Fig 1. Growth of iPSC in RocketCell™ iPSC Xeno-Free Growth Medium on CytoGrow™ Vitronectin-coated VitroPrime™ Spread-Attach Plates – Weekend Free Feeding Schedule.​

A. iPSCs grown on a CytoGrow™ vitronectin-coated VitroPrime™ 6-well plate with RocketCell™ iPSC Xeno-Free Growth Medium for 4-7 days.
B. iPSC growth was quantified by percent confluence over time, with images collected every 4 hours and analyzed relative to the initial timepoint (T = 0 + 24 hrs).​​

Fig 2. CytoGrow™ Vitronectin-coated VitroPrime™ Spread-Attach Plates support canonical iPSC morphology.

iPSCs grown on Supplier C plasticware coated with Vitronectin or Geltrex were passaged using the PBS/EDTA method, and plated in the presence of Rock Inhibitor (2 uM Thiazovivin) on CytoGrow™ Vitronectin-coated VitroPrime™ 24-well plates, or Supplier C 24-well plates coated with Geltrex. The following day, the medium was changed without Rock Inhibitor, and this was repeated every other day for up to 7 days. The cultures were inspected and photographed using an Olympus CKX41 with a Canon EOS Rebel T3i Digital SLR.

Micrographs were optimized using Adobe Lightroom. Scale bar is 100 microns. iPSCs, known to grow in colonies, have small, compact cell cytoplasm, prominent nuclei, and nucleoli, as described in both cultures grown on Vitronectin VitroPrime™ and Geltrex Supplier C plates. This demonstrates that the RocketCell™/CytoGrow™/VitroPrime™ platform is a suitable system for expanding iPSCs.​

Figure 3: IPSCs grown in RocketCell™ iPSC Xeno-Free Growth Medium on CytoGrow™ Vitronectin-coated 24-well plates were processed for indirect immunofluorescence using standard techniques.

Samples were stained with a combination of (LEFT) anti-SSEA1 and SSEA4, followed by fluorescently labeled second-step antibodies; goat-anti-mouse IgM-Alexa Fluor 488 or IgG3-Alexa Fluor 594 or (RIGHT) anti-Oct4 and anti-Lin28 followed by goat-anti-rabbit-Alexa Fluor 594, and –mouse IgG1-Alexa Fluor 488. Samples were then counterstained for 10 min using Hoechst 33342 (2.5 ug/mL). Samples were visualized using a Keyence BZ-X system. These results demonstrate that the IPSC Growth system is capable of maintaining canonical pluripotent markers while preventing differentiation, as little to no SSEA1 staining was observed (see white arrows on left panel)​.

Figure 3A: IPSCs grown in RocketCell™ iPSC Xeno-Free Growth Medium on CytoGrow™ Vitronectin-coated 24-well plates were processed for indirect immunofluorescence using standard techniques.

Samples were stained witha combination of anti-SSEA1 (ThermoFisher, mouse IgM) and SSEA4 (ThermoFisher, mouse IgG3), followed by fluorescently labeled second-step antibodies; goat-anti-mouse IgM-Alexa Fluor 488 or IgG3-Alexa Fluor 594(ThermoFisher). Samples were then counterstained for 10 min using Hoechst 33342 (2.5 ug/mL). Samples were visualized using a Keyence BZ-X system. These results demonstrate that the IPSC Growth system is capable of maintaining canonical pluripotent markers while preventing differentiation, as little to no SSEA1 staining was observed (see white arrows in the left panel).

Figure 3B: IPSCs grown in RocketCell™ iPSC Xeno-Free Growth Medium on CytoGrow™ Vitronectin-coated 24-well plates were processed for indirect immunofluorescence using standard techniques.

Samples were stained with a combination of anti-Oct4 and anti-Lin28 followed by goat-anti-rabbit-Alexa Fluor 594, and –mouse. IgG1-Alexa Fluor 488. Samples were then counterstained for 10 min using Hoechst 33342 (2.5 ug/mL). Samples were visualized using a Keyence BZ-X system. These results demonstrate that the RocketCell™ iPSC Xeno-Free Growth Medium can maintain expected and canonical pluripotent markers.​

Figure 1: Indirect Immunofluorescence of Pluripotency Marker on IPSCs Grown using 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.​

Figure 2: Direct Immunofluorescence Staining of IPSCs (HFF-1VL, TheWell Bioscience) Grown in RocketCell™ 3D iPSC Xeno-Free Complete Growth Kit. ​

iPSCs (100k/well) were grown for 7 days in 24-well VitroPrime™ 3D imaging plates before fixation and staining with directly labeled pluripotency markers Lin28 (Alexa 488), Podocalyxin (Alexa 594), and Nanog (Alexa 647). Images were captured using a Leica MICA confocal microscope. These results show that the RocketCell™ 3D iPSC Xeno-Free Complete Growth Kit provides a supportive 3D environment for maintaining pluripotent stem cells, while the use of directly labeled antibodies enables faster and more efficient hydrogel-based sample processing.​