Optimized Recombinant Production of Secreted Proteins Using Human Embryonic Kidney (HEK293) Cells Grown in Suspension

[Abstract] Recombinant proteins are an essential milestone for a plethora of different applications ranging from pharmaceutical to clinical, and mammalian cell lines are among the currently preferred systems to obtain large amounts of proteins of interest due to their high level of post-translational modification and manageable large-scale production. In this regard, human embryonic kidney 293 (HEK293) cells constitute one of the main standard lab-scale mammalian hosts for recombinant protein production since these cells are relatively easy to handle, scale-up, and transfect. Here, we present a detailed protocol for the cost-effective, reproducible, and scalable implementation of HEK293 cell cultures in suspension (suitable for commercially available HEK293 cells, HEK293-F) for high-quantity recombinant production of secreted soluble multi-domain proteins. In addition, the protocol is optimized for a Monday-to-Friday maintenance schedule, thus simplifying and streamlining the work of operators responsible for cell culture maintenance. Graphic abstract:

Here, we present the optimized protocols, currently in use in our lab, for the maintenance and transfection of HEK293-F cells, a commercially available HEK293 cell line suitable for high-density culture and transient transfection in suspension (Nettleship et al., 2015). This protocol allows the successful production of a variety of challenging recombinant protein targets, as described in our previous publications (Banushi et al., 2016;Scietti et al., 2018;Angiolini et al., 2019;Chiapparino et al., 2020). Our protocol offers a comprehensive description of all the procedures needed to establish and operate a recombinant protein production facility using these cells in an affordable but highly reproducible lab-scale setting. The proposed optimized Monday-to-Friday schedule further facilitates the lab procedures without the need for monitoring cells during the weekend. Each section describes the step-by-step procedures for cell handling, in addition to a series of technical notes to facilitate troubleshooting and improve reproducibility.  cultured in suspension inside polycarbonate square-bottomed bottles of different volumes or in multiwell polystyrene plates. When required, cells are centrifuged using a benchtop centrifuge at 1,000 × g for 10 min at room temperature. Scrupulous monitoring of the incubation settings is required since small changes will strongly affect both cell growth and transfection efficiency. CO2 supply, shaking orbit, and shaking speed are the most critical parameters. In particular, the latter is adjusted to prevent aggregation but at the same time avoid cell death caused by excess speed or accumulation of cell debris at the airmedium interface. In our setting, HEK293-F cells are maintained in New Brunswick S41i shaking incubators set at 37°C with 5% CO2. These incubators have a shaking orbit of 2.5 cm: after extensive benchmarking for identification of the best compromise between cell viability and aggregation, we noticed that a shaking speed between 120 and 130 rpm was optimal for HEK293-F cell growth in both bottles and flat-bottomed multi-well plates. The various sections describe methods that, in our opinion, may also be used for specific maintenance/transfection of other mammalian cell lines; however, this has not been tested thoroughly as with HEK293-F cells.

Materials and Reagents
A. Cell thawing and culture initiation 1. Pre-warm the Freestyle 293 medium supplemented with 0.1% penicillin-streptomycin (hereafter referred to as Medium) to 37°C in a water bath (for a 1 L bottle, this may take a couple of hours).  Table 1.

Notes:
a. In our experience, the optimal seeding concentration for HEK293-F cells is 2 × 10 6 cells/ml.   3. Grow the cells in the shaking incubator using the same settings as described in Step A4.

Every Friday, count the cells and calculate the total cell number and viability as described in
Step A5.

Note: It is strongly recommended to split the cells to concentrations no lower than 0.3 × 10 6 cells/ml, as we noticed that this may eventually lead to a drastic decrease in transfection efficiency even when the cells appear to grow normally.
6. Culture the cells in the shaking incubator using the same settings as described in Step A4.
C. Cell cryopreservation HEK293-F cells should be stored in liquid nitrogen. Prepare new aliquots of HEK293-F cells for cryopreservation as follows: 1. Seed at least 100 ml cells from a maintenance stock as described in Procedure B at a final concentration of 0.5 × 10 6 /ml and culture them as described in Step A4.

After 24 h, count the cells as indicated in
Step A5 and calculate the total cell number and viability: the cell concentration should have reached 1 × 10 6 /ml. 8 www.bio-protocol.org/e3998

Note: HEK293-F cells are extremely sensitive to the concentration used for cryopreservation.
We carried out extensive testing using multiple cell concentrations to assess the quality of the reconstituted HEK293-F cell batches after thawing. We noticed a remarkable increase in cell viability and transfection efficiency when the cells were frozen at a concentration of 10 × 10 6 cells/ml. When using higher cell concentrations in cryostocks, we did not observe changes in duplication rates but did see significantly lower transfection efficiencies. For this reason, we strongly recommend not to exceed this cell concentration.
3. Freshly prepare the freezing medium composed of Medium supplemented with 10% DMSO.
Filter the freezing medium through a 0.2 µm filter.
4. Harvest the cells by centrifugation at 1,000 × g for 10 min at room temperature, discard the supernatant, and gently resuspend the cell pellet in the freezing medium to a final concentration of 10 × 10 6 cells/ml. 5. Rapidly aliquot 1 ml suspension into the cryovials and transfer to a pre-conditioned controlledrate freezing apparatus to progressively lower the cell temperature by 1°C per min.
6. Transfer the cell aliquots to -80°C for 24 h. 7. The following day, transfer the cells to the liquid nitrogen tank for long-term storage.

D. Preparation of polyethyleneimine for cell transfection
Discovered in the early 1970s for its ability to precipitate DNA (Atkinson and Jack, 1973), polyethyleneimine (PEI) was shown 20 years later to be capable of delivering genetic material into mammalian cell lines (Boussif et al., 1995;Goula et al., 1998;Pollard et al., 1998;Ringenbach et al., 1998;Longo et al., 2013). This polymer has a high density of positive charge that allows it to condense DNA molecules. The DNA-PEI complexes have a net positive charge and can bind to the cell membrane by interacting non-specifically with negatively charged glycoproteins, proteoglycans, and sulfated proteoglycans located on the cell surface (Pham et al., 2006).
In our protocol, we use linear polyethyleneimine (PEI) due to its ease of preparation, long-term stability, low cost, high transfection efficiency, and very low cell toxicity.
A 1 mg/ml PEI solution (1× stock) can be prepared as follows: 1. Dissolve 100 mg PEI MAX in ultrapure water in a 100 ml glass beaker by gently stirring.

E. Preparation of protein hydrolysate supplement for cell transfection
Supplementation of peptones (protein hydrolysates) to the cell culture media after transfection is known to produce a significant increase in production yield, especially for secreted proteins (Pham et al., 2003;Pham et al., 2005). In our setup, we found that supplementation of Primatone RL, a meat protein enzymatic hydrolysate, boosts the recombinant protein production by an average of 2.5-fold.
A 6% Primatone RL (10× stock) solution can be prepared as follows: 1. Prepare a 100 ml aliquot of Medium in the hood.
2. Take the Medium aliquot out of the hood and dissolve 6 g Primatone RL.
Note: During Primatone RL preparation, special care should be taken to avoid contamination.

Since this compound consists of a very thin powder, it is strongly recommended not to pour the
Medium directly on it from the stock, but instead to prepare a dedicated aliquot in a separate container and dissolve the Primatone powder outside the sterile environment. 2. Seed the cells in the appropriate container at a final concentration of 0.5 × 10 6 /ml, adjusting the volume according to Table 1.
Note: It is strongly recommended to seed the cells at 0.5 × 10 6 /ml and further incubate for at least an additional 24 h prior to transient transfection. As discussed in (B), avoid two consecutive cell splits within 24 h as this will strongly impact transfection efficiency.

Incubate the cells as described in
Step A4.   To perform the control transfection with eGFP, follow the protocol steps (Steps F1-F8), then: 1. Incubate the transfected cells as described in Step A4 for 72 h.
2. Observe the cells under the microscope and collect images using both brightfield and green fluorescence.
Note: When using an automatic cell counter instead of manual counting with a Neubauer chamber, use an untransfected sample to set the threshold for GFP signal detection in the instrument. The advantage of the CellDrop™ automatic cell counter is that it does not require any consumables, such as slides, typically used by other systems.
3. Transfection efficiency is calculated by counting the green cells (blue light imaging) and the total cells (white light imaging), and applying the following formula:

Transfection efficiency % = ((total number of green cells)/(total cell number)) × 100
4. The expected efficiency should be between 65% and 80%. Lower transfection efficiencies may suggest problems with some of the reagents or indicate that the cell batch may need to be replaced.