Magnetic nanoparticles
Commercial green fluorescent MNPs produced by Chemicell (4415 nano-screenMAG-ARA, Berlin, Germany) were used. These MNPs have a magnetite core covered by a lipophilic green fluorescent dye and a polysaccharide matrix of glucuronic acid, a derivate of glucose, for additional functionalization. Because of the carboxyl group of the coating polymer, the particles become anionic in solution. Zeta potential measurements of these MNPs suspended in cell culture media revealed a negative charge on the particles; this finding was also observed in the presence of serum supplement. MNPs were diluted the day before use with three different serum-supplemented media: For primary cell cultures MNPs were diluted in Dulbecco's modified Eagle medium (DMEM) medium with 10% fetal calf serum (FCS), 6 g/l D-glucose and 1% penicillin/streptomycin (pen/strep), resulting in a hydrodynamic MNP diameter of 190 nm (± 2 nm) and a zeta potential of -6.1 mV. PC12 cells were cultured with MNPs diluted in Roswell Park Memorial Institute (RPMI) medium with 10% FCS, 50 ng/ml NGF (Sigma, St. Louis, USA), 2 mM L-glutamine and 1% pen/strep, yielding a hydrodynamic MNP diameter of 199 nm (± 1 nm) and a zeta potential of -40.0 mV. For the organotypic co-cultures, MNPs were diluted in 50% Eagle's minimal essential medium (MEM), 25% Hank's balanced salt solution (HBSS), 25% FCS, 33.3 mM D-glucose, 1% pen/strep and 100 ng/ml recombinant rat glia cell line-derived neurotrophic factor (GDNF, R&D systems, Minneapolis, USA) giving a hydrodynamic diameter of 185 nm (± 1 nm) and a zeta potential of -12.8 mV.
Animal care
All animal experiments were carried out in accordance with the guidelines of the German Animal Welfare Act. The study was approved by the Animal Care and Use Committees of Saxony-Anhalt. Formal approval to conduct the experiments described was obtained from the animal subjects review board of our institution and can be provided upon request. All efforts were made to minimize the number of animals used and their suffering.
Cerebellar cultures
5-8 day neonatal rats were decapitated and the cerebellum was separated. Meninges were removed in serum-free DMEM containing 6 g/l D-glucose and 1% pen/strep. Subsequently, the cerebelli were mechanically dissociated (18 and 23 gauge needles) in serum-free DMEM and centrifuged at 1500 rpm for 5 min. Cells were resuspended in serum-supplemented DMEM with 10% FCS, 6 g/l D-glucose, 1% pen/strep, counted and seeded for immunocytochemistry in 12-well plates with 1*106 cells per well on poly-D-lysine-coated coverslips (18 or 15 mm). For viability assay, cerebellar cells were seeded in poly-D-lysine-coated Petri dishes (35 mm) with a cell density of 2.5*105 cells per dish.
Cells were incubated at 37°C in a humidified 6% CO2 atmosphere and medium was changed 24 h after preparation to remove cell debris.
For the uptake quantification based on immunocytochemistry, the medium was replaced with MNP medium (50 μg/ml MNPs) at different time points depending on cell type and different proliferation patterns of the various cell types. For example, the number of neurons decreases over the first week in cerebellar cultures [23, 24] and oligodendroglia differentiate from precursor cells after more than one week [25–27]. Quantification of our mixed cerebellar cultures respective the culture composition revealed that they are composed of 45.5% (± 16.7%) astroglia, 32.1% (± 19.2%) microglia, 24.3% (± 14.6%) neurons and no oligodendroglia on day in vitro (DIV) 5. On DIV 7, they contain 35.2% (± 17.3%) microglia, 32.5% (± 17.2%) astroglia, 13.7% (± 10.4%) neurons and 2.1% (± 3.4%) oligodendroglia. On DIV 13, they are composed of 48.4% (± 28.4%) microglia, 38.3% (± 24.8%) astroglia, 1.5% (± 2.8%) neurons and 3.0% (± 4.1%) oligodendroglia.
To quantify the uptake of neurons, MNP medium was added on DIV 4. MNP uptake in astroglia and microglia was analyzed on DIV 7 and in oligodendroglia on DIV 13. Simultaneously, MNP-free medium was added to control cells.
For viability assay 10, 50 or 100 μg/ml MNP-containing medium was added to the cultures on DIV 6. At the same time, MNP-free medium was added to control cells. Cells were incubated for 24 h, washed with phosphate buffered saline (PBS) and used further for immunocytochemistry or for viability assay.
Schwann cell/fibroblast cultures
5-7 day old rats were decapitated, the spinal ganglia were taken out and collected in a solution of 2 ml serum-free DMEM (6 g/l D-glucose, 1% pen/strep), 40 μl collagenase (0.05%), 100 μl hyaluronidase (0.1%) and 2 ml dispase II (1.25 U/ml). In this enzyme-supplemented medium the isolated spinal ganglia were incubated at 37°C in a 6% CO2 humidified atmosphere for ca. 3.5 h to dissociate the cells enzymatically. The cells were additionally dissociated mechanically using injection needles (18 and 23 gauge needles) and centrifuged at 1500 rpm for 5 min. Afterwards the cells were resuspended in serum-supplemented medium (DMEM, 10% FCS, 6 g/l D-glucose, 1% pen/strep).
For immunocytochemistry, 18 or 15 mm coverslips in 12-well plates were coated with laminin (0.05 mg/ml) before seeding 5*104 cells per well.
For viability assay cells were seeded on laminin-coated Petri dishes (35 mm) with a cell density of 2.5*105 cells per dish.
Cells were incubated at 37°C in a humidified 6% CO2 atmosphere and the medium was changed 24 h after preparation to remove cell debris. Culture composition was quantified and revealed 62.7% (± 17.0%) Schwann cells and 37.3% (± 17.0%) fibroblasts on DIV 7. For immunocytochemistry and viability assay, the medium was replaced with MNP medium on DIV 6. MNP medium contained 50 μg/ml MNPs for immunocytochemistry and 10, 50 and 100 μg/ml MNPs for the viability assay. MNP-free medium was added to control cells. After 24 h, cells were washed with PBS and used further.
Cell line
PC12 cells were seeded on poly-D-lysine coated culture flasks and cultured with RPMI medium supplemented with 10% FCS, 2 mM L-glutamine and pen/strep. After 24 h, cells were differentiated with 50 ng/ml NGF for 6 days. Differentiated cells were seeded afterwards in 12-well plates with 1.25*105 cells per well on poly-D-lysine coated coverslips and kept in differentiation medium for 3 days before the medium was replaced with differentiation medium supplemented with 50 μg/ml MNPs. After 24 h, cells were washed with PBS and fixed for immunocytochemistry.
Organotypic spinal cord co-cultures
Organotypic spinal cords co-cultured with peripheral nerve grafts were prepared according to Vyas et al. [28] with slight modifications. For the cultures, neonatal rats of postnatal day 3-4 were used. Rats were decapitated, the spinal cords excised and roots and meninges removed in dissection buffer (HBSS, 3.4 mM NaHCO3, 10 mM HEPES, 33.3 mM D-glucose, 5.8 mM MgSO4, 0.03% bovine serum albumin (BSA), 1% pen/strep).
The lumbar part of the spinal cord was cut into 350 μm transverse sections with a McIlwain tissue chopper (Mickle Laboratory Engineering, Gomshall, UK). About 10 usable sections could be obtained from one lumbar spinal cord part. Three slices were cultured on one Millicell membrane insert (Millipore, Billerica, USA) placed in 6-well plates. Each well contained 1 ml of medium composed of 50% MEM, 25% HBSS, 25% FCS, 33.3 mM D-glucose, 1% pen/strep and 100 ng/ml GDNF to keep the motor neurons in the culture alive.
To enhance the motor neuronal survival and to guide sprouting neurites, a co-culture of the spinal cord slice with a peripheral nerve graft as a reconstructed ventral root was chosen.
As peripheral nerve graft, pieces of ulnar and median nerves were harvested from the same animals and one graft was opposed to the ventral surface of each spinal cord slice.
To check the contribution of MNPs in the tissue slices, 100 μg/ml MNPs were added to the medium for the whole culture time. Co-cultures were incubated at 37°C in humidified 6% CO2 atmosphere. The medium was changed 24 h after preparation and afterwards every second day.
Immunocytochemistry of cell cultures
Staining procedure
After 24 h MNP incubation, cells were washed once with PBS to remove free-floating MNPs and fixed for 30 min with 4% paraformaldehyde (PFA). Fixing was followed by three times washing with PBS. Unspecific binding sides were blocked for 1 h with 10% FCS, 0.3% Triton-X in PBS. Subsequently, cells were incubated with the primary antibody overnight at 4°C. Cell type specific primary antibodies were chosen: primary neurons were stained with mouse monoclonal anti-microtubule associated protein-2 (MAP2, 1:1000, Sternberger Monoclonals, Baltimore, USA), PC12 cells with rabbit polyclonal anti-ß-III-tubulin (1:1000, Covance, Princeton, USA), microglial cells with mouse monoclonal anti-CD11b/c (1:400, BD Pharmigen, Franklin Lakes, USA) or with rabbit polyclonal anti- ionized calcium binding adaptor molecule 1 (IBA-1, 1:1000, Wako Pure Chemicals Industries, Osaka, Japan), astroglia with rabbit polyclonal anti- glial fibrillary acidic protein (GFAP, 1:500, Progen, Heidelberg, Germany) and oligodendroglia with mouse monoclonal anti-galactocerebroside (1:250, Chemicon, Billerica, USA). Schwann cells were stained with rabbit polyclonal anti-S100 (1:200, DAKO, Glostrup, Denmark) and fibroblasts with mouse monoclonal anti-fibronectin (1:200, Abcam, Cambridge, UK) antibody. Primary antibodies were diluted in PBS containing 0.3% Triton-X and 1% FCS.
After incubation overnight, cells were washed three times with PBS and incubated for 3 h with an anti-rabbit or anti-mouse Alexa Fluor 546 Dye secondary antibody (Invitrogen, Carlsbad, USA) diluted 1:200 in PBS. Cells were washed again three times with PBS and cell nuclei were counterstained with 4', 6-diamidino-2-phenylindole (DAPI, 1 μg/ml diluted in PBS, Roche Applied Science, Indianapolis, USA). After washing again with PBS, coverslips were embedded with Immu-Mount (Thermo Scientific, Waltham, USA).
Quantification
To quantify the cellular uptake cell type specific antibodies were used to stain cell lines followed by counterstaining all nuclei with DAPI. Control coverslips (4-5) and 10 coverslips of the MNP-incubated cells were used and 4-5 random images per coverslip (each corner and middle) were taken. Images were acquired with an AxioImager microscope (Zeiss, Jena, Germany). Total cell number (given by the DAPI-staining), the number of cell type specific stained cells (for example neurons) and the number of cells which were co-localized with the green fluorescent MNPs were counted with the AxioVision Rel. 4.8 Imaging software by Zeiss. Values were calculated as a percentage to the total cell number providing the percentage specific cell types in the cultures. Total uptake was calculated as percentage of specific cell type with MNP co-localization to the total number of this specific cell type.
In PC12 cells, the total cell number, the number of clearly differentiated cells (cells with ß-III-tubulin expression and neurite growth) and the number of differentiated cells co-localized with MNPs was quantified and calculated in a similar manner.
Statistical analysis was performed with Graph Pad Prism 4 (GraphPad Software, La Jolla, USA) using an unpaired t-test for the comparison of control and MNP group. For each cell type n = 20 images from 4-5 coverslips were chosen. For the comparison of the MNP uptake between the different cell types in the cerebellar cultures a one-way ANOVA followed by a Bonferroni post-hoc test was performed (n = 40 images from 10 coverslips). The comparison of the MNP uptake between primary neurons/PC12 cells and between Schwann cells/fibroblasts was done with an unpaired t-test (both n = 40 images from 10 coverslips). In all statistical tests a p-value ≤ 0.05 was considered to be statistically significant.
Immunohistochemistry of organotypic co-culture
Staining procedure
Cultures were fixed after one week by replacing the medium with 4% PFA overnight. For immunohistochemistry, the membranes of inserts were separated from the carrier and cultures attached on membranes were stained free-floating according to the protocol described above. As primary antibodies mouse monoclonal anti-pan-neuronal neurofilament marker (1:1000, Sternberger Monoclonals), rabbit polyclonal anti-ß-III-tubulin (1:1000, Covance), mouse monoclonal anti-CD11b/c (1:400, BD Pharmigen), rabbit polyclonal anti-GFAP (1:500, Progen), rabbit polyclonal anti-S100 (1:200, DAKO) and rabbit polyclonal anti-myelin basic protein (1:200, Chemicon) were used to visualize the main cell types.
Imaging
Organotypic co-cultures were imaged with a TCS SPE DMI4000 confocal microscope by Leica (Wetzlar, Germany) and edited in the Leica Application Suite 2.3. Figure 1 shows a phase contrast image of a spinal cord co-culture. This picture was taken with a cell culture microscope DMI3000 by Leica and was edited with Photoshop CS4 (Adobe Incorporated Systems, San Jose, USA) to create a collage of the whole co-culture. Images of motor neurons were taken at the ventral part of the spinal cord as marked in Figure 1 by the upper white box. Images of other cell types were taken in the vicinity of the intersection of the spinal cord slice and the peripheral nerve graft, marked by the other boxes in Figure 1.
Electron microscopy
Cultures were fixed for 24 h with 4% PFA. They were washed with 0.1 M cacodylate buffer and incubated for 1 h with 1% osmium tetroxide, washed again with 0.1 M cacodylate buffer and 50% ethanol and incubated with 2% uranyl acetate for 1 h. Subsequently, cells were dehydrated in an ascending ethanol series and embedded in Durcupan™ ACM Fluka (Sigma, St. Louis, USA). After hardening at 68°C, cell culture plastic was removed and cells were cut in ultra-thin slices of 70 nm. Images were taken with an EM 900 transmission electronic microscope by Zeiss and edited with Adobe Photoshop CS4.
Viability assay of cell cultures
To reveal changes in cell viability of primary cell cultures incubated with MNPs, an MTS (3-(4.5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) proliferation assay (CellTiter 96 Aqueous one solution cell proliferation assay by Promega, Madison, USA) was used. To avoid previously reported and discussed interferences of nanoparticles with colorimetric assays and absorbance measurement [29], the experimental protocol of the MTS assay was slightly changed. Cells of both primary cell cultures were incubated on DIV 6 for 24 h with MNP medium containing different concentrations of MNPs (10, 50 and 100 μg/ml, each with n = 10 for Schwann cell/fibroblast cultures and n = 12 for cerebellar cultures). Control cells received MNP-free medium. After 24 h of incubation, the medium was removed and cells were washed once with sterile PBS. New MNP-free medium and MTS reagent were added. Cells were incubated for 3 h at 37°C in humidified 6% CO2 atmosphere. All values were normalized by the mean of the blank.
After incubation the absorbance was measured with a Tecan M200 microplate reader (Tecan, Männedorf, Switzerland) at 490 nm. Two values per Petri dish were determined and the mean used for statistical analysis. Statistical analysis was performed with GraphPad Prism 4 using a one-way ANOVA followed by Dunnett's post-hoc test. A p-value ≤ 0.05 was considered to be statistically significant.
To check for integrity of the cell cultures, staining of cells with nuclear fast red-aluminium sulphate and Prussian blue was performed. Prussian blue stained iron-(III) oxide and visualized remaining MNPs (Figure 2). Images for each group were acquired with an AxioImager microscope (Zeiss).