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 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 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 'MyoTACTIC', 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.
Skeletal muscle is one of the most abundant tissues in the human body and supports key body functions such as locomotion, heat homeostasis and metabolism1. Historically, animal models and two-dimensional (2D) cell culture systems have been used to study biological processes and disease pathogenesis, as well as for testing pharmacological compounds in the treatment of skeletal muscle diseases2,3. While animal models have greatly improved our knowledge of skeletal muscle in health and in disease, their translational impact has been hampered by high costs, ethical considerations and interspecies differences2,4. In turning to human cell-based systems to study skeletal muscle, 2D cell culture systems are favorable due to their simplicity. However, there is a limitation. This format often fails to recapitulate the cell-cell and cell-extracellular matrix interactions that occur naturally within the body5,6. Over the last several years, three dimensional (3D) skeletal muscle models have emerged as a powerful alternative to whole animal models and conventional 2D culture systems by allowing the modeling of physiologically and pathologically relevant processes ex vivo7,8. Indeed, a plethora of studies have reported strategies to model human skeletal muscle in a bioartificial 3D culture format1. One limitation for many of these studies is that active force is quantified following the removal of the muscle tissues from the culture platforms and attachment to a force transducer, which is destructive and hence, limited to serving as an endpoint assay9,10,11,12,13,14,15,16,17,18,19,20,21. Others have designed culture systems that allow for non-invasive methods of measuring active force, but not all are amenable to high content molecule testing applications7,8,9,10,14,18,22,23,24,25,26,27,28,29.
This protocol describes a detailed method to fabricate human muscle microtissues (hMMTs) in the skeletal muscle (Myo) microTissue Array deviCe To Investigate forCe (MyoTACTIC) platform; a 96 well plate device that supports the bulk production of 3D skeletal muscle microtissues30. The MyoTACTIC plate fabrication method enables generation of a 96 well polydimethylsiloxane (PDMS) culture plate and all corresponding well features in a single casting step, whereby each well requires a relatively small number of cells for microtissue formation. Microtissues formed within MyoTACTIC contain aligned, striated, and multinucleated myotubes that are reproducible from well to well of the device, and upon maturation, can respond to chemical and electrical stimuli in situ30. Herein, the technique to manufacture a PDMS MyoTACTIC culture plate device from a polyurethane (PU) replica, an optimized method to implement immortalized human myogenic progenitor cells to fabricate hMMTs, and the functional assessment of engineered hMMT force generation and calcium handling properties are outlined and discussed.
1. PDMS MyoTACTIC plate fabrication
NOTE: PDMS MyoTACTIC plate fabrication requires a PU negative mold, which can be manufactured as previously described30. The computer-aided design (CAD) SolidWorks file for the MyoTACTIC plate design has been made available on GitHub (https://github.com/gilbertlabcode/MyoTACTIC-SolidWork-CAD-file).
2. Culture of immortalized human myoblast progenitor cells
NOTE: The immortalized myoblasts used in this protocol were obtained from the Institut de Myologie (Paris, France)31.
3. Seeding engineered hMMTs with MyoTACTIC
4. Electrical stimulation and analysis of hMMT-induced post deflection
5. Calcium transient analysis using electrical stimulation
NOTE: For calcium handling experiments, immortalized myoblasts were stably transduced with the MHCK7-GCAMP6 reporter as previously described11,12. Transduced cells were FACS sorted for GFP to obtain the positive population, and then used to fabricate hMMTs. Alternative methods for calcium imaging such as using ratio-metric dyes like Fura-2 AM and Indo-1 or fluorescence lifetime imaging of calcium indicators (e.g., Fluo-4 or Oregon Green BAPTA1) may be amenable to our system.
Described herein are methods to cast a 96-well PDMS-based MyoTACTIC culture platform from a PU mold, to fabricate arrays of hMMT replica tissues, and to analyze two aspects of hMMT function within the culture device-force generation and calcium handling. Figure 1 offers a schematic overview of the preparation of MyoTACTIC culture wells before hMMT seeding. PDMS is a widely used silicone-based polymer, that can be easily molded to create complex devices32. A PDMS-based protocol was designed to cast an unlimited number of 96-well culture devices from a manufactured negative PU mold30. The final cured PDMS positive plate is flexible and can be easily cut into smaller-sized functional units with the aid of a single edge razor blade (Figure 1). This allows the user to scale the device to meet the hMMT replica needs specific to their experimental design. These smaller functional units are also beneficial for overcoming a limitation of working with an ECM scaffold that polymerizes rapidly, such as fibrin hydrogels, by easily tuning the number of wells to match the cell seeding speed of the user. Moreover, PDMS is readily autoclavable and it can be stored for an indefinite period. Finally, from a logistics standpoint, a full MyoTACTIC plate can be covered by any standard 96-well plate lid, and the dimension and profile of MyoTACTIC plate portions are amenable to incubation within a 10 cm culture plate, to ensure hMMT culture sterility. Pluronic F-127 solution coating ahead of cell seeding serves a dual role of providing an additional measure of culture well sterilization, and as a non-ionic surfactant, preventing cell adhesion in favor of uniform cell – ECM remodeling (Figure 1).
When the immortalized myogenic progenitor cell population is ready for experimentation, the cells are then encapsulated in a hydrogel comprised of fibrinogen and basement membrane extract. The cell-ECM mixture is then evenly deposited in the oval pool at the bottom of each MyoTACTIC well, and then over a 14-day culture period, the cells spatially self-organize across these two vertical posts to form a 3D microtissue populated with a bed of aligned, multinucleated, striated myotubes that resemble aspects of native tissue organization (Figure 2a,g,h). Over the remodeling process, uniaxial tension is generated between the two anchorage points, and this guides the tissue self-organization process to in turn drive the formation of a compact tissue. During the differentiation period, the width of the hMMTs constructed from myoblasts derived from healthy patients remains fairly consistent. However, in events of errors that interfere with hMMT remodeling, this uniformity is lost (Figure 2a-f), making this simple metric useful in hMMT quality control assessment. For example, overzealous mixing of the cell – ECM suspension can result in the formation of bubbles. Bubbles carried over to the MyoTACTIC well impede hMMT remodeling. In such instances, bubbles displace some or all of the myoblasts from the area occupied by the bubble, eventually forming a crevice in the final hMMT (Figure 2b; see red arrow). Another example relates to the integrity of the Pluronic F-127 coating in the wells. Pluronic F-127 is a non-ionic surfactant that prevents cells from adhering to the MyoTACTIC well. If the coating is scraped off during aspiration, myoblasts will adhere to the surface of the MyoTACTIC wells, forming new anchorage points and resulting in myotubes that are not aligned across the two posts (Figure 2c). Additional errors can also result in aberrant hMMT remodeling, as seen in Figure 2d–f. Cell vitality post seeding can also be evaluated using calcein / propidium iodide-based staining assays. When these technical errors are avoided, hMMTs are populated by a bed of aligned and multinucleated myotubes that extend across the length of each hMMT (Figure 2g). The myotubes are fairly uniform in size across their length and between different hMMTs (Figure 2h–i). Myotubes are characterized by the presence of sarcomere striations which are visualized by immunostaining for sarcomeric α – actinin (SAA; Figure 2h).
Functional properties can also be examined in hMMTs beginning around 7 days of differentiation. The experimental set up to complete these functional studies is illustrated in Figure 3a. The microscopy setup is pictured on top of a vibraplane table; however, this is not required to complete functional investigations. In functional studies, electrodes generated as described in the electrical stimulation section of the protocol are inserted into the PDMS, such that electrodes reside behind the posts. It is important to have good visualization of the post region before inserting the electrode as the need to reinsert may result in displacement of the tissue from the post. Proper insertion of the electrodes is also important to obtain sharp focus of the post so that post-deflection can subsequently be tracked with the python script. Figure 3b shows a representative displacement of the MyoTACTIC well plate post in response to low (twitch, 0.5 Hz) and high (tetanus, 20Hz) frequency hMMT electrical stimulation. Quantification of post displacement can be used to calculate the induced contractile force of hMMTs (Figure 3c). When this information is combined with hMMT cross-sectional area, specific force can be determined30. Moreover, calcium transients play an important role in muscle contraction. Stable transduction of skeletal muscle myoblasts with a genetically encoded calcium indicator such as GCaMP612,30,33,34, enables live surveillance of calcium transients12,30,34. Calcium transients were analyzed using the afore described electrical stimulation setup up to stimulate day 12 hMMTs generated with immortalized myoblasts expressing the MHCK7-GCAMP6 reporter (Figure 4a), and quantified as mean peak fluorescent intensity during low (twitch, 0.5 Hz) and high (tetanus, 20Hz) frequency electrical stimulation (Figure 4b). It is observed that hMMT calcium handling properties measured across different experiments are relatively consistent (Figure 4b). Quantification methods for post deflection and calcium handling are outlined in Supplementary Video 1 and Supplementary Video 2.
Figure 1: Preparation of MyoTACTIC PDMS plate prior to hMMT seeding. The 96-well platform referred to as MyoTACTIC is fabricated by first curing polydimethylsiloxane (PDMS) within a negative polyurethane (PU) mold. The PDMS-based culture platform is then carefully peeled from the negative PU mold. In an academic setting, the PDMS device is then cut into smaller units containing 6 ± 2 functional wells. These wells are then placed in a sterilization pouch, autoclaved and stored until use. Up to one day before use, the desired number of device portions are placed within a 10 cm culture plate and Pluronic F-127 is added to each well. The 10 cm dish lid is added, the edge is sealed with parafilm before incubating the plate at 4° for 2-24 h. Please click here to view a larger version of this figure.
Figure 2: MyoTACTIC supports hMMT self-organization and formation of aligned myotubes. (a) (top) Schematic timeline of hMMT self-organization and subsequent myotube maturation over a 14-day culture period. (bottom) Representative 4x phase-contrast images to illustrate the kinetics of immortalized myoblasts self-organization across the two vertical posts that result in the formation of a 3D hMMT. Scale bar, 500 µm. (b–f) Representative 4x phase-contrast images of hMMTs on day 12 of differentiation showing hMMT outcomes caused by (b) a bubble within the cell – ECM at time of seeding (red arrow points to bubble induce hMMT damage), (c) incorrect aspiration of the Pluronic F-127 solution coating, and (d–f) hMMT over-remodeling that can signal improper ECM gelation, lack of ACA activity in culture media, improper care of myoblast parental line, etc. Scale bar, 500 µm. (g) Representative tiled and flattened confocal image of a Day 12 hMMT taken at 10x magnification. hMMTs are fixed directly in the PDMS mold, then carefully removed and placed in a 96 well culture plate for staining. Sarcomeric α-actinin (SAA) shown in magenta, and Hoechst 33342 nuclear counterstain shown in cyan. Scale bar, 500 µm. (h) Representative confocal image of a Day 12 hMMT at 40x magnification. hMMT myotubes and nuclei are visualized by immunostaining for SAA (magenta) and counterstaining with Hoechst 33342 (cyan). Scale bar, 50 µm. (i) Dot plot graph of mean hMMT myotube diameter at day 12 of differentiation. Values are reported as mean ± SEM. n = 8 hMMTs from 3 biological replicates, distinguished by symbol shapes, whereby a minimum of 30 myotubes per hMMT are analyzed. Please click here to view a larger version of this figure.
Figure 3: In situ measurement of contractile force within the MyoTACTIC platform. (a) Representation of the electrical stimulation arrangement (left). Although pictured in this setup, a vibraplane table is not required. A smartphone camera and mount were outfitted to the eyepiece of an inverted microscope equipped with a fluorescence lamp for the video recordings of hMMTs electrical stimulation post displacements (left). 25 G needles were wrapped with tin-coated copper wires (bottom right), connected to a BNC to alligator clip connector cable (top right) and were affixed up to an arbitrary waveform generator. Placement of electrodes during electrical stimulation is shown in bottom right. (b) Representative snapshots taken from post deflection videos acquired from hMMTs on Day 12 of differentiation. Videos were captured at 10x magnification. Solid white vertical lines show post placement when hMMTs are relaxed, while dotted white vertical lines show post displacement following high (tetanus 20 Hz) or low (0.5 Hz) frequency stimulation. Scale bar, 100 µm. (c) Dot plot graph of mean hMMT twitch (0.5 Hz)- and tetanus (20 Hz)-induced contractile force on Day 12 of differentiation. Values are reported as mean ± SEM. n = 9 hMMTs from 3 biological replicates, distinguished by symbol shapes. Please click here to view a larger version of this figure.
Figure 4: In situ measurement of calcium handling within the MyoTACTIC platform. (a) Representative images from a 4x magnification video recording of spontaneous GCaMP6+ calcium transients and calcium transients in response to low (twitch contraction, 0.5 Hz) and high (tetanus contraction, 20 Hz) frequency electrical stimulation. hMMTs are outlined by a dotted yellow line. Scale bar, 200 µm. (b) Dot plot graph showing quantification of mean peak fluorescent intensity per hMMT following low (twitch contraction, 0.5 Hz) and high (tetanus contraction, 20 Hz) frequency electrical stimulation. Values are reported as mean ± SEM. n = 9 hMMTs from 3 biological replicates, distinguished by symbol shapes. Please click here to view a larger version of this figure.
Supplemental Video 1: Demonstration of methods to analyze post-deflection data. A representative video of post deflection analysis at day 12 of differentiation being tracked by the custom script during tetanus stimulation. First the ROI size is selected directly within the post tracking script. Next the video is opened by selecting the play button to run the script. A sharp focused region of the post is selected as the ROI and the blue post tracking box is placed. Enter is pressed to lock ROI and pressed again to run the script. "y" is entered as response to the prompt "Multiple contraction video? [Y/N]", then 'n' is entered as a response to the prompt "Export post locations as .csv file? [Y/N]". Post deflection as relative displacement in pixels is the final output. Please click here to download this Video.
Supplemental Video 2: Demonstration of methods to analyze GCaMP6 calcium transient data. A representative video of epifluorescence calcium handling analysis at day 12 of differentiation during tetanus stimulation. First, a video of calcium transients (saved as a series of tiff images) has been opened in ImageJ and the user has navigated through frames until the hMMT is visible. Then, the required analysis measurement "mean grey value" is confirmed and the hMMT is outlined using the polygonal drawing tool. This outline is saved as the region of interest and the fluorescent intensity of each video slice is analyzed using multi-measure. These measurements are copied to the spreadsheet "Calcium Handling Template" with frame data and calcium transient peak values filled in and confirmed. Please click here to download this Video.
This manuscript describes methods to fabricate and analyze a 3D hMMT culture model that can be applied to studies of basic muscle biology, disease modeling, or for candidate molecule testing. The MyoTACTIC platform is cost-friendly, easy to manufacture, and requires a relatively small number of cells to produce skeletal muscle microtissues. hMMTs formed within the MyoTACTIC culture platform are comprised of aligned, multinucleated, and striated myotubes, and respond to electrical stimuli by initiating calcium transients that trigger contraction (Figure 2, Figure 3, Figure 4). Prior studies showed that hMMTs offer a similar response to biochemical stimuli and can reach maturation levels matching those reported in larger format 3D skeletal muscle models12,30.
A critical theme throughout the MyoTACTIC plate fabrication and hMMT generation is ensuring that bubbles are prevented, and if formed have been removed. To facilitate single step casting of an operable PDMS mold, a PU mold had been generated downstream of the initial 3D-printed plastic mold previously described by Afshar et al.30. The PU mold that has been generated allows for users to produce a 96 well footprint of PDMS mold whereby a maximum of 96 wells are functional (contain two posts), as dictated by the PU mold fabrication step. During fabrication of the PDMS MyoTACTIC plate, new users often miss smaller bubbles that remain behind in the liquid PDMS, even after degassing. Bubbles that localize to regions of the PU mold corresponding to the anchoring flexible post structures will result in post breakage upon separation of the PU mold from the PDMS culture plate, and in the process, leaves behind a small piece of cured PDMS in the PU mold. Compressed air can be used to remove these cured PDMS remnants, and a failure to do so will effectively reduce the number of functional wells available for future plate castings. Therefore, it is critical to ensure all bubbles have been removed before the PDMS plate is cured as an advantage of this platform is that is it a stand-alone device, whereby all of the features of the plate are cast in a single step rather than requiring each individual microtissue anchoring point to be introduced to the culture wells manually35,36,37. Most academic laboratory studies do not require an entire MyoTACTIC culture plate. To maximize use of each PMDS plate, it is manually cut into groups of 6 ± 2 MyoTACTIC wells. To improve this process a PU mold that lets users peel functional units of 6 ± 2 MyoTACTIC wells can entirely eliminate this plate cutting step. Moreover, large bubbles introduced into the cell-ECM solution while mixing or during the tissue seeding procedure will impair proper tissue formation. These large bubbles displace cells from the region the bubble occupies, often resulting in hMMTs with regions with few myotubes that will succumb to contractile stress and snap during electrical stimulation.
A notable advantage of MyoTACTIC is the ability to quantify active force generation and calcium handling properties in situ. This is a challenge faced by many other 3D culture systems, where investigating 3D tissue contractile force is implemented as an endpoint assay following removal of the tissue from the culture device12,37. Therefore, MyoTACTIC is well-suited to supporting longitudinal studies, for example, understanding the temporal effects of drug treatment on skeletal muscle. One limitation of the method described herein to quantify active force generation and calcium handling properties in situ is the manual placement of electrodes, which limits the use of this system for high content molecule testing applications. A possible solution would be to engineer a clear lid of electrodes set up as a parallel circuit that can be directly inserted into a standardized set of functional wells enabling electrical stimulation of multiple tissues at once. Alternatively, use of a myogenic progenitor cell line stably expressing a channelrhodopsin construct would allow muscle cell membrane depolarization, and hence, tissue contraction induced by blue light exposure. Moreover, active force is captured in videos as post deflection and quantification of active contractile force can be conducted in an unbiased manner by using a custom semi-automated python script to track post deflection in short videos. Therefore, unbiased longitudinal assessment of stimulated contractile force is enabled by MyoTACTIC30. To improve the efficiency of data analysis, smartphone applications that are designed to measure force while simultaneously capturing videos of post deflection, are ideal to increase throughput.
Finally, the value of this platform has also been previously described in its ability to accurately predict drug response. Similar to clinical outcomes, we have previously reported that treatment of hMMTs (made with primary myoblasts) with myotoxic compounds (dexamethasone, cerivastatin) induced myotube atrophy and decreased active contractile force30. Moreover, the predictive value of MyoTACTIC generated hMMTs was validated by showing that a clinically relevant dosage of a chemotherapeutic used to treat pancreatic cancer, a disease that is often associated with cachexia2, significantly reduced hMMT quality and contractile force30. Therefore, the simple fabrication of MyoTACTIC and its ease of use in the generation of 3D hMMTs shows many advantages for high-content data capture as demonstrated by its reliability with regards to structural and functional outputs. A general limitation of PDMS-based cell culture devices is that PDMS adsorbs proteins. The method described herein makes use of serum-containing culture media, which overcomes this limitation by serving to 'block' the device and allow the effects of molecule and drug treatments to be observed. However, owing to this limitation, definitive conclusions about molecule doses are to be avoided, and dose response curves are encouraged. Conversely, this platform is not suitable for serum-free microtissue culture as the additives will adsorb to the PDMS, thereby hindering tissue health and development. In the future, integrating techniques such as 3D bioprinting and/or automated liquid handling will improve throughput in manufacturing hMMT cultures.
The authors have nothing to disclose.
We would like to thank Mohammad Afshar, Haben Abraha, Mohsen Afshar-Bakooshli, and Sadegh Davoudi for contributing to the invention of the MyoTACTIC culture platform and establishing the fabrication and analysis methods described herein. HL received funding from a Natural Sciences and Engineering Research Council (NSERC) Training Program in Organ-on-a-Chip Engineering and Entrepreneurship Scholarship and a University of Toronto Wildcat graduate scholarships. PMG is the Canada Research Chair in Endogenous Repair and received support for this study from the Ontario Institute for Regenerative Medicine, the Stem Cell Network, and from Medicine by Design, a Canada First Research Excellence Program. Schematic diagrams were created with BioRender.com.
0.9% Saline Solution, Sterile | House Brand | 1010 | 10 mL aliquots of the solution are made and stored at 4°C |
25G Needle | BD, Medstore, University of Toronto | 2548-CABD305127 | |
6-Aminocaproic Acid, ≥99% (titration), Powder | Sigma – Aldrich | A2504-100G | A 50 mg / mL stock solution is generated by dissolving 5 mg of 6-aminocaproic acid powder in 100 mL of autoclaved, distilled water. The solution is vaccum filtered and 10 mL aliquots are stored at 4°C |
6.35 mm ID Tubing | VWR | 60985-528 | |
AB1167 Myoblast Cell Line | Institut de Myologie (Paris, France) | ||
Arbitrary Waveform Generator | Rigol | DG1022Z | |
Basement Membrane Extract (Geltrex) | Thermo Fisher Scientific | A14132-02 | Stored as aliquots of 50 µL or 100 µL at -80°C |
Benchtop Vacuum Chamber | Sigma – Aldrich | D2672 | |
BNC to Aligator Clip Cable | Ordered from Amazon | ||
Culture Plastics | Sarstedt | Includes culture plates, serological pipettes, etc | |
Dimethyl Sulfoxide | Sigma – Aldrich | D8418-250ML | |
DPBS, Powder, No Calcium, No Magnesium | Thermo Fisher Scientific | 21600069 | |
Dulbecco's Modified Eagle Medium (DMEM) (1X) | Gibco | 11995-065 | This is a high glucose DMEM with L-glutamine and sodium pyruvate |
Fetal Bovine Serum | Fisher Scientific | 10437028 | |
Fibrinogen from Bovine Plasma | Sigma – Aldrich | F8630-5G | Aliquots ranging from 7 – 10 mg of fibrinogen powder are made and stored at -20°C |
Filtropur Syringe Filter, 0.22um Pore Size | Sarstedt | 83.1826.001 | |
Horse Serum | Gibco | 16050-122 | |
Human Recombinant Insulin | Sigma – Aldrich | 91077C | Stock solution is 100X and made by dissolving 1 mg of human recombinant insulin in 1 mL of DMEM and 1 µL of NaOH 10N. Solution is filtered and stored as 1 mL aliquots at 4°C |
Image Acquisition Software | Olympus | cellSens Dimension | |
Image Processing Software | National Institutes of Health | ImageJ | |
Isotemp Oven | Thermo Fisher Scientific | 201 | |
Microscope | Olympus | IX83 | |
Microscope – Camera Mount | Labcam | Labcam for iPhone | Ordered from Amazon |
Penicillin-Streptomycin (10,000 U/mL) | Gibco | 15140-122 | |
Plastic Disposable Syringes, 1cc | BD | 2606-309659 | |
Plastic Disposable Syringes, 50cc | BD | 2612-309653 | |
Pluronic F-127, Powder, BioReagent | Sigma – Aldrich | P2443-250G | A 5% stock solution of pluronic acid is made by dissolving 5 g of pluronic acid powder in 100 mL of chilled, autoclaved, distilled water. The solution is vaccum filtered and 10 mL aliquots are stored at 4°C |
Polydimethylsiloxane (Sylgard 184 Silicone Elastomer Kit) | Dow | 4019862 | Kits are also available at Thermo Fisher Scientific, Sigma – Aldrich, etc. |
Polyurethane Negative Mold | In House | ||
Release Agent | Mann Release Technologies | 200 | |
Rotary Vane Vacuum Pump | Edwards | A65401906 | |
Scalpel | Almedic, Medstore, University of Toronto | 2586-M36-0100 | |
Single Edge Razor Blade | VWR | 55411-050 | |
Skeletal Muscle Cell Basal Medium | Promocell | C-23260 | 30 mL aliquotes are generated and at stored at 4°C. |
Skeletal Muscle Cell Growth Medium (Ready-to-use) | Promocell | C-23060 | 42 mL aliquots are generated and stored at 4°C. |
Smartphone (iPhone) | Apple | SE | |
Standard Duty Dry Vacuum Pump | Welch | 2546B-01 | |
Sterilization Bag | Alliance | 211-SCM2 | |
Thimble | Igege | Ordered from Amazon | |
Thrombin from human plasma | Sigma – Aldrich | T6884-250UN | 100 units of thrombin is dissolved in 1 mL of a 0.1% BSA solution. 10 µL aliquots are prepared and stored at – 20°C. |
Tin coated copper wire | Arco | B8871K48 | Ordered from Amazon |
Trypan Blue Solution, 0.4% | Thermo Scientific | 15250061 | |
Trypsin-EDTA, 0.25% | Thermo FIsher Scientific | 25200072 | |
Vacuum Chamber 2 | SP Bel-Art | F42027-0000 |