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Size-controllable networked neurospheres as a 3D neuronal tissue model for Alzheimer's disease studies
Biomaterials 34 (2013) 2938e2946
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Size-controllable networked neurospheres as a 3D neuronal tissue model for Alzheimer’s disease studies Yoon Jung Choi a,1, JiSoo Park a,1, Sang-Hoon Lee a, b, * a b
Department of Biomedical Engineering, Korea University, Seoul 136-703, Republic of Korea KU-KIST Graduate School of Converging of Sciences & Technologies, Korea University, Seoul 136-701, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 November 2012 Accepted 5 January 2013 Available online 29 January 2013
Intensive in vitro studies on the neurotoxicity of amyloid beta have been conducted for decades; however, a three-dimensional neuronal tissue model for Alzheimer’s disease has not yet been achieved. In this study, we developed size-controllable networked neurospheres comprised of cerebral cortical neuronal cells that mimics the cytoarchitecture of the cortical region of the brain. The toxicity of amyloid beta on the neurosphere model was assessed quantitatively and qualitatively. Decreased cell viability after amyloid beta exposure was demonstrated using MTT and live/dead assays. Neurite degeneration after amyloid beta exposure was evident in both SEM and fluorescence images. Ultrastructural features of apoptotic neurons were analyzed and quantitative analysis of synapsin II concentration and an acetylcholine assay were also performed. The three-dimensional neurospheres, produced using a concave microwell array, are a potential in vitro model for Alzheimer’s disease studies. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Neurosphere 3D model Amyloid beta Alzheimer’s disease PDMS microconcave wells Neural networks
1. Introduction Alzheimer’s disease, the most common neurodegenerative disease [1], has been studied for decades, with the general consensus now being that amyloid beta protein is the cause [2]. The cerebral cortex, often denoted as gray matter, is the outermost layer of the cerebrum of the mammalian brain. As a major center of the central nervous system (CNS), the cerebral cortex is largely involved in higher brain functions such as conscious memory, awareness, thought, and language. Substantial evidence demonstrates that the cerebral cortex is the region most affected by amyloid beta [3e5]. Progressive deterioration in cognitive function, which is a remarkable pathological feature of Alzheimer’s disease that distinguishes it from other forms of senile dementia, is derived from amyloid beta-induced neuron loss [6e8]. To gain an understanding of the neuron loss induced by amyloid beta, two dimensional (2D) models with limited planar structures have been employed [2,6]. However, such models lack the unique pathophysiological characteristics present in three dimensional (3D) in vivo neuronal tissue. There is a lack of linkages between the neuronal cells in all directions, and the positioning of the cells does * Corresponding author. Department of Biomedical Engineering, Korea University, Seoul, Republic of Korea. Tel.: þ82 2 940 2773; fax: þ82 2 921 6818. E-mail address: [email protected] (S.-H. Lee). 1 These authors contributed equally. 0142-9612/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2013.01.038
not accurately represent in vivo conditions [9]. In general, the distance of the synaptic cleft between cells is increased in 2D culture models. As multiple messenger molecules that travel through the synaptic cleft transmit messages between the neuronal cells [10,11], the increased size of this impedes the signaling. Therefore, the presence of a cell gap comparable in size to that of in vivo cells is highly important for understanding how neuronal cells interact with each other [12]. Apoptosis caused by amyloid beta is highly regulated by cell signaling [13e15] and so the mimicking of the in vivo cell gap size could play a critical role in understanding the pathophysiological phenomena attributed to amyloid beta. Due to these significant requirements, adequate 3D ultrastructural models of neural tissue are extremely rare. In this study, we propose a networked neurosphere model that represents the cerebral cortex, the region of the brain most vulnerable to amyloid beta. By simply seeding neuronal progenitor cells into concave microwells fabricated in PDMS [16e18], cells were self-aggregated and formed neurospheres. These were cultured within the wells for 10 days, where the neural progenitor cells became fully differentiated and neural networks were generated. After the formation of the neurospheres, they were cultured in the presence of 5 mM amyloid beta protein solution to validate the applicability of the model to Alzheimer’s disease studies. Exposure to the amyloid beta solution led to neuronal death and the appearance of pathophysiological features of Alzheimer’s disease. Using transmission electron microscopy (TEM), ultrastructural
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features consistent with the apoptosis of neurospheres were detected. Morphological changes were analyzed immunohistochemically and investigated using scanning electron microscopy (SEM). Synapsin II and acetylcholine were assessed to validate the neurotoxicity of amyloid beta. 2. Materials and methods
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2.4. Cell proliferation assay The neurospheres cultured in Neurobasal medium for 7 days and exposed to 5 mM synthetic amyloid beta protein (1-42, SigmaeAldrich, USA) for an additional 3 days were placed into the wells of a 96-well plate (100 mL medium/well; Corning, USA) and thereafter 10 mL of MTT cell viability solution were added. The neurospheres were incubated at 37 C in a humidified 5% CO2 atmosphere for 45 min. Absorbance at 450 nm was measured using a microplate reader (SpectraMax M2; Molecular Devices, CA, USA).
2.1. Isolation of prenatal rat cortical neurons 2.5. Fixation and immunocytochemistry Prenatal rat cortical neurons were isolated from cerebral cortical regions of E-6 rat embryos (DBL, Incheon, South Korea), which consists of 6 horizontal layers. The surgical procedures used in this experiment were authorized by the Institutional Animal Care and Use Community (IACUC) of Texas Southern University. Neural progenitor cells were maintained in Neurobasal media (Gibco) supplemented with B-27 Supplement (Gibco), 0.5 mM L-glutamine, and 1% antibiotics containing 10,000 units penicillin (Gibco) and streptomycin. 2.2. Cell seeding and formation of neurospheres A schematic depiction of neurosphere formation and networks between spheres is illustrated in Fig. 1B. Neurospheres were formed by using poly(dimethylsiloxane) (PDMS)-based concave microwells with 300 mm, 400 mm, 500 mm, and 800 mm diameters, fabricated using standard soft lithography techniques. The neural progenitor cell suspension was seeded on top of the concave molds and was subsequently cultured for a total of 10 days. In Group 1, the neurons were cultured for 10 days in normal medium and in Group 2, the neurons were cultured for the first 7 days in normal medium and for the next three days in medium containing 5 mM amyloid beta. To verify that the neurospheres consisted of cells from six horizontal layers, the spheroid was immunofluorescence stained for the transcription factors Brn2 and Satb2 (specific to layers II and III), CTIP2 (specific to layer V), and Tbr1 (specific to layer VI) and we observed that the cell in spheroid were from six layers. 2.3. Cell viability test The viability of the neural cells cultured in the concave molds was assessed using a live/dead assay kit (Invitrogen, CA). 1 mL of calcein AM solution and 5 mL of ethidium homodimer-1 solution were dissolved in 2 mL of Neurobasal medium, which was then added to the cells followed by incubation at 37 C for 40 min. The stained cells were observed using a confocal laser scanning microscope (LSM 5 Exciter, Carl Zeiss Germany) and the resulting acquired images were analyzed using the ImageJ software (NIH, Bethesda, MD).
The neurospheres formed in the concave microwells were fixed with 4% paraformaldehyde (PFA) for 20 min at 4 C and then washed with 0.1% BSA in phosphate buffered saline (PBS). The cells were incubated in 0.1% Triton X-100 in PBS for 20 min at room temperature and then washed with 0.1% BSA in PBS. The cells were blocked for 30 min at 4 C to reduce non-specific protein adsorption and then probed with primary antibodies: neurofilament H (Millipore), beta III tubulin (Invitrogen), TBR1 (Abcam), Ctip (Abcam), Tuj1 (Covance), Satb2 (Abcam), and synapsin (Abcam) overnight at 4 C. Cells were rinsed with 0.1% BSA in PBS, and incubated with the secondary antibody (Alexa Fluor 488 or 594; Invitrogen) for 90 min at 4 C. Fluorescence images were collected using a confocal laser scanning microscope (LSM 5 Exciter, Carl Zeiss, Germany) after counterstaining the cell nuclei with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI; Invitrogen). 2.6. Scanning electron microscopy (SEM) The neurospheres were fixed with 2.5% glutaraldehyde in deionized water for 1 h, and then rinsed with deionized water 3e5 times. They were then soaked in 1% osmium tetroxide in deionized water for 1 h for secondary fixation. The fixed neurospheres were dehydrated through a series of ethanolewater mixtures (25%, 50%, 75%, 95%, and 100%) for 10 min each. After dehydration, the neurospheres were immersed in tetra butyl alcohol for 30 min 2e3 times at room temperature, frozen at 70 C, and then freeze-dried to remove the tetra butyl alcohol. The spheroids were mounted on top of a sample holder using carbon tape, coated with palladium alloy, and observed under a scanning electron microscope (JEOL Ltd, Tokyo, Japan). 2.7. Transmission electron microscopy (TEM) The neurospheres were fixed using a solution containing both 4% PFA and 2.5% glutaraldehyde. The samples were sliced and observed under a transmission electron microscope (Hitachi High-Technologies, Tokyo).
Fig. 1. Neural network formation among neurospheres comprised of cortical neuronal cells. A. Neuronal cells isolated from cortical region of prenatal rat were used as building blocks of neurosphere. B. (a) Schematic diagram of formation of neurospheres and their networks in the PDMS microconcave wells. (b) Neurites extension and their connection among the neurospheres. (c) Calcein AM stained neurospheres and their networks. Scale bar is 300 mm.
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2.8. Cryosectioning of neurospheres C
The neurospheres were fixed with 4% (w/v) PFA at 4 for 1 h, rinsed gently three times with PBS, and then immersed in 20% sucrose in PBS at 4 C for 1 h. Samples were embedded in Tissue-Tek O.C.T. compound (Sakura Finetek Japan, Tokyo) and kept as frozen blocks at 80 C. Frozen sections were cut to a thickness of approximately 14 mm using a cryostat and placed on Histobond adhesive glass slides (Marienfeld, Germany). Thereafter, the glass slides containing the samples were dried for 30 min and subsequently washed three times with PBS (5 min each). Subsequent procedures were conducted as described in Section 2.5 for immunofluorescence staining, beginning with incubation of samples with Block Ace. 2.9. Functional assessment The concentration of acetylcholine in the neurospheres was determined using the EnzyChrom Acetylcholine Assay Kit (EACL-100) (BioAssay), according to the manufacturer’s instructions. Briefly, the neurospheres were lysed and centrifuged and acetylcholinesterase was subsequently added to the supernatant. H2O2, the hydrolysis product of acetylcholine after catalytic reaction with acetylcholinesterase, reacts with a specific dye to form a pink colored product. The acetylcholine concentration in the neurospheres is directly proportional to the color intensity at 570 nm. The color intensity was read using a Multimode Plate Reader (PerkinElmer). 2.10. Toluidine blue staining The neurospheres were fixed using a solution containing both PFA and glutaraldehyde. A grinding machine (EXAKT cutting/grinding systems, EXAKT Advanced Technologies GmbH, Norderstedt, Germany) was used to section the neurospheres to a thickness of 1 mm and they were subsequently stained with toluidine blue. Microscopic observation was carried out using a light microscope (BX50, Olympus Optical, Osaka, Japan). 2.11. Statistical analysis All quantitative data were expressed as means standard errors of the mean. All collected data were analyzed using unpaired t-tests or one-way analyses of variance (ANOVA) with Scheffe’s post hoc test using the statistical software SPSS, Version 10.0 (SPSS, Chicago, IL, USA). P-values below 0.005 were considered statistically significant.
3. Results 3.1. Neurospheres and their neural networks formed in the PDMS microconcave wells Neural progenitor cells were isolated from prenatal rat cerebral cortical regions of brain (Fig. 1A) and thereafter cultured within
micro concave microwells of four different diameters, 300, 400, 500, and 800 mm respectively. Layer II, III, V, and VI neurons (Fig. 1Ba) aggregated and then formed uniform-sized neurospheres. Outgrowing neurites during the time passage connected neighboring spheres and formed multi-neural networks (Fig. 1Bb and Bc). SEM images and optical images of the neurospheres taken on day 10 are shown in Supplementary Figs. 1e3, where neurites can be seen to extend from somas and become entangled and tightly interconnected. Outreached neurites formed a thin layer of neural networks and on day 13, sheet-like neural network were formed (Supplementary Fig. 3C). Transcription factors, Brn2 and Satb2 (specific to layers IIeIV), CTIP2 (specific to layer V), and Tbr1 (specific to layer VI) were shown to be expressed by the cells in the neurosphere model [19] (Fig. 2), indicating that the platform can mimic the cerebral cortex with up to six organized horizontal layers [20]. To determine the size variations of the neurospheres over time, their diameters were measured after different time periods (Fig. 3A). On day 0 after seeding, the average sizes of the neurospheres were 147.79, 260.36, 382.77 and 424.33 mm in the 300, 400, 500, and 800 mm diameter microwells, respectively, indicating that the size of the neurospheres increased according to the width of the cavity. During the first 3 days, the size of neurospheres decreased but then increased at days 5e10. Cell viability in the neurospheres was assessed by using an MTT assay on days 1, 2, 3, 6, and 10 after seeding in the microwells, with measured values normalized to that of day 0 (Fig. 3B). The average cell viabilities on days 1, 2, 3, 6, and 10 were 97.65%, 96.89%, 92.73%, 82.60% and 75.08%, respectively. A live/dead assay was also performed in order to visualize the cell viability (Fig. 3C). Confocal images demonstrated a high level of green fluorescence (live cells) and a low level of red fluorescence (dead cells) within the neurospheres cultured for different time periods, validating the applicability of this platform for 3D cell culture. 3.2. Decrease in viability derived from amyloid beta exposure Two experimental groups were employed to study how neurospheres comprised of cortical neuronal cells were affected by amyloid beta. The neurospheres were cultured in the absence (Group 1)
Fig. 2. Neurospheres mimicking six horizontal layers of cortical region of brain. Neurospheres immunostained against the transcription factors, brn2, satb2, ctip2, and tbr1 of the different cortical layers. Scale bars are 100 mm.
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Fig. 3. Size distributions and viability of neurospheres over time in culture. (A) Size distributions of Group 1 neurospheres cultured in 300 mm, 400 mm, 500 mm, and 800 mm concave microwells and their change with time (*p < 0.005, **p < 0.001, two-tailed test). (n ¼ 15) (B) Analysis of the viability of Group 1 neurospheres over 10 days by MTT assay. (C) Cell viability of Group 1 neurospheres cultured for (a) 2 days, (b) 3 days, (c) 6 days, and (d) 10 days in concave microwells, analyzed by live/dead assay. Calcein AM stains live cells (green) and ethidium homodimer stains dead cells (red). Scale bars are 50 mm (*p < 0.005, **p < 0.001, two-tailed test) (n ¼ 15). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
and presence (Group 2) of 5 mM amyloid beta. A live/dead assay was performed for further assessment of viability and visualization (Fig. 4A). Group 1 can be seen to have a relatively higher level of green fluorescence (live cells) than that of Group 2. Quantification of the results of the live/dead assay shows that Group 2 had 19.07% lower viability than that of Group 1, with average viabilities of Group 1 and Group 2 being 72.19% and 58.43%, respectively (Fig. 4B). An MTT assay was also used to assess the viability of the two groups, with the values normalized to day 0 being 76.56% and 57.94% for Groups 1 and 2, respectively (Fig. 4C). The viability of the Group 2 neurospheres was 24.34% less than that of Group 1. 3.3. Morphological changes and ultrastructural features of cell apoptosis induced by amyloid beta To examine the neurospheres microscopically, toluidine blue staining was employed. Fig. 5A shows the microsectioned neurospheres stained with toluidine blue. Group 1 neurospheres had a circular shape with few apoptotic neurons while Group 2
neurospheres had a more irregular shape with rough edges and apoptotic neurons over the entire structure. Group 2 neurospheres had a substantial number of orange colored apoptotic neurons and fragmented nuclei. To analyze neurospheres immunohistochemically, they were fluorescently stained for neurofilament and tubulin (Fig. 5B). Group 1 neurospheres were seen to have smooth surfaces and maintained a stable spheroidal formation, whereas Group 2 neurospheres had rough surfaces with damaged neurites and distorted and atrophied neurospheres overall. From the staining, the ratio of cytoplasm to nuclei of the neurospheres was quantitatively analyzed using a computerized image processing program, a custom Image J (Fig. 5C). The area of cytoplasm (marked by tubulin and neurofilament) was divided by that of the nuclei (marked by DAPI), and it was seen that the ratio for Group 2 (with amyloid beta) was 34.52% lower than that of Group 1 (Fig. 5Ca). The same procedure was performed for cryosectioned neurospheres and, as expected, Group 2 had a 45. 51% lower ratio of cytoplasm to nuclei than that of Group 1 (Fig. 5Cb). A comparison of SEM images of Groups 1 and 2 showed significant differences as shown in Fig. 6, with the surfaces
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Fig. 4. Analysis of the viability of Group 1 and Group 2 neurospheres over 10 days. (A) Cell viability of (a) Group 1 and (b) Group 2 on day 10, analyzed by live/dead assay. Calcein AM stains live cells (green) and ethidium homodimer stains dead cells (red). Scale bar is 100 mm. (B) Quantification of live/dead assay (**p < 0.001, two-tailed test) (n ¼ 4) and (C) MTT assay were performed (**p < 0.001, two-tailed test) (n ¼ 7). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 5. Overall structure of Group 1 and Group 2 neurospheres. (a) Microsectioned neurospheres stained with toluidine blue. Group 1 neurosphere shows a circular shape with few apoptotic neurons. (b) Group 2 neurosphere shows an irregular shape with rough edges and apoptotic neurons over the entire structure. White arrows point to healthy neuronal cell bodies and yellow arrows point to apoptotic neuronal cell bodies. Scale bars are 50 mm. (B) Confocal microscope images of immunostained (a) 3D Group 1 neurosphere, (b) 3D Group 2 neurosphere, (c) cryosectioned Group 1 neurosphere, and (d) cryosectioned Group 2 neurosphere. (Nuclei (DAPI, blue), neurofilament (anti-neurofilament, red), and tubulin (antib3 tubulin, green)). Scale bars are 50 mm. (C) Ratio of cytoplasm to nucleus of Group 1 and Group 2. (a) three dimensional neurosphere (**p < 0.001, two-tailed test) (n ¼ 8) (b) dissectioned neurosphere (**p < 0.001, two-tailed test) (n ¼ 12). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 6. Ultrastructure of neurospheres of Group 1 and Group 2. SEM images of (A) A Group 1 neurosphere on day 10 and (B) A Group 2 neurosphere on day 10. White triangular arrows indicate neurites extended out from the microwells. Scale bar is 200 mm. Magnified SEM images of (C) Group 1 neurosphere and (D) Group 2 neurosphere. Black triangular arrows indicate soma of neurons and white triangular arrows indicate neurites of neurons. Scale bar is 50 mm. Surface of (E) a Group 1 neurosphere and (F) a Group 2 neurosphere. White triangular arrows indicate robust neurites while white arrows indicate damaged neurites. Scale bar is 5 mm.
of Group 1 neurospheres appearing smooth with some regions where extended neurites were visible. In contrast, the surfaces of the Group 2 neurospheres were observed to be rough and the cells were atrophied. It was observed that less extended neurites in Group 2 compared to Group 1. In addition, the magnified SEM image of Group 2 showed some neurite damage (Fig. 6E and F). As shown in Fig. 7A and B, Group 1 neurospheres had large number of neurons with healthy cell bodies while most of neurons in Group 2 were apoptotic cells. TEM images of Group 2 cells present typical characteristics of apoptotic neurons, with patches of condensed chromatin laid against the nuclear membrane visible. Irregular nucleus vacuoles, membrane blebbing, and cell shrinkage, which are the representative features of apoptosis, were also observed (Fig. 7C). These results demonstrate that amyloid beta induced apoptosis, which resulted in ultrastructural changes of the neurosphere [21,22]. 3.4. Decrease in synapsin II and acetylcholine To analyze the release of neurotransmitters by the neurons in the spheroids of both groups, the cells were immunostained for synapsin II. Fluorescence images of neurospheres and cryosectioned neurospheres of the two groups show that Group 2 had less synapsin II than Group 1 (Fig. 8A). Synapsin II protein was evenly distributed over the entire Group 1 neurosphere, while it was barely visible in the Group 2 neurosphere, indicating neuritic disruption. Fig. 8B shows quantification of the area ratio of synapsin II to nuclei which showed neurospheres of Group 2 had 80.10% less synapsin II than Group 1; the average ratio of synapsin II to nuclei of Group 1 and Group 2 were 0.540 and 0.1074, respectively. Cryosectioned Group 2 neurospheres had 81.52% less synapsin II than Group 1. To test the pathophysiological features of Alzheimer’s disease, the concentration of acetylcholine in both groups was measured. As illustrated in Fig. 8C, the concentration in Group 2 was 31.75% less than that in Group 1.
4. Discussion We have demonstrated the formation of uniform-sized neurospheres as a 3D neural tissue model by using concave microwells, and shown their potential for use in applications such as drug screening and toxicity testing. The neural progenitor cells in each microwell aggregated by cellecell contacts. In contrast to other cells (e.g. embryonic stem cells, hepatocytes, or beta cells), the detailed image of the spheroid in Supplementary Figs. 1e3 shows that some of the neurites extended out from the microwells, forming a thin layer of neural network with neighboring spheroids. Due to the neuronal processes, neural interconnections was easily formed in microconcave wells [23]. Especially, the smooth surface of concave wells seems to play positive roles in networking among neurospheres. Neurospheres cultured in concave microwells with different diameters for 10 days showed the same trend in size change over time, with the size decreasing over the first 3 days then gradually increasing over the following 7 (Fig. 3A). The size increase is likely due to the differentiation of the neural progenitor cells into mature neurons, which then extend axons and dendrites, increasing the volume of the neurospheres [24]. Fluorescence images of neurospheres expressing transcription factors representative of six horizontal layers of the cerebral cortex showed the potential of the 3D neurospheres to model the in vivo structure (Fig. 2). 2D culture of neural progenitor cells can also contain cerebral cortical neurons of six horizontal layers, but their network is limited to planar 2D interconnections, which is significantly different to the cerebral cortex neural network in vivo. Moreover, cellecell contacts and their topology are known to have significant roles in mechanical signaling for determining cell fates. 2D and 3D cell culture environments lead to quite different cellecell contacts, resulting in different mechanical signals passing between the cells [25e28]. This implies that neurosphere, as a 3D model, will provide more accurate results in in vitro tests, such as for drug screening or toxicity testing, that is more equivalent to the actual neural tissue in vivo.
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Fig. 7. Apoptosis derived from amyloid beta. TEM images of (A) a Group 1 neurosphere and (B) a Group 2 neurosphere on day 10. White triangular arrows indicate robust neurites while white arrows indicate damaged neurites. Black triangular arrows indicate healthy cell bodies of neurons while black arrows indicate apoptotic neurons. Scale bars are 2 mm (C) TEM images of Group 2 neurospheres on day 10 showing characteristic features of apoptotic neurons. (a) Nuclear changes after exposure to amyloid beta; the patches of condensed chromatin lie against the nuclear membrane (arrowhead) and organelles are intact. (b) The cell body becomes smaller with an irregular shaped nucleus (arrowhead) and vacuoles. (c) The nuclear membrane of the condensed nucleus becomes stuck in the nucleus which makes it fragment, the plasma membrane shows blebbing (arrowhead), and the neuron is shrunk. (d) Shrunken apoptotic neuron with few remaining attached blebs (arrowhead) and damaged neurites. Scale bars are 2 mm.
Fig. 8. Functional assessment of Group 1 and Group2 neurospheres. (A) Confocal microscope images of immunostained (a) 3D Group 1 neurosphere, (b) 3D Group 2 neurosphere, (c) cryosectioned Group 1 neurosphere, and (d) cryosectioned Group 2 neurosphere against nuclei (DAPI, blue) and synapsin (Synapsin II, red). Scale bars are 100 mm. (B) The ratio of synapsin to nuclei of Group 1 and Group 2 (upper: three dimensional neurosphere (n ¼ 3), lower: cryosectioned neurosphere (n ¼ 10)) (*p < 0.005, **p < 0.001, two-tailed test) (C) Analysis of acetylcholine concentration of Group 1 and Group 2. A higher concentration of acetylcholine was detected in Group 1 than Group 2, as expected from the in vivo features of Alzheimer’s disease (**p < 0.001, two-tailed test) (n ¼ 30). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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To date, a large number of in vitro studies of the toxicity of amyloid beta have been conducted, but only in planar and 2D environments. Here, we tested the neurotoxicity of amyloid beta in a 3D in vivo mimicking model and observed the effects not only on the surface, but also inside the neurospheres using SEM, TEM, toluidine blue staining, and fluorescence imaging. From the staining, the ratio of cytoplasm to nuclei of the neurospheres was quantitatively analyzed (Fig. 5C). The ratio for Group 2 was lower than that of Group 1, indicating that amyloid beta exposure causes atrophy of neuronal cells. These experimental observations demonstrated that amyloid beta induces the degeneration of neurites and apoptosis, even deep in the core of the neurospheres. The results demonstrate that neurospheres could be a promising tool for toxicity testing or drug screening, as a useful alternative to in vivo tests. Synapsins II associate as endogenous substrates to the surface of synaptic vesicles and act as key modulators of neurotransmitter release across the pre-synaptic membrane of axonal neurons in the nervous system. Synapsin II encodes a neuronspecific phosphoprotein that selectively binds to small synaptic vesicles in the pre-synaptic nerve terminal [29]. A significant reduction in synapsin II levels after amyloid beta exposure (Fig. 8A and B) suggests that amyloid beta disrupts neurites, leading to neuritic degeneration. Reduced synthesis of acetylcholine shown in Fig. 8C, a neurotransmitter in both the peripheral and central nervous system, due to a deficit in cholinergic neuronal cells is one of pathophysiological features of Alzheimer’s disease [2,30,31]. In contemplating future perspectives, neurospheres could be used for transplantation as a building block for enhancement of specific neurotransmitters in vivo. Increased acetylcholine secretion after transplantation or replacement of neurosphere to the neuron tissue in Alzheimer’s disease will slow down the rate of disease progression and alleviate symptom. Furthermore neurosphere model can also be applied to other neurodegenerative disease treatment. For example, Parkinson’s disease, which is induced by a loss of dopaminergic neurons in the substantia nigra, could be cured by transplantation of neurospheres made of dopaminergic neurons into the necessary region of the brain. The transplanted neurospheres would release dopamine, which is an essential neurotransmitter for normal movement, alleviating the symptoms of the disease. However, the amount of dopamine released in the brain is also very important; if too much dopamine is released there could be a loss of balance between the two hemispheres. The ability to control the size of the neurospheres by using the microwells described in this work would go some way to overcoming this problem. 5. Conclusion Our neurosphere model had a biomimetic cytoarchitecture with robust connections among the cells and maintained high viabilities during the culture period. Neural networks among neurospheres also formed in our suggested model. The formed neurospheres consisted of cortical neuronal cells that represented 6 layers of the cortical region of the brain. The model provided cell gaps of a comparable size to those present between cells in vivo, providing an excellent in vitro platform for studying the neurotoxicity of amyloid beta. As apoptosis due to amyloid beta is highly regulated by signaling molecules, the provision of biomimetic gaps between the cells is extremely important for understanding the neurotoxicity of amyloid beta. When the neurospheres were exposed to amyloid beta, some of the pathophysiological features of Alzheimer’s disease were evident, validating the applicability of the model for studying this condition. The neurosphere model could also be used for studies and a potential treatment of other neurodegeneration diseases. Simply by seeding specific neuronal cells
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targeted to the particular study into the concave microwells, it would be possible to investigate the effects of specific chemicals such as drugs, therapeutic materials, or disease agents. Furthermore, the neurospheres could be used as building blocks to produce organ-like structures. In summary, the neurospheres have great potential as a highly versatile 3D model with a number of applications, including drug screening, brain (or neuron) regeneration, and well-organized neural studies under in vivo-mimicking environments. Acknowledgment This research was supported by the Converging Research Center Program funded by the Ministry of Education, Science and Technology, Republic of Korea (2012K001360). Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.biomaterials.2013.01.038. References [1] Hardy J, Selkoe DJ. The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science 2002;297:353e6. [2] Kar S, Slowikowski SPM, Westaway D, Mount HTJ. Interactions between b-amyloid and central cholinergic neurons: implications for Alzheimer’s disease. J Psychiatry Neurosci 2004;29:427. [3] Schmechel DE, Saunders AM, Strittmatter WJ, Crain BJ, Hulette CM, Joo SH, et al. Increased amyloid beta-peptide deposition in cerebral-cortex as a consequence of apolipoprotein-E genotype in late-onset Alzheimer-disease. Proc Natl Acad Sci U S A 1993;90:9649e53. [4] Selkoe DJ. Translating cell biology into therapeutic advances in Alzheimer’s disease. Nature 1999;399:A23e31. [5] Greenberg SM, Briggs ME, Hyman BT, Kokoris GJ, Takis C, Kanter DS, et al. Apolipoprotein E ε4 is associated with the presence and earlier onset of hemorrhage in cerebral amyloid angiopathy. Stroke 1996;27:1333e7. [6] Estus S, Tucker HM, van Rooyen C, Wright S, Brigham EF, Wogulis M, et al. Aggregated amyloid-b protein induces cortical neuronal apoptosis and concomitant “apoptotic” pattern of gene induction. J Neurosci 1997;17:7736e45. [7] Chui HC, Victoroff J, Margolin D, Jagust W, Shankle R, Katzman R. Criteria for the diagnosis of ischemic vascular dementia proposed by the State of California Alzheimer’s Disease Diagnostic and Treatment Centers. Neurology 1992;42:473. [8] Arendt T, Bigl V, Arendt A, Tennstedt A. Loss of neurons in the nucleus basalis of Meynert in Alzheimer’s disease, paralysis agitans and Korsakoff’s disease. Acta Neuropathol (Berl) 1983;61:101e8. [9] Cullen DK, Wolf JA, Vernekar VN, Vukasinovic J, LaPlaca MC. Neural tissue engineering and biohybridized microsystems for neurobiological investigation in vitro (part 1). Crit Rev Biomed Eng 2011;39:201e40. [10] Grewal SS, York RD, Stork PJS. Extracellular-signal-regulated kinase signalling in neurons. Curr Opin Neurobiol 1999;9:544e53. [11] Fields RD, Stevens B. ATP: an extracellular signaling molecule between neurons and glia. Trends Neurosci 2000;23:625e33. [12] Delmas P, Brown DA. Junctional signaling microdomains: bridging the gap between the neuronal cell surface and Ca2þ stores. Neuron 2002;36:787. [13] Tamagno E, Parola M, Guglielmotto M, Santoro G, Bardini P, Marra L, et al. Multiple signaling events in amyloid beta-induced, oxidative stress-dependent neuronal apoptosis. Free Radic Biol Med 2003;35:45. [14] Awasthi A, Matsunaga Y, Yamada T. Amyloid-beta causes apoptosis of neuronal cells via caspase cascade, which can be prevented by amyloid-beta-derived short peptides. Exp Neurol 2005;196:282e9. [15] Morishima Y, Gotoh Y, Zieg J, Barrett T, Takano H, Flavell R, et al. {beta}-Amyloid induces neuronal apoptosis via a mechanism that involves the c-jun n-terminal kinase pathway and the induction of fas ligand. Sci Signal 2001;21:7551. [16] Lee SA, Choi YY, Park DY, Jang JY, Kim DS, Lee SH. Functional 3D human primary hepatocyte spheroids made by co-culturing hepatocytes from partial hepatectomy specimens and human adipose-derived stem cells. PLoS One 2012;7:e50723. [17] Wong SF, No DY, Choi YY, Kim DS, Chung BG, Lee SH. Concave microwell based size-controllable hepatosphere as a three-dimensional liver tissue model. Biomaterials 2011;32:8087e96. [18] Choi YY, Chung BG, Lee DH, Khademhosseini A, Kim JH, Lee SH. Controlledsize embryoid body formation in concave microwell arrays. Biomaterials 2010;31:4296e303. [19] Seuntjens E, Nityanandam A, Miquelajauregui A, Debruyn J, Stryjewska A, Goebbels S, et al. Sip1 regulates sequential fate decisions by feedback
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[25] Mahoney MJ, Anseth KS. Three-dimensional growth and function of neural tissue in degradable polyethylene glycol hydrogels. Biomaterials 2006;27:2265e74. [26] Patwari P, Lee RT. Mechanical control of tissue morphogenesis. Circ Res 2008; 103:234e43. [27] Chen CS, Mrksich M, Huang S, Whitesides GM, Ingber DE. Geometric control of cell life and death. Science 1997;276:1425e8. [28] Ingber DE. Tensegrity: the architectural basis of cellular mechanotransduction. Annu Rev Physiol 1997;59:575e99. [29] Bähler M, Benfenati F, Valtorta F, Greengard P. The synapsins and the regulation of synaptic function. Bioessays 2005;12:259e63. [30] Watanabe T, Yamagata N, Takasaki K, Sano K, Hayakawa K, Katsurabayashi S, et al. Decreased acetylcholine release is correlated to memory impairment in the Tg2576 transgenic mouse model of Alzheimer’s disease. Brain Res 2009;1249:222e8. [31] Xu Y, Yan J, Zhou P, Li J, Gao H, Xia Y, et al. Neurotransmitter receptors and cognitive dysfunction in Alzheimer’s disease and Parkinson’s disease. Prog Neurobiol 2012;97:1e13.
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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Size-controllable networked neurospheres as a 3D neuronal tissue model for Alzheimer’s disease studies Yoon Jung Choi a,1, JiSoo Park a,1, Sang-Hoon Lee a, b, * a b
Department of Biomedical Engineering, Korea University, Seoul 136-703, Republic of Korea KU-KIST Graduate School of Converging of Sciences & Technologies, Korea University, Seoul 136-701, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 November 2012 Accepted 5 January 2013 Available online 29 January 2013
Intensive in vitro studies on the neurotoxicity of amyloid beta have been conducted for decades; however, a three-dimensional neuronal tissue model for Alzheimer’s disease has not yet been achieved. In this study, we developed size-controllable networked neurospheres comprised of cerebral cortical neuronal cells that mimics the cytoarchitecture of the cortical region of the brain. The toxicity of amyloid beta on the neurosphere model was assessed quantitatively and qualitatively. Decreased cell viability after amyloid beta exposure was demonstrated using MTT and live/dead assays. Neurite degeneration after amyloid beta exposure was evident in both SEM and fluorescence images. Ultrastructural features of apoptotic neurons were analyzed and quantitative analysis of synapsin II concentration and an acetylcholine assay were also performed. The three-dimensional neurospheres, produced using a concave microwell array, are a potential in vitro model for Alzheimer’s disease studies. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Neurosphere 3D model Amyloid beta Alzheimer’s disease PDMS microconcave wells Neural networks
1. Introduction Alzheimer’s disease, the most common neurodegenerative disease [1], has been studied for decades, with the general consensus now being that amyloid beta protein is the cause [2]. The cerebral cortex, often denoted as gray matter, is the outermost layer of the cerebrum of the mammalian brain. As a major center of the central nervous system (CNS), the cerebral cortex is largely involved in higher brain functions such as conscious memory, awareness, thought, and language. Substantial evidence demonstrates that the cerebral cortex is the region most affected by amyloid beta [3e5]. Progressive deterioration in cognitive function, which is a remarkable pathological feature of Alzheimer’s disease that distinguishes it from other forms of senile dementia, is derived from amyloid beta-induced neuron loss [6e8]. To gain an understanding of the neuron loss induced by amyloid beta, two dimensional (2D) models with limited planar structures have been employed [2,6]. However, such models lack the unique pathophysiological characteristics present in three dimensional (3D) in vivo neuronal tissue. There is a lack of linkages between the neuronal cells in all directions, and the positioning of the cells does * Corresponding author. Department of Biomedical Engineering, Korea University, Seoul, Republic of Korea. Tel.: þ82 2 940 2773; fax: þ82 2 921 6818. E-mail address: [email protected] (S.-H. Lee). 1 These authors contributed equally. 0142-9612/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2013.01.038
not accurately represent in vivo conditions [9]. In general, the distance of the synaptic cleft between cells is increased in 2D culture models. As multiple messenger molecules that travel through the synaptic cleft transmit messages between the neuronal cells [10,11], the increased size of this impedes the signaling. Therefore, the presence of a cell gap comparable in size to that of in vivo cells is highly important for understanding how neuronal cells interact with each other [12]. Apoptosis caused by amyloid beta is highly regulated by cell signaling [13e15] and so the mimicking of the in vivo cell gap size could play a critical role in understanding the pathophysiological phenomena attributed to amyloid beta. Due to these significant requirements, adequate 3D ultrastructural models of neural tissue are extremely rare. In this study, we propose a networked neurosphere model that represents the cerebral cortex, the region of the brain most vulnerable to amyloid beta. By simply seeding neuronal progenitor cells into concave microwells fabricated in PDMS [16e18], cells were self-aggregated and formed neurospheres. These were cultured within the wells for 10 days, where the neural progenitor cells became fully differentiated and neural networks were generated. After the formation of the neurospheres, they were cultured in the presence of 5 mM amyloid beta protein solution to validate the applicability of the model to Alzheimer’s disease studies. Exposure to the amyloid beta solution led to neuronal death and the appearance of pathophysiological features of Alzheimer’s disease. Using transmission electron microscopy (TEM), ultrastructural
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features consistent with the apoptosis of neurospheres were detected. Morphological changes were analyzed immunohistochemically and investigated using scanning electron microscopy (SEM). Synapsin II and acetylcholine were assessed to validate the neurotoxicity of amyloid beta. 2. Materials and methods
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2.4. Cell proliferation assay The neurospheres cultured in Neurobasal medium for 7 days and exposed to 5 mM synthetic amyloid beta protein (1-42, SigmaeAldrich, USA) for an additional 3 days were placed into the wells of a 96-well plate (100 mL medium/well; Corning, USA) and thereafter 10 mL of MTT cell viability solution were added. The neurospheres were incubated at 37 C in a humidified 5% CO2 atmosphere for 45 min. Absorbance at 450 nm was measured using a microplate reader (SpectraMax M2; Molecular Devices, CA, USA).
2.1. Isolation of prenatal rat cortical neurons 2.5. Fixation and immunocytochemistry Prenatal rat cortical neurons were isolated from cerebral cortical regions of E-6 rat embryos (DBL, Incheon, South Korea), which consists of 6 horizontal layers. The surgical procedures used in this experiment were authorized by the Institutional Animal Care and Use Community (IACUC) of Texas Southern University. Neural progenitor cells were maintained in Neurobasal media (Gibco) supplemented with B-27 Supplement (Gibco), 0.5 mM L-glutamine, and 1% antibiotics containing 10,000 units penicillin (Gibco) and streptomycin. 2.2. Cell seeding and formation of neurospheres A schematic depiction of neurosphere formation and networks between spheres is illustrated in Fig. 1B. Neurospheres were formed by using poly(dimethylsiloxane) (PDMS)-based concave microwells with 300 mm, 400 mm, 500 mm, and 800 mm diameters, fabricated using standard soft lithography techniques. The neural progenitor cell suspension was seeded on top of the concave molds and was subsequently cultured for a total of 10 days. In Group 1, the neurons were cultured for 10 days in normal medium and in Group 2, the neurons were cultured for the first 7 days in normal medium and for the next three days in medium containing 5 mM amyloid beta. To verify that the neurospheres consisted of cells from six horizontal layers, the spheroid was immunofluorescence stained for the transcription factors Brn2 and Satb2 (specific to layers II and III), CTIP2 (specific to layer V), and Tbr1 (specific to layer VI) and we observed that the cell in spheroid were from six layers. 2.3. Cell viability test The viability of the neural cells cultured in the concave molds was assessed using a live/dead assay kit (Invitrogen, CA). 1 mL of calcein AM solution and 5 mL of ethidium homodimer-1 solution were dissolved in 2 mL of Neurobasal medium, which was then added to the cells followed by incubation at 37 C for 40 min. The stained cells were observed using a confocal laser scanning microscope (LSM 5 Exciter, Carl Zeiss Germany) and the resulting acquired images were analyzed using the ImageJ software (NIH, Bethesda, MD).
The neurospheres formed in the concave microwells were fixed with 4% paraformaldehyde (PFA) for 20 min at 4 C and then washed with 0.1% BSA in phosphate buffered saline (PBS). The cells were incubated in 0.1% Triton X-100 in PBS for 20 min at room temperature and then washed with 0.1% BSA in PBS. The cells were blocked for 30 min at 4 C to reduce non-specific protein adsorption and then probed with primary antibodies: neurofilament H (Millipore), beta III tubulin (Invitrogen), TBR1 (Abcam), Ctip (Abcam), Tuj1 (Covance), Satb2 (Abcam), and synapsin (Abcam) overnight at 4 C. Cells were rinsed with 0.1% BSA in PBS, and incubated with the secondary antibody (Alexa Fluor 488 or 594; Invitrogen) for 90 min at 4 C. Fluorescence images were collected using a confocal laser scanning microscope (LSM 5 Exciter, Carl Zeiss, Germany) after counterstaining the cell nuclei with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI; Invitrogen). 2.6. Scanning electron microscopy (SEM) The neurospheres were fixed with 2.5% glutaraldehyde in deionized water for 1 h, and then rinsed with deionized water 3e5 times. They were then soaked in 1% osmium tetroxide in deionized water for 1 h for secondary fixation. The fixed neurospheres were dehydrated through a series of ethanolewater mixtures (25%, 50%, 75%, 95%, and 100%) for 10 min each. After dehydration, the neurospheres were immersed in tetra butyl alcohol for 30 min 2e3 times at room temperature, frozen at 70 C, and then freeze-dried to remove the tetra butyl alcohol. The spheroids were mounted on top of a sample holder using carbon tape, coated with palladium alloy, and observed under a scanning electron microscope (JEOL Ltd, Tokyo, Japan). 2.7. Transmission electron microscopy (TEM) The neurospheres were fixed using a solution containing both 4% PFA and 2.5% glutaraldehyde. The samples were sliced and observed under a transmission electron microscope (Hitachi High-Technologies, Tokyo).
Fig. 1. Neural network formation among neurospheres comprised of cortical neuronal cells. A. Neuronal cells isolated from cortical region of prenatal rat were used as building blocks of neurosphere. B. (a) Schematic diagram of formation of neurospheres and their networks in the PDMS microconcave wells. (b) Neurites extension and their connection among the neurospheres. (c) Calcein AM stained neurospheres and their networks. Scale bar is 300 mm.
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2.8. Cryosectioning of neurospheres C
The neurospheres were fixed with 4% (w/v) PFA at 4 for 1 h, rinsed gently three times with PBS, and then immersed in 20% sucrose in PBS at 4 C for 1 h. Samples were embedded in Tissue-Tek O.C.T. compound (Sakura Finetek Japan, Tokyo) and kept as frozen blocks at 80 C. Frozen sections were cut to a thickness of approximately 14 mm using a cryostat and placed on Histobond adhesive glass slides (Marienfeld, Germany). Thereafter, the glass slides containing the samples were dried for 30 min and subsequently washed three times with PBS (5 min each). Subsequent procedures were conducted as described in Section 2.5 for immunofluorescence staining, beginning with incubation of samples with Block Ace. 2.9. Functional assessment The concentration of acetylcholine in the neurospheres was determined using the EnzyChrom Acetylcholine Assay Kit (EACL-100) (BioAssay), according to the manufacturer’s instructions. Briefly, the neurospheres were lysed and centrifuged and acetylcholinesterase was subsequently added to the supernatant. H2O2, the hydrolysis product of acetylcholine after catalytic reaction with acetylcholinesterase, reacts with a specific dye to form a pink colored product. The acetylcholine concentration in the neurospheres is directly proportional to the color intensity at 570 nm. The color intensity was read using a Multimode Plate Reader (PerkinElmer). 2.10. Toluidine blue staining The neurospheres were fixed using a solution containing both PFA and glutaraldehyde. A grinding machine (EXAKT cutting/grinding systems, EXAKT Advanced Technologies GmbH, Norderstedt, Germany) was used to section the neurospheres to a thickness of 1 mm and they were subsequently stained with toluidine blue. Microscopic observation was carried out using a light microscope (BX50, Olympus Optical, Osaka, Japan). 2.11. Statistical analysis All quantitative data were expressed as means standard errors of the mean. All collected data were analyzed using unpaired t-tests or one-way analyses of variance (ANOVA) with Scheffe’s post hoc test using the statistical software SPSS, Version 10.0 (SPSS, Chicago, IL, USA). P-values below 0.005 were considered statistically significant.
3. Results 3.1. Neurospheres and their neural networks formed in the PDMS microconcave wells Neural progenitor cells were isolated from prenatal rat cerebral cortical regions of brain (Fig. 1A) and thereafter cultured within
micro concave microwells of four different diameters, 300, 400, 500, and 800 mm respectively. Layer II, III, V, and VI neurons (Fig. 1Ba) aggregated and then formed uniform-sized neurospheres. Outgrowing neurites during the time passage connected neighboring spheres and formed multi-neural networks (Fig. 1Bb and Bc). SEM images and optical images of the neurospheres taken on day 10 are shown in Supplementary Figs. 1e3, where neurites can be seen to extend from somas and become entangled and tightly interconnected. Outreached neurites formed a thin layer of neural networks and on day 13, sheet-like neural network were formed (Supplementary Fig. 3C). Transcription factors, Brn2 and Satb2 (specific to layers IIeIV), CTIP2 (specific to layer V), and Tbr1 (specific to layer VI) were shown to be expressed by the cells in the neurosphere model [19] (Fig. 2), indicating that the platform can mimic the cerebral cortex with up to six organized horizontal layers [20]. To determine the size variations of the neurospheres over time, their diameters were measured after different time periods (Fig. 3A). On day 0 after seeding, the average sizes of the neurospheres were 147.79, 260.36, 382.77 and 424.33 mm in the 300, 400, 500, and 800 mm diameter microwells, respectively, indicating that the size of the neurospheres increased according to the width of the cavity. During the first 3 days, the size of neurospheres decreased but then increased at days 5e10. Cell viability in the neurospheres was assessed by using an MTT assay on days 1, 2, 3, 6, and 10 after seeding in the microwells, with measured values normalized to that of day 0 (Fig. 3B). The average cell viabilities on days 1, 2, 3, 6, and 10 were 97.65%, 96.89%, 92.73%, 82.60% and 75.08%, respectively. A live/dead assay was also performed in order to visualize the cell viability (Fig. 3C). Confocal images demonstrated a high level of green fluorescence (live cells) and a low level of red fluorescence (dead cells) within the neurospheres cultured for different time periods, validating the applicability of this platform for 3D cell culture. 3.2. Decrease in viability derived from amyloid beta exposure Two experimental groups were employed to study how neurospheres comprised of cortical neuronal cells were affected by amyloid beta. The neurospheres were cultured in the absence (Group 1)
Fig. 2. Neurospheres mimicking six horizontal layers of cortical region of brain. Neurospheres immunostained against the transcription factors, brn2, satb2, ctip2, and tbr1 of the different cortical layers. Scale bars are 100 mm.
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Fig. 3. Size distributions and viability of neurospheres over time in culture. (A) Size distributions of Group 1 neurospheres cultured in 300 mm, 400 mm, 500 mm, and 800 mm concave microwells and their change with time (*p < 0.005, **p < 0.001, two-tailed test). (n ¼ 15) (B) Analysis of the viability of Group 1 neurospheres over 10 days by MTT assay. (C) Cell viability of Group 1 neurospheres cultured for (a) 2 days, (b) 3 days, (c) 6 days, and (d) 10 days in concave microwells, analyzed by live/dead assay. Calcein AM stains live cells (green) and ethidium homodimer stains dead cells (red). Scale bars are 50 mm (*p < 0.005, **p < 0.001, two-tailed test) (n ¼ 15). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
and presence (Group 2) of 5 mM amyloid beta. A live/dead assay was performed for further assessment of viability and visualization (Fig. 4A). Group 1 can be seen to have a relatively higher level of green fluorescence (live cells) than that of Group 2. Quantification of the results of the live/dead assay shows that Group 2 had 19.07% lower viability than that of Group 1, with average viabilities of Group 1 and Group 2 being 72.19% and 58.43%, respectively (Fig. 4B). An MTT assay was also used to assess the viability of the two groups, with the values normalized to day 0 being 76.56% and 57.94% for Groups 1 and 2, respectively (Fig. 4C). The viability of the Group 2 neurospheres was 24.34% less than that of Group 1. 3.3. Morphological changes and ultrastructural features of cell apoptosis induced by amyloid beta To examine the neurospheres microscopically, toluidine blue staining was employed. Fig. 5A shows the microsectioned neurospheres stained with toluidine blue. Group 1 neurospheres had a circular shape with few apoptotic neurons while Group 2
neurospheres had a more irregular shape with rough edges and apoptotic neurons over the entire structure. Group 2 neurospheres had a substantial number of orange colored apoptotic neurons and fragmented nuclei. To analyze neurospheres immunohistochemically, they were fluorescently stained for neurofilament and tubulin (Fig. 5B). Group 1 neurospheres were seen to have smooth surfaces and maintained a stable spheroidal formation, whereas Group 2 neurospheres had rough surfaces with damaged neurites and distorted and atrophied neurospheres overall. From the staining, the ratio of cytoplasm to nuclei of the neurospheres was quantitatively analyzed using a computerized image processing program, a custom Image J (Fig. 5C). The area of cytoplasm (marked by tubulin and neurofilament) was divided by that of the nuclei (marked by DAPI), and it was seen that the ratio for Group 2 (with amyloid beta) was 34.52% lower than that of Group 1 (Fig. 5Ca). The same procedure was performed for cryosectioned neurospheres and, as expected, Group 2 had a 45. 51% lower ratio of cytoplasm to nuclei than that of Group 1 (Fig. 5Cb). A comparison of SEM images of Groups 1 and 2 showed significant differences as shown in Fig. 6, with the surfaces
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Fig. 4. Analysis of the viability of Group 1 and Group 2 neurospheres over 10 days. (A) Cell viability of (a) Group 1 and (b) Group 2 on day 10, analyzed by live/dead assay. Calcein AM stains live cells (green) and ethidium homodimer stains dead cells (red). Scale bar is 100 mm. (B) Quantification of live/dead assay (**p < 0.001, two-tailed test) (n ¼ 4) and (C) MTT assay were performed (**p < 0.001, two-tailed test) (n ¼ 7). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 5. Overall structure of Group 1 and Group 2 neurospheres. (a) Microsectioned neurospheres stained with toluidine blue. Group 1 neurosphere shows a circular shape with few apoptotic neurons. (b) Group 2 neurosphere shows an irregular shape with rough edges and apoptotic neurons over the entire structure. White arrows point to healthy neuronal cell bodies and yellow arrows point to apoptotic neuronal cell bodies. Scale bars are 50 mm. (B) Confocal microscope images of immunostained (a) 3D Group 1 neurosphere, (b) 3D Group 2 neurosphere, (c) cryosectioned Group 1 neurosphere, and (d) cryosectioned Group 2 neurosphere. (Nuclei (DAPI, blue), neurofilament (anti-neurofilament, red), and tubulin (antib3 tubulin, green)). Scale bars are 50 mm. (C) Ratio of cytoplasm to nucleus of Group 1 and Group 2. (a) three dimensional neurosphere (**p < 0.001, two-tailed test) (n ¼ 8) (b) dissectioned neurosphere (**p < 0.001, two-tailed test) (n ¼ 12). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 6. Ultrastructure of neurospheres of Group 1 and Group 2. SEM images of (A) A Group 1 neurosphere on day 10 and (B) A Group 2 neurosphere on day 10. White triangular arrows indicate neurites extended out from the microwells. Scale bar is 200 mm. Magnified SEM images of (C) Group 1 neurosphere and (D) Group 2 neurosphere. Black triangular arrows indicate soma of neurons and white triangular arrows indicate neurites of neurons. Scale bar is 50 mm. Surface of (E) a Group 1 neurosphere and (F) a Group 2 neurosphere. White triangular arrows indicate robust neurites while white arrows indicate damaged neurites. Scale bar is 5 mm.
of Group 1 neurospheres appearing smooth with some regions where extended neurites were visible. In contrast, the surfaces of the Group 2 neurospheres were observed to be rough and the cells were atrophied. It was observed that less extended neurites in Group 2 compared to Group 1. In addition, the magnified SEM image of Group 2 showed some neurite damage (Fig. 6E and F). As shown in Fig. 7A and B, Group 1 neurospheres had large number of neurons with healthy cell bodies while most of neurons in Group 2 were apoptotic cells. TEM images of Group 2 cells present typical characteristics of apoptotic neurons, with patches of condensed chromatin laid against the nuclear membrane visible. Irregular nucleus vacuoles, membrane blebbing, and cell shrinkage, which are the representative features of apoptosis, were also observed (Fig. 7C). These results demonstrate that amyloid beta induced apoptosis, which resulted in ultrastructural changes of the neurosphere [21,22]. 3.4. Decrease in synapsin II and acetylcholine To analyze the release of neurotransmitters by the neurons in the spheroids of both groups, the cells were immunostained for synapsin II. Fluorescence images of neurospheres and cryosectioned neurospheres of the two groups show that Group 2 had less synapsin II than Group 1 (Fig. 8A). Synapsin II protein was evenly distributed over the entire Group 1 neurosphere, while it was barely visible in the Group 2 neurosphere, indicating neuritic disruption. Fig. 8B shows quantification of the area ratio of synapsin II to nuclei which showed neurospheres of Group 2 had 80.10% less synapsin II than Group 1; the average ratio of synapsin II to nuclei of Group 1 and Group 2 were 0.540 and 0.1074, respectively. Cryosectioned Group 2 neurospheres had 81.52% less synapsin II than Group 1. To test the pathophysiological features of Alzheimer’s disease, the concentration of acetylcholine in both groups was measured. As illustrated in Fig. 8C, the concentration in Group 2 was 31.75% less than that in Group 1.
4. Discussion We have demonstrated the formation of uniform-sized neurospheres as a 3D neural tissue model by using concave microwells, and shown their potential for use in applications such as drug screening and toxicity testing. The neural progenitor cells in each microwell aggregated by cellecell contacts. In contrast to other cells (e.g. embryonic stem cells, hepatocytes, or beta cells), the detailed image of the spheroid in Supplementary Figs. 1e3 shows that some of the neurites extended out from the microwells, forming a thin layer of neural network with neighboring spheroids. Due to the neuronal processes, neural interconnections was easily formed in microconcave wells [23]. Especially, the smooth surface of concave wells seems to play positive roles in networking among neurospheres. Neurospheres cultured in concave microwells with different diameters for 10 days showed the same trend in size change over time, with the size decreasing over the first 3 days then gradually increasing over the following 7 (Fig. 3A). The size increase is likely due to the differentiation of the neural progenitor cells into mature neurons, which then extend axons and dendrites, increasing the volume of the neurospheres [24]. Fluorescence images of neurospheres expressing transcription factors representative of six horizontal layers of the cerebral cortex showed the potential of the 3D neurospheres to model the in vivo structure (Fig. 2). 2D culture of neural progenitor cells can also contain cerebral cortical neurons of six horizontal layers, but their network is limited to planar 2D interconnections, which is significantly different to the cerebral cortex neural network in vivo. Moreover, cellecell contacts and their topology are known to have significant roles in mechanical signaling for determining cell fates. 2D and 3D cell culture environments lead to quite different cellecell contacts, resulting in different mechanical signals passing between the cells [25e28]. This implies that neurosphere, as a 3D model, will provide more accurate results in in vitro tests, such as for drug screening or toxicity testing, that is more equivalent to the actual neural tissue in vivo.
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Fig. 7. Apoptosis derived from amyloid beta. TEM images of (A) a Group 1 neurosphere and (B) a Group 2 neurosphere on day 10. White triangular arrows indicate robust neurites while white arrows indicate damaged neurites. Black triangular arrows indicate healthy cell bodies of neurons while black arrows indicate apoptotic neurons. Scale bars are 2 mm (C) TEM images of Group 2 neurospheres on day 10 showing characteristic features of apoptotic neurons. (a) Nuclear changes after exposure to amyloid beta; the patches of condensed chromatin lie against the nuclear membrane (arrowhead) and organelles are intact. (b) The cell body becomes smaller with an irregular shaped nucleus (arrowhead) and vacuoles. (c) The nuclear membrane of the condensed nucleus becomes stuck in the nucleus which makes it fragment, the plasma membrane shows blebbing (arrowhead), and the neuron is shrunk. (d) Shrunken apoptotic neuron with few remaining attached blebs (arrowhead) and damaged neurites. Scale bars are 2 mm.
Fig. 8. Functional assessment of Group 1 and Group2 neurospheres. (A) Confocal microscope images of immunostained (a) 3D Group 1 neurosphere, (b) 3D Group 2 neurosphere, (c) cryosectioned Group 1 neurosphere, and (d) cryosectioned Group 2 neurosphere against nuclei (DAPI, blue) and synapsin (Synapsin II, red). Scale bars are 100 mm. (B) The ratio of synapsin to nuclei of Group 1 and Group 2 (upper: three dimensional neurosphere (n ¼ 3), lower: cryosectioned neurosphere (n ¼ 10)) (*p < 0.005, **p < 0.001, two-tailed test) (C) Analysis of acetylcholine concentration of Group 1 and Group 2. A higher concentration of acetylcholine was detected in Group 1 than Group 2, as expected from the in vivo features of Alzheimer’s disease (**p < 0.001, two-tailed test) (n ¼ 30). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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To date, a large number of in vitro studies of the toxicity of amyloid beta have been conducted, but only in planar and 2D environments. Here, we tested the neurotoxicity of amyloid beta in a 3D in vivo mimicking model and observed the effects not only on the surface, but also inside the neurospheres using SEM, TEM, toluidine blue staining, and fluorescence imaging. From the staining, the ratio of cytoplasm to nuclei of the neurospheres was quantitatively analyzed (Fig. 5C). The ratio for Group 2 was lower than that of Group 1, indicating that amyloid beta exposure causes atrophy of neuronal cells. These experimental observations demonstrated that amyloid beta induces the degeneration of neurites and apoptosis, even deep in the core of the neurospheres. The results demonstrate that neurospheres could be a promising tool for toxicity testing or drug screening, as a useful alternative to in vivo tests. Synapsins II associate as endogenous substrates to the surface of synaptic vesicles and act as key modulators of neurotransmitter release across the pre-synaptic membrane of axonal neurons in the nervous system. Synapsin II encodes a neuronspecific phosphoprotein that selectively binds to small synaptic vesicles in the pre-synaptic nerve terminal [29]. A significant reduction in synapsin II levels after amyloid beta exposure (Fig. 8A and B) suggests that amyloid beta disrupts neurites, leading to neuritic degeneration. Reduced synthesis of acetylcholine shown in Fig. 8C, a neurotransmitter in both the peripheral and central nervous system, due to a deficit in cholinergic neuronal cells is one of pathophysiological features of Alzheimer’s disease [2,30,31]. In contemplating future perspectives, neurospheres could be used for transplantation as a building block for enhancement of specific neurotransmitters in vivo. Increased acetylcholine secretion after transplantation or replacement of neurosphere to the neuron tissue in Alzheimer’s disease will slow down the rate of disease progression and alleviate symptom. Furthermore neurosphere model can also be applied to other neurodegenerative disease treatment. For example, Parkinson’s disease, which is induced by a loss of dopaminergic neurons in the substantia nigra, could be cured by transplantation of neurospheres made of dopaminergic neurons into the necessary region of the brain. The transplanted neurospheres would release dopamine, which is an essential neurotransmitter for normal movement, alleviating the symptoms of the disease. However, the amount of dopamine released in the brain is also very important; if too much dopamine is released there could be a loss of balance between the two hemispheres. The ability to control the size of the neurospheres by using the microwells described in this work would go some way to overcoming this problem. 5. Conclusion Our neurosphere model had a biomimetic cytoarchitecture with robust connections among the cells and maintained high viabilities during the culture period. Neural networks among neurospheres also formed in our suggested model. The formed neurospheres consisted of cortical neuronal cells that represented 6 layers of the cortical region of the brain. The model provided cell gaps of a comparable size to those present between cells in vivo, providing an excellent in vitro platform for studying the neurotoxicity of amyloid beta. As apoptosis due to amyloid beta is highly regulated by signaling molecules, the provision of biomimetic gaps between the cells is extremely important for understanding the neurotoxicity of amyloid beta. When the neurospheres were exposed to amyloid beta, some of the pathophysiological features of Alzheimer’s disease were evident, validating the applicability of the model for studying this condition. The neurosphere model could also be used for studies and a potential treatment of other neurodegeneration diseases. Simply by seeding specific neuronal cells
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targeted to the particular study into the concave microwells, it would be possible to investigate the effects of specific chemicals such as drugs, therapeutic materials, or disease agents. Furthermore, the neurospheres could be used as building blocks to produce organ-like structures. In summary, the neurospheres have great potential as a highly versatile 3D model with a number of applications, including drug screening, brain (or neuron) regeneration, and well-organized neural studies under in vivo-mimicking environments. Acknowledgment This research was supported by the Converging Research Center Program funded by the Ministry of Education, Science and Technology, Republic of Korea (2012K001360). Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.biomaterials.2013.01.038. References [1] Hardy J, Selkoe DJ. The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science 2002;297:353e6. [2] Kar S, Slowikowski SPM, Westaway D, Mount HTJ. Interactions between b-amyloid and central cholinergic neurons: implications for Alzheimer’s disease. J Psychiatry Neurosci 2004;29:427. [3] Schmechel DE, Saunders AM, Strittmatter WJ, Crain BJ, Hulette CM, Joo SH, et al. Increased amyloid beta-peptide deposition in cerebral-cortex as a consequence of apolipoprotein-E genotype in late-onset Alzheimer-disease. Proc Natl Acad Sci U S A 1993;90:9649e53. [4] Selkoe DJ. Translating cell biology into therapeutic advances in Alzheimer’s disease. Nature 1999;399:A23e31. [5] Greenberg SM, Briggs ME, Hyman BT, Kokoris GJ, Takis C, Kanter DS, et al. Apolipoprotein E ε4 is associated with the presence and earlier onset of hemorrhage in cerebral amyloid angiopathy. Stroke 1996;27:1333e7. [6] Estus S, Tucker HM, van Rooyen C, Wright S, Brigham EF, Wogulis M, et al. Aggregated amyloid-b protein induces cortical neuronal apoptosis and concomitant “apoptotic” pattern of gene induction. J Neurosci 1997;17:7736e45. [7] Chui HC, Victoroff J, Margolin D, Jagust W, Shankle R, Katzman R. Criteria for the diagnosis of ischemic vascular dementia proposed by the State of California Alzheimer’s Disease Diagnostic and Treatment Centers. Neurology 1992;42:473. [8] Arendt T, Bigl V, Arendt A, Tennstedt A. Loss of neurons in the nucleus basalis of Meynert in Alzheimer’s disease, paralysis agitans and Korsakoff’s disease. Acta Neuropathol (Berl) 1983;61:101e8. [9] Cullen DK, Wolf JA, Vernekar VN, Vukasinovic J, LaPlaca MC. Neural tissue engineering and biohybridized microsystems for neurobiological investigation in vitro (part 1). Crit Rev Biomed Eng 2011;39:201e40. [10] Grewal SS, York RD, Stork PJS. Extracellular-signal-regulated kinase signalling in neurons. Curr Opin Neurobiol 1999;9:544e53. [11] Fields RD, Stevens B. ATP: an extracellular signaling molecule between neurons and glia. Trends Neurosci 2000;23:625e33. [12] Delmas P, Brown DA. Junctional signaling microdomains: bridging the gap between the neuronal cell surface and Ca2þ stores. Neuron 2002;36:787. [13] Tamagno E, Parola M, Guglielmotto M, Santoro G, Bardini P, Marra L, et al. Multiple signaling events in amyloid beta-induced, oxidative stress-dependent neuronal apoptosis. Free Radic Biol Med 2003;35:45. [14] Awasthi A, Matsunaga Y, Yamada T. Amyloid-beta causes apoptosis of neuronal cells via caspase cascade, which can be prevented by amyloid-beta-derived short peptides. Exp Neurol 2005;196:282e9. [15] Morishima Y, Gotoh Y, Zieg J, Barrett T, Takano H, Flavell R, et al. {beta}-Amyloid induces neuronal apoptosis via a mechanism that involves the c-jun n-terminal kinase pathway and the induction of fas ligand. Sci Signal 2001;21:7551. [16] Lee SA, Choi YY, Park DY, Jang JY, Kim DS, Lee SH. Functional 3D human primary hepatocyte spheroids made by co-culturing hepatocytes from partial hepatectomy specimens and human adipose-derived stem cells. PLoS One 2012;7:e50723. [17] Wong SF, No DY, Choi YY, Kim DS, Chung BG, Lee SH. Concave microwell based size-controllable hepatosphere as a three-dimensional liver tissue model. Biomaterials 2011;32:8087e96. [18] Choi YY, Chung BG, Lee DH, Khademhosseini A, Kim JH, Lee SH. Controlledsize embryoid body formation in concave microwell arrays. Biomaterials 2010;31:4296e303. [19] Seuntjens E, Nityanandam A, Miquelajauregui A, Debruyn J, Stryjewska A, Goebbels S, et al. Sip1 regulates sequential fate decisions by feedback
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