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Abstract

Selective administration of mesenchymal stromal cells to the mesenteric arteries is a potential technique to overcome pulmonary trapping and increase the density of transplanted cells in extensive mural inflammation of the intestine, such as in inflammatory bowel disease and graft-versus-host disease. We injected 5 × 10^{6 111}In-oxine-labeled human decidual stromal cells (DSCs) to the rabbit superior mesenteric artery (SMA) using clinical routine catheters guided by an angiographical system under sterile conditions. We used longitudinal single-photon emission tomography at 6 h and at 1, 2, and 5 days to assess trafficking and distribution of DSCs. We used digital subtraction angiography, computed tomography, and hematoxylin and eosin stainings to determine biodistribution of cells and to assess safety end points. We found that selective injection of human DSCs to the rabbit SMA does not result in acute embolic complications. Furthermore, we found that IV administration resulted in extensive retention of the radiolabeled DSCs in the lungs, corroborating previous studies on pulmonary trapping. In sharp contrast, selective injections to the SMA resulted in uptake distributed in the intestine supplied by the SMA and in the liver, indicating that this approach could significantly increase the fraction of injected DSCs reaching the target tissue.

Keywords

Cell transplantation Intra-arterial Decidual stromal cells (DSCs)Inflammatory bowel disease (IBD)

Introduction

Crohn's disease (CD) and ulcerative colitis are the two primary diseases within the inflammatory bowel disease (IBD) spectrum (23). The distinguishing pathological condition of IBD is a chronic and uncontrolled inflammation of the intestinal mucosa (38). CD is characterized by transmural, patchy granulomatous inflammation of any part of the gastrointestinal tract, though most prominently featured in the ileocecal region. Ulcerative colitis, on the other hand, is confined to the large bowel, engaging primarily the superficial layers of the mucosa (17).

Despite intense progress in the pharmacological treatment of IBD, including anti-tumor necrosis factor antibodies, many patients are still suffering from poor quality of life. Several studies aimed at cell transplantation as a means of mediating immunosuppression, and anti-inflammatory effects have also been performed, and there are now techniques for producing clinical-grade mesenchymal stem cells (MSCs) for potential therapy of CD and other conditions (34). Since 1995, the development of guidelines for entry criteria and transplantation protocols for IBD and design of clinical trials for intravenously and intralesionally injected MSCs have been performed. The best therapeutic outcome is achieved in patients treated by intralesional injection, as reviewed by Dalal et al. (12). The results may be explained by the fact that endogenous homing of MSCs has proven insufficient, with less than 1% of delivered cells being found in target tissues (39). Thus, it is anticipated that the delivery route of MSCs should be tailored to the lesion being treated as suggested in a review by Kean et al. (28).

Another disorder that is characterized by intestinal inflammation is acute graft-versus-host disease (GVHD), a life-threatening condition that can develop in patients undergoing allogeneic hematopoietic stem cell transplantation. Bone marrow-derived MSCs have been used to treat steroid-refractory acute GVHD (4,31,43), but this therapy has failed to increase the long-term survival (40).

The optimal administration route of cell therapy, using MSCs, is unclear. The intravenous (IV) route may not be optimal for cell delivery due to pulmonary trapping, a phenomenon elegantly described in a landmark article by Fischer et al. (18). Mesenchymal cells are known to accumulate in the lungs following IV injection (6,36), presumably due to their size (44). Pulmonary accumulation of IV-injected cells has been suggested to cause adverse effects, such as embolus formation and posttransplantation pneumonitis (11,19), and is considered as a side effect that limits transplantation efficacy and therapeutic effectiveness (6,18,36). Thus, specific delivery of cells to affected organs could potentially increase their therapeutic effect and reduce pulmonary side effects.

With a selective intra-arterial (IA) injection by catheter-based technique, it is possible to reach a high concentration in the gastroenteral capillary bed at first passage, an approach that has been performed in one patient with IBD and reported in an abstract (13). Modern imaging-based interventional techniques now provide alternatives to open surgical access, and arteries and veins can be regarded as “internal routes” to essentially anywhere in the body. However, the cellular retention of the targeted organ may be influenced by many different factors, including type of organ and cell, as well as technical infusion parameters. In vivo tracking of cells carrying radioligands is a viable approach to further understand the possibilities and limitations of different administration regimes (42). The physical half-life of ¹¹¹In allows single photon emission tomography (SPECT) scanning of labeled cells several days after injection, thus presenting means to evaluate graft biodistribution (33).

Stromal cells with therapeutic potential can be isolated from several different tissues, including bone marrow, fat, and placenta, as reviewed by Kaipe et al. (26). The majority of clinical studies involving MSCs have used bone marrow-derived MSCs (31), which are the best characterized stromal cells, but adipose-derived stromal cells have also successfully been used in the treatment of CD (20). Recently, decidual stromal cells (DSCs) from term placental fetal membranes were introduced as a therapy for acute intestinal GVHD (41). DSCs of mesenchymal origin are easily isolated from the maternal layers of the placental membranes, the decidua, and expanded without invasive procedures, and possess potent immunosuppressive capacities by inhibiting proinflammatory T-cell responses and promoting regulatory T cells (16). Compared to bone marrow-derived MSCs, DSCs also express higher levels of adhesion markers, including very late antigen-4 (27), a characteristic feature that may be of importance for migration of cells over the vessel wall toward the inflamed tissue (32). However, like MSCs, intravenously administered DSCs first target the lungs and thereafter primarily relocate to the liver and spleen, with little evidence of homing to inflamed tissue (15).

The aim of this study was to evaluate the biodistribution and safety after superselective mesenteric artery administration of ¹¹¹In-labeled DSCs in rabbits. We hypothesized that superselective IA injections do not cause acute embolic complications and that the distribution of cells following IA injection is significantly more concentrated to the target tissue compared to IV injections.

Materials and Methods

Cell Preparation

Term placentas were obtained from healthy mothers, with informed consent after cesarean section. Ethical approval was obtained from the Institutional Ethical Review Board. The donors were seronegative for HIV, hepatitis B and C, and syphilis.

DSCs were isolated as previously described (41). Briefly, the placenta was washed in phosphate-buffered saline (Life Technologies, Gaithersburg, MD, USA) to remove contaminating blood. The fetal membrane, which is composed of both fetal (amnion and chorion) and maternal (decidua) layers, was dissected mechanically from the placental structure, trypsinized (HyClone, Logan, UT, USA), and was thereafter cut into 3- to 4-cm² pieces. The explants were spread out and incubated in T175 flasks in Dulbecco's modified Eagle's medium (DMEM) (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal calf serum (FCS) (Thermo Fisher Scientific) and penicillin–streptomycin (complete DMEM) (Thermo Fisher Scientific). The tissue explants were removed from the flasks when colonies of fibroblast-like stromal cells appeared. When the DSCs were about 90-95% confluent, the cells were harvested with trypsin/ethylenediaminetetraacetic acid (EDTA; Thermo Fisher Scientific), washed in complete DMEM, and seeded in new T175 flasks at 2.9 × 10³ cells/cm² in complete DMEM. The cells were cultured to passage 3 or 4 and frozen slowly in complete DMEM containing 10% dimethyl sulfoxide (DMSO; WAK-Chemie Medical GmbH, Steinbach, Germany). As previously described, the DSCs were positive for CD29, CD44, CD73, CD105, and HLA class I, but negative for CD34, CD14, CD45, CD31, and HLA class II (41). The origin of the DSCs was analyzed by microsatellite polymorphism using capillary electrophoresis, which showed that the cells were only of maternal origin. Altogether, this shows that the cells are of mesenchymal origin and derive from the decidua of the placental layers and hence can be termed DSCs.

Labeling Cells with ¹¹¹Indium

Labeling was performed essentially according to the instructions for cell labeling provided by the ¹¹¹In-oxinate supplier (Mallinckrodt Medical B.V., Le Petten Holland). Five million DSCs cells were centrifuged and resuspended in 1 ml complete DMEM. ¹¹¹In-oxinate (15.2-16.3 MBq) in tris buffer (0.4 ml) was added and allowed to react for 20 min at 37°C with occasional swirling to maintain suspension. The tubes were centrifuged, the unreacted radioactivity in the supernatant removed, 1 ml FCS in phosphate-buffered saline/EDTA was added, and the cells were resuspended with swirling. The centrifugation was repeated. Thereafter measurements of the radioactivity in the cells and supernatants were carried out. Labeling efficiency (total activity in cells/total activity in cells + total activity in supernatants) was 76 ± 8 (mean % ± 1 SD). The labeled cells were resuspended in 0.9 ml physiological saline supplemented with 10% FCS for the transplantation procedure. The mean injected activity was 9.3 ± 2.1 (MBq ± 1 SD).

Intra-arterial Administration of DSCs

All animal studies were conducted according to the Karolinska Institute guidelines for experiments on rabbits. The study was approved by the regional ethics committee for animal research.

Six adult male New Zealand White rabbits weighing 3.4-4.0 kg (Lidkopings Kaninfarm HB, Lidkoping, Sweden) were used in the study. Four received DSCs in the superior mesenteric artery (SMA), and two received DSCs in the ear vein. The animals were not subjected to any form of immunosuppression. Procedures were performed in sterile conditions and under general anesthesia with the intubated rabbits connected to a servo ventilator. Anesthesia was induced by subcutaneous injection of 0.5 ml/kg Hypnorm (fentanyl citrate 0.315 mg/ml, fluanisone 10 mg/ml; Janssen Pharmaceuticals, Belgium) followed by a combination of 0.4-0.6 ml diazepam (5 mg/ml; Actavis Group PTC, Iceland) and 0.4-1 ml Propofol-lipuro (20 mg/ml; Braun AB, Sweden). Maintenance of anesthesia was achieved by continuous IV infusion of Propofol-lipuro (20 mg/ml) at a rate of 20 ml/h and IV injection of 0.1 ml fentanyl (10 μg/ml; Braun AB) every 30 min.

A 4F pediatric introducer (Terumo Medical Corporation, Tokyo, Japan) was inserted into the right femoral artery through a surgical incision and a 4F catheter (Cobra Hydroglide; Cook, Bloomington, IN, USA). A 1.2F microcatheter (Magic; Balt Extrusion, Montmorency, France) was navigated to the proximal SMA, and in that position 5 million DSCs dispersed in 1 ml saline with 10% FCS were infused over 3 min. IV cell injections were performed through an IV line in the ear of the rabbit, using the same parameters as for IA transplantation.

Planar Scintigraphy and SPECT/CT Imaging of ¹¹¹Indium-Labeled Cells

The animals were placed prone in a dual-headed gamma camera with medium energy collimators (Ecam dual head, Siemens, Erlangen, Germany). Planar scintigraphy (10 min, 256 matrix) and SPECT (64 projections, 40 s per view, 128 matrix) was acquired at 6 h (IA group, n = 3; IV group n = 1), day 1 (30 h; IA group, n = 3; IV group n = 1), day 2 (53 h; IA group, n = 2; IV group n = 2), and day 5 (125 h; IA group, n = 4; IV group n = 2) after the cell injections.

CT of the thorax and abdomen was performed in all animals for coregistration and evaluation of adverse reactions using a Siemens Symbia True Point 16 hybrid system (Siemens). To facilitate coregistration, we used a vacuum mattress to fix the position of the animals under imaging and during manual transport between the SPECT-camera and the CT.

Image Processing and Analysis

A Hermes workstation (Hermes Medical Solutions AB, Stockholm, Sweden) was used for reconstruction of the SPECT (4 iter, 8 sub, 3D Gaussian postfilter 0.8 cm FWHM), coregistration of SPECT and CT, and evaluation of images. Using the planar images, regions of interest (ROI) were manually drawn to outline the uptake (counts per pixel) in lungs, liver, kidney, intestine, bladder, and skeletal muscle. All ROI data were decay corrected. Interpretation of uptake was performed by comparing uptake ratios defined as the number of counts per pixel for each organ divided by the number of counts per pixel for skeletal muscle.

Evaluation of the coregistered SPECT/CT images was performed in three orthogonal planes. Interpretation of CT was performed using OsiriX imaging software (OsiriX Foundation, Geneva, Switzerland).

Tissue Analysis

After the last imaging end point at 5 days after cell injections, all animals were sacrificed using a lethal dose of IV pentobarbital (APL, Stockholm, Sweden). Tissue specimens were taken from lungs, liver, and small intestine and stored in 4% formaldehyde (HistoLab, Gothenburg, Sweden). Specimens from the small intestine were taken corresponding to the uptake on the SPECT images. Tissue specimens were embedded in paraffin and cut in 15-μm sections for hematoxylin and eosin staining (HistoLab) according to Mayer's protocol (5).

Results

Selective catheterization of the SMA in rabbits (n = 4) was achieved using clinical routine catheters guided by an angiographical system under sterile conditions. Digital subtraction angiography confirmed the vascular anatomy of the SMA supplying the small intestine, the ascending colon, and the transverse colon (Fig. 1A). Single-cell suspensions of 5 × 10⁶ ¹¹¹In-DSCs were injected manually, and follow-up digital subtraction angiography showed normal flow rates without signs of vessel occlusion. Two rabbits receiving IV injections of 5 × 10⁶ ¹¹¹In-DSCs served as controls. All animals showed normal gait, motility, and exploratory behavior throughout the study until sacrifice at day 5.

Figure 1.

Selective IA injection of 5 × 10⁶ human decidual stromal cells (DSCs) labeled with ¹¹¹In-oxine into the superior mesenteric artery (SMA) versus IV injection into the ear vein. (A) Digital subtraction angiography (DSA) of the SMA in a rabbit model. Distribution of radioactivity using volume rendered single-photon emission computed tomography (SPECT) coregistered with computed tomography (CT) 6 h after (B) SMA injection; distribution of radioactivity is found in areas corresponding to a) the small bowel, b) the ascending, and c) transverse colon, and in d) the liver and (C) IV injection; distribution of radioactivity is mainly concentrated to e) the lungs. In both IA and IV animals, a small amount of radioactivity is seen in kidneys and bladder.

Six hours after ¹¹¹In-DSC injections, animals in the IA group showed uptake in the small intestine, the ascending and transverse colon, and in the liver (Fig. 1B). Uptake within the intestine corresponded to the vascular bed injected at digital subtraction angiography (Fig. 1B). At days 1, 2, and 5 after cell injections, the distribution of uptake was essentially the same. In the IA-injected group, the uptakes were detected in the intestine and liver (Fig. 2), whereas in the IV-injected animals, the uptake was found mainly in the lungs (Fig. 1C). Increasing amounts of uptakes were detected in the kidneys and bladder, presumably resulting from filtration of radiotracer released from cells succumbing to host-versus-graft rejection (Figs. 2 and 3D). The ROI analysis showed a rapid decline of the uptake in lungs and intestine between imaging at 6 h and at day 1, whereas the decrease in activity between days 1 and 5 was lower (Fig. 3A, D).

Figure 2.

Longitudinal in vivo scintigraphy of radioactivity distribution after selective IA versus IV cell injection. Planar scintigraphy images at 6, 30, and 125 h after (A–C) IV injection and after (D–F) superior mesenteric artery (SMA) injection of 5 × 10⁶ human decidual stromal cells (huDSCs), labeled with ¹¹¹In-oxine in a rabbit model. The radioactivity distribution is essentially the same between time points for both groups, indicating retention of cells in the intestine and liver in the IA group and in the lungs in the IV group. Increased radioactivity in kidneys and bladder is seen presumably reflecting radiotracer release from cells followed by renal filtration. Letters indicate regions corresponding to a) small intestine, b) ascending colon, c) transverse colon, d) liver, e) lungs, f) right kidney, g) left kidney, h) bladder, and i) spleen.

Figure 3.

Region of interest (ROI) analysis of organ radioactivity after selective IA (n = 4) versus IV cell injection (n = 2). ROIs were manually drawn using images from planar scintigraphy acquired at 6, 30, 53, and 125 h after IV versus IA injections of 5 × 10⁶ human decidual stromal cells (huDSCs), labeled with ¹¹¹In-oxine, in a rabbit model. Relative uptake ratios were calculated as the uptake in the organ divided by the uptake in skeletal muscle. (A, C) From the graphs showing relative uptake in lungs and intestine, a rapid decline in radioactivity is seen between 6 and 30 h, whereas the decrease in activity between 30 and 125 h is lower.

All animals underwent imaging with whole-body CT for evaluation of potential adverse reactions. Coregistration with SPECT was performed to define organs with uptake (Fig. 4A, C, and E). No signs of acute embolic complications were detected in any of the abdominal or thoracic organs. No effusion was detected in the abdomen or the pleural cavity. No evidence of tissue injury was detected in hematoxylin and eosin stainings of tissue specimens obtained from small intestine, lungs, and liver (Fig. 4B, D, and F).

Figure 4.

Safety analysis using CT and immunohistochemistry after selective IA versus IV cell injection. All rabbits underwent imaging with nonenhanced whole-body CT at 125 h after injection of 5 × 10⁶ human decidual stromal cells (huDSCs), labeled with ¹¹¹In-oxine, to the superior mesenteric artery (SMA) (n = 4) and the ear vein (n = 2). CT scans were examined for signs of adverse reactions. Coregistration with SPECT was performed to define organs with uptake (A, C, and E). No signs of organ injury were detected. No effusion was detected within the abdomen or the pleural cavity. No evidence of tissue injury was detected in hematoxylin and eosin stainings of tissue specimens obtained from lungs (A), liver (B), and small intestine (C).

Discussion

In the last 10 years, several clinical trials on cell therapy have demonstrated safety and efficacy in the treatment of IBD (9,10,14,37). Several factors influence the effectiveness of the therapy, such as cell type, the amount of cells administered, and timing and route of administration. MSCs and hematopoietic stem cells are the most common immunomodulatory cell types used. Recent in vitro studies on DSCs isolated from term placentas indicate that DSCs have potential to suppress alloreactivity by systemic paracrine immunosuppression and promotion of local regulatory T cells (16). Furthermore, DSCs have been tested in humans with severe GVHD with promising results (41).

Cell therapy of IBD using systemic administration requires homing or migration of cells to sites of inflammation. Selective recruitment of IV-injected human MSCs by the inflamed rodent colon has been reported (22). However, mounting evidence suggest that pulmonary trapping and pooling of cells in the spleen and liver limit therapeutic cell exposure to the target sites following IV injection (18). One way to circumvent pulmonary trapping and increase the density of therapeutic cells in inflamed tissue of, for example, the intestines would be by selective injection of cells to the mesenteric arteries. This approach was proven feasible by Dinesen et al. in a single patient with severe CD (13). IA infusion of MSCs was given in a small pilot study to treat severe hepatic or gut GVHD (2). MSCs were infused in the hepatic, gastroduodenal, superior-, and inferior mesenteric arteries. One patient had slightly improved hepatic GVHD and was free from gastrointestinal symptoms, one had a transient response, and one did not respond.

In the present study, we aimed for a systematic description of cell distribution and possible side effects of selective IA cell delivery using imaging-based and histological methods. With 6 days of longitudinal in vivo imaging, we found extensive pulmonary retention of radioactivity following IV injection. Using ROI analysis of the lungs we detected a decline of radioactivity during the first 30 h that did not correspond with the increase in radioactivity in the urinary system. We found the radioactivity distribution in IA animals to be significantly different from that of IV injection. Animals receiving SMA-selective injections showed radioactivity corresponding to the vascular supply of the SMA. Furthermore, substantial amounts of radioactivity were found in the liver. The interpretation of this finding is that a fraction of cells passes the intestinal capillaries without interaction with the endothelium succeeded by portal transport to the liver, where they are retained. When using the same labeling protocol on platelets and leukocytes in humans, stable labeling of cells with minimal excretion of activity in urine and feces is anticipated. We observed a considerable excretion of radiotracer from the renal system. Our interpretation of this is that the excreted radioactivity in the urine comes from labeled DSCs that succumbed to host-versus-graft rejection, as it has been shown that the detected signal from ¹¹¹In-oxine can be attributed to viable cells (8,30). In this study we did not investigate potential effects of ¹¹¹In-oxine on cell viability. However, radiolabeling human mesenchymal cells with ¹¹¹In-oxine demonstrated no effect on cell viability, character, or plasticity, whereas metabolic activity and migration were significantly reduced (21,30). We believe that the evidence for retained cell viability is sufficient for the main end points of this study. However, a potential impact on migratory properties may interfere when conducting experiments, including therapeutic effect as an end point, and appropriate cell assays should then be performed. The animals in this study were not subjected to any kind of immunosuppression. We were primarily interested in testing a rabbit model in a clinical angiographical setup to assess basic safety end points, such as mechanical obstruction of capillaries from solitary cells or cell aggregates.

Selective IA infusion is associated with very low risks—although higher than for IV injection. The overall incidence of vascular complications in the adult population is less than 1% (24,35). Infectious complications following endovascular procedures are exceedingly rare, and there are protocols for antibiotic prophylaxis to immunocompromised patients (7). Dinesen et al. did not report any adverse side effects after two injections, separated by 4 weeks, of 10⁵ MSCs/kg and 10⁶ MSCs/kg in one patient (13). However, considering the consequences of acute occlusion of the SMA and that regions of the bowel are prone to ischemia due to end-artery anatomy (1), a preclinical safety study preceding human studies is well motivated when using cell types and protocols not tested for IA injections. In humans, a dose of 1-2 × 10⁶ cells/kg of MSCs of DSCs seems sufficient to obtain a clinical effect following IV administration (31,41). The cell dose used in the rabbit is comparable to this. We injected 5 million DSCs to the SMA in rabbits weighing 3.5 kg. The animals were observed for 6 days, a time span in which severe ischemia would be fatal, and moderate ischemia would have been detected by clinical signs of distress and changes in stool production. On day 6, whole-body CT did not reveal any secondary signs of occlusion of the SMA or any other adverse tissue reactions. Tissue specimens obtained from parts of the intestine, showing high amounts of radioactivity, and from liver and lungs showed normal histology on hematoxylin and eosin stainings, further demonstrating the safety of the procedure.

Owing to the low immunogenicity of MSCs and DSCs, allogenic cells have often been used in cell therapy. The risk of alloimmunization in immunosuppressed GVHD patients appears to be low, but immunocompetent patients may over time develop anti-HLA antibodies after stromal cell treatment (25). Likewise, splenocytes from mice challenged with human MSCs and DSCs elicit higher cellular responses to human peripheral blood mononuclear cells compared to naive mice, but MSCs and DSCs still fail to induce a proliferative response in cells from immunized mice (25). Human MSCs have, nevertheless, been shown to promote therapeutic effects in several animal models (3,29), and there is little evidence of immediate rejection. Thus, the xenogenicity of human DSCs in the present study has likely only played a minor, if any, role.

The present study provides proof-of-concept evidence of successful integration of a rabbit model in a clinical setting of IA cell delivery to the intestine, demonstrating the benefit of ¹¹¹In cell labeling for determination of cell trafficking and biodistribution. The study is limited by the small study population and by the fact that xenogeneic cells are administered to the normal rabbit. Future studies should include IA administration of allogeneic cells to a rabbit model of intestinal disease, as this situation may affect vascular integrity and cell trafficking.

Conclusion

In this study, we show that selective IA injection of 5 × 10⁶ human DSCs to the normal rabbit SMA does not cause acute embolic complications in a small study population when using clinical routine catheters guided by an angiographical system under sterile conditions. Furthermore, we show that the use of selective injection of DSCs result in an almost dichotomist state of cell distribution. Following IV injections, the vast majority of injected cells are lodged in the lung as opposed to the selective IA injection, where the majority of cells are in the intended target region.

Footnotes

Acknowledgments

This study was supported by the Swedish Society of Medicine, Söderbergska Stiftelsen, Uppdrag Besegra Stroke (supported by the Swedish Heart-Lung Foundation, Karolinska Institutet, Friends of Karolinska Institutet USA and the Swedish order of St John), Åhlén-stiftelsen, Thurings stiftelse, Tore Nilsons stiftelse, and KERIC. The authors declare no conflicts of interest.

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Intra-arterial Administration of Placenta-Derived Decidual Stromal Cells to the Superior Mesenteric Artery in the Rabbit: Distribution of Cells,Feasibility,and Safety

Abstract

Keywords

Introduction

Materials and Methods

Cell Preparation

Labeling Cells with 111Indium

Intra-arterial Administration of DSCs

Planar Scintigraphy and SPECT/CT Imaging of 111Indium-Labeled Cells

Image Processing and Analysis

Tissue Analysis

Results

Discussion

Conclusion

Footnotes

Acknowledgments

References

Labeling Cells with ¹¹¹Indium

Planar Scintigraphy and SPECT/CT Imaging of ¹¹¹Indium-Labeled Cells