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Optimizing the production of suspension cells using the G-Rex "M" series.

Bajgain P, Mucharla R, Wilson J, Welch D, Anurathapan U, Liang B, Lu X, Ripple K, Centanni JM, Hall C, Hsu D, Couture LA, Gupta S, Gee AP, Heslop HE, Leen AM, Rooney CM, Vera JF - Mol Ther Methods Clin Dev (2014)

Bottom Line: Broader implementation of cell-based therapies has been hindered by the logistics associated with the expansion of clinically relevant cell numbers ex vivo.A multicenter study confirmed that this fully optimized cell culture system can reliably produce a 100-fold cell expansion in only 10 days using 1L of medium.The G-Rex M series is linearly scalable and adaptable as a closed system, allowing an easy translation of preclinical protocols into the good manufacturing practice.

View Article: PubMed Central - PubMed

Affiliation: Center for Cell and Gene Therapy, Baylor College of Medicine, Houston Methodist Hospital, Texas Children's Hospital , Houston, Texas, USA.

ABSTRACT
Broader implementation of cell-based therapies has been hindered by the logistics associated with the expansion of clinically relevant cell numbers ex vivo. To overcome this limitation, Wilson Wolf Manufacturing developed the G-Rex, a cell culture flask with a gas-permeable membrane at the base that supports large media volumes without compromising gas exchange. Although this culture platform has recently gained traction with the scientific community due to its superior performance when compared with traditional culture systems, the limits of this technology have yet to be explored. In this study, we investigated multiple variables including optimal seeding density and media volume, as well as maximum cell output per unit of surface area. Additionally, we have identified a novel means of estimating culture growth kinetics. All of these parameters were subsequently integrated into a novel G-Rex "M" series, which can accommodate these optimal conditions. A multicenter study confirmed that this fully optimized cell culture system can reliably produce a 100-fold cell expansion in only 10 days using 1L of medium. The G-Rex M series is linearly scalable and adaptable as a closed system, allowing an easy translation of preclinical protocols into the good manufacturing practice.

No MeSH data available.


Related in: MedlinePlus

Measuring glucose as a surrogate for culture performance. Panel (a) shows the glucose concentration in culture medium, as measured using a standard glucometer, and the inverse correlation between glucose and cell number. Panel (b) shows the formula we developed to calculate the number of cells in the culture based on the glucose concentration in the culture medium. Panel (c) shows the cell number obtained by: (i) hemocytometer counting, (ii) flow cytometry, and (iii) the glucose consumption formula.
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fig4: Measuring glucose as a surrogate for culture performance. Panel (a) shows the glucose concentration in culture medium, as measured using a standard glucometer, and the inverse correlation between glucose and cell number. Panel (b) shows the formula we developed to calculate the number of cells in the culture based on the glucose concentration in the culture medium. Panel (c) shows the cell number obtained by: (i) hemocytometer counting, (ii) flow cytometry, and (iii) the glucose consumption formula.

Mentions: Traditionally, in order to accurately quantify cell numbers, one must first generate a homogenous cell suspension from which to sample. However, since the G-Rex accommodates large media volumes (e.g., 10 ml/cm2), cell resuspension is challenging. To address this issue, we sought to identify an alternate marker that could be used to predict cell growth. We initiated G-Rex cultures (n = 3) using optimal conditions (1.25 × 105 K562 cells/cm2 with 10 ml medium/cm2) and measured glucose in the medium by sampling 20 µl of the culture supernatant daily using a standard glucometer. At the same time points, we resuspended the cultures and quantified cell numbers by cell counting using trypan blue exclusion. As shown in Figure 4a, the glucose concentration in the G-Rex devices progressively decreased over the culture period (250.3 ± 1.5, 229.7 ± 2.9, 158.3 ± 0.6, 45.7 ± 1.5 mg/dl on days 0, 3, 6, and 9, respectively), which inversely correlated with an increase in cell numbers determined by cell counting (0.125 × 106, 0.9 ± 0.1 × 106, 5.43 ± 0.3 × 106, and 12.87 ± 0.6 × 106 cells/cm2 on days 0, 3, 6, and 9, respectively). Based on this inverse correlation, we developed a formula to calculate cell number based solely on glucose measurements. The formula is as follows:


Optimizing the production of suspension cells using the G-Rex "M" series.

Bajgain P, Mucharla R, Wilson J, Welch D, Anurathapan U, Liang B, Lu X, Ripple K, Centanni JM, Hall C, Hsu D, Couture LA, Gupta S, Gee AP, Heslop HE, Leen AM, Rooney CM, Vera JF - Mol Ther Methods Clin Dev (2014)

Measuring glucose as a surrogate for culture performance. Panel (a) shows the glucose concentration in culture medium, as measured using a standard glucometer, and the inverse correlation between glucose and cell number. Panel (b) shows the formula we developed to calculate the number of cells in the culture based on the glucose concentration in the culture medium. Panel (c) shows the cell number obtained by: (i) hemocytometer counting, (ii) flow cytometry, and (iii) the glucose consumption formula.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4362380&req=5

fig4: Measuring glucose as a surrogate for culture performance. Panel (a) shows the glucose concentration in culture medium, as measured using a standard glucometer, and the inverse correlation between glucose and cell number. Panel (b) shows the formula we developed to calculate the number of cells in the culture based on the glucose concentration in the culture medium. Panel (c) shows the cell number obtained by: (i) hemocytometer counting, (ii) flow cytometry, and (iii) the glucose consumption formula.
Mentions: Traditionally, in order to accurately quantify cell numbers, one must first generate a homogenous cell suspension from which to sample. However, since the G-Rex accommodates large media volumes (e.g., 10 ml/cm2), cell resuspension is challenging. To address this issue, we sought to identify an alternate marker that could be used to predict cell growth. We initiated G-Rex cultures (n = 3) using optimal conditions (1.25 × 105 K562 cells/cm2 with 10 ml medium/cm2) and measured glucose in the medium by sampling 20 µl of the culture supernatant daily using a standard glucometer. At the same time points, we resuspended the cultures and quantified cell numbers by cell counting using trypan blue exclusion. As shown in Figure 4a, the glucose concentration in the G-Rex devices progressively decreased over the culture period (250.3 ± 1.5, 229.7 ± 2.9, 158.3 ± 0.6, 45.7 ± 1.5 mg/dl on days 0, 3, 6, and 9, respectively), which inversely correlated with an increase in cell numbers determined by cell counting (0.125 × 106, 0.9 ± 0.1 × 106, 5.43 ± 0.3 × 106, and 12.87 ± 0.6 × 106 cells/cm2 on days 0, 3, 6, and 9, respectively). Based on this inverse correlation, we developed a formula to calculate cell number based solely on glucose measurements. The formula is as follows:

Bottom Line: Broader implementation of cell-based therapies has been hindered by the logistics associated with the expansion of clinically relevant cell numbers ex vivo.A multicenter study confirmed that this fully optimized cell culture system can reliably produce a 100-fold cell expansion in only 10 days using 1L of medium.The G-Rex M series is linearly scalable and adaptable as a closed system, allowing an easy translation of preclinical protocols into the good manufacturing practice.

View Article: PubMed Central - PubMed

Affiliation: Center for Cell and Gene Therapy, Baylor College of Medicine, Houston Methodist Hospital, Texas Children's Hospital , Houston, Texas, USA.

ABSTRACT
Broader implementation of cell-based therapies has been hindered by the logistics associated with the expansion of clinically relevant cell numbers ex vivo. To overcome this limitation, Wilson Wolf Manufacturing developed the G-Rex, a cell culture flask with a gas-permeable membrane at the base that supports large media volumes without compromising gas exchange. Although this culture platform has recently gained traction with the scientific community due to its superior performance when compared with traditional culture systems, the limits of this technology have yet to be explored. In this study, we investigated multiple variables including optimal seeding density and media volume, as well as maximum cell output per unit of surface area. Additionally, we have identified a novel means of estimating culture growth kinetics. All of these parameters were subsequently integrated into a novel G-Rex "M" series, which can accommodate these optimal conditions. A multicenter study confirmed that this fully optimized cell culture system can reliably produce a 100-fold cell expansion in only 10 days using 1L of medium. The G-Rex M series is linearly scalable and adaptable as a closed system, allowing an easy translation of preclinical protocols into the good manufacturing practice.

No MeSH data available.


Related in: MedlinePlus