Preparation of stock solution
1. Dissolve the reagents according to manual instructions. The concentrations of stock solutions and their final concentration in the medium are shown below:
Insulin 10 mg/ml (final concentration 10 μg/ml), Apo-transferrin 55 mg/ml (final concentration 5.5 μg/ml), Sodium selenite 10 μg/ml (final concentration 1 ng/ml), L-Ascorbic acid 2-phosphate 50 mg/ml (final concentration 100 μg/ml), Activin A 100 μg/ml (final concentration 5-40 ng/ml), Human LIF 100 μg/ml (final concentration 10 ng/ml), CHIR99021 10 mM (final concentration 1 μM), (S)-(+)-Dimethindene maleate
20 mM (final concentration 2 μM), Minocycline Hydrochloride 20 mM (final concentration 2 μM), Y-27632 100 mM (final concentration 5 μM), IWR-1-endo (optional) 5 mM (final concentration 0.5 μM).
2. Store the cytokines in a refrigerator at - 80°C, except that insulin is stored at 4°C. Store other dissolved reagents in a refrigerator at - 20°C. Reagents are stored in small quantity to avoid repeated freezing and thawing. The thawed reagents can be stored at 4°C for no more than 2 weeks.
Preparation of xeno-free human EPS cell medium
1. Thaw packed stock solutions at room temperature.
2. For preparing 50 ml xeno-free human EPS cell culturing medium, add the reagents according to the information shown below:
DMEM/F12 (liquid) 25 ml, Neurobasal (liquid) 25 ml, Insulin 50 μl, Apo-transferrin 5.5 μl, Sodium selenite
5 μl, L-Ascorbic acid 2-phosphate 100 μl, Activin A 10 μl (20 ng/ml), Human LIF 5 μl, CHIR99021 5 μl, (S)-(+)-Dimethindene maleate 5 μl, Minocycline Hydrochloride 5 μl, Y-27632 2.5 μl, Ethanolamine (liquid) 2.5 μl, Catalase (liquid) 10 μl.
3. Prepared xeno-free hEPS medium could be kept at 4°C for up to 1 week.
Preparation of LN-521 coated plates
Thaw 1 vial of LN-521 (100 μg/ml) at 4°C. Prepare the LN-521 solution (1:40) in DPBS with calcium and magnesium. Add the solution into the wells (for a 24-well plate: 350 μl per well; for a 12-well plate: 700 μl per well). Incubate the coated plates 37°C for at least 2 hours. Alternatively, the coated plates could be placed at 4°C overnight. Do not allow the culture surface to dry as the matrix will become inactivated. The coated plates should be used as soon as possible and stored at 4°C for no more than one week.
Culture and passaging of xeno-free human EPS cells
1. Warm the xeno-free hEPS medium at room temperature and TrypLE Select at 37°C.
2. Dilute TrypLE Select with DPBS with a 1:1 ratio. Keep the diluted TrypLE Select warm at 37°C.
3. Change the culture medium every 2-3 days. To change the medium, remove the supernatant and add fresh xeno-free human EPS cell culturing medium. Passage xeno-free human EPS cells every 3-4 days.
4. Cell passaging is conducted when xeno-free human EPS cells reach 85%-95% of confluence. Wash xeno-free human EPS cells with DPBS without calcium and magnesium for once. After aspirating the DPBS solution, add 0.5X TrypLE Select solution (for a 12-well plate: about 500 μl per well) and incubate the cells at 37°C for 3 min in the incubator.
5. Remove the 0.5X TrypLE Select solution and add xeno-free human EPS cell culturing medium into the well (for a 12-well plate: 1 ml per well). Pipet the digested cells into single cells and centrifuge at room temperature. Aspirate the suspension and resuspend the cells using xeno-free human EPS cell culturing medium.
6. Remove the LN-521 solution from the LN-521 coated plate and add xeno-free human EPS cell culturing medium (for a 12-well plate, 1 ml culturing medium). Seed the cells into the new wells at the density of 20,000-30,000 cells/cm2.
7. Change the culturing medium the next day. When the density of the seeded cells exceeds 50,000 cells/cm2, the medium needs to be changed every day.
Adaptation of feeder-cultured human EPS cells to xeno-free culture system
1. Warm the medium and TrypLE Select at room temperature.
2. Remove the supernatant of feeder-cultured human EPS cells, wash once with DPBS, dissociate the cells with 0.5X TrypLE Select.
3. Centrifuge the cells and resuspend them with N2B27-LCDM medium (culturing medium for feeder cultured human EPS cells), and seed the cells onto the LN-521 coated plates at desired densities. The ratio is usually from 1:6 to 1:10.
4. Replace the medium with xeno-free hEPS medium 24 hours later.
5. For the first 3-5 passages, lower the split ratio (1:3) so that cell viability could be improved. The density of the seeded cells is about 40,000 cells/cm2. In addition, 1%-5% of xeno-free KSR is recommended to be added during the adaptation so that cell viability and proliferation could be improved.
6. After 5-7 passages, the cells could proliferate well in xeno-free human EPS cell culturing medium.
Deriving xeno-free human EPS cells from Human fetal fibroblast (HFF)
1. Perform sendai virus transduction (CytoTuneTM-iPS 2.0 Sendai Reprogramming Kit ) according to the user’s manual.
2. 5-7 days post transduction, replate the infected HFFs onto LN-521 coated 6-well plates at the density of 1*105/cm2.
3. Replace the cultured medium with xeno-free human EPS cell culturing medium plus 5% xeno-free KSR. Change the medium every 3 days.
4. 14 days post transduction, human EPS cell-like colonies emerge. Picked up the colonies. Dissociate the colonies into single cell using TrypLE Select.
5. After 4-7 days, colonies could be visually observed. After 3-5 passages, xeno-free human EPS cell lines can be stably maintained in xeno-free human EPS cell culturing medium without KSR.