Engineering of cardiac microtissues by microfluidic cell encapsulation in thermoshrinking non-crosslinked PNIPAAm gels

The synthesis was carried out at room temperature. In an evacuated, and subsequently, N₂ flooded 250 ml round bottom flask with magnetic stirrer, 150 ml deionized water and 10 g (88 mmol) N-isopropylacrylamide were added. After dissolving the solid monomer while stirring, 120 mg (0.53 mmol) ammonium peroxodisulfate was added. After homogenization, 61.5 mg (0.53 mmol) Tetramethylethylenediamine (TEMED) was added, and the reaction was carried out for 2 h while stirring. After reaction time, the viscosity of the reaction solution was increased, and the product was precipitated in deionized water at 60 °C. The precipitate was vacuum filtered through a Büchner funnel with standard filter paper, washed with 80 °C hot deionized water and freeze-dried for further use.

The freeze-dried polymer was weighed and then sterilized by supercritical carbon dioxide in presence of peracetic acid. Freeze-dried PNIPAAm was added to a 100 ml Erlenmeyer flask and the flask was packed into a Tyvek sterilization bag and sealed. For sterilization, a Novagenesis bench top device (Novasterilis, NY) with 1.0 l vessel was used. A sterilizing supplement containing peracetic acid (NovaKill^™, Novasterilis, NY) was added according to the manufacturer's instructions. A Tyvek sealed flask with PNIPAAm was placed in the vessel. The vessel was filled with liquid carbon dioxide and a booster pump was used to raise the vessel pressure to 1500 PSI. The temperature was set to 35 °C, and the magnetic stirrer was activated. Sterilization was performed for 90 min. The pressure was released slowly, and remaining traces of NovaKill^™ were removed under vacuum. Dulbecco's phosphate buffered solution without calcium and magnesium (DPBS -/-, Gibco) was then added to create a PNIPAAm solution at a concentration of 1.8 wt/vol%. PNIPAAm was left on a magnetic stirrer at 50 rounds per minute (rpm) for 24 h to allow the freeze-dried PNIPAAm to solve.

A microfluidics chip was generated at the workshop of the medical faculty, University of Cologne. The exact dimensions of the chip are illustrated by the technical drawing in figure 1. The chip was connected to the tubing system via Luer-Lock Connectors (Carl Roth, Karlsruhe, Germany). To create a constant flow at preset rates, two infusion pumps (Landgraf Laborsysteme HLL GmbH, Langenhagen, Germany) were used. Pump 1 was loaded with two 20 ml syringes, both containing rapeseed oil. Rapeseed oil is used because it is environmentally friendly; furthermore, it easily separates from the aqueous phase due to lower density. From these syringes, fluorinated ethylene propylene (FEP) inflow tubes with an inner diameter of 1.58 mm ran to the Y-shaped side port channels of a flow-focusing microfluidic chip. The exact dimensions of the chip are provided in figure 1(A).

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The central channel was connected to a 20 ml syringe mounted on pump 2 and used to inject PNIPAAm solutions or PNIPAAm enriched cell suspension into the microfluidic chip. Homogenous droplets of the PNIPAAm containing solution (water phase) are generated as oil flows into the chip through the side ports. The resulting PNIPAAm containing droplets were transported in a continuous oil flow and enter a tube that is heated to 37 °C by a counter-streaming tube heater (see figure 1 for illustrations). After 40 cm in the heated tube, the gelled PNIPAAm spheroids left the tubing system under a sterile hood heated to 39 °C and were transferred to a 37 °C humidified cell culture incubator with 5% CO₂.

Parts of the microfluidics system were sterilized for 30 min in hydrogen peroxide vapor followed by low-pressure oxygen plasma treatment for 30 min. Briefly, the dry parts of the microfluidic were sealed in Tyvek pouches and brought into the plasma chamber. A −20 °C cold aliquot of 30% H₂O₂ was opened and placed in the chamber. The device was rapidly evacuated to 0.15 mbar and the pump was stopped to allow H₂O₂ to evaporate. After 30 min, the chamber was again evacuated to 0.2 mbar. Oxygen was automatically injected to keep the working pressure at 0.2 mbar. Plasma was ignited and samples were sterilized in plasma for 30 min. The process was performed in a Plasma Flecto 30 (Plasma Technologies, Germany).

To produce cell-free polymer beads, the middle inflow tube of the microfluidic setup was connected to a 20 ml syringe containing PNIPAAm solution. This solution was created by mixing the 1.8 wt/vol% solution of PNIPAAm in DPBS -/- and Dulbecco's Modified Eagle's Medium (DMEM) containing 4.5 g glucose l⁻¹ (Thermo Fisher Scientific, Germany) at room temperature (20 °C–22 °C) at 50/50 ratio to obtain a cell suspension containing a final PNIPAAm concentration of 0.9 wt/vol%. The syringes connected to the outer inflow tubes contained rapeseed oil.

The PNIPAAm beads were produced by setting the flow rate of the PNIPAAm solution to 0.025 ml min⁻¹ and the flow rates of the oil to 0.1 ml min⁻¹. The beads were collected in a 10 cm cell culture dish (Sarstedt Laborbedarf, Nümbrecht, Germany) containing rapeseed oil pre-warmed to 39 °C.

The beads were measured with light microscopy and NIS-Elements software (Nikon Europe, Amsterdam, Netherlands). To visualize the beads' surface, the sample was mounted on a heated stage on an upright light microscope (Carl Zeiss Microscopy Deutschland GmbH) equipped with dipping objectives and Orca Flash 4.0 V2 camera (Hamamatsu Photonics Deutschland, Herrsching am Ammersee, Germany). The sample was illuminated from the side by two gooseneck LED lights to allow visualization of the surface.

Human renal carcinoma cells (cell line 786-O) engineered to express an enhanced green fluorescent protein (eGFP) were used for the initial establishment of the methods. The unmodified cell line was kindly provided by the Clinic II for Internal Medicine, University Hospital of Cologne. The cells were transfected by nucleofection with the eGFP control plasmid (pmaxGFP^™) provided by Lonza (Lonza Group Ltd, Basel, Switzerland) along with the nucleofector kit. Stably transfected green fluorescent cells were enriched by limiting dilution and selection of GFP positive colonies. The cells were cultured in complete DMEM (4.5 g) glucose l⁻¹ with 15% fetal calf serum (FCS) (Sigma-Aldrich, Taufkirchen, Germany). MSCs were prepared as described previously [8] and cultured in complete DMEM containing (1 g) glucose l⁻¹ (Thermo Fisher Scientific) and 15% FCS. For a generation of miPS-CMs, TαP4 [28] cultivated on mitomycin inactivated embryonic feeder cells in Iscove's modified Dulbecco's medium (IMDM) supplemented with 15% FCS and 1000 units ml⁻¹ leukemia inhibitory factor (ORF Genetics, Reykjavik, Iceland). Start of differentiation was counted as day 0. On day 0 miPSCs were dissociated into single cells by 0.05% trypsin/EDTA (Gibco by Thermo Fisher Scientific) and 10⁶ cells were re-suspended in 12 ml IMDM with 20% FCS and 200 µg ml⁻¹ L-ascorbic acid phosphate magnesium salt n-hydrate (Wako Chemicals GmbH, Neuss, Germany). The cell suspension was transferred to a 10 cm bacteriological petri dish (Sarstedt Laborbedarf, Nümbrecht, Germany) and cultured in a humidified incubator on a rocking table at 40 rpm for two days. In suspension, miPSC form cell spheroids and/or clusters, referred to as EBs. On day 2, EBs were collected and diluted to 800 EBs per 10 cm bacteriological petri dish in 14 ml IMDM with 20% FCS and 50 µg ml⁻¹ ascorbic acid. Plates were left on a rocking table inside the cell culture incubator. On day 7, EBs from two plates were collected to one plate and the medium was changed to IMDM with 5% FCS. On day 8, EBs from two plates were pooled to one plate and the medium was changed (IMDM 5% FCS). Puromycin (Invivogen, Toulouse, France) was added to a final concentration of 10 µg ml⁻¹ to eliminate all non-CMs. Steps from day 8 were repeated on day 9. The medium was changed daily with IMDM plus 5% FCS and 10 µg ml⁻¹ puromycin. On weekends IMDM with 20% FCS was used instead to maintain cells without medium change.

Puromycin-selected iPS cell-derived CMs (iPSC-CMs) were dissociated on day 12. The day before dissociation, 10 cm cell culture dishes were coated with 10 µg ml⁻¹ fibronectin in PBS overnight at 4 °C. One plate per 10–15 × 10⁶ CMs was prepared. Dissociation was performed on the shaker at 37 °C for 30–35 min. Trypsin was blocked with undiluted FCS and cells were dissociated by gentle pipetting (10–15 repetitions). (Note: Rigorous pipetting destroys the CMs.) miPSC-CMs were spun down at 180 g and resuspended in PBS. The cells were filtered through a 40 µm cell sieve to obtain a single-cell suspension. miPS-CMs were counted in a Neubauer counting chamber, spun down at 180 g for 5 min, and re-suspended in IMDM with 10% FCS supplementation.

On day 13 of differentiation, miPSC-CMs were harvested by trypsinization and viable cells were counted after trypan blue (Gibco by Thermo Fisher) staining. For further processing, the cells were re-suspended in IMDM supplemented with 10% FCS and chilled on ice. MSCs and 786-0 were treated the same way but resuspended in their respective culture medium.

For the encapsulation of cells, a cell suspension with a total absolute cell count of 2 × 10⁶ cells was prepared. If miPSC-CMs and MSCs were encapsulated together, their suspensions were combined to a cell ratio of 4:1 (miPSC-CMs:MSCs). After centrifugation of the cell suspension, the supernatant was discarded carefully. The cells were resuspended in 100 µl of cell culture medium and 100 µl PNIPAAm 1.8 wt/vol%. This polymer-cell suspension was then transferred to a 1 ml syringe (BD, Heidelberg, Germany). The cell suspension in PNIPAAm solution was injected into the middle inflow tube manually. (Hence, after loading the microfluidic system the cell suspension is now in place in the tube connected to the middle port of the chip. Next, the encapsulation suspension is transported forward into the chip for cell encapsulation.) Flow rates were set at 0.1 ml min⁻¹ for the lateral inflow tubes and 0.425 ml min⁻¹ for the middle inflow tube. For the middle channel, the oil is used to push forward the PNIPAAm containing cell suspension. Once the PNIPAAm-cell suspension reached the microfluidic chip, the flow rate of the middle inflow tube was reduced to 0.025 ml min⁻¹. The recurrent flow tube heater was operated at 37 °C. Polymer beads leaving the tube heater in their hydrogel phase were collected in 10 cm culture dishes with pre-warmed IMDM 10% FCS inside a heated sterile cabinet. For handling and transport of the spheres, caution was taken to keep temperature of the medium above the critical temperature of 32 °C. After 24 h of incubation at 37 °C and 5% CO₂, the polymer beads were cooled to room temperature by letting them stand under a sterile hood for 15 min. The culture medium was carefully discarded and replaced by DPBS -/- at room temperature. This process was repeated twice for washing the cell aggregates with DPBS -/- and removing the PNIPAAm which had been solved in the culture medium.

A cell suspension with a density of 250 cells µl⁻¹ was prepared. The cell suspension contained 80% miPSC-CMs and 20% MSCs. Five square shaped polystyrene plates were prepared by adding 15 ml DPBS per plate. With a multichannel pipette, the cell suspension was dispersed in droplets of 20 µl each onto the lids of the square plates. The lids were carefully placed back on the plates and the hanging drops were incubated for 24 h in a humidified incubator at 37 °C, 5% CO₂. During this incubation time cell spheroids formed at the tips of the hanging drop. For harvesting of the spheroids the lids were flushed with cell culture medium and spheroids were transferred to a non-adhesive 10 cm dish. The dishes were further cultured on a rocking table inside a cell culture incubator.

For cell counting, a single cluster in 30 µl DPBS -/- was transferred to a well of a 96-well plate (Greiner bio-one) manually by a microliter pipette. Then, 70 µl 0.05% trypsin/EDTA were added. After 2 min of incubation at 37 °C, the cells were separated mechanically by repeated pipetting with a 200 µl pipette with a 'yellow tip'. After 2 more minutes of incubation at 37 °C, the obtained single cells were counted by trypan blue staining using a hematocytometer.

A live-dead cell assay kit based on calcein AM and ethidiumhomodimer-1 (Thermo Fisher Life Technologies, Germany) was used according to the manufacturer's instructions. Cell aggregates in 200 µl IMDM were incubated with 200 µl staining solution containing calcein AM 1:1000, ethidium homodimer-1 1:500, and 5 µg ml⁻¹ Hoechst dye for 30 min. Staining was evaluated immediately after the incubation by fluorescence microscopy. For detection of apoptosis in sections of microtissues a Click-iT Plus TUNEL assay conjugated with Alexa Fluor 488 (Invitrogen, #C10617) was used.

For immunofluorescence staining, cell aggregates were unleashed from the PNIPAAm hydrogel, the aggregates were fixed and permeabilized with 100% methanol (Sigma-Aldrich, Taufkirchen, Germany) at −20 °C for 10 min. The aggregates were washed with DPBS -/- and then incubated with 3% bovine serum albumine (BSA) (Sigma-Aldrich) for 1 h at room temperature. After washing with DPBS -/- once more, the cell aggregates were incubated with primary anti-α-actinin monoclonal IgG (Sigma-Aldrich, clone EA-53, isotype IgG1) in 3% BSA for 16 h at 4 °C. For staining of MSCs monoclonal anti-vimentin antibodies produced in mouse (Sigma-Aldrich, clone LN-6, isotype IgM) were used. After a further washing step, secondary detection was performed by goat anti-mouse IgG Alexa Fluor 647 and goat anti-mouse IgM Alexa Fluor 555 (both from Thermo Fisher Scientific). Conjugated monoclonal anti-CD29 IgG (Miltenyi Biotec, Bergisch Gladbach, Germany) was used to stain total cells. About 1 µg Hoechst 33342 dye (Sigma-Aldrich) in 3% BSA were added to the aggregates in order to stain nuclear DNA. Samples were incubated for 60 min in the dark at room temperature. After multiple washing steps, the stained aggregates were analyzed with a fluorescence microscope (Axiovert 200, Carl Zeiss Microscopy Deutschland GmbH) using AxioVision 4.5 software as well as by an SP8 confocal microscope (Leica Microsystems).

Intracellular AP measurements with sharp glass microelectrodes (20–50 MΩ resistance when filled with 3 mol l⁻¹ KCl; World Precision Instrument, Sarasota, USA) have been performed as described previously [29, 30]. APs of TαP derived CMs were measured in cell cluster containing CMs and MSCs. Recordings were performed at two different time points, 24 h (directly after removal of PNIPAAm) and 72 h (24 h in PNIPAAm plus 48 h in culture) after preparing the cell cluster. APs of iPSC-CM were measured without external stimulation. The recording electrode was connected to an SEC-10LX amplifier (npi electronic, Tamm, Germany), and the signal was acquired with the Pulse software (HEKA, Lambrecht/Pfalz, Germany). Data were analyzed with Mini Analysis (Synaptosoft, Fort Lee, USA). All data from AP measurements are presented as mean ± SEM. Differences of AP parameters were tested for statistical significance by Student's t-test. A two-sided p-value <0.05 was considered statistically significant. GraphPad Prism version 8 (GraphPad Software, San Diego, California, USA) was used for all calculations and creating AP graphs.

Differences between the experimental groups were determined by using one-way analysis of variance. An unpaired and Bonferroni corrected t-test was used as post-hoc test. A difference was considered significant when p < 0.05. All values are presented as mean ± standard deviation (mean ± SD). Statistical analysis and graphing were performed using Microsoft Excel.

The microfluidic method was used to encapsulate murine mesenchymal stromal cells (MMSCs) inside PNIPAAm beads. Therefore, a total number of 2.0 × 10⁶ mMSCs was resuspended in 100 µl DMEM with 15% FCS mixed with 100 µl 1.8% PNIPAAm suspension and injected into the system for cell encapsulation. When the mMSC-PNIPAAm beads left the heated tubing system, they were dropped into liquid nitrogen, transferred as frozen beads to a centrifuge tube inside a −20 °C pre-chilled aluminum block and transferred to an ultralow freezer at −80 °C until further processing. Next, the mMSC-PNIPAAm beads were freeze dried for 24 h. To remove the remaining rapeseed oil, 20 ml of dry ethanol were added to the tube with the mMSC-PNIPAAm beads, mixed gently, and agitated on a shaker at room temperature for 4 h at 300 rpm. Beads were separated from ethanol by gravity. To remove residual ethanol, the samples were kept in a vacuum desiccator for 2 h. Because of some remaining oil, the washing step with the absolute ethanol was repeated. For the second washing step, the samples were incubated with 2 ml dry ethanol overnight on a shaker and vacuum dried in a desiccator. Samples were mounted on a stub using a double sided adhesive carbon tape and sputter coated with a 12 nm gold layer. Examination was done by a FEI Quantus 250 scanning electron microscope.

The synthesis was carried out at room temperature. In an evacuated and subsequently, N₂ flooded 50 ml round bottom flask with magnetic stirrer, 2 g (18 mmol) N-isopropylacrylamide and 30 ml deionized water were added. Then 20 mg acryloxyethyl thiocarbamoyl rhodamine B was added. After dissolving the solid monomer under stirring, 24 mg (0.1 mmol) ammonium peroxodisulfate were added. Following homogenization, 12.3 mg (0.1 mmol) TEMED was added, and the reaction was carried out for 2 h while stirring. With the increasing reaction time, the viscosity of the reaction solution increases, and the product was precipitated in deionized water at 60 °C. The precipitate was vacuum filtered through a Büchner funnel with standard filter paper, washed with 80 °C hot deionized water and dried overnight in the vacuum drying oven at 40 °C.

For the experiment 1.8% PNIPAAm-co-rhodamine solution was diluted with 1.8% unlabeled PNIPAAm solution in a ratio of 1:10. This solution is referred to as 'Rhodamine-PNIPAAm'. Rhodamine-PNIPAAm was used to encapsulate mMSC with the microfluidic method. Therefore 2.0 × 10⁶ cells in total were resuspended in 100 µl of DMEM with 15% FCS and mixed with 100 µl of the rhodamine-PNIPAAm before injected into the system. After the mMSC-PNIPAAm beads were generated and collected in 10 cm dishes filled with 15% DMEM. Beads were incubated for 24 h at 37 °C, 5% CO₂ before the rhodamine-PNIPAAm was washed out. To remove the rhodamine-PNIPAAm capsule, the cell-PNIPAAm beads were incubated in a petri dish with 10 ml DPBS at room temperature. The plate was gently swirled. PBS was removed as complete as possible and replaced by fresh PBS (room temperature). This step was repeated in total six times to remove dissolved PNIPAAM and rapeseed oil. The washing procedures took about 10 min in total. For further analysis, the washed cell spheroids were transferred to a petri dish filled with fresh cell culture medium and incubated on a rocking table inside a cell culture incubator for 24 h. Spheroids were stained with Hoechst dye and analyzed under a fluorescent microscope.

In order to produce PNIPAAm spheroids, a microfluidics chip was designed (figure 1(A)). The chip is equipped with two ports that transport the hydrophobic phase (rapeseed oil) at an angle of 60° to the central channel. A central port is used to inject the PNIPAAm containing an aqueous phase into the oil stream (figure 1(B)). At the tip of the cannula, a spherical droplet is formed and buds off once the forces produced by the flow of the hydrophobic phase exceed the retraction forces at the interphase of the hydrophobic phase and aqueous solution (figure 1(C)). Using a flow of 25 µl min⁻¹ for the aqueous phase, a production rate of 28 droplets per minute was estimated. Droplets moved forward to the exit of the chip and entered a tube with a heated mantle inside a heated sterile cabinet (figures 1(D) and (E)). Heating the droplets to above the lower critical solute temperature (LCST) of 32 °C resulted in the phase change of the PNIPAAm and precipitation of solid and hydrophobic PNIPAAm. The droplets turned from fully transparent liquid drops to opaque hydrogel spheres at this stage. A schematic drawing of the setup is provided in figure 1(F).

For a demonstration of the change in transparency, droplets were imaged in a non-heated (room temperature) tube (figure 2(A)) and collected in a multiwell plate filled with 37 °C warm medium (figure 2(B)). For the further experiments, the setup outlined in figure 1(F) was used and operated with cell-free PNIPAAm solution. The resulting transmission light microscopy revealed that a spherical shape of the PNIPAAm beads was generated by this setup (figure 2(C)). Dark field microscopy showed that the polymer had formed 3D interwoven meshes at the surface of the spheroids (figure 2(D)). When PNIPAAm solution without cells was injected into the system, round beads with a mean diameter of 718 ± 28 µm (n = 115) formed (figure 2(E)). After 4 h of incubation above the LCST spheroids reduced in size to a mean diameter of 516 ± 25 µm (n = 35) (figure 2(F)).

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In the next step, the ability of PNIPAAm to encapsulate cells and promote the formation of cell aggregates was tested. Initial experiments were conducted with eGFP expressing 786-0 renal carcinoma cells. First, we experimented to check whether incubation of the cells within the hydrogel capsule is necessary to form cell aggregates. 786-0 cells were encapsulated and the PNIPAAm capsules were transferred to room temperature shortly after they had been collected at the outlet of the production unit. Directly after transfer to room temperature, capsules appeared non-transparent to light (figure 3(A)). After 2 min at room temperature, the hydrogel started to become transparent (figure 3(B)). After 4 min at room temperature, the capsules dissolved as PNIPAAm underwent phase transition, and encapsulated cells were released (figure 3(C)). No formation of aggregates was observed at this stage. Therefore, an incubation of the cells inside the hydrogel shell was necessary to form aggregates. The number of cells released per capsule when dissolved immediately after encapsulation was estimated as 3636 ± 1575 single cells (n = 22).

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After 24 h of incubation in PNIPAAm, cell aggregates formed and showed a round, spheroid-like shape (figures 3(D) and (E)). Cell aggregates were obtained 24 h after encapsulation by washing out the hydrogel shell and showed a mean diameter of 525 ± 116 µm (n = 20) (figure 3(F)). These aggregates contained 6346 ± 1753 cells (n = 26) (figure 3(G)). That is 1.7-fold more than directly after the encapsulation, pointing to cell proliferation in the first 24 h. Incubation of the cells inside the hydrogel shell for 48 or 72 h instead, resulted in a significantly (p < 0.001) reduced cell count of 3932 ± 1209 (n = 22) and 4467 ± 1932 (n = 30), respectively. In line with this observation, the clusters obtained after 48 and 72 h of incubation had a significantly (p < 0.001 and p = 0.01) smaller diameter of 407 ± 82 µm (n = 26) and 445 ± 97 µm (n = 30), respectively, as compared to cell clusters released after 24 h.

Encapsulation experiments in this work were performed using a 1.8% PNIPAAm stock solution. The PNIPAAm stock solution is diluted 1 + 1 with the cell suspension to result in a final PNIPAAm concentration of 0.9% in the microfluidics. In order to test if an increase of the PNIPAAm concentration reduces the time for microtissue formation PNIPAAm stock solutions of 1.8%, 2.4% and 3.0% were tested for encapsulation of MSCs (supplementary figure 1 (available online at stacks.iop.org/BF/14/035017/mmedia)). When 1.8% PNIPAAm solution was used, no compact microtissues were formed after 4 h. In contrast, 24 h were sufficient for microtissue formation. About 2.4% and 3.0% PNIPAAm facilitated microtissue formation within 4 h. To further reduce the duration of encapsulation, MSCs were encapsulated in 2.4% for 1 or 3 h, respectively. While 1 h did not result in compact microtissues, encapsulation for 3 h produced microtissues that appear less compact as compared to approaches with longer encapsulation times. An increase of the PNIPAAm stock solution to 3.6% did not produce microtissues in either 1 or 3 h.

In some applications it could be necessary to fine tune not only the cellular composition of the microtissues but also its absolute size. To address the relation between flow parameters and final size of microtissues, we varied the flow rate of rapeseed oil while keeping the flow rate of the cell suspension constant. It was found that an increase of the hydrophobic phase flow rate results in a decrease in the diameter of the resulting microtissues (supplementary figure 2).

In a proof-of-concept approach to generate functional cardiac microtissues, purified miPSC-CMs and MSCs were used. When miPSC-CMs were encapsulated together with MSCs at a 4:1 ratio, the diameter of the microtissues that were released after 24 h in the hydrogel shell had an average diameter of 341 ± 31 µm (n = 20) (figure 4(A)). If the aggregates were recovered from the hydrogel after 48 or 72 h, they showed an increased diameter of 363 ± 74 µm (n = 39) and 391 ± 51 µm (n = 73) respectively. When microtissues were released from PNIPAAm after 24 h and further cultivated for 24 h (video 1) or 48 h (video 2) they retained their spherical shape and were found to contract autonomously and regularly.

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Hydrogel beads dissolved directly after the encapsulation of iPSC-CMs and MSCs contained 5020 ± 1741 cells per microtissue (n = 25) (figure 4(B)). After 24 h of incubation in the hydrogel shell, the clusters contained 4485 ± 1574 cells (n = 34). While this decrease in cell number was not significant (p = 0.23), aggregates obtained after 48 and 72 h of incubation in the polymer showed significant (p < 0.001) cell count decreases to 3477 ± 1331 (n = 44) and 3044 ± 1211 (n = 57) cells per aggregate.

Staining of live cells with calcein dye (green) and dead cells with ethidiumhomodimer (red) was performed and qualitatively analyzed. All nuclei were stained with Hoechst dye (blue). After release of cardiac microtissues from the hydrogel shell the majority of cells were found to be alive and only a few nuclei positive for ethidiumhomodimer were identified (figures 4(C)–(F)). To exclude that cell death occurs with delay another fraction of the microtissues were released from the polymer shell after 24 h and cultured for two days on a rocking table inside a cell culture incubator. Live-dead staining again revealed only single dead cells as indicated by ethidiumhomodimer positive nuclei (figures 4(G)–(N)).

As the aggregates obtained after 24 h of incubation in PNIPAAm contained the highest cell counts, further analysis focused on this group. Once released from the polymer shell, the cardiac microtissues showed spontaneous contractions. Directly following release from the hydrogel shell, microtissues were labeled with antibodies against the cardiac protein α-actinin and the common surface marker CD29 (ß1-integrin) known to be highly expressed by MSCs and imaged by confocal microscopy (figures 5(A)–(D)). It was noted that the surface presented itself as rough with cells standing out above the general surface level. As yet, tunnels or canals reaching into the interior of the spheroids, which could result from the removal of PNIPAAm, could not be observed. This description of the surface is in line with the impression gained by transmission light microscopy. As far as the antibodies reached into the microtissues, the different cell types appeared to be homogenously intermingled.

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At the time point of release from the polymer, the miPSC-CMs were at day 14 of differentiation. Confocal imaging of z-disc protein α-actinin revealed nascent striation of their sarcomeres (figure 5(E)). To further investigate the CMs' fate within the spheroids, PNIPAAm was washed out of some microtissues after 24 h of incubation. These microtissues were then further incubated under continuous agitation to prevent adhesion of the microtissues to each other. After 24 and 48 h of shaker culture, the microtissues still exhibited a round shape. The surface appeared smoothened. After 24 h the clusters' diameter was reduced to (309 ± 25) µm (n = 12). This is significantly (p = 0.007) less than at the time of release from the hydrogel. Aggregates that were incubated on a shaker for 48 h had a diameter of (341 ± 21) µm (n = 10), which does not differ significantly (p = 0.94) from the diameter at the time of release from the hydrogel. The clusters which had been released from the hydrogel and further incubated on a shaker were fixed and stained with antibodies against α-actinin and CD29 and examined by confocal microscopy. CMs at day 15 showed improved striation (figure 5(F)). miPSC-CMs at day 16 showed further signs of the sarcomeric organization (figure 5(G)), indicating an ongoing process of sarcomere maturation. Confocal z-stacking was conducted on microtissues after removal of the hydrogel shell and planar projections were generated (figure 5(H)). The surface projection supports the conclusion that the microtissues exhibit rough surfaces right after removal of PNIPAAm.

Sharp electrode AP measurements in miPSC-CMs/MSC microtissues (figure 6) were performed to analyze electrophysiological properties after 48 h (24 h + 24 h (24 h in the PNIPAAm shell + 24 h in culture post PNIPAAm washout), n = 6), 96 h (24 h + 72 h, n = 8), 8 days (n = 11) and 15 days (n = 6). Hanging drop clusters generated from equal cell numbers as found per PNIPAAm capsule were measured after 48 h (24 h + 24 h, n = 7) and 72 h (24 h + 48 h, n = 3).

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In PNIPAAm capsule-derived microtissues, CMs showed typically cardiac AP-shape of young, unmature CMs. Analyses of AP properties showed significant differences between 48 h, 96 h, 8 days and 15 days in in amplitude (48 h: 53.8 ± 1.2 mV; 96 h: 48.26 ± 2.69 mV; 8 days: 58.14 ± 1.7 mV; 15 days: 37.46 ± 5.06 mV, p < 0.001) AP duration at 50% of repolarization (APD50; 48 h: 36.05 ± 1.55 ms; 96 h: 28.70 ± 0.86 ms; 8 days: 20.32 ± 0.34 ms; 15 days: 19.17 ± 4.94 ms; p < 0.001), AP duration at 90% of repolarization (APD90; 48 h: 61.98 ± 4.57 ms; 96 h: 58.25 ± 1.31 ms; 8 days: 54.45 ± 1.96 ms; 15 days: 45.00 ± 9.93 ms; p < 0.001) the ration of AP duration at 50% of repolarization and the AP duration of 90% of repolarization (APD 50/90; 48 h: 58.79 ± 2.02%; 96 h: 49.2 ± 0.65%; 8 days: 37.50 ± 0.94%; 15 days: 42.97 ± 5.66%, p < 0.001), the upstroke velocity (V_max; 48 h 1.28 ± 0.14 V s⁻¹; 96 h: 3.7 ± 0.7 V s⁻¹; 8 days: 13.52 ± 2.6 V s⁻¹; 15 days: 4.18 ± 0.81 V s⁻¹, p < 0.001) and spontaneous contraction frequency (48 h: 0.6 ± 0.2 Hz; 96 h: 3.4 ± 0.2 Hz; 8 days: 4.78 ± 0.18 Hz; 15 days: 5.87 ± 0.81 Hz, p < 0.001). Comparison of AP properties revealed no significant differences between 48 h, 96 h, 8 days and 15 days old microtissues in the maximal diastolic potential (MDP; 96 h: −59.08 ± 2.13 mV; 96 h: −55.89 ± 1.78 mV; day 8: −57.31 ± 0.83 mV; day 15: −51.38 ± 6.95, p = 2.476; table 1).

Analyses of AP properties in hanging drop cluster (supplementary figure 3 and table 2) revealed significant differences between 48 h and 72 h cluster in amplitude (48 h: 41.59 ± 4.29 mV; 72 h: 23.90 ± 3.3 mV, p = 0.037), MDP (48 h: −48.03 ± 2.76 mV; 72 h: −37.3 ± 1.97 mV, p = 0.045), APD50 (48 h: 33.04 ± 3.04 ms; 72 h: 40.54 ± 2.6 ms, p = 0.013) and APD90 (48 h: 60.63 ± 3.04 ms; 72 h: 78.69 ± 5.43 ms, p = 0.014). Comparison of AP properties revealed no significant differences between 48 h and 72 h in APD50/90 (48 h: 54.75 ± 0.97%; 72 h: 51.55 ± 0.36%, p = 0.074), V_max (48 h: 2.94 ± 0.56 V s⁻¹; 72 h: 1.04 ± 0.26 V s⁻¹) and frequency (48 h: 3.3 ± 0.48 Hz; 72 h: 2.4 ± 0.2 Hz, p = 0.201).

During the analysis of the AP recording, it was noticeable that the success rate of AP measurements in hanging drop and PNIPAAm capsule-derived microtissues dropped at the latest time points analyzed. The success rate is defined as the ratio between successful and unsuccessful AP recording attempts. In PNIPAAm capsule-derived microtissues the success rate of AP measurements was 60% (48 h: 6 out of 10 attempts were successful) at 48 h, 61.5% at 96 h (8 out of 13 attempts were successful), 73.3% at 8 days (11 out of 15 attempts were successful) and 20.1% at 15 days (6 out of 29 attempts were successful). In hanging drop clusters the success rate of AP measurements was 63.6% (7 out of 11 attempts were successful) at 48 h and 27.3% (3 out of 11 attempts were successful) at 72 h. Increasing amount of dead cells inside the older cell clusters might led to the higher unsuccessful attempts of AP measurements.

In order to identify potential segregation of the cells during longer culture of cardiac microtissues, stainings for vimentin as a marker for MSCs and cardiac α-actinin were performed. The staining was combined with a TUNEL assay to test for increased apoptosis rates. It was found that there is no segregation of CMs and MSCs in microtissues cultured for 96 or 168 h after PNIPAAm removal. After 96 h few apoptotic cells were observed while after 168 h DNA fragmentation was found widespread throughout the microtissues (figure 7). In hanging drop derived microtissues massive apoptosis was detected at early timepoints (supplementary figure 3).

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In order to detect residues of PNIPAAM inside the microtissue we generated a rhodamine labeled PNIPAAm variant. Encapsulation of the cells with PNIPAAm-co-rhodamine resulted in bright red fluorescent capsules (not shown). MSCs were encapsulated and after 24 h the PNIPAAm-co-rhodamin shell was removed by washing at room temperature. After further culture for another 24 h at 37 °C the microtissues were sliced and analyzed for PNIPAAm residues. Fluorescent microscopy revealed inclusions of PNIPAAm-rhodamin entrapped inside the microtissues (figure 8).

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When counting the numbers of droplets generated in the microfluidic system, we estimated a production rate of 28 droplets per minute at a flow rate of 25 µl min⁻¹ resulting in an average volume of 0.89 µl per droplet, equivalent to a sphere with a diameter d = 1.19 mm. To calculate the size of the droplet, the formula for sphere volumes was applied and resolved for the radius:

$$\begin{equation*}V = \left( {\frac{4}{3}} \right)\pi \times {r^3} = 0.89 \times {10^{ - 6}}{\text{ l}}\end{equation*}$$

$$\begin{equation*}{r^3} = \frac{{0.89 \times {{10}^{ - 6}}}}{{\left( {\frac{4}{3}} \right)\pi }} = 212{\text{5 }} \times { }{10^{ - 7}}.\end{equation*}$$

The calculation results in a radius of 0.005 97 dm. This corresponds well with the channel diameter of the microfluidics chip (1.3 mm). When injecting PNIPAAm solution without cells into the microfluidics chip and heating above the LCST, spheroids were generated and size of the spheroids was estimated. Most of the spheroids fall within the range of 695–740 µm. With a mean diameter of 718 µm, the volume of the spheroids was calculated as 0.194 µl. After 4 h the PNIPAAm spheroids shrunk to 490–549 µm, corresponding to a volume of 0.062–0.087 µl. It can be concluded that the volume was reduced by about 2.7-fold during the shrinking process. When this volume change is extrapolated to the density of cell suspensions in experiments where cells are encapsulated, the cell density increases from 1 × 10⁷ cells ml⁻¹ to about 2.7 × 10⁷ cells ml⁻¹.