SIM HF Expression Medium (For Hi5, Serum free)

1. Rapidly thaw (less than 2 minute) avail of frozen Hi5cells in a 27℃ water bath;

2. Decontaminate the vial and transfer the thawed cells into a sterile centrifuge tube with 5-10 mL fresh medium;

3. Centrifuge the cells at 200 × g for 5 minutes, remove the supernatant slowly.

4. Suspend the cells with fresh medium in sterile shake flask, and culture in an orbital shaker at 150 rpm;

5. Subculture the cells when the cell density is ≥2-3× 10⁶ viable cells/mL.

1. Seed cells into pre-warmed fresh SIM HF Expression Medium at a final density of 0.4-0.5×10⁶ viable cells/mL. Incubate the cells at 27±1℃ rotating at 150 rpm(The speed may vary among different shakers. Resuscitated cells are recommended for adaptive culture at a lower rotational speed);

2. Subculture the cells again, once cell density reaches 2-3 ×10⁶ viable cells/mL.

1. Determine the density of cells in logarithmic phase with a viability ≥ 90%;

2. Prepare cell freezing medium which is composed of 45% fresh SIM HF Expression Medium, 45% conditioned medium(cell culture supernatant of the cells used for cryopreservation)and 10% DMSO;

3. Centrifuge (200 × g, 5minutes) and remove all the supernatant. Resuspend the cells with suitable volume of cell freezing medium to viable cell density;

4. Aliquot the cells to cryovials and place to controlled-rate freezing apparatus. Put the apparatus in -80℃ refrigerator;

5. Transfer the tubes to liquid nitrogen for long-term storage.

(1).Direct adaptation

In general, Hi5 cells can be directly adapted from serum-reduced media or other serum-free media into SIM HF Expression Medium. Cells used for adaptation is required to be in the logarithmic phase with a viability ≥ 90%.

1. Subculture the cells directly into 20 mL of fresh SIM HF Expression Medium at a final density of 0.5-0.7×10⁶ viable cells/mL;

2. Incubate the cells at 27℃, rotating at 150 rpm;

3. 3-4 days later, determine the cell density and viability. Generally, the cell density should be > 2×10⁶ cells/mL, which means that direct adaptation is successful.

(2). Sequential adaptation

The cell inoculation density was also 0.5-0.7×10⁶ cells/mL. In the passage process, the ratio between the primary used medium and SIM HF Expression Medium in the mixed medium gradually decreased from 90:10 to 75:25, 50:50, 25:75, 10:90, 0:100.

Note: In the process of cell adaptation, the first three generations of cells may not be good, but generally the cell state will improve from the fourth generation.