Cryopreservation and CO₂-independent culture of 3D cardiac progenitors for spaceflight experiments

Abstract

Space experimentation of cardiomyocyte differentiation from human induced pluripotent stem cells offers an exciting opportunity to explore the potential of these cells for disease modeling, drug discovery and regenerative medicine. Previous studies on the International Space Station were done with 2D non-cryopreserved cultures of cardiomyocytes being loaded and cultivated in spaceflight culture modules with CO₂. Here we report the development of methods of cryopreservation and CO₂-independent culture of 3D cardiac progenitors. The cryopreservation allows preparation and pretesting of the cells before spaceflight, makes it easier to transport the cell culture, reduces the impact of strong gravitational force exerted on the cells during the launch of spaceflight, and accommodates a more flexible working schedule for the astronauts. The use of CO₂-independent medium with supplements supports cell growth and differentiation without a CO₂ incubator. With these methods, we conducted a spaceflight experiment through the SpaceX-20 mission to evaluate the effect of microgravity on the survival and differentiation of 3D cardiac progenitors. Our cryopreserved cardiac progenitor spheres were successfully cultivated in a spaceflight culture module without CO₂ for 3 weeks aboard the International Space Station. Beating cardiomyocytes were generated and returned to the earth for further study.

1. INTRODUCTION

The International Space Station (ISS) U.S. National Laboratory and other national and space agencies are increasingly exploring space microgravity through innovative biomedical engineering to study biology and disease [1, 2]. In this aspect, understanding the growth and cardiomyocyte differentiation from human induced pluripotent stem cells (hiPSCs) is of great interest given the potential application of these cells in disease modeling, drug development and regenerative medicine [3–5]. For example, a recent spaceflight experiment shows that cardiomyocytes derived from hiPSCs (hiPSC-CMs) have increased metabolic feature [6]. Another spaceflight experiment shows that cardiovascular progenitor cells cultured on the ISS have increased proliferative potential [7–9]. A ground-based study we conducted shows that simulated microgravity increases the induction, proliferation and survival of cardiac progenitors from hiPSCs [10], providing a rationale to further study the effect of spaceflight on the growth and differentiation of cardiac progenitors derived from hiPSCs.

Our planned spaceflight study requires cultivating 3D cardiac progenitors on the ISS for approximately 3 weeks inside a cell culture module that does not have the supply of CO₂. The use of cell culture modules that require CO₂, although is the standard on the Earth, makes experimentation on the ISS more difficult, because it requires carrying not only medium for cell growth but also CO₂ tanks on a spaceflight that take space/mass allowances. Furthermore, to facilitate cell maintenance duties for the astronauts, it would be better if cells would be cultivated in the same atmospheric conditions common to spaceflight without additional CO₂.

Experiments performed on the ISS are often done with live cells and as a consequence are demanding to the astronauts’ schedule. Resupply flights can be delayed due to technical issues or non-ideal weather conditions. These delays could negatively affect the outcome of the experiments aboard the ISS, especially if certain types of cells (e.g. progenitor cells) need experiments to be performed at specific and key time points (e.g. before full maturation). Furthermore, normally researchers have to prepare several batches of cell cultures at different time points close to the launch date to accommodate possible flight alterations.

To overcome these issues, we tested - culture conditions on the ground to support cell growth and differentiation of cardiac progenitors without the supply of CO₂ and examined cell viability, purity and yield of cardiomyocytes. We also optimized the method of cryopreservation, thawing, propagation and differentiation of cardiomyocytes from cardiac progenitor spheres.

Our optimized samples of cardiac progenitor spheres recently traveled to the ISS via the SpaceX-20 mission. The method we developed allowed astronauts to directly thaw cardiac progenitors onboard the ISS. The astronauts successfully conducted the space experiment and sent the live cultures in a CO₂-independent medium back to us.

2. MATERIALS AND METHODS

2.1. Cell culture and cardiomyocyte differentiation

SCVI-273 hiPSCs (Stanford Cardiovascular Institute) and IMR90 hiPSCs (WiCell Research Institute) [11] were cultured in a feeder-free hiPSC condition on Matrigel-coated plates, and fed daily with mTeSR^™1-defined medium.

For growth factor-induced cardiomyocyte differentiation, cardiac differentiation was induced with growth factors when compact colonies reached almost 100% confluence [12, 13]. Cells were treated with 100 ng/mL activin A from differentiation day 0 until day 1 and 10 ng/mL bone morphogenic protein-4 (BMP4) from day 1 until day 4 in RPMI/B27 insulin-free medium (RPMI 1640 with 2% B27 minus insulin).

For small molecule-induced cardiomyocyte differentiation, cardiac differentiation was induced with small molecules targeting the Wnt pathway when compact colonies reached ~90% confluence [14]. On day 0 of differentiation, cells were initially treated with 6 μM CHIR99021 in RPMI/B27 insulin-free medium. After 48 h, RPMI/B27 insulin-free medium was used for another 24 h. On day 3 of differentiation, cells were again treated with 5 μM IWR1 in RPMI/B27 insulin-free medium for another 48 h.

Differentiation cultures from IMR90 hiPSCs were used for qRT-PCR, flow cytometry, and TMRM assay, and those from SCVI-273 hiPSCs were used for Ca²⁺ transient assays. Both of these cell lines were used for the other experiments and the results were comparable.

2.2. Formation of cardiac progenitor spheres

Cardiac progenitor spheres were generated in Aggrewell 400 plates (STEMCELL Technologies) [15]. Cells were dissociated using 0.25% trypsin-EDTA (ThermoFisher Scientific), centrifuged and resuspended for cell counting. Cells were seeded into the Aggrewell 400 plates at various concentrations, ranging from 1.2×10⁶ cells/well (1,000 cells/microwell) to 2.4×10⁶ cells/well (2,000 cells/microwell), and cultured in RPMI/B27 medium (RPMI 1640 with 2% B27 supplement with insulin) and 10 μM Rock inhibitor Y-27632 to facilitate cell survival following dissociation [16]. After 24 h, cardiac spheres were either cryopreserved or transferred to a suspension culture in RPMI/B27 or the CO₂-independent medium. Medium was changed every other day; cardiac spheres typically started beating spontaneously by days 8–12 and were cultured for subsequent experiments. Imaging of spheres was performed using an inverted microscope (Axio Vert.A1), and the images obtained were analyzed by ImageJ software to compare the size of the spheres between conditions.

2.3. Cryopreservation and thawing of cardiac progenitor spheres

For cryopreservation, cardiac progenitor spheres were collected and resuspended in 0.5 mL cryopreservation medium (90% FBS and 10% DMSO with 10 μM Rock inhibitor) and transferred into either cryosyringes or cryovials. In the optimized protocol, cardiac spheres were initially precooled at 4°C for up to 25 min in order to maximize cryopreservation efficiency, then stored at −80˚C in a Corning LX CoolCell Freezing System.

For thawing, cryovials or cryosyringes containing cardiac spheres were placed at 37°C for 5 min. Cardiac spheres were then transferred into low-adhesion dishes containing either RPMI/B27 or the CO₂-independent medium with 10 μM Rock inhibitor. Medium was partially changed (half volume) every other day. Cardiac spheres typically started beating spontaneously by days 10–12 (3–5 days after thawing) and were cultured for subsequent experiments.

2.4. Immunocytochemical analysis

Immunocytochemical analysis was conducted as described [17]. Cells were dissociated using 0.25% trypsin-EDTA and plated onto a Matrigel-coated 96-well culture plate at a density of 5×10⁴ cells/well and cultured for 2 days before fixation. On the day of the immunocytochemical staining, cells were washed with D-PBS and fixed in 4% (vol/vol) paraformaldehyde at room temperature for 15 min and permeabilized in cold methanol for 2 min at room temperature. The cells were then blocked with 5% normal goat serum (NGS) in D-PBS at room temperature for 1 h and incubated with the primary antibodies (Supplementary Table 1) in 3% NGS overnight at 4°C. After the incubation with the primary antibodies, the cells were washed 3 times with D-PBS for 5 min each with gentle agitation to remove unbound primary antibodies. The cells were then incubated with the corresponding conjugated secondary antibodies (Supplementary Table 2) (1:1000) at room temperature for 1 h in the dark, followed by 3 times wash with D-PBS. The nuclei were counterstained with Hoechst 33342. Imaging was performed using an inverted microscope (Axio Vert.A1).

For high resolution images of cardiac marker α-actinin, cells were seeded onto Matrigel-coated 25×25×1 mm glass coverslips (~10,000 cells per coverslip), allowed to adhere overnight, then fixed, permeabilized and stained. The slides were then placed in a chamber on an Olympus FV1000 inverted confocal IX81 microscope; single cell images at 40x magnification were then processed and exported using FV10-ASW 3.0.

2.5. Cell viability assay

Cardiac spheres were dissociated using 0.25% trypsin-EDTA, and stained with Trypan blue, a dye routinely used to assess cell viability by dye exclusion. The total number of viable cells (Trypan blue negative) and total number of dead cells (Trypan blue positive) were counted using a hemocytometer under an inverted microscope (Axio Vert.A1). In another cell viability analysis, cardiac spheres were stained using LIVE/DEAD cell viability assay (ThermoFisher Scientific). Cells were washed 2 times with warm D-PBS and incubated with 0.25 μM calcein AM (which labels live cells) and 1 μM ethidium homodimer-1 (which labels dead cells) for 30 min at 37°C in the dark. After the incubation, cells were washed twice with warm D-PBS and resuspended in appropriate medium, then immediately imaged with an inverted microscope (Axio Vert.A1).

2.6. Quantitative real-time RT-PCR (qRT-PCR)

RNA was isolated from cardiac spheres cultivated either in RPMI/B27 or the CO₂-independent medium for 7 days using RNeasy Mini Kit (Qiagen) as per the manufacturer’s instructions. Briefly, cells were lysed in 350 μL lysis buffer supplemented with 1% β-mercaptoethanol, and stored at −80°C until further processing. Upon collecting RNA, samples were reverse-transcribed using 100 U of Superscript III enzyme and random primers in 20 μL reaction mixture containing Vilo reaction buffer as per the manufacturer’s instructions (SuperScript VILO cDNA Synthesis Kit by Life Technologies). cDNA was further processed in a Bio-Rad thermal cycler upon incubation with the reaction mixture at following temperature cycles: 25°C for 10 min, 42°C for 2 h, and 25°C for 5 min. The reaction mixture was then diluted 15 times before further use for quantitative real-time PCR (qRT-PCR). Human-specific PCR primers (Supplementary Table 3) for the genes examined were retrieved from an open access website (http://pga.mgh.harvard.edu/primerbank/). Thermocycler reaction was set up as follows using the iTaq SyBr green master mix: Initial denaturation step at 95°C for 10 min, 40 cycles of 2 steps with 15 s of denaturation at 95°C followed by 1 min of annealing at 60°C using Applied Biosystems 7500 real-time PCR systems. All samples were normalized to the level of the housekeeping gene RPS18. Relative expression levels compared with control samples were presented as fold changes calculated by the 2–ΔΔCt method.

2.7. High-content imaging analysis

Cardiac spheres were dissociated using 0.25% trypsin-EDTA and plated onto a Matrigel-coated 96-well culture plate at a density of 5×10⁴ cells/well and cultured for 2 days to recover. After immunocytochemical staining for either cardiac marker α-actinin or NKX2.5, cells were imaged using an ArrayScan XTI Live High Content Platform (Life Technologies). Images of Hoechst and α-actinin, NKX2.5 or Tom20 positive cells were acquired and quantitatively analyzed using ArrayScan XTI Live High Content Platform (Life Technologies). Twenty fields of images/well were selected and 3–5 replicate wells per condition were imaged using a 10x objective. Acquisition software Cellomics Scan (ThermoFisher Scientific) was used to capture images, and data analysis was performed using Cellomics View Software (ThermoFisher Scientific). Images were analyzed with mask modifier for Hoechst and NKX2.5-positive cells restricted to the nucleus, and Tom20 and α-actinin were quantified with mask modifier within a circular area extending the nuclear region. The percentage of α-actinin or NKX2.5-positive cells in each treatment was used as readout.

2.8. Mitochondrial membrane potential assay

Cardiac spheres were dissociated using 0.25% trypsin-EDTA and plated onto a Matrigel-coated 96-well culture plate at a density of 5×10⁴ cells/well and cultured for 2 days to recover. Changes in mitochondrial membrane potential were quantified by using TMRM-Mitochondrial Membrane Potential Assay Kit (Abcam) as per the manufacturer’s instructions—TMRM (tetramethylrhodamine, methyl ester) is a cell permeant, positively-charged dye that accumulates in active mitochondria due to their negative charge; depolarized or inactive mitochondria have decreased membrane potential and thus reduced TMRM accumulation. Cells were incubated with Hoechst and TMRM in PBS with 0.2% BSA for 15 min, then washed with PBS with 0.2% BSA, imaged using an ArrayScan XTI Live High Content Platform (Life Technologies) and analyzed using Acquisition software Cellomics Scan (ThermoFisher Scientific).

2.9. Calcium imaging

Cardiac spheres were dissociated using 0.25% trypsin-EDTA and plated onto a Matrigel-coated 96-well culture plate at a density of 5×10⁴ cells/well and cultured for 2 days to recover. Live cell imaging of intracellular Ca²⁺ transient was performed using Fluo-4 AM, a cell permeant-fluorescent Ca²⁺ dye, as described [18]. Cells were incubated with 10 μM Fluo-4 AM for 20 min at 37°C followed by a 20 min wash at room temperature in Tyrode’s solution (148 mM NaCl, 4 mM KCl, 0.5 mM MgCl₂·6H₂O, 0.3 mM NaPH₂O₄·H₂O, 5 mM HEPES, 10 mM D-Glucose, 1.8 mM CaCl₂·H₂O, pH adjusted to 7.4 with NaOH). Fluorescence was imaged over time using an ImageXpress Micro XLS System (Molecular Devices) at 20x objective and 30 frame per second. Fluorescence was measured from the entire cell region with excitation at 488 nm and emission at >500 nm. Analysis of Ca²⁺ recordings was performed with Clampfit 10.0 software (Molecular Devices), and Ca²⁺-transient parameters analyzed include beating frequency, maximum rise slope and maximum decay slope.

2.10. Spaceflight experiment

3D cardiac progenitors were generated in Aggrewell 400 plates on differentiation day 6 and cryopreserved on day 7. The cryopreserved samples were sent to the ISS through the SpaceX-20 mission, a mission launched by the aerospace company SpaceX on March 6, 2020 for the delivery of cargo and supplies to the ISS (https://www.issnationallab.org/launches/spacex-crs-20/). Following culture for 22 days on the ISS, cell culture samples were returned to the ground via warm storage. Upon arrival at Emory University, cardiac spheres were transferred immediately to an incubator and allowed to recover overnight. The following day cardiac spheres were transferred from the collection bags into low adhesion dishes. Medium was changed from the CO₂-independent medium to standard cardiomyocyte culture medium RPMI/B27. Cardiac spheres were then maintained in a standard incubator.

2.11. Statistics and data presentation

Data were analyzed in GraphPad Prism 7.04. Comparisons were conducted via one-way ANOVA test followed by multiple comparison procedures (Dunnett’s method) or via an unpaired, two-tailed Student’s t test with significant differences defined by *, P < 0.05; **, P < 0.01; ***, P < 0.001 and ****, P < 0.0001. Data are presented as mean ± SD. Sample sizes were given for each experiment in figure legends.

3. RESULTS

3.1. Differentiation of cardiac progenitors without CO₂ supply

To prepare for our experiment on the ISS with the cell culture hardware that does not have the supply of CO₂, we prepared 3D cardiac progenitors (Supplementary Results and Supplementary Fig. 1) and evaluated culture conditions that can support cell survival and differentiation of cardiac progenitors without CO₂. Initially we directly cultured 3D cardiac progenitors in the standard RPMI/B27 medium supplemented with additional components (including buffering agent HEPES, ascorbic acid, non-essential amino acids, and GlutaMAX) to verify if this formulation could support the differentiation of hiPSCs at 37°C without CO₂ (Supplementary Fig. 2). However, cells did not look healthy after 5 days. Spheres cultured in an incubator with CO₂ survived well, whereas the spheres without CO₂ appeared loosely aggregated with apoptotic/necrotic cells floating in the medium. The color of the medium in the two sets of culture also appeared strikingly different: in the standard culture the medium turned yellow which was normal in routine culture, but in the culture without CO₂ it was visibly pink as the medium was not utilized by cells.

We then tested whether a commercially available basal CO₂-independent medium could support the growth and differentiation of hiPSCs. The commercial CO₂-independent medium is a non-HEPES proprietary medium that was formulated for transporting cells or tissue under atmospheric conditions without CO₂ supply. We also supplemented the medium with other components described in Table 1 in addition to B27, including HEPES, ascorbic acid, GlutaMAX and non-essential amino acids.

Table 1.

Composition of CO₂-independent medium for cardiac progenitors to differentiate into cardiomyocytes

Ingredients	Company Cat. No.	Volume (100 mL)
base CO2-independent medium	Gibco 18045-088	Make up to 100 mL
B27 supplement with Insulin (50X)	Gibco 17504-044	2 mL
FBS	Hyclone SH30396-03	3 mL
HEPES (1M)	Gibco 15630-080	1.5 mL
GlutaMAX-I (100X)	Gibco 35050-061	1 mL
L-ascorbic acid 2-phosphate	Sigma A8960-5G	25 μL
sesquimagnesium salt hydrate (100 mg/mL, stock)
non-essential amino acids (100X)	Gibco 11140-050	1 mL
penicillin/streptomycin (100X)	Gibco 15140-122	1 mL

We performed 3D cardiomyocyte differentiation to compare the CO₂-independent medium without the supply of CO₂ with normal culture condition (RPMI/B27 medium in a CO₂ incubator). 3D cardiac progenitors were switched to the CO₂-independent medium containing the supplements (Table 1). Control cardiac progenitor spheres in RPMI/B27 were kept in conventional culture conditions under 5% CO₂ supply. Cells in both culture conditions were observed daily and did not show differences in morphological appearances till day 20 (Fig. 1A). The typical beating of cardiomyocytes was observed as early as day 9 in both the culture conditions and the cells continued beating until harvesting on day 20. LIVE/DEAD staining showed that the cell viability was similar in the culture grown in the CO₂-independent medium in an incubator without CO₂ and the culture grown in regular RPMI/B27 medium in an incubator with CO₂ (Fig. 1A). The diameters of the spheres in these conditions were also similar (Fig.1B). High-throughput ArrayScan analysis of NKX2.5 showed that the purity of hiPSC-CMs was comparable between these conditions (Fig. 1C). In addition, the supplements added to the CO₂-independent medium increased the viable cell counts compared with the medium without the supplements (Supplementary Results and Supplementary Fig. 3). These results indicate that long-term cell survival and differentiation of cardiac spheres can be supported without CO₂ supply using the CO₂-independent medium.

Fig. 1.

Differentiation of 3D cardiac progenitors in the CO₂-independent medium in conditions without CO₂ compared with those cultured in the standard RPMI/B27 medium with CO₂. (A) Cell morphology and viability of the 3D cardiac progenitors derived from SCVI-273 hiPSCs. LIVE/DEAD staining showed viable cells with green fluorescence from calcein AM staining and dead cells with red fluorescence from ethidium homodimer. Scale bar = 100 μm. (B) Average size of the cardiac spheres (n = 99 spheres) in the indicated cultures derived from IMR90 hiPSCs. (C) Representative images of immunocytochemistry of NKX2.5 of the differentiation cultures derived from SCVI-273 hiPSCs. Scale bar = 100 μm. (D) Purity of cardiomyocytes analyzed by ArrayScan (n = 4 culture wells; 20 images/well) showing NKX2.5 positive cells from differentiation cultures derived from SCVI-273 hiPSCs.

3.2. Characterization of hiPSC-CMs from cardiac progenitors maintained in the CO₂-independent medium

We examined the sarcomeric structures and gene expression of the hiPSC-CMs derived from cardiac progenitors maintained in the CO₂-independent medium. Sarcomeric structures within cardiomyocytes were similarly developed in both RPMI/B27 medium and the CO₂-independent medium as detected by immunostaining using antibodies against α-actinin, a protein in the sarcomeric Z line (Fig. 2A). Compared with the cells maintained in the standard medium RPMI/B27, cells maintained in the CO₂ independent medium expressed similar levels of genes related to cardiac structural parameters (MYH6, MYH7, MYL7, ACTN1, TNNI1 and TNNT2), cardiac hormone (ANF), Ca²⁺ handling and ion channels (RYR2, ATP2A2, CASQ2, and SCN5A), and cardiac transcription factors (ISL1, GATA4, and GATA6) (Fig. 2B).

Fig. 2.

Effect of the CO₂-independent medium on the expression of key cardiac genes. 3D cardiac progenitors derived from IMR90 hiPSCs were cultured in either the standard RPMI/B27 medium or the CO₂-independent medium for 7 days. (A) Immunocytochemical analysis showing the expression of myocyte structural protein α-actinin. Scale bar = 10 μm. (B) qRT-PCR panel showing relative mRNA expression levels of gene associated with cardiac structure, cardiac hormone, Ca²⁺ handling proteins, ion channels and cardiac transcription factors (n = 3 culture samples; 3 PCR reactions/sample).

We also evaluated if the CO₂-independent medium affected mitochondrial features of hiPSC-CMs derived from cardiac progenitors maintained in the CO₂-independent medium. To examine the mitochondrial content, we performed immunocytochemistry using antibodies against Tom20, a mitochondrial marker. As analyzed by high-content imaging, hiPSC-CMs cultures in the CO₂-independent medium had slightly higher levels of mean fluorescence intensity of Tom20 compared with the parallel cultures in RPMI/B27 medium (Fig. 3A & 3B). We also examined mitochondrial membrane potential by TMRM, a cell-permeant dye that accumulates in active mitochondria with intact membrane potentials. As analyzed by high-content imaging, hiPSC-CMs cultures in the CO₂-independent medium had 33.1% increased levels of mean fluorescence intensity of TMRM compared with the parallel cultures in RPMI/B27 medium (Fig. 3C & 3D).

Fig. 3.

Effect of the CO₂-independent medium on mitochondrial features of hiPSC-CMs. Differentiated 3D cardiac progenitors derived from IMR90 hiPSCs were cultured in either the standard RPMI/B27 medium or the CO₂-independent medium for 7–14 days. (A) Representative images of immunocytochemical analysis showing the expression of mitochondrial import receptor subunit Tom20. Scale bar = 100 μm. (B) Detection of Tom20 by ArrayScan (n = 5 culture wells; 20 images/well). (C) Representative images of mitochondrial membrane potential assay, showing fluorescence intensity of TMRM positive cells. Scale bar = 100 μm. (D) Detection of TMRM by ArrayScan (n = 5 culture wells; 20 images/well). (E) qRT-PCR panel showing relative mRNA expression levels of gene associated with mitochondrial function (n = 3 culture samples; 3 PCR reactions/sample).

In addition, we examined if the CO₂-independent medium affected the expression of genes associated with mitochondrial function. The genes examined include OPA1 which encodes a mitochondrial protein (OPA1 mitochondrial dynamin like GTPase) that localizes to the inner mitochondrial membrane and helps regulate mitochondrial stability and energy output; MFN2 (mitofusin 2) which encodes a mitochondrial membrane protein that participates in mitochondrial fusion and contributes to the maintenance and operation of the mitochondrial network; COQ10A (Coenzyme Q10A) which is required for the function of coenzyme Q in the respiratory chain; and NDUFB5 (NADH:ubiquinone oxidoreductase subunit B5). Among these genes, all had higher levels of expression in hiPSC-CMs cultured in the CO₂-independent medium than in the RPMI/B27 medium (Fig. 3E). These results suggest that hiPSC-CMs in the CO₂-independent medium may have improved mitochondrial properties, which might be contributed by medium supplements that are reported to facilitate the metabolic and functional maturation of hiPSC-CMs [19].

3.3. Cryopreserved cardiac progenitors from hiPSCs efficiently differentiate into cardiomyocytes

To facilitate our ground-based hardware testing and space experiments, we attempted to establish an effective cryopreservation method for hiPSC-derived cardiac progenitors. If feasible, this will allow enough time to characterize the quality of cardiac progenitors before a particular batch of progenitors is sent to the ISS or for other ground-based experiments. Sending cryopreserved cells to the ISS also allows flexible timing to start the experiment at the ISS and overcome possible negative impact on the cells that may be caused by high g-force during launching.

We first examined if cardiac progenitors can be efficiently cryopreserved with high post-thawing viability and high efficiency of cardiomyocyte differentiation. Cardiac progenitors from 2D cultures on differentiation day 4 were cryopreserved at 5×10⁶ cells/vial in a cryopreservation medium containing 10% DMSO. After cell thawing and culturing in the standard culture condition, cell survival and cardiomyocyte differentiation efficiency were compared with parallel continuous cultures without cryopreservation (control) (Supplementary Fig. 4). After thawing, the cryopreserved cardiac progenitors attached well to Matrigel-coated plates. Beating cells were detected on differentiation day 9 in cultures derived from control cells and cryopreserved cells. On differentiation day 15, cells in cultures without cryopreservation appeared densely packed with debris throughout the well but cultures from cryopreserved cells had visibly less debris. Similarly, flow cytometry detected 73.5% viable cells (EMA negative cells) in cryopreserved cultures and 50.9% viable cells in cultures without cryopreservation (control). The lower cell viability in cultures without cryopreservation might be due to the high density after long-term culture. The purity of cardiomyocytes (α-actinin positive cells) in cultures with and without cryopreservation was comparable.

3.4. Rock inhibitor improved cryopreservation of intact 3D cardiac progenitors

We next examined if 3D cardiac progenitor spheres could be cryopreserved and thawed directly in suspension culture and further differentiate into cardiomyocytes. We also examined if cell survival and differentiation of 3D cardiac progenitors could be improved with Y-27632, an inhibitor of Rho-associated, coiled-coil containing protein kinase (Rock) known to be able to improve the survival of many cell types. Cardiac progenitor spheres were cryopreserved and thawed into suspension culture in the CO₂-independent medium with or without Rock inhibitor in cryoprotectant and thawing medium. In the following days, the culture from cryopreserved spheres without Rock inhibitor contained more cell debris with sphere sizes that were smaller than those in the culture without cryopreservation (Fig. 4A). By contrast, the sphere morphology appeared comparable throughout the course of differentiation in cryopreserved spheres with the Rock inhibitor compared with those in cultures without cryopreservation (Fig. 4A). After 1 week in culture, the cell number and purity were compared with parallel culture without cryopreservation. ArrayScan analysis showed the cardiomyocyte purity was comparable among the culture conditions (~86–89% NKX2.5 positive cells; ~76% α-actinin positive cells) (Fig. 4B & 4C). However, the total viable cell count was ~2.5-fold higher in the cryopreserved culture with the Rock inhibitor than in the cryopreserved culture without Rock inhibitor (Fig. 4D). These results suggest that cryopreserved intact cardiac progenitor spheres survived and differentiated following thawing and that the Rock inhibitor improved the survival of cryopreserved 3D cardiac progenitors.

Fig. 4.

The effect of Rock inhibitor on the survival of cryopreserved 3D cardiac progenitors. Cells derived from IMR90 hiPSCs on differentiation day 6 were used for the formation of cardiac spheres, cryopreserved, and revived in medium with and without Rock inhibitor into suspension culture. Cultures without cryopreservation were used as a control. Cells on differentiation day 15 were compared with cultures without cryopreservation (control) for morphology, viability and cardiomyocyte purity. (A) Morphology of cryopreserved 3D cardiac progenitors thawed in medium with and without Rock inhibitor. Scale bar = 200 μM. (B) Representative images of immunocytochemical analysis showing the expression cardiac marker NKX2.5 and α-actinin. Scale bar = 100 μm. (C) Purity of cardiomyocytes analyzed by ArrayScan showing the percentage of α-actinin and NKX2.5-positive cells (n=5 culture wells; 20 images/well). (D) Total number of viable cells and dead cells recovered from cardiac spheres as analyzed by Trypan blue exclusion (n = 3 cultures).

3.5. The effect of cell seeding density for sphere formation on cryopreservation of 3D cardiac progenitors

To evaluate the effect of cell seeding density for sphere formation on cryopreservation of cardiac progenitor spheres, we generated spheres with various amounts of cells plated into Aggrewell 400 plates ranging from 1,000 cells/sphere to 2,000 cells/sphere. Frozen cardiac progenitor spheres from these preparations were then cultured in the CO₂-independent medium for 4 days. To examine cell viability, we performed LIVE/DEAD staining with calcein AM and ethidium. Cultures with a higher cell seeding density per sphere showed more ethidium-positive cells than cultures with lower seeding density per sphere (Fig. 5A). Three days after thawing, spheres in all cultures had similar size (Fig. 5B). Further differentiation of these thawed cardiac progenitor spheres resulted in cardiomyocytes with similar differentiation efficiency: more than 90% cells were positive for NKX2.5 in all the cultures (Fig. 5C). These results suggest that cryopreservation outcomes are comparable with cardiac spheres generated from 1,000 to 2,000 cells/sphere.

Fig. 5.

The effect of cell seeding density for sphere formation on cryopreservation of 3D cardiac progenitors. Day 6 cardiac progenitors derived from SCVI-273 hiPSCs were seeded at indicated densities to form spheres and cryopreserved on day 7. Cells were then thawed and cultured in the CO₂-independent medium. (A) Representative images of LIVE (green)/DEAD (red) assay. (B) Average size of cardiac spheres (CS) (n=10 spheres) after thawing (day 0) and after 3 days in CO₂-independent medium. (C) Representative images and summary of cardiomyocyte purity (percentage of NKX2.5-positive cells) analyzed by ArrayScan (n=3 culture wells; 20 images/well).

3.6. Pre-incubation with cryopreservation medium improved cell survival of cryopreserved 3D cardiac progenitors

We examined if pre-incubation of cardiac progenitor spheres with cryopreservation medium prior to freezing could improve cell survival. Cardiac spheres were collected and resuspended in freezing medium and either immediately cryopreserved at −80°C (control) or pre-incubated for various durations at 4°C before being cryopreserved at −80°C. Cardiac spheres were then thawed at 37°C for 5 min and transferred into low adhesion dishes in the CO₂-independent medium with 10 μM Rock inhibitor and cultivated for 7 days. Cardiac spheres were then dissociated and cell viability was tested by Trypan blue staining. We observed a substantial increase in the total viable cells from cardiac spheres with pre-incubated for 10 min (Fig. 6) compared with the control without pre-incubation. Increasing the pre-incubation time to 20 to 30 min resulted in even higher survival rate of the frozen cultures. These results suggest that pre-incubation with cryopreservation medium improved cell survival of cryopreserved 3D cardiac progenitors.

Fig. 6.

The effect of pre-incubation prior to cryopreservation on the survival of cryopreserved 3D cardiac progenitors. 3D cardiac progenitors derived from IMR90 hiPSCs were resuspended in cryopreservation medium and pre-incubated at 4°C before cryopreservation, thawed and cultured in the CO₂-independent medium for 7 days. Total number of viable cells and dead cells recovered from cardiac spheres as analyzed by Trypan blue exclusion (n = 3 cultures).

3.7. Serum supplement in CO₂-independent medium improved post-thawing cell survival and differentiation of 3D cardiac progenitors and supported differentiation of functional cardiomyocytes

We examined if cell viability post-thawing of cardiac progenitor spheres could be further improved by adding 3% of FBS or knockout serum replacement (KO-SR) to the CO₂-independent medium. Cryopreserved 3D cardiac progenitors were thawed and cultured in the CO₂-independent medium with or without serum supplements. Beating cardiomyocytes were observed as early as day 9–10 in all culture conditions. The total viable cells showed an improvement with the serum-supplemented formulations compared with the CO₂-independent medium without the supplements (Fig. 7A). The purity of the cardiomyocytes based on NKX2.5 staining was ~85% in all the culture conditions (Fig. 7B).

Fig. 7.

The effect of supplemental serum on the survival of 3D cardiac progenitors and the function of subsequently derived cardiomyocytes. (A) Total number of viable cells in cultures derived IMR90 hiPSCs as analyzed by Trypan blue exclusion (n=3 cultures). (B) Purity of cardiomyocytes (% NKX2.5 positive cells) in cultures derived IMR90 hiPSCs analyzed by ArrayScan (n=5 culture wells; 20 images/well). (C) Percentage of cells exhibiting regular Ca²⁺ transients or spontaneous Ca²⁺ waves and the representative traces of Ca²⁺ transients in cultures derived SCVI273 hiPSCs (n=66–68 single cells). (D) Summary of Ca²⁺ transient parameters in cultures derived SCVI273 hiPSCs (n=64–66 single cells).

To examine the function of hiPSC-CMs following cryopreservation and culture in the CO₂-independent medium, we examined Ca²⁺ transients by calcium imaging using ImageXpress Micro XLS System. As shown in Fig. 7C, 94% of the cells in the serum-free CO₂-independent medium had regular Ca²⁺ transients and all cells examined in the CO₂-independent medium with FBS or KO-SR had normal Ca²⁺ transients. Abnormal Ca²⁺ transients including peaks with single notch, ectopic beat or tachyarrhythmia were found in 6% of the cells in the serum-free CO₂-independent medium, which is within the range we typically observed in hiPSC-CMs cultured in the standard medium of RPMI/B27 [18]. Furthermore, analysis of Ca²⁺ transient parameters indicated that cells had similar beating rate, maximum rise slope and maximum decay slope among these cultures (Fig. 7D), which are also within the range we typically observed in hiPSC-CMs cultured in the standard medium of RPMI/B27 [20].

Overall, these results indicate that 3D cardiac progenitors are able to survive post-thawing and further differentiate into functional beating cardiomyocytes in the CO₂-independent medium with serum or serum replacement.

3.8. Recovery of live cells following cell culture of cryopreserved 3D cardiac progenitors without CO₂ on the International Space Station

For the spaceflight experiment, we generated 3D cardiac progenitors using Aggrewell 400 plates on differentiation day 6 and cryopreserved them on day 7 (Fig. 8A). These samples were flown to the ISS through the SpaceX-20 mission. The astronauts on board the ISS thawed and cultured the cardiac progenitor spheres at 37°C with the final formulation of the CO₂-independent medium. Live cultures were returned to the ground via warm storage after being cultured for 22 days on the ISS. We then examined the cell morphology and viability of the returned samples. We observed that the cardiac spheres had robust beating activity and the majority of the cells in spheres were alive (positive for the green calcein AM staining), with minimal staining for ethidium (red) (Fig. 8B). These results demonstrate the feasibility of cryopreservation and CO₂-independent culture of 3D cardiac progenitors for spaceflight experiments.

Fig. 8.

Survival and differentiation of cryopreserved 3D cardiac progenitors following the spaceflight to the International Space Station (ISS). (A) Schematic diagram of the experimental design. SCVI273 and IMR90 hiPSCs were directed for cardiac differentiation and cardiac progenitors were aggregated into cardiac progenitor spheres on differentiation day 6. The 3D cardiac progenitors were subsequently cryopreserved and sent to the ISS through the SpaceX-20 mission. On the ISS, cardiac progenitor spheres were thawed and cultured in suspension with the CO₂-independent medium in the MVP modules without CO₂ for 22 days. Scale bar = 100 μm. (B) Cell morphology and viability detected by LIVE (green)/DEAD (red) staining were monitored after the cells were returned to the ground. Scale bar = 100 μm.

4. DISCUSSION

In this study we have developed methods of cryopreservation and CO₂-independent culture of 3D cardiac progenitors for spaceflight experiments—3D cultures have the advantage of enhanced cell-cell and cell-matrix interaction and improved cardiomyocyte maturation [10, 21–24]. The feasibility of both CO₂-independent medium and cryopreservation has greatly facilitated our spaceflight project to evaluate the impact of microgravity on cardiac differentiation. Our cryopreserved spheres were successfully thawed and further cultivated for 3 weeks on board the ISS. Initial results showed healthy and beating cardiomyocytes generated from the progenitor cells just as we routinely observe at the end of the cardiomyocyte differentiation on the ground. With modifications in medium composition, these protocols might be adapted for other cell types as well.

The cell preparation, cell culture conditions on the ISS, culture format and cell types differ in our study compared with previous studies. Previous studies on the ISS were done with non-cryopreserved cultures being loaded and cultivated in self-contained modules with CO₂ [6–9], In our study, cryopreserved cells were sent to the ISS, thawed and cultured by the astronauts on the ISS in a CO₂-independent condition. Our study also cultured cells in a 3D suspension culture format instead of the 2D format in previous studies. In addition, we used a different cell type; the starting cells in our study were cardiac progenitors or early-stage immature cardiomyocytes derived from hiPSCs whereas previous studies used cardiovascular progenitors derived from human heart tissues [7–9] and late-stage hiPSC-CMs [6]. We are currently analyzing cellular, molecular and functional changes in hiPSC-CMs following the spaceflight.

A CO₂-independent culture of 3D cardiac progenitors is a prerequisite for the cell culture module that does not provide CO₂. A CO₂-independent culture is also highly desirable for this and other types of cells that are to be experimented on the ISS. Without the need of CO₂ supply, a CO₂ tank would not need to be carried on a spaceflight and thus precious space/mass allowances can be devoted for other purposes. In addition, this new technology can facilitate practical operations of stem cell-cardiomyocytes for ground-based applications such as live-cell shipping and live-cell imaging for long durations where CO₂ supply is limited.

The CO₂-independent culture medium we used in this study not only supports the long-term survival but also the differentiation of cardiac progenitors. Based on cell viability and cardiomyocyte purity, differentiation of the 3D cardiac progenitors in the CO₂-independent medium in an incubator without CO₂ is comparable to those cultured in conventional medium with CO₂. We note that in a previous study, a CO₂-independent medium was successfully used to culture human mesenchymal stem cells and to induce osteogenic differentiation [25].

We found that cardiac spheres can be efficiently frozen and thawed with minimal loss of cell viability. While single cells from cultures could be frozen at −80°C immediately after being resuspended in freezing medium, cardiac spheres required a pre-incubation of 20–30 min at 4°C for the best outcome. Pre-incubation of the spheres with cryopreservation medium can increase the survival of the cardiac progenitors by several fold. The benefit of the pre-incubation with cryopreservation medium was observed for cryopreservation of other cell types [26, 27].

The cryopreservation protocol we developed in this study provides advantages for preflight, launch and on-orbit operations. It allows enough time to prepare the cells quite far in advance, which is important as spaceflight launch can be delayed for various reasons. It also offers the possibility to pre-test aliquots of cardiac progenitor cell preparations to ensure that they can be efficiently differentiated into cardiomyocytes in standard conditions before being sent to the ISS; this is important as differentiation outcomes can vary from batch to batch; therefore cell preparations of the highest quality (as well as optimum culture condition) should be used given limited opportunities of the spaceflight experiments and the costly nature of spaceflight missions. In addition, cryopreserved samples can avoid the aggregation of spheres during spaceflight launch that would otherwise occur for suspension samples. In addition, cryopreservation will also accommodate a more flexible working schedule for the astronauts to prepare the modules and thaw the cells when it best suits their timetable onboard the ISS.

Furthermore, cryopreservation of cardiac spheres also has a broad impact for the application of hiPSC-CMs in regenerative medicine, given significant advantages of transplantation of cryopreserved cells in pre-clinical and clinical settings [28, 29]. Cryopreservation is preferable for cell therapy since it allows enough time for pre-testing the quality of cell preparations prior to transplantation and confirmation of sufficient quantity of cells for transplantation. It will also facilitate transportation of the cells to operation sites.