Figure1: 3D Expansion of Human iPSCs in RocketCell™ 3D iPSC Xeno-Free Complete Growth Kit​

A: Representative time-lapse micrographs show human iPSCs transitioning from single cells and small aggregates into uniform 3D spheroids within the VitroGel® STEM hydrogel (seeded cells from a 2D source on Day 0). The spheroids exhibit consistent morphology and reach diameters of approximately 85–100 μm after several days in culture. B: Quantitative analysis of spheroid growth demonstrates a steady increase in total spheroid area over time, reaching an average of nearly 300% expansion after 6.5 days, confirming robust and reproducible 3D proliferation under xeno-free culture conditions.​

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Figure 2. Quantitative Assessment of 3D iPSC Growth in RocketCell™ 3D iPSC Xeno-Free Complete Growth Kit​

A: Alamar Blue absorbance assay showing a progressive increase in metabolic activity of iPSCs cultured in 3D, indicating robust cell proliferation and viability over the culture period. B: Corresponding cell count analysis confirming steady growth and expansion of iPSC spheroids within the VitroGel® STEM hydrogel, demonstrating the kit’s capability to support sustained, xeno-free 3D cell proliferation. These findings demonstrate that metabolic assays, such as Alamar Blue, can be used with IPSCs embedded in VitroGel® STEM hydrogel to estimate cell expansion, compared with recovering cells from the hydrogel and using traditional cell counting methods. These figures highlight that IPSCs can respond differently to the 3D environment, so grow better than others. The overall pattern of the two assays are similar. and it is well known that Alamar Blue assays can saturate. We see that at Day 11 in the CD34-eIPS line more than other.​​​

Figure 3. Imaging of RFP-labeled human iPSCs (HFF-1VL-RFP, TheWell Bioscience) grown in RocketCell™ 3D iPSC Xeno-Free Complete Growth Kit. ​

iPSCs (100k/well) from 2D cultures were plated in 24-well, VitroPrime™ 3D Imaging Plates. After 7 days, the dishes were imaged on the Keyence BZX system (A, B) or on the Leica MICA (C, D). The Keyence system uses widefield illumination with a 1D sectioning filter to aid 3D imaging and allows 3D imaging of the IPSC spheroids in both brightfield (A) and fluorescence (B). In contrast, the MICA uses a traditional laser to capture in higher detail and render 3D models. On image D, the depth captured is 500 microns, within a field approximately 800 x 900 microns.​

Figure 4: Live Cell Surface Immunofluorescent Staining of Human IPSCs (HFF-1VL, TheWell Bioscience) Grown in RocketCell™ 3D iPSC Xeno-Free Complete Growth Kit. ​

IPSCs (100k/well) from 2D sources were plated in 24-well VitroPrime™ 3D Imaging Plates. After 7 days, the medium was removed and replaced with media containing a diluted anti-EPCAM-PE-Alexa594-labeled antibody. The mixture was incubated for 1 hour at 37 °C, and then the well was rinsed three times with 5 min incubation with 0.5 mL of Growth Media. The well was imaged on a Leica MICA confocal microscope (A). The rendered image (B) is 300 microns deep, with a field approximately 800 x 900 microns.​

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Figure 5: Vital Dyes for Monitoring the Health of IPSC (HFF-1VL-RFP, TheWell Bioscience) Grown in RocketCell™ 3D iPSC Xeno-Free Complete Growth Kit. ​

IPSCs (100k/well) from 2D sources were plated in 24-well VitroPrime 3D Imaging Plates. After 7 days, the medium was removed and replaced with media containing diluted Calcein AM and DAPI. After 30 min, the dyes were removed, the well was washed with growth media, and placed back into the incubator for an additional 30 min before imaging on a Leica MICA confocal microscope (A,B). The rendered image (B) is 400 microns in depth, with a tiled field of approximately 1.4 mm x 1.1 mm.​

Figure 6: Cyto3D® Live-Dead Assay for Monitoring the Health of IPSC (HFF-1VL, TheWell Bioscience) Grown in RocketCell™ 3D iPSC Xeno-Free Complete Growth Kit. ​

IPSCs (100k/well) from 2D sources were plated in 24-well, VitroPrime™ 3D Imaging Plates. After 7 days, the medium was removed and replaced with media containing 1:50 diluted Cyto3D® Live-Dead Assay solution. After 30 min, the well was imaged on a Leica MICA confocal microscope (A, B). The rendered image (B) is 600 microns in depth, with a tiled field of approximately 1.4 mm x 1.1 mm. The data also demonstrates that the Cyto3D® protocol does not require any wash steps and can expedite imaging protocols needed to determine the vitality of live cultures.​

Figure 7: Indirect Immunofluorescence of Pluripotency Marker on IPSCs Grown RocketCell™ 3D iPSC Xeno-Free Complete Growth Kit. ​

iPSCs (100k/well) were grown for 7 days in 24-well VitroPrime™ 3D imaging plates, fixed, and stained with pluripotency markers Tra-1-60, Lin28, and Oct4 using Alexa Fluor 488, 594, and 647 secondary antibodies. Images were captured using a Leica MICA confocal microscope. These results confirm that the RocketCell™ 3D iPSC Growth Kit provides a supportive 3D microenvironment for maintaining pluripotency.​