A Comprehensive Protocol for Culturing H1 Human Embryonic Stem Cells

Reagents and Consumables for the Entire Workflow

Category	Cat. No.	Product Name	Specification
Stem Cells	abs90289	hESC(H1) Human Embryonic Stem Cells	1 mL
Pluripotent Stem Cell Medium	abs90487	hESC/iPSC Cell Culture Kit	500 mL
Coating Solution	abs9496	Matrigel (iPSC-qualified, Phenol Red-free)	1.5 mL × 4
Basal Medium	abs9560	DMEM/F-12 Medium	500 mL
Passaging Dissociation Solution (Clumps)	abs90493	hESC/iPSC Passaging Working Solution (Enzyme-free)	500 mL
DPBS	abs970	D-PBS Buffer (1×, Ca²⁺/Mg²⁺-free)	500 mL
Stem Cell Freezing Medium	abs9412	ES/iPS Cell Cryopreservation Medium	100 mL
Cell Consumables	abs7033	Cell Culture Plate (Standard Clear 6-well)	1 case
	abs7034	Cell Culture Plate (Standard Clear 12-well)	1 case
	abs7035	Cell Culture Plate (Standard Clear 24-well)	1 case
	abs7053	10 mL Disposable Serological Pipette	1 case
	abs7054	25 mL Disposable Serological Pipette	1 case
	abs7164	2 mL Internal-thread Cryovial	1 case
	abs7289	2 mL Low-temperature Metal Ice Box (24-well, flat-bottom)	1 pc

I. Key Points for Recovery

1. Preparation of hESC/iPSC Complete Medium

Table 1. hESC/iPSC Cell Culture Kit

Product Information	Specification	Storage
hESC/iPSC Basal Medium	450 mL	2–8 °C, 12 months
hESC/iPSC Growth Supplements A	50 mL	–20 °C or –80 °C, 12 months
hESC/iPSC Supplement C (Y27632)	1 mL (5 mM)	–20 °C or –80 °C, 12 months

Table 2. Preparation Instructions for hESC/iPSC Complete Medium

Component	500 mL	100 mL	50 mL
hESC/iPSC Basal Medium	450 mL	90 mL	45 mL
hESC/iPSC Growth Supplements A	50 mL	10 mL	5 mL

1. Thaw Supplement A overnight at 4 °C; do not thaw in 37 °C incubator/water bath. Do not exceed 2 freeze-thaw cycles. Aliquot into 5 mL tubes.
2. Thaw Supplement C overnight at 4 °C; do not thaw in 37 °C incubator/water bath. Do not exceed 2 freeze-thaw cycles. Aliquot into 100 µL tubes. Y27632 is used only on the first day of recovery/passaging and for cryopreservation.
3. Refer to Table 1, mix the components aseptically to prepare hESC/iPSC complete medium. Store at 4 °C and use within 2 weeks. Complete medium can be aliquoted and stored at –20 °C for up to 6 months.
4. Before use, pre-warm complete medium to room temperature until no longer cold to touch.

2. Matrigel Coating (6-well plate example)

1. Thaw Matrigel overnight at 4 °C on ice. Pre-chill 200 µL and 1 mL 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 ice-cold DMEM/F-12. Coating density: 300 µL/cm²; 6-well plate = 10 cm²/well, use 3 mL per well, spread evenly. Use remaining coating solution within 2 weeks at 4 °C.
3. Pre-warm coated plates in incubator.
4. Let coated plate sit at room temperature for 1 h; do not allow surface to dry.
5. Aspirate coating solution and immediately seed stem-cell suspension.

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

1. Pre-heat water bath to 37 °C; place Matrigel-coated 6-well plate in biosafety cabinet for ~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); add 18 µL Supplement C to 9 mL DMEM/F-12 (final 10 µM); bring 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 ice crystals are almost gone (pea-size).
4. Wipe vial with 75 % ethanol; transfer to biosafety cabinet; pipette cell suspension into 15 mL tube, add 10 mL Y27-containing DMEM/F-12 drop-wise with gentle mixing, centrifuge 160 ×g for 5 min.
5. Aspirate coating solution from wells, gently add 2 mL hESC/iPSC complete medium + Supplement C per well.
6. Aspirate supernatant leaving ~50 µL, flick tube 3–4× to resuspend, add 1 mL complete medium + Supplement C drop-wise, flick again, seed the 1 mL suspension into the 2 mL medium in well.
7. Cross-shake plate 3× (left-right + up-down = 1×), incubate at 37 °C, 5 % CO₂, saturated humidity; repeat cross-shake once more.
8. Change to fresh hESC/iPSC complete medium after 18–24 h, then daily (2 mL/well). If confluency >50 %, increase volume to 3–4 mL/well.

II. Key Points for Passaging

1. Conditions and Ratio

(1) Passaging conditions:

• Cells reach ~85 % confluency (Fig. 1C–D), usually every 3–4 days.
• Colonies overly dense or large.
• Increased differentiation observed.

Note: Do not continuously culture >5 days even if colonies are small.

(2) Splitting ratio:

• Generally 1:5–1:12 according to growth status and experimental needs.
• If 85 % confluency and uniform colonies (Fig. 1B–D), use 1:10.
• Reduce ratio if density is low; increase if high.

Note: 1:10 means 1 well → 10 wells (6-well plate).

2. Dissociation

1. Equilibrate Matrigel-coated 6-well plate, hESC/iPSC Passage Solution, and DPBS to room temperature (~25 °C) in biosafety cabinet for ~1 h.
2. Prepare hESC/iPSC complete medium + Supplement C (10 µM) at 2 mL/well + 1 mL extra, bring to ~25 °C.
3. Aspirate spent medium, rinse with 1 mL/well Ca²⁺/Mg²⁺-free DPBS, aspirate.
4. Add 1 mL/well hESC/iPSC Passage Solution to completely cover surface.
5. Incubate at 37 °C for 5–6 min.

   ① Observe under microscope: most cells should become bright and rounded but still attached; extend time if needed, but <10 min total.

   ② Keep plate in direct contact with metal shelf, do not stack.

6. Return plate to biosafety cabinet without shaking, tilt and aspirate Passage Solution.
7. Immediately add 2 mL/well pre-warmed complete medium + Supplement C, gently pipette once to detach cells; collect and dispense once more.

   ① Limit to 1–2 gentle pipettings; 10–15 % cells remaining is normal. Extend dissociation if majority remain attached (<10 min).

   ② Work quickly, process ≤4 wells at a time to avoid prolonged exposure (<15 min).

8. Aspirate Matrigel solution from new plate, add 2 mL/well pre-warmed complete medium + Supplement C.
9. Label plate with cell line, passage number, split ratio, date, operator. Distribute cell suspension evenly according to desired ratio.
10. Cross-shake 3×, incubate at 37 °C, 5 % CO₂, saturated humidity; repeat cross-shake once more.
11. Change to fresh complete medium after 18–24 h, then daily. Passage or freeze 4–5 days later.

Absin’s Pick of the Week

Cat. No.	Product Name	Specification
abs90289	hESC(H1) Human Embryonic Stem Cells	1 mL
abs90487	hESC/iPSC Cell Culture Kit	500 mL
abs9496	Matrigel (iPSC-qualified, Phenol Red-free)	1.5 mL × 4
abs9560	DMEM/F-12 Medium	500 mL
abs90493	hESC/iPSC Passaging Working Solution (Enzyme-free)	500 mL
abs970	D-PBS Buffer (1×, Ca²⁺/Mg²⁺-free)	500 mL
abs9412	ES/iPS Cell Cryopreservation Medium	100 mL
abs7033	Cell Culture Plate (Standard Clear 6-well)	1 case
abs7034	Cell Culture Plate (Standard Clear 12-well)	1 case
abs7035	Cell Culture Plate (Standard Clear 24-well)	1 case
abs7053	10 mL Disposable Serological Pipette	1 case
abs7054	25 mL Disposable Serological Pipette	1 case
abs7164	2 mL Internal-thread Cryovial	1 case
abs7289	2 mL Low-temperature Metal Ice Box (24-well, flat-bottom)	1 pc

 

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