APPLICATION
Bioprocess analysis with the LUNA-FX7™
#Bioprocess monitoring #biomanufacturing #cell therapies #bioactive production #automatic calculation #elapsed time #growth rate #doubling time #growth curves
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INTRODUCTION
The biomanufacturing industry is experiencing rapid growth due to improvements in cell
therapies, such as CAR-T cell therapy, and increasing bioactive production1. It is expected to
grow at least 12 to 17% annually over the next decade2. In response to these demands,
biomanufacturers have been expanding the production capacity while maintaining quality and
regulatory compliance. Furthermore, it is even more critical to monitor cell growth and health
accurately while managing multiple cell batches. To provide biomanufacturers with the
flexibility, accuracy, and power to accommodate these analysis demands, we recently developed
the LUNA-FX7™ Automated Cell Counter.
The LUNA-FX7™ Automated Cell Counter provides a new bioprocess monitoring feature as well as a
Quality Control mode with unique validation slides that can be utilized for quality outcomes.
The Bioprocess feature enables scientists to monitor, record, and analyze an individual batch of
bioprocessing activities. Once counting is performed under the Bioprocess mode, the LUNA-FX7™
automatically generates growth rates, cell doubling time, and trend charts. Furthermore, entire
cell counting data may easily be saved and transferred via a USB flash drive, Wi-Fi, or
Ethernet. Notably, the 21 CFR Part 11 compliant “CountWire™” software packages allow more
advanced remote access and data management for multiple LUNA-FX7™ devices connected to the same
network. By providing a convenient means to trace individual batches and monitor various
operations securely, the LUNA-FX7™ can facilitate a more efficient workflow. Here, we
demonstrate how the LUNA-FX7™ monitors the cell growth of three suspension cell lines using the
Bioprocess option. Each cell line was applied to the three counting modes, Fluorescence Cell
Counting mode, Bright Field Total Cell Counting mode, and Bright Field Cell Counting & Viability
mode.
MATERIALS AND METHODS
Cell preparation and counting of batch run simulation
Data visualization and extrapolation
Three suspension cell lines, HL60, K562, and U937, were used in this assessment. All cell lines
were grown in RPMI 1640 culture media supplemented with 10% fetal bovine serum (100 units/mL).
Batch runs were simulated by seeding 10 mL of initial concentrations at 6 x 104 cells/mL in 100
mm cell culture dishes and grown for seven days. During batch runs, counts were performed twice
a day for all seven days utilizing the Bioprocess feature. For each count, 100 µl of cells were
sampled. Each sample was counted via three different cell counting modes: Fluorescence Cell
Counting mode, Bright Field Total Cell Counting mode, and Bright Field Cell Counting & Viability
mode. For brightfield viable cell counting, a 1:1 mix of 0.4% Trypan blue stain (Cat# B13101)
was used. For fluorescent cell counting, a 2:18 mix of Acridine Orange/Propidium Iodide (AO/PI)
(Cat# F23001) was used.
Bioprocess operations
The LUNA-FX7™, the most advanced model in the LUNA™ Family comes equipped with the Bioprocess
software plus an expanded 1 TB hard drive. First, a unique protocol name for an individual batch
is assigned to start bioprocess monitoring. In this manner, multiple batches are easily
monitored and stored in the profiles of status. For this demonstration, the three protocols for
bioprocess monitoring were named as the cell lines – HL60, K562, and U937* 1, respectively.
Actual protocol parameters may be customized to specific cell lines or remain unchanged. Here,
the default protocol parameters were used for all cell lines. Prior to counting, the assigned
protocol for a specific batch is loaded. For each counting, load the target protocol for the
bioprocess, count the cells, and save the results with a check on the Bioprocess saving option.
The LUNA-FX7™ automatically calculates the growth rate and the doubling time, generating updated
growth curves for individual batches based on the counting in the last two intervals (Figure 1).
The on-screen chart is flexible; the y-axis can be toggled to total cell concentration, viable
cell concentration, and viability; simultaneously, the x-axis is changeable to time, date, and
month (More details in the User Manual).
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Figure 1. On-screen growth chart and results of the K562 cell in the Bioprocess Review. (A) The growth chart is showing the cumulative results of cell concentration over date. (B) Summarized results show the start date, the number of measurements, current growth rate, doubling time, current concentration, and viability.
The LUNA-FX7™ automatically records and calculates bioprocess indicators such as growth rate,
viability, and doubling time. Thus, the report is generated including the trend charts of the
data and tables of cell counting status (Figure 2). Further, the cell status also includes
cumulative tracking at each time point, along with immediate or overall cell growth rates. The
data can be exported as a CSV file for more compiled analysis if needed (Table 1).
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Table 1.
Table 1. The report in Fluorescence Cell Counting Mode. The
example CSV report from the HL60 batch run. The report contains date, time,
concentration and viability. The data of escape time, growth rate, and doubling time are
calculated from the last count (Blue shadows) and the run’s total time (Green shadows).
*Growth rate per hour = (LN V2 – LN V1) / (t2 – t1) **Doubling time in hours = LN (2) / Growth rate per hour LN = Natural log, t1, t2 = Unit of hour, V1 = Cell concentration in cells/mL at elapsed time t1 in hours, V2 = Cell concentration in cells/mL at elapsed time t2 in hours
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Figure 2. Bioprocessing data output. Images and charts from fluorescent counting of HL60 cells during a 136-hour batch run. (A) Tagged images at each sampling of the batch run. (B) The plots of cell concentration and viability curves over date were accordingly generated in the LUNA-FX7™.
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LUNA-FX7™ Automated Cell Counter
CONCLUSION
The Bioprocess option in the LUNA-FX7™ reduces unnecessary effort by automating the recording
and analyzing status indicators such as cell growth and viability, simultaneously performed
three cell culture batches of 3 different counting modes. Remarkably, the automated calculations
of doubling times, growth curves, and viability status provide near-real information to monitor
and forecast bioprocess production timelines reliably and accurately. When combined with the
CountWire™ software package, the Bioprocess feature allows team members to monitor multiple
culture batches in real-time across multiple facilities.
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