Biowire platform for maturation of human pluripotent stem cell-derived cardiomyocytes

Methods xxx (2015) xxx–xxx

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Biowire platform for maturation of human pluripotent stem cell-derived cardiomyocytes Xuetao Sun a, Sara S. Nunes a,b,c,⇑ a

University Health Network, Toronto General Research Institute, 101 College St., Canada Institute of Biomaterials and Biomedical Engineering, University of Toronto, Canada c Heart & Stroke/Richard Lewar Centre of Excellence, University of Toronto, Canada b

a r t i c l e

i n f o

Article history: Received 15 July 2015 Received in revised form 31 October 2015 Accepted 3 November 2015 Available online xxxx Keywords: Stem cells Cardiomyocyte Tissue engineering Maturation Biomimetic cues

a b s t r a c t Human pluripotent stem cells (hPSCs)-derived cardiomyocytes (hPSC-CMs) represent a potential indefinite cell supply for cardiac tissue engineering and possibly regenerative medicine applications. However, these cells are immature compared with adult ventricular cardiomyocytes. In order to overcome this limitation, an engineered platform, called biowire, was devised to provide cultured cardiomyocytes important biomimetic cues present during embryo development, such as threedimensional cell culture, extracellular matrix composition, soluble factors and pacing through electrical stimulation, to induce the maturation of hPSC-CMs in vitro. Ó 2015 Elsevier Inc. All rights reserved.

1. Introduction A variety of approaches have been utilized to repair/regenerate an injured, diseased or aged heart. These can be implemented through intrinsically enhancing innate pathways of repair/ regeneration, directing cellular reprogramming, or extrinsically generating appropriate microenvironments to promote survival/ function of cardiomyocytes. One of the most actively pursued strategies for heart repair has been cell replacement/transplantation therapies, which has been aided by cardiac tissue engineering and biomaterials delivery. So far, nearly all possible cell sources have been investigated in animal models for their potential in cardiac regeneration [1]. Of these cell types, human pluripotent stem cells (hPSCs)-derived cardiomyocytes (hPSC-CM) represent a potentially unlimited cell source for cardiac tissue engineering. However, these cells are immature compared with adult ventricular cardiomyocytes [2,3]. Immature human embryonic stem cells-derived cardiomyocytes (hESC-CMs) display automaticity, slow conduction and pose arrhythmic risk in large animals after engraftment [4]. Furthermore, hESC/iPSC-CM can be used to develop in vitro models of human cardiac tissue for investigation of various aspects of human cardiac diseases and drug screening,

though, the fact that existing cardiac stem cell differentiation protocols [5–7] only produce cardiomyocytes associated with fetal-like maturity challenges the significance of modeling the behavior of the adult myocardium using hPSC-CMs [8]. It has been reported adult-like phenotype could be promoted through cyclic mechanical [9] or electrical stimulation [10] of neonatal rat cardiomyocytes. Maturation of hPSC-CMs could also be promoted using gel compaction and cyclic mechanical stimulation [11,12]. However, the maturation state of these hPSC-CMs was only moderate and coupled with minimal electrophysiological and calcium handling enhancements. Thus, in order to overcome these limitations, we devised a system termed ‘biological wire’ (biowire) to provide both structural cues and electrical field stimulation to mature hPSC-CMs [13]. This microfabricated platform allows generation of aligned cardiac tissues, which can be subjected to electrical stimulation to promote structural and electrophysiological maturation of cardiomyocytes. The details regarding the generation of such biowires will be described here.

2. Materials and methods 2.1. Materials

⇑ Corresponding author at: University Health Network, 101 College St., MaRS, TMDT 3-904, Toronto, ON M5G 1L7, Canada. E-mail address: [email protected] (S.S. Nunes).

All solutions are prepared using ultrapure water (MilliQ, 18.2 MX cm at 25 °C) and analytical grade reagents.

http://dx.doi.org/10.1016/j.ymeth.2015.11.005 1046-2023/Ó 2015 Elsevier Inc. All rights reserved.

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 Master components: SU-8 50 (MicroChem Corp.), SU-8 2050, 4inch silicon wafer, propylene glycol monomethyl ether acetate (PGMEA) (Doe & Ingalls Inc.).  Electrical stimulation chamber components: 3 mm Diameter carbon rods, 0.2 mm platinum wires.  Tools: Dremel, drill bit 1, drill bit size 2.  Polydimethylsiloxane (PDMS) (Sylgard 184, Dow Corning)  Collagenase type I solution: 0.2% collagenase type I (Sigma) (w/v) and 20% fetal bovine serum (FBS) (v/v) in phosphatebuffered saline (PBS) with Ca2+ and Mg2+. Sterilized with 0.22 lm filter and make 12 ml aliquots. Store at 20 °C.  DNase I (CalBiochem): 1 mg/ml DNase I stock solution in water. Filter sterile and store 0.5 ml aliquots at 20 °C.  0.25% trypsin/EDTA (Gibco)  Wash medium: Iscove’s Modified Dulbecco’s Medium (IMDM) with 1% penicillin/streptomycin.  Stop medium: Wash medium:FBS (1:1).  StemPro-34 culture medium (Thermo Fisher Scientific) (Per 50 ml): add 1.3 ml supplement, 500 ll of 100 L-Glutamine, 250 ll of 30 mg/ml transferrin, 500 ll of 5 mg/ml ascorbic acid, 150 ll of 26 ll/2 ml monothioglycerol (MTG), and 500 ll (1%) penicillin/streptomycin.  Collagen gel (final concentrations): 2.1 mg/ml of rat tail collagen type I (BD Biosciences) in 24.9 mM glucose, 23.8 mM NaHCO3, 14.3 mM NaOH, 10 mM HEPES, in 1xM199 media with 10% of growth factor-reduced Matrigel (BD Biosciences).

2.2.1. Design and fabrication of master 1. Design the device using AutoCAD (Autodesk, Inc) (Fig. 1A, left). Print the device feature on two film masks (CADART) corresponding to the two layers of the master. 2. Pour SU-8 50 onto a 4-inch silicon wafer and spin at 2000 RPM for 30 s. Then bake on hotplate preheated to 95 °C for 10 min before exposing to UV light at a dose of 200 mJ/ cm2 to make the base layer. 3. Pour SU-8 2050 onto the silicon wafer and spin at 2500 RPM for 30 s. Then Bake at 95 °C for 15 min before cooling to room temperature (r.t). 4. Repeat step 3 two more times. Then expose the coated wafer to UV light in a dose of 270 mJ/cm2 under the first-layer mask to make the first layer including the suture channel and the chamber with thickness of 185 lm. Then Bake at 95 °C for 15 min. 5. Repeat step 3 twice to make the second layer of SU-8 2050. 6. Use a mask aligner to align the second-layer mask to the features on the first layer before exposing to UV at a total dose of 240 mJ/cm2. The exposure time is determined by the required total UV dose and the UV light output intensity measured on that specific day prior to use. Bake at 95 °C for 15 min. 7. Immerse the wafer in PGMEA and stir for 30 min on an orbital shaker to develop the wafer. 8. Cast PDMS mixture (cross-linker to base in a ratio of 1:10) with minimal air bubbles onto the master with all features covered. 9. Bake for 2 h at 70 °C before cutting out the PDMS biowire template and trimming to a desired length and width (Fig. 1A, right). Autoclave the PDMS biowire template. 10. Under sterile condition, transfer the PDMS biowire template to a Petri dish, use tweezers to hold a piece of sterile surgical silk suture (6-0) of desired length in the center of channel of the PDMS microwells by placing it in the grooves located at both ends of the channel (Fig. 1B I, Supplemental video 1). Expose to UV light overnight for sterilization.

2.2.2. Electrical stimulation chamber 1. Cut rectangular pieces (2 cm (length)  0.85 cm (width)  0.35 cm (thickness)) out of a sheet of polycarbonate. Use a pair of rectangular pieces as frame of the electrical stimulation chamber. 2. Use a dremel attached with a drill bit of 3 mm diameter to make two holes, which are 2 cm apart along the center line of each polycarbonate frame piece. 3. Make a pair of 1.5 cm-long carbon rods. Use a dremel tool attached with a 1 mm diameter drill bit to make a hole through the carbon rod about 3 mm from the end of the carbon rod. String platinum wire through the hole and wrap the wire tightly against the carbon rod. Attach a clip to the other end of the platinum wire for the connection with wire from the cardiac stimulator (see below). 4. Place the carbon rods into the polycarbonate frame pieces and then mount the assembled frame with carbon rods into a well of a six-well plate. Critical step: The carbon rods should be placed 1–2 cm apart or at the smallest possible distance to prevent high energy stimulation and cell death. 5. Pour 2 ml of PDMS into the well. Make sure that the bottom of the polycarbonate frames is immersed in PDMS while the PDMS surface level is close to but does not reach the bottom of the carbon rods. Bake the six-well plate at 70 °C for 2 h. This will stabilize the carbon rods securely during biowire cultivation. 6. Take the electrical stimulation chamber out of the six-well plate by holding the polycarbonate frame first and then twisting and pulling the chamber. Autoclave for sterilization. Critical step: The connections between the platinum wire and the carbon rod should be tested once the stimulation chamber is created for the first time. Link the stimulation chamber to the electrical stimulator and then set a desired voltage. Place a volt meter lead onto each of the two carbon rods. The outputted voltage from the stimulation chamber should be the same voltage being read on the volt meter. Any wrong readings might be due to the platinum wire coated by PDMS or other faulty connection in the system. 2.2.3. hPSC-CM dissociation and biowire seeding 1. Cardiomyocytes are derived from hPSC to form embryoid bodies (EBs) as per [5]. EBs from day 20–34 of differentiation are used to make biowires. Transfer EBs from the low cluster plate to a conical tube. Centrifuge at 125 RCF for 5 min, at r.t. Carefully remove supernatant, and then re-suspend the pellet with pre-warmed 37 °C collagenase type I containing 1% DNAse I. Incubate in the incubator at 37 °C for 2 h. Gently shake tube every 30 min. Critical step: Incubation time with collagenase is determined by the age of the hPSC-CMs and differentiation protocol (monolayer vs EBs). This protocol recommends a 2 h incubation for day 21 EBs. For monolayers, 30 min digestion is recommended. For younger or older cells, collagenase digestion time should be experimentally determined considering cell viability and contractile function post digestion. 2. Prepare collagen gel right before collagenase digestion is completed. Pre-cool all gel components to 4 °C by placing on ice. Then, mix all gel components except the collagen and Matrigel into a sterile 1.5 ml Eppendorf tube. Add collagen type I and mix by pipetting up and down. Then add reduced growth factor Matrigel, mix well and keep on ice.

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Fig. 1. Biowire culture platform. (A) Schematic (left) and actual (right) PDMS mold of biowire. (B) To set up biowire, suture was placed centrally in the channel (I). hESCcardiomyocyte suspension in collagen type I gel were seeded around the suture into the channel (II) and incubated in desired media (III). Details were illustrated in both schematic and actual inset images. (C–E) After 7 days of culture in biowire, cells remodeled the gel and contracted around the suture. (Reproduced from [13]). (C) Brightfield images of hESC-cardiomyocytes on indicated days of pre-culture in biowire template. (D) Quantification of gel compaction on the indicated days of culture. (E) Hematoxylin and eosin (H&E) and Masson’s trichrome (MT) staining of biowire sections (double-headed arrows represent suture axis).

3. Add 5 ml of wash medium to the cells and spin at 125 RCF for 5 min. Aspirate supernatant, resuspend in 2 ml of Trypsin solution, and incubate at 37 °C for 5 min (precisely). Critical step: Trypsin can cause damage to the cells. Keep at a maximum of 5 min. 4. Quench trypsin with 1 ml of stop medium containing 3% (v/v) DNase I. 5. Pass EB suspension through a 5 ml syringe attached to a 20-gauge needle 3–5 times.

Critical step: Carefully attach the syringe to the needle tip by holding only the very end of the syringe. Avoid touching syringe body since this can introduce contaminants when the entire syringe is placed into the 15 ml conical tube. The number of plunging to dissociate the cells is determined by the age of the cells, differentiation protocol, and the force exerted to disrupt the cells. This can be assessed visually, by observing the presence of large clumps of cells (requires more disruption). Needle disruption should be kept to a minimum to avoid cell death. Bubble formation should be avoided. Troubleshooting: If, after disruption using the needle, there are still multiple visible clumps, it is preferable to increase the

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drop of media can skew the ratio of cell:gel and prevent proper gel polymerization. It is recommended to use a glass Pasteur pipette connected to a vacuum line for best results.

incubation time in collagenase in subsequent isolations instead of increasing the time in trypsin or the number of plunging. 6. Add 7 ml of wash medium and spin the cells at 280 RCF for 5 min. Remove supernatant, resuspend cells in desired culture medium, and place cells on ice. Count cells using a haemocytometer. Take appropriate volume equal to desired number of cells in suspension, and spin at 280 RCF for 5 min. Discard the supernatant.

7. Add 3.5 ll of collagen gel mixture for every 0.5  106 cells. Mix the gel cell slurry well by gently pipetting up and down, avoiding the formation of air bubbles. 8. Take 4 ll of the cardiac cell suspension (8 ll/cm of channel length) with a P10 pipette tip and deliver into the 0.5 cm long channel so that it surrounds the suture (Fig. 1B II, Supplemental video 2). Use sterile forceps to adjust the position of the suture before the gel polymerizes.

Critical step: Supernatant should be completely removed at this step, prior to the addition of gel. Given the small volumes, a single

Electrical stimulator

Biowire

Platinum wire

Carbon rod

Fig. 2. The biowire was transferred to the electrical stimulation chamber during the second week of cultivation with the biowire placed perpendicular to the carbon electrodes.

α-actinin/actin

cardiac troponin T 20 μm

20 μm

B

Control

A

20 μm

3Hz

20 μm

20 μm

20 μm

Cardiomyocyte proliferation (%)

25

**

20

* 15

# &

10 5 0 3Hz

6Hz

6Hz

EBd20 EBd34 Control

Fig. 3. Physiology of cardiomyocytes cultured in the biowire platform. (A) Representative confocal images of non-stimulated (control) and electrically stimulated biowires (3Hz and 6-Hz regimens) showing cardiomyocyte alignment and frequent Z disks (double-headed arrows represent suture axis). Green, a-actinin; red, actin. Scale bar, 20 lm. (B) Cardiomyocyte proliferation in biowires was lower than in EBs. Proliferation was assessed by double staining for sarcomeric a-actinin and Ki67 (n = 3–4 per condition, average ± s.d.). ⁄, ⁄⁄, # and & represent statistically significant difference compared to EBd34. (Reproduced from [13]).

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Fig. 4. Electrical stimulation promoted improvement in Ca2+ handling properties. (A–C) Representative traces of Ca2+ release in response to caffeine in nonstimulated control cells (A), cells subjected to the 3-Hz regimen (B) and 6-Hz regimen (C). (D) Caffeine-induced change of peak fluorescence intensity among experimental groups (mean ± s.e.m. after normalizing the peak fluorescence intensity before administration of caffeine; control versus 3 Hz, P = 1.1  10 6; control versus 6 Hz, P = 2.1  10 7; 3 Hz versus 6 Hz, P = 0.003; n = 10 (control), n = 6 (3 Hz) and n = 9 (6 Hz)). (E) Representative fluorescence recording of Ca2+ transients before and after administration of caffeine at 5 mM (arrow) in cells subjected to the 6-Hz regimen. (Reproduced from [13]).

Fig. 5. Electrical pacing improves electrophysiological properties and automaticity. Electrophysiological properties in single cardiomyocytes isolated from biowires or embryoid bodies were recorded with patch clamp. (A) hERG tail current density. (B) IK1 current density measured at 100 mV. (C) Upregulation of potassium inwardlyrectifying channel gene (KCNJ2). (D) Ratio of cells displaying spontaneous beating (automaticity) or no spontaneous beating (no automaticity). (Reproduced from [13]).

9. Add enough sterile PBS to coat the bottom of the Petri dish but not too much to avoid touching the polymerizing gel. Incubate the microwells with seeded cells at 37 °C for 30 min for polymerization. Avoid touching the seeded cells and gel in the PDMS microwells.

10. Remove the PBS and add 12 ml of culture medium to completely cover the cells in the gel-seeded microwells (Fig. 1B III). Culture the biowires for 1 week, changing media every other day. For microwells bigger than 1 cm, polymerization may take longer and more frequent media

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changes may be required due to the increased number of cells. Critical step: PDMS microwells should stay at the bottom of the Petri dish and should not float. To ensure this, make sure the Petri dish is dry and push the PDMS wells down using a sterile forceps before seeding the cells. 2.2.4. Electrical stimulation for Biowire cultivation 1. Under sterile conditions, open a six-well plate and add 2 ml of sterile PBS to each well. Use blunt-tipped sterile tweezers to place the autoclaved electrical stimulation device into the PBS-primed well. The operation should be gentle until the electrical stimulation chamber covers the bottom of the well. Remove PBS from the well by aspiration. 2. Add 5 ml of pre-warmed culture medium to each well to completely cover the carbon rods. Then use sterile tweezers to transfer the biowires to the electrical stimulation chamber by placing them perpendicular to the carbon rods (Fig. 2). Critical step: Ensure all biowires are in place and submersed completely with media (without tipping over or floating) in the electrical stimulation chamber. The orientation of all biowires should be perpendicular to the carbon rods. 3. Mount the six-well plate lid on top of the plate and leave the platinum wires reaching outside the plate. Then place the six-well plate into the incubator and connect wires from the electrical stimulator and the platinum wire.

(Fig. 4) and cardiomyocyte electrophysiology maturation (Fig. 5) were improved in biowires. Mature cardiomyocytes respond to caffeine by inducing an abrupt release of Ca2+ and such calcium transient could be monitored [3]. hESC-derived cardiomyocytes in electrically stimulated, but not in non-stimulated control cells, were responsive to caffeine by inducing an increase in cytosolic Ca2+ (Fig. 4A–C). Quantification of Ca2+ transient amplitudes showed that electrically stimulated cells exhibited significantly higher amplitude intensity in response to caffeine than non-stimulated controls, in a stimulation frequency-dependent manner (Fig. 4D and E). Meanwhile, hERG current and inward rectifier current (Ik1) densities were not only improved by culture in biowire but were further improved by electrical stimulation compared to non-stimulated biowire cells (Fig. 5A and B). Improvements in Ik1 current seen were compatible with induction of potassium inwardly-rectifying channel gene (KCNJ2) in the biowire conditions (Fig. 5C). Automaticity (spontaneous beating activity) was greater in EB-derived cardiomyocytes compared to control biowires (Fig. 5D), which was comparable to that in biowires subjected to the 6-Hz regimen. Taken together, these results suggested that biowires and electrical stimulation promoted maturation of hPSC-CMs [13]. Acknowledgements Authors wish to acknowledge the following funding sources: This work was supported by a grant-in-aid from the Heart and Stroke Foundation of Canada (G-14-0006265) and by an operating grant from the Canadian Institutes of Health Research (137352) to SSN. Appendix A. Supplementary data

Critical step: The lid should be closed gently to avoid any damage to the platinum wire. In case lid does not close fully, tape lid down to ensure that the lid sits properly on the plate. When closing the incubator doors, place the thinner portion of the wires from the electrical stimulator at the door closing points while leaving the thicker portions of wires outside of the incubator. This allows proper sealing of the incubator. 4. Start electrical stimulator with following settings: biphasic repeating pulse, 1-ms pulse duration, 3 V/cm, and 1 pulse per second (PPS) for the pacing frequency. Increase PPS every 24 h to the following values: 1.83, 2.66, 3.49, 4.82, 5.15, and 6. Change medium every other day. 5. After 7 days, PPS should be set to 1 if further culture is wanted. Change medium every other day. 3. Anticipated results During the first week after seeding cells in the biowire chamber, the collagen gel matrix was contracted due to cell remodeling (Fig. 1C and D, Supplemental video 3). The cells aligned along the axis of the suture (Fig. 1E). An example of the matured hPSC-CMs generated using biowire is shown in Fig. 3. After 2 weeks in culture, cells throughout the biowires exhibited multiple parameters comparable to mature cardiomyocytes. These include: (1) Biowired cardiomyocytes showed strong expression of cardiac contractile proteins sarcomeric a-actinin and actin as evidenced by immunostaining (Fig. 3A). Sarcomeric banding of the contractile apparatus and myofibrillar alignment along the suture axis was similar to the structure in the adult heart (Fig. 3A). (2) These hESC-derived cardiomyocytes cultured in biowires also exhibited lower proliferation rates than those of EBs (Fig. 3B). (3) Calcium handling properties

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