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Abstract

As the drug discovery process evolves and demands more challenging and relevant assays, we have experienced a recent and significant escalation in the number of cell-based high-throughput assays for both small molecule and target identification screens. This has resulted in an increased need for the reproducible production of high quality cells in large quantities. Historically, manual cell culture was the only option available for providing cells in sufficient numbers for small molecule ultra-high-throughput screens (uHTSs), representing a technology gap in our automated cell culture process.

Recently, we have applied an automated solution in the form of a novel 10-layer tissue culture flask, the HYPERFlask (Corning, Lowell, MA). This technology, when introduced as an upgrade to the SelecT (an automated cell culture system manufactured by The Automation Partnership, Ltd., Royston, England), provides a new approach to automating production of the high number of cells required for uHTS and consequently a highly desirable alternative to manual cell culture.

The HYPERFlask has a surface area of 1720 cm² and can yield up to 3 × 10⁸ cells after 3 days in culture. This can be compared to a typical yield of 2 × 10⁷ cells from a T175 flask (with a surface area of 175 cm²), the current standard flask type for automated cell culture on the SelecT. Cells grown in both flask types are of comparable quality, as demonstrated by equivalent cell viability, yield per cm², functional response, and pharmacology.

Keywords

HYPERFlask cell culture SelecT high-throughput assays cell-based assays

Introduction

Cell-based assays have become increasingly prevalent in many stages of drug discovery and target identification processes.^1,2 Traditional high-throughput screens used purified proteins or membrane preparations that were amenable to large-scale production, but limited to use in a minimal variety of assays.^{3 –6} As the field of screening matured, new technologies made high-throughput cell-based assays a possibility, and the demand for large numbers of functional cells grew exponentially.^{7 –15} Supplying cells in sufficient quantity and quality for ultra-high-throughput screens (uHTSs) presents a significant challenge for existing automated cell culture systems, primarily due to the extremely high numbers of cells required. This demand can range from hundreds of millions to billions of cells every day for 5–15 consecutive days depending on the format of the assay. Both system capacity and process time are limiting in these conditions, forcing the use of manual cell culture to support uHTS cell-based assays.

Automated cell culture using the SelecT^{16 –18} offers several advantages over manual cell culture (Fig. 1). It removes many of the variables involved in growing cells. By maintaining cells with robotic protocols, greater consistency is achieved by alleviating variables when cells are transferred from one cell culture scientist to another. Automated cell culture also enables cell passaging and plating over weekends, allowing the delivery of plated cells for assay on Mondays. Importantly, when compared to manual cell culture, automated cell culture generates key parameters from the cell populations. These data are captured and can be applied to enhance the quality control of cell production. For example, percent viability, average cell size, and the presence of cell debris and aggregates can be compared to assay performance to identify the optimal cell conditions. Because cell counting is automated, 20 cell counts are routinely taken from each cell sample to generate a highly accurate number. Another key capability of the automated cell counter on the SelecT is that it produces and retains an image of the counted cells that can be critically important in trouble-shooting cell-based assays and avoids subjectivity in retrospective analysis of cells provided for assay.

Figure 1.

SelecT. The SelecT, designed by The Automation Partnership, is the first automated tissue culture system. A consortium of six major pharmaceutical companies were involved in the system design. The SelecT automates cell seeding, harvesting, and passaging. It has a capacity of 182 T175 flasks and 420 plates.

The biggest disadvantage to using the SelecT for automated cell culture is the linear process of the system. The SelecT has only one arm, and is therefore only capable of handling one flask at a time, which can lead to long processing times. This was a significant driver for implementing the HYPERFlask. Another consideration is that the SelecT can only manipulate a 10-mL pipette, which has a maximum aspiration/dispense speed of 5 mL/s. Therefore, it can be difficult to dissociate a clumpy cell line into individual cells. These few disadvantages are outweighed by the quality control, consistency, and reliability obtained from using the SelecT.

We have used the SelecT to successfully support cell production for target identification and high content screening assays. Target identification and high content screens require moderate cell numbers, between 10⁷–10⁸ cells each assay day.^{19 –24} However, for our process, automated production of cells in sufficient quantities to support cell-based uHTS screens (10⁸–10⁹ cells each assay day) has been limited by both speed and capacity of our current automation. Recent development of the HYPERFlask²⁵ in combination with a compatible upgrade of SelecT automation presented an opportunity to expand the capabilities of automated cell culture for uHTS applications.

The HYPERFlask is an innovative tissue culture flask that drastically increases the surface area of cell growth, while maintaining a footprint almost identical to that of a standard automation-compatible T175 flask (Fig. 2). The HYPERFlask consists of 10 essentially identical, interconnected “cassettes,” each containing a novel membrane that has been treated with CellBIND (Corning, Lowell, MA) for improved cell adherence. The membrane is gas permeable allowing exchange of oxygen and carbon dioxide through the base of the membrane. Gas enters the HYPERFlask through the sides and subsequently underneath each cell layer of the flask, allowing gas exposure to a large surface area within the flask (Fig. 3).

Figure 2.

HYPERFlask compared to standard T175 flask. The HYPERFlask (left) consists of 10 polystyrene growth surfaces each treated with CellBIND. The 10 layers have a combined surface area of 1720 cm². Between each of the 10 interconnected growth surfaces is an air gap that allows passive gas exchange. The flask is barcoded and compatible with the SelecT. The standard T175 flask (right) has a growth surface area of 175 cm² and gas exchange occurs through a vented cap.

Figure 3.

Diagram of gas exchange in a HYPERFlask. The cells grow on an ultra thin gas-permeable layer. Between each layer is an air space that allows gas exchange along the length of the flask. Each of the individual layers is known as a “cassette.”

Here, we demonstrate that the HYPERFlask is capable of producing approximately 10 times the number of cells when compared to a standard T175 flask. Furthermore, the quality of the cells grown in the HYPERFlask was equivalent to that of cells grown in a standard T175 flask when assessed by viability and assay performance in functional cell-based uHTS assays.

Materials and Methods

Cell Lines

Three recombinant Chinese hamster ovary (CHO) cell lines and a recombinant human embryonic kidney (HEK 293) cell line expressing various targets of interest were generated in house. A nonrecombinant HeLa S3 cell line was obtained from American Type Culture Collection (ATCC) (Manassas, VA).

Cell Culture Conditions

Manual validation of the HYPERFlask was performed using a recombinant CHO cell line that overexpressed an enzyme of interest (CHO #1). Cells were maintained in F12K, 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 0.5 mg/mL hygromycin, and 0.25 mg/mL Geneticin. A second recombinant CHO cell line, that overexpressed a target enzyme (CHO #2) was grown in Iscove's Dulbecco's modified eagle medium (DMEM), 10% FBS, 2 mM l-glutamine, 1 mg/mL Geneticin, and 1 × sodium hypoxanthine and thymidine (HT) supplement for automated validation. The functional validation was performed with CHO cells stably transfected with a nuclear receptor fused to a ProLabel tag (CHO #3). This cell line was maintained in F-12 Nutrient Mixture, 10% FBS, and 1% penicillin/streptomycin/l-glutamine. HeLa S3 cells were grown in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. HEK 293 cells were grown in DMEM, 10% FBS, and 1 mg/mL Geneticin.

All cell lines were grown at 37 °C in a humidified environment containing 5% CO₂. All incubation steps contained in protocols below were performed in this environment. The following products were purchased from Invitrogen Corporation (Carlsbad, CA): FBS (catalog #10082), penicillin/streptomycin (catalog #15140), hygromycin (catalog #10687), Geneticin (catalog #10131), Iscove's DMEM (catalog #12440), L-glutamine (catalog #25030), HT supplement (catalog #11067), F-12 Nutrient Mixture (catalog #11765), penicillin/streptomycin/l-glutamine (catalog #10378), and DMEM (catalog #11965). F12K was purchased from ATCC (catalog #30–2004). For dissociation, Accutase was purchased from Innovative Cell Technologies (San Diego, CA) (catalog #AT-104). Trypsin (catalog #25300) and Trypsin LE (catalog #12605) were both purchased from Invitrogen Corporation.

Manual Protocols

HYPERFlask Seeding . Two T175 flasks were harvested on SelecT and cells were resuspended at a concentration of 1.7 × 10⁶ cells/mL. The cell suspension (10.2 mL) was diluted in 100 mL of media to generate a large enough volume for equal seeding throughout all 10 layers of the HYPERFlask. HYPERFlasks were seeded manually with a total of 1.7 × 10⁷ cells at a concentration of 9.9 × 10³ cells/cm². Adding the cell suspension to the flask involved placing it in a clamp provided by Corning for this specific purpose. The clamp held the flask at an angle that minimized the generation of bubbles during filling. (Note: this clamp was necessary only for the beta-testing version of the HYPERFlask and is no longer required for current HYPERFlask protocols.) The cell suspension was poured slowly down the side of the neck of the flask, and equilibrated by tilting flask 90° such that the flat surface of the flask is perpendicular to the surface of the biological safety cabinet (BSC). Equilibration ensured that all 10 layers of the flask received an equal volume of cell suspension. The flask was returned to the clamp and an additional 400 mL of media was then poured into the flask. The flask was tilted to an upright position and an additional 50 mL of media was added. This completely filled the HYPERFlask and brought the level of the media to the bottom of the threads for the cap. When removed from the clamp, air bubbles caught in the layers of the flask traveled to the air dam and were trapped in the neck of the flask. The cells were grown in the HYPERFlask for 4 days.

HYPERFlask Harvesting . Media was decanted and the HYPERFlask washed by addition of 100 mL of Dulbecco's phosphate buffered saline (PBS) followed by equilibration and swirl steps where the large flat surface of the flask was turned parallel to the surface of the BSC. PBS was removed and 50 mL of 0.05% Trypsin added, equilibrated, and swirled. The flask was incubated for 5 min at 37 °C and then banged twice by hand to detach all cells. To inactivate the Trypsin, 100 mL of growth media was added to the flask, equilibrated, and rotated to dislodge cells from the flask. Effective cell harvest was confirmed by inspection of the harvested flask by microscope at 4x magnification. The cell suspension was decanted into a collection vessel. PBS was added to the flask to harvest remaining cells, bringing the cell suspension to a final volume of 250 mL. Cells were counted on the SelecT and cell viability determined by Trypan Blue exclusion using a Cedex automated cell counter (Innovatis AG, Bielefeld, Germany).

Crystal Violet Staining . Media was decanted from the HYPERFlask, and 200 mL of Crystal Violet (Fisher Scientific, Waltham, MA) added to the flask. After a 3-min incubation at room temperature, the Crystal Violet was removed followed by two PBS washes of approximately 200 mL. The HYPERFlask was broken apart into individual cassettes and examined visually for homogeneity of cell distribution.

Automated Protocols

SelecT Automation . The Automation Partnership developed modifications of the Staubli arm movements (Staubli Robotics, Faverges, France) on the SelecT that were necessary to accommodate the HYPERFlask. Although similar in footprint, the HYPERFlask is not identical to the T175. New moves included an “equilibrate” step necessary for even distribution of liquid and a modified “pour” step where the HYPERFlask is rotated 90°, with the neck facing downward, after which the flask is rocked back and forth several times to maximize the removal of liquid from all layers of the flask. A “HYPERswirl” motion was also introduced that is similar to the existing SelecT swirl motion, with an added 180° rotation and swirl. In addition, the media dispense procedure was modified for the HYPERFlask because the flask must approach the cocktail bar at a 20° angle from vertical, and then be repositioned perpendicular to the floor to fill the flask. Finally, the presentation of the HYPERFlask to the barcode reader was adjusted to rotate the flask 90° to access the barcode located on the bottom surface of the flask. This is now a commercially available upgrade to the SelecT.

HYPERFlask Seeding . Cells were grown in three T175 flasks and harvested at 90% confluence using the SelecT. Harvested cells were counted, diluted to 1.05 × 10⁶ cells/mL in a final volume of 50 mL and pipetted into a HYPERFlask. The HYPERFlask was then filled with media and cells (approximately 550 mL total volume) and returned to the incubator for 3 days.

HYPERFlask Harvesting . Accutase (75 mL) was added to the HYPERFlask and the flask was incubated for 5 min at 37 °C on the SelecT. When incubation was complete, dissociated cells were poured into a T175 collection flask containing 75 mL of media. An additional rinse with 50 mL of media was added to the HYPERFlask and these cells were combined with those already collected. Cells were triturated to generate a single-cell suspension, then counted in automated mode on the SelecT.

Nunclon TripleFlask Seeding . The same cell suspension that was used to seed the HYPERFlask was also used to seed the Nunclon TripleFlasks. The flasks were seeded at 1.5 × 10⁷ cells/flask and filled with 160 mL of media. Cells were incubated for 3 days.

Nunclon TripleFlask Harvesting . Accutase (15 mL) was added to the Nunclon TripleFlask and the flask was incubated for 10 min at 37 °C on the SelecT. After the incubation, 25 mL of growth media was added to the flask to collect cells. Cells were triturated and counted in automated mode on the SelecT.

Functional Validation

Recombinant CHO cells stably expressing a human nuclear receptor fused to a ProLabel tag (CHO #3) were grown in a HYPERFlask and in T175 flasks using identical seeding densities to compare the functionality of the cells.

PathHunter Assay. A PathHunter Nuclear Translocation Assay²⁶ (DiscoveRx Corporation, Fremont, CA) was used to evaluate the functional response of the cells. The assay is a single-addition, homogeneous assay measuring the effects of agonist or antagonist compounds on target protein translocation to the nucleus. In this system, a target protein of interest, fused to an inactive β-galactosidase (β-gal) enzyme fragment (ProLabel tag), is expressed in the cytoplasm. A complementary fragment of β-gal (Enzyme Activator, or EA) is expressed only in the nucleus. On protein activation and translocation to the nucleus, the two fragments of β-gal interact forming an active enzyme complex capable of hydrolyzing a chemiluminescent substrate that then generates measurable light.

In 3456 well assays, compounds were preplated in 5 nL drops before the addition of cells and agonist. This was followed by a 3-h incubation at 37 °C in a humidified atmosphere containing 5% CO₂. Detection reagent was added and plates were incubated 1 h before reading on the ViewLux (PerkinElmer Life and Analytical Sciences, Inc., Waltham, MA) for a 90-s exposure.

PathHunter Reagents . The PathHunter detection reagents were purchased from DiscoveRx Corporation (Fremont, CA). Assay plates (3456 well) were purchased from Aurora Discovery (Carlsbad, CA).

Results

Manual Validation of HYPERFlask

The recombinant CHO cell line (CHO #1) was selected for these experiments as an example of a robustly adherent cell line with a doubling time representative of a typical cell line used in uHTS assays.

Two HYPERFlasks were each seeded with 1.7 × 10⁷ cells at a concentration of 9.9 × 10³ cells/cm². The cell suspension was harvested from the HYPERFlask and counted using the Cedex counter on the SelecT. Yields of 3.5 × 10⁸ and 3.8 × 10⁸ cells were obtained with 99.2% viability in both cases, based on Trypan Blue exclusion. This yield was compared to the same cell line, seeded at equivalent density, grown in a T175 flask and harvested on the SelecT (Fig. 4). The T175 yielded 3.0 × 10⁷ cells. In this experiment, the HYPERflask gave a 12-fold increase in yield when compared to the T175 flask.

Figure 4.

In manual protocols, cells harvested from HYPERFlask show greater than 10-fold cell yield when compared to a T175 flask. Yields of viable recombinant Chinese hamster ovary (CHO) cells (CHO #1) harvested from a HYPERFlask (n = 2) and a T175 flask (n = 3) are represented.

Automated Validation of HYPERFlask

Seeding Volume and Density . By varying the volume of liquid in the HYPERFlask when developing the seeding protocol, we established that a minimum seeding volume of 50 mL was necessary for even distribution of cells in the HYPERFlask. We had previously observed that a seeding volume of 9 mL resulted in very low cell yields (4.4 × 10⁷ total cells) due to an uneven dispersal of cells between layers. This was confirmed when cell distribution was evaluated by staining the HYPERFlask with Crystal Violet and breaking the HYPERFlask into the individual component cassettes (data not shown).

Seeding Density . Standard T175 protocols generally require a seeding density of 25,000–30,000 cells/cm² (4.4 × 10⁶ to 5.25 × 10⁶ total cells/flask) to reach 90% confluence after 3 days of growth. Similarly, for the HYPERFlask experiments, seeding at a cell density of 25,000 cells/cm² (4.4 × 10⁷ total cells/flask) resulted in 90% confluence after 3 days in culture. This was confirmed by staining with Crystal Violet (Fig. 5). For all cell types studied, seeding densities determined for T175 flasks could be reliably used to generate HYPERFlask protocols.

Figure 5.

Homogeneous distribution of cells is illustrated by Crystal Violet Staining of HYPERFlask cassettes. HeLa cells were seeded at 25,000 cells/cm and incubated for 3 days in a HYPERFlask. The cells were stained using Crystal Violet, then cassettes were separated at the manifold and examined visually and under 10× magnification. Crystal Violet staining demonstrates that cells were evenly seeded using SelecT.

Optimization of Cell-Dissociation Protocols . For each cell type tested, cell dissociation conditions diverged from those typically used for automated culture using T175 flasks. Incubation with TrypLE (Invitrogen Corporation, Carlsbad, CA) for 5–10 min is standard for CHO and HeLa cell lines in T175 flasks on the SelecT. However, we observed significant lysing of the cells dissociated from the HYPERFlask in these conditions (Fig. 6). Lysed cells released genomic DNA and healthy cells aggregated around the DNA, resulting in cell clumps that the automated cell counter did not include in generating cell counts. Consequently, cell yields were low and not representative of the number of cells grown in the HYPERFlask. We diluted the TrypLE threefold with PBS and decreased incubation time to 3 min, but this failed to resolve the issue (data not shown).

Figure 6.

Cell lysing captured during cell count of Chinese hamster ovary cells on SelecT using Cedex. This image illustrates that cell lysing was occurring when cells from a HYPERFlask were dissociated with TrypLE. Circle A shows healthy cells. Circle B highlights genomic DNA resulting from cell lysis.

When cell lines are not robust under dissociation with TrypLE, we have previously used Accutase successfully. Accutase was therefore tested in automated HYPERFlask protocols and cell lysing was no longer observed.

Yields and Viability from HYPERFlask

Comparison of T175, TripleFlask, and HYPERFlask . HEK 293 cells, a recombinant CHO cell line (CHO #2) and HeLa S3 cells were used to evaluate cell culture comparing the T175 to the HYPERFlask. In each case, cell yield and viability were compared to that observed with equivalent seeding densities and growth parameters used in the automated T175 protocol (Fig. 7). In all cases, viability was acceptable and greater than 90%. Yields were sevenfold to 12-fold higher in the HYPERFlask (Table 1).

Table 1.

Comparison of cells grown in T175 flasks, TripleFlasks, and HYPERFlasks

	T175		TripleFlask			HYPERFlask
Cell line	Yield (×10⁶)	Viability (%)	Yield (×10⁶)	Viability (%)	Yield (×10⁶)	Viability (%)	HYPERFlask/T175 (fold)	HYPERFlask/TripleFlask (fold)
CHO#2	30.1	99.8	82	98.1	209	96.6	6.9	2.55
HeLa S3	38.8	99.3	—	—	373	91.5	10.6	—
HEK 293	23.1	99.1	—	—	290	95.2	12.6	—
CHO#3	—	—	69	98.1	250	97.4	—	3.63

Figure 7.

Comparison of cell yields from a range of cell lines grown in HYPERFlasks or T175 flasks. A variety of cell lines were harvested from HYPERFlasks and T175 flasks. In all cases, a significant increase in yield was observed when cells were grown in HYPERFlask.

The Nunclon TripleFlask (Nalge Nunc International, Rochester, NY) was also evaluated in this comparison, because this flask was the original solution for high density automated cell culture on SelecT. The Nunclon TripleFlask is a 500-cm², three-layered flask with similar footprint to a T175. Using automated protocols, two recombinant CHO cell lines were grown and harvested from HYPERFlasks and yields compared with those obtained from Nunclon TripleFlasks. Yields from HYPERFlask averaged threefold higher than those obtained from Nunclon Triple-Flasks, with comparable high viability of harvested cells (Table 1).

Throughput. Automated processing times for harvesting a single T175 flask, a Nunclon TripleFlask, and a HYPERFlask were compared. Harvesting a HYPERFlask took approximately 8 min longer than harvesting a T175 flask. This additional time is primarily a result of the time required to completely fill a HYPERFlask. A HYPERFlask takes only 2 min longer to harvest when compared to a Nunclon TripleFlask (Fig. 8).

Figure 8.

Comparison of harvesting times and cell yields. Chinese hamster ovary #3 cells were seeded at 30,000 cells/cm into three flask types and incubated for 3 days. Flasks were harvested on SelecT, and harvesting time and cell yield compared. A T175 flask takes the shortest amount of time to harvest, at 22 min, however the HYPERFlask takes just 8 min longer with a ∼ 10-fold increase in cell yield.

Functional Validation

To compare the function of cells grown in HYPERFlasks with those grown in T175s in automated cell culture, we implemented a functional translocation assay using the recombinant CHO cell line (CHO #3). Each population of cells was characterized in terms of signal-to-background ratio of control agonist. EC₅₀ values of control and reference agonists were also compared. In addition, we monitored the consistency of these parameters over time as cells were stirred by a stir bar in a collection flask (stir flask) at room temperature before assay.

During uHTS, it is considered advantageous that the cells are stable in their response for 3–4 h in a stir flask after dissociation and before plating for assay. To test functional stability, cells were plated and assayed immediately after dissociation (t = 0), and after stirring in a flask and being dispensed at 1-h intervals (t = 1–4). Cells from both T175 and HYPERFlask showed comparable results and were stable for at least 4 h postdissociation, without impacting the assay window (Fig. 9).

Figure 9.

Stability test with cells cultured in T175 flasks (blue) or HYPERFlask (red). Cells from a T175 flask and a HYPERFlask were tested for functional stability over a 4-h period. Cells were plated in 3456 well plates and assayed at hourly intervals using the Path-Hunter assay according to Methods. The signal-to-basal ratio is plotted as a function of time (h).

In addition to evaluating the function of cells grown in the HYPERFlask and T175 with a single concentration of agonist, we performed dose–response titrations of several reference compounds. Figure 10 illustrates that we observed comparable potencies and efficacies for all compounds tested.

Figure 10.

Dose–response curves of control compounds (agonist, antagonist, and partial agonist) using cells from T175 (left) and HYPERFlask (right). A 16-point dose titration for each compound was performed in a white 3456 well plate using the PicoRaptr (Aurora Discovery). Cells and PathHunter reagents were added according to Methods. Luminescence was read using the ViewLux.

Discussion

We have demonstrated that automation of the HYPERFlask is a viable solution to satisfying the requirements of uHTS for the supply of large quantities of cells without compromising cell quality. In addition, for some cell types, the flask exceeded the 10-fold increase in cell yield predicted by the approximately 10-fold increase in surface area of HYPERFlask when compared to a T175 flask. One possible explanation for this increased yield may lie in the novel architecture of the flask. Gas exchange occurs below the entire growth surface of each cassette of the HYPERFlask. This is likely to allow highly efficient gas exchange, supporting high cell numbers. In comparison, gas exchange in a T175 flask is via five small holes on the vented cap. An additional feature of the HYPERFlask is a CellBIND treatment on cell culture surfaces that increases the flask's wettability.²⁵ This may also contribute to increased yield of cells by maintaining cell contact and adherence during culture.

In validating a range of cell types and morphologies, we addressed the variety of cell lines commonly used in uHTS screens. CHO K1, HeLa S3, and HEK 293 cells have all been grown and harvested successfully. Some optimization was necessary for these cell types, particularly in the cell dissociation step. Although the cell lines described here were not amenable to dissociation with TrypLE, this cannot be assumed for all cell types and we would consider TrypLE an appropriate reagent for dissociating cell lines that may have a tendency to clump, or are extremely adherent and require long incubation protocols.

At higher cell densities, clumping of harvested cells emerged as an issue that was subsequently resolved by seeding and harvesting cells at lower cell density. Further optimization of dissociation conditions or off-line resuspension of harvested cells may also address this issue. One observed limitation of the SelecT is that trituration protocols are restricted to the use of a 10-mL pipette, at a relatively slow pipetting speed of 5 mL/s. Because HYPERFlask harvest protocols generate large cell volumes that may contain clumps, this on-line trituration method may not adequately break up clumps for some cell types.

This does raise the question of applications of these flasks for cell lines with more challenging morphologies, particularly cell types such as the hepatocellular carcinoma cell line HepG2 that are prone to clumping using standard tissue culture techniques. In the cell types investigated so far, this has not been an issue. Although decreased cell density in the HYPERFlask may compromise yield, transition of a cell line into HYPERFlask can still be considered, because savings will still be realized in improved SelecT incubator capacity and process times.

Another important advantage driving application of the HYPERFlask to uHTS is the rapid preparation time for harvesting. In cell-based assays, cells are continuously stirred in plating protocols and this is a key parameter used in designing cell delivery schedules for screening. Minimizing the time between cell dissociation and plating is desirable and is predicted to result in assays requiring fewer batches of cells. Benefits to cell culture workflow are immediate—fewer cell deliveries per assay day will be required. Decreasing variables in assay protocols is always desirable, thus increased assay stability should also be an important downstream improvement.

Considerations in choosing cell lines that would benefit from culture in the HYPERFlask center around acceptable yields. Assuming a 10-fold increase in yield, protocols should require seeding from three or fewer flasks. Once this threshold is reached, multiple T175 flasks would need to be maintained on the SelecT for seeding of HYPERFlasks. This would lead to a substantial occupation of incubator space for scale-up and would not be an efficient use of the HYPERFlask. Similarly, compromises in yield should be limited—a sevenfold increase when compared to a T175 flask is acceptable, in most cases. Anything less than this would indicate moving cell culture into Nunclon TripleFlasks as an alternative approach.

The advantages and applications of high density cell culture in HYPERFlask are numerous. The implementation of HYPERFlask in combination with cell culture automation using the SelecT has allowed us to achieve the standard of quality control desirable for uHTS. In addition to live cell assays, we plan to use automated high density cell culture to generate large batches of cells for preparation of frozen vials to support frozen cell assays. This will minimize the number of batches of frozen cells in assay protocols. Similarly, high volumes of cells required for membrane preparations are appropriately supported by high-density cell culture, releasing significant incubator capacity for other processes and allowing more efficient use of our automated cell culture system.

It can be noted that, to date, the SelecT is the only automated cell culture system capable of handling the HYPERFlask. There is one commercially available alternative to SelecT, the Cellerity, an automated cell culture system manufactured by Tecan (Mannedorf/Zurich, Switzerland).²⁷ The Cellerity automation is limited to the use of RoboFlasks for cell culture.²⁵ RoboFlasks have dimensions that conform to Society for Biomolecular Screening format; therefore, the Cellerity is incapable of using the HYPERFlask.

In general, any process requiring large cell numbers that are sensitive to process time or batch-to-batch variability would benefit from the enhanced quality control of automated cell culture and would be a candidate for implementation of HYPERFlask automation on the SelecT.

Acknowledgments

We are grateful to Matt Golding and Stephen Guy (The Automation Partnership), Todd Upton (Corning), and Julie Kerby and Mathew Leveridge (Stem Cell Sciences), for their contributions to the protocols described in this manuscript.

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Automated Application of a Novel High Yield,High Performance Tissue Culture Flask

Abstract

Keywords

Introduction

Materials and Methods

Cell Lines

Cell Culture Conditions

Manual Protocols

Automated Protocols

Functional Validation

Results

Manual Validation of HYPERFlask

Automated Validation of HYPERFlask

Yields and Viability from HYPERFlask

Functional Validation

Discussion

Acknowledgments

References