A Comprehensive Protocol for Culturing H9 Embryonic Stem Cells

Reagents & Consumables for the Complete Workflow

I. Key Points for Thawing

1. Preparation of hESC/iPSC Complete Medium

1. Thaw Supplement A overnight at 4 °C; do NOT use 37 °C incubator or water bath. Limit freeze-thaw cycles of A to ≤ 2; aliquot 5 mL per tube.
2. Thaw Supplement C overnight at 4 °C; do NOT use 37 °C incubator or water bath. Limit freeze-thaw cycles of C to ≤ 2; aliquot 100 µL per tube. Y27632 is added only on the first day of recovery/passage and for cryopreservation.
3. Refer to Table 1: aseptically combine components to prepare hESC/iPSC complete medium; store at 4 °C and use within 2 weeks. Complete medium may be aliquoted and stored at –20 °C for up to 6 months.
4. Pre-warm complete medium to room temperature (no longer feels cold) before use.

2. Matrigel® Coating (6-well plate example)

abs9496 – 1.5 mL × 4

1. Thaw Matrigel® overnight at 4 °C on ice. Pre-chill 200 µL and 1 mL pipette tips at –20 °C overnight.
2. Dilute Matrigel® stock 1:100 with ice-cold DMEM/F-12. Example: add 1.5 mL Matrigel® to 150 mL cold DMEM/F-12; coating density = 300 µL cm⁻². A 6-well plate (10 cm² per well) requires 3 mL per well. Spread evenly to cover the entire surface. Use remaining coating solution within 2 weeks at 4 °C.
3. Pre-warm the coated plate in the incubator.
4. Incubate the coated plate 1 h at room temperature; prevent the surface from drying.
5. Aspirate coating solution and immediately seed stem-cell suspension.

3. Thawing hESC/iPSC (6-well plate example)

1. Pre-heat water bath to 37 °C; place Matrigel®-coated 6-well plate in biosafety cabinet ~1 h to equilibrate to room temperature (15–30 °C).
2. Add 6 µL Supplement C to 3 mL hESC/iPSC complete medium (final 10 µM). Prepare 9 mL DMEM/F-12 + 18 µL Supplement C (10 µM). Equilibrate both to room temperature (15–30 °C). Do NOT pre-warm media in 37 °C water bath.
3. Thaw 1 vial of frozen cells in 37 °C water bath with gentle agitation for ≤ 2 min; remove when only a tiny ice crystal (mung-bean size) remains.
4. Wipe vial with 75 % ethanol; transfer to biosafety cabinet. Move cell suspension to a 15 mL conical tube. Drop-wise add 10 mL DMEM/F-12 + Y27 while gently swirling. Centrifuge 160 × g, 5 min.
5. Aspirate coating solution from wells. Gently add 2 mL complete medium + Supplement C per well by wall-running.
6. Aspirate supernatant leaving ~50 µL. Flick tube 3–4× to resuspend cells. Drop-wise add 1 mL complete medium + Supplement C, flick 3–4×, and seed the 1 mL drop-wise into the 2 mL medium already in the well.
7. Mix by horizontal cross-swirling 3× (1 cycle = left-right + up-down). Incubate at 37 °C, 5 % CO₂, saturated humidity; repeat cross-swirling 3× after placement.
8. Change to fresh complete medium 18–24 h later, then daily (2 mL per well for 6-well plate). When confluency > 50 %, increase volume to 3–4 mL per well.

II. Key Points for Passaging

1. Criteria & Split Ratios

Figure 1. Morphology of hiPSC cultured in hESC/iPSC complete medium: (A, B) low-power view on days 2 & 4; (C, D) high-power view on days 2 & 4.

(1) Passage Criteria:

• Confluency ~85 % (Figure 1C–D); usually passage every 3–4 days;
• Colonies overly dense or large;
• Increased differentiation within colonies.

(2) Split Ratio:

Adjust split ratio from 1:5 to 1:12 according to growth status and experimental needs.

• Normal colonies, ~85 % confluency, uniform size (Figure 1B–D): recommended 1:10;
• Lower density → decrease ratio;
• Higher density → increase ratio.

2. Dissociation

Figure 2. hiPSC morphology during dissociation: (A) 5 min; (B) 6 min.

3. Equilibrate Matrigel®-coated 6-well plate, hESC/iPSC Passage Solution, and DPBS to room temperature (~25 °C) in biosafety cabinet for ~1 h.
4. Prepare 2 mL + 1 mL complete medium per well to be seeded; add Supplement C to 10 µM and equilibrate to ~25 °C.
5. Aspirate spent medium; gently rinse with 1 mL DPBS (Ca²⁺/Mg²⁺-free) per well and aspirate.
6. Add 1 mL hESC/iPSC Passage Solution per well to completely cover the surface.
7. Incubate at 37 °C for 5–6 min.
   Note: ① After 5–6 min inspect under microscope—cells should appear bright and round but still attached. If most cells are not yet bright, extend incubation (total < 10 min). ② Keep plate in direct contact with incubator shelf to ensure even heating; do not stack plates.
8. Return plate to biosafety cabinet gently without shaking; tilt and aspirate Passage Solution.
9. Immediately add 2 mL pre-warmed complete medium + Supplement C per well. Gently flush once with the medium in the pipette tip, then re-triturate once to detach cells.
   Note: ① Limit trituration to 1–2 gentle strokes. 10–15 % cells remaining attached is normal; if majority remain, extend dissociation (< 10 min). ② Work quickly—keep cells in Passage Solution < 15 min and process ≤ 4 wells per batch.
10. Aspirate residual Matrigel® solution; add 2 mL pre-warmed complete medium + Supplement C per well.
11. Label plate with cell line, passage number, split ratio, date, operator. Mix cell suspension gently and distribute according to predetermined ratio.
12. Mix by horizontal cross-swirling 3×; incubate at 37 °C, 5 % CO₂, saturated humidity; repeat cross-swirling 3× after placement.
13. Change to fresh complete medium 18–24 h later, then daily. Passage or freeze again after 4–5 days.

Absin Bioscience Inc. Email: worldwide@absin.cn

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