Numerous bioprocess applications rely on adherent cell culture, including viral vector and vaccine manufacturing, and also stem cell culture for cell and gene therapy.1 Manual handling of adherent cell culture vessels in a scale-out process, on the other hand, presents issues during production, particularly as the number and size of vessels grows.
Image Credit: Shutterstock.com/Hakat
The transition from process development to production scale necessitates consistency and standardization, which is aided by some type of automation.1,2
The Automated Manipulator Platform from Corning is a programmable manipulator intended to automate liquid handling and manipulation during the scaling up of adherent cell culture procedures in Corning® CellSTACK® and HYPERStack® containers. The manipulator platform streamlines fill, empty, and harvest procedures for a more reproducible process by automating the handling of several containers during essential liquid handling steps.
The use of automated handling is particularly crucial during the last stages of harvest since these operations involve the most manipulations and are consequently subject to the greatest degree of unpredictability.
A single HYPERStack 36-layer vessel, for example, necessitates a detailed procedure that includes positioning the vessel at specific angles to empty spent medium, fill and equilibrate with harvest solution, shake the vessel to help release cells from the surface, and eventually gather the harvested cells.3,4
In this study, researchers assessed an automated 3-dimensional (3D) harvest — with integrated left/forward and right/reverse rotations to agitate vessels at compound angles — against a typical manual harvest method using HEK293T cells cultivated in Corning HYPERStack 36-layer vessels.
Materials and methods
Prior to scaling into a Corning CellBIND® surface-treated CellSTACK 10-stack culture container (Corning 3320), HEK293T Cells (ATCC® CRL-3216TM) were grown in complete medium, consisting of Dulbecco’s Modified Eagle Medium (DMEM; Corning 10-013-CM) with 5% fetal bovine serum (FBS; Corning 35-010-CV).
The cells were grown to 70% to 80% confluence, with a targeted final yield of 5.4 × 108 cells.
A 50 L collection bag (Corning 91-200- 48) was loaded with 36 L of complete medium the day before sowing. The medium and six Corning CellBIND surface-treated HYPERStack 36-layer vessels were warmed overnight at 37 °C.
Cells were collected from the CellSTACK 10-stack chamber for 10 to 15 minutes at room temperature with TrypLETM Select enzyme (1×) (Thermo Fisher 12563029) and 0.1% Poloxamer 188 (Corning 13-901-CI) after a 1× Dulbecco’s Phosphate-Buffered Saline (DPBS; Corning 21-031-CM) wash.5
Following cell enumeration, the collection bag was inoculated with cell suspension to seed the 6 HYPERStack 36-layer vessels (with a surface area of 1.8 × 104 cm2 each) at 5 × 103 cells/cm2 or 9 × 107 cells/vessel cells/vessel.
By gently massaging the bag, the contents of the bag was well mixed. The HYPERStack 36-layer vessels were locked onto the Corning Automated Manipulator Cart (Corning 6652) and placed into the Corning Automated Manipulator Rack (Corning 6655). (Figure 1).
Figure 1. Corning Automated Manipulator rack for Corning HYPERStack 36-layer cell culture vessels. (A) Corning Automated Manipulator rack loaded with Corning HYPERStack 36-layer vessels raised to the home position in the Corning Automated Manipulator. (B) Loaded rack (Corning 6655) atop the cart (Corning 6652) with a schematic of vessel positioning inside of the rack (inset). Image Credit: Corning Life Sciences
Following filling and equilibration, the filled rack was loaded into the manipulator, moved into the horizontal seeding position, filled by gravity fill, and then positioned upright to depressurize.3,4 Seeded vessels were incubated in a humidified incubator at 37 °C/5% CO2 until they reached 80% to 100% confluence (Figure 2).
Figure 2. Confluent HEK293T monolayer culture. Representative images of confluent HEK293T monolayers on the day of harvest. The manipulator harvest yields from the corresponding Corning HYPERStack 36-layer vessels were 1.4 × 105 cells/cm2 (left) and 1.6 × 105 cells/cm2 (right). Images were acquired with a handheld USB microscope (Bysameyee Microscope 1000×). Image Credit: Corning Life Sciences
On the day of harvest, 600 mL of complete medium were added to roller bottles (Corning 431644) that had disposable tubing sets (Corning 10043) attached, and the bottles were warmed to 37 °C. Following that, three of the six HYPERStack vessels were loaded into the manipulator rack’s top right, top left, and bottom middle locations (Figure 1B, Positions 1, 3, and 5).
As placeholders, the remaining spots were filled with empty HYPERStack vessels. The rack was then prepared for manipulator operation.
The manipulator harvest program was run to completion to shift vessels in position for the subsequent liquid-handling steps. The media from the three vessels was emptied into a 50 L collection bag. After the spent medium was removed, each of the three containers was loaded with 600 mL of warm TrypLE Select enzyme (1×).
A sequence of rotations and rocking left-to-right consistent with manual operations was used to distribute TrypLE Select enzyme (1×) uniformly through the HYPERStack vessel layers.3,4 After a 5-minute incubation period, the vessels were rocked numerous times from left to right at 60°/seconds
The TrypLE Select enzyme (1×) was then transferred to each layer, and the vessels were incubated for an additional 5 minutes. The vessels were agitated forcefully by a series of 3D shaking motions at a compound angle (left and forward to right and reverse) at 60°/second to detach cells from the vessel surface.
At the end of the program, the harvested cells and protease were manually gathered into the three individual roller bottles containing complete media.
The remaining three vessels were manually treated one at a time in accordance with established processes.3,4 Each vessel received 600 mL of warm TrypLE Select enzyme (1×), which was diluted with 600 mL of the warm complete medium after harvest. The experiment was conducted three times independently, each replicated with a different operator.
On the day of harvest, one of the HYPERStack vessels was placed into the manipulator rack’s top right position (Figure 1B, Position 3). As placeholders, the remaining spots were filled with empty HYPERStack vessels. The rack was prepared for manipulator use. The manipulator harvest programs were run to completion to shift vessels in position for the subsequent liquid-handling steps.
The HYPERStack vessel’s media was transferred into a collection bag. After the spent medium was removed, the HYPERStack vessel was filled with 600 mL of warm TrypLE Select enzyme (1×). At the end of the program, the collected cells and protease were diluted in a single roller container with 600 mL of complete media.
This procedure was performed for each of the subsequent automated harvest protocols, with processing rates ranging from 60°/seconds to 30°/seconds, as described in the Basic Harvest section. The remaining vessel was manually processed with 600 mL of warm TrypLE Select enzyme (1×), which was diluted using a complete medium after harvest, according to known protocols.3,4
Before collecting samples for enumeration, all harvests were recirculated using a peristaltic pump at 750 mL/minute to disperse big aggregates. The experiment was repeated four times independently.
Results and discussion
The Corning HYPERStack vessel's unique design allows for high density 2D cell culture but needs specialized handling steps to achieve high yields. With precise movement control, the manipulator shifts 6 empty vessels into seeding position in one motion, allowing the vessels to be filled by a single large volume of cell seeding suspension.
The only thing the operator needs to do to start controlling the fluid flow is open and close the vent filter clamps on each vessel.
After seeding, the vessels are rotated to allow for medium equilibration and depressurization before proceeding with cell expansion incubation. Automation of the seeding process reduces labor and eliminates a lot of the in-process human variation for both seeding and, more significantly, harvest.
The value of automation is most apparent during cell harvest, which offers a far bigger ergonomic challenge and can pose a greater concern to operator safety. Each HYPERStack vessel needs multiple empty and fill processes with agitation during harvest to disperse harvest reagents and detach cells from the vessel surface.
This level of agitation varies significantly and largely depends on how firmly the cells are adhered to the culture surface and is, consequently, influenced by the cell type and the application. The manipulator can be programmed to perform a variety of agitations, ranging from a very gentle back-and-forth rocking motion to an aggressive shaking motion in all three axes.
In contrast, there are limitations to the capability of an operator to manually manipulate the vessel safely to dissociate and harvest cells. Furthermore, consistency during manual processing is strongly reliant on sufficient operator training and adherence to a tight standard operating procedure (SOP).
Manual process SOPs do not assure that variations will not occur from day to day. Whilst the automated manipulator, once tuned for usage with various cell types, will perform the same set of motions prescribed by the chosen program, boosting harvest uniformity regardless of the operator.
Human error in production is influenced by fatigue and training.6 To capture the intrinsic variability associated with the hand processing of numerous HYPERStack vessels, harvests were done using the automated manipulator and compared to harvests performed by three different operators in the present study.
As could be expected in a production situation, the three operators in this study had various levels of training and expertise with manual HYPERStack vessel harvests: basic, intermediate, and expert. Overall harvest yields for both manual and manipulator harvests ranged from 2 × 109 cells to 3 × 109 cells, with manipulator harvests deviating less from the mean (Figure 3A).
Figure 3. Automated harvests decrease the variability associated with manual Corning HYPERStack 36-layer vessel handling. (A) Total cell yield of manual (dark blue) and manipulator (orange) harvests by 3 different operators. Each data point represents the mean ± SD of 3 vessels. Mean cell viability (open circle) for each operator is plotted on the right axis. (B) Coefficient of variance calculated for each operator’s 3 manual (dark blue) and 3 manipulator (orange) harvest yields. N = 3 vessels per operator per harvest mode. Image Credit: Corning Life Sciences
Evaluation of the coefficient of variance indicates that the automated manipulator harvests produced more consistent yields (Figure 3B). This amount of variation is for laboratory-scale processing of three vessels. With manual processing in a large-scale manufacturing run of more than 3 to 6 vessels, it is realistic to expect the variance to be amplified.
Figure 4. Optimization of manipulator speed of movement can improve harvest yields. Box plots comparing manual harvest (dark blue) with manipulator harvests at varying speeds of agitation: 60°/seconds (orange), 50°/seconds (green), 40°/seconds (gray), and 30°/seconds (light blue). Whiskers mark the minimum and maximum values, box boundaries mark the 25th and 75th percentiles, and the line marks the median. Mean cell viability (open circle) for each speed is plotted on the right axis. N = 4 vessels per condition. Image Credit: Corning Life Sciences
It is still possible to increase harvest by optimizing the parameters, even with constant automated manipulator harvests. The automated manipulator can accurately regulate the movement of the HYPERStack vessels in 0.5° increments at rates of up to 1°/second to optimize harvests; the speed of motion was altered from aggressive (60°/seconds) to moderate (30°/seconds) in the second phase of the trial.
Even though there was a wide range of harvest values between repetitions, slowing the 3D agitation to 30°/seconds during harvest yielded the highest mean yield (Figure 4). Interestingly, irrespective of processing speed, cell viability was greater than 95%. The range of harvest values increased with slower agitation speeds, indicating an inverse association with harvest speed.
The varying pace of agitation during automated manipulator harvests may reflect day-to-day and operator-to-operator variability and may reflect some of the variability related to manual processing. Determining the ideal manipulator harvest parameters for applications requiring HEK293T cell culture, such as those used in this research, may require a balance between yield and consistency.
Nevertheless, the automated manipulator allows for parameter adjustment (i.e., angles, rotation, agitation speed, and timing) to optimize harvests for a certain cell type and application. With manual manipulators, it is not possible to refine the HYPERStack vessel harvest process with that level of precision in a reproducible manner.
- Programming allows for exact control of angles, rotations, and speed, making it easier to manipulate Corning® HYPERStack® 36-layer vessels for filling, depressurizing, emptying, and harvesting.
- The automated manipulation of Corning HYPERStack 36-layer vessels for seeding and harvesting processes eliminates the labor and strain caused by manual handling.
- The Corning Automated Manipulator System decreases harvest variability from operator to operator and day to day for consistent cell yields.
- Corning Life Sciences. The race to billions: how amplifying adherent cultures can get you there. July 14, 2021. Retrieved from https://www.biopharminternational.com/view/the-race-to-billions-how-amplifying-adherentcultures-can-get-you-there.
- Doulgkeroglou MN, et al. Automation, monitoring, and standardization of cell product manufacturing. Front Bioeng Biotechnol 2020;8:811. doi: 10.3389/fbioe.2020.00811.
- Corning HYPERStack Cell Culture Vessel Closed System User Guide (CLS-AN-364).
- Corning HYPERStack Cell Culture Vessels Guidelines for Use (CLS-AN-603DOC).
- Corning CellSTACK Culture Chambers Instructions for Use (CLS-BP-007DOC).
- Tulip. Augmented worker: how digital technology can power your workforce. Retrieved from https://tulip.co/ebooks/augmented-worker/.
About Corning Life Sciences
A division of Corning Incorporated, Corning Life Sciences is a leading global manufacturer of cell culture products and solutions that enable academic, biotech and biopharma scientists to harness the power of cells to create life-changing innovations. Corning supports a range of application areas including core cell culture, 3D cell culture, bioprocess, cancer research, primary and stem cell research, drug screening, cell and gene therapy, disease modeling, lab automation and more.
Whether your goal is stem cell expansion or viral vector production, Corning Life Sciences platforms, including HYPERStack® vessels that maximize cell growth area in a small footprint, the high-yield Ascent® Fixed Bed Reactor platform, microcarriers, and closed system solutions can help get you there. Choose from hundreds of vessels, the widest selection of cell culture surfaces, and custom media in a variety of single-use technology configurations. Learn more at www.corning.com/lifesciences.