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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.


Results from our multicenter study validating our optimized G-Rex culture conditions. Panel (a) shows a schematic diagram of the G-Rex 100M device and protocol for cell culture. Panel (b) shows a comparison between the expansion of K562 cells obtained at Baylor College of Medicine and Celgene Corporation using an initial seeding density of 1.25 × 105 cells/cm2 in a G-Rex 100M with 1 l of medium. Panel (c) shows a comparison between the expansion of K562 cells obtained at our Center (Baylor), Celgene Corporation, and City of Hope when G-Rex 100M devices were seeded at 2.5 × 105 cells/cm2 in 1L of medium. Panel (d) shows the final cell output obtained when G-Rex 100M devices were seeded at either 1.25 × 105 cells/cm2 (Baylor and Celgene Corporation) or 2.5 × 105 cells/cm2 (Baylor, Celgene Corporation, and City of Hope) in 1L of medium. Panel (e) shows the fold expansion of K562 cells in the experiments described in panel d.
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fig5: Results from our multicenter study validating our optimized G-Rex culture conditions. Panel (a) shows a schematic diagram of the G-Rex 100M device and protocol for cell culture. Panel (b) shows a comparison between the expansion of K562 cells obtained at Baylor College of Medicine and Celgene Corporation using an initial seeding density of 1.25 × 105 cells/cm2 in a G-Rex 100M with 1 l of medium. Panel (c) shows a comparison between the expansion of K562 cells obtained at our Center (Baylor), Celgene Corporation, and City of Hope when G-Rex 100M devices were seeded at 2.5 × 105 cells/cm2 in 1L of medium. Panel (d) shows the final cell output obtained when G-Rex 100M devices were seeded at either 1.25 × 105 cells/cm2 (Baylor and Celgene Corporation) or 2.5 × 105 cells/cm2 (Baylor, Celgene Corporation, and City of Hope) in 1L of medium. Panel (e) shows the fold expansion of K562 cells in the experiments described in panel d.

Mentions: Based on these observations, Wilson Wolf Manufacturing designed the G-Rex M series (as seen in Figure 5a), a novel cell culture platform built to incorporate these ideal culture conditions. To determine whether the results achieved in G-Rex 100M in our Center could be reproduced in other institutions, we conducted a multicenter study supported by the NHLBI-Production Assistance for Cellular Therapies (PACT) program. We provided our optimized culture recommendations to both academic institutions (City of Hope and University of Wisconsin–Madison) and a biotech company (Celgene) and asked them to repeat our G-Rex expansion experiments. As illustrated in Figure 5b, when the group at Celgene cultured K562 cells at an initial seeding density of 1.25 × 105 cells/cm2 in a G-Rex 100M (100 cm2 surface area) with 1L of medium (10 ml/cm2), the total cell number achieved by day 12 of culture was 1.56 × 109 (15.6 × 106 cells/cm2, n = 1), which was similar to our findings (12.9 ± 0.6 × 106 cells/cm2, n = 3). Next, to confirm that the maximum cell output remained fixed regardless of the initial seeding density, K562 cells were cultured at 2.5 × 105 cells/cm2 in a G-Rex 100M in 1L of medium and, as shown in Figure 5c, after 12 days of culture, our group, City of Hope, and Celgene all reported similar total cell numbers (1.30 × 109 (n = 3), 1.46 × 109 (n = 1), and 1.35 × 109 (n = 1), respectively). As expected, regardless of initial seeding density, maximum number of cells obtained was the same, confirming that the highest expansion is achieved by using the lowest seeding density possible (Figure 5d,e). These data, collected at different institutions, validate the use of the G-Rex as a highly reproducible platform for the culture of suspension cells and confirms that our optimized conditions produce highly predictable results.


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)

Results from our multicenter study validating our optimized G-Rex culture conditions. Panel (a) shows a schematic diagram of the G-Rex 100M device and protocol for cell culture. Panel (b) shows a comparison between the expansion of K562 cells obtained at Baylor College of Medicine and Celgene Corporation using an initial seeding density of 1.25 × 105 cells/cm2 in a G-Rex 100M with 1 l of medium. Panel (c) shows a comparison between the expansion of K562 cells obtained at our Center (Baylor), Celgene Corporation, and City of Hope when G-Rex 100M devices were seeded at 2.5 × 105 cells/cm2 in 1L of medium. Panel (d) shows the final cell output obtained when G-Rex 100M devices were seeded at either 1.25 × 105 cells/cm2 (Baylor and Celgene Corporation) or 2.5 × 105 cells/cm2 (Baylor, Celgene Corporation, and City of Hope) in 1L of medium. Panel (e) shows the fold expansion of K562 cells in the experiments described in panel d.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5: Results from our multicenter study validating our optimized G-Rex culture conditions. Panel (a) shows a schematic diagram of the G-Rex 100M device and protocol for cell culture. Panel (b) shows a comparison between the expansion of K562 cells obtained at Baylor College of Medicine and Celgene Corporation using an initial seeding density of 1.25 × 105 cells/cm2 in a G-Rex 100M with 1 l of medium. Panel (c) shows a comparison between the expansion of K562 cells obtained at our Center (Baylor), Celgene Corporation, and City of Hope when G-Rex 100M devices were seeded at 2.5 × 105 cells/cm2 in 1L of medium. Panel (d) shows the final cell output obtained when G-Rex 100M devices were seeded at either 1.25 × 105 cells/cm2 (Baylor and Celgene Corporation) or 2.5 × 105 cells/cm2 (Baylor, Celgene Corporation, and City of Hope) in 1L of medium. Panel (e) shows the fold expansion of K562 cells in the experiments described in panel d.
Mentions: Based on these observations, Wilson Wolf Manufacturing designed the G-Rex M series (as seen in Figure 5a), a novel cell culture platform built to incorporate these ideal culture conditions. To determine whether the results achieved in G-Rex 100M in our Center could be reproduced in other institutions, we conducted a multicenter study supported by the NHLBI-Production Assistance for Cellular Therapies (PACT) program. We provided our optimized culture recommendations to both academic institutions (City of Hope and University of Wisconsin–Madison) and a biotech company (Celgene) and asked them to repeat our G-Rex expansion experiments. As illustrated in Figure 5b, when the group at Celgene cultured K562 cells at an initial seeding density of 1.25 × 105 cells/cm2 in a G-Rex 100M (100 cm2 surface area) with 1L of medium (10 ml/cm2), the total cell number achieved by day 12 of culture was 1.56 × 109 (15.6 × 106 cells/cm2, n = 1), which was similar to our findings (12.9 ± 0.6 × 106 cells/cm2, n = 3). Next, to confirm that the maximum cell output remained fixed regardless of the initial seeding density, K562 cells were cultured at 2.5 × 105 cells/cm2 in a G-Rex 100M in 1L of medium and, as shown in Figure 5c, after 12 days of culture, our group, City of Hope, and Celgene all reported similar total cell numbers (1.30 × 109 (n = 3), 1.46 × 109 (n = 1), and 1.35 × 109 (n = 1), respectively). As expected, regardless of initial seeding density, maximum number of cells obtained was the same, confirming that the highest expansion is achieved by using the lowest seeding density possible (Figure 5d,e). These data, collected at different institutions, validate the use of the G-Rex as a highly reproducible platform for the culture of suspension cells and confirms that our optimized conditions produce highly predictable results.

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.