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Original Research |
1 Department of Surgery, Duke University Medical Center, Box 3815, Durham, NC
27710.
2 Department of Biomedical Engineering, Duke University Medical Center, Durham,
NC.
3 Department of Anesthesiology, Duke University Medical Center, Durham,
NC.
4 Department of Radiology, Duke University Medical Center, Durham, NC.
Received May 16, 2006;
accepted after revision September 12, 2006.
Imaging studies were performed at the Duke Center for In Vivo Microscopy,
an NCRR/NCI National Resource (P41 RR005959/R24 CA092656).
Abstract
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MATERIALS AND METHODS. Adipose-derived stem cells harvested from mice inbred for green fluorescent protein were labeled with SPIO ferumoxide particles through the use of poly-L-lysine. Adipose-derived stem cell viability, iron staining, and proliferation were measured after SPIO labeling, and the sensitivity of MRI in the detection of SPIO-labeled adipose-derived stem cells was assessed ex vivo. Adult mice (n = 12) were subjected to unilateral MCAO. Two weeks later, in vivo 7-T MRI was performed to guide stereotactic transplantation of SPIO-labeled adipose-derived stem cells into brain tissue adjacent to the infarct. After 24 hours, the mice were sacrificed for high-resolution ex vivo 7-T or 9.4-T MRI and histologic study.
RESULTS. Adipose-derived stem cells were efficiently labeled with SPIO particles without loss of cell viability or proliferation. Using MRI, we guided precise transplantation of adipose-derived stem cells. MR images of mice given injections of SPIO-labeled adipose-derived stem cells had hypointense regions that correlated with the histologic findings in donor cells.
CONCLUSION. MRI proved useful in transplantation of adipose-derived stem cells in vivo. This imaging technique may be useful for studies of CNS stem cell therapies.
Keywords: brain • ischemia • MRI • stem cells • transplantation
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MRI, which can offer critical information in stem cell studies, requires labeling of donor cells for stem cell localization [20]. Labeling is performed most commonly with superparamagnetic iron oxide (SPIO) nanoparticles. Because of the small crystal size (7-10 nm), SPIO particles exhibit magnetic moments that align in an applied magnetic field, produce microscopic field gradients that dephase the neighboring proton magnetic moments, and reduce T2* relaxation time. The most common method of SPIO labeling is use of a cationic transfection agent, such as poly-L-lysine [20, 21]. These agents are relatively easy to use, but cellular toxicity and effects on differentiation can be troublesome.
MRI may improve the use of SPIO-labeled adipose-derived stem cells in a middle cerebral artery occlusion (MCAO) model. This model is commonly used for study of cerebral ischemia in rodents [22, 23]. Although the variability of infarct size limits use of this model, MRI can be used for precise definition of the area of infarction and for evaluation of the implantation of SPIO-labeled stem cells. Several investigators [21, 24-26] have found it feasible to image SPIO-labeled stem cells in the spine and brain, but the efficacy of this technology with adipose-derived stem cells is unknown. We hypothesized that MRI can be used to define a target adjacent to the infarct for stem cell transplantation and to localize transplanted SPIO-labeled adipose-derived stem cells. The goals of the current study were to establish methods of labeling adipose-derived stem cells with SPIO particles, to use MRI to guide transplantation of adipose-derived stem cells in MCAO-injured mice, and to use MRI to localize donor adipose-derived stem cells in the injured brain.
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Cell Labeling and Viability
We first performed a series of in vitro experiments to evaluate the effect
of ferumoxide (Feridex, Berlex Laboratories) and poly-L-lysine
hydrobromide (PLL; MW, 388 kd; Sigma) on adipose-derived stem cell viability,
iron labeling, and proliferation. Adipose-derived stem cells were labeled with
SPIO particles by incubation with a complex of ferumoxide and PLL according to
a previously published protocol
[27]. Duplicate cultures of
adipose-derived stem cells were labeled for 24 hours with various
concentrations of ferumoxide (iron, 0, 12.5, or 25 µg/mL), PLL (0, 0.188,
0.375, or 0.75 µg/mL), or both agents. Control adipose-derived stem cells
were cultured in control medium without ferumoxide or PLL.
A diluted solution of ferumoxide particles (Feridex; stock elemental iron, 11.2 mg/mL) was added to culture medium without cells and mixed well. PLL was added from a 1.5-mg/mL stock solution for reconstitution of a desired final concentration of elemental iron. The medium was gently mixed for 60 minutes at room temperature to allow complexing of ferumoxide particles to PLL. Labeling was initiated by replacement of the initial control medium with fresh control medium containing ferumoxide-PLL complexes for 24 hours. Cells were then washed twice in phosphate-buffered saline solution (PBS) to remove excess iron-PLL complexes and then were placed in fresh control medium for an additional 24 hours for recovery. After cell recovery, cells were harvested with trypsin digest or were examined in situ for viability and iron staining.
For assessment of cell viability, cultures were washed three times with PBS and stained with trypan blue (Sigma). Viable cells were considered those that did not stain with trypan blue. Two independent observers using light microscopy (10x magnification) enumerated cell viability in 10 separate fields. To assess iron staining, we used Prussian blue (ferric hexacyanoferrate and hydrochloric acid, Sigma) to determine the percentage of cells stained with iron. Cultures were incubated for 30 minutes with 2% potassium ferrocyanide (Perls' Prussian blue reagent) in 3.7% hydrochloric acid, washed again, and counterstained with nuclear fast red. A positive test result was defined as the presence of brown iron particles visible within cell cytoplasm. All experiments were performed on duplicate cultures of adipose-derived stem cells at each concentration of PLL and ferumoxide, and each experiment was repeated three times.
To assess adipose-derived stem cell proliferation, we used the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay (ATCC) as described [28]. Adipose-derived stem cells were seeded at a density of 10,000 cells/well in a 96-well plate and were allowed to grow to 60% confluence in 5% CO2 at 37°C in control medium. In the three experimental groups, the medium was changed to include ferumoxide, PLL, or ferumoxide-PLL complex. Control cells included control medium alone. On the basis of the results of our initial viability studies (see Results), we used ferumoxide at a concentration of 12.5 µg/mL and PLL at a concentration of 0.375 µg/mL for proliferation studies. After 1-, 3-, 5-, and 7-day culture periods, adipose-derived stem cell proliferation was measured with the MTT assay according to the manufacturer's recommendations. To each culture on the 96-well plate, we added 10 µL of MTT and incubated the cells for 4 hours at 37°C. We then added 100 µL of lysis solution and incubated the sample at room temperature for 2 hours. The absorbance of each sample was recorded at 570 nm in an enzymelinked immunosorbent assay reader and expressed as optic density (OD) after blank subtraction. All experiments were performed in quadruplicate. We compared the proliferation rate (difference between ODs) from days 3-7 in each experimental group with that of the control group. Comparison of CIs showed differences in proliferation rates between each experimental group and the control group.
MRI of SPIO-Labeled Cells In Vitro
To develop techniques for MRI in adipose-derived stem cell transplantation,
we performed a series of phantom studies to identify the sensitivity of MRI in
the detection of small numbers of adipose-derived stem cells implanted in
agar. Cultures of adipose-derived stem cells were incubated with
ferumoxide-PLL complex. On the basis of the results of our initial viability
studies (see Results), we used ferumoxide at a concentration of 12.5 µg/mL
and PLL at a concentration of 0.375 µg/mL. Adipose-derived stem cell
cultures were incubated with ferumoxide-PLL complex for 24 hours, washed twice
with PBS to ensure removal of unbound ferumoxide-PLL complexes, and recovered
for 24 hours in control medium before being released by trypsin digest and
enumerated. Cell dilutions were made to suspend various numbers of
adipose-derived stem cells (0, 10, 100, 500, 1,000, and 5,000 cells) in 5
µL of control medium. The cell aliquot was imbedded by use of a pipetter in
agar (4% weight/weight) in plastic Petri dishes (Corning) for MRI. All
experiments were performed in duplicate, and each experiment was repeated
three times.
MRI was performed with a 7-T MRI microscopy system consisting of a superconducting 21-cm horizontal-bore magnet (Magnex, Varian) interfaced with a console (Excite, GE Healthcare). The Petri dish sample was placed on top of a 3.0-cm-diameter radiofrequency surface coil. A series of multislice T2-weighted and T2*-weighted images (TR/TE, 250/10-40 in increments of 5 milliseconds; matrix size, 128 x 128; field of view, 4.0 cm; slice thickness, 1 mm) were acquired with standard spin-echo and gradient-echo sequences.
Middle Cerebral Artery Occlusion
To develop techniques for the use of MRI in guiding the transplantation of
adipose-derived stem cells in MCAO-injured mice and to localize donor
adipose-derived stem cells in the injured brain, we produced MCAO injury in
mice as described [29].
Briefly, 8- to 10-week-old male Balb/C mice (Jackson Laboratories) (n
= 12) were anesthetized with 1.0-1.5% halothane in 50%
O2/N2, intubated, and mechanically ventilated. Rectal
temperature was monitored and servo-regulated with surface heating and cooling
at 37.0°C throughout the procedure. Inspired halothane concentration was
kept between 0.6% and 1.0%. The right common carotid artery was identified
through a midline cervical skin incision. The external carotid artery was
transected, and the internal carotid artery was dissected for visualization of
the origin of the pterygopalatine artery. A 7-0 nylon monofilament suture,
tapered at the tip and coated with silicone, was inserted into the external
carotid artery stump and advanced approximately 11 mm into the internal
carotid artery. The mice were subjected to 60 minutes of MCAO, after which the
occlusive filament was removed, the wounds were closed, and the mice were
extubated and placed in an O2-enriched environment
(FIO2, 50%) for 1 hour. Two of the 12 mice died within
several days of MCAO.
In Vivo MRI
Two weeks after MCAO injury, the surviving mice (n = 10) were
imaged with 7-T MRI. The mice were anesthetized with ketamine (20-60 mg/kg)
and xylazine (2-5 mg/kg) administered intraperitoneally and then placed supine
with the head above a 1.0-cm-diameter surface coil. After endotracheal
intubation, anesthesia was maintained with 2-3% isoflurane. A custom-made
ventilator was used to deliver the isoflurane. The mice were mechanically
ventilated at a rate of 90 breaths/min at a tidal volume of 0.4 mL. An airway
pressure tracing was monitored with a solid-state pressure transducer on the
breathing valve, and ECG recording was performed with electrodes taped to the
footpads of the mice. Body temperature was recorded with a rectal thermistor
that was also used to control heat lamps that maintained core body temperature
at 36.5°C.
Multislice T2-weighted spin-echo images (TR, 1.0; matrix size, 128 x 128; field of view, 3.5 cm; thickness of consecutive slices, 1 mm; number of signals averaged, 4) were obtained in the anatomic coronal orientation. We performed all sequences within 40-60 minutes per mouse. Two of these 10 mice did not have a visible infarct and were not subjected to stem cell transplantation.
MRI images were reviewed to assess the infarct and to determine the stereotactic coordinates for transplantation of donor adipose-derived stem cells into either the hippocampus (n = 4) or the striatum (n = 4) immediately adjacent (within 0.5 mm) to the edge of the infarct. Briefly, we identified the coronal image section showing both the hippocampus and the infarct, usually within 0.2 mm rostral or caudal to the bregma. Bregma refers to the junction of the sagittal and coronal sutures at the top of the skull and is a common, easily identifiable landmark on the mouse skull with which to guide stereotactic transplantation. Using the MRI images, we correlated surrounding major structures (lateral ventricles, hippocampus, central aqueduct) with images in a standard mouse brain anatomic atlas [30]. With these coordinates, we defined the stereotaxic coordinates for transplantation of donor adipose-derived stem cells into the area immediately adjacent to the infarct.
Stereotactic Transplantation
Within 24 hours after in vivo MRI, the mice were anesthetized with
tribromoethanol (10 mg/kg intraperitoneally) and placed in a standard
stereotaxic device for transplantation of adipose-derived stem cells. On the
basis of stereotaxic coordinates defined on in vivo MRI, mice received
transplants into either the hippocampus (n = 4) or the striatum
(n = 4) in the area within 0.5 mm of the mature infarct. A Hamilton
syringe was used to inject donor cells (500, 1,000, 2,000, or 5,000
cells/mouse) within a 0.5-µL volume over a 20-minute period to minimize
backflow of cells along the injection track. Each cell dose was administered
to two mice. Two control MCAO-injured mice received transplants of 1,000
unlabeled adipose-derived stem cells into the striatum.
High-Resolution Ex Vivo MRI
Twenty-four hours after adipose-derived stem cell transplantation, the mice
were sacrificed for high-resolution ex vivo MRI and histologic examination for
confirmation of placement of donor cells in the area adjacent to the infarct.
The mice were anesthetized with xylazine (10 mg/kg) and ketamine (90 mg/kg)
administered intraperitoneally. Transcardial perfusion of 20 mL of warm
(37°C) PBS was followed by 20 mL of PBS with 10% buffered formalin and 5%
gadoteridol (ProHance, Bracco Diagnostics). To avoid physically perturbing the
brain, we removed the entire mouse head after at least 24 hours of fixation
and placed it inside a 2.0-cm-diameter cylindric holder.
High-resolution ex vivo MRI was performed with either a 7-T (n = 4) or a 9.4-T (400 MHz, vertical bore Oxford superconducting magnet) (n =4) system with a 2.0-cm-diameter birdcage radiofrequency coil. We used both 7-T and 9.4-T magnets to determine whether differences existed in the quality of MR signal and ease of localizing donor cells. An isotropic 100-µm-resolution image (100/7; matrix size, 512 x 256 x 256; number of signals averaged, 2) was obtained with a standard 3D spin-echo sequence. We examined the MR images in the coronal, axial, and sagittal planes to identify the hypointense region corresponding to the donor cell transplant. In some cases, 3D images were volume rendered with VoxelView Ultra 2.5 software (Vital Images) on a Reality Engine2 workstation (Silicon Graphics). Volume-rendered images in all three orientations and reformatted slices were captured on the workstation for annotation with Adobe PhotoShop 4.0 v4.0.1 PowerPC software (Adobe Systems).
Tissue Histology
After ex vivo MRI, brain samples were cryosectioned at 30-µm thickness
through the entire coronal rostral-caudal extent of the infarct. Donor
adipose-derived stem cells (GFP+) were detected by
immunohistochemical analysis with a peroxidase-conjugated anti-GFP marker as
described [17]. In addition,
brain sections were treated with Prussian blue to detect iron and with Nissl
stain. Prussian blue staining (ferric hexacyanoferrate and hydrochloric acid)
was performed by incubation of sections for 30 minutes with 2% potassium
ferrocyanide (Perls' Prussian blue reagent) in 3.7% hydrochloric acid. The
sections were washed again and counterstained with nuclear fast red.
To ensure that the coronal brain sections were in the same coronal plane as the MR images, we correlated MRI and histologic sections with images in a standard mouse brain atlas [30] to determine rostral-caudal distances from the bregma.
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Adipose-Derived Stem Cells Can Be Labeled with SPIO In Vitro with High Efficiency
Using Prussian blue staining, we found distinctive blue cytoplasmic
inclusions clustered in the nuclei of adipose-derived stem cells labeled with
ferumoxide-PLL complex. As the concentration of PLL was increased from 0 to
0.375 µg/mL in the presence of ferumoxide, the percentage of iron-labeled
cells increased proportionally (Fig.
1). At increasing levels of PLL, there was a progressive loss of
cell viability, measured by trypan blue exclusion, with relatively high levels
of both iron staining and viability at a PLL concentration of 0.375 µg/mL.
Therefore, for all further experiments, we used a PLL concentration of 0.375
µg/mL to maximize both iron uptake and cell viability. Similar experiments
performed with variation in the ferumoxide concentration showed that a
ferumoxide concentration of 12.5 µg/mL was optimal for maximizing iron
uptake while minimizing cell toxicity, and this concentration of ferumoxide
was used for all further experiments.
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To test the sensitivity of MRI in the detection of small numbers of adipose-derived stem cells, we labeled adipose-derived stem cells with ferumoxide-PLL complex, immobilized labeled cells in agar, and imaged this plate with 7-T MRI. In these phantom studies, we found that T2-weighted images revealed clear differences in signal intensity between labeled and unlabeled cells and that the number of labeled cells correlated with the decrease in MR signal intensity (Fig. 3A, 3B, 3C, 3D, 3E, 3F). These results showed low MR signal intensity at cell numbers greater than 10, suggesting that MRI can depict as few as 10 labeled adipose-derived stem cell cells with nominal pixels, at least in vitro. Quantitative assessment of the sensitivity of MRI in the detection of adipose-derived stem cells with use of signal strength was not addressed in our study but may be useful for evaluation of detection of adipose-derived stem cells compared with other stem cell populations [31].
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We correlated major structures (e.g., lateral ventricles, hippocampus, central aqueduct) between the MR images and images in a standard mouse brain anatomy atlas [30] (Fig. 4). From these coordinates, we calculated the stereotaxic coordinates for transplantation of donor adipose-derived stem cell into either the striatum or hippocampus next to the infarct. We reanesthetized the mice and injected ferumoxide-PLL-labeled adipose-derived stem cells into the appropriate regions.
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Donor Adipose-Derived Stem Cells Can Be Localized with MRI
To confirm the accuracy of MRI for localization of stem cells, we used
various stains for visual correlation of histologic findings and MR signal
intensity in the MCAO-injured brain after adipose-derived stem cell
transplantation. In all seven mice with a hypointense region at the site of
adipose-derived stem cell infusion, MRI findings correlated well with
histologic evidence of donor cells (Fig.
6A,
6B,
6C,
6D,
6E). For these experiments,
the intrinsic GFP fluorescence of the donor adipose-derived stem cell cells
was used to assist with donor cell visualization at immunohistochemical
analysis, which showed GFP+ donor cells in the area corresponding
to the loss of MR signal intensity. Findings at histologic examination of
corresponding brain sections confirmed that MRI findings correlated with the
deposition of donor cells: At immunohistochemical analysis sections showed
cells containing iron (Prussian blue) and GFP. MRI was sensitive for
relatively low numbers of cells. We colocalized MRI signal intensity and
histologic findings in mice receiving as few as 500 cells. In the single mouse
in which no change in MRI signal intensity was seen after transplantation, no
donor cells were seen at histologic examination. This finding, which suggested
a technical problem with cell implantation in this mouse, was an indication of
the specificity of MRI. These results suggest that MRI can be used, at least
immediately after transplantation, to localize small numbers of donor labeled
stem cells.
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Stem cell studies in animal stroke models are limited by variability in stroke patterns that results in difficulty placing donor stem cells into the ischemic border zone immediately adjacent to the injury. A variety of techniques have been described for localization of ischemic tissue in rodents, including laser Doppler flowmetry [22] and measurement of global and regional cerebral blood flow [34]. These techniques, however, do not easily lend themselves to guidance of stem cell transplantation. We based our work on the leading studies of Jiang and collaborators, who defined parameters for the use of MRI in tracking neuronal progenitor cells in animal stroke models [35]. Our study appears to be the first report of MRI guidance of transplantation of donor adipose-derived stem cells within a brain injury model.
The fate of transplanted adipose-derived stem cells and their effect on the functional outcome of cerebral ischemia were not addressed in our study and remain important issues. The efficient labeling of adipose-derived stem cells with ferumoxide concurs with results of stem cell studies showing the usefulness of SPIO preparations for labeling of mesenchymal stem cells [36], embryonic stem cells [21], and neural progenitor cells [24]. We recognize that our short-term study did not specifically address the long-term differentiation of labeled adipose-derived stem cells in vivo. However, other stem cell studies have shown both conservation and limited differentiation after SPIO labeling [24, 36]. In most studies of embryonic stem cell and neuronal stem cell transplantation into the brain, donor cells generally have differentiated along glial lineages [37, 38]. In contrast, transplantation of stem cells into neurogenic regions, such as the subventricular zone, may increase differentiation toward neurons [37]. Results [18] have shown that donor adiposederived stem cells transplanted into either the normal or injured brain may lead to both neuronal and glial differentiation. These topics require further study. Before clinical use of adipose-derived stem cells, characterization of the effect of SPIO labeling on long-term adipose-derived stem cell differentiation and function is needed.
We recognize that before in vivo MRI is used to track the activity of stem cells over time after transplantation, it is essential to define the resolution and validity of an MRI protocol. To design our initial imaging protocol, we chose ex vivo rather than in vivo MRI for posttransplantation localization of stem cells. The resolution of our ex vivo studies was 0.043 x 0.043 x 0.043 mm (7.9 x 10-5 mm3), nearly 1,000 times greater spatial resolution than in our in vivo studies and providing a much more sensitive method for detection. Therefore, the use of ex vivo MR histologic examination allows much higher confidence in localization because of correlation with conventional histologic findings and detection of far fewer cells than would be possible in vivo.
Results of studies by Heyn and collaborators [39] have suggested that single SPIO-labeled stem cells can be detected in vivo. Several authors [20, 25, 26] have performed longitudinal studies to track labeled stem cells in vivo. The spatial resolution of our in vivo studies at 0.273 x 0.273 x 1.0 mm (7.4 x 10-1 mm3) is on the same scale as that in the previous in vivo studies. Our current work validates the use of MRI for adipose-derived stem cell localization with sensitive ex vivo imaging and provides the foundation for subsequent longitudinal in vivo studies. Those studies will be conducted with a larger number of cells and longer in vivo scan times to achieve the required sensitivity.
Labeling adipose-derived stem cells with SPIO particles enhances cell-to-background contrast and renders the cells visible on MRI. The intensity and area of the low MRI signal intensity from the SPIO particles depend on the concentration of iron oxide particles per cell and the image acquisition parameters. In the current study, our phantom experiments showed that MRI can depict as few as 10 cells labeled with a relatively low concentration of SPIO particles (12.5 µg/mL). In contrast, a higher iron content for cell labeling, for example, up to 1,200 µg/mL, has been used in other stem cell MRI studies [21]. Frank et al. [40] reported that iron in a concentration of 25 µg/mL was well tolerated by various stem cells and allowed MRI detection. However, we agree with Magnitsky et al. [26] that given the wide variation of labeling techniques, imaging hardware (radiofrequency coils), and acquisition parameters (TE, imaging matrix, and acquisition time) used by various investigators, it is difficult to compare the quantitative results of stem cell labeling among laboratories.
With our tissue fixation and imaging protocol, both 7-T and 9.4-T MRI yielded high-resolution ex vivo images of donor stem cells. Each image element (pixel) in an MR image represents the signal intensity from a volume (voxel) of tissue. As described earlier, 0.043 x 0.043 x 0.043 mm voxels are encoding a volume of 7.9 x 10-5 mm3. For context, such a voxel is almost equal in size to a single Purkinje neuron in the mouse cerebellum [41]. We consider our method of tissue fixation important for rendering high-quality images, as reported previously [33]. This method of fixation exploits differences in tissue magnetization, which both uniformly reduces the T1 intensity of all tissues, resulting in a 10-fold increase in signal intensity, and the detection of T2 differences in tissues with short TRs. Furthermore, the MRI data set we used yields 3D images (matrix size, 256 x 256 x 512) that can be viewed and sectioned in any plane (coronal, horizontal, and sagittal) without loss of spatial resolution.
Although histochemical examination showed the presence of iron-positive cells in tissue sections that corresponded to hypointense regions on MR images, we recognize that several endogenous sources of iron can result in similar appearances. The areas of iron also costained for GFP. We do not believe, however, that the iron originated in hemorrhage and iron degradation product release from iron-containing proteins, such as hemoglobin, ferritin, and hemosiderin, from endogenous cell uptake. Long-term studies of donor cells in vivo, including phenotype analysis, would be helpful for assessment of the role of phagocytic processes in iron sequestration that may affect MRI.
We conclude that adipose-derived stem cells can be safely and efficiently labeled with ferumoxide particles and that MRI can be used to guide transplantation of donor stem cells in MCAO-injured mice and localization of ferumoxide-PLL-labeled adipose-derived stem cells in MCAO-injured mice. As shown in this immediately posttransplantation model, these techniques can assist the study of stem cell implantation. Use of these labeling techniques may facilitate long-term longitudinal study of donor stem cells after transplantation.
Acknowledgments
We thank Boma Fubara for excellent technical assistance.
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) and percentage iron staining
(
) in presence of ferumoxide (12.5 µg/mL of elemental iron). Error
bars represent SE. Increasing concentration of PLL resulted in both increased
iron staining and decreased cell viability. These data showed PLL
concentration of 0.375 µg/mL as providing acceptable level of iron staining
with minimal loss of cell viability. Findings led to choice of this
concentration for future experiments.
), ferumoxide (
), and PLL-ferumoxide complex
(
). Results show initial decrease in proliferation of cells grown with
PLL-ferumoxide complex, although within 7 days, all experimental groups showed
proliferation equivalent to that of control cells.
