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Mar 2019

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Optimized Recombinant Production of Secreted Proteins Using Human Embryonic Kidney (HEK293) Cells Grown in Suspension
利用悬浮培养的人胚胎肾(HEK293)细胞优化分泌蛋白的重组生产   

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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:


Schematic overview of the workflow described in this protocol


Keywords: Recombinant protein production (重组蛋白的生产), HEK293 cells (HEK293细胞), Suspension cultures (悬浮培养物), Extracellular proteins (细胞外蛋白质), Cell transfection (细胞转染), Polyethyleneimine (聚乙烯亚胺)

Background

Large amounts of high quality recombinant proteins are essential reagents for a variety of fields ranging from in vitro biochemical investigations to pharmaceutical and clinical applications (Reeves et al., 2002; Olsen et al., 2003; Swiech et al., 2012; Forneris et al., 2017; Amanat et al., 2020). Mammalian cells are particularly preferred in this context due to their ability to perform complex post-translational modifications (PTMs) of proteins, and presently large-volume cultures of mammalian cells constitute a well-established methodology to obtain milligram-to-gram quantities of recombinant proteins (Wurm, 2004; Durocher et al., 2007; Chaudhary et al., 2011; Dyson, 2016; Arena et al., 2018). Among the most used mammalian cell lines, human embryonic kidney 293 (HEK293) cells have become the standard choice for research-scale production due to their relative ease of manipulation and high transfection efficiency (Thomas and Smart, 2005; Nettleship et al., 2015; Arena et al., 2018). The HEK293 cell line was derived in 1973 from HEK cells grown in tissue culture by the introduction of a sheared adenovirus 5 DNA into wild type cells, resulting in a partially dysregulated cell cycle and subsequent immortalization of the cell line (Graham et al., 1977).


Although the literature offers many protocols to efficiently obtain high quantities of proteins of interest using mammalian cells as a recombinant host (Aricescu et al., 2006; Durocher et al., 2007; Aricescu and Owens, 2013; Portolano et al., 2014; Arena et al., 2018; L’Abbé et al., 2018), the difficulties associated with establishing scalable and reproducible setups in a cost-effective manner remain evident. 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.


Materials and Reagents

  1. Pipette tips (Sarstedt, catalog numbers: 70.1130 [10 ml], 70.760.002 [200 ml], 70.762 [1,000 ml]), to be autoclaved before use

  2. Serological pipettes (Sarstedt, catalog numbers: 86.1685.001 [25 ml], 86.1254.001 [10 ml], 86.1253.001 [5 ml], 86.1252.001 [2 ml])

  3. Polycarbonate square-bottomed bottles (Triforest Labware, catalog numbers: BCP0250 [250 ml], BCP0500 [500 ml], BCP2000 [2 L])

  4. 6-well polystyrene plates (Sarstedt, catalog number: 83.3920)

  5. Filtropur 0.2 μm sterile filters (Sarstedt, catalog number: 83.1826.00)

  6. 1.6 ml CryoPure tubes (Sartstedt, catalog number: 72.380.992)

  7. 50 ml syringe without needle, luer-lock (VWR, catalog number: TERUSS-50L1)

  8. 10 ml syringe without needle, luer-lock (VWR, catalog number: 613-0973)

  9. HEK293-F cells (Gibco, catalog number: R79007)

  10. Freestyle 293 Expression Medium (Gibco, catalog number: 12338-026)

  11. Penicillin-Streptomycin (Merck, catalog number: P0781)

  12. Trypan Blue (Merck, catalog number: T8154; starting concentration: 0.4%, dilute 1:1 with PBS to reach a final working concentration of 0.2%)

  13. Dimethylsulfoxide (Sigma-Aldrich, catalog number: D8414)

  14. PEI MAX polyethyleneimine 40K powder (Polysciences, catalog number: 24765-1)

  15. Primatone RL (Sigma Aldrich, catalog number: P4963)

  16. OptiMEM I (1×) + Glutamax-1 (Gibco, catalog number: 51985-034)

  17. pTT3-eGFP (Addgene, catalog number: 154346)

Equipment

  1. Microvolume pipettes (Gilson, models: Pipetman P1000, P200, P100, P20)

  2. Automated pipettor for serological pipettes (VWR, model: Pipetboy pro Integra)

  3. BSL-2 laminar flow hood (Thermo Fisher, model: MSC Advantage)

  4. Large-volume benchtop centrifuge with swing-out rotor (Beckman Coulter, model: Allegra X-15R with SX4750 rotor)

  5. CO2 incubator with embedded shaker (Eppendorf, model: New Brunswick S41i, catalog number: S41I230011)

  6. –80°C ultrafreezer (Thermo Fisher, model: TDE 300FV)

  7. CoolCell™ controlled rate freezing apparatus (VWR, catalog number: 479-0492)

  8. Water bath (Exacta Optech, model: WNB22)

  9. Neubauer chamber (Merck, catalog number: BR717820)

  10. Automated cell counter (DeNovix Inc., model: Celldrop)

  11. Liquid nitrogen storage tank (Taylor-Wharton, model: VHC-35)

Procedure

All procedures described herein are performed with HEK293-F cells, which are maintained in Freestyle 293 Expression Medium, i.e., a chemically defined, protein-free medium optimized for growth and recombinant protein production in HEK293-F cells kept in suspension culture. Maintenance medium is supplemented with penicillin-streptomycin at a final concentration of 0.1% (v/v). All cell handling procedures are carried out under sterile conditions in a standard BSL-2 laminar flow hood. Cells are cultured in suspension inside polycarbonate square-bottomed bottles of different volumes or in multi-well 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 air–medium 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.


  1. 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).

      Note: Although the addition of antibiotic to the Medium is generally not recommended, penicillin-streptomycin concentrations up to 10 ml/L (equal to 1%) are compatible with cell cultures and, based on several tests carried out using different antibiotic concentrations (0 ml/L–10 ml/L), do not impact cell growth or transfection efficiency. According to these results, we recommend supplementing the cell cultures with 1 ml/L (0.1%) penicillin-streptomycin to minimize the risk of contamination.

    2. Rapidly thaw an aliquot of frozen HEK293-F cells (each aliquot should contain approximately 10 × 106 cells/ml) in the 37°C water bath. This operation should take less than 1 min.

    3. Directly dispense the cells into a 250 ml bottle containing 20 ml Medium to reach a final concentration of 0.5 × 106 cells/ml.

      Note: HEK293-F cells are extremely sensitive and prone to contamination during the thawing step; therefore, the greatest care must be taken during this step. It is strongly advised to aspirate the entire cell volume from the frozen vial in a single pipetting operation and dispense the cells directly into the pre-warmed Medium in the culture bottle. Any further manipulation can increase the risk of contamination.

    4. Incubate the cells at 37°C, 5% CO2 with 120 rpm shaking, taking care to loosen the bottle cap to allow gas exchange.

    5. Check the cell growth rate daily and assess the viability using the Trypan Blue assay as follows:

      1. Harvest approximately 1 ml cells from the bottle.

      2. Dilute 15 µl cells 1:1 with 0.2% Trypan Blue.

      3. Count live cells using a Neubauer chamber (or an automatic cell counter, Figure 1) and assess cell viability using the following formula:

        Mean number of cells counted in 4 squares × 2 (dilution factor) × 104



      Figure 1. Example of automatic cell counting. Comparison of cells after treatment with Trypan Blue counted using the CellDrop™ automatic cell counter (Denovix Inc.). The brightfield image (left) was processed by the DenovixEasyApps software for identification of live cells (green circles, as highlighted by the green arrow, total count in the image = 3.17 × 106/ml) and dead cells (red circles, as highlighted by the red arrow, total count in the image = 5.75 × 105/ml). Scale bar, 100 µm.

      Note: Under good culture conditions, suspension-cultured HEK293-F cells display an average doubling time of 24 h; however, 24 h after thawing, the cells typically do not show any signs of duplication. Starting from 48 h post-thawing, the cells should duplicate every 24 h, reaching a final concentration of approximately 2 × 106 cells/ml on day 3. At this timepoint, it is recommended to replace the Medium by centrifuging the cells at 1,000 × g for 10 min at room temperature and resuspending in fresh Medium.


    6. 72 h after thawing, the cells should have reached a final concentration of approximately 2 × 106 cells/ml: harvest the cells by centrifugation at 1,000 × g for 10 min at room temperature.

    7. Discard the supernatant and gently resuspend the cell pellet in fresh, pre-warmed Medium using a volume sufficient to reach a final concentration of 0.5 × 106 cells/ml.

    8. Culture the cells in the shaking incubator using the same settings as stated in Step A4, following the schedule for cell maintenance described in Procedure B.

      Note: An effective weekly schedule suggestion includes thawing cells on a Monday (day 0), as this will allow the operator to monitor cell growth (as well as possible contamination) daily throughout the first week. Day 3 (72 h after thawing) should therefore correspond to a Thursday, on which cells should be split to a final concentration of 0.5 × 106 cells/ml. Splitting again the following day (Friday) will guarantee exponential growth and optimal cell density over the weekend without intervention from the operator.


  2. Cell maintenance

    Subculture the cells three times a week as follows:

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

      Note: Avoid daily cell splitting: in our experience, this will drastically slow down cell growth and eventually result in a significant reduction in transfection efficiency.

    2. Split cells into a new bottle. Seed cells at a final concentration of 0.5 × 106 cells/ml in the required volume of Medium following the guidelines suggested in Table 1.

      Notes:

      1. In our experience, the optimal seeding concentration for HEK293-F cells is 2 × 106 cells/ml. It is detrimental to allow the cells to overgrow since they consume all the nutrients in the Medium and dead cell debris accumulates, posing a problem for subsequent transfection procedures. Cell debris can be easily visualized as the formation of a sticky ring around the walls of the culture bottle (Figure 2).

      2. A fundamental parameter to consider when performing cell splitting is the air/Medium ratio used for cell culture. HEK293-F cells are extremely sensitive to this parameter: we recommend filling the culture bottles with Medium to no more than a quarter of their total volume capacity to ensure proper oxygen exchange and prevent excess aggregation. At the same time, too low a culture volume will cause cell death due to increased mechanical stress induced by shaking, which may be solved by reducing the shaking speed but may interfere with the need to standardize the incubator settings to enable multiple cultures at the same time. The suggested optimal culture volumes (Table 1) were selected after extensive testing based on the assessment of cell viability and accumulation of cell aggregates and debris using bottles of several sizes and shapes, in addition to multi-well plates, with the incubator settings as described in Step A4.

      3. When splitting cells, it is not necessary to perform washes with PBS or other buffers to remove cell debris. Such operations are typically avoided in this protocol to limit the risk of contamination and minimize stressful operations related to cell handling.


        Table 1. Recommended cell culture volumes and associated containers. Overview of the minimal, maximal, and optimal cell culture volumes in different containers used for cell culture.

        Container type Minimal volume Maximal volume Optimal volume
        250 ml bottle 10 ml 50 ml 25 ml
        500 ml bottle 50 ml 100 ml 80 ml
        2 L bottle 100 ml 500 ml 250 ml
        6-well plate 2 ml 3 ml 2 ml



      Figure 2. Sticky ring of dead cells. The formation of a sticky ring on the lower part of the bottle constitutes the typical presentation of cell debris accumulation.


    3. Grow the cells in the shaking incubator using the same settings as described in Step A4.

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

      Note: Although HEK293-F are immortalized cells, we observed a sort of senescence, with a drastic reduction in the transfection efficiency of the old batches as compared with the newer ones. This may negatively impact the yield of recombinant protein production, in particular for recombinant targets with production yields below 1 mg/L using “fresh” HEK293-F cells. For this reason, we encourage the initiation of a new cell culture batch by thawing a fresh aliquot of HEK293-F cells at passage 25–30 (i.e., every 11 weeks). This will ensure that the cells subject to transient transfection will never be older than passage 30–32.

    5. Split cells into a new bottle, seeding them at a final concentration of 0.3 × 106 cells/ml in the required volume of Medium.

      Note: It is strongly recommended to split the cells to concentrations no lower than 0.3 × 106 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.


  3. 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 × 106/ml and culture them as described in Step A4.

    2. 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 × 106/ml.

      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 × 106 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 × 106 cells/ml.

    5. Rapidly aliquot 1 ml suspension into the cryovials and transfer to a pre-conditioned controlled-rate 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.


  4. 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.

    2. Carefully add 1 M NaOH dropwise to the PEI solution until the final pH is between 6.90 and 7.10.

      Note: Immediately after adding the water, the pH of the PEI MAX solution will be around 2. Due to the lack of a buffering component in the solution, titration with 1 M NaOH should be carried out very carefully to reach neutral conditions.

    3. Transfer the PEI solution into a graduated cylinder and adjust the final volume to 100 ml to reach a final concentration of 1 mg/ml.

    4. Sterile-filter the solution using a 0.2 µm filter.

    5. Aliquot the solution into 500 µl stocks and store at 4°C.

      Note: PEI MAX solutions are stable for roughly 6 months. We recommend checking the transfection efficiencies periodically and preparing fresh PEI MAX solution when required.


  5. 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.

    3. Vigorously vortex the solution to completely dissolve the meat hydrolysate.

    4. Place the Primatone solution in the hood, sterile-filter using a 0.2 µm filter, and store at 4°C.

      Note: Use the solution within one month.

    5. The final working concentration (0.6%) should be added to the cell cultures at least 4 h after transfection.


  6. Cell transfection

    The following protocol was standardized for basic transient cell transfection for the recombinant production of secreted extracellular protein targets:

    1. 24 h prior to cell transfection, count the cells 1:1 as indicated in Step A5 and calculate the total cell number and viability.

    2.  Seed the cells in the appropriate container at a final concentration of 0.5 × 106/ml, adjusting the volume according to Table 1.

      Note: It is strongly recommended to seed the cells at 0.5 × 106/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.

    3.  Incubate the cells as described in Step A4.

    4.  The following day, count the cells as indicated in Step A5, and calculate the total cell number and viability (cells should have doubled and their final concentration should have reached 1 × 106/ml).

    5.  Prepare the transfection mix as follows:

      1. Use 1 µg pure plasmid DNA for every 1 × 106 cells.

      2. Use a 1:5 DNA:PEI MAX ratio.

      3. Calculate the final transfection volume (in µl) as follows:


        Transfection volume = total µg DNA × 25 µl/µg


        Note: We performed several experiments to elucidate the optimal volume for cell transfection. We established that 25 µl per µg DNA represents a good ratio for an efficient transfection mix.

      4. Prepare two different sterile containers, each containing half the calculated transfection volume of OptiMEM I (1×) + Glutamax-1 medium.

      5. Dispense the plasmid DNA into one container and PEI MAX into the other.

      6. Transfer the DNA–OptiMEM solution dropwise into the PEI–OptiMEM solution to obtain the transfection mix.

    6. Vortex the transfection mix for 5 s, three times.

    7. Incubate the transfection mix at room temperature for 10 min.

      Note: The incubation time of the DNA–PEI mix prior to transient transfection may also heavily impact protein production yield. We recommend testing different incubation times (5, 10, and 15 min) in a small-scale expression trial of the protein target of interest.

    8. Add the transfection mix dropwise to the cell suspension while gently shaking the culture bottle in a circular motion.

    9. 4 h post-transfection, add Primatone RL, prepared as previously described, at a final concentration of 0.6%.

      Note: Ensure that the transfection procedure is performed in the morning: this will allow the addition of Primatone RL (Step F9) during working hours.

    10. Incubate the transfected cells as described in Step A4 for the appropriate number of days in accordance with the recombinant target protein of interest.

    11. Periodically check cell viability (if cell viability drops below 70%, this may indicate that the recombinant protein is toxic and may therefore require early harvesting).

      Notes:

      1. Proper cell harvesting time is of fundamental importance when producing recombinant proteins in HEK293-F cells. Our rule-of-thumb based on extensive empirical testing is that for secreted recombinant protein targets, the optimal harvesting time for maximum protein yield is 6–7 days post-transfection. Such an extended culture time is not recommended in the case of intracellular or transmembrane proteins, which are typically harvested between 48 h and 72 h post-transfection. Nevertheless, continuous monitoring of cell viability is mandatory, particularly for new targets, to prevent the accumulation of cell debris in the Medium caused by unexpected toxicity associated with recombinant protein production. We noticed that, depending on the plasmid used for transfection, HEK293-F cells can be particularly resilient to the presence of the plasmid and may still continue to grow after the transfection procedure.

      2. A negative control experiment for the comparative assessment of cell viability can be performed by transfecting cells with a plasmid lacking a mammalian cell promoter, e.g., a “dummy” DNA cloning vector.

    12. 6 days after transfection, count the cells 1:1 as indicated in Step A5 and calculate the total cell number and viability.

      Note: For an optimized Monday-to-Friday schedule, organize the transfection days for Tuesday and Thursday: this will allow both splitting the cells for transfection (Step F1) and harvesting at 6–7 days post-transfection (Step F12) on weekdays.

    13. Transfer the cell culture to conical centrifuge tubes and harvest the culture media by centrifugation at 1,000 × g for 15 min at 10°C.

    14. Keep the supernatant and proceed according to your protein purification protocol.


  7. Evaluation of transfection efficiency using eGFP

    As discussed in (B), HEK293-F cells become senescent after a number of passages; therefore, it is essential to periodically monitor the transfection performance. We recommend checking transfection efficiency with a positive control at least twice a month. Mock transfections using expression plasmids containing the gene for enhanced green fluorescent protein (eGFP, e.g., pTT3-eGFP) can be used for this purpose. Measurement of the transfection efficiency evaluates the number of cells that have successfully incorporated the recombinant DNA and are therefore capable of producing the recombinant protein(s) of interest. The ratio between the number of cells displaying green fluorescence and the total number of cells yields the transfection efficiency value (Figure 3).



    Figure 3. Evaluation of transfection efficiency. Comparison between untreated cells (left) and those transfected with either GFP (center) or dummy DNA (right). For each sample, the comparative evaluation was performed using the CellDrop™ automatic cell counter (Denovix Inc.). The brightfield images (top) were subjected to GFP fluorescence counting (excitation = 470 nm ± 20 nm; emission = 525 nm ± 25 nm) using the DenovixEasyApps software. When unexpected objects different from cells are present in the counting chamber (e.g., air bubbles, as indicated by the black arrow in the right panel), the instrument automatically excludes them from the final cell count. Scale bar, 100 µm.


    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.


Acknowledgments

We thank Dr. F. Magnani (University of Pavia) and Dr. S. Wulhfard for useful discussions and suggestions, in addition to all members of the Armenise-Harvard laboratory of Structural Biology for continuous support, testing, and benchmarking of protein production conditions.

  This project received funding from Fondazione Giovanni Armenise-Harvard (grant id. CDA2013 to FF), the Italian Association for Cancer Research (AIRC, “My First AIRC Grant” id. 20075 to FF), Fondazione Cariplo (grant ids. 2014-0881 and 2015-0768 to FF), the Mizutani Foundation for Glycoscience (grant id. 200039), the Italian Ministry of Education, University and Research (MIUR) (Rita Levi-Montalcini Award 2012 to FF), and Dipartimenti di Eccellenza Program (grant id. 2018-2022, to the Dept. of Biology and Biotechnology "L. Spallanzani", University of Pavia). AC2 was the recipient of a Marie Curie individual fellowship from the European Union’s Horizon 2020 research and innovation program (grant agreement COTETHERS - n. 745934). None of the funding sources played a role in study design, collection, analysis, and interpretation of data, writing of the report, or the decision to submit this article for publication. Author Contributions: FF supervised the set-up and development of the cell culture infrastructure. MC established the initial cell culture setup, with support from MP and AC1. SF and AC2 optimized the culture setup and standardized the culture and transfection procedures. SF carried out the comparative analysis presented in this work and elaborated on the proposed Monday-to-Friday schedule. SF and FF organized the protocol sections and wrote the paper, with contributions from all authors.

Competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper.

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简介


[摘要]重组蛋白是一个对于不同的应用范围从药物到临床过多必不可少里程碑,和哺乳动物细胞系是目前优选的系统,以获得大量的目标蛋白质中由于它们的高电平后-翻译后修饰一个nd可管理的大规模生产。在这方面,人胚胎肾293(HEK293)细胞构成的一个主标实验室规模的哺乳动物宿主小号用于重组蛋白质生产,因为这些细胞是相对容易处理,按比例放大,并且转染。在这里,我们提出了一个详细的协议用于具有成本效益的,可再现的,在悬浮液和可扩展执行的HEK293细胞培养物(适于市售的HEK293细胞,HEK293-F),用于高量重组生产分泌的可溶性多域蛋白。此外,该协议为星期一到星期五的维护计划进行了优化,从而简化和STREA毫升进不去负责细胞培养维护人员的工作。

图形摘要:

该协议中描述的工作流程的示意图


[背景]高品质的重组蛋白的大的量用于所必需的多种试剂的字段范围从体外生化研究药物和临床应用(Reeves的等人,2002;奥尔森等人,2003; Swiech等人,2012; Forneris等人,2017; Amanat等人,2020)。哺乳动物细胞是特别优选的在这方面是由于它们的能力进行复杂的后-翻译后修饰的蛋白质和哺乳动物细胞的当前大体积培养物构成一个完善的方法来获得毫克-到-的重组克量蛋白质(Wurm,2004; Durocher等,2007; Chaudhary等,2011; Dyson,2016; Arena等,2018)。其中最常用的哺乳动物细胞系,人胚肾293(HEK293)细胞已成为科研规模生产标准的选择,因为它们相对容易操作和较高的转染效率分析的Ÿ (托马斯和Smart,2005; Nettleship等, 2015; Arena等人,2018)。第Ë HEK293细胞系在1973年来源于在组织培养中生长的HEK细胞通过引入剪切的腺病毒5的DNA中,以野生型细胞,导致在部分d YS调节细胞周期和细胞系的后续永生化(格雷厄姆等等人,1977)。

虽然文献提供了许多协议,以有效地获得高quantit IES使用哺乳动物细胞作为目的蛋白质的一个重组宿主(Aricescu等人,2006;迪罗谢等人,2007; Aricescu和欧文斯,2013;波尔托拉诺。等人,2014 ;竞技场等人,2018; L'阿贝。等人,2018) ,相关的困难与以具有成本效益的方式建立可扩展的和可重复的设置仍然明显。这里,我们提出的优化的协议,目前在我们的实验室使用,对于HEK293-F细胞的维持和转染,市售适合于高密度文化性可用的HEK293细胞系Ë和瞬时转染在悬浮液(Nettleship等人, 2015)。该协议允许小号的成功生产的各种挑战重组蛋白质靶的,如在我们以前的出版物中描述(Banushi等人,2016; Scietti等人,2018;安焦利尼。等人,2019; Chiapparino 。等人,2020 )。我们的协议提供了所有的全面描述的建立,需要的程序运行在一个负担得起的,但高度可重复的实验室规模的环境中使用这些细胞的重组蛋白生产设施。所提出的优化周一至周五的时间表进一步促进了实验室程序,而不需要期间监测细胞的周末。每个部分均介绍了细胞处理的分步过程,此外还提供了一系列技术说明,以帮助进行故障排除并提高可重复性。

关键字:重组蛋白的生产, HEK293细胞, 悬浮培养物, 细胞外蛋白质, 细胞转染, 聚乙烯亚胺



材料和试剂


移液管头(Sarstedt的,目录号š :70.1130 [ 10毫升] ,70.760.002 [ 200米1] ,70.762 [ 1 ,000米1] ),在使用前进行高压灭菌
血清移液器(Sarstedt ,目录号s :86.1685.001 [ 25 m l] ,86.1254.001 [ 10 m l] ,86.1253.001 [ 5 m l] ,86.1252.001 [ 2 m l] )
聚碳酸酯方形底部ED瓶(Triforest医学实验,目录号小号:BCP0250 [ 250 ml的] ,BCP0500 [ 500 ml的] ,BCP2000 [ 2大号] )
6孔聚苯乙烯板(Sarstedt ,目录号:83.3920)
菲罗普尔0.2μ中号无菌过滤器(Sarstedt的,目录号:83.1826.00)
1.6毫升CryoPure吨ubes (Sartstedt ,目录号:72.380.992)
50毫升不带针的注射器,鲁尔锁(VWR,目录号:TERUSS-50L1)
10 ml不带针的注射器,鲁尔锁(VWR,目录号:613-0973)
HEK293-F细胞(Gibco ,目录号:R79007)
Freestyle 293表达培养基(Gibco ,目录号:12338-026)
青霉素-链霉素(Merck,目录号:P0781)
台盼蓝(Merck,目录号:T8154;起始浓度:0.4%,用PBS稀释1:1以达到0.2%的最终工作浓度)
二甲基亚砜(Sigma-Aldrich,目录号:D8414)
PEI MAX聚乙烯亚胺40K粉末(Polysciences ,目录号:24765-1)
Primatone RL(西格玛奥德里奇(Sigma Aldrich),目录号:P4963)
OptiMEM I(1 × )+ Glutamax-1(Gibco ,目录号:51985-034)
pTT3-eGFP(Addgene ,目录号154346)


设备


微量移液器(Gilson,型号:Pipetman P1000,P200,P100,P20)
血清移液器的自动移液器(VWR,型号:Pipetboy pro Integra)
BSL-2层流罩(Thermo Fisher,型号:MSC Advantage)
大-体积台式离心机用水平转子(Beckman Coulter公司,型号:Allegra的X-15R与SX4750转子)
带有嵌入式振荡器的CO 2培养箱(Eppendorf,型号:New Brunswick S41i,目录号:S41I230011)
- 80℃ ultrafreezer (热费舍尔,型号:TDE 300FV)
冷却Ç ELL ™受控速率冷冻装置(VWR,目录号:479-0492)
水浴(Exacta Optech ,型号:WNB22)
Neubauer c hamber(默克,目录号:BR717820)
自动细胞计数器(DeNovix公司。,型号:Celldrop )
液氮储罐(泰勒-沃顿,型号:VHC-35)


程序


本文所述的所有程序均用HEK293-F细胞进行,该细胞保存在Freestyle 293表达培养基中,即一种化学定义的无蛋白培养基,该培养基针对悬浮培养的HEK293-F细胞中的生长和重组蛋白生产进行了优化。维持培养基补充有p enicillin-小号以0.1%的终浓度treptomycin(V / V)。所有细胞处理程序均在无菌条件下在标准BSL-2层流通风橱中进行。细胞在内部聚碳酸酯方形底部悬浮培养编不同体积的瓶或在多井聚苯乙烯板。需要时,在室温下使用台式离心机以1,000 × g离心细胞10分钟。由于细微的变化会强烈影响细胞生长和转染效率,因此需要对孵育设置进行严格的监控。CO 2供给,摇动轨道,和摇动速度是最关键的参数。特别是,对后者进行了调整以防止聚集,但同时避免了由于过快的速度或空气碎片在空气-介质界面上积聚而导致的细胞死亡。在我们的环境中,将HEK293-F细胞保存在温度为5%CO 2的37°C的New Brunswick S41i摇动培养箱中。这些孵化器具有SHAK为2.5cm ING轨道:细胞生存力和聚集之间的最佳折衷的识别广泛的基准测试后,我们注意到一个120和130的转速W之间振荡速度作为最佳的HEK293-F细胞的生长在两个瓶和平底多孔板。各部分描述的方法该,在我们看来,也可用于特定的维护/转染的其它哺乳动物细胞系; ħ H但是,这没有被彻底的测试与HEK293-F细胞中。


细胞解冻和培养启动
预温热补充有0.1%的293自由式介质p enicillin-小号treptomycin(以下简称为培养基)到在水浴中37℃(对于1升的瓶子,这可能需要几个小时)。
注意:尽管抗生素添加一般不推荐到培养基中,p enicillin-小号treptomycin浓度高达10毫升/ L(等于1%)与细胞培养物和相容的,基于多次测试进行了使用不同的抗生素浓度(0 ml / L – 10 ml / L),不影响细胞生长或转染效率。根据这些结果,我们建议补充细胞培养物用1ml / L(0.1%)p enicillin-小号treptomycin以最小化污染的风险。           

在37°C水浴中迅速解冻冰冻的HEK293-F细胞等分试样(每个等分试样应含有约10 × 10 6个细胞/ ml)。此操作应少于1分钟。
将细胞直接分配到装有20 ml培养基的250 ml瓶中,使最终浓度为0.5 × 10 6个细胞/ ml。
注意:HEK293-F细胞非常敏感,在解冻步骤中容易被污染;因此,在大EST必须谨慎考虑在此步骤。强烈建议吸出整个细胞体积从冷冻小瓶在一个单一的吸液操作,并直接分配细胞进入预热培养基在培养瓶中。任何进一步的操作ç的增加污染的风险。


在37°C,5%CO 2和120 rpm摇动下孵育细胞,注意松开瓶盖以进行气体交换。
检查细胞每日生长速率和驴小号生存能力使用台盼乙略测定如下:
从瓶子里收获大约1毫升的细胞。
用0.2%锥虫蓝1:1稀释15 µl细胞。
计数使用活细胞纽鲍尔室(或自动细胞凑NTER,图1)的d驴小号细胞活力使用了以下公式:


平均数的数细胞在4个方格×2(稀释因子)计数×10 4




图1.自动细胞计数的例子。用处理后的细胞的比较Ť rypan乙使用略计数细胞d ROP ™自动细胞计数器(Denovix Inc.)中。的明场图像(左)被处理通过所述DenovixEasyApps软件用于活细胞的识别(绿色圆圈,由绿色箭头突出显示,总计数我n中的图像= 3.17 × 10 6 / ml)和死细胞(红色圆圈,如红色箭头突出显示,总计数我n中的图像= 5.75 × 10 5 /毫升)。比例尺,100 µm。


注意:在良好文化性ë条件下,悬浮培养HEK293-F细胞显示24小时的平均倍增时间; ^ h H但是,24小时后解冻时,细胞通常不表现出任何迹象小号的重复。从融化后48小时开始,细胞应每24小时复制一次,并在第3天达到终浓度约2 × 10 6细胞/ ml 。在这个时间点,建议通过以1,000 × g下在室温下10分钟并重新悬浮荷兰国际集团在新鲜培养基中。


解冻后72小时,细胞应该达到约2的终浓度× 10 6个细胞/ ml的:通过离心收获细胞以1000 ×克为10分钟在室温下。
弃去上清液,然后将细胞沉淀轻轻悬浮在新鲜的预热培养基中,其体积应足以使最终浓度达到0.5 × 10 6细胞/ ml。
培养使用相同的设置作为振荡培养箱将细胞表示在步骤A4中,以下的时间表细胞维持在步骤B中描述
注意:有效的每周计划建议包括在周一(第0天)解冻细胞,因为这将使操作员可以在整个第一周内每天监控细胞的生长(以及可能的污染)。因此,第3天(解冻后72小时)应对应于星期四,在该星期四,应将细胞分裂至终浓度为0.5 × 10 6细胞/ ml。第二天(星期五)再次分裂,将保证周末有指数增长和最佳细胞密度,而无需操作员干预。


细胞米aintenance
每周将细胞传代培养3次,如下所示:


每蒙德AY和星期三,计数细胞并计算所述中所述的总细胞数和存活力步骤A5 。
注意:避免每天小区分裂:根据我们的经验,这将大大减缓细胞的生长,并最终导致作为ignificant减少的转染效率。           

将细胞分裂成一个新的瓶子。种子细胞的终浓度为0.5 × 10 6个细胞/ ml,该培养基的体积应符合表1中所建议的要求。
笔记:


根据我们的经验,HEK293-F细胞的最佳接种浓度为2 × 10 6细胞/ ml。这是不利的允许细胞以长满因为他们消费Ë所有营养素中的中型和死细胞碎片累积小号,POS荷兰国际集团为后续的转染程序的问题。细胞碎片Ç一个被容易地可视化作为围绕培养瓶的壁(图2)粘性环的形成。
一个基本参数时需要考虑执行小区分裂是用于细胞文化性空气/培养基比ë 。HEK293-F细胞对这个参数非常敏感:填写推荐荷兰国际集团与培养瓶中,以不超过四分之一的的总体积容量,以确保适当的氧气交换和防止过度聚集。与此同时,太低一个文化性ë体积将引起细胞死亡由于通过摇动引起的增加的机械应力,这可以通过减小摇动速度来解决,但可以与需要干扰到standardiz Ë的培养箱设置,以使多个培养物在同时。所建议的最佳文化性ë体积(表1)的基础上广泛的试验之后选定的细胞生存力和细胞聚集体和多种尺寸和形状的使用碎片瓶的积累的评估,除了对多孔板,与所述培养箱的设置如在步骤A4中描述。
分裂细胞时,无需用PBS或其他缓冲液进行洗涤以去除细胞碎片。在该协议中通常避免此类操作,以限制污染的风险并使与细胞处理相关的压力操作最小化。


表1 。推荐的细胞培养体积和相关容器。Ó的概述的最小的,最大的,在用于细胞培养不同的容器和最佳的细胞培养物体积。


图2.死细胞的粘环。在瓶子的下部的粘性环的形成构成的典型呈现的细胞碎片积累。


成长使用相同的设置作为振荡培养箱将细胞描述在步骤A4。
每个星期五,计数细胞并计算的如步骤A5中描述的总细胞数和存活力。
注:虽然HEK293-F是永生化细胞,我们观察到一种衰老的,在转染效率急剧降低的旧批次为相比与较新的。这可能会对重组蛋白的产量产生负面影响,特别是对于使用“新鲜” HEK293-F细胞生产的产量低于1 mg / L的重组靶标。因此,我们鼓励通过在第25至30代(即每11周)解冻新鲜的HEK293-F细胞等分试样来开始新的细胞培养批次。这将确保细胞受到瞬时转染决不会比旧的通道30 - 32。


将细胞分成一个新瓶,以所需体积的培养基以0.3 × 10 6个细胞/ ml的最终浓度播种。
注意:强烈建议对细胞分裂,以浓度不低于0.3 × 10 6个细胞/ ml,因为我们注意到,这可能最终会导致急剧d ecrease在转染效率甚至当该细胞出现于GRO瓦特正常LY。           

培养使用相同的设置作为细胞在振荡培养箱describ编辑在步骤A4。


              细胞冷冻保存
HEK293-F细胞应储存在液氮中。如下准备用于冷冻保存的HEK293-F细胞的新等分试样:


按步骤B中所述从维持原液中播种至少100 ml细胞,终浓度为0.5 × 10 6 / ml,并按照步骤A4中所述进行培养。
24小时后,计数如步骤A5所指示,将细胞计算的总细胞数目和生存力:细胞浓度应该已经达到1 × 10 6 / ml的。
注意:HEK293-F细胞对冷冻保存所用浓度极为敏感。我们使用多种细胞浓度进行了广泛的测试,以评估融化后重构的HEK293-F细胞批次的质量。当细胞以10 × 10 6细胞/ ml的浓度冷冻时,我们发现细胞活力和转染效率显着提高。当在冷冻库中使用较高的细胞浓度时,我们没有观察到重复率的变化,但是确实看到了明显较低的转染效率。对于日我的理由为何,我们强烈建议不要以超过日我的蜂窝浓度。


新鲜制备由添加了10%DMSO的培养基组成的冷冻培养基。通过0.2 µm过滤器过滤冷冻介质。
收获细胞,通过离心在1 ,000 ×克为10英里Ñ在室温下,弃去上清液,并在冷冻介质中的细胞沉淀轻轻悬浮于10的最终浓度× 10 6个细胞/ ml 。
迅速等分试样1 ml的悬浮液中的冷冻管并转移到经调节的预控-速率冷冻装置以逐渐降低电池温度由每分钟1℃。
将细胞等分试样转移至– 80°C 24小时。
在下面的当天,将细胞转移到液氮罐长期-term存储。
           

用于细胞转染的聚乙烯亚胺的制备
在70年代初期发现了它的能力沉淀DNA (Atkinson和杰克,1973),聚乙烯亚胺(PEI)中的溶液中所示,20年后是帽能够的递送荷兰国际集团遗传物质引入哺乳动物细胞系(Boussif等人,1995;够辣等人,1998; Pollard等人,1998; Ringenbach等人,1998; Longo等人,2013)。该聚合物具有高密度的正电荷,可使其凝结DNA分子。DNA – PEI复合物具有净正电荷,可以通过与细胞表面带有负电荷的糖蛋白,蛋白聚糖和硫酸化蛋白聚糖非特异性相互作用而与细胞膜结合(Pham等,2006)。


在我们的协议中,我们使用线性聚乙烯亚胺(PEI)由于其易于制备,长期稳定性,低成本,高转染效率,和非常低的细胞毒性。


可以如下制备1 mg / ml的PEI溶液(1 ×储备液):


在由gentl 100ml玻璃烧杯中,溶解100毫克PEI MAX在超纯水中ÿ搅拌。
小心地向PEI溶液中滴加1 M NaOH ,直到最终pH在6.90和7.10之间。
注:加入后,立即在水中,PEI MAX溶液的pH值将围绕2.由于在缺乏在溶液中的缓冲组分,用1M滴定氢氧化钠应该非常小心地进行,以达到中性条件。


将PEI溶液转移到带刻度的量筒中,并将最终体积调整为100 ml,以达到1 mg / ml的最终浓度。
使用0.2 µm过滤器无菌过滤溶液。
将溶液分装到500 µl储备液中,并储存在4°C下。
注意:PEI MAX解决方案稳定约6个月。我们建议您检查转染效率周期性和prepar荷兰国际集团在需要时新鲜PEI MAX的解决方案。           



用于细胞转染的蛋白水解物补充剂的制备
小号补编第通货膨胀蛋白胨(蛋白质水解物)向细胞培养物的培养基转染已知产生在成品率显著增加之后,尤其是对于分泌的蛋白质(范。等人,2003 ;范等人,2005)。在我们的设置中,我们发现,补充的通货膨胀Primatone RL,肉蛋白质酶解液,助力重组蛋白生产平均的2.5倍。


可以如下制备6%Primatone RL(10 ×原液)的溶液:


在通风橱中准备100毫升等分的培养基。
从烟囱中取出中等份量,溶解6 g Primatone RL 。
注意:在Primatone RL制备过程中,应特别注意避免污染。由于该化合物利弊派非常薄的粉末,强烈建议不要直接在它从库存倾的介质,但代替在一个单独的容器中准备专用等分试样并溶解Primatone无菌环境以外的粉末。


剧烈涡旋溶液以完全溶解肉水解物。
放置的Primatone溶液在发动机罩,用0.2μm过滤器无菌过滤,并储存在4℃ 。
注意:在一个月内使用该解决方案。


转染后至少4小时,应将最终工作浓度(0.6%)加入细胞培养物中。


细胞吨ransfection
以下协议已标准化用于基本瞬时细胞转染,以重组生产分泌的细胞外蛋白靶标:


24 ħ之前细胞转染,计数与c厄尔1:如步骤A5所指示,并计算1的总细胞数目和生存力。
              将细胞接种到合适的容器中,终浓度为0.5 × 10 6 / ml,并根据表1调整体积。
注意:强烈建议种子的细胞以0.5 × 10 6 / ml,并用于至少进一步孵育的另外24小时之前,瞬时转染。如(B)中所述,避免在24小时内连续分裂两个细胞,因为这会严重影响转染效率。


              按照步骤A4所述孵育细胞。
              在下列天,计数如步骤A5所指示的细胞,并计算所述总细胞数目和活力(细胞应了一倍和它们的最终浓度应达到1 × 10 6 / ml)中。
              如下准备转染混合物:
              使用1微克纯质粒DNA为每1 × 10 6细胞。
使用一个1:5的DNA :PEI MAX比。
              计算最终转染体积(以µl为单位),如下所示:


转染体积= TOT人微克DNA×25微升/微克


注意:我们进行了几次实验,以阐明细胞转染的最佳体积。我们确定每微克DNA 25微升代表有效转染混合物的良好比例。


              准备两个不同的无菌容器,每个容器包含计算出的OptiMEM I(1 × )+ Glutamax-1培养基转染体积的一半。
              分配在质粒DNA到一个容器和PEI MAX在到其他。
              将DNA – OptiMEM溶液逐滴转移至PEI – OptiMEM溶液中,以获得转染混合物。
              将转染混合物涡旋3秒钟,持续5 s 。
              在室温下孵育转染混合物10分钟。
注意:在瞬时转染之前,DNA – PEI混合物的孵育时间也可能会严重影响蛋白质的产量。我们建议测试不同的温育时间(5,10 ,和15分钟)在感兴趣的蛋白质靶的小规模的表达试验。


              添加转染混合物滴明智细胞悬浮液中,同时轻轻晃动培养瓶在圆形莫重刑。
              转染后4小时,添加如前所述制备的Primatone RL,终浓度为0.6%。
注:确保将转染程序进行早晨:这将允许在阿迪和灰Primatone RL在工作时间(步骤F9)。


              孵育转染的细胞在S作为描述TEP FO A4 - [R的适当根据目的重组靶蛋白的天数。
              定期检查细胞生存能力(如果细胞生存能力降至70%以下,则表明重组蛋白具有毒性,因此可能需要尽早收获)。
注意小号:


在HEK293-F细胞中产生重组蛋白时,正确的细胞收获时间至关重要。我们基于广泛的经验测试的经验法则是,对于分泌的重组蛋白靶标,获得最大蛋白产量的最佳收获时间是转染后6–7天。这样的延长的文化性ê在不建议时间的细胞内的情况下,或跨膜蛋白,其48之间通常收获ħ和72小时后转染。然而,细胞生存力的连续监测是强制性的,特别是对于新的目标,以防止所述细胞碎片引起的相关联的意外毒性介质堆积与重组蛋白的生产。我们注意到,这取决于用于转染的质粒,HEK293-F细胞可能特别有弹性的存在质粒仍可继续到转染程序之后成长。
对于阴性对照实验的细胞存活率的比较评估可以通过转染细胞来执行与质粒缺乏哺乳动物细胞启动子,例如,一个“虚拟” DNA克隆载体。
              转染后6天,计数细胞1:1作为在步骤A5所指示,并计算所述总细胞数目和生存力。
注:为了获得最佳周一TO-周五的日程安排,组织的转天的周二和周四:这将使双方分割的转染(步骤F1)和收获细胞在6 -平日后第7天转染(步骤F12) 。


              将细胞培养物转移到锥形离心管中,并在10°C下以1,000 × g离心15分钟以收获培养基。
              保留上清液,并根据蛋白质纯化方案进行操作。


评价牛逼ransfection Ë使用fficiency Ë GFP
如在(B)中所讨论的,HEK293-F细胞成为若干通道的后衰老; 因此,定期有必要监视的转染性能。我们建议每月至少两次使用阳性对照检查转染效率。使用含有该基因的表达质粒转染的模拟为增强型绿色荧光蛋白(ê GFP ,例如,的pTT3-EGFP)可以被用于该目的。的测定的转染效率评价细胞的数量已经成功incorporat编重组DNA和是有前能够生产感兴趣的重组蛋白(多个)。显示绿色荧光的细胞数与细胞总数之比得出转染效率值(图3)。


图3 。评估转染效率。比较之间未处理细胞(左)和那些与任一GFP(中心)或虚设DNA(右)进行转染。对于每个样品,比较评价是使用进行细胞d ROP ™自动细胞计数器(Denovix Inc.)中。的明场图像(顶部)为对象编到GFP荧光计数(激发= 470nm的 ± 20纳米; Ë任务= 525纳米± 25nm的使用)DenovixEasyApps软件。当来自小区的不同意外对象存在于计数室(例如,气泡,如所指示的黑色箭头我n中的右图),仪器自动排除他们从最终细胞计数。比例尺,100 µm。


要使用e GFP进行对照转染,请遵循方案步骤(步骤F1 - F8),然后:


如步骤A4所述将转染的细胞孵育72小时。
观察在显微镜下的细胞,并收集图像全光照克既明场和绿色荧光。
注意:当使用自动细胞计数器,而不是与人工计数一个纽鲍尔室,使用Ñ未转染的样品设置在仪器GFP信号检测的阈值。所述的优点细胞d ROP ™自动细胞计数器是,它不需要任何消耗品,如滑梯,通常使用的其他系统。


通过计算绿色细胞(蓝光成像)和总细胞(白光成像),并使用以下公式,计算转染效率:


转染效率(%)=((TOT人数绿色细胞)/ (总细胞数))×100


预期效率应在65%至80%之间。较低的转染效率可能会建议问题与一些试剂或表明细胞批次可能需要为replac版。


一个uthor贡献


FF监督了细胞培养基础设施的设置和开发。MC在MP和AC1的支持下建立了最初的细胞培养设置。SF和AC2优化了培养设置,并标准化了培养和转染程序。SF开展了这项工作中提出的比较分析,阐述对所提出的周一TO-周五的日程安排。SF和FF组织了协议部分,并在所有作者的贡献下撰写了论文。


致谢


我们感谢F.博士马尼亚尼(帕维亚大学)和S博士Wulhfard有用的讨论和建议,除了对所有成员Armenise结构生物学持续支持,测试的实验室-Harvard ,和蛋白质生产条件标杆。


该项目获得了资金来自基金会乔瓦尼Armenise -Harvard(GRANT ID 。CDA2013到FF),意大利癌症研究协会(AIRC,“我的第一个劳资关系委员会授予的” ID。20075至FF),基金会Cariplo(GRANT ID小号。2014- 0881和2015-0768至FF)时,水谷基金会Glycoscience (GRANT ID。200039),教育,大学和研究(MIUR)意大利教育部(丽塔·莱维-蒙塔尔奇尼大奖2012至FF) ,和Dipartimenti迪义大利足球卓越联赛计划(授予ID 。2018年- 2022年,以生物帕维亚大学系和生物技术“L斯帕兰扎尼”)。AC2获得了欧盟Horizon 2020研究与创新计划(授权协议COTETHERS-n。745934)的Marie Curie个人研究金。资金来源无起了研究设计,收集,分析角色,和解释数据,报告的撰写,或提交这篇文章发表的决定。


利益争夺


作者宣称,他们已经没有任何已知的竞争性的经济利益或个人关系可能具有影响d工作报告中提出。


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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Faravelli, S., Campioni, M., Palamini, M., Canciani, A., Chiapparino, A. and Forneris, F. (2021). Optimized Recombinant Production of Secreted Proteins Using Human Embryonic Kidney (HEK293) Cells Grown in Suspension. Bio-protocol 11(8): e3998. DOI: 10.21769/BioProtoc.3998.
  2. Angiolini, F., Belloni, E., Giordano, M., Campioni, M., Forneris, F., Paronetto, M. P., Lupia, M., Brandas, C., Pradella, D., Di Matteo, A., Giampietro, C., Jodice, G., Luise, C., Bertalot, G., Freddi, S., Malinverno, M., Irimia, M., Moulton, J. D., Summerton, J., Chiapparino, A., Ghilardi, C., Giavazzi, R., Nyqvist, D., Gabellini, D., Dejana, E., Cavallaro, U. and Ghigna, C. (2019). A novel L1CAM isoform with angiogenic activity generated by NOVA2-mediated alternative splicing. Elife 8: e44305.
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