Radiofrequency field.

A 465-kHz sinusoidal signal was provided by a signal generator and applied through an amplifier (both Ultraflex, Ronkonkoma, New York) to a 2-turn solenoid coil (radius 2.5 cm) to produce a field strength of 32 or 29 mT. Samples were placed within the solenoid.

Application of static magnetic force.

A solenoid magnetic microneedle was fabricated by winding a 24-G copper wire around a 1/4-inch Permalloy-80 rod 1,200 times and the tip lathed to hemisphere with a diameter of 100 μm. Current through the coil was controlled by a Beckman 3 A/30 V adjustable power supply. The needle tip was placed ∼30 μm from the cells being pulled, and the magnetic force was produced by a 1.8-A current.

Force calibration.

It has been estimated that the force to mechanically open an ion channel is approximately 0.2–0.4 pN33. Therefore, we measured the force from magnetic treatment on the cells using a method described by Kim et al. Briefly, iron-loaded, ferritin-expressing cells were fixed in paraformaldehyde, added to aqueous buffer and subjected to a static magnetic field. The cells accelerate toward the magnet and their velocity is proportional to the magnitude of the magnetic force. This is modeled as a creeping flow around a sphere, where the drag of the fluid on the particle is equal to the magnetic force on the cell34. With U being the cell velocity, μ being the kinematic viscosity of the buffer, and D cell being the cell diameter, this linear relationship is:

HEK cells were loaded with transferrin for 3 d then transfected and collected 24 h later. After fixation, the cells were added to buffer and subjected to magnetic field. The movement of the cells was recorded for 5 s in duplicate. From these two image stacks, the position of 3 cells were tracked over 10 frames for a total of ∼60 cell velocities recorded. For all cells expressing ferritin, the magnetic force was over 10 pN. Cells loaded with iron but not expressing ferritin showed magnetic forces less than 1 pN possibly owing to random Brownian motion of the fixed cells in the liquid. As 10 pN is greater than the force required to open an ion channel, this experiment shows that the magnetic treatment is applying sufficient force to observe channel openings.

Plasmids.

TRPV1 (in pcDNA3.1) was a kind gift from W. Liedkte (Duke University, North Carolina) and cloned into pEGFP-N1 (Clontech, Mountain View, California). A GFP-binding nanobody sequence was synthesized by Integrated DNA technologies (Coralville, Iowa) and fused to the N terminus of TRPV1 to create αGFP-TRPV1. A fusion protein of mouse ferritin heavy and light chains was made by inserting a synthesized flexible linker region containing a FLAG tag sequence (ARGGGGSDYKDDDDKGGGGSRV) between the ferritin chains and cloning downstream of the EF1α promoter in pCR2.1. Mouse ferritin heavy chain was obtained from ATTC (Manassas, VA) in pCMV; sport6 and mouse ferritin light chain 1 was obtained from Invitrogen (Carlsbad, CA) in pYX-Asc. pCR2.1 with EF1α-ferritin chimera was modified by cloning a myristoylation signal to the N terminus of ferritin light chain to create myrferritin or the addition of GFP sequence from Pegfp-n1 to the N terminus of ferritin light chain to create GFP-ferritin. TRPV1 followed by a 2A sequence and ferritin, myrferritin or αGFP-TRPV1 followed by a 2A sequence and GFP-ferritin were cloned into MSCV-hygro plasmids and calcium-responsive furin insulin was cloned into MSCV-puro plasmid (Clontech) for retrovirus production using Phoenix packaging cells. These sequences were also cloned into pVQ Ad CMV KNpA for generation of replication-deficient adenovirus. The fidelity of PCR products and cloning was confirmed by DNA sequencing.

Cell culture and in vitro studies.

Human embryonic kidney cells (HEK 293T (ATCC CRL-3216), mycoplasma testing and STR profiling performed by ATCC) and Phoenix ecotropic packaging cells (Stanford University) were cultured in Dulbecco's modified eagle medium with 10% FBS (Gibco, Carlsbad, California) at 37 °C and 5% CO 2 . Mouse mesenchymal stem cells (MSC) (Gibco, tested for cell-surface markers for MSC by flow cytometry and mycoplasma testing performed by Gibco) were grown in DMEM/F12 medium with 10% FBS at 37 °C and 5% CO 2 .

Stable cell lines were produced by retroviral infection of MSCs using the Phoenix system. Briefly, Phoenix eco cells (2 × 106 cells per 6-cm dish) were transfected with MSCV-puro or hygro plasmids as above. After 24 h, the medium was replaced and the cells placed at 32 °C. Medium was aspirated after a further 24 h, spun to remove cell debris and the supernatant added to MSCs (plated at 1 × 106 cells per 6-cm dish) using a 1:2 dilution in RPMI medium/10% FBS with polybrene (4 μg ml−1, Sigma-Aldrich, St. Louis). Cells were incubated at 32 °C for a further 24 h and then the medium was replaced with DMEM/F12 medium/10% FBS. Selection medium was added 48 h after infection.

Stably transfected MSC (2 × 106 cells in 60 μl medium) were seeded onto 5 × 5 × 5-mm gelatin scaffolds (Gelfoam). Cells were maintained at 37 °C for 4 h, then 450 μl DMEM/F12 medium/10% FBS was added. Cell-scaffold constructs were then maintained at 37 °C for 5 d before implantation.

For immunocytochemistry and RF studies, cells were cultured on 12-mm cover glass (Fisher Scientific, Pittsburgh) coated with collagen (BD Biosciences, Bedford, Massachusetts) and poly-D-lysine (Millipore, Billerica, Massachusetts). Cells were transfected with appropriate constructs 24 h after plating using lipofectamine 2000 (Invitrogen, Carlsbad, California). Medium was replaced 18 h after transfection and holotransferrin (2 mg ml−1, Sigma) added to the cells. Cells were studied 72–96 h after transfection or subculture.

RF-dependent release of calcium-dependent human insulin. 24 h before the study, cells were placed in 1% FBS medium at 32 °C to minimize TRPV1 and calcium-dependent pathway activation. On the day of study, cells were preincubated for 30 min in 500 μl PBS, then incubated in 300 μl of calcium imaging buffer at room temperature (control) or in an RF field at room temperature. The supernatant was removed after 60 min, spun to remove cell debris and frozen at −80 °C until assay. Each study was repeated on two (TRPV1/ferritin) or three occasions (TRPV1/myrferritin and αGFP-TRPV1/GFP-ferritin) each with four replicates. For gene-expression analysis, cells from the supernatant and cover glass were lysed and the lysate stored at −80 °C until RNA purification. Each study was repeated on two (TRPV1/ferritin) or three occasions (TRPV1/myrferritin and αGFP-TRPV1/GFP-ferritin) each with 4 replicates. Control studies with insulin reporter alone, αGFP-TRPV1 and insulin reporter, or GFP-ferritin and insulin reporter were performed on two occasions with four replicates. For studies of apoptosis, three groups were studied: (i) cells were placed at room temperature for 1 h on three occasions, (ii) cells treated with RF for 1 h and then placed at room temperature for a further two occasions or (iii) cells treated with RF for 1 h on three occasions each separated by 1 h. At the end of the study, cells were lysed and the lysate stored at −80 °C until RNA purification. Each study was repeated on 2 occasions each with 4 replicates.

Magnet-dependent release of calcium-dependent human insulin. Cells were prepared as described above. Cells were incubated in 300 μl of calcium imaging buffer at room temperature (control) or treated with a static magnetic field for 5 s every 2 min for 1 h at room temperature. To produce a constant magnetic field, a neodymium-iron-boron permanent magnet was used (K&J Magnetics, Pipersville, Pennsylvania) producing a magnetic flux density of around 5 kiloGauss near the cell surface. The supernatant was removed after 60 min, spun to remove cell debris and frozen at −80 °C until assay. Each study was repeated on 3 occasions, each with 4 replicates. For gene-expression analysis, cells from the supernatant and cover glass were lysed and the lysate stored at −80 °C until RNA purification. Each study was repeated on two occasions, each with four replicates. Control studies with insulin reporter alone, αGFP-TRPV1 and insulin reporter, or GFP-ferritin and insulin reporter were performed on two occasions with 4 replicates. For studies of apoptosis, three groups were studied: (i) cells were placed at room temperature for 1 h on three occasions, (ii) cells treated with magnet for 1 h and then placed at room temperature for a further two occasions or (iii) cells treated with magnet for 1 h on three occasions each separated by 1 h. Each study was repeated on two occasions, each with four replicates.

Calcium imaging.

Transfected cells were washed three times in PBS, then loaded with Fluo-4 3 μM (Invitrogen) in the presence of sulfinpyrazone 500 μM (Sigma) or 1 mM probenecid for 45–60 min at room temperature. Cells were washed again in PBS, then incubated for 15–30 min in sulfinpyrazone in PBS. Cells were then imaged in calcium imaging buffer. Imaging was performed using a Deltavision personal DV imaging system (Applied Precision, Issawaq, Washington) equipped with a custom-made ceramic lens. Cells were imaged before and during RF, bar magnet or solenoid magnet treatment or before and after treatment with 150 μM 2-aminoethoxydiphenyl borate. Imaging was performed on at least three occasions for each condition.

The majority of cells tested with the solenoid magnetic microneedle did not respond. This is in keeping with the findings of Hughes et al.35 who noted that only a small number of cells with ion channel–tethered particles could be activated by a static magnetic field. Ion channels are sensitive to the direction of an applied force, and Hughes et al. propose that a single force direction in a static magnetic field could only open channels sensitive to that direction. Under 40× magnification, a total of approximately 2,000 cells were tested. The image stack generated for each pulling was analyzed in ImageJ. Cell fluorescence was tracked, and a 10% increase in maximum cell fluorescence over baseline (F max /F 0 ) (to exclude basal calcium oscillations) was marked as a channel opening event and a responding cell.

Immunocytochemistry and immunohistochemistry.

Immunocytochemistry and IHC were used to detect expression of TRPV1, GFP and FLAG-tagged ferritin and to quantify apoptotic cells in cells and tissue. Cells or tissue were washed in PBS and then fixed for 15 min in 2% paraformaldehyde (Electron Microscopy Services, Hatfield, Pennsylvania) or 10% formalin (Sigma) at 4 °C overnight then placed in 30% sucrose in PBS at 4 °C for a further 24 h. 20 μm cryosections were cut from tissue and placed directly on glass slides, then heated at 55 °C for 1 h and stored at −80 °C before staining. Cells or tissue sections were incubated for 1 h in blocking buffer (3% BSA (Sigma) and 2% goat serum (Sigma) in PBS with 0.1% Triton-X (Sigma)). Slides were incubated in primary antibody (rabbit anti-TRPV1 1:500 (AB9554 (ref. 36), Chemicon), mouse anti-FLAG 1:1,000 (Anti-FLAG M2 mouse mAb no. F1804, Sigma; ref. 37), chicken anti-GFP 1:1,000 (ab13970 (ref. 38), Abcam), rabbit anti-activated caspase-3 1:250 (G7481, Promega39)) in blocking buffer overnight at 4 °C. Slides were washed three times in PBS before incubation in secondary antibody (goat anti-rabbit 594 (A1012) or goat anti-rabbit 488 (A11008), goat anti-chicken 488 (A11039), goat anti-mouse 350 (A11045), all 1:1,000) in blocking buffer for 2 h. Slides were washed a further three times in PBS before mounting using Fluoromount (Southern Biotech, Birmingham, Alabama).

Images were acquired using a Zeiss Axioplane microscope and captured digitally with separate band-pass filters using the multichannel module of the AxioVision Zeiss software. Additional images were acquired using confocal microscopy (LSM 510 laser scanning confocal microscope; Carl Zeiss MicroImaging). Quantification of active caspase-3 immunostaining was performed by an investigator blinded to the treatment group.

Animals and in vivo studies.

Male athymic NCr-nu/nu (nude) mice (NCI-Frederick, 6–8 weeks) or male C57BL/6 mice (8–9 weeks) were used and housed under controlled light conditions (12 h light/12 h dark) and temperature (22 °C), single-caged and fed ad libitum on standard mouse chow. Animal care and experimental procedures were performed with the approval of the Animal Care and Use Committee of Rockefeller University (protocol 11421) under established guidelines. In all cases, mice were randomized according to body weight after STZ treatment. The investigator was not blinded to the treatment group. The sample size required was estimated to be n = 6–8 per group on the basis of previous studies examining the effects of RF treatment on gene expression and protein release using exogenous nanoparticles.

Study 1. Nude mice were treated for 5 d with low-dose streptozotocin. Two days later, MSCs seeded onto gelatin scaffolds were implanted into the flank of anesthetized nude mice bilaterally. Radiofrequency studies were performed 4 weeks later. Mice received intraperitoneal iron dextran (50 μl of 100 mg/ml) 5 and 3 d before the study. Mice were fasted overnight before all studies. On the study day, mice with MSC implants (calcium-dependent insulin alone (control), TRPV1/myrferritin with calcium-dependent insulin or αGFP-TRPV1/GFP-ferritin with calcium-dependent insulin, n = 6–8/group) were anesthetized with inhaled isoflurane. After 30 min, mice were treated with an RF field for 60 min. Tail vein samples were taken at −30, 0 min before and at 15, 30, 45, 60, 75, 90 and 120 min after the onset of RF treatment. Retro-orbital blood was taken using EDTA-coated capillary tubes at −30 and 60 min for plasma insulin measurement. After 120 min, approximately half the mice in each group were sacrificed and the implants removed. Each tumor was divided in two and one half snap frozen in liquid nitrogen for RNA extraction and the other half placed in 10% formalin for IHC. Tissue was harvested from the remaining mice 24 h later after identical anesthesia but no RF treatment. For control group, n = 4 no RF, 7 RF-treated; for TRPV1/myrferritin, n = 4 no RF, 5 RF-treated; and for αGFP-TRPV1/GFP-ferritin, n = 7 no RF, 5 RF-treated.

Study 2. C57BL/6 mice were treated for 5 d with low-dose streptozotocin. Two days later, replication-deficient adenoviruses expressing LacZ, TRPV1/myrferritin with calcium-dependent insulin or αGFP-TRPV1/GFP-ferritin with calcium-dependent insulin were injected into the jugular vein of anesthetized C57BL/6 mice. Iron was given as above. After 4 weeks, mice were studied as described in study 1. For hepatic insulin gene expression, control group: n = 3 no RF, 5 RF-treated; TRPV1/myrferritin: n = 3 no RF, 3 RF-treated; αGFP-TRPV1/GFP-ferritin: n = 3 no RF, 3 RF treated. For plasma insulin levels, blood glucose and cumulative change in blood glucose, control group: n = 7, TRPV1/myrferritin: n = 5 and αGFP-TRPV1/GFP-ferritin: n = 6.

Study 3. C57BL/6 mice were treated with low-dose streptozotocin and replication-deficient adenovirus expressing LacZ, TRPV1/myrferritin with calcium-dependent insulin or αGFP-TRPV1/GFP-ferritin with calcium-dependent insulin (n = 6/group) as described above. Iron was given as above. After 3 weeks mice were treated as above. One week later, mice were treated with a lower RF field strength (29 mT) with monitoring as above. After an additional week, mice were treated as described in study 2 but at 60 min after the onset of RF treatment, mice were sacrificed and blood taken by cardiac puncture for assessment of insulin and C-peptide. Hepatic tissue was harvested and frozen in liquid nitrogen to assess apoptotic gene expression. A set of STZ-treated, adenovirus-injected mice (n = 4–6) that had not received previous RF treatment were anesthetized and liver harvested and snap frozen to assess apoptotic gene expression.

Study 4. C57BL/6 mice were prepared (control, n = 7, αGFP-TRPV1/GFP-ferritin, n = 6). Mice received two doses of intraperitoneal iron dextran (50 μl of 100 mg/ml) 5 and 3 d before the first study and then 3 d before each subsequent study. The first RF study was performed 2 weeks after virus injection and weekly thereafter until 6 weeks. The study protocol was as described for study 1 on each occasion.

Study 5. C57BL/6 mice were prepared as for study 2. After 4 weeks, half the mice were studied using magnet stimulation using an identical protocol to study 1 but with a static magnetic field for 5 s every 2 min for 1 h as above and half remained untreated. One week later, the previously treated mice were assessed without magnet treatment and the previously untreated mice were treated with a static magnetic field (n = 8).

Study 6. C57BL/6 mice were treated with low-dose streptozotocin as described above. After 4 weeks, mice were fasted overnight, then anesthetized with inhaled isoflurane. After 15 min, mice were treated with an intraperitoneal injection of either HumulinR (0.1 U kg−1) or an equal volume of PBS. Tail vein samples were taken at −15 and 0 min before treatment and at 15, 30, 45, 60, 90 and 120 min after injection. Retro-orbital blood was taken using EDTA-coated capillary tubes at −15 and 60 min for plasma human and mouse insulin measurement to examine the effects of isoflurane on endogenous insulin release and the effects of IP HumulinR on blood glucose and human insulin levels. PBS treated group, n = 7; HumulinR treated group, n = 5.

Assays.

Proinsulin was measured in cell supernatants by ELISA (Alpco, Salem, New Hampshire) according to manufacturer's protocol. Blood glucose was determined using a Breeze 2 glucometer (Bayer, Leverkusen, Germany). Blood was spun for 10 min and plasma was collected. Plasma levels of human insulin were determined in mouse plasma by human-specific ELISA (Alpco). Plasma levels of human C-peptide were determined in mouse plasma by human-specific ELISA (Mercodia, Winston-Salem, North Carolina).

Real-time PCR.

Total RNA was isolated by homogenizing tissue in TRIzol reagent (Invitrogen) or cells in buffer RLT and purifying the RNA using Qiagen RNA prep kit. Complimentary DNA was synthesized using Qiagen omniscript RT kit. Real-time PCR was performed using the TaqMan system (Applied Biosystems, Foster City, California) according to the manufacturer's protocol.

Statistics.

Data over 2 s.d. outside the mean were excluded from further analysis as determined before the studies. All data were tested for Gaussian distribution and variance. Data with normal distribution and similar variance were analyzed for statistical significance using two-tailed, unpaired Student's t-test unless otherwise indicated. Data with normal variation and unequal variance were analyzed by two-tailed Welch's t-test. Paired data were analyzed by paired t-test. Data which were not normally distributed were analyzed by two-tailed Mann-Whitney test. P values are as indicated. Data are shown as mean ± s.e.m. unless otherwise stated.