Cryopreservation of Induced Pluripotent Stem Cell-Derived Dopaminergic Neurospheres for Clinical Application

Maintenance and neural differentiation of human iPSCs

This study was approved by the ethical committees of Kyoto University (Kyoto, Japan) and Sumitomo Dainippon Pharma (Osaka, Japan). Human iPSC lines 1231A3 and S17 were used in the study. 1231A3 was generated under feeder-free conditions from the donor’s peripheral blood using episomal vectors. S17 was established by reprogramming the donor’s peripheral blood using Sendai viral vectors (CytoTune-iPS 2.0LG; ID Pharma) under GMP compliance at Sumitomo Dainippon Pharma [18]. Human iPSCs were maintained and differentiated as described in detail [19]. When we began neural differentiation, the iPSCs were dissociated into single cells after 10 min of incubation with 0.5×TrypLE Select and were plated at 5.3×10⁵ cells/cm² onto iMatrix511 (Nippi)-coated plates with differentiation media containing GMEM supplemented with 8% KSR, 0.1 mM MEM nonessential amino acids (all Thermo Fisher Scientific), 1 mM sodium pyruvate (Sigma-Aldrich), and 0.1 mM 2-mercaptoethanol. 0.5×TrypLE Select was prepared by mixing TrypLE select (Thermo Fisher Scientific) and 0.5 mM EDTA/PBS. We added 100 nM LDN193189 (STEMGENT) and 500 nM A83-01 (Wako) to induce neuronal differentiation efficiently. We also added 2μM purmorphamine (Wako) and 100 ng/mL FGF8 (Wako) from day 1 to day 7 and 3μM CHIR99021 (Wako) from day 3 to induce floor plate cells.

Cell sorting and culture

To apply fluorescence-activated cell sorting, cultured cells were dissociated using 0.5×TrypLE select and stained with PE-conjugated anti-CORIN antibody (100 ng/mL; Catalent/BD) for 20 min. Dead cells and debris were excluded by 7-AAD staining. The analysis was performed using a FACS Aria II or III cell sorter (BD Biosciences) and Gigasort (Cytonome). After cell sorting on culture day 12, the sorted cells were replated on low cell adhesion U bottom 96-well plates (Sumitomo Bakelite) at a density of 3×10⁴ cells per well and in neural differentiation medium containing neurobasal medium supplemented with B27 supplement, 2 mM Glutamax-I (all Thermo Fisher Scientific), 10 ng/mL GDNF, 200μM ascorbic acid, 20 ng/mL BDNF (all Wako), 400μM dbcAMP (Sigma-Aldrich), and 100 nM LDN193189. Unsorted cells were replated at a density of 1.5×10⁴ cells per well in a neural differentiation medium without LDN193189. We changed the medium every 3 days, and 30μM of Y-27632 (Wako) was added to the first medium. For prolonged culture, cells were cultured in the neural differentiation medium.

Cell transplantation

Animals were cared for and handled according to the Guidelines for Animal Experiments of Sumitomo Dainippon Pharma. Adult male 6-OHDA-lesioned F344 NJcl–rnu/rnu rats (CLEA Japan) were used. Cell transplantation was performed by the stereotactic injection of spheres (A,+1.0; L, –3.0; V, –5.0 and –4.0 from bregma; and TB, 0 (2μL; 200,000 cells/μL); or A,+1.0; L, –3.5 and 2.5; V, –5.5 and –4.5 from bregma; and TB, 0 (4μL; 200,000 cells/μL)) into the right striatum. The number of injected cells was determined by the cell count before cryopreservation. The animals were anesthetized and perfused transcardially with PBS, followed by 4% paraformaldehyde.

Behavioral analysis

The methamphetamine-induced rotation assay was performed pre-transplantation and every 4 weeks from 8 weeks after the transplantation using video-monitored rotational bowls and video tracking software (EthoVision XT; Noldus). A dose of 2.5 mg/kg of methamphetamine (Sumitomo Dainippon Pharma) was injected intraperitoneally, and the rotations were recorded for 90 min.

Cryopreservation

Spheres collected on day 28 were placed in cryovials with 1 mL ice-cold cryopreservation medium (see Table 1) and kept on ice until freezing. For the cryopreservation, the vials were transferred into a freezing container (BICELL; NIHON FREEZER), one of three programmed freezers: PDF-150 or 250 (STREX), Cryomed (Thermo Fisher Scientific), or Proton Freezer (Ryoho Freezer Systems). Six cooling profiles (shown in Fig. 3) were used. BICELL was transferred into a deep freezer (–80°C) and kept for more than 4 h. For the programmed freezers, the vials were frozen at –0.5 or –1°C/min until –40°C and then at about – (3–5)°C/min until –80°C. In the shock cooling method, the following steps were taken beginning at –4°C: freezing at –25°C/min until –35°C and then heating at +10°C/min until –12°C. Vials in Proton Freezer were kept in the chamber for 30–60 min until completely frozen. Proton Freezer combines a static magnetic field (SMF), alternating electric field (AEF), and intense airflow. After freezing, the cryovials were stored in the vapor phase of a liquid nitrogen tank. The frozen cells were quickly thawed at 37°C and diluted ten times with neurobasal medium. After supernatant removal, the cells were rinsed with PBS or saline and used for each assay or transplantation. To estimate the cell number after cryopreservation, approximately 50 aggregates were dissociated and counted by a hemocytometer to calculate the cell concentration one day after thawing and before freezing.

Quantitative RT-PCR

Total RNA was extracted using an RNeasy Mini Kit or RNeasy Micro Kit (Qiagen), and cDNA was synthesized using the SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific). Quantitative PCR were carried out with the Fast SYBR Green PCR Master Mix in StepOne (Applied Biosystems). The data were assessed using the delta-Ct method and normalized by the GAPDH expression. The primer sequences used are shown in Supplementary Table 1.

Immunofluorescence studies

For the in vitro studies, the cultured cells were fixed with 4% paraformaldehyde. For the in vivo studies, fixed frozen brains were sliced at 40μm thickness. The slices were immunologically stained using the free-floating method. The primary antibodies used are listed in Supplementary Table 2. The cells were visualized using a fluorescence microscope (BZX710 and BZX810; Keyence, AxioScan; Zeiss) and one of three confocal laser microscopes (Fluoview FV1200; Olympus, and LSM880 and LSM800; Zeiss). The number of immunoreactive cells was quantified in every 6th section throughout the grafts and corrected using the Abercrombie method. The mean intensity of the IBA1 staining was measured in the graft areas and is shown as a ratio to the contralateral striatum. Image processing and analysis were done using Photoshop (Adobe Systems) and Fiji software.

Neurite extension assay

Floating spheres on day 28 were plated on 24-well plates coated with iMatrix511 for five days and fixed with 4% paraformaldehyde. The spheres were stained with PE-conjugated anti-PSA-NCAM antibody (1 : 100; Milteny) and visualized using the fluorescence microscope. The area covered by PSA-NCAM positive neurites was measured using Photoshop and WinRoof (Mitani Corporation).

Electrophysiological analysis

S17-derived DA neurospheres on day 28 were cultured on plates coated with poly-l-ornithine, fibronectin, and laminin (O/F/L) until whole-cell patch-clamp recordings. 1231A3-derived DA neurospheres were dissociated with papain before plating. The cells were maintained in physiological saline solution with the following composition: 125 mM NaCl, 2.5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 26 mM NaHCO₃, 1.25 mM NaH₂PO₄, and 17 mM glucose. Patch pipettes were made from borosilicate glass capillaries (GC150TF-10; Clark) and had a 3–4 MW resistance when filled with an internal solution composed of 140 mM KCl, 10 mM HEPES, and 0.2 mM EGTA (pH 7.3). Recordings with a voltage clamp and current clamp were made with a patch-clamp amplifier (Axopatch 200B; Molecular Devices). The giga-seal resistances were in the range of 10–20 GΩ. The current signals from the amplifier were filtered at 1 kHz and stored and analyzed on a 64-bit computer (Cooler Master). All experiments were performed at room temperature.

Multielectrode array (MEA)

Floating spheres on day 28 were cultured on laminin-coated HD-MEA chips (MaxWell Biosystems) until the recordings. The recordings were performed using the Activity Scan Assay and Network Assay modules on MaxLab Live software (MaxWell Biosystems). The entire HD-MEA was scanned using the Activity Scan Assay module, and electrodes on the spheres were selected. Spontaneous neuronal activities were recorded from the selected electrodes for 30 sec using the Network Assay module. After inactive electrodes (< 0.5 Hz) were removed, the mean values of spike amplitudes were calculated for each sphere.

Dopamine release assay

Floating spheres on day 28 were cultured on O/F/L-coated 12- or 24-well plates for 28 days, washed twice with low KCl (4.7 mM) solution, and incubated in low KCl solution for 15 min. The medium was subsequently replaced with high KCl solution (60 mM) for 15 min. The solution was then collected, and the concentration of dopamine was determined by LC-MS/MS using QTRAP6500 or Triple Quad 6500 (AB Sciex) coupled to Nexera X2 (Shimadzu). Cells that remained on the plate were harvested in PBS and sonicated. The DNA concentration of the cell lysate was measured using the Quant-iT dsDNA Assay Kit (Thermo Fisher Scientific) and used to compensate the dopamine concentration.

Statistical analysis

Statistical analyses were performed using a commercially available software package (GraphPad Prism 8; GraphPad Software). Data were analyzed by a one-way ANOVA and Tukey’s post hoc analysis groups, an unpaired t-test, or a two-way ANOVA with Tukey’s multiple comparisons test, as indicated in the figure legends.

Induction of midbrain DA progenitors from iPSCs

We induced DA progenitors from a research-grade human iPSC line (1231A3) based on a protocol with dual SMAD inhibition and floor plate induction [20]. To screen for the best cryopreservation conditions, we did not employ cell sorting to simplify the procedure (Fig. 1). The induced spheres were mainly comprised of DA progenitors (FOXA2/LMX1A; 86.1±5.1%) and contained some neural stem cells (SOX1; 4.7±1.6% and PAX6; 2.7±0.8%) on day 28 (Supplementary Figure 1).

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Fig. 1

Schematic overview of the protocol steps. Cryopreserved×1 and Cryopreserved×2 are defined in the main text. iPSCs, induced pluripotent stem cells.

Cryopreservation with Bambanker hRM (BBK) yields good post-thaw recovery and neurite extension

First, we determined the best CPA among six clinically available ones (Table 1). After freezing the day-28 spheres at –0.5°C/min in each CPA, we thawed them and compared the cell viability and neurite extensions. We chose day 28, because we are transplanting fresh day-28 to -30 cells for Parkinson’s disease patients in our clinical trial [19]. Some cells died during the freeze/thaw procedure due to apoptosis and necrosis [21], but the survived cells started to proliferate within 24 to 48 h after thawing [22–24]. Previous report indicated that measuring cell viability at 24 h post-thawing is essential to evaluate the quality and efficiency of a cryopreservation process [25]. Therefore, we examined the percentage of viable cells (viability) at 24 h, finding that BBK (63±4%) gave a significantly higher percentage than STEM-CELL BANKER DMSO free (SCB DMSO-free; 21±7%) or CryoStor CS5 (CS5; 16±6%) (Fig. 2A). SCB (49±16%), SaF (49±6%), and CS10 (45±14%) ranked second best in terms of cell recovery without any significant difference among them.

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Fig. 2

Effects of cryopreservation media on iPSC-derived neurospheres (A): Viability and (B): neurite extension of spheres from unsorted cells cryopreserved at –0.5°C/min on day 28 using the cryopreservation media shown in table 1 (n = 4). Viability and neurite extensions were analyzed on day 1 (24 hours) and day 5, respectively. One-way ANOVA with Tukey’s multiple comparisons test; ^*p < 0.05, ^***p < 0.001, ^****p < 0.0001 versus Bambanker hRM. C: Immunostaining of neurites for PSA-NCAM. Scale bars, 1 mm. Data are shown as means±SD.

Neurite extensions are diminished by cell damage, such as neurotoxic injury and disease-related genetic aberration [26–28]. Therefore, we measured the area covered by neurites extended from the spheres five days after plating on day 28 to evaluate cell function. With BBK, the area was 49±14% that of fresh spheres, a percentage significantly larger than with SCB DMSO-free or CS5 (Fig. 2B, C). Adding that BBK has already been registered in a Japanese drug master file, we concluded that BBK is the best CPA to cryopreserve DA progenitor spheres.

Proton Freezer provides the optimal freezing condition

Next, to determine the optimal freezing protocol, we froze the spheres in BBK by six different cooling conditions and two equilibration times (Fig. 3). Intracellular ice formation is a significant cause of cell death, and the control of cell dehydration and ice nucleation is a critical parameter that affects cell viability. The slower the cells are cooled, the stronger the cell dehydration and less intracellular ice. On the other hand, excessive cellular dehydration increases the solute concentration and damages cells [29]. Therefore, an optimal cooling rate is needed. Furthermore, during the freezing process, the CPA temperature drops below the freezing point before ice is formed (i.e., supercooling). When freezing starts, the temperature rises due to the release of latent heat and returns to the freezing point. In this process, intracellular freezing occurs because the cells freeze at once without dehydration [30, 31]. Therefore, it is essential to minimize supercooling.

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Fig. 3

Time-temperature curves of the sample (red line), freezing chamber (blue line), and program (gray line). Bambanker hRM was used as the sample. The temperature change caused by latent heat release is magnified in the lower graphs.

BICELL is a conventional freezing container. We tested BICELL and two cooling speeds, –0.5°C/min and –1°C/min, using a programmed freezer. Next, we tried the shock cooling method. This method induces ice nucleation by a transient temperature drop and suppresses supercooling [32]. The final candidate was Proton Freezer. This freezer can rapidly cool the sample from –4°C to –30°C at –5°C/min, an intermediate speed between conventional slow cooling and vitrification. In addition, Proton Freezer includes a SMF and AEF, which align the orientation of water molecules, minimizing the ice crystals and preventing cell destruction. Finally, because the equilibration time in CPA before freezing is another critical issue to be determined, we compared 15- and 60-minute equilibration times.

We evaluated the viability and neurite extension of cells cryopreserved in BBK. At 15 minutes equilibration, there was no significant difference in viability at 24 hours between the six conditions (Fig. 4A). On the other hand, Proton Freezer showed more robust neurite extensions than the other conditions, suggesting only minor damage to the cells (Fig. 4B). At 60 min equilibration, Proton Freezer resulted in higher cell viability and neurite extensions than all other conditions (Fig. 4C, D). Neither cell viability nor neurite extensions showed a significant difference between the 15- and 60-minute equilibrations in BBK (Supplementary Figure 2).

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Fig. 4

Effects of the freezing program and equilibration time on iPSC-derived neurospheres. A, C) Viability and (B, D) neurite extensions of spheres from unsorted cells cryopreserved on day 28 under the different freezing programs after 15 min (A, B) and 60 min (C, D) equilibration in Bambanker hRM (n = 4). Viability and neurite extensions were analyzed on day 1 (24 h) and day 5, respectively. One-way ANOVA with Tukey’s multiple comparisons test; ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001 versus Proton Freezer. Data are shown as means±SD.

Based on these results, we concluded that the combination of BBK and Proton Freezer is suitable for the cryopreservation of neurospheres.

Cryopreserved iPSC-derived neurospheres maintain their characteristics and functions

Next, we investigated the characteristics and functions of cryopreserved spheres derived from a clinical-grade human iPSC line (S17). We sorted the cells expressing CORIN (a floor plate marker in the developing brain) on day 12 to enrich DA progenitors, cultured the sorted cells as floating spheres until day 28, and froze them in BBK using Proton Freezer (Fig. 1). We thawed the spheres and found that the cell viability and the area covered by the neurites were 63±19% and 35±21%, respectively, compared to those of fresh spheres. To determine DA neurons, the expressions of related proteins and genes were examined seven days after thawing. Immunocytochemistry revealed that the cryopreserved spheres (D28 + 7) showed similar expressions of a floor plate marker, FOXA2, and DA neuron markers, NURR1 and TH (Fig. 5A, B), compared to fresh spheres (D35). There was no difference in the expression of two neural stem cell markers, SOX1 and PAX6, and a proliferating cell marker, KI67 (Fig. 5A, C). Quantitative PCR analyses revealed that the expression levels of several DA markers, including FOXA2, LMX1A, EN1, NURR1, PITX3, and TH, were unaffected by the cryopreservation method (Fig. 5D). In all conditions, the expression levels of the pluripotent markers POU5F1 and NANOG were significantly reduced compared to undifferentiated iPSCs (Fig. 5E).

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Fig. 5

Characterization of cryopreserved spheres derived from S17 in vitro. A) Immunostaining of the spheres on day 35. FOXA2/DAPI (left), NURR1/TH (center), and SOX1/KI67/PAX6/DAPI (right). Scale bars 100μm. B, C) The percentages of FOXA2⁺, NURR1⁺, TH⁺ (B) and SOX1⁺, PAX6⁺ and KI67⁺ (C) cells per total cells on day 35 (n = 4). D, E) Gene expressions of the spheres relative to GAPDH measured by quantitative RT-PCR (n = 4). D28, cells cultured for 28 days; D28 + 7, cells cultured for 7 days after 28 days cryofreezing; D35, fresh cells cultured for 35 days. The expression level of D28 (D) and undifferentiated cells (D0) (E) was set to 1. There were no significant differences between D35 and D28 + 7 by one-way ANOVA with Tukey’s multiple comparisons test (D). One-way ANOVA with Tukey’s multiple comparisons test; ^*p < 0.05, ^****p < 0.0001 versus D0 (E). F) Immunostaining of post-thawed iPSC-derived DA neurons for TUBB3, TH, and FOXA2 on day 49. Scale bars, 50μm. G) Representative induced action potentials of post-thawed iPSC-derived DA neurons on day 68. H) Box plots display the value in each sphere of spike amplitudes on day 42. I) The dopamine release on day 56 induced by high potassium stimulation (n = 4). Data are shown as means±SD.

To confirm the DA function of the spheres, we conducted electrophysiological analysis and dopamine release measurements. When the cryopreserved spheres were cultured on the plate for further maturation, most of the cells differentiated into TH⁺TUBB3⁺ double positive DA neurons on day 49 (Fig. 5F). Repeated action potentials were detected by whole-cell current-clamp recording on day 68 (Fig. 5G). We also measured spontaneous electrophysiological activity by MEA. No difference was observed in spike amplitudes between fresh and cryopreserved cells (Fig. 5H). Dopamine secretion was measured by LC/MS/MS. The amount of dopamine released by the cryopreserved spheres on day 56 was comparable to that of the fresh spheres (Fig. 5I).

These results suggest that cryopreserved spheres can differentiate into mature and functional DA neurons as fresh spheres do. Regarding the expression of markers and functional activities, similar results were obtained using another iPSC line (1231A3) (Supplementary Figure 3).

Cryopreserved spheres improve the behavior of 6-OHDA lesioned rats

We transplanted fresh or cryopreserved spheres into 6-OHDA-lesioned rats and investigated cell survival and pharmacological efficacy. We set two dose groups for the cryopreserved spheres: cryopreserved×1 (4×10⁵ cells) and cryopreserved×2 (8×10⁵ cells). We analyzed the methamphetamine-induced rotation every four weeks. Each group with the cell transplantation showed improved abnormal behavior after 20 weeks (Fig. 6A).

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Fig. 6

Graft survival and function of cryopreserved spheres. A) The methamphetamine-induced rotations of rats that received the grafts. (n = 4–7). Two-way ANOVA with Tukey’s multiple comparisons test; ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001 versus vehicle group. B) Immunostaining of representative grafts for HNA (green) and DAPI (blue). C) DAB staining of representative grafts for TH. The right panels are magnified images of the left panels. Scale bars, 50μm. D) The number of survived HNA⁺ cells in the grafts (n = 4–7). E) Number of survived TH⁺ cells in the grafts (n = 4–7). F) The percentages of TH⁺ cells per survived human cells (n = 4–7). One-way ANOVA with Tukey’s multiple comparisons test; ^*p < 0.05, ^**p < 0.01 (D–F). G) Immunostaining of the grafts derived from cryopreserved cells for FOXA2, TH, and HNA (upper) and GIRK2, CALB, and TH (lower). Scale bars, 50μm. Data are shown as mean±SD.

An immunofluorescence study at 24 weeks revealed that the numbers of survived human cells (HNA⁺ cells) in the graft were 51,944±22,462 (13.0±5.6% of injected cells), 27,046±8,490 (6.8±2.1% of injected cells) and 63,108±25,834 (7.9±3.2% of injected cells) for the fresh, cryopreserved×1 and cryopreserved×2 groups, respectively (Fig. 6B, D), and the numbers of TH⁺ cells in the graft were 4,601±1,189 (1.2±0.3% of injected cells), 1,306±480 (0.3±0.1% of injected cells) and 3,988±1,961 (0.5±0.2% of injected cells), respectively (Fig. 6C, E). These results suggest that the survival rate of TH⁺ neurons from cryopreserved spheres is about half that compared to fresh ones. The percentages of TH⁺ cells per survived human cells in the graft were relatively low in cryopreserved spheres compared to fresh ones (Fig. 6F). TH⁺ cells exhibited neurite outgrowth in every group (Fig. 6C) and were positive for FOXA2 (Fig. 6G). Finally, we observed GIRK2⁺ A9-subtype DA neurons had a large cell body with good arborization and Calbindin (CALB)⁺ A10-subtype DA neurons had a relatively small cell body in the grafts (Fig. 6G).

To examine the immune response to the grafts, we performed staining for IBA1, a marker of microglia (Supplementary Figure 4). Activated microglia were observed around the grafts, but there was no difference in the density of microglia between the grafts of fresh and cryopreserved cells.