In Vitro Technologies

Permits and Restrictions	View Permits View Restrictions
Organism	Homo sapiens, human
Tissue	Kidney
Product Format	frozen
Morphology	Lymphocyte-like; single cells to small aggregates
Culture Properties	suspension
Biosafety Level	2 [Cells contain adenovirus] Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.
Applications	This cell line is optimal for transient transfection and protein expression
Storage Conditions	liquid nitrogen vapor phase

Derivation	HEK293T/17 SF cells are a derivative of the 293T (293tsA1609neo) cell line (ATCC CRL-11268), adapted to serum-free medium and suspension.
Antigen Expression	SV40 T antigen
Comments	The cells constitutively express the temperature-sensitive SV40 T antigen that allows for episomal replication of transfected plasmids containing the SV40 origin of replication. This feature increases protein expression levels by permitting more plasmid copies to persist in the transiently transfected cells. Expression vectors containing the human cytomegalovirus (CMV) promoter have been shown to achieve high levels of protein expression in 293T/17 cell line. Transient transfections can be performed at small and large scale. High transfection efficiencies and protein yields have been demonstrated in this cell line. ATCC recommends passaging thawed cells at least twice prior to transfection to ensure optimal viability. Prior to transfection (24 hours), seed cells at a density of 8 x 10⁵ cells/mL.

Derivation

HEK293T/17 SF cells are a derivative of the 293T (293tsA1609neo) cell line (ATCC CRL-11268), adapted to serum-free medium and suspension.

Antigen Expression

SV40 T antigen

Comments

The cells constitutively express the temperature-sensitive SV40 T antigen that allows for episomal replication of transfected plasmids containing the SV40 origin of replication. This feature increases protein expression levels by permitting more plasmid copies to persist in the transiently transfected cells. Expression vectors containing the human cytomegalovirus (CMV) promoter have been shown to achieve high levels of protein expression in 293T/17 cell line.

Transient transfections can be performed at small and large scale. High transfection efficiencies and protein yields have been demonstrated in this cell line. ATCC recommends passaging thawed cells at least twice prior to transfection to ensure optimal viability. Prior to transfection (24 hours), seed cells at a density of 8 x 10⁵ cells/mL.

Complete Growth Medium	The base medium for this cell line is IS 293-V (Irvine Scientific No. 91107). To make complete medium, add: 200 mM L-Alanyl-L-Glutamine, 200 mM Solution in 0.85% NaCl (ATCC® No. 30-2115™) to a final concentration of 8 mM 10% Poloxamer 188 (Sigma P5556) to a final concentration of 0.1% 10uL/mL of ITS Solution (Insulin, Transferrin, Selenious Acid; ATCC® No. 30-2606™) This medium is formulated for use with a 5-8% CO2 air atmosphere.
Subculturing	Subculture cells at log phase (when cells are ready for passaging, i.e., every 2-3 days, and are approximately 2 x 10⁶ cells/mL). Pre-warm fresh growth medium prior to use. Swirl the flask gently to evenly distribute cells in medium. Remove a small volume of cells from the flask and perform cell count. 1. Seed at 5x10⁵ cells/mL for a 2 day subculture and 4x10⁵ cells/mL for a 3 day subculture (weekend) 2. To maintain high cell viability, prior to seeding, centrifuge cells for 5min at 170x g 3. Discard spent media and re-suspend cell pellet in pre-warmed fresh complete growth media 4. Pipette cells gently to break aggregates Note: Slight aggregates may be observed, but they are easily dispersed with minimal pipetting and do not impact the performance of the cell line. Alternately, appropriate amount of fresh media maybe added directly into the flask to adjust cell seeding density. However, cell viability might be slightly compromised and decreased by 5%.
Cryopreservation	Cells should be frozen at a high concentration (e.g., 5-7 x 10⁶ cells/mL) and at a low passage number. The cells should be ≥ 85% viable prior to freezing. 1. Prepare 2X freezing medium (Complete Growth Medium supplemented with 15% DMSO) and store at 2°C to 8°C until ready to use. 2. Determine the viable number of cells and percent viability. Calculate the required volume of freezing medium based on the desired viable cell density per vial. 3. Centrifuge the cell suspension at 170 × g for 5 to 10 minutes. Carefully aspirate & discard supernatant. 4. Resuspend the cell pellet in Complete Growth Medium, and then add equal volume of the cold 2X freezing medium (prepared in step 1). 5. Transfer the cell suspension into cryovials (1 mL/vial). Continue to gently mix the cell suspension to avoid cell clumping and to keep the suspension at a homogeneous state. 6. Freeze the cells gradually at a rate of -1°C/min until the temperature reaches -70°C to -80°C. If a controlled rate freezer is not available, an isopropanol freezing container also may be used (e.g., Mr. Frosty). Store cells at -80°C overnight. Follow manufacturer instructions for freezing cells in chambers. 7. The cells should not be left at -80°C for more than 24 to 48 hours. Once at -80°C, frozen cryovials should be transferred to the vapor phase of liquid nitrogen for long-term storage.

Complete Growth Medium

The base medium for this cell line is IS 293-V (Irvine Scientific No. 91107). To make complete medium, add:

200 mM L-Alanyl-L-Glutamine, 200 mM Solution in 0.85% NaCl (ATCC® No. 30-2115™) to a final concentration of 8 mM
10% Poloxamer 188 (Sigma P5556) to a final concentration of 0.1%
10uL/mL of ITS Solution (Insulin, Transferrin, Selenious Acid; ATCC® No. 30-2606™)

This medium is formulated for use with a 5-8% CO2 air atmosphere.

Subculturing

Subculture cells at log phase (when cells are ready for passaging, i.e., every 2-3 days, and are approximately 2 x 10⁶ cells/mL). Pre-warm fresh growth medium prior to use. Swirl the flask gently to evenly distribute cells in medium. Remove a small volume of cells from the flask and perform cell count.

1. Seed at 5x10⁵ cells/mL for a 2 day subculture and 4x10⁵ cells/mL for a 3 day subculture (weekend)

2. To maintain high cell viability, prior to seeding, centrifuge cells for 5min at 170x g

3. Discard spent media and re-suspend cell pellet in pre-warmed fresh complete growth media

4. Pipette cells gently to break aggregates

Note: Slight aggregates may be observed, but they are easily dispersed with minimal pipetting and do not impact the performance of the cell line. Alternately, appropriate amount of fresh media maybe added directly into the flask to adjust cell seeding density. However, cell viability might be slightly compromised and decreased by 5%.

Cryopreservation

Cells should be frozen at a high concentration (e.g., 5-7 x 10⁶ cells/mL) and at a low passage number. The cells should be ≥ 85% viable prior to freezing. 1. Prepare 2X freezing medium (Complete Growth Medium supplemented with 15% DMSO) and store at 2°C to 8°C until ready to use. 2. Determine the viable number of cells and percent viability. Calculate the required volume of freezing medium based on the desired viable cell density per vial. 3. Centrifuge the cell suspension at 170 × g for 5 to 10 minutes. Carefully aspirate & discard supernatant. 4. Resuspend the cell pellet in Complete Growth Medium, and then add equal volume of the cold 2X freezing medium (prepared in step 1). 5. Transfer the cell suspension into cryovials (1 mL/vial). Continue to gently mix the cell suspension to avoid cell clumping and to keep the suspension at a homogeneous state. 6. Freeze the cells gradually at a rate of -1°C/min until the temperature reaches -70°C to -80°C. If a controlled rate freezer is not available, an isopropanol freezing container also may be used (e.g., Mr. Frosty). Store cells at -80°C overnight. Follow manufacturer instructions for freezing cells in chambers. 7. The cells should not be left at -80°C for more than 24 to 48 hours. Once at -80°C, frozen cryovials should be transferred to the vapor phase of liquid nitrogen for long-term storage.

Population Doubling Time	24 hours

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HEK293T/17 SF Suspension Cells Item ID: ATCACS4500

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