SIM HF Expression Medium (For Hi5, Serum free)

Product description
SIM HF Expression Medium is an optimized serum-free medium used to support the growth of High FiveTMcell.It is for research use only not for use in human or animals. This medium contains L-glutamine and is ready-to-use.
Product features
• Specifically optimized for the culture and transient transfection of Hi5 cells
• Strict quality control, stable performance, cost-effective
• Serum-free, protein-free
Product parameters
Appearance Clear Yellow
Osmotic pressure 340-400 mOsm/kg
pH 6.0-6.5
Sterility No Microbial Growth Detected
endotoxin < 10 EU/mL
Period of validity 12 months from the date of manufacture
Storage conditions 2-8 ℃; Protect from light
* SIM HF medium is yellow transparent liquid. Do not use the medium, once it becomes cloudy or precipitated.
Culture Conditions
• Media: SIM HF Expression Medium
• Cell Line: BTI-TN-5B1-4 (Hi5) cells
• Culture Type: Suspension
• Culture Vessel: Shake flask, bioreactor, etc.
• Temperature Range: 26℃ to 28℃
Recovery

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.

Subculture

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.

Cryopreservation

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.

Cell adaptation

(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.

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