This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.
Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues.
To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRPα and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.
Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.
Cardiac tissue engineering has advanced greatly in the last decade, with multiple groups publishing results of fully functional, beating tissues made from both murine cardiomyocytes1-6 and, more recently, human stem cell-derived cardiac myocytes7-12. The cardiac tissue engineering field is driven by two primary and essentially independent goals: 1) to develop exogenous grafts that can be transplanted into failing hearts to improve function4-6; and 2) to develop in vitro models for studying physiology and disease, or as screening tools for therapeutic development2,7.
Three-dimensional (3-D) cell culture is considered essential for developing next generation screening tools, as the 3-D matrix reflects a more natural cardiac microenvironment than traditional 2-D monolayer cell culture; indeed some aspects of cell biology are fundamentally different in 2-D vs. 3-D cultures13,14. Additionally, engineered cardiac tissues are constructed from completely defined components: an extracellular matrix, and a cell population. For traditional engineered human cardiac tissues, while the extracellular matrix composition (usually fibrin9 or collagen7,8,10) is strictly controlled, the input cell composition is less well defined, with the entire mixture of cells from a directed cardiac differentiation of either embryonic stem cells (ESC7,9) or induced pluripotent stem cells (iPSC10,12) being added to the tissues. Depending on the specific cell line and the efficiency of the differentiation protocol used, the resulting percentage of cardiomyocytes can range from less than 25% to over 90%, the specific cardiomyocyte phenotype (i.e., ventricular-, atrial-, or pacemaker-like) can also vary, even the non-cardiomyocyte fraction can be highly heterogeneous15,16 and alter the maturity of the differentiated cardiac myocytes17.
Recent cardiac tissue engineering work has attempted to control the input population of cells, with either a cardiac reporter human embryonic stem cell line8 or cell surface markers18 being used to isolate the cardiac myocyte component of the differentiation. While initially a tissue composed of only cardiac myocytes would seem to be the ideal, this is in fact not the case; hECTs composed solely of cardiac myocytes fail to compact into functional tissues, with some groups finding a 3:1 ratio of cardiac myocytes:fibroblasts producing the highest twitch force8. By using various cell selection methods, including surface markers for live cell sorting, it is possible to create hECTs with defined cell populations. While markers of non-cardiac stromal cells have been available for some time, such as the putative fibroblast marker CD9019,20, surface markers of cardiac myocytes have been more difficult to identify. SIRPα was among the first cardiac surface markers identified for human cardiac myocytes18 and has been shown to be highly selective for the cardiac lineage. Recently, we have found that double-sorting for SIRPα+ and CD90– cells yields nearly pure cardiomyocytes, with the CD90+ population exhibiting a fibroblast-like phenotype (Josowitz, unpublished observations). Based on these collected findings, herein we describe creating hECTs using a 3:1 combination of SIRPα+/CD90– cardiomyocytes and CD90+ fibroblasts.
The ability to engineer a completely defined human cardiac tissue is essential not only for creating robust screening tools, but also for developing model systems to investigate emerging cell- and gene-based cardiac therapies. In particular, numerous cell therapies for heart failure, utilizing cell types including mesenchymal stem cells (MSC)21, cardiac stem cells22 and bone marrow mononuclear cells23-25, have been tested in clinical trials. While many of the initial results have been promising21,23,25, the initial benefit often diminishes over time26-29. A similar trend has been reported in murine engineered cardiac tissues, which display a significant functional benefit due to MSC supplementation, but the benefit is not sustained during long-term culture1. Underlying the sub-optimal performance is our limited knowledge of the mechanisms governing cell therapies. A deeper understanding of how therapeutic cells exert their beneficial influence, as well as potential negative consequences of myocyte-nonmyocyte interactions, would enable the development of improved therapies yielding clinically significant and sustained benefits, with minimal side effects, for patients with heart failure.
Here, we describe the use of defined hECTs to interrogate mechanisms of cell-based therapy. The controlled tissue composition is essential to identify specific factors impacting cardiomyocyte performance. Directly supplementing hECTs with the therapeutic cell type of interest (e.g., MSCs), can reveal the effects on cardiac myocyte performance, as we have demonstrated in rat ECTs1.
The following multi-step protocol begins with directed cardiac stem cell differentiation, followed by fabrication of the multi-tissue bioreactor, and concluding with a description of tissue construction and functional analysis. Our experiments are performed using the NIH-approved H7 human embryonic stem cell (hESC) line. However, the following protocols have also been tested using an additional hESC line and three induced pluripotent stem cell (hiPSC) lines with similar results. We have found that efficiency in cardiomyocyte differentiation and success in hECT fabrication can be cell line dependent, particularly for hiPSC lines derived from individual patients. By following this protocol, two 6-well dishes are plated with a total of 1.68 million hESCs (140,000 cells per well), which yields approximately 2.5 million myocytes after differentiating for 20 days and sorting, enough to make six defined tissues.
Note: Perform all cell manipulations in aseptic conditions using a HEPA-filtered class II biological safety cabinet and sterilize all solutions by filtering them through a 0.2 µm filter. Perform tissue construction and function testing in either the same aseptic conditions or a laminar flow hood.
1. Seeding of H7 hESCs in Preparation for Cardiac Differentiation
2. (Day 4-24) Differentiation of Human Embryonic Stem Cells to Cardiomyocytes30,31
3. (Day 24, Differentiation Day 20) Isolation of Cardiac Myocytes and Fibroblast-like Cells
4. Human Cardiac Tissue Engineering
To obtain cardiac myocytes, a slightly modified version of the Boheler and Lian differentiation methods is used30,31. It is imperative that the differentiation starts during the log-phase of cell growth, but also that the starting population is sufficiently confluent to obtain a useable number of cells after sorting (approximately 75% is optimal). Typically, for H7 hESCs, plating at a density of 140,000 hESCs per well of a 6-well dish in essential 8 media and 5% CO2 incubator maintained at 37 °C yields sufficiently confluent cultures after 4 days to begin the differentiation, as illustrated in Figure 1A–D. Since insulin inhibits cardiac specification during the differentiation process32, insulin free media (differentiation media I, Table 1) is used for the first 10 days of differentiation. Once differentiation is initiated via the application of the GSK3 inhibitor CHIR99021, significant cell death will occur (Figure 1E). As a consequence it is essential to change the media every day. On day 2 of differentiation, the cells are removed from CHIR99021 and rested in differentiation media I (with no added small molecules) for 24 hr. After the 24 hr rest in differentiation media, the cells are treated with IWR-1 for 48 hr, after which they begin to self-organize into clusters (Figure 1F) that beat as early as seven days from starting the differentiation. Since cardiac specification has occurred after the application of IWR-1, the media is changed to media containing insulin (differentiation media II, Table 1). By 18 days of differentiation, the colonies of beating cells have connected and partially detached from the cell surface, forming robustly beating “webs” throughout the dish (Video Figure 1).
On day 20, the beating monolayer is gently dissociated via enzymatic methods to obtain single cells for live fluorescent associated cell sorting (FACS). Using the differentiation method described above, we conservatively harvest approximately 65% of the population as SIRPα+ and 10% as CD90+ (Figure 2). Generally 1-2 million SIRPα+ cells are obtained per 6-well plate.
After reaggregation for 48 hr in a 3:1 ratio of SIRPα+:CD90+ cells, the defined cell aggregates are mixed with a collagen-based hydrogel and pipetted into narrow wells in the baseplate of the multi-tissue bioreactor. Typically, before removal from the dish, small clusters of 5-10 cells will be seen beating on the surface of the dish. The defined tissue bioreactor consists of three components: a master mold to cast the PDMS posts; a frame to hold two PDMS racks of posts; and a black polytetrafluorethylene baseplate containing six wells for the tissue mix (Figure 3A). The entire tissue construction process is performed on ice and in aseptic conditions. After adding the liquid cell-matrix mixtures to the wells in the baseplate, the PDMS posts are attached to the polysulfone frame, and then inverted so that the posts align with the wells in the baseplate (Figure 3B). After 48 hr of incubation, the tissues should be sufficiently compacted that the baseplate can be removed. The baseplate is most easily detached by extracting the entire system from the media and gently removing any excess media from between the PDMS and baseplate. If all of the media is not removed, surface tension of the remaining fluid can pull the tissues off the posts. Once the media is removed, gently push the baseplate back against the PDMS posts to help loosen the tissue from the baseplate. Then gradually push the baseplate off by gently moving one side up a few millimeters, then the other side up the same amount. Repeat this process until the baseplate is removed, with all 6 hECTs attached to their respective PDMS end-posts (Figure 4A). Removing the baseplate should take no more than one minute. Replace the system into the cell culture media, inverted, so that all the tissues are covered by the cell culture media.
Approximately one day after removing the baseplate, weak localized contractions should be observable in the defined hECTs under bright field microscopy. After 7 days, the tissues have matured and compacted (Figure 4B-C) with aligned sarcomeres (Figure 4D). The tissues visibly deflect the PDMS posts with an average of 5-15 µN of force (Figure 4E). Twitch force measurements for the defined engineered cardiac tissues can be measured by real-time tracking of the post tip deflection with a high speed camera and custom data acquisition software, and applying the post deflection to beam bending theory after determining the Young’s modulus of the silicone posts using a force transducer, as explained in more detail previously.1,7 Maintaining sterility during data acquisition ensures the ability to measure changes in tissue function over time. We have maintained functional tissues for several weeks, as explained in more detail elsewhere1. Our preliminary findings indicate a substantial enhancement of hECT contractile force when tissues are supplemented with 10% human mesenchymal stem cells at both 1 Hz and 2 Hz pacing frequencies (Figure 4F).
Figure 1: Representative images of H7 hESCs during the expansion and cardiac differentiation process. The expansion phase should last 4 days, with growth of the colonies expanding from 24 (A), to 48 (B), 72 (C) and 96 hr (D). After 96 hr of growth the differentiation is begun, resulting in substantial cell death during the first 48 hr of CHIR99021 treatment (E). After two days of IWR-1 treatment, reorganization occurs (F) to form clusters of cells that beat as early as day 7. Further organization into a robustly beating “web” occurs from day 7 to day 20, when the monolayers are dissociated for creating engineered cardiac tissues.
Video Figure 1: Representative cardiac monolayer after 17 days of differentiation. Please click here to view this video. Spontaneous beating within the monolayer is usually first observed between day 7 and day 10, and by 17 days of differentiation the monolayer has formed interconnected “webs” that beat robustly.
Name | Composition | Differentiation days used |
Differentiation Media I | RPMI 1640 B27 Supplement (minus insulin) Penicillin-Streptomycin |
D0-D1 (with CHIR99021) D2 D3-D5 (with IWR-1) D5-D7 |
Differentiation Media II | RPMI 1640 B27 Supplement (with insulin) Penicillin-Streptomycin |
D7-D20 |
Table 1: Composition of Differentiation Medias.
Figure 2: Representative live cell sorting results. Two samples were prepared: an unstained control and a stained control using 1:500 dilution of the SIRPα-PE/Cy7 antibody and 1:250 dilution of the CD90-FITC antibody. After staining, the cells were sorted at 20 psi in sorting buffer composed of PBS, 10% neonatal bovine serum, 10 µM ROCK inhibitor Y-27632, and 1 µg/ml DAPI. Both cell populations were gated in the forward and side scatter profiles to exclude debris (A, blue line) then single cells were selected by comparing the pulse width and height (pulse width analysis) in both the forward scatter (B, blue line) and side scatter channels (C, blue line). Next, live cells were selected by gating the DAPI– population (D, blue line). The final cell populations for collection were determined by examining the FITC and PE-Cy7 expression levels of the live populations. The unstained control (E, left) was used to establish the appropriate gate to select for the FITC+ (CD90) and SIRPα-PE/Cy7+ (cardiomyocyte) population present in the stained sample (E ,right). The FITC and SIRPα-PE/Cy7+poplations were collected in separate 15 ml centrifuge tubes.
Figure 3: Schematic of the multi-tissue bioreactor and the cardiac tissue engineering process. Construction of the multi-tissue bioreactor requires three components (A): 1) a polytetrafluoroethylene master mold 9 x 33 x 5 mm3, with 6 evenly spaced holes 0.5 mm in diameter; 2) a 25 x 35 x 11 mm3 polysulfone frame to hold the PDMS posts during tissue construction and culture; and 3) a 20 x 40 x 5 mm3 black polytetrafluorethylene baseplate with 6 wells of 6 x 1 x 1 mm3 dimensions, spaced 4 mm apart along its length. To create human engineered cardiac tissues (B), the PDMS posts are fabricated by PDMS soft lithography using the polytetrafluorethylene master mold, two of which are then pressed onto the tabs of the polysulfone frame to form 6 pairs of force-sensing cantilever posts. The tissue solution is pipetted into the wells in the black polytetrafluorethylene baseplate. The frame and posts are then inverted and aligned on the polytetrafluorethylene baseplate so that one pair of posts enters each well containing the tissue mix. After 48 hr, the baseplate is removed and the defined tissues are cultured on the posts, remaining inverted in NBS media.
Figure 4: Representative images and functional measurements of defined human engineered cardiac tissues. The multi-tissue bioreactor system holds six tissues in parallel (A, arrowheads). Defined tissues are self-assembled within seven days after tissue creation (top view, B and oblique side view, C). (D). Portion of a defined hECT stained for cardiac troponin-T (cTnT). (E) Representative twitch tracing shows raw force over time during 1 Hz pacing by electrical field stimulation. (F) Twitch tracing during stimulation at 1 Hz (0-2 sec) and 2 Hz (2-4 sec) pacing, with arrow marking change in frequency, for both unsupplemented defined hECTs (solid line) and tissues supplemented with 10% hMSCs (dotted line).
Construction of defined human engineered cardiac tissues (hECT) can provide a more consistent and reliable model of human cardiac myocyte function. Critically, all cellular and extracellular components in the system are known and can be manipulated as desired, thus removing the confounding influence of other unknown cell types resulting from the differentiation process. To balance rapid cell growth and high yield, it is preferable that the differentiation starts at 75% confluence of the hESCs, ideally four days after plating. Additionally, the use of ROCK inhibitor Y-27632 during both the cell dissociation and reaggregation after sorting greatly enhances cell viability. The presence of spontaneous beating of myocytes during the differentiation process is an important indicator of the efficiency and health of the differentiation. If spontaneous beating is not observed this may indicate poor differentiation efficiency, either due to ineffective reagents or a loss of pluripotency of the stem cells.
During tissue construction, the PDMS posts must align properly with the channels in the baseplate to create well-formed tissues that last long enough for measuring contractile function. To enhance the ease of device alignment, and strengthen tissue formation around the posts where they are susceptible to breaking, it is possible to add extra width (approximately one post diameter on each side of the post) to the ends of the channels, as demonstrated by the “dog bone”-shaped channels in Figure 3A. Without proper device alignment the tissue will either detach from the posts shortly after culture, or will remain in the well when the baseplate is removed. If a well-formed tissue falls off during baseplate removal, it is possible to gently reattach the tissue to the posts using fine forceps under a dissecting microscope, although it is easy to damage the delicate hECTs if adequate care is not taken.
A major strength of the described defined system is the ability to interrogate the contribution of direct cell-cell interaction mechanisms in cardiac cell therapies based on specific control of cell composition. The injection of cells in animals is imprecise and subject to low viability and retention33. Additionally, the effect of the injected cells on the target tissue is complicated by interactions with other physiologic systems, such as the immune system. As such, understanding the contribution of direct cell-cell interactions in cardiac cell therapies is challenging to address in model organisms. Therefore, an advantage to the described method is that cell-cell interactions are analyzed in a biomimetic three-dimensional environment, preserving key aspects of the cardiac niche, and in the presence of the specific cell types of interest — namely, the cardiac myocytes and fibroblasts. The utility of using the defined hECT system is demonstrated with the supplementation of 10% hMSCs derived from human marrow34,35 and used in clinical trials to the cell mixture during tissue creation. Those tissues that were supplemented with the hMSCs exhibited larger contractile force than the unsupplemented defined human tissues (Figure 4F). Since the tissue environment and composition has been controlled, the enhancement of tissue function with MSC supplementation reflects an inherent effect of hMSCs on cardiomyocyte function. Another strength to the system is that since the tissues are smaller than used previously,1,7 cell use is more efficient, an important consideration when using directed differentiations of pluripotent stem cells as the cell source.
Beyond studying mechanisms of cell therapy, the defined hECT system would enable the examination of cellular basis of cardiovascular cell-cell interactions. Perturbation of the biology of the cells prior to tissue construction, for example with shRNAs, would permit the investigation of molecular mechanisms governing cell-cell and cell-matrix interactions. An additional advantage to the tissue engineering system is the formation of aligned myofibrils capable of functional contractions. Thus, using the defined hECT system, the dynamics of myofibril formation, cardiomyocyte development and the organization of the tissue architecture can all be investigated via modification of the components of the extra-cellular matrix or molecular manipulation of the cells. The human and 3-D nature of the hECT system additionally increases the translatability of the findings.
While the described system offers a powerful method for understanding basic questions of cardiovascular biology, it is not without limitations. First, the engineered tissues exhibit an immature phenotype, consistent with published twitch force data from newborn myocardial samples7. While the phenotypic immaturity could be a confounding factor in evaluating cell interaction mechanisms, there is evidence that diseased cardiomyocytes revert to a fetal gene program7,36-38. If findings from the hECT system prove to be relevant in the context of a failing heart, this may be advantageous for testing cardiac therapies. The protocol also uses a hESC qualified basement membrane matrix during tissue construction. The composition of this matrix can vary from lot to lot, and while defined basement matrix alternatives are available they have yet to be evaluated in the context of hECTs.
Other limitations include the lack of a vascular system and no systems-level interaction effects. Given the size of the tissues, metabolic demands are met by diffusion alone so no vascular system is needed to ensure cell viability. However, endothelial cell signaling may be an important factor in specific cell therapies. If this is the case, a specific number of endothelial cells can simply be added to the defined cell mix during tissue construction. While systems-level interactions are important for complete understanding of cell mechanisms, the engineered tissue system offers a reductionist approach to simplify the problem in order to address the mechanism systematically. Complexity can be added to the defined tissue system as needed through the introduction of systems-related effects, such as leukocytes to incorporate an immune response, or by adding neighboring tissue types to interrogate paracrine signaling.
As an investigative tool, the defined human engineered cardiac tissue system offers the benefits of species-specific human cells, a biomimetic 3-D culture environment with controlled biocomplexity, straightforward and longitudinal monitoring of contractile function, and a defined cellular and matrix composition. Future directions for the hECT system include manipulating the therapeutic cell type of interest with siRNA or shRNA prior to introduction in the tissue in order to investigate specific molecular details of cell-cell interaction. Additionally, rather than using hESCs to form the cardiac cells, it is possible to use induced pluripotent stem cells (iPSCs) from patients with an inherited mutation in an effort to model related cardiac disease manifestations in the engineered tissues. Thus, our method of creating human engineered cardiac tissues with defined cell populations promises to open new avenues for studying cardiac cell therapies to help accelerate the development of novel treatments for patients with heart disease.
The authors have nothing to disclose.
This work was supported by NIH (1F30HL118923-01A1) to T.J.C., NIH/NHLBI PEN contract HHSN268201000045C to K.D.C., the research grant council of Hong Kong TRS T13-706/11(K.D.C), NIH (R01 HL113499) to B.D.G., the American Heart Association (12PRE12060254) to R.J., and Research Grant Council of HKSAR (TBRS, T13-706/11) to R.L. Additional funding was provided to T.J.C. by NIH DRB 5T32GM008553-18 and as a traineeship on NIDCR-Interdisciplinary Training in Systems and Developmental Biology and Birth Defects T32HD075735. The authors also wish to gratefully acknowledge Arthur Autz at The Zahn Center of The City College of New York for assistance with machining the bioreactor and Mamdouh Eldaly for technical assistance. We also thank Dr. Kenneth Boheler for advice on cardiac differentiation, and Dr. Joshua Hare for generously providing human mesenchymal stem cells.
Cell Culture | Company | Catalog Number | Comments |
Amphotericin B | Sigma-Aldrich | A2411 | Prepare a 2.5 mg/ml stock in DMSO and filter-sterilize |
B27 with Insulin | Life Technologies | 17505055 | |
B27 without Insulin | Life Technologies | A1895601 | |
CHIR99021 | Stemgent | 04-0004 | Create 6 μM stock, then aliquot and store at -20 °C. |
Essential 8 Media | Life Technologies | A1517001 | |
H7 Human Embryonic Stem Cells | WiCell | WA07 | |
hESC Qualified Matrix, Corning Matrigel | Corning | 354277 | Thaw on ice at 4 °C overnight then aliquot 150 μl into separate tubes and store at -20 °C. |
IWR-1 | Sigma-Aldrich | I0161 | Create 10 mM stock and aliquot. Store at -20 °C |
Neonatal Calf Serum | Life Technologies | 16010159 | |
Non-enzymatic Dissociation Reagent: Gentle Cell Dissociation Reagent | Stem Cell Technologies | 7174 | |
Penicillin-Streptomycin | Corning | 30-002-CI | |
RPMI 1640 | Life Technologies | 11875-093 | Keep refrigerated |
Y-27632 (ROCK Inhibitor) | Stemgent | 04-0012 | Resuspend to a 10 mM stock concentration, aliquot and store at -20 °C. Avoid freeze thaw cycles. |
Cell Sorting | Company | Catalog Number | Comments |
4’,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI) | Life Technologies | D1306 | |
CD90-FITC | BioLegend | 328107 | |
Enzymatic Dissociation Reagent: Cell Detach Kit I (0.04 % Trypsin/ 0.03% EDTA, Trypsin neutralization solution and Hanks Buffered Salt Solution) | PromoCell | C-41200 | |
Fetal Bovine Serum | Atlanta Biologics | S11250 | |
SIRPα-PE/Cy7 | BioLegend | 323807 | |
Tissue Construction | Company | Catalog Number | Comments |
0.25% Trypsin/0.1% EDTA | Fisher Scientific | 25-053-CI | Optional: For collection of supplemental cells of interest |
10x MEM | Sigma-Aldrich | M0275-100ML | |
10X PBS Packets | Sigma-Aldrich | P3813 | |
Collagen, Bovine Type I | Life Technologies | A10644-01 | Keep on ice |
DMEM/F12 | Life Technologies | 11330057 | |
Dulbecco’s Modified Eagles Medium (DMEM), High Glucose | Sigma-Aldrich | D5648 | |
Polydimethylsiloxane (PDMS) | Dow Corning | Sylgard 184 | |
Sodium HEPES | Sigma-Aldrich | H3784 | |
Sodium Hydroxide | Sigma-Aldrich | 221465 | |
Materials | Company | Catalog Number | Comments |
1.5 ml microcentrifuge tubes | Fisher Scientific | NC0536757 | |
15 ml polyproylene centrifuge tube | Corning | 352096 | |
5 ml Polystyrene Round-Bottom Tube | Corning | 352235 | With integrated 35 μm cell strainer |
50 ml polyproylene centrifuge tube | Corning | 352070 | |
6-well flat bottom tissue-culture treated plate | Corning | 353046 | |
Cell Scraper, Disposable | Biologix | 70-2180 | |
Polysulfone | McMaster-Carr | ||
Polytetrafluoroethylene (Teflon) | McMaster-Carr | ||
Equipment | Company | Catalog Number | Comments |
Dissecting Microscope | Olympus | SZ-61 | Or similar, must have a mount for the high speed camera to attach |
Electrical Pacing System | Astro-Med, Inc | Grass S88X Stimulator | |
High Speed Camera | Pixelink | PL-B741U | Or similar, but must be capable of 100 frames per second for accurate data acquisition |
Plate Temperature Control | Used to maintain media temperature during data acqusition. | ||
Custom Materials | Company | Catalog Number | Comments |
LabView Post-tracking Program | available upon request from the authors |