HiDef® B8 Stem Cell Growth Medium

View PDF

Preparation of Complete HiDef® B8 Medium (standard vial)

Use appropriate aseptic technique to prepare HiDef® B8 complete medium (DMEM/F12 basal medium + HiDef-B8 400X Supplement). The following example is for preparing 501.25 mL of HiDef-B8 complete medium. If preparing other volumes, adjust accordingly. NOTE: Thaw supplements or complete medium 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 HiDef® B8 400X Supplement and warm to room temperature. Mix thoroughly via inversion or gentle pipetting. It is normal to observe turbidity after thaw. Immediate dissolution should still be observed when added to basal medium (Step 2).
2. Add 1.25 mL of HiDef® B8 400X Supplement (the full volume provided by the manufacturer) to 500 mL of DMEM/F12. Mix thoroughly and avoid foaming.
3. If prepared aseptically, HiDef® B8 complete medium is ready for use. Store complete HiDef® B8 medium at 2 - 8°C for two weeks. Once prepared, do not freeze/thaw HiDef® B8 complete medium.

Preparation of Complete HiDef® B8 Medium (miniature vial)

Defined Bioscience now offers HiDef® B8 in a miniature format, enabling convenient preparation of small volumes for pilot studies, optimization experiments, and low-throughput workflows. Each miniature 400x Supplement vial is sufficient to prepare 50.125 mL of HiDef-B8 complete medium. Miniature 10-packs collectively yield 500 mL of complete medium, and complete medium kits include a single bottle of DMEM/F12, sufficient for all miniature vials.
1. Thaw the HiDef-B8 400x Supplement and allow it to warm to room temperature. Mix thoroughly by gentle inversion or pipetting. Turbidity may be observed after thaw; this is normal and should fully resolve upon dilution in basal medium (Step 2).
2. Add 0.125 mL of HiDef-B8 400x Supplement (the full vial volume) to 50 mL of DMEM/F12 in a sterile 50-mL conical tube or equivalent vessel. Mix thoroughly, avoiding foaming.
3. When prepared aseptically, HiDef-B8 complete medium is ready for immediate use. Store at 2-8 °C for up to two weeks. Do not freeze or refreeze prepared HiDef-B8 complete medium.

General Pluripotent Stem Cell Adherent Culture Guide

• Use an incubator temperature range of 37 +/- 1°C with humidified atmosphere of 5% CO2. Ensure that proper gas exchange is achieved in culture vessels. Reduced O2 tri-gas incubators are encouraged, but not required.
• Split cultures when PSC colonies become too dense, when PSCs show increased differentiation, and/or when colonies cover ~85% of the surface area of the culture vessel, usually every three to five days.
• For standard culture, cells can be passaged at a ratio of up to 1:20 every 4 days after achieving ~70-80% confluence using 0.5 mM EDTA in DPBS (without Ca2+ and Mg2+). The split ratio can vary, though it is generally between 1:2 and 1:4 for newly derived PSCs and between 1:3 and 1:20 for established cultures. Occasionally, cells may recover at a different rate and the split ratio will need to be adjusted.
• A general rule is to observe the last split ratio and adjust the ratio according to the appearance of PSC colonies. If the cells look healthy and the colonies have enough space, split using the same ratio. If the colonies are overly dense and crowding, increase the ratio; if they are sparse, decrease the ratio.
• Newly derived PSC lines may contain a fair amount of differentiation through the first 3-5 passages. It is not necessary to remove differentiated material prior to passaging. By propagating/splitting the cells, the overall culture homogeneity should improve throughout the early passages.
• For complete transition to the HiDef® B8 medium, a minimum two-passage adaptation phase is recommended.

Key Characteristics

HiDef® B8 complete medium is a specially formulated defined medium that maintains human pluripotent stem cells in feeder-free and serum-free conditions with reduced hands-on cell culture effort and lower frequency feeding required.

• Feeder-free, serum-free, defined cell culture medium for human PSCs
• Versatile - allows enzyme-free passaging from multiple substrates and supports enzymatic single-cell passaging
• Supports the stable expansion and maintenance of PSC lines for >50 passages without karyotypic abnormalities
• Less frequent cell feeding eliminates the requirement for weekend labor. Take the weekend off!

References

1- Romero‐Tejeda, H. H. Kuo, X. Gao, J. M. DeKeyser, K. A. Fetterman, E. A. Pinheiro, C. J. Weddle, H. Fonoudi, M. V. Orman, M.M. Jouni, M. Blancard, T. Magdy, C. L. Epting, A. L. George Jr., P. W. Burridge, Stem Cell
Rep. 2020, 14, 256.

What is HiDef® B8? 

A complete, serum-free, feeder-free medium for human PSCs, available as a complete kit or supplement-only.

What makes it unique? 

Maintains PSCs for >50 passages without karyotypic abnormalities, supports weekend-free feeding, and is compatible with multiple matrices and passaging methods.

How is it stored? 

Store the 400X supplement at -20°C; prepared complete medium can be kept at 2–8°C for up to 2 weeks.

Can I switch from other media directly? 

Yes. PSCs can be transitioned to HiDef-B8 either 1. directly or 2. by gradual adaptation, in active cell cultures or at time of thawing. Please see our transition guide for further details.

How do the new miniature vials work? 

Defined Bioscience now offers the miniature vial format for smaller-volume workflows. Each miniature supplement vial is sufficient to make 50.125 mL of HiDef-B8 complete medium, or one-tenth of the amount of standard vials. Please see our user guide for further details.