A novel quantitative volumetric spreading index (VSI) is defined that depends on the total distance between object voxels and the contact surface plane in three‐dimensional (3D) space. The VSI, which ranges from 0 to 1, is rotationally invariant around the z‐axis. VSI can be used to quantify the degree of individual cell spreading, which is important for analysis of cell interactions with their environment. The VSIs of astrocytes cultured on a nanofibrillar surface and three different comparative planar surfaces have been calculated from confocal laser scanning microscope z‐series images, and the effects of both culture surface and immunoreactivity on the degree of cell spreading were investigated. VSI calculations indicated a statistical correlation between increased reactivity, based on immunolabeling for glial fibrillary acidic protein, and decreased cell spreading. Further results provided a quantitative measure for the increased spreading of quiescent‐like and reactive‐like astrocytes on planar substrates functionalized with poly–L–lysine. © 2017 International Society for Advancement of Cytometry
CELL cultures are used for studying cell behaviors in controlled environments. Cell spreading, also known as cell flattening, is a result of cell and surface interactions initiated by cell adhesion. The degree of cell spreading is an important component of cell interactions with the environment; however, only a few methods to rigorously quantify individual cell spreading are available. In some cell spreading studies, cells were categorized by human observation as belonging to two to four classes depending on the degree of spreading, and the percentage in each class was determined to calculate a cell spreading index (1) (2) (3) . In other studies, cell spreading area (4) (5) (6) and cell perimeter measurements (7) were used to quantify the cell spreading. Although these methods are practical and easy to implement, the quantitative degree of single cell volumetric spreading is partly ignored. Farooque et al. (8) proposed a dimensionality matrix to assess gyration tensor ellipsoids that are fit to each cell, and then classified the ellipsoids as 1D, 2D, or 3D. This measure can be used to quantitatively estimate the cell spreading behavior for cell types that are dominated by the cell soma response. However, for process-bearing cells such as neurons and astrocytes, the cell process extension response beyond the cell soma is also important. A cell shape index recently proposed by Tiryaki et al. quantifies both stellation and spreading together but is sensitive to cell surface area changes (9) .
In the present work, a new volumetric spreading index (VSI) is defined that depends on the total distances between object voxels and the contact surface plane.
The new VSI does not directly depend on the object surface area, is rotationally invariant around the z-axis and has a range between 0 and 1 with clear and intuitive interpretation.
In this investigation, the new VSI is first introduced and investigated for sensitivity to changes in discretization and changes in scale. It is then used to quantify the degree of spreading by astrocytes on substrates with different properties that were previously shown to influence spreading (10) .
Nanofibrillar Scaffolds And Comparative Culture Surfaces
Poly-L-lysine-functionalized planar glass (PLL glass), unfunctionalized planar Aclar (Aclar), PLL-functionalized planar Aclar (PLL Aclar), and polyamide nanofibrillar scaffolds were prepared (9) . Glass (12 mm, No. 1 coverglass; Fisher Scientific, Pittsburgh, PA) or Aclar coverslips (12 mm; Ted Pella, Redding, CA) were placed in a 24-well tissue culture plate (one coverslip/ well) and covered with 1 ml of PLL solution (50 mg PLL/mL in dH 2 O) overnight. The coverslips used for the cultures were then rinsed with dH 2 O and sterilized with 254 nm UV light using a Spectronics Spectrolinker XL-1500 (Spectroline Corporation, Westbury, NY). The polyamide nanofibrillar scaffolds electrospun on Aclar substrates were obtained from Donaldson (Minneapolis, MN) and Corning Life Sciences (Lowell, MA). The nanofiber diameter range of nanofibrillar scaffolds is from $100 to $300 nm. Promising in vivo and in vitro results have been obtained for astrocytes in contact with these nanofibrillar scaffolds, as implants or as culture surfaces (10) (11) (12) .
Primary Quiescent-Like And Reactive-Like Astrocyte Cultures
Primary quiescent-like astrocyte cultures were prepared from new born Sprague Dawley (postnatal day 1 or 2) rats (9,10). All procedures were approved by the Rutgers Animal Care and Facilities Committee (IACUC Protocol #02-004). The rat pups were sacrificed by decapitation, and the cerebral hemispheres were isolated aseptically. The cerebral cortices were dissected out, freed of meninges, and collected in Hank’s buffered saline solution (HBSS; Mediatech, Herndon, VA). The cerebral cortices were then minced with sterile scissors and digested in 0.1% trypsin and 0.02% DNase for 20 min at 378C. The softened tissue clumps were then triturated by passing several times through a fine bore glass pipette to obtain a cell suspension. The cell suspension was washed twice with culture medium (Dulbecco’s Modified Eagle’s Medium [DMEM]; Life Technologies, Carlsbad, CA 1 10% fetal bovine serum [FBS] ; Life Technologies) and filtered through a 40-mm nylon mesh. For culturing, the cell suspension was placed in 75 cm 2 flasks (one brain/flask in 10 mL growth medium) and incubated at 378C in a humidified CO 2 incubator. After 3 days of incubation, the growth media were removed, cell debris was washed off, and fresh medium was added. The medium was changed every 3-4 days. After reaching confluency (7 days), the cultures were shaken to remove macrophages and other loosely adherent cells.
To obtain reactive-like astrocytes, 0.25 mM dibutyryl cyclic adenosine monophosphate (dBcAMP) was added to the culture medium of 7-day-old semiconfluent quiescent astrocyte cultures and the serum concentration was reduced to 1%. The cultures in dBcAMP containing medium were incubated for additional 7-8 days with a media change every 3-4 days. The morphology of the cells was observed on alternate days under a phase contrast microscope. In the control cultures, the cells were fed with DMEM 1 1% FBS (without dBcAMP).
Quiescent-like and reactive-like astrocytes were harvested at the same time point using 0.25% trypsin/ethylene-diaminetetraacetic acid (EDTA; Sigma-Aldrich, St. Louis, MO) and reseeded at a density of 30,000 cells/well directly on 12-mm Aclar or PLL Aclar coverslips, PLL glass coverslips, or on Aclar coverslips coated with nanofibers in 24-well plates in astrocyte medium containing dBcAMP (0.5 ml). After culturing the astrocytes on the aforementioned substrates for 24 h, they were fixed with 4% paraformaldehyde for 10 min. Parallel cultures were immunostained with glial fibrillary acidic protein (GFAP), an identification marker for astrocytes, and >95% were found to be GFAP-positive.
Confocal Laser Scanning Microscope Imaging
Confocal z-series images were captured using an Olympus FluoView 1000 CLSM system attached to an Olympus IX81 automated inverted microscope platform equipped with a 403 oil immersion objective (NA 5 1.3). The z-series were acquired under identical imaging conditions, including the same high voltage, offset and gain settings, same objective, and same resolution (1024 3 1024 pixels). The intensity data were collected from samples in three dimensions. The z-step size was set to 1.13 lm as described in Ref. (11) . Fifty cells were analyzed for each culture surface and immunoreactivity situation, making a total of 400 astrocytes. GFAP immunostained images were used for the quantitative cell spreading analysis.
Quantitative Astrocyte Spreading Assessment By Vsi
A face-connected voxel model was created for each cell in the CLSM data. The CLSM imaging conditions were carefully set so that the background pixel illumination was almost zero. The raw CLSM GFAP data was segmented using simple thresholding (T > 0). After segmentation of each z-slice, thresholding was used to isolate individual cells and cell clusters, with further segmentation of cells in clusters performed by hand. The volumetric data for each cell was obtained using the assembly of z-series slices and was represented by the means of a spatial occupancy array, defined in Bribiesca (13) . The unit voxel dimensions for images taken with a 403 objective was 0.3097 mm 3 0.3097 mm 3 1.13 mm in x-y-z. Since there is approximately fourfold difference between the axial and lateral resolution, the 3D data were upsampled to obtain nearly cubic unit voxels using cubic interpolation. Implementation was done using the “interp3” command of MATLAB.
The new VSI is defined as the exponential of negative total voxel distance to the contact surface plane where the total distance is normalized according to a cube:
EQUATION (1): Not extracted; please refer to original document.
where v is an object voxel, V c is the set of all object voxels, N is the total number of object voxels, z is the minimum distance between voxel v and the contact surface plane, and u is the unit voxel length. An object is considered as a series of z-slices that are parallel to the xy-plane. The exponential formulation ensures that VSI is between 0 and 1. The normalization factor
N ffiffiffiffi N 3 p 21 À Á =2
is the total voxel distances of a cube with edge length ffiffiffiffi N 3 p unit voxels to the contact surface plane. A cube has equal edge lengths, and therefore, the VSI should be constant and equal to e 21 for any cube.
VSI ranges from 0 to 1 and has an inverse correlation with the total distance between object voxels and the contact surface plane. The MATLAB codes for a tutorial example VSI calculation are given in Section 1 of Supporting Information (14) .
VSI calculations for a set of 10 test objects each having 25 voxels in different “spreading” arrangements are shown in Figure 1 . The VSI is, correctly, equal to 1 when all voxels are in a single plane. The VSI increases as the “spreading” increases, and it is close to 0 for the 1D voxel configuration normal to the xy-plane.