We thank Prof. Robert Campbell at the University of Tokyo for sharing the material and valuable discussion; Dr. Chia-Lin Ho in Molecular Device for technical support.

Left ventricular non-compaction cardiomyopathy (LVNC) patient iPSC-derived cardiomyocytes (iPSC-CM) were provided by the National Taiwan University Hospital. The collection of patient samples for reprogramming to iPSC and the maintenance protocols14 for research purposes followed the WMA Declaration of Helsinki (ethical principles for medical research involving human subjects) and were approved by Institutional Review Board: Research Ethics Committee Office at NTUH (#201612099RINC). Other iPSC-CM&#39;s were obtained from commercial vendors. The use of iPSC&#160;derivatives does not require specific permissions in our institution.
1. iPSC derived cardiomyocytes preparation
<ol>
	<li>Prepare the multi-well plate before iPSC-CM is removed from cold storage for thawing. Coat each well of the 96-well microplate with 100 &#181;L of 50 &#181;g/mL fibronectin/0.2% gelatin solution (see Table of Materials) to cover all surfaces thoroughly.</li>
	<li>Incubate the plate at 37 &#176;C for 1 h in a humidified incubator.</li>
	<li>Warm the medium (see Table of Materials) to room temperature for 30 min before cell thawing. 
	NOTE: The present protocol describes room temperature as 25 &#176;C.</li>
	<li>Transfer the iPSC-CM from the liquid nitrogen storage tank to a 37 &#176;C water bath. 
	NOTE: iPS-CMs are typically shipped on dry ice by academic or commercial sources. They are transferred to liquid nitrogen storage on receipt until use.</li>
	<li>Remove the vial before the ice melts entirely and spray the vial with 75% ethanol.</li>
	<li>Take the vial to a Class II biosafety cabinet and gently transfer the contents to a new 15 mL conical tube containing 9 mL of room temperature medium.</li>
	<li>Centrifuge the cells at 200 x g for 3 min at room temperature.</li>
	<li>Discard the supernatant by pipette and gently resuspend the cells in 1 mL of medium with 10 &#181;M of ROCK inhibitor (Y27632) (see Table of Materials). 
	NOTE: Avoid repeated pipetting of thawed cells to ensure maximum cell recovery.</li>
	<li>Remove the coating solution from the well and add 50 &#181;L of medium with 10 &#181;M of ROCK inhibitor quickly.</li>
	<li>Confirm the number of viable cells using the trypan blue exclusion method with a hemocytometer15.</li>
	<li>Plate cells in coated 96-well plate at a density of 100,000-150,000 cell/cm2,as per manufacturer&#39;s instructions16.</li>
	<li>After 24 h, ensure that the cells attached to the surface of the plate are in contact with other cells. 
	NOTE: Single cells survive poorly in long-term culture and typically behave more variably.</li>
	<li>Replace pre-warmed maintenance medium every 2 days. 
	&#8203;NOTE: For long-term drug treatment, the LVNC iPSC-CM were changed with half medium replacements with, or without, small molecule drugs every 2 days.</li>
</ol>
2. Expression of genetically encoded calcium indicators
<ol>
	<li>After plating the cells for 72 h, thaw the GECIs viral kits (see Table of Materials) on ice.</li>
	<li>Prepare a 2x viral kit stock solution with the maintenance medium.</li>
	<li>Prepare the cells for infection by adjusting the medium volume to 100 &#181;L per well. Add 100 &#181;L of the pre-mixed viral solution and incubate for 8-16 h at 37 &#176;C.</li>
	<li>Replace with 200 &#181;L pre-warmed medium after incubation.</li>
	<li>Visualize the GECIs expression 24 h post-transduction using an imaging system (see Table of Materials). 
	NOTE: The expression level reaches the maximum at 48-72 h post-transduction. Cells can be imaged from the 24 h time-point. However, the signals obtained from GECIs sustain for more than one month.</li>
	<li>Maintain cells in the humidified incubator before functional assays are performed.</li>
	<li>Infect the iPSC-CMs (LVNC patient-derived iPSC-CMs) with ChR2-K-GECO viral kit (see Table of Materials) at day 25 post differentiation using the infection protocol (step 2.1-2.6).</li>
</ol>
3. Dye-based calcium imaging using Fluo-4
<ol>
	<li>Dissolve Fluo-4 powder (see Table of Materials) in DMSO as a stock solution (5 mM).</li>
	<li>Dilute the stock solution to a concentration of 10 &#181;M Fluo-4 in Tyrode&#39;s buffer. 
	NOTE: Tyrode&#39;s buffer consists of 1.0 g/L of sodium bicarbonate, 0.2 g/L of calcium chloride, 0.1 g/L of magnesium chloride, 0.2 g/L of potassium chloride, 8.0 g/L of sodium chloride, 0.05 g/L of sodium phosphate monobasic, and 1.0 g/L of D-glucose.</li>
	<li>Replace half medium with 10 &#181;M of Fluo-4 solution (giving a final concentration of 5 &#181;M).</li>
	<li>Incubate the cells at 37 &#176;C for 30 min.</li>
	<li>Wash the cells with pre-warmed Tyrode&#39;s buffer and replace them with the medium before image acquisition.</li>
	<li>Start recording with the stream acquisition protocol. 
	&#8203;NOTE: Stream acquisition is continuously acquiring images without interval time. The beating frequency of iPSC-CM is about 1 Hz, and this stream acquisition protocol is used for collecting beating patterns.</li>
</ol>
4. Image acquisition and functional assays for toxicity test and phenotypic screening
<ol>
	<li>Switch on all the devices of the High-Content Imaging System (see Table of Materials).</li>
	<li>Verify that the chamber temperature reaches 37 &#176;C, with 5% CO2 supplementation. Keep the system at equilibrium for 2 h before image acquisition. 
	NOTE: Thermal drift is a significant problem for long-term live-cell observations, 1 &#176;C can change the focal plane by ~1 &#181;m, so allowing the lens, and stage, to reach the target temperature is essential before image acquisition.</li>
	<li>Open the imaging software (see Table of Materials) and choose the plate setting for a 96-well plate.</li>
	<li>Place an unlidded 96-well plate into the chamber and load with the live-cell sealing ring on top. 
	NOTE: The live-cell sealing ring is an adapter for reaching environmental equilibrium.</li>
	<li>Choose the 20x (Figure 1 and Figure 2), 60x (Figure 3 and Figure 4), and 20x (Figure 5) water immersion objective lens according to the spatial resolution required.</li>
	<li>Adjust the focus to take clear images before experimental acquisition.</li>
	<li>Select 3-5 regions randomly per well for calcium activity recording. 
	NOTE: Every region must include at least 20 cells (n &#8805; 20).</li>
	<li>Choose appropriate filters for different indicators. FITC 475/536 for mNG-GECO, mtGCEPIA3, and Oria1-G-GECO; Cy5 631/692 for NIR-GECO2; ER 530/593 for ER-LAR-GECO; Texas Red 560/624 for K-GECO.</li>
	<li>Set the appropriate light-emitting diode (LED) power for each imaging channel used. 
	NOTE: When optimizing LED power for imaging acquisition by signal (detected intensity of object) to noise (detected intensity of background) ratio, the S/N ratio should be higher than 2. In this protocol, 10% LED power for TRITC and FITC; 20% for Cy5 was typical.</li>
	<li>Set the camera exposure time to a maximum of 40 ms (25 Hz) and a recording duration of 30 sfor stream acquisition to observe calcium transients reported by mNG-GECO and K-GECO probes expressed in cardiomyocytes (Figure 1-3 and Figure 5). Increase the LED power if the signal/noise is &#60;2 at higher frame rates.</li>
	<li>For optogenetic stimulation (Figure 2 and Figure 3C,D), optimize the power (typically 5%-10%) and frequency of blue light at 1 Hz. 
	NOTE: Generally, human iPSC-CM beats spontaneously around ~ 1 Hz, and the operator needs to pace faster than the spontaneous rate.</li>
	<li>If sequential (Figure 3) or extended (Figure 4) measurements are planned, avoid phototoxicity at all costs. This may mean reducing illumination power intensity and compensating for this by increasing camera exposure time, for example, to 100 ms with the time-lapse protocol used for three-channel real-time observation of subcellular Ca2+ signals (Figure 4).</li>
	<li>Use the asynchronous dispense protocol for 10 mM caffeine addition via the auto microfluidics system (see Table of Materials) (Figure 4). 
	NOTE: The asynchronous dispense protocol allows image acquisition to occur while drugs such as caffeine are added, without gaps between images.</li>
	<li>Start acquisition.</li>
</ol>
5. Small molecule preparation
<ol>
	<li>Resuspend the E4031, dofetilide, and verapamil in DMSO and dilute them with Tyrode&#39;s buffer. The final concentration of DMSO in the medium was 0.1% (v/v).</li>
	<li>Prepare the compound solutions at the double (i.e., 2x) the desired working concentration and equilibrate at 37 &#176;C before addition. 
	NOTE: Minimizing temperature fluctuations following medium addition is vital as contractility is temperature-dependent, and the effect of changing temperature is rapid.</li>
	<li>Arrange compounds to the corresponding target well.</li>
	<li>Add 100 &#181;L of the double strength (2x) compounds via the auto microfluidics system (see Table of Materials) into the wells and start acquisition after the desired (typically 15 min) incubation period.</li>
</ol>
6. Imaging processing and analysis
<ol>
	<li>Isolate the signal area from the background by detecting the feature of the pulse over time-revolution automatically with the software.</li>
	<li>Use the Calcium peak analysis software (see Table of Materials) to analyze Beating frequency (BPM), Peak signal (Amplitude), Calcium transient duration 50% (CTD50), Calcium transient duration 90% (CTD90), rise time, and decay time (Figure 1C). 
	NOTE: In these settings, photobleaching was apparent over the first 10 s, and the signals are stabilized after that. Thus, the calcium peak was analyzed from 11-30 s. In three-channel recordings (Figure 4), the cell contour is bordered manually for measuring intensity change.</li>
</ol>

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LumiSTAR has filed for patent protection of the use of K-GECO, NIR-GECO, and LAR-GECO.

To successfully perform high-throughput screening, signals must be evenly distributed across the culture vessel, guaranteeing that randomly selected regions of interest (ROIs) can be applied automatically during imaging acquisition. Although chemical calcium indicators easily meet this requirement, genetically encoded probes have historically produced patches, rather than sheets, of signal limiting their use. The use of a viral transduction system delivers high27,28 and equivalent expression levels of multiple indicators among the target cells compared to conventional transfection methods. Different cell types may require different promoters, and the choice of gene delivery system may need to change accordingly29,30,31. In heterogeneous cell models, transduction and expression efficiency may be different in specific cell types. On the one hand, this may limit the application to particular cells, but the phenomenon can be exploited to distinguish particular cells in a co-culture system32.
The main impact of this approach is the liberation from the one-to-one relationship between the experimenter and the sample by automation. In a standard multi-well experiment, for collecting data at four positions within a single well, we estimate it takes about 15 h at the microscope to gather 30 s videos from a 96 well plate. In the automated system, this takes ~4 h. Furthermore, analysis of the data manually takes far longer than data acquisition. The analysis system used here is about 200 times faster than the manual equivalent. The net result is an improved workflow and high cost and time savings.
In contrast to primary cardiomyocytes loaded with calcium dyes that survive in the order of hours, GECIs expressed in iPSC-CM&#39;s via viral delivery systems can yield a signal in an experimental model that lives for a few months. These models can be maintained in a multi-well format and tracked simultaneously for serial analysis in high-content imaging systems developed for small molecule screening. This produces a simple human experimental platform to test drug effects over durations closer to those experienced by patients (weeks or months) rather than the minutes typically used in toxicology assays.
The system can be expanded to deliver multi-parameter measurements by including GECIs with distinct emission properties from green to near infra-red. Such experiments require proper excitation light and filter sets in the experimental design to keep signals separate. Broad application in a drug screening context requires robust and efficient analysis platforms in addition to drug dispensing. Although the model described here focuses on the use of multiple dynamic calcium indicators, this could be modified to structural markers of cell form or function. These are typically easier to visualize as they show little beat-to-beat variability compared to the hundred-fold variation in intracellular calcium concentration. This approach may be particularly valuable for rare-disease where the iPSC intermediate derived from patients may contain all the genetic drivers and modifiers of a particular condition which can be preserved in a scaleable cell model, facilitating drug discovery in various diseases based on cellular phenotypes that can be visualized independently of the underpinning biological mechanism which may be challenging to elucidate.

Human-based induced pluripotent stem cells (iPSC)-derived models are considered a promising solution to the 3Rs aims (Replacement, Reduction, and Refinement) set out for animal studies1,2. iPSC-derived cardiomyocytes have been used for disease modeling and drug discovery as they recapitulate essential aspects of human biology3. Calcium imaging, typically with chemical dyes, has been used to observe cellular activity before and after drug treatment4,5. However, chemical dye-based calcium-sensing probes directly inhibit the Na, K-ATPase and disrupt cellular function6. Thus, tracking the same cells over hours or days has been problematic when chemical dyes are used. Here, a range of genetically encoded calcium indicators (GECIs)7,8,9,10 are utilized across a broad excitation/emission and calcium affinity spectrum to form a cardiac toxicity testing platform offering real-time multi-organelle measurements over extended periods of time11.
To further develop the high-throughput calcium-based screening concept in the iPSC-CM model beyond the previously demonstrated academic context using genetically encoded tools for optical control and calcium imaging by co-expression of a red-shifted calcium indicator, R-GECO or K-GECO with an optogenetic tool, ChR212,13, off-the-shelf viral kits have been generated. By transitioning imaging into a high-content instrument equipped with an auto-microfluidic system, single-channel pipetting of compound addition is layered on top of live-cell imaging. Finally, both crucial aspects of the experimental pathway, image processing, and analysis are improved and automated.

<table><tbody><tr><td>96-well plate</td><td>Perkin Elmer</td><td>6055302</td><td></td></tr><tr><td>auto microfluidic system</td><td>Molecular Device</td><td></td><td>A device has a single channel pipetter that can be used to add compound automatically</td></tr><tr><td>Caffeine</td><td>Sigma-Aldrich</td><td>C0750</td><td></td></tr><tr><td>Cardiosight-S l iPSC derived cardiomyocytes</td><td>NEXEL</td><td>C-002</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Millipore Sigma</td><td>1096780100</td><td></td></tr><tr><td>Dofetilide</td><td>Sigma-Aldrich</td><td>PZ0016</td><td></td></tr><tr><td>E4031</td><td>Tocris</td><td>1808</td><td></td></tr><tr><td>ER-LAR-GECO viral kit</td><td>LumiSTAR</td><td>AA001a</td><td>Red-shifted GECI packed into adeno-viral vector</td></tr><tr><td>Fibronectin</td><td>Sigma-Aldrich</td><td>F1141</td><td></td></tr><tr><td>Fluo-4 AM</td><td>Invitrogen</td><td>F14201</td><td>Chmical calcium sensitive dye</td></tr><tr><td>Gelatin</td><td>Sigma-Aldrich</td><td>G1890</td><td></td></tr><tr><td>ImageXpress Micro Confocal High-Content Imaging System</td><td>Molecular Device</td><td></td><td></td></tr><tr><td>iPSC-CMs Maintenance Medium</td><td>NEXEL</td><td>CMS-002</td><td>iPSC-CMs Medium (Cardiosight-S medium) + Cardiosight-S Supplement</td></tr><tr><td>iPSC-CMs Medium (Cardiosight-S medium)</td><td>NEXEL</td><td>CMS-002</td><td></td></tr><tr><td>K-GECO viral kit</td><td>LumiSTAR</td><td>AA005a</td><td>Red-shifted GECI packed into adeno-viral vector</td></tr><tr><td>LumiCAL software</td><td>LumiSTAR</td><td>LUCS01a</td><td>Software for analysis of calcium peak in cardiomyocytes</td></tr><tr><td>mNG-GECO viral kit</td><td>LumiSTAR</td><td>AL008a</td><td>Brighter green GECI packed into lenti-viral vector</td></tr><tr><td>mt-GCEPIA3 viral kit</td><td>LumiSTAR</td><td>AL011a</td><td>GECI targgeting on mitochondria packed into lenti-viral vector</td></tr><tr><td>NIR-GECO viral kit</td><td>LumiSTAR</td><td>AV004a</td><td>Near infrared GECI packed into viral vector</td></tr><tr><td>Orai1-GGECO viral kit</td><td>LumiSTAR</td><td>AL010a</td><td>GECI targgeting on Orai1 packed into lenti-viral vector</td></tr><tr><td>Tyrode&#039;s salts</td><td>Sigma-Aldrich</td><td>T2145</td><td></td></tr><tr><td>Verapamil hydrochloride</td><td>Sigma-Aldrich</td><td>V4629</td><td></td></tr><tr><td>Y-27632 dihydrochloride</td><td>Tocris</td><td>1254</td><td></td></tr></tbody></table>

high-throughput optical controlling and recording calcium signal in ipsc-derived cardiomyocytes for toxicity testing and phenotypic drug screening

Understanding how excitable cells work in health and disease and how that behavior can be altered by small molecules or genetic manipulation is important. Genetically encoded calcium indicators (GECIs) with multiple emission windows can be combined (e.g., for simultaneous observation of distinct subcellular events) or used in extended applications with other light-dependent actuators in excitable cells (e.g., combining genetically encoded optogenetic control with spectrally compatible calcium indicators). Such approaches have been used in primary or stem cell-derived neurons, cardiomyocytes, and pancreatic beta-cells. However, it has been challenging to increase the throughput, or duration of observation, of such approaches due to limitations of the instruments, analysis software, indicator performance, and gene delivery efficiency. Here, a high-performance green GECI, mNeonGreen-GECO (mNG-GECO), and red-shifted GECI, K-GECO, is combined with optogenetic control to achieve all-optical control and visualization of cellular activity in a high-throughput imaging format using a High-Content Imaging System. Applications demonstrating cardiotoxicity testing and phenotypic drug screening with healthy and patient-derived iPSC-CMs are shown. In addition, multi-parametric assessments using combinations of spectral and calcium affinity indicator variants (NIR-GECO, LAR-GECO, and mtGCEPIA or Orai1-G-GECO) are restricted to different cellular compartments are also demonstrated in the iPSC-CM model.

The observation of spontaneous calcium oscillation in mNG-GECO10 expressed by iPSC-derived cardiomyocytes (iPSC-CMs) with or without drug treatments is demonstrated in Figure 1 and Animated Video 1. Kinetic traces obtained using mNG-GECO show that small molecular ion channel inhibitors, verapamil, dofetilide, and E4031, affected calcium transients (Figure 1B) as expected. The typical calcium transient shown in Figure 1C can be analyzed by Calcium peak analysis software to derive Peak signal (Amplitude), Calcium transient duration 50% (CTD50), Calcium transient duration 90% (CTD90), rise time, and decay time. Verapamil, an L-type calcium blocker17, completely inhibits the calcium influx in the cells as expected (Figure 1B). Dofetilide and E4031 are hERG channel inhibitors18,19,20, listed as experimental class III antiarrhythmic drugs. The decay time is prolonged with both Dofetilide (p &#60; 0.05) and E4031 treatment (p &#60; 0.001) when compared with the vehicle group. Prolongation of CTD50 (p &#60; 0.01) and CTD90 (p &#60; 0.001) with E4031 treatment was observed compared with vehicle only (Table 1), these results are consistent with reported effects on the QT interval, a clinically relevant measurement of repolarization determined from the surface electrocardiogram from a long time between the start of the Q-wave and the end of T-wave, in previous studies12,21.
A difficulty of CTD assessment in spontaneously beating cells is the variability beat rate imposes on the CTD. This is a particular problem for the iPSC-CM model, where each well of a 96 well plate can have its own beat rate even though all cells come from the same parent vial. It is possible to impose a beat rate by optical or electrical pacing. To evaluate the dose-response of E4031 under standardized paced conditions, ChR2 and K-GECO7 expressing iPSC-CMs were optical controlled using 1 Hz 470 nm pulses of light and observed using the red K-GECO signal (Figure 2A and Supplementary Figure 1). Progressive reductions of peak amplitude (p &#60; 0.01) and increase in decay time (p &#60; 0.05) of the calcium transients occur with increasing concentrations of E4031 (Figure 2B1). A dose-dependent effect of E4031 was most apparent for the reductions in peak amplitude (Figure 2B1,B2).
Although short-term studies lasting minutes are typically used for cardiotoxicity assessments, there is a blind spot for chronic toxicity assessments which parallel patient exposures to drugs over weeks, months, or years. It would be advantageous to study disease, or toxicity, effects over the intervals reflecting the treatment durations relevant to clinical practice. We utilized our all-optical control and detection system to study the iPSC-derived cardiomyocytes from a patient with left ventricular non-compaction (LVNC). iPSC-CMs were transduced with a K-GECO viral kit (step 2.2-2.6) post differentiation Day 25. The K-GECO signal was then tracked every 1-2 weeks in the same wells, with or without additional drug treatments. Both spontaneous calcium activity (Figure 3A,3B) and calcium dynamics under optical pacing (Figure 3C,D, step 4.11) were obtained using the high-Content Imaging System (step 4.1-4.12) and processed by calcium peak analysis software (step 6). As demonstrated in Figure 3A,C, clear calcium transients remain visible a month after viral transduction, with or without the additional light used for optical pacing. This work identified a drug that appears to increase beat rate, regularise contraction interval, and increase the transient calcium amplitude in the LVNC iPSC-CM model (Figure 3A,B). There are two important observations to make from this data. Firstly, from the therapeutic perspective, the drug effect appears durable at Day 63 and Day 70-time points. Secondly, and of relevance to a field which in general assays compound effects during brief windows (&#60;1 min) at earlier time-points (typically &#60;50 days); the disease phenotype modifying effects of the compound are only apparent after Day 60, suggesting it may be easy to discount drugs which have slow mechanisms of action in standard screening protocols.
The variability of beat frequency, and the subsequent impact on calcium transient duration, is not just a well-to-well problem at any given time point. It also changes as iPSC-CM are maintained in culture, which varies within a well over time (Figure 3B). To impose consistency within sequential experiments, 1 Hz optical pacing can be applied with or without drug treatment (Figure 3C,D). Here although the Day 63 results show the encouraging trends in the calcium transient with a significantly higher amplitude, shorter CTD90, and shorter decay time; by the 70 day time-point - and indicative of the need for chronic assessments during the drug screening process for rare disease - early after depolarizations (EAD) are detected. The value of adding a pacing protocol to disease phenotyping, or small molecule evaluation, can be qualitatively assessed by comparison of the results presented in Figure 3B,D for beat rate, calcium transient amplitude, CTD90, and decay time.
An advantage of a GECI, compared to chemical dye, based methodology to visualize the calcium transient is the ability to restrict the probe to a specific compartment within a cell. This can be further expanded by multiplexing multiple color and/or affinity variants in single cells. Hence, several GECIs including ER-LAR-GECO9, mtGCEPIA22,23 (or Oria1-G-GECO), and NIR-GECO28,24 have been developed for expression singly, or in combination, in cell models for real-time calcium activity measurement arising in the endoplasmic reticulum / sarcoplasmic reticulum (ER/SR), mitochondria (or CRAC channel) and cytosol, respectively. They can be combined in the iPSC-CM model (Figure 4A,C) to study the interaction between different intracellular calcium stores. For example, 10 mM caffeine treatment can be used to empty the SR calcium store. Here a decrease of ER-LAR-GECO signal (indicative of loss of calcium from the SR) paralleled a decline of the NIR-GECO signal (indicative of an increase in cytosolic calcium concentration) as expected. How other intracellular compartments are affected by these fluctuations is not well studied. Still, the inclusion of a green mitochondrial-targeted mtGCEPIA3 shows that calcium uptake occurs in mitochondria in these conditions (Figure 4A,B).
Similarly, visualizing the interplay between different calcium currents can be seen by including a probe, Orai1-G-GECO25, to reveal the calcium release-activated channel (CRAC) Ca2+ current (Figure 4C,D). In the iPSC-CM model, this signal increases with the spontaneous cytosolic calcium transient (Figure 4D1,D2). In keeping with this, treatment with caffeine evokes a large calcium transient in both the cytosol and from the Orai1 channel.
It is acknowledged that most calcium transient imaging in cardiomyocyte models has been determined using calcium dyes26. A genetically encoded calcium indicator was compared to a chemical dye in the iPSC-derived cardiomyocyte model to enable a side-by-side comparison of different calcium imaging approaches. K-GECO expressing iPSC-CMs were imaged alongside Fluo-4 loaded cells. K-GECO expressing cardiomyocytes showed consistent beating behavior over time (Figure 5A,C). However, Fluo-4 loading impacted both the beat frequency and CTD in this model (Figure 5B,C), suggesting Fluo-4 itself might influence the results of such experiments. This will be especially true after long-term exposure to the imaging probe, which can occur in the multiwall imaging format if well-by-well imaging occurs with a minute dwell time per well.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63175/63175fig01.jpg" /> 
Figure 1: Small molecule ion channel inhibitors applied to iPSC-derived cardiomyocytes. (A-B) Time-lapse images of iPSC-CM expressing mNG-GECO taken at 25 Hz. Scale bar = 50 &#181;m. (B) Representative Ca2+ oscillations after compound addition. Fluorescent signals obtained from mNG-GECO expressing cells are presented as a fluorescence ratio F/F0, where F0 is defined as basal intensity and F the intensity detected at each time point. (C) Calcium peak analysis software can extract parameters including half-maximal width (CTD50), 90% transient duration (CTD 90), rise time, and decay time. <a href="https://www.jove.com/files/ftp_upload/63175/63175fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63175/63175fig02.jpg" /> 
Figure 2: Dose-response example using the hERG inhibitor E4031 within an optical pacing system. (A) Traces of optical stimulation with 10% blue light in different concentrations of E4031. Time-lapse images of iPSC-derived cardiomyocytes expressing K-GECO taken at 25 Hz. (B1) Representative traces of calcium transients obtained with different compound doses. Peak analysis and dose-response of E4031 in amplitude (B2) and Decay time (B3). Blue bars indicate 1 Hz 470 nm pulse light stimulation. <a href="https://www.jove.com/files/ftp_upload/63175/63175fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63175/63175fig03.jpg" /> 
Figure 3: All-optical platform applied for long-term drug phenotypic screening in LVNC patient-derived cardiomyocytes. (A) Representative trace of spontaneous beating activity in patient-derived cardiomyocytes (post differentiation Day 70) with (red) or without (blue) 5 &#181;M drug treatment. (B) Peak analysis of spontaneous beating activity with or without 5 &#181;M drug treatment. (C) Intensity traces of drug response in patient-derived iPSC-CM under 1 Hz optical stimulation. (D) Peak analysis of patient-derived cardiomyocytes with 1 Hz optical stimulation. <a href="https://www.jove.com/files/ftp_upload/63175/63175fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63175/63175fig04.jpg" /> 
Figure 4: Three channels subcellular Ca2+ imaging in the iPSC-CM model. (A-B) iPSC-CMs were transduced with subcellular calcium probes for the cytoplasm, NIR-GECO2 (red), the endoplasmic reticulum ER-LAR-GECO (yellow), and mtGCEPIA3 (cyan), a mitochondrial Ca2+ indicator. (B) Three-channel time-lapse calcium imaging before and after 10 mM caffeine treatment. (C-D) iPSC-CMs transduced with cytoplasmic, NIR-GECO2 (red), endoplasmic reticulum ER-LAR-GECO (yellow), and CRAC channel indicators Orai1-G-GECO (cyan) were visualized. (D) Three-channel time-lapse calcium imaging was captured. (D1) Spontaneous beat-to-beat activity was observed with NIR-GECO2 and Orai1-G-GECO. (D2) Calcium efflux from the ER (decrease of ER-LAR-GECO signal) accompanied an increase in the cytosolic calcium signal upon caffeine treatment. <a href="https://www.jove.com/files/ftp_upload/63175/63175fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63175/63175fig05.jpg" /> 
Figure 5: Comparison of the genetically encoded calcium indicator (K-GECO) to the chemical calcium-sensitive dye (Fluo-4) in iPSC-CMs. (A-B) Representative calcium traces of K-GECO (A) and Fluo-4 (B) are presented over time. (C) Calcium transient analysis of K-GECO transduced, or Fluo-4 loaded iPSC-CM. <a href="https://www.jove.com/files/ftp_upload/63175/63175fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>BPM</td>
			<td>CTD50 (s)</td>
			<td>CTD90 (s)</td>
			<td>Amplitude (F/F0)</td>
			<td>Rise time</td>
			<td>Decay time (s)</td>
		</tr>
		<tr>
			<td>Vehicle (0.1% DMSO)</td>
			<td>112.3657+/-10.95</td>
			<td>0.25+/-0.04</td>
			<td>0.41+/-0.01</td>
			<td>1.99+/-0.02</td>
			<td>0.07+/-0.01</td>
			<td>0.28+/-0.01</td>
		</tr>
		<tr>
			<td>Verapamil (1 uM)</td>
			<td>0</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Dofetilide (5 nM)</td>
			<td>103.42+/-9.87**</td>
			<td>0.23+/-0.03</td>
			<td>0.46+/-0.03</td>
			<td>1.91+/-0.03</td>
			<td>0.07+/-0.01</td>
			<td>0.35+/-0.02*</td>
		</tr>
		<tr>
			<td>E4031 (30 nM)</td>
			<td>75.73+/-12.08***</td>
			<td>0.37+/-0.04**</td>
			<td>0.58+/-0.08***</td>
			<td>1.55+/-0.02**</td>
			<td>0.11+/-0.04***</td>
			<td>0.35+/-0.07**</td>
		</tr>
	</tbody>
</table>
Table 1: Effects of compound addition to transient calcium parameters in the iPSC-CM model. Significance values are indicated by *(p&#160;&#8804; 0.05), **(p &#60; 0.01) and ***(p &#60; 0.001).
Animated Video 1: Spontaneous calcium activity was observed by mNG-GECO in iPSC-derived cardiomyocytes with vehicle only. A Pseudo-coloured movie of a grey-scale image is provided. <a href="https://www.jove.com/files/ftp_upload/63175/fig1-video.avi" target="_blank">Please click here to download this Video.</a>
Supplementary Figure 1: Schematic representation of the adenoviral-vector. This includes channel rhodopsin ChR2 as an actuator, with a red fluorescent calcium indicator K-GECO as a calcium reporter for an all-optical assay. The light-sensitive ion channel allows excitable cells to be depolarized by light in the blue-green range of the visible spectrum. 470 nm light was used in these experiments. Calcium transients in excitable cells can simultaneously be imaged by K-GECO, which requires green excitation light, producing red emissions. <a href="https://www.jove.com/files/ftp_upload/63175/Sup r1.pdf" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about High-Throughput Optical Controlling and Recording Calcium Signal in iPSC-Derived Cardiomyocytes for Toxicity Testing and Phenotypic Drug Screening at JoVE.com

High-Throughput Optical Controlling and Recording Calcium Signal in iPSC-Derived Cardiomyocytes for Toxicity Testing and Phenotypic Drug Screening

The present protocol describes all-optical control and observation of triggered cellular activity in iPSC-derived cardiomyocytes (iPSC-CMs) for high throughput drug screening and toxicity testing. Multi-parametric quantification of phenotypic patterns in time, and space, are shown. Long-term effects of drugs over hours, or sequential measurements over days, are demonstrated.

Understanding how excitable cells work in health and disease and how that behavior can be altered by small molecules or genetic manipulation is important. ...

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Research

JoVE Journal

Medicine

3.1K Views.  LumiSTAR Biotechnology, Inc.. We demonstreren talloze methoden voor alle optische controle en observatie van geactiveerde cellulaire activiteit in iPSC-afgeleide cardiomyocyten voor high-throughput geneesmiddelenscreening en toxiciteitstests. Maak multiparametrische kwantificering van de fenotypische patronen in tijd en ruimte mogelijk, maak het mogelijk om het effect van geneesmiddelen gedurende uren of dagen te observeren. Deze methode kan worden toegepast op verschillende soorten cellen, zoals neuro's of bètacellen, v...

Video: High-Throughput Optical Controlling and Recording Calcium Signal in iPSC-Derived Cardiomyocytes for Toxicity Testing and Phenotypic Drug Screening

Human iPSC-Derived Cardiomyocyte Networks on Multiwell Micro-electrode Arrays for Recurrent Action Potential Recordings

Cardiac safety screening is of paramount importance for drug discovery and therapeutics. Therefore, the development of novel high-throughput electrophysiological approaches for hiPSC-derived cardiomyocyte (hiPSC-CM) preparations is much needed for efficient drug testing. Although multielectrode arrays (MEAs) are frequently employed for field potential measurements of excitable cells, a recent publication by Joshi-Mukherjee and colleagues described and validated its application for recurrent action potential (AP) recordings from the same hiPSC-CM preparation over days. The aim here is to provide detailed step-by-step methods for seeding CMs and for measuring AP waveforms via electroporation with high precision and a temporal resolution of 1 &#181;s. This approach addresses the lack of easy-to-use methodology to gain intracellular access for high-throughput AP measurements for reliable electrophysiological investigations. A detailed work flow and methods for plating of hiPSC-CMs on multiwell MEA plates are discussed emphasizing critical steps wherever relevant. In addition, a custom-built MATLAB script for rapid data handling, extraction and analysis is reported for comprehensive investigation of the waveform analysis to quantify subtle differences in morphology for various AP duration parameters implicated in arrhythmia and cardiotoxicity.

This article contains a set of protocols for the development of human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) networks cultured on multiwell MEA plates to reversibly electroporate the cell membrane for action potential measurements. High-throughput recordings are obtained from the same cell sites repeatedly over days.

Cardiac safety screening is of paramount importance for drug discovery and therapeutics. Therefore, the development of novel high-throughput ...

Bioengineering

Optical Imaging of Isolated Murine Ventricular Myocytes

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances in calcium sensitive dyes have permitted the robust optical recording of single cell calcium dynamics, recording of robust transmembrane optical voltage signals has remained difficult. Arguably, this is because of the low single to noise ratio, phototoxicity, and photobleaching of traditional potentiometric dyes. Therefore, single cell voltage measurements have long been confined to the patch clamp technique which while the gold standard, is technically demanding and low throughput. However, with the development of novel potentiometric dyes, large, fast optical responses to changes in voltage can be obtained with little to no phototoxicity and photobleaching. This protocol describes in detail how to isolate adult murine myocytes which can be used for cellular shortening, calcium, and optical voltage measurements. Specifically, the protocol describes how to use a ratiometric calcium dye, a single-excitation calcium dye, and a single excitation voltage dye. This approach can be used to assess the cardiotoxicity and arrhythmogenicity of various chemical agents. While phototoxicity is still an issue at the single cell level, methodology is discussed on how to reduce it.

We present the methodology for the isolation of murine myocytes and how to obtain voltage or calcium traces simultaneously with sarcomere shortening traces using fluorescence photometry with simultaneous digital cell geometry measurements.

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances ...

Measuring Fast Calcium Fluxes in Cardiomyocytes

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to extracellular agents is predominantly regulated by the phospholipase C&beta;- G&alpha;q pathway localized on the plasma membrane which is stimulated by agents such as acetylcholine 3,4. We have recently found that plasma membrane protein domains called caveolae5,6 can entrap activated G&alpha;q 7. This entrapment has the effect of stabilizing the activated state of G&alpha;q and resulting in prolonged Ca2+ signals in cardiomyocytes and other cell types8. We uncovered this surprising result by measuring dynamic calcium responses on a fast scale in living cardiomyocytes. Briefly, cells are loaded with a fluorescent Ca2+ indicator. In our studies, we used Ca2+ Green (Invitrogen, Inc.) which exhibits an increase in fluorescence emission intensity upon binding of calcium ions. The fluorescence intensity is then recorded for using a line-scan mode of a laser scanning confocal microscope. This method allows rapid acquisition of the time course of fluorescence intensity in pixels along a selected line, producing several hundreds of time traces on the microsecond time scale. These very fast traces are transferred into excel and then into Sigmaplot for analysis, and are compared to traces obtained for electronic noise, free dye, and other controls. To dissect Ca2+ responses of different flux rates, we performed a histogram analysis that binned pixel intensities with time. Binning allows us to group over 500 traces of scans and visualize the compiled results spatially and temporally on a single plot. Thus, the slow Ca2+ waves that are difficult to discern when the scans are overlaid due to different peak placement and noise, can be readily seen in the binned histograms. Very fast fluxes in the time scale of the measurement show a narrow distribution of intensities in the very short time bins whereas longer Ca2+ waves show binned data with a broad distribution over longer time bins. These different time distributions allow us to dissect the timing of Ca2+fluxes in the cells, and to determine their impact on various cellular events.

We present a method to isolate rapid (microsecond) calcium events from slower fluxes in living cells using laser scanning confocal microscopy. The method measures fluorescence intensity fluctuations of calcium indicators by recording line scans of several hundred pixels in a cell. Histogram analysis allows us to isolate the time scales of different calcium fluxes.

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to ...

Biology

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

High-Throughput Cardiotoxicity Screening Using Mature Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Monolayers

Human induced stem cell-derived cardiomyocytes (hiPSC-CMs) are used to replace and reduce the dependence on animals and animal cells for preclinical cardiotoxicity testing. In two-dimensional monolayer formats, hiPSC-CMs recapitulate the structure and function of the adult human heart muscle cells when cultured on an optimal extracellular matrix (ECM). A human perinatal stem cell-derived ECM (maturation-inducing extracellular matrix-MECM) matures the hiPSC-CM structure, function, and metabolic state in 7 days after plating. 
Mature hiPSC-CM monolayers also respond as expected to clinically relevant medications, with a known risk of causing arrhythmias and cardiotoxicity. The maturation of hiPSC-CM monolayers was an obstacle to the widespread adoption of these valuable cells for regulatory science and safety screening, until now. This article presents validated methods for the plating, maturation, and high-throughput, functional phenotyping of hiPSC-CM electrophysiological and contractile function. These methods apply to commercially available purified cardiomyocytes, as well as stem cell-derived cardiomyocytes generated in-house using highly efficient, chamber-specific differentiation protocols. 
High-throughput electrophysiological function is measured using either voltage-sensitive dyes (VSDs; emission: 488 nm), calcium-sensitive fluorophores (CSFs), or genetically encoded calcium sensors (GCaMP6). A high-throughput optical mapping device is used for optical recordings of each functional parameter, and custom dedicated software is used for electrophysiological data analysis. MECM protocols are applied for medication screening using a positive inotrope (isoprenaline) and human Ether-a-go-go-related gene (hERG) channel-specific blockers. These resources will enable other investigators to successfully utilize mature hiPSC-CMs for high-throughput, preclinical cardiotoxicity screening, cardiac medication efficacy testing, and cardiovascular research.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer an alternative to using animals for preclinical cardiotoxicity screening. A limitation to the widespread adoption of hiPSC-CMs in preclinical toxicity screening is the immature, fetal-like phenotype of the cells. Presented here are protocols for robust and rapid maturation of hiPSC-CMs.

Human induced stem cell-derived cardiomyocytes (hiPSC-CMs) are used to replace and reduce the dependence on animals and animal cells for preclinical ...

Contractions of Human-iPSC-derived Cardiomyocyte Syncytia Measured with a Ca-sensitive Fluorescent Dye in Temperature-controlled 384-well Plates

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CM) are a useful model of human cardiac physiology and pharmacology. Various methods have been proposed to record this spontaneous activity and to evaluate drug effects, but many of these methods suffer from limited throughput and/or physiological relevance. We developed a high-throughput screening system to quantify the effects of exogenous compounds on hiPSC-CM's beating frequency, using a Ca-sensitive fluorescent dye and a temperature-controlled imaging multi-well plate reader. We describe how to prepare the cell plates and the compound plates and how to run the automated assay to achieve high sensitivity and reproducibility. We also describe how to transform and analyze the fluorescence data to provide reliable measures of drug effects on spontaneous rhythm. This assay can be used in drug discovery programs to guide chemical optimization away from, or toward, compounds affecting human cardiac function.

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells are useful models of human cardiac physiology and pharmacology. Here we present a high-throughput screening system to quantify the effects of exogenous compounds on beating frequency, using a Ca-sensitive fluorescent dye and a temperature-controlled imaging multi-well plate reader.

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CM) are a useful model of human cardiac ...

Generation of Ventricular-Like HiPSC-Derived Cardiomyocytes and High-Quality Cell Preparations for Calcium Handling Characterization

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a valuable human source for studying the basic science of calcium (Ca2+) handling and signaling pathways as well as high-throughput drug screening and toxicity assays. Herein, we provide a detailed description of the methodologies used to generate high-quality iPSC-CMs that can consistently reproduce molecular and functional characteristics across different cell lines. Additionally, a method is described to reliably assess their functional characterization through the evaluation of Ca2+ handling properties. Low oxygen (O2) conditions, lactate selection, and prolonged time in culture produce high-purity and high-quality ventricular-like cardiomyocytes. Similar to isolated adult rat cardiomyocytes (ARCMs), 3-month-old iPSC-CMs exhibit higher Ca2+ amplitude, faster rate of Ca2+ reuptake (decay-tau), and a positive lusitropic response to &#946;-adrenergic stimulation compared to day 30 iPSC-CMs. The strategy is technically simple, cost-effective, and reproducible. It provides a robust platform to model cardiac disease and for the large-scale drug screening to target Ca2+ handling proteins.

Here we describe and validate a method to consistently generate robust human induced pluripotent stem cell-derived cardiomyocytes and characterize their function. These techniques may help in developing mechanistic insight into signaling pathways, provide a platform for large-scale drug screening, and reliably model cardiac diseases.

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a valuable human source for studying the basic science of calcium (Ca2+) ...

A Cardiac Microphysiological System for Studying Ca2+ Propagation via Non-genetic Optical Stimulation

In vitro cardiac microphysiological models are highly reliable for scientific research, drug development, and medical applications. Although widely accepted by the scientific community, these systems are still limited in longevity due to the absence of non-invasive stimulation techniques. Phototransducers provide an efficient stimulation method, offering a wireless approach with high temporal and spatial resolution while minimizing invasiveness in stimulation processes. In this manuscript, we present a fully optical method for stimulating and detecting the activity of an in vitro cardiac microphysiological model. Specifically, we fabricated engineered laminar anisotropic tissues by seeding human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) generated in a 3D bioreactor suspension culture. We employed a phototransducer, an amphiphilic azobenzene derivative, named Ziapin2, for stimulation and a Ca2+ dye (X-Rhod 1) for monitoring the system&#39;s response. The results demonstrate that Ziapin2 can photomodulate Ca2+ responses in the employed system without compromising tissue integrity, viability, or behavior. Furthermore, we showed that the light-based stimulation approach offers a similar resolution compared to electrical stimulation, the current gold standard. Overall, this protocol opens promising perspectives for the application of Ziapin2 and material-based photostimulation in cardiac research.

Using light to control cardiac cells and tissue enables non-contact stimulation, thereby preserving the natural state and function of the cells, making it a valuable approach for both basic research and therapeutic applications.

In vitro cardiac microphysiological models are highly reliable for scientific research, drug development, and medical applications. Although widely ...

Technical Applications of Microelectrode Array and Patch Clamp Recordings on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Drug-induced cardiotoxicity is the leading cause of drug attrition and withdrawal from the market. Therefore, using appropriate preclinical cardiac safety assessment models is a critical step during drug development. Currently, cardiac safety assessment is still highly dependent on animal studies. However, animal models are plagued by poor translational specificity to humans due to species-specific differences, particularly in terms of cardiac electrophysiological characteristics. Thus, there is an urgent need to develop a reliable, efficient, and human-based model for preclinical cardiac safety assessment. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as an invaluable in vitro model for drug-induced cardiotoxicity screening and disease modeling. hiPSC-CMs can be obtained from individuals with diverse genetic backgrounds and various diseased conditions, making them an ideal surrogate to assess drug-induced cardiotoxicity individually. Therefore, methodologies to comprehensively investigate the functional characteristics of hiPSC-CMs need to be established. In this protocol, we detail various functional assays that can be assessed on hiPSC-CMs, including the measurement of contractility, field potential, action potential, and calcium handling. Overall, the incorporation of hiPSC-CMs into preclinical cardiac safety assessment has the potential to revolutionize drug development.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as a promising in vitro model for drug-induced cardiotoxicity screening and disease modeling. Here, we detail a protocol for measuring the contractility and electrophysiology of hiPSC-CMs.

Drug-induced cardiotoxicity is the leading cause of drug attrition and withdrawal from the market. Therefore, using appropriate preclinical cardiac safety ...

Subtype-specific Optical Action Potential Recordings in Human Induced Pluripotent Stem Cell-derived Ventricular Cardiomyocytes

Cardiomyocytes generated from human induced pluripotent stem cells (iPSC-CMs) are an emerging tool in cardiovascular research. Rather than being a homogenous population of cells, the iPSC-CMs generated by current differentiation protocols represent a mixture of cells with ventricular-, atrial-, and nodal-like phenotypes, which complicates phenotypic analyses. Here, a method to optically record action potentials specifically from ventricular-like iPSC-CMs is presented. This is achieved by lentiviral transduction with a construct in which a genetically-encoded voltage indicator is under the control of a ventricular-specific promoter element. When iPSC-CMs are transduced with this construct, the voltage sensor is expressed exclusively in ventricular-like cells, enabling subtype-specific optical membrane potential recordings using time-lapse fluorescence microscopy.

Here we present a method to optically image action potentials, specifically in ventricular-like induced pluripotent stem cell-derived cardiomyocytes. The method is based on the promoter-driven expression of a voltage-sensitive fluorescent protein.

High-Throughput Optical Controlling and Recording Calcium Signal in iPSC-Derived Cardiomyocytes for Toxicity Testing and Phenotypic Drug Screening

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