FreeStyle 293 Expression System

The Invitrogen FreeStyle 293 Expression System is designed to allow large-scale transfection of suspension 293 human embryonic kidney cells in a defined, serum-free medium. The system includes Invitrogen FreeStyle 293-F cells that have been adapted to serum-free, suspension culture in Invitrogen FreeStyle 293 Expression Medium. Transfection and expression experiments may be performed directly in FreeStyle 293 Expression Medium without the need to change media. The complete FreeStyle 293 Expression Kit provides enough reagents to perform 16 transfections in a 30 ml volume, but larger volume transfections may be performed using simple scale-up of reagents.

Components of the FreeStyle 293 Expression System

The FreeStyle 293 Expression System includes the following major components:

• FreeStyle 293-F cells: This cell line is adapted to high density, serum-free suspension culture in FreeStyle 293 Expression Medium and is capable of producing high levels of recombinant protein (see more information below).
• FreeStyle 293 Expression Medium: This is a defined, serum-free medium formulated specifically to allow growth and large-scale transfection of suspension FreeStyle 293-F cells.
• Invitrogen 293fectin Transfection Reagent: This transfection reagent provides high transfection efficiency in suspension FreeStyle 293-F cells.

The FreeStyle 293-F cell line is supplied with the FreeStyle 293 Expression System and is derived from the 293 cell line (see below). FreeStyle 293-F cells are adapted to suspension culture in FreeStyle 293 Expression Medium. Frozen cells are supplied in and may be thawed directly into FreeStyle 293 Expression Medium (see Thawing and Establishing Cells).

Parental Cell Line

The 293 cell line is a permanent line established from primary embryonal human kidney transformed with sheared human adenovirus type 5 DNA (Graham et al., 1977; Harrison et al., 1977). The E1A adenovirus gene is expressed in these cells and participates in transactivation of some viral promoters, allowing these cells to produce very high levels of protein.

The FreeStyle 293-F cell line supplied with the FreeStyle 293 Expression System is a variant of the 293 cell line that has been adapted to suspension growth in FreeStyle 293 Expression Medium. The 293-F cell line was obtained from Robert Horlick at Pharmacopeia.

Characteristics of FreeStyle 293-F Cells

The FreeStyle 293-F cell line exhibits the following characteristics:

• Prepared from low passage Master Cell Bank cultures derived from parental 293-F cells that were re-cloned by limiting dilution. The 293 clonal derived cultures are maintained in serum-free conditions for only 30 to 35 total passages.
• Adapted to high density, serum-free, suspension growth and are maintained in FreeStyle 293 Expression Medium.
• Demonstrates high transfection efficiencies with 293fectin.
• Suspension cultures may be transfected in FreeStyle 293 Expression Medium without the need to change media.
• Permits transfection of cells at large volumes. 

Note:  Other 293 cell lines may be used with the FreeStyle 293 Expression System. Before these cell lines may be used for transfection studies, however, they must be adapted to serum-free, suspension culture in FreeStyle 293 Expression Medium and evaluated for transfection and expression.

Introduction

FreeStyle 293 Expression Medium is a defined, serum-free medium specifically developed for the high-density, suspension culture and transfection of 293 cells. The medium contains NO human or animal origin components.

Features of the Medium

FreeStyle 293 Expression Medium exhibits the following features:

• An optimized, serum-free, protein-free formulation designed to support the high-density culture and transfection of 293 cells (e.g. FreeStyle 293 cells) in suspension. The medium is not recommended for adherent 293 cell culture.
• Prepared ready-to-use, with no supplementation required.
• Contains no human or animal-origin products.
• Formulated with Gibco™ GlutaMAX™-I (see below) to increase stability and maximize shelf life.
• Supports the large-scale, high-density growth of FreeStyle 293-F cells in bioreactors.

GlutaMAX-I

GlutaMAX-I media contain the dipeptide, L-alanyl-L-glutamine, a stabilized form of L-glutamine. With GlutaMAX-I media:

• L-glutamine does not degrade in storage or during incubation
• Ammonia build-up is minimized
• Glutamine delivery is controlled
• L-glutamine does not need to be added at the time of use.

Note: GlutaMAX-I is only removed from the medium by cell metabolism. There is no accumulation of toxic metabolites due to spontaneous breakdown.

Growth Characteristics of FreeStyle 293-F Cells in the Medium

Typically, FreeStyle 293-F cells cultured in FreeStyle 293 Expression Medium demonstrate the following:

• Doubling time in the range of 20-25 hours Note: The doubling time can exceed 25 hours during the first few passages after the cells have been thawed.
• Cell densities of up to 3 x 10⁶ cells/ml in shaker or spinner culture
• Cell densities of up to 4 x 10⁶ cells/ml in bioreactor culture

Note: Individual culturing and passaging techniques coupled with cellular heterogeneity inherent within the FreeStyle293-F cell population may result in experimental variability.

General cell handling

Follow the general guidelines below to grow and maintain FreeStyle 293-F cells.

• All solutions and equipment that come in contact with the cells must be sterile. Always use proper sterile technique and work in a laminar flow hood.
• Before starting experiments, be sure to have cells established and also have some frozen stocks on hand. We recommend using early-passage cells for your experiments. Upon receipt of the cells, grow and freeze multiple vials of the FreeStyle 293-F cell line to ensure that you have an adequate supply of early-passage cells.
• For general maintenance of cells, pass FreeStyle 293-F cells when they reach a density of approximately 1–3 x 10⁶ viable cells/mL (generally every 3-4 days).
• Use trypan blue exclusion to determine cell viability (see below). Log phase cultures should be >90% viable.
• When thawing or subculturing cells, transfer cells into pre-warmed medium.

Caution:  As with other human cell lines, when working with FreeStyle 293-F cells, handle as potentially biohazardous material under at least Biosafety Level 2 containment.

Preparing media

For suspension growth and transfection applications, use:

• FreeStyle 293 Expression Medium as is. No supplementation is required.
• Antibiotics are not recommended; however, 5 mL/L of Antibiotic-Antimycotic (Cat. No. 15240) containing penicillin, streptomycin, and amphotericin B may be used when required.

Important:  FreeStyle 293 Expression Medium is extremely sensitive to light. For optimal results, use and store media protected from light.

Determining cell density and viability

Follow the procedure below to determine viable and total cell counts.

1. Transfer a small aliquot of the cell suspension to a microcentrifuge tube.
2. Determine viability and the amount of cell clumping using the trypan blue dye exclusion method.
3. Vigorously vortex for 10-30 seconds to break up cell clumps.
4. Determine cell density electronically using a Coulter Counter or manually using a hemacytometer.

Thawing and establishing cells

Introduction

Follow the protocol below to thaw FreeStyle 293-F cells to initiate cell culture. The FreeStyle 293-F cell line is supplied in a vial containing 1 mL of cells at 1 x 10⁷ viable cells/mL in 90% FreeStyle 293 Expression Medium and 10% DMSO. Thaw FreeStyle 293-F cells directly into the FreeStyle 293 Expression Medium supplied with the kit.

Materials needed

You will need to have the following reagents on hand before beginning:

• FreeStyle 293-F cells (supplied with the kit; store frozen cells in liquid nitrogen until ready to use)
• FreeStyle 293 Expression Medium (supplied with the kit; pre-warmed) Note: We do not recommend adding antibiotics to media as this may negatively impact cell growth.
• 125 mL polycarbonate, disposable, sterile Erlenmeyer flask
• Orbital shaker in 37°C incubator with a humidified atmosphere of 8% CO₂
• Room temperature table-top centrifuge and sterile centrifuge tubes
• Reagents to determine viable and total cell counts
• Sterile, 50 mL conical tubes

Thawing procedure

Store frozen cells in liquid nitrogen until ready to use. To thaw and establish cells:

1. Remove the cryovial of cells from the liquid nitrogen and thaw quickly in a 37°C water bath.
2. Just before the cells are completely thawed, decontaminate the outside of the vial with 70% ethanol. Triturate and transfer the entire contents of the cryovial into a 125 mL polycarbonate, disposable, sterile Erlenmeyer shaker flask containing 30 mL of pre-warmed FreeStyle 293 Expression Medium
3. Incubate cells in a 37°C incubator containing a humidified atmosphere of 8% CO₂ in air on an orbital shaker platform rotating at 125 rpm. Loosen the cap of the flask a quarter turn from snug to allow oxygenation/aeration.
4. Once the culture has reached greater than 1 x 10⁶ viable cells/mL (typically 3 to 5 days), transfer the cell suspension aseptically into a centrifuge tube and vortex for 10 seconds.
5. Determine viable and total cell counts.
6. Subculture the FreeStyle 293-F cells by seeding shaker flasks at 3 x 10⁵ viable cells/mL in pre-warmed FreeStyle 293 Expression Medium. We generally use 125 or 250 mL polycarbonate, disposable, sterile, Erlenmeyer flasks containing 20 or 40 mL total working volume of cell suspension, respectively.

Important note: Subculture cells a minimum of two additional times before use in transfection experiments to allow opportunity for recovery from thawing. To subculture cells, see the procedure below.

Subculturing cells

Passaging cells

Subculture cells when the density is approximately 2–3 x 10⁶ viable cells/mL, typically every 3-4 days. When maintaining FreeStyle 293-F cells, we generally use a 125 or 250 mL polycarbonate, disposable, sterile Erlenmeyer flask containing 25 to 40 mL or 50 to 80 mL total working volume of cell suspension, respectively. Note: Glass flasks without baffles may be used, but thorough cleaning after each use is essential to avoid potential toxicity which is more problematic in serum-free cultures.

1. Determine viable and total cell counts.
2. Using the cell density determined in Step 1, calculate the split ratio needed to seed the new shaker flask at 3 x 10⁵
3. Dilute the cells in fresh, pre-warmed FreeStyle 293 Expression Medium to give a final cell density of 0.1–0.2 x 10⁶ viable cells/mL in the desired final volume.
4. Incubate flasks in a 37°C incubator containing a humidified atmosphere of 8% CO₂ in air on an orbital shaker platform rotating at 135 rpm.
5. Repeat Steps 1-5 as necessary to maintain or expand cells. Monitor the degree of cell clumping (see below).

Recommendation:  FreeStyle 293-F suspension cultures may grow as 2 to 10 cell clusters. Vigorous vortexing for 10–30 seconds may be required at each subculture for a number of passages until the cultures grow predominantly as single cells.

Scaling up cell culture

It is possible to scale up the FreeStyle 293-F cultures in spinner flasks or bioreactors. The appropriate spinner or impeller speed and seeding density should be determined and optimized for each system. In our lab, the optimum spinner speed was 100–130 rpm and 70–100 rpm impeller speed in Celligen™  stirred tank bioreactors. We recommend seeding cells at 3–5 x 10⁵ viable cells/mL.

Note: If the split ratio of cells to fresh media is less than  1:2, you may want to spin down the cell suspension and resuspend the cell pellet in fresh, pre-warmed FreeStyle 293 Expression Medium prior to inoculating the spinner or bioreactor culture. Monitor cell viability and the degree of cell clumping. Note that extensive cell clumping may reduce transfection efficiency. Recommendation:  At high stirring speeds (i.e. greater than 130 rpm) and/or depending on the impeller design, you may want to supplement the FreeStyle 293 Expression Medium with additional Pluronic® F-68 (2.5-5 mL/L of 10% Pluronic® F-68, Cat. No. 24040) to avoid sheer stress in the culture.

Pluronic® is a registered trademark of BASF Corporation. Celligen™ is a registered trademark of New Brunswick Corp.

Freezing cells

Introduction

You may freeze FreeStyle 293-F cells directly in FreeStyle 293 Expression Medium. When freezing the FreeStyle 293-F cell line, we recommend the following:

• Freeze cells at a density of 5–8 x 10⁶ viable cells/mL.
• Use a freezing medium composed of 90% fresh FreeStyle 293 Expression Medium and 10% DMSO. Guidelines to prepare freezing medium and to freeze cells are provided in this section.

Preparing freezing medium

Prepare freezing medium immediately before use.

1. In a sterile, conical centrifuge tube, mix together the following reagents for every 1 mL of freezing medium needed:  FreeStyle 293 Expression Medium 0.9 mL DMSO 0.1 mL
2. Filter-sterilize the freezing medium and place the tube on ice until use. Discard any remaining freezing medium after use.

Freezing cells

Before starting, label cryovials and prepare freezing medium. Keep the freezing medium on ice.

1. Grow the desired quantity of FreeStyle 293-F cells in shaker flasks, harvesting when the cell density reaches 0.5 to 1 x 10⁶ viable cells/mL. Transfer cells to a sterile, conical centrifuge tube.
2. Determine the viable and total cell counts and calculate the volume of freezing medium required to yield a final cell density of 5-8 x 10⁶ viable cells/mL.
3. Centrifuge cells at 100 x g for 5 minutes at room temperature and carefully aspirate the medium.
4. Resuspend the cells in the pre-determined volume of chilled freezing medium.
5. Place cryovials in a microcentrifuge rack and aliquot 1 mL of the cell suspension into each cryovial.
6. Freeze cells in an automated or manual, controlled-rate freezing apparatus following standard procedures. For ideal cryopreservation, the freezing rate should be a decrease of 1°C per minute.
7. Transfer frozen vials to liquid nitrogen for long-term storage.

Note: You may check the viability and recovery of frozen cells 24 hours after storing cryovials in liquid nitrogen by following the procedure outlined in Thawing and Establishing Cells

Transfecting cells

Introduction

To transfect suspension FreeStyle 293-F cells, you will use the cationic lipid-based transfection reagent, 293fectin included with the kit. Unlike some other serum-free media formulations, FreeStyle 293 Expression Medium does not inhibit cationic lipid-mediated transfection. FreeStyle 293 Expression Medium is specifically formulated to allow high efficiency transfection of suspension FreeStyle 293-F cells without the need to change or add media. Transient transfection experiments may be performed in a large volume, allowing larger-scale protein production.

293fectin 

293fectin is a proprietary formulation suitable for transfection of nucleic acids into eukaryotic cells. In the FreeStyle 293 Expression System, use of 293fectin to transfect FreeStyle 293-F cells provides the following advantages:

• 293fectin demonstrates high transfection efficiency in suspension FreeStyle 293-F cells (cultured in FreeStyle 293 Expression Medium)
• DNA-293fectin complexes can be added directly to cells in culture medium
• It is not necessary to remove complexes or change or add medium following transfection. 293fectin is available separately. For more information, see our website or call Technical Service.

Opti-MEM I

Gibco Opti-MEM I Reduced Serum Medium is included with the FreeStyle 293 Expression System to facilitate optima  formation of DNA-293fectin complexes. Opti-MEM I is a modification of Eagle's Minimal Essential Medium, buffered with HEPES and sodium bicarbonate, and supplemented with hypoxanthine, thymidine, sodium pyruvate  L-glutamine, trace elements, and growth factors. The protein level is minimal (15 μg/mL) with insulin and transferrin being the only protein supplements. Phenol red is included at a reduced concentration as a pH indicator. Opti-MEM I Reduced Serum Medium is available separately.

Positive control

pCMV SPORT-βgal is provided as a positive control vector for transfection and expression in FreeStyle 293-F cells. The gene encoding β-galactosidase is expressed in FreeStyle 293-F cells under the control of the human cytomegalovirus (CMV) promoter. Successful transfection will result in β-galactosidase expression that is easily assayed. For a map of pCMV SPORT-βgal.

Assay for β-galactosidase activity

You may evaluate β-galactosidase expression by activity assay using cell-free lysates (Miller, 1972). We offer the β-Gal Assay Kit (Cat. No. K1455-01) for fast and easy detection of β-galactosidase expression.

Plasmid preparation

Plasmid DNA for transfection into eukaryotic cells must be clean, sterile and free from phenol and sodium chloride. Contaminants may kill the cells, and salt will interfere with complexing, decreasing transfection efficiency. We recommend isolating plasmid DNA using one of the Invitrogen PureLink HiPure Plasmid Kits (Cat. No. K2100-14 or K2100-16).

Note:  Make sure your DNA preparation is sterile, for instance by performing filtration through a 0.22 μm filter before use.

Materials to have on hand

You will need to have the following reagents on hand before beginning:

• Suspension FreeStyle 293-F cells cultured in FreeStyle 293 Expression Medium Recommendation: Calculate  the number of cells that you will need for your transfection experiment and expand cells accordingly. Make sure that the cells are healthy and greater than 90% viable before proceeding to transfection.
• Purified plasmid DNA of interest
• 293fectin (supplied with the kit; store at +4°C until use)
• Opti-MEM I Reduced Serum Medium (supplied with the kit; pre-warmed)
• FreeStyle 293 Expression Medium (supplied with the kit; pre-warmed) Note: Do not add antibiotics to media during transfection as this may decrease transfection activity.
• 125 mL polycarbonate, disposable, sterile Erlenmeyer flasks
• Orbital shaker in 37°C incubator with a humidified atmosphere of 8% CO₂
• Room temperature table-top centrifuge and sterile, conical centrifuge tubes
• Reagents to determine viable and total cell counts
• Sterile, disposable, polycarbonate snap-cap tubes
• Vortex mixer

Optimal conditions for 30 mL transfection

We generally perform transfection experiments in a 30 mL volume. To transfect suspension FreeStyle 293-F cells, we recommend using the following optimized conditions:

• Final transfection volume: 30 mL
• Number of cells to transfect: 3 x 10⁷ cells (final cell density of 1 x 10⁶ cells/mL)
• Amount of plasmid DNA: 20–40 μg (we typically use 30 μg)
• Amount of 293fectin: 40-80 μl (we typically use 60 μl). Use 2 μl 293fectin per 1 μg of plasmid DNA transfected

Note:  If you are using other 293 cells, you may want to test varying amounts of 293fectin (e.g. 30, 40, 50, 60, 80 μl) with 30 μg plasmid DNA to determine the optimal conditions for transfection.

Transfection procedure

Follow the procedure below to transfect suspension FreeStyle 293-F cells in a 30 mL volume. Remember that you may keep the cells in FreeStyle 293 Expression Medium during transfection. We recommend including a positive control (pCMV SPORT-βgal) and a negative control (no DNA, no 293fectin) in your experiment.

1. The day before transfection (day 1), determine the number of cells that you will need for your experiment. Remember that for each 30 mL transfection, you will need 3 x 10⁷ cells in 28 mL of FreeStyle 293 Expression Medium. Tip: To transfect cells on day 2, seed cells at a density of 6~7 x 10⁵ viable cells/mL. To transfect on day 3, seed cells at a density of 3~4 x 10⁵ cells/mL.
2. On the day of transfection, transfer a small aliquot of the cell suspension to a microcentrifuge tube and determine viability and the amount of cell clumping using the trypan blue dye exclusion method. Vigorously vortex for 45 seconds to break up cell clumps and determine total cell counts using a Coulter Counter or a hemacytometer. Viability of cells must be over 90%. Important: For optimal transfection results, make sure that you have a single cell suspension. It may be necessary to vortex the cells for 10 to 30 seconds.
3. Calculate the volume of cell suspension containing the number of cells needed for one transfection (for each 30 mL transfection, you will need 3 x 10⁷ cells). Place the shaker flask containing cells in a 37°C incubator on an orbital
4. For each transfection sample, prepare lipid-DNA complexes by performing the following:
5. Dilute 30 μg of plasmid DNA in Opti-MEM I to a total volume of 1 mL. Mix gently.
6. Dilute 60 μl of 293fectin in Opti-MEM I to a total volume of 1 mL. Mix gently and incubate for 5 minutes at room temperature. Note: Longer incubation times may result in decreased activity.
7. After the 5 minute incubation, add the diluted DNA to the diluted 293fectin to obtain a total volume of 2 mL. Mix gently.
8. Incubate for 20–30 minutes at room temperature to allow the DNA– 293fectin complexes to form.
9. While the DNA-293fectin complexes are incubating, remove the cell suspension from the incubator and add the appropriate volume of cell suspension (see step 3) into each sterile, disposable 125 mL Erlenmeyer shaker flask. Add fresh, pre-warmed FreeStyle 293 Expression Medium up to a total volume of 28 mL for a 30 mL transfection.
10. After the DNA-293fectin complex incubation is complete, add the 2 mL of DNA-293fectin complex to each shaker flask from Step 4. To the negative control flask, add 2 mL of Opti-MEM I instead of DNA-293fectin complex. Each flask should contain a total volume of 30 mL, with a final cell density of approximately 1 x 10⁶ viable cells/mL
11. Incubate the cells in a 37°C incubator with a humidified atmosphere of 8% CO² in air on an orbital shaker rotating at 125 rpm.
12. Harvest cells or media (if recombinant protein is secreted) at approximately 48 hours post-transfection and assay for recombinant protein expression.

Optimizing protein expression

Expression levels may vary depending on the nature of your recombinant protein; therefore, you may want to perform a time course (i.e. harvest cells or media at 24, 48, 72, 96 hours post-transfection) to optimize expression of your recombinant protein.

Scaling up transfections

It is possible to perform transfection experiments in a larger (e.g. 1 liter) volume. If you wish to transfect suspension FreeStyle 293-F cells in a larger volume, scale up the volume of each reagent accordingly. The table below lists suggested conditions to use when transfecting FreeStyle 293-F cells in a 1 liter or 3.8 liter volume. The optimized conditions to use when transfecting FreeStyle 293-F cells in a 30 mL volume are listed as a reference. Note that transfection conditions may vary depending on the type of culture vessel used and the growth conditions of your cells; therefore, you may want to perform pilot studies to optimize your transfection conditions.

Note: The transfection efficiency may decrease as the volume increases if the FreeStyle 293-F cells are not growing as a single-cell suspension (i.e. if significant cell clumping is observed).

1. Graham FL, Smiley J, Russell WC, Nairn R (1977) Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol 36:59–74.
2. Harrison T, Graham F, Williams J (1977) Host-range Mutants of Adenovirus Type 5 Defective for Growth in HeLa Cells. Virology 77:319–329.
3. Miller JH (1972) Experiments in Molecular Genetics (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory).