HiDef® S8 Stem Cell Suspension Medium

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Preparation of Complete HiDef® S8 Medium (standard vial)

Use appropriate aseptic technique to prepare HiDef® S8 complete medium (DMEM/F12 basal medium + HiDef® S8 400X Supplement). The following example is for preparing 501.25 mL of HiDef® S8 complete medium. If preparing other volumes, adjust accordingly.

NOTE: Thaw supplements at room temperature (15–25°C) and use immediately. Do not thaw in a 37°C water bath, overnight at 4°C, or on ice.

1. Thaw the HiDef® S8 400X Supplement and allow it to warm to room temperature. Mix thoroughly by gentle inversion or pipetting. Turbidity may be observed after thawing; this is normal and should fully resolve upon dilution in basal medium (Step 2).
   
2. Add 1.25 mL of HiDef® S8 400X Supplement (the full vial volume) to 500 mL of DMEM/F12. Mix thoroughly while avoiding foaming.
   
3. When prepared aseptically, HiDef® S8 complete medium is ready for immediate use. Store at 2-8°C for up to two weeks. Do not freeze or refreeze prepared HiDef® S8 complete medium.

Preparation of Complete HiDef® S8 Medium (miniature vial)

Defined Bioscience also offers HiDef® S8 in a miniature format, enabling convenient preparation of small volumes for pilot studies, optimization experiments, and low-throughput suspension culture workflows. Each miniature 400X Supplement vial prepares 50.125 mL of complete HiDef® S8 medium. Miniature 10-packs collectively yield 501.25 mL of complete medium.

1. Thaw the HiDef® S8 400X Supplement Mini and allow it to warm to room temperature. Mix thoroughly by gentle inversion or pipetting. Turbidity may be observed after thawing; this is normal and should fully resolve upon dilution in basal medium (Step 2).
2. Add 0.125 mL of HiDef® S8 400X Supplement Mini (the full vial volume) to 50 mL of DMEM/F12 in a sterile 50 mL conical tube or equivalent vessel. Mix thoroughly while avoiding foaming.
3. When prepared aseptically, HiDef® S8 complete medium is ready for immediate use. Store at 2-8°C for up to two weeks. Do not freeze or refreeze prepared HiDef® S8 complete medium.
   

General Pluripotent Stem Cell Suspension Culture Guide

Use an incubator temperature of 37 ± 1°C with 5% CO₂, ~40% dissolved oxygen (DO), and a target pH of approximately 7.2 for stirred-tank suspension culture.

Maintain sufficient agitation to keep aggregates uniformly suspended while minimizing shear stress. Appropriate agitation speeds depend on the vessel and application; example operating ranges include 240-250 rpm for 2-L stirred-tank bioreactors and 70-90 rpm for small-scale suspension systems such as DASBox bioreactors or ultra-low attachment culture plates on orbital shakers.

Seed cultures at an application-dependent density. A typical starting point for single-cell inoculation is 1-3 × 10⁵ cells/mL with cell viability greater than 85%. For improved survival following dissociation, Defined Bioscience recommends supplementing cultures with Ready-CEPT® during inoculation. Optional surfactants such as polyvinyl alcohol (PVA) or Pluronic® F-68 may further improve aggregate uniformity and viability where appropriate.

Healthy PSC aggregates cultured in HiDef® S8 typically maintain diameters of 100-250 μm. Larger aggregates may be encouraged by reducing agitation, while smaller aggregates can be maintained by increasing agitation. Aggregate diameters exceeding approximately 400 μm may develop necrotic centers and reduce expansion efficiency.

For harvesting, collect aggregates by gentle settling or low-speed centrifugation. PSC-compatible dissociation reagents may be used to generate small aggregates or single cells for serial passaging. Following suspension expansion, aggregates or dissociated cells can be replated into adherent culture, including HiDef® B8, where they exhibit normal morphology within approximately 24 hours.

For stirred-tank bioreactors, perfusion or media exchange may begin approximately 24 hours after inoculation, with exchange rates increased as cell density rises. For orbital suspension culture in ultra-low attachment plates, allow aggregates to settle by gravity before aspirating medium during exchanges.

Key Characteristics

HiDef® S8 complete medium is a specially formulated defined medium that supports feeder-free, serum-free expansion of human pluripotent stem cells in high-density suspension culture while enabling scalable manufacturing workflows.

• Defined, serum-free, feeder-free medium for high-density human PSC suspension culture
• Supports microcarrier-free expansion in stirred bioreactors and other suspension systems
• Enables rapid biomass generation while maintaining high viability and pluripotency
• Compatible with single-cell inoculation and Ready-CEPT® supplementation
• Supports efficient transition back to adherent culture following suspension expansion
• Scalable from milliliter-scale research workflows to multi-liter bioreactor production

What is HiDef® S8?

A complete, serum-free, defined medium for high-density, microcarrier-free suspension expansion of human pluripotent stem cells (PSCs), provided as a 400X supplement for preparation with the user's preferred DMEM/F12 basal medium.

What makes it unique?

HiDef® S8 enables high-yield PSC expansion in stirred suspension culture without microcarriers, supporting single-cell or small aggregate inoculation, rapid biomass generation, and expansion to tens of millions of PSCs per mL while maintaining high viability, uniform aggregate morphology, and pluripotency. Cells also readily transition back to adherent culture following suspension expansion.

How is it stored?

Store the HiDef® S8 400X Supplement at -70°C, protected from light. Prepared complete medium may be stored at 2-8°C for up to 2 weeks. Do not refreeze prepared medium.

Can I switch from adherent culture directly?

Yes. PSCs maintained in traditional adherent culture can be transitioned directly into suspension using HiDef® S8 either at routine passage or immediately following thawing. Defined Bioscience recommends supplementing cultures with Ready-CEPT® during inoculation to maximize post-dissociation survival. Please refer to the User Guide for additional recommendations regarding agitation, inoculation density, and adaptation.

How do the new miniature vials work?

Defined Bioscience offers HiDef® S8 in a miniature vial format for small-scale suspension cultures, optimization experiments, and pilot studies. Each miniature 400X Supplement vial prepares 50.125 mL of complete HiDef® S8 medium, one-tenth the volume of a standard vial. A 10-pack of miniature vials prepares a total of 501.25 mL of complete medium. Please see the User Guide for complete preparation instructions.