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

doi:10.21203/rs.3.rs-4934775/v1

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

https://doi.org/10.21203/rs.3.rs-4934775/v1

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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

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 worldwide¹. 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 costs¹. 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 diseases². 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 samples³. 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 models⁶. 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 conditions¹⁹. Lack of the complexity of the human brain's cellular environment, traditional 2D cultures may not fully recapitulate certain aspects of neuronal physiology and pathology²⁰. 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 limited⁷. 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.

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. 1A). 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. 1B). 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. 1C). 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 2A 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. 2B). 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. 2A). 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 2B 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. 2C,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 3A illustrates the expression of VGLUT1, a key indicator for glutamatergic neurons²¹. 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 neurons²², 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. 3C). 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. 3D). 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. 4A) further supports that culturing cells in 3D PAMCELL™ with 5% hiFBS creates an optimal environment for SH-SY5Y differentiation and maturation.

Figure 4B, 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. 3A) 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 5A 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 (H₂O₂) as a byproduct. The H₂O₂ 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 5B 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 hyperactivity²³. Figure 5C 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.

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. 3A), 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 reaction⁷. Additionally, B27 was found to reduce TH expression⁷, 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 report³³. 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.

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 Eapp (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.

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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Scheme 1 is available in the Supplementary Files section

No competing interests reported.

TableS1antibody.docx
TableS2Primers.docx
TableS4.docx
TableS5.docx
tables3spheroidsize.docx
Scheme1.png
Scheme 1: Timeline for differentiation protocol.

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You are reading this latest preprint version

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

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Version 1

Abstract

Figures

Introduction

Results

Morphological analysis of differentiated SH-SY5Y cells in 2D and 3D culture systems

Flow Cytometry Analysis of serum concentration and the effect of RA in maturation media

Immunocytochemical and mRNA expression analysis of neuronal differentiation and maturation

Glutamate detection in maturated neural spheroid using cyclic voltammetry

Discussion

Materials and Methods

Cell culture and differentiation

Immunocytochemistry

Flow cytometry analysis (FACS)

RNA extraction, cDNA conversion and qRT-PCR

Glutamate Oxidase (GO) electrodes preparation for glutamate level detection

Statistical analysis

Declarations

References

Schemes

Additional Declarations

Supplementary Files

Status:

Version 1