This work was partially supported by funding from the Food and Drug Administration (U01 FD006676-01 awarded to the Health and Environmental Sciences Institute) and by funding from the National Institutes of Health (HL151094 to Dr. Geisse). We thank Dr. Alec S. T. Smith for his assistance with the preparation of this manuscript.

<ol>
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All authors are employees and equity holders in Curi Bio Inc., the company that commercializes the Mantarray hardware and associated software.

This study describes methods for generating 3D engineered cardiac and skeletal muscle tissues within a 24-well consumable casting kit. By following these methods, it is possible to consistently achieve a complete array of 24 tissues with no casting failures for subsequent drug screening. Critical considerations for achieving such an outcome are ensuring that during casting all the steps are performed on ice to prevent premature polymerization of the hydrogels, removal of cell dissociation reagent prior to tissue casting, thorough mixing of the cell and hydrogel suspension for each tissue, replacement of pipette tips between tissues, and the use of heat-inactivated FBS (if used at all). Also, it is important to ensure that the post lattice is not moved once casting starts and is transferred gently once hydrogels have formed.
Major modifications include the use of different cell types to achieve cardiac versus skeletal EMTs and the doping of hydrogels with variable concentrations of basement membrane proteins to promote cellular maturation and tissue stability. The beneficial effects of such doping must be tested on a case-by-case basis, but has been shown to improve functional outcomes and tissue longevity under certain circumstances14,16,22. It is also noteworthy that the cell densities listed are a guide and may need to be optimized for different cell lines. Alternative hydrogel compositions could also be considered as a means to modify the structural and functional properties of the achieved EMTs23,24,25. The native muscle microenvironment also contains supporting cell types to promote vascularization, innervation, and matrix deposition to support myocytes in form and function26,27. While the system described here currently incorporates fibroblasts into 3D cardiac tissues, additional cell types may create a more physiological relevant model to study the safety and efficacy of therapeutic compounds in vitro. Previously, a range of supporting cell types have successfully been integrated into 3D-engineered tissues presenting an exciting template for future study using the magnetic sensing contractility platform28,29,30.
Troubleshooting for this protocol centers on the formation of unreliable or inconsistent tissues during the casting process. Care must be taken to avoid bubble formation in the hydrogels as they are cast while still facilitating even distribution of the cells during mixing. Optimization experiments will likely be needed for each new cell type to identify ideal cell densities, cell ratios, and matrix composition.
A major limitation for this technique is the significant number of cells required to establish a full plate of 24 EMTs. For the data presented here, 15 million cardiomyocytes and 18 million skeletal myoblasts were used per plate. Certain researchers may not have access to such large pools of cellular material, which may inhibit their ability to use this platform to its fullest extent. If end-users do not have access to magnetic sensing hardware, measurements of post deflections need to be performed optically, which significantly reduces throughput and prevents the simultaneous recording of muscle contractions across multiple wells. However, the Mantarray hardware overcomes these limitations to offer the first commercial system capable of continuous, non-invasive analysis of EMT contraction simultaneously across multiple constructs.
Magnetic sensing across 24 wells facilitates longitudinal studies of EMT functional development in real-time and allows accurate measurement of acute responses to chemical, environmental, or genetic manipulation. While magnetic sensing has several advantages such as simultaneous measurement across multiple tissues, and does not require complicated data analysis, optical detection methods do enable simultaneous measurement of physiological metrics such as calcium flux or voltage mapping. However, data sets such as those illustrated in the results section demonstrate the breadth of applications this technology has in the drug development space. Given that few assays on the market offer the means to perform a direct assessment of contractile output in engineered muscle, these methods hold the potential to revolutionize the preclinical development pipeline.

Induced pluripotent stem cell (iPSC) models are increasingly becoming key players in the preclinical pipeline for therapeutic discovery and development, as well as basic biological research and disease modeling1,2,3,4,5. Contractile tissues, such as cardiac and skeletal muscle derived from iPSCs hold great potential for improving the predictive power of human in vitro studies as direct assessment of muscle contractile force and kinetics are quantitative metrics to study overall tissue function4,6,7,8. Typically, measurements of contractile force have been obtained either indirectly by optical tracking of substrate deflection9,10 or directly by attachment of cells/tissues to a force transducer4,11,12. These methods, while accurate, are inherently low-throughput and typically require highly skilled operators to collect and analyze data.
Previous work has shown that magnetic field sensing circumvents these obstacles and provides an alternate method to assess engineered muscle function simultaneously across multiple tissue constructs13. The Mantarray (Magnetometric Analyzer for eNgineered Tissue ARRAY) 3D Contractility Platform builds on this technology using a device capable of measuring the contractility of engineered muscle tissues in a highly-parallel manner that leverages the complexity of 3D cellular models with higher-throughput screening14. The platform enables label-free, quantitative, real-time monitoring of contractile function in cardiac and skeletal muscle tissues in or outside of a standard cell-culture incubator, eliminating the need for optical-based contractile imaging and analysis. This technology facilitates direct comparison of healthy and diseased cell lines and enables measurement of a drug&#39;s effect on contractile tissues, establishing quantifiable, in vitro, safety, and efficacy data for new and existing therapeutic compounds.
Engineered 3D muscle tissues can be fabricated between two posts in a highly reproducible manner using the Mantarray consumable, 24-well casting plate (Figure 1). One post is rigid, while the other post is flexible and contains a small magnet. When the tissue construct contracts, it displaces the flexible post and the embedded magnet. The EMT plate is positioned within the instrument, and post displacement is measured via an array of magnetic sensors on a circuit board below the plate holder. The measured changes in the magnetic field are converted into absolute contractile force using a mathematical algorithm. The instrument employs rapid data sampling rates to enable the collection of detailed information about the functional capacity and maturity of the cell type(s) being assayed, including contraction frequency, velocity, and decay time. These functional measurements can be obtained across all 24 wells simultaneously with the magnetic sensing platform or individually and sequentially using traditional optical methods.
This study describes a highly reproducible method for engineering 3D skeletal muscle and cardiac microtissues in a fibrin-based hydrogel. During a brief, 80-min reaction, thrombin catalyzes the conversion of fibrinogen into fibrin, providing a scaffold for muscle cells to develop in suspended culture15. Stromal cells help remodel the matrix and tissues become contractile as muscle cells form a syncytium within the hydrogel. The contractility of these tissues was analyzed using the magnetic sensing approach, both before and after compound exposure, validating this modality for use in dose-response drug studies. Primary human myoblasts from a healthy donor biopsy were obtained commercially and cultured in 2D according to the vendor&#39;s protocols. Cells were expanded using a skeletal muscle growth medium through three passages to generate sufficient cell numbers to fabricate 3D tissues. Stromal cells and hiPSC-derived cardiomyocytes were cultured according to the vendor&#39;s protocol for 3 days to allow recovery from cryopreservation before casting cells into tissues. Representative results are provided illustrating the types of data sets that can be collected using the magnetic sensing platform. Common pitfalls associated with the generation of engineered tissues using these methods are also addressed.

<table><tbody><tr><td>100 &amp;micro;m cell strainer&amp;nbsp;</td><td>CELLTREAT</td><td>229485</td><td></td></tr><tr><td>100 mm cell culture dish</td><td>ThermoFisher</td><td>150466</td><td></td></tr><tr><td>50 mL Steriflip filter</td><td>MilliporeSigma&amp;nbsp;</td><td>SCGP00525</td><td></td></tr><tr><td>500 mL filter flask</td><td>MilliporeSigma&amp;nbsp;</td><td>S2GVU05RE</td><td></td></tr><tr><td>6-aminocaproic acid</td><td>Sigma</td><td>A2504</td><td></td></tr><tr><td>B27</td><td>Gibco</td><td>17504044</td><td></td></tr><tr><td>Cardiosight Maintenance Medium</td><td>NEXEL</td><td>CM-002A</td><td></td></tr><tr><td>Cardiosight Plating Medium</td><td>NEXEL</td><td>CM-020A</td><td></td></tr><tr><td>C-Pace EM stimulator&amp;nbsp;</td><td>IonOptix&amp;nbsp;</td><td>EM</td><td></td></tr><tr><td>Curi Bio Muscle Differentiation Media Kit</td><td></td><td>Primary - DIFF</td><td></td></tr><tr><td>Curi Bio Muscle Maintenance Media Kit</td><td>Curi Bio</td><td>Primary - MAINT</td><td></td></tr><tr><td>DAPI</td><td>Invitrogen</td><td>D1306</td><td></td></tr><tr><td>DMEM, high glucose, GlutaMAX</td><td>Gibco</td><td>10566-016</td><td></td></tr><tr><td>Dnase</td><td>Sigma</td><td>11284932001</td><td></td></tr><tr><td>DPBS</td><td>Gibco</td><td>14190-250</td><td></td></tr><tr><td>Dystrophin antibody&amp;nbsp;</td><td>Abcam</td><td>ab154168</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Thermo Scientific</td><td>10082147</td><td>Must be heat-inactivated</td></tr><tr><td>Fibrinogen (Bovine)</td><td>Sigma</td><td>E8630</td><td></td></tr><tr><td>Glutaraldehyde</td><td>Sigma</td><td>354400</td><td></td></tr><tr><td>Ham&#039;s F10</td><td>Gibco</td><td>11550043</td><td></td></tr><tr><td>Hemacytometer&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>Z359629</td><td></td></tr><tr><td>HS-27A Fibroblasts</td><td>ATCC</td><td>CRL-2496</td><td></td></tr><tr><td>Human Skeletal Muscle Myoblasts&amp;nbsp;</td><td>Lonza&amp;nbsp;</td><td>CC-2580</td><td></td></tr><tr><td>Luer Lock 0.2 &amp;micro;m syringe filter</td><td>Corning</td><td>431219</td><td></td></tr><tr><td>Luer Lock 10 mL syringe</td><td>BH Supplies</td><td>BH10LL</td><td></td></tr><tr><td>Mantarray Instrument</td><td>Curi Bio</td><td>MANTA-24-B1</td><td>System</td></tr><tr><td>Mantarray Plate Kits</td><td>Curi Bio</td><td>MA-24-SKM-5</td><td>Pack of 5 kits</td></tr><tr><td>Mantarray stimulation lid&amp;nbsp;</td><td>Curi Bio</td><td>EM</td><td></td></tr><tr><td>Matrigel (ECM)</td><td>Corning</td><td>356231</td><td></td></tr><tr><td>Nexel Cardiosight-S, Cardiomyocytes</td><td>NEXEL</td><td>C-002</td><td></td></tr><tr><td>Optical Microscope</td><td>Nikon Ti2E</td><td>MEA54000</td><td></td></tr><tr><td>Pan Myosin Heavy Chain antibody&amp;nbsp;</td><td>DSHB</td><td>MF-20</td><td></td></tr><tr><td>Poly(ethyleneimine)</td><td>Sigma</td><td>P3143</td><td></td></tr><tr><td>ROCK inhibitor</td><td>StemCell Technologies</td><td>Y-27632</td><td></td></tr><tr><td>RPMI</td><td>Gibco</td><td>11875-093</td><td></td></tr><tr><td>Skeletal Muscle Growth Medium (SkGM-2)</td><td>Lonza</td><td>CC-3245</td><td></td></tr><tr><td>Standard 24-well plates</td><td>Greiner</td><td>M8812&amp;nbsp;</td><td>Other manufacturer&#039;s plates will not fit</td></tr><tr><td>Standard 6-well plates</td><td>ThermoFisher</td><td>140675</td><td></td></tr><tr><td>Stromal medium (DMEM + 20% FBS)</td><td></td><td></td><td></td></tr><tr><td>T175 Filter Flask</td><td>ThermoFisher</td><td>159910</td><td></td></tr><tr><td>T225 Filter Flask</td><td>ThermoFisher</td><td>159934</td><td></td></tr><tr><td>Thrombin</td><td>Sigma</td><td>T4648</td><td></td></tr><tr><td>Trypan Blue solution, 0.4%</td><td>ThermoFisher</td><td>15250061</td><td></td></tr><tr><td>TrypLE Select Enzyme (10x)</td><td>Thermo Scientific</td><td>A1217702</td><td></td></tr><tr><td>TrypLE Select Enzyme (1x)</td><td>Thermo Scientific</td><td>12563011</td><td></td></tr></tbody></table>

preclinical drug testing in scalable 3d engineered muscle tissues

Accurately modeling healthy and disease conditions in vitro is vital for the development of new treatment strategies and therapeutics. For cardiac and skeletal muscle diseases, contractile force and kinetics constitute key metrics for assessing muscle function. New and improved methods for generating engineered muscle tissues (EMTs) from induced pluripotent stem cells have made in vitro disease modeling more reliable for contractile tissues; however, reproducibly fabricating tissues from suspended cell cultures and measuring their contractility is challenging. Such techniques are often plagued with high failure rates and require complex instrumentation and customized data analysis routines. A new platform and device that utilizes 3D EMTs in conjunction with a label-free, highly-parallel, and automation-friendly contractility assay circumvent many of these obstacles. The platform enables facile and reproducible fabrication of 3D EMTs using virtually any cell source. Tissue contractility is then measured via an instrument that simultaneously measures 24 tissues without the need for complex software analysis routines. The instrument can reliably measure micronewton changes in force, allowing for dose-dependent compound screening to measure the effect of a drug or therapeutic on contractile output. Engineered tissues made with this device are fully functional, generating twitch and tetanic contractions upon electrical stimulation, and can be analyzed longitudinally in culture over weeks or months. Here, we show data from cardiac muscle EMTs under acute and chronic dosing with known toxicants, including a drug (BMS-986094) that was pulled from clinical trials after patient fatalities due to unanticipated cardiotoxicity. Altered skeletal muscle function in engineered tissues in response to treatment with a myosin inhibitor is also presented. This platform enables the researcher to integrate complex, information-rich bioengineered model systems into their drug discovery workflow with minimal additional training or skills required.

Cells were cast into engineered muscle tissues in the 2-post consumable plate (Figure 1). Successful EMTs will appear uniform, and the matrix will be evenly distributed between the posts (Figure 2A). The matrix should also wrap around both posts, producing equivalent anchor points for the tissue. Failures in casting are rare with this method and are usually obvious with a visual inspection. Unsuccessful EMT production can range from catastrophic failures, such as tissue detachment from the posts (Figure 2B) to more subtle structural flaws, such as air bubbles and loose attachment to the posts (Figure 2C,D). Tissues with minor flaws may still be viable, but the data from these tissues should be examined carefully to ensure it is comparable to uncompromised EMTs. For example, air bubbles within an EMT may be squeezed out as the tissue compacts over time, rendering a fully functional construct without contractile deficiencies. These tissues must be evaluated on a case-by-case basis, however, as the location of the air bubbles may affect functional recovery. Air bubbles generated at the posts, for instance, may affect tissue attachment, which could impede long-term adherence to the post.
Tissues begin to compact within the first 24 h as cells remodel the matrix within the hydrogel (Figure 3). Compaction is a gradual process and usually proceeds over the first 2-4 weeks of culture. Overall, tissue compaction is consistent between technical and biological replicates (Figure 4). It is normal for some cell lines to compact the matrix more than others as tissues mature over time. The percentage of myogenic cells within a construct influences the rate and overall degree of EMT compaction. Total myogenic content for both cardiac and skeletal muscle cell lines should be above 80% to minimize variation between engineered tissues. This is particularly important when comparing contractile forces and kinetics across cell lines.
Within the first few days after casting, cardiomyocytes begin to spontaneously beat in culture, rhythmically bending the flexible post with each muscle contraction. Skeletal muscle constructs contract in response to electrical stimulation by day 7 after starting differentiation. Field stimulation was applied to skeletal muscle tissues via an external stimulator attached to a custom 24-well electrode lid. The lid, fabricated with a pair of carbon electrodes for each well, sits on top of the 24-well plate of tissues, simultaneously stimulating each EMT to induce muscle contractions. Tissues were paced using a 10 V stimulus for 10 ms pulse durations at 1 Hz during functional measurements. Contractile tissues indicate skeletal myoblasts that have fused, forming myotubes complete with functional sarcomeres and contractile machinery. Skeletal EMTs stain positive for myosin heavy chain (MyHC) and dystrophin is localized to the myotube membrane revealing a classic ring shape in cross-sectional immunohistochemical analysis (Figure 5). Once EMTs are functional, contractility can be measured daily in the magnetic sensing instrument, tracking force and kinetics as the constructs develop and mature over time. Both cardiac and skeletal muscle tissues remain contractile for weeks to months in 3D culture (Figure 6), and they can be used for a wide range of contractility studies.
The magnetic detection approach can be used to simultaneously measure acute and chronic effects of structural cardiotoxicants, such as doxorubicin (Figure 7) and BMS-986094 (Figure 8), as well as other drugs that affect muscle contractility. Optical tracking methods of contraction detection can also be used, but care must be taken when studying acute drug effects as measurements must be taken sequentially. The extended longevity of cardiac and skeletal EMTs in 3D culture enables long-term drug studies in these tissues. This permits users to explore the effects of repeat dosing, as well as continued, long-term exposure to compounds that may show cardiotoxic effects over time as occurs with doxorubicin. Doxorubicin (dox) is an anti-cancer chemotherapy drug17. The amount of drug administered to patients varies, depending on the type of cancer, age of the patient, height and weight of the patient, as well as other factors. For this reason, it is important to test the effect of dox across a wide range of concentrations and delivery schedules. Here, cardiac EMTs were treated over the course of 27 days with three separate concentrations (0.1 &#181;M, 1 &#181;M, and 10 &#181;M) of dox (Figure 7). The groups were stratified further by treating EMTs at each concentration with either a bolus treatment or continuous administration with a medium change every 72 h. Wells given bolus treatments of dox were exposed to the drug at three separate time points, allowing for recovery between dosing. The two highest doses of bolus and continuous exposure showed an immediate and prolonged cessation of contractile force generation throughout the study. The middle and lowest concentrations had varying effects on the tissues, depending on the administration method. In the lowest concentration of the drug, the bolus group showed no difference from the controls. However, contractile force diminished after 2 weeks of continuous exposure. The mid-range concentration of the drug had an interesting effect. While continuous dosing reduced force over the first couple of days of treatment and lasted throughout the experiment, the bolus group showed a recovery of contractile force back to control levels when the drug was washed out after 3 days. However, the second bolus of the drug caused a full cessation of force, followed by no recovery (Figure 7), indicating that repeatedly dosing at this concentration may have a cardiotoxic effect in patients treated with this drug. The broad scope of this study, in both time and experimental conditions, highlights the utility of 3D engineered tissues in toxicity screening, as they remain contractile and responsive to chemical exposure over extended periods of time, allowing for long-term drug studies within a single set of muscle tissues. This facilitates not only identifying compounds that may have a cardiotoxic effect with chronic exposure but also detecting potential cardiotoxic timing of administration.
In vitro toxicity testing in engineered human muscle tissues is one way to help keep human patients safe in clinical trials. BMS-986094 is a nucleotide polymerase (NS5B) inhibitor used to treat hepatitis C. The drug was in Phase II clinical development when Bristol-Myers Squibb discontinued development due to several cases of unexpected heart failure in patients18,19. Here, BMS-986094 was applied to cardiac EMTs to test whether 3D engineered muscle tissues would develop a cardiotoxic reaction to the drug (Figure 8). Three different concentrations of the drug were applied, and tissues were monitored over 13 days. Contractile force dropped with the addition of the drug in a dose-dependent manner (Figure 8A). Twitch frequency was also significantly affected as the beat rate slowed and eventually stopped as expected with continued exposure to the cardiotoxic compound (p &#60; 0.05, Figure 8B). These results demonstrate how 3D-engineered human muscle tissues can be used to facilitate bringing new drugs to the market and flag compounds that eventually fail due to cardiotoxicity. Moreover, this technology could potentially save lives by exposing dangerous drugs before they are put into patients in clinical trials.
The ability to measure the effect of acute and chronically applied drugs on human contractile tissue is a vital first step when investigating therapeutics for safety and efficacy. It is important to know, however, that the concentration of drugs applied are physiologically relevant and appropriate for in vitro testing. Skeletal muscle tissues were used to establish an IC50 value for 2,3-Butanedione monoxime (BDM) in a full dose-response curve. This drug is a well-characterized ATPase inhibitor of skeletal muscle myosin-II20. BDM inhibits muscle contractions by preventing myosin cross-bridge formation with the actin filament in sarcomeres21. Results shown here reveal a dose-dependent decrease in absolute force when the drug is applied and complete recovery of contractile force when the drug is washed out, indicating the transient effect is preventing muscle contractions and not merely killing cells within the tissue (Figure 9A). Furthermore, a full dose-response curve was measured across the seven concentrations examined, establishing an IC50 of 3.2 mM in these human microtissues (Figure 9B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64399/64399fig01.jpg" /> 
Figure 1: EMT casting in the 2-post Mantarray consumable 24-well plate. (A) Myogenic and stromal cells were cultured on 2D surfaces prior to tissue casting. (B) Cells are lifted from 2D surfaces and mixed together with extracellular matrix proteins to form hydrogels in the individual plate casting wells shown in the inset. (C) 24-well plate containing engineered tissues in every well. (D) Representative tissues showing relaxed and contracted engineered muscle, comparing displacement of the magnetic post (green bars). <a href="https://www.jove.com/files/ftp_upload/64399/64399fig01large.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/64399/64399fig02.jpg" /> 
Figure 2: Successful and unsuccessful EMT casting. (A) Ideal engineered muscle tissue 24 h post-casting uniformly compacted around the posts with homogenous cell/matrix composition throughout the tissue. (B) Failed EMT showing detachment of the hydrogel from the flexible post. (C) EMT containing air bubbles throughout the tissue. (D) Unequal tissue deposition around both posts. Tissue is loosely anchored to the flexible post on one side. Scale bars are 1 mm. <a href="https://www.jove.com/files/ftp_upload/64399/64399fig02large.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/64399/64399fig03.jpg" /> 
Figure 3: Compaction in engineered muscle tissue over time. (A) EMT construct shown 1 day after casting. Tissues are transferred into differentiation medium, beginning day 0 of cell fusion and hydrogel compaction. (B-E) The same EMT at day 7 through day 21 showing a slightly shorter overall length between the two posts over time and smaller width when measured through the middle section of the EMT. Scale bars are 1 mm. <a href="https://www.jove.com/files/ftp_upload/64399/64399fig03large.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/64399/64399fig04.jpg" /> 
Figure 4: EMT diameter over time. Four plates of tissues were tracked over 21 days, comparing EMT diameter throughout compaction. Each tissue was measured through the mid-section every week using optical microscopy. Time points show consistent EMT size between plates. Maximal compaction is reached at day 21 as matrix remodeling is stabilized. The table shows the standard deviation (% of total) of compaction within each plate of tissues and the average deviation for all plates. Colored bars are individual plates. Error bars are SD of EMTs within plates. <a href="https://www.jove.com/files/ftp_upload/64399/64399fig04large.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/64399/64399fig05.jpg" /> 
Figure 5: Immunohistochemistry of the engineered skeletal muscle tissues. EMTs were fixed on day 10 of culture and embedded in paraffin. Thin cross-sections (7 &#956;m) were stained with antibodies against myosin heavy chain and dystrophin prior to imaging. Green = MyHC, red = Dystrophin, blue = DAPI. Objective magnification is 40X; scale bar is 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64399/64399fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64399/64399fig06.jpg" /> 
Figure 6: Contractile force in engineered muscle tissues over time. (A) Average absolute twitch force measured from cardiac EMTs from day 7 through day 63 in culture; n = 3 per group. (B) Average absolute twitch force in skeletal EMTs derived from a primary cell line on day 7 through day 53 in culture; n = 3. Error bars are SD for both graphs. <a href="https://www.jove.com/files/ftp_upload/64399/64399fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/64399/64399fig07.jpg" /> 
Figure 7: Acute and chronic doxorubicin treatment in the engineered muscle tissue. Three separate dose concentrations of dox, 0.1 &#181;M, 1 &#181;M, and 10 &#181;M, were delivered either in bolus or administered continuously to engineered muscle tissues over the course of 27 days. Bolus doses of the drug were added at media changes on days 0, 12, and 24, noted by the green arrows on the X-axis. The drug was added to the media at every media change for continuous dosing, noted by the black and green arrows on the X-axis. The percent change in force from baseline values (pre-drug treatment) is on the Y-axis, and the time in days of treatment is on the X-axis. Light orange = DMSO continuous control, dark orange = DMSO bolus control, light green = 0.1 &#181;M dox continuous, dark green = 0.1 &#181;M dox bolus, light blue = 1 &#181;M dox continuous, dark blue = 1 &#181;M Dox bolus, light yellow = 10 &#181;M dox continuous, dark yellow = 10 &#181;M dox bolus. Error bars are SD; n = 3 per condition. <a href="https://www.jove.com/files/ftp_upload/64399/64399fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/64399/64399fig08.jpg" /> 
Figure 8: Chronic treatment with BMS-986094 in engineered muscle tissue. EMTs were treated with 0.4 &#181;M (green), 2 &#181;M (dark blue), and 10 &#181;M (light blue) BMS-986094 over 13 days. (A) Contractile twitch force (Y-axis) decreases at all drug concentrations in the first 2 days, whereas the control tissues in DMSO continue to get stronger over time (X-axis). (B) The cardiac beat rate, or twitch frequency, ceases in a dose-dependent like manner in tandem with a cessation of contractile force shown in graph A. Control tissues in DMSO (gray) maintain a regular beat rate throughout the experiment. Error bars are SD; n = 3 per condition. <a href="https://www.jove.com/files/ftp_upload/64399/64399fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/64399/64399fig09.jpg" /> 
Figure 9: Dose-response to BDM in engineered skeletal muscle tissues. (A) Absolute twitch force decreases in a dose-dependent manner when primary cell-derived EMTs are exposed to 2,3-Butanedione monoxime (BDM) on day 16 in 3D culture. Absolute twitch force returns to near baseline values when the drug is washed out. (B) Absolute twitch force normalized to baseline values decreases in a dose-dependent manner when exposed to BDM yielding a full dose-response curve and IC50 value; n = 4 per dose. Error bars are SD. <a href="https://www.jove.com/files/ftp_upload/64399/64399fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Preclinical Drug Testing in Scalable 3D Engineered Muscle Tissues at JoVE.com

Preclinical Drug Testing in Scalable 3D Engineered Muscle Tissues

This protocol provides methods for generating 3D engineered cardiac and skeletal muscle tissues and describes their use in preclinical drug screening modalities. The described methods utilize a magnetic sensing system to facilitate the simultaneous assessment of 24 tissues in parallel.

Accurately modeling healthy and disease conditions in vitro is vital for the development of new treatment strategies and therapeutics. For cardiac and ...

preclinical-drug-testing-in-scalable-3d-engineered-muscle-tissues

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Bioengineering

3.2K Views.  Curi Bio Inc.. This protocol provides methods for generating 3D engineered cardiac and skeletal muscle tissues and describes their use in preclinical drug screening modalities. The described methods utilize a magnetic sensing system to facilitate the simultaneous assessment of 24 tissues in parallel.

Video: Preclinical Drug Testing in Scalable 3D Engineered Muscle Tissues

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of anti-cancer therapeutics in a physiologically representative microenvironment. We outline the formation of patient derived cancer stem cell spheroids, as well as, the manipulation of these spheroids for thorough analysis following drug treatment. Specifically, we describe collection of spheroid morphology, proliferation, viability, drug toxicity, cell phenotype and cell localization data. This protocol focuses heavily on analysis techniques that are easily implemented using the 384-well hanging drop platform, making it ideal for high throughput drug screening. While we emphasize the importance of this model in ovarian cancer studies and cancer stem cell research, the 384-well platform is amenable to research of other cancer types and disease models, extending the utility of the platform to many fields. By improving the speed of personalized drug screening and the quality of screening results through easily implemented physiologically representative 3D cultures, this platform is predicted to aid in the development of new therapeutics and patient-specific treatment strategies, and thus have wide-reaching clinical impact.

This protocol describes generation of patient-derived spheroids, and downstream analysis including quantification of proliferation, cytotoxicity testing, flow cytometry, immunofluorescence staining and confocal imaging, in order to assess drug candidates&#8217; potential as anti-neoplastic therapeutics. This protocol supports precision medicine with identification of specific drugs for each patient and stage of disease.

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of ...

Cancer Research

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific mutational background. The model is generated on a decellularized porcine scaffold that reproduces tissue-specific characteristics regarding extracellular matrix composition and architecture including the basement membrane. We standardized a protocol that allows artificial tumor tissue generation within 14 days including three days of drug treatment. Our article provides several detailed descriptions of 3D read-out screening techniques like the determination of the proliferation index Ki67 staining&#39;s, apoptosis from supernatants by M30-ELISA and assessment of epithelial to mesenchymal transition (EMT), which are helpful tools for evaluating the effectiveness of therapeutic compounds. We could show compared to 2D culture a reduction of proliferation in our 3D tumor model that is related to the clinical situation. Despite of this lower proliferation, the model predicted EGFR-targeted drug responses correctly according to the biomarker status as shown by comparison of the lung carcinoma cell lines HCC827 (EGFR -mutated, KRAS wild-type) and A549 (EGFR wild-type, KRAS-mutated) treated with the tyrosine-kinase inhibitor (TKI) gefitinib. To investigate drug responses of more advanced tumor cells, we induced EMT by long-term treatment with TGF-beta-1 as assessed by vimentin/pan-cytokeratin immunofluorescence staining. A flow-bioreactor was employed to adjust culture to physiological conditions, which improved tissue generation. Furthermore, we show the integration of drug responses upon gefitinib treatment or TGF-beta-1 stimulation - apoptosis, proliferation index and EMT - into a Boolean in silico model. Additionally, we explain how drug responses of tumor cells with a specific mutational background and counterstrategies against resistance can be predicted. We are confident that our 3D in vitro approach especially with its in silico expansion provides an additional value for preclinical drug testing in more realistic conditions than in 2D cell culture.

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

We present a three-dimensional (3D) lung cancer model based on a biological collagen scaffold to study sensitivity towards non-small-cell-lung-cancer-(NSCLC)-targeted therapies. We demonstrate different read-out techniques to determine the proliferation index, apoptosis and epithelial-mesenchymal transition (EMT) status. Collected data are integrated into an in silico model for prediction of drug sensitivity.

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific ...

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these procedures in basic research and preclinical drug development, it is important to develop protocols for automated generation and analysis under standardized conditions. Here, we present a technique to generate engineered heart tissue (EHT) from cardiomyocytes of different species (rat, mouse, human). The technique relies on the assembly of a fibrin-gel containing dissociated cardiomyocytes between elastic polydimethylsiloxane (PDMS) posts in a 24-well format. Three-dimensional, force-generating EHTs constitute within two weeks after casting. This procedure allows for the generation of several hundred EHTs per week and is technically limited only by the availability of cardiomyocytes (0.4-1.0 x 106/EHT). Evaluation of auxotonic muscle contractions is performed in a modified incubation chamber with a mechanical interlock for 24-well plates and a camera placed on top of this chamber. A software controls a camera moved on an XYZ axis system to each EHT. EHT contractions are detected by an automated figure recognition algorithm, and force is calculated based on shortening of the EHT and the elastic propensity and geometry of the PDMS posts. This procedure allows for automated analysis of high numbers of EHT under standardized and sterile conditions. The reliable detection of drug effects on cardiomyocyte contraction is crucial for cardiac drug development and safety pharmacology. We demonstrate, with the example of the hERG channel inhibitor E-4031, that the human EHT system replicates drug responses on contraction kinetics of the human heart, indicating it to be a promising tool for cardiac drug safety screening.

Here, we show the generation of human engineered heart tissue from induced pluripotent stem cells (hiPSC)-derived cardiomyocytes. We present a method to analyze contraction force and exemplary alteration of contraction pattern by the hERG channel inhibitor E-4031. This method shows high level of robustness and suitability for cardiac drug screening.

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided a unique opportunity to study the complex interplay among different cardiovascular cell types that drives tissue development and disease. In the area of cardiac tissue models, several sophisticated three-dimensional (3D) approaches use induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to mimic physiological relevance and native tissue environment with a combination of extracellular matrices and crosslinkers. However, these systems are complex to fabricate without microfabrication expertise and require several weeks to self-assemble. Most importantly, many of these systems lack vascular cells and cardiac fibroblasts that make up over 60% of the nonmyocytes in the human heart. Here we describe the derivation of all three cardiac cell types from iPSCs to fabricate cardiac microtissues. This facile replica molding technique allows cardiac microtissue culture in standard multi-well cell culture plates for several weeks. The platform allows user-defined control over microtissue sizes based on initial seeding density and requires less than 3 days for self-assembly to achieve observable cardiac microtissue contractions. Furthermore, the cardiac microtissues can be easily digested while maintaining high cell viability for single-cell interrogation with the use of flow cytometry and single-cell RNA sequencing (scRNA-seq). We envision that this in vitro model of cardiac microtissues will help accelerate validation studies in drug discovery and disease modeling.

Here, we describe an easy-to-use methodology to generate 3D self-assembled cardiac microtissue arrays composed of pre-differentiated human-induced pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells. This user-friendly and low cell requiring technique to generate cardiac microtissues can be implemented for disease modeling and early stages of drug development.

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided ...

Assessing Functional Metrics of Skeletal Muscle Health in Human Skeletal Muscle Microtissues

Three-dimensional (3D) in vitro models of skeletal muscle are a valuable advancement in biomedical research as they afford the opportunity to study skeletal muscle reformation and function in a scalable format that is amenable to experimental manipulations. 3D muscle culture systems are desirable as they enable scientists to study skeletal muscle ex vivo in the context of human cells. 3D in vitro&#160;models closely mimic aspects of the native tissue structure of adult skeletal muscle. However, their universal application is limited by the availability of platforms that are simple to fabricate, cost and user-friendly, and yield relatively high quantities of human skeletal muscle tissues. Additionally, since skeletal muscle plays an important functional role that is impaired over time in many disease states, an experimental platform for microtissue studies is most practical when minimally invasive calcium transient and contractile force measurements can be conducted directly within the platform itself. In this protocol, the fabrication of a 96-well platform known as &#39;MyoTACTIC&#39;, and en masse production of 3D human skeletal muscle microtissues (hMMTs) is described. In addition, the methods for a minimally invasive application of electrical stimulation that enables repeated measurements of skeletal muscle force and calcium handling of each microtissue over time are reported.

This manuscript describes a detailed protocol to produce arrays of 3D human skeletal muscle microtissues and minimally invasive downstream in situ assays of function, including contractile force and calcium handling analyses.

Three-dimensional (3D) in vitro models of skeletal muscle are a valuable advancement in biomedical research as they afford the opportunity to study ...

Engineering and Characterization of an Optogenetic Model of the Human Neuromuscular Junction

Many neuromuscular diseases, such as myasthenia gravis (MG), are associated with dysfunction of the neuromuscular junction (NMJ), which is difficult to characterize in animal models due to physiological differences between animals and humans. Tissue engineering offers opportunities to provide in vitro models of functional human NMJs that can be used to diagnose and investigate NMJ pathologies and test potential therapeutics. By incorporating optogenetic proteins into induced pluripotent stem cells (iPSCs), we generated neurons that can be stimulated with specific wavelengths of light. If the NMJ is healthy and functional, a neurochemical signal from the motoneuron results in muscle contraction. Through the integration of optogenetics and microfabrication with tissue engineering, we established an unbiased and automated methodology for characterizing NMJ function using video analysis. A standardized protocol was developed for NMJ formation, optical stimulation with simultaneous video recording, and video analysis of tissue contractility. Stimulation of optogenetic motoneurons by light to induce skeletal muscle contractions recapitulates human NMJ physiology and allows for repeated functional measurements of NMJ over time and in response to various inputs. We demonstrate this platform&#39;s ability to show functional improvements in neuromuscular connectivity over time and characterize the damaging effects of patient MG antibodies or neurotoxins on NMJ function.

We describe a reproducible, automated, and unbiased imaging system for characterizing neuromuscular junction function using human engineered skeletal muscle tissue and optogenetic motoneurons. This system allows for the functional quantification of neuromuscular connectivity over time and detects diminished neuromuscular function caused by neurotoxins and myasthenia gravis patient serum.

Many neuromuscular diseases, such as myasthenia gravis (MG), are associated with dysfunction of the neuromuscular junction (NMJ), which is difficult to ...

Generation, High-Throughput Screening, and Biobanking of Human-Induced Pluripotent Stem Cell-Derived Cardiac Spheroids

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We recently published a cost-effective strategy for the massive expansion of hiPSC-CMs in two dimensions (2D). Two major limitations are cell immaturity and a lack of three-dimensional (3D) arrangement and scalability in high-throughput screening (HTS) platforms. To overcome these limitations, the expanded cardiomyocytes form an ideal cell source for the generation of 3D cardiac cell culture and tissue engineering techniques. The latter holds great potential in the cardiovascular field, providing more advanced and physiologically relevant HTS. Here, we describe an HTS-compatible workflow with easy scalability for the generation, maintenance, and optical analysis of cardiac spheroids (CSs) in a 96-well-format. These small CSs are essential to fill the gap present in current in vitro disease models and/or generation for 3D tissue engineering platforms. The CSs present a highly structured morphology, size, and cellular composition. Furthermore, hiPSC-CMs cultured as CSs display increased maturation and several functional features of the human heart, such as spontaneous calcium handling and contractile activity. By automatization of the complete workflow, from the generation of CSs to functional analysis, we increase intra- and inter-batch reproducibility as demonstrated by high-throughput (HT) imaging and calcium handling analysis. The described protocol allows modeling of cardiac diseases and assessing drug/therapeutic effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow. In addition, the study describes a straightforward procedure for long-term preservation and biobanking of whole-spheroids, thereby providing researchers the opportunity to create next-generation functional tissue storage. HTS combined with long-term storage will substantially contribute to translational research in a wide range of areas, including drug discovery and testing, regenerative medicine, and the development of personalized therapies.

Presented here is a set of protocols for the generation and cryopreservation of cardiac spheroids (CSs) from human-induced pluripotent stem cell-derived cardiomyocytes cultured in a high-throughput, multidimensional format. This three-dimensional model functions as a robust platform for disease modeling, high-throughput screenings, and maintains its functionality after cryopreservation.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We ...

Preclinical Drug Testing in Scalable 3D Engineered Muscle Tissues

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Chapters in this video

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Tissue Engineering of a Human 3D in vitro Tumor Test System

Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

Assessing Functional Metrics of Skeletal Muscle Health in Human Skeletal Muscle Microtissues

Engineering and Characterization of an Optogenetic Model of the Human Neuromuscular Junction

Generation, High-Throughput Screening, and Biobanking of Human-Induced Pluripotent Stem Cell-Derived Cardiac Spheroids