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, 0.05 ng/µL BDNF, 1% PS): RA free and RA treated (10 µM). Additionally, in the differentiation stage (stage I) 3D culture cells were examined with 2 different serum concentrations (2.5% and 5%) due to the difference in metabolic demands. Cellular morphology was observed every day under an 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 6-day stage II, 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.
Morphology of differentiated SH-SY5Y cells in 2D and 3D PAMCELL cultures with/without RA in maturation media and different serum conditions. (A) Bright-field images of SH-SY5Y in 2D culture on day 1, day 6, and day 12. The arrow (black) indicates the S-type cells, which is flat and stretch on surface. 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 concentrations. Diameters of 50 random spheroids were measured. Significant differences between different cultured conditions were evaluated. (ns = not significant, *p < 0.05, ***p < 0.001, unpaired Student’s t-test).
Flow cytometry analysis of serum concentration and the effect of RA in maturation media
The observation of cellular morphology was a confirmation of the presence of mature-like 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, and 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 cells cultured with 5% hiFBS after a 12-day maturation period, with and without RA (Fig. 2C, D). On 2D cultures, the proportion of MAP2-positive cells increased from 61.33 to 93.25% when RA was added to the maturation media. VGLUT1 also expressed the same result from 79.43% cells population to 93.25%. In 3D cells cultured in 5% hiFBS, the presence of RA also led to enhanced signals, with the mean MAP2 and VGLUT1 signals increasing fourfold and sixfold, respectively. These results suggest that RA not only inhibited unwanted proliferation but also promoted the maturation of SH-SY5Y cells in both 2D and 3D culture conditions. The contrast in signals between TH and VGLUT1 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 more 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.
Differentiation and maturation of SH-SY5Y cells in 2D and 3D cultures assessed by flow cytometry analysis. (A) FACS analysis on day 6 of differentiation. The black arrow indicated the population of undifferentiated cells (B) FACS analysis of cells cultured in maturation media without RA. The black arrow indicates the population of undifferentiated (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.
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 neurons23. VGLUT1 was strongly expressed across all conditions, confirming the differentiation protocol’s effectiveness towards a glutamatergic pathway. Served as vesicular glutamate transporters that transported glutamate into synaptic vessels in neurotransmission, VGLUT1 is localized in axon ends and is an important marker for presynaptic terminal development. Additionally, glutamate is an impactful regulator on generation of contact dendrites23,24,25. Thus, in the 3D 5% hiFBS condition, VGLUT1 was prominently expressed on the outer layer of the spheroid, suggesting a distinct orientation of the neurons and their connections.
Expression of mature neuronal markers by 2D, 3D 2.5% hiFBS and 3D 5% hiFBS after 12-day maturation in presence of RA. (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.
MAP2, essential for dendritic elongation and structural integrity of neurons26, 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. Additionally, different media conditions directly affected the ability to outgrow the neurites of spheroids. While neurites in 5% hiFBS-growth spheroids had average 53.43 ± 17.39 μm in length, spheroid treated in 2.5 hiFBS only extended neurites with 30.66 ± 10.93 μm in length. These suggest that spheroids in 5% hiFBS are at a more mature stage of neuronal development than lower serum concentration one. (Supplementary Table S6 and Figure S2).
The expression of TUJ1 reinforced similar observations (Fig. 3D). 3D 2.5% hiFBS spheroid only expressed 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.
Synapsin plays a key role in the regulation of neurotransmitter release and serves as a reliable marker for presynaptic vesicles27. Similarly, EN1 (Engrailed-1) is crucial for interneuron communication and has been shown to play an important role in neuronal survival. The deletion of EN1 results in the death of neurons in the ventral nuclei of the lateral lemniscus during development28. These roles of Synapsin and EN1 suggest that their expression across the spheroids indicate synaptic activities inside the spheroid. (Fig. 3B, E).
Additionally, Fig. 3F highlights the height of spheroids as visualized through depth-coded imaging with TUJ1 staining, showing that the representative spheroid had an approximate height of 70 μm along the z-axis and a diameter of around 100 μm in the x and y axes. The spheroid formation image also illustrates the uniformity of spheroids, further supporting the consistency of the model used.
The 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, and D explore the impact of RA on mRNA expression levels in all 3-culture platforms and conditions. The expression levels 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 to 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 influences the expression levels of EN1 and GLUL in the 3D culture platform. Additionally, the overexpression level of GLUL and VGLUT1 (Fig. 3A) indicates that the cells were expressed glutamatergic phenotype and were ready for further application.
The mRNA levels of SNCA were further evaluated to elucidate the effects of retinoic acid (RA) and serum concentrations on neuronal differentiation and maturation in SH-SY5Y cells (Fig. 4E). In the absence of RA treatment during stage II, SNCA levels on day 12 across all three conditions did not show significant improvements compared to day 6. However, upon RA treatment, SNCA levels in 2D cultures on day 12 were found to be 2.4 times higher than those measured on day 6 and 1.5 times higher than in cells nurtured without RA. For 3D cultures in 2.5% hiFBS, SNCA levels on day 12 with RA treatment were approximately 1.5 times higher than those on day 6 and day 12 without treatment. Notably, in 3D cells cultured in 5% hiFBS, RA treatment led to a substantial increase in SNCA levels, with values recorded at 2.9 times higher than on day 6 and 1.5 times higher than on day 12 without treatment.
Relative 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. (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, *p < 0.05, **p < 0.01, ***p < 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. (E) Serum concentration and RA effect on SNCA expression level. The vertical axis shows the relative gene expression levels as the means ± SE (n = 03). Bars represent the means of three replicates ± SD.
Glutamate detection in mature-like neural spheroid using cyclic voltammetry
After confirming the protocol for mature-like neurons with glutamatergic phenotype, we proceeded to measure the levels of glutamate released as a neurotransmitter. Figure 5A illustrates the design of the electrochemical Glutamate Oxidase (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 electrochemical 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 starving 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 is likely caused by the glutamate communication of spheroids. The current continued increase in 2-day starving spheroids and 4-day starving spheroids suggests the loss in glutamate homeostasis in spheroid led to glutamatergic hyperactivity29. 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 1136 ± 69 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 NDD modeling and drug discovery.
Concept and result of cyclic voltammetry of enzymatic glutamate sensor. (A) Scheme of GO enzymatic sensor. (B) Detection of glutamate in 3D 5% hiFBS in different conditions. (C) Violin plot of current density of spheroid in different conditions (ns = not significant, *p < 0.05, **p < 0.01, ****p < 0.0001, unpaired Student’s t-test, n = 5).
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- Source: https://www.nature.com/articles/s41598-024-80369-3