleIdentity":"rs-4934775","link":"https://doi.org/10.1038/s41598-024-80369-3","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-11-25 15:58:03","publishedOnDateReadable":"November 25th, 2024"},"versionCreatedAt":"2024-09-27 15:08:58","video":"","vorDoi":"10.1038/s41598-024-80369-3","vorDoiUrl":"https://doi.org/10.1038/s41598-024-80369-3","workflowStages":[]},"version":"v1","identity":"rs-4934775","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4934775","identity":"rs-4934775","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}