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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 37  |  Issue : 2  |  Page : 82-88

Comparison of medium supplements in terms of the effects on the differentiation of SH-SY5Y human neuroblastoma cell line


1 Department of Biochemistry and Health Sciences Institute, Dokuz Eylul University, Faculty of Medicine, İzmir, Turkey
2 Department of Histology and Embryology, Dokuz Eylul University, Faculty of Medicine, İzmir, Turkey
3 Department of Biochemistry, Dokuz Eylul University, Faculty of Medicine, İzmir, Turkey

Date of Submission23-Mar-2019
Date of Decision15-May-2019
Date of Acceptance13-Sep-2019
Date of Web Publication29-Jun-2020

Correspondence Address:
Semra Koçtürk
Department of Biochemistry, Faculty of Medicine, Dokuz Eylül University, 35340 Balçova, Izmir
Turkey
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/NSN.NSN_15_20

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  Abstract 


Objective: Human SH-SY5Y cell line has been frequently used for in vitro experiments in neuroscience-related research. To reflect a better neuronal characteristic the cell line needs to a differentiation. To compare the results obtained from in vitro models, the similarity of the phenotype and characteristic of the cells has great importance. However, many studies have been performed using the different medium ingredients which affect the differentiation progress of the cells. Therefore, we aimed to compare generally used differentiation mediums, contain only retinoic acid (RA) and supplemented with different ingredients, in the aspect of neuron-like phenotype characteristics and stability. Materials and Methods: The effects of medium ingredients on differentiation levels were evaluated using morphological changing, neurite length, and immunofluorescence detection of neuronal markers such as NFH, β-III Tubulin, and microtubule-associated protein 2 (MAP2). The stability of differentiated cells was followed microscopically at the 7th, 10th, and 14th days by morphological changings and neurite length using Neuron J software. Results: The results revealed that the cells pretreated with RA for 5-day treatment and followed by 5-day treatment with the mix medium and brain-derived neurotrophic factor (BDNF), provided significantly higher neurite length than the other groups (P < 0.001). In this group, the expressions of β-tubulin III, MAP2, and NF-H were also significantly higher than the control group (P < 0.05, P < 0.001, and P < 0.05, respectively) and differentiated cells were stable until the 7th day. Conclusion: The results demonstrated that enriched mediums are necessary for a better differentiation of SH-SY5Y cells. We recommend 10-day treatment period and using of RA, BDNF, dc-AMP, KCI together in SH-SY5Y cell differentiation.

Keywords: Differentiation, microtubule-associated protein 2, NF-H, SH-SY5Y cell, β-III tubulin


How to cite this article:
Serdar BS, Erkmen T, Ergür BU, Akan P, Koçtürk S. Comparison of medium supplements in terms of the effects on the differentiation of SH-SY5Y human neuroblastoma cell line. Neurol Sci Neurophysiol 2020;37:82-8

How to cite this URL:
Serdar BS, Erkmen T, Ergür BU, Akan P, Koçtürk S. Comparison of medium supplements in terms of the effects on the differentiation of SH-SY5Y human neuroblastoma cell line. Neurol Sci Neurophysiol [serial online] 2020 [cited 2020 Sep 25];37:82-8. Available from: http://www.nsnjournal.org/text.asp?2020/37/2/82/288416




  Introduction Top


Neuroblastoma (SH-SY5Y) cell line has been frequently used cells for in vitro experiments, but it lacks some of the features of functionally mature neurons.[1],[2] Many studies have demonstrated that differentiated and undifferentiated SH-SY5Y cells have important differences that affect morphology, polarization, neurites lengths, and proliferation rates of the cells.[2],[3] For instance, undifferentiated SH-SY5Y cells consist of neuroblast-like cells (N-type), epithelial-like cells (S-type), and lack of many of features that define neurons such as expression of neuron-specific markers.[2],[4] Therefore, the differentiation of SH-SY5Y cell line has great importance for research in neuroscience. Studies have revealed that SH-SY5Y cells are differentiated by a treatment with a differentiation medium which provides to the cells gaining some biochemical characteristics and expression of neuronal markers such as β-III tubulin, microtubule-associated protein 2 (MAP-2), neurofilaments heavy/light polypeptide (NF-H, NF-L), growth-associated protein-43 (GAP-43), and neuronal nuclei (NeuN).[2],[4],[5] Evidence of the differentiation is accepted to determine three or four of the neuronal markers in differentiated cells. For instance, β-III tubulin is a microtubule element found in mature neurons and involved in axon maintenance.[6] The other marker MAP2 is a major component of microtubules in dendrites and have a role in determining and stabilizing dendritic shape during neuron development.[7] Neurofilaments heavy polypeptide is a type of intermediate filaments existing in axons and its expression allows the cytoskeleton network that provides a gradient between the distal and proximal side of the cells.[8],[9] GAP-43 is another nervous tissue-specific cytoplasmic protein and its expression is increased during neuronal growth, development, and axonal regeneration.[10] NeuN is accepted as a neuron-specific marker in the literature, but its function is unknown.[11]

Currently, various reagents have been used in the differentiation process but which medium provides better differentiation for the neuron-like phenotype has not been clearly elucidated yet. Retinoic acid (RA) is commonly used reagent in differentiation and many studies show that RA and the addition of Vitamin D3, phorbol esters, dibutyryl-cyclic AMP (db-cAMP), cholesterol, estradiol, 12-Otetradecanoyl-phorbol-13-acetate, brain-derived neurotrophic factor (BDNF), and nerve growth factor to the medium provides better differentiation of SH-SY5Y cells that reflects neuron-like phenotype.[5],[12],[13] Besides, the treatment period of the cells is another important factor in differentiation. In the literature, RA treatment periods show variability between 3 and 5 days and >10 days.[4],[5] Encinas et al. have emphasized the importance of RA treatment period on differentiation. They have argued that short-term treatment (up to 5 days) has a capacity to induce the proliferation of neuroblast-like N-type cells, but longer period, (>10 days) leads to promote the proliferation of epithelial-like S-type cells.[4] Some of the researchers also emphasized that the combination of RA with other agents such as BNDF, cholesterol, estradiol, dc-AMP has a beneficial effect on the expression of neuronal markers.[1],[14],[15],[16] That there are different recommendations in the literature about the ingredients of differentiation medium creates a need an investigation, which determines the best differentiation medium ingredients for SH-SY5Y cells differentiation.

In the present study, the aim was to examine the effects of medium ingredients and the treatment periods on the differentiation of SH-SY5Y cells. We have compared generally used differentiation mediums-containing RA only, BDNF, and a mix medium composed of different ingredients such as dc-AMP, glutamax I, and KCI. Differentiation has been evaluated using the morphological changes, neurite length, expression of β-III tubulin, MAP2, and NF-H and the stability of neurite length. Our results showed that RA-Mix-BDNF medium has the best differentiation capacity on the SH-SY5Y cells and we suggest using the medium for neuroscience studies.


  Materials and Methods Top


The study was approved by the Dokuz Eylül University Noninvasive Research Ethics Board (acceptance code: 2018/18-33).

Cell culture and differentiation mediums

Human neuroblastoma cell line SH-SY5Y (American Type Culture Collection [ATCC] −2266) was purchased from (ATCC; Manassas, USA). Cells were cultured in DMEM: F12 (Biochrom; Berlin, Germany) supplemented with FBS, penicillin, streptomycin in humidified 5% CO2 incubator at 37°C. Six groups are composed; Group 1: Control, undifferentiated SH-SY5Y cells, Group 2: Cells treated with RA (10 μM) for 5-day, Group 3: Cells treated with 10 μM RA (Sigma; St Louis, USA) for 10-day, Group 4: Cells pre-treated with RA for 5-day followed with 50 ng/ml BDNF (Sigma; St Louis, USA) for 5 days, Group 5: Cells pre-treated with RA for 5 days followed with mix medium (Neurobasal (Thermo Fisher Scientific; Massachusetts, ABD), B27 (1X) (Thermo Fisher Scientific; Massachusetts, ABD), 2 mM dc-AMP (Sigma; St Louis, USA), 20 mM KCI (Fisher; Michigan, USA), 2 mM Glutamax I (Thermo Fisher Scientific; Massachusetts, ABD) for 5 days, Group 6: Cells pre-treated with RA for 5 days followed mix medium (Neurobasal, B27 (1X), db-cAMP (2 mM), KCI (20 mM), Glutamax I (2 mM) and BDNF (50 ng/ml) for 5 days. Differentiation of period was 10 days for all of the groups except for Group 2 which was 5 days. The culture medium was changed every 3–4 days and supplemented with fresh medium. Cellular morphology of the groups was examined with an Olympus CKX40 digital camera at ×40. Early passages [1],[2],[3],[4],[5],[6],[7],[8],[9],[10] of the cells were used in all experiments.

Measurement of neurite length

Neurite length was measured with the Neuron J analysis software (ImageJ software, NIH) which provides counting of the neurites. The cultured cells were observed using a phase-contrast microscope on the 10th day and 5th days. Six different locations were counted for each well and mean values were evaluated with software.

Immunofluorescence assay

Immunofluorescence assay was performed to investigate the expression of MAP2, NFH, and β-III tubulin neuronal-specific markers. The immunofluorescence protocol was modified from the method described previously.[17] The undifferentiated and differentiated SH-SY5Y cells were plated at a density of 3 × 104 cells/coverslip on 12 mm glass coverslips precoated with Poly-D-lysine (Sigma; St Louis, USA). At the 10th day of the differentiation, the cells were fixed with cold methanol for 30 min at room temperature and rinsed three times with phosphate-buffered saline (PBS Biochrom; Berlin, Germany). After washing, cells were permeabilized with 0.2% Triton X-100 (Sigma; St Louis, USA) in PBS for 5 min and then incubated in blocking solution (1% BSA in PBS) at room temperature. Then, the cells were incubated for overnight at 4°C with primary antibodies separately (MAP2 (Abcam; Cambridge, UK), β-III Tubulin (Santa Cruz; Texas, USA) and NF-H (Abcam; Cambridge, UK). At the end of the incubation period, all samples were washed with PBS and incubated with secondary antibody at room temperature for 1 h. After nuclei were stained with Hoechst (Sigma; St Louis, USA), samples were immediately analyzed with a Leica DMIL fluorescence microscope.

Assessment of stabilization period of the differentiation

We also examine the stability period of the differentiated cells by morphological and neurite length parameters which provide information about how many days of the cells carry neuronal characteristics. Cellular morphology and neurite length measurements were performed as described previously at the last day of differentiation period and accepted as the 0th day of stabilization period after then, it monitored on the 7th, 10th, and 14th days.

Statistical analysis

Statistical analyses were performed using GraphPad Prism software version 8.00, Inc., (La Jolla; CA, USA). Multiple groups were compared using one way-ANOVA and Tukey Multiple comparison test and values of P < 0.05 were considered statistically significant. All the experiments were performed at least in triplicate.


  Results Top


Morphology assessment of the groups

Our morphological assessment results showed that control cells (Group 1) tend to grow in clusters and had no neuritis, but in the other groups (Group 2–6), the neurites have been tending to extend [Figure 1]. In Group 2 and 3, cells treated with RA for 5 and 10 days, respectively, displayed neurites with branches morphology compared to the control group. Furthermore, Group 6, cells pretreated with RA for 5 days and followed with BDNF included-mix medium (Glutamax I, Neurobasal, KCI, dc-AMP) for 5 days, the branching rate and connections of neurites were higher than the other groups [Figure 1]f. Our morphological assessment clearly revealed that used mediums, ingredients, and treatment periods have an important effect on the differentiation level of SH-SY5Y cells.
Figure 1: Cellular morphology and network of undifferentiated and differentiated SH-SY5Y cells with different treatment periods. (a) undifferentiated (b) treated with RA for 5 days (c) treated with RA for 10 days (d) pre-treated RA for 5 days then treated with BDNF for 5 days e) pre-treated with RA for 5 days then treated with mix medium for 5 days (f) pre-treated with RA for 5 days then treated with mix medium and BDNF for 5 days. Images were obtained at 40X magnification using by phase contrast microscopy

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Determination of neurite length

The neurites lengths of the groups were calculated at the 10th and 5th day. As shown in [Figure 2] differentiated cells treated with mix medium + BDNF displayed significant longer neurite extensions than the other groups (P < 0.001), except for RA + Mix group (P > 0.05). When we compared the groups with each other, significant differences were found between the groups (P < 0.001, P < 0.05) [Figure 2]. However, medium ingredients contain Neurobasal, B27, KCI, Glutamax I, dc-AMP, and BNDF with differentiated cells was obtain the highest length of neurites in six groups. This data showed that BDNF increased neurite length according to treated only mix medium and effect on differentiation.
Figure 2: Effects of different treatments on neurite lengths of SH-SY5Y cells. Data showed mean ± S.D. **P < 0.001, *P <0.05

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Immunofluorescence analysis of neuronal specific markers

Immunofluorescence assays show that β-III tubulin was found in low amounts in undifferentiated cells, but in Group 6, its expression tends to increase with neuronal morphology [P < 0.05, [Figure 3] and [Figure 4]. The intensity of MAP2 expression is significantly stronger in Group 6th an in the undifferentiated group [P < 0.001, [Figure 5] and [Figure 6]. NF-H expression is significantly higher in Group 6th an in the undifferentiated group [P < 0.05, [Figure 7] and [Figure 8].
Figure 3: Immunofluorescence staining for the neuronal markers β-III tubulin. a.Nuclei stained with Hoechst in undifferentiated SH-SY5Y group b. β-Tubulin III in undifferentiated cells c. Merged image d. Nuclei stained with Hoescht in differentiated SH SY5Y cells in group 6 e. β-Tubulin III in differentiated SH SY5Y cells in group 6. f. Merged image group six. Images were obtained at 40X magnification using an immunofluorescence microscope. Scale bar: 150 μm. Blue: Hoechst; Green: β-Tubulin III

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Figure 4: β-III tubulin signal intensity calculated by using Image-J software. Data showed mean ± S.D. *P < 0.05 vs. Control group (n = 9)

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Figure 5: Immunofluorescence staining for the neuronal markers MAP2. (a) Nuclei stained with Hoechst in undifferentiated group (b) MAP2 in undifferentiated cells. (c) Merged image (d) Nuclei stained with Hoescht in differentiated SH-SY5Y in group 6. (e) MAP2 in differentiated SH SY5Y cells in group 6. (f) Merged image in group 6. Images were obtained at 40X magnification using an immunofluorescence microscope. Scale bar: 150 μm. Blue: Hoechst; Green: MAP2

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Figure 6: MAP-2 signal intensity calculated by using Image-J software. Data showed mean ± S.D. **P < 0.001 vs. Control group (n = 9)

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Figure 7: Immunofluorescence staining for the neuronal markers neurofilamnet heavy polypeptide. (a) Nuclei stained with Hoechst in undifferentiated group (b) NF-H in undifferentiated cells. (c) Merged image. (d) Nuclei stained with Hoescht in differentiated SH-SY5Y in group 6. (e) NF-H in differentiated SH-SY5Y in group 6. (f) Merged image in group 6. Images were obtained at 40X magnification using an immunofluorescence microscope. Scale bar: 150 μm. Blue: Hoechst; Green: NF-H

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Figure 8: NF-H signal intensity calculated by using Image-J software. Data showed mean ± S.D. *P < 0.05 vs. Control group (n = 9)

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Stability

We also investigated the stability period of neuronal morphology and neurite length in differentiated cells [Figure 9] and [Figure 10]. Our result demonstrated that there was no significant difference between 0th and 7th day results in term of neurite length [Figure 10]. However, neurite lengths of the 10th and 14th days were significantly lower than the 0th day (P < 0.05 and P < 0.001, respectively). Obtained data were clearly revealed that differentiated cells were stable up to the 7th day.
Figure 9: Morphological changings after differentiation periods. (a) 0th day, after differentiation protocols (b) 7th day, after differentiation protocols (c) 10th day, after differentiation protocols (d) 14th day, after differentiation protocols. Images were obtained at 40X magnification using by phase contrast microscopy

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Figure 10: Neurite length changings at 0th, 7th, 10th and 14th days after differentiation periods. Images were obtained at 40X magnification using by phase contrast microscopy and Neuron J software. Data showed mean ± S.D. **P < 0.001, *P <0.05

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  Discussion Top


To reach accurate results, the cells reflecting mature neuron-like phenotype or behavior have great importance in nervous system-related studies. With this awareness, we aimed to compare the generally used differentiation mediums with their differentiation performance for SH-SY5Y cells. Several attempts have been made to differentiate SH-SY5Y cells until today.[1],[2],[3],[4],[5],[11],[13],[14],[15],[16] Although RA-induced differentiation of SH-SY5Y cells has been accepted and generally used protocol, the ingredients of mediums and treatment period in differentiation have been still controversial.[4],[5],[14],[16],[18],[19],[20],[21] Combinational uses of BDNF and RA has been suggested by many studies for the differentiation,[1],[2],[4],[13],[22],[23],[24],[25] but only in one study, the researcher has claimed that there was no enhancing effect of combinational uses of RA and BDNF on differentiation.[15] Shipley et al. advocated that pretreatment of the cells and combinational uses of some mediums have a beneficial effect on differentiation. In their study, they had pre-treated the cells with RA for 10 days and followed with differentiation media for 8 days, which consists of Neurobasal, B27 (1X), KCI (20 mM), Glutamax I (2 mM), dc-AMP (2 mM), and BDNF (50 ng/ml).[3] They have suggested the protocol to generate neuronal cultures which have been proved by the high expression levels of NF-H and MAP2 in the cells. Shipley et al. have contributed to the literature about the ingredients of differentiation medium, but their results lack comparison of the medium ingredients each other. The treatment period is another important factor in differentiation. The study has shown that a neuron-like type phenotype (N-type) is needed to ensure a better differentiation of SH-SY5Y cells and treatment of RA with a longer period (>10 days) leads to promote the proliferation of epithelial-like S-type cells.[4] Therefore, there is a need to examine the effects of ingredients of the medium and RA treatment period together on the differentiation of the cells. We think that our study contributes to the literature by comparing medium ingredients (RA, RA-BDNF, RA-Mix, RA-Mix-BDNF) and in terms of the time period (5 and 10 days). Our results were controversial to the many studies suggesting RA treatment only. For instance, we found a significant difference in neurite extensions between only RA treated, Mix and Mix-BDNF-treated groups. Different ingredients of the differentiation mediums have been examined by the studies and their results indicated that combinational treatment plays an important role in the differentiation of SHSY5Y cells. For instance, Teppola et al. showed that when SH-SY5Y cells treated with a combination of RA, cholesterol, and BDNF, neurites lengths, branching and network formation of the cells were higher than estradiol or RA treatment alone.[15] This finding also supported by the study of Agholme et al. and they have suggested the RA + BDNF and RA + BNDF + cholesterol treatments to provide longer neurites than the treatment with RA alone.[1] Currently, Paik et al. also demonstrated that combinational usage of RA and somatostatin has an enhancing effect on neurite outgrowth.[16]

The increased expression of some proteins, such as MAP2, β-III tubulin, GAP-43 and NFH are also the characteristics of differentiated cells but variation in the protein markers can be seen related to the in vitro disease model. For instance, different markers can be chosen to prove the in vitro disease model such as Parkinson or Alzheimer. Avola et al. confirmed the SH-SY5Y cell differentiation by increased expression of MAP2, β-III tubulin, and tyrosine hydroxylase for an in vitro Parkinson disease model.[26] However, Jämsä et al. analyzed the cell differentiation by increased the level of total glycogen synthase kinase 3 beta as well as the expression of the phosphorylation state of tau in Alzheimer disease model.[14] Some of the researchers also emphasized the importance of the treatment period in differentiation. Schneider L et al. showed that the expression of MAP2 was increased by 50% after 5 days of treatment with RA.[27] Another study result demonstrated that RA-induced differentiated SH-SY5Y cells clearly exhibit neuronal specific markers Tau, β-III Tubulin, and MAP2 at the 3th and 7th days of the treatment.[16] Encinas et al. results also exhibited higher expression of NF-H and MAP2 in differentiated cells, treated with RA for 5 days and subsequently treated by BDNF for 5 days.[4] Constantinescu et al. found higher expression of MAP2 and β-III tubulin in the differentiated cells treated with 10 μM RA for 14 days.[11] Filograna et al. also demonstrated that RA and staurosporine-treated in SH-SY5Y and BE (2)-M17 neuroblastoma cell for 7 days. Their study stated that these agents increased the expression of neurofilament and β-III tubulin of differentiated of cells.[28] If all of the literature evaluated, a variation on the suggested treatment periods and the confirmation methods can be clearly seen. Therefore, we decide to compare the mediums by assessing its effects on morphology and neurite length. The differentiation marker assessment has been performed in the group which has the highest neurite length which implies the best morphological differentiation. In this group, the marker expressions (β-III tubulin, MAP2, and NF-H) were found significantly higher compared to control groups (P < 0.05, P < 0.001, and P < 0.05, respectively) which are similar to Shipley et al. study.[3]

Stability period of the differentiation characteristics needs to take in consideration in the studies. Shipley et al. exhibited that differentiated SH-SY5Y cell showed stability until 14th days.[3] Therefore, we examined whether the differentiated cells maintain their characteristics for up to 14 days. We clearly showed that neurite length is stable until the 7th day after the differentiation, but the stability of the differentiation may be examined by different markers and methods.


  Conclusion Top


To obtain more accurate and as close as to in vivo results, we suggest using a differentiation method that provides differentiation of neuron-like phenotype. According to our results, we recommend to the researcher our treatment period (10 days) and ingredients (RA, BDNF, dc-AMP, KCI, B27) for SH-SY5Y cell differentiation.

Acknowledgment

This research was supported in part by a grant (2017.KB.SAG.004) from Dokuz Eylül University Scientific Research Project Unit. We also would like to express our gratitude to Professor Kemal S. Korkmaz for guidance in immunofluorescence assays.

Financial support and sponsorship

This research was supported in part by a grant (2017.KB.SAG.004) from Dokuz Eylül University Scientific Research Project Unit. We also would like to express our gratitude to Professor Kemal S. Korkmaz for guidance in immunofluorescence assays.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10]



 

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