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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.


Identifying the optimal volume of medium to support maximal cell expansion. Panel (a) shows the maximum cell output per cm2 that was achieved in G-Rex devices that were seeded at an initial seeding density of 0.125 cells/cm2 and supplemented with different volumes of medium per cm2. Panel (b) shows the expansion of cultures that received a total of 10 ml medium/cm2 provided in (i) four increments of 2.5 ml/cm2, (ii) two increments of 5 ml/cm2, or (iii) 10 ml/cm2 up-front.
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fig3: Identifying the optimal volume of medium to support maximal cell expansion. Panel (a) shows the maximum cell output per cm2 that was achieved in G-Rex devices that were seeded at an initial seeding density of 0.125 cells/cm2 and supplemented with different volumes of medium per cm2. Panel (b) shows the expansion of cultures that received a total of 10 ml medium/cm2 provided in (i) four increments of 2.5 ml/cm2, (ii) two increments of 5 ml/cm2, or (iii) 10 ml/cm2 up-front.

Mentions: Having identified the optimal initial seeding density, we next wanted to define the optimal volume of medium that would support maximal cell output. Thus, we initiated cultures with 1.25 × 105 K562 cells/cm2 and supplemented the devices (n = 3 per condition) with various medium volumes ranging from 0.5 to 20 ml/cm2 on day 0. From that point on, medium was not replenished and culture performance was assessed daily by cell counting. As shown in Figure 3a, when using from 0.5 to 10 ml of medium per cm2, there was a direct correlation between volume and cell expansion. Thereafter, however, there was no benefit conferred by higher medium volumes (Figure 3a). We next explored how best to provide this medium volume to the cells. Figure 3b shows the different feeding schedules tested, which included (i) a total of 10 ml of medium per cm2 divided into four feedings (2.5 ml/cm2 added on days 0, 6, 12, and 18), (ii) 10 ml provided in two feedings (5 ml/cm2 added on days 0 and 12), and (iii) 10 ml/cm2 added up-front. Figure 3b shows that, irrespective of the feeding schedule, the maximum cell density achieved was similar (schedule (i) 11.4 ± 1.3 × 106 cells/cm2; schedule (ii) 11.8 ± 0.8 × 106 cells/cm2; schedule (iii) 12.9 ± 0.6 × 106 cells/cm2 (n = 3)). However, cultures that received all 10 ml/cm2 of medium up-front (schedule (iii)) grew exponentially and reached their maximum cell density by day 9–10 of culture, whereas addition of medium in a staggered fashion resulted in an interrupted growth pattern where the cells fluctuated between log and lag phase growth, prolonging the time until maximal cell output was achieved. Thus, we have demonstrated that 10 ml of medium per cm2 administered at culture setup results in the shortest time required to achieve maximum cell numbers.


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)

Identifying the optimal volume of medium to support maximal cell expansion. Panel (a) shows the maximum cell output per cm2 that was achieved in G-Rex devices that were seeded at an initial seeding density of 0.125 cells/cm2 and supplemented with different volumes of medium per cm2. Panel (b) shows the expansion of cultures that received a total of 10 ml medium/cm2 provided in (i) four increments of 2.5 ml/cm2, (ii) two increments of 5 ml/cm2, or (iii) 10 ml/cm2 up-front.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Identifying the optimal volume of medium to support maximal cell expansion. Panel (a) shows the maximum cell output per cm2 that was achieved in G-Rex devices that were seeded at an initial seeding density of 0.125 cells/cm2 and supplemented with different volumes of medium per cm2. Panel (b) shows the expansion of cultures that received a total of 10 ml medium/cm2 provided in (i) four increments of 2.5 ml/cm2, (ii) two increments of 5 ml/cm2, or (iii) 10 ml/cm2 up-front.
Mentions: Having identified the optimal initial seeding density, we next wanted to define the optimal volume of medium that would support maximal cell output. Thus, we initiated cultures with 1.25 × 105 K562 cells/cm2 and supplemented the devices (n = 3 per condition) with various medium volumes ranging from 0.5 to 20 ml/cm2 on day 0. From that point on, medium was not replenished and culture performance was assessed daily by cell counting. As shown in Figure 3a, when using from 0.5 to 10 ml of medium per cm2, there was a direct correlation between volume and cell expansion. Thereafter, however, there was no benefit conferred by higher medium volumes (Figure 3a). We next explored how best to provide this medium volume to the cells. Figure 3b shows the different feeding schedules tested, which included (i) a total of 10 ml of medium per cm2 divided into four feedings (2.5 ml/cm2 added on days 0, 6, 12, and 18), (ii) 10 ml provided in two feedings (5 ml/cm2 added on days 0 and 12), and (iii) 10 ml/cm2 added up-front. Figure 3b shows that, irrespective of the feeding schedule, the maximum cell density achieved was similar (schedule (i) 11.4 ± 1.3 × 106 cells/cm2; schedule (ii) 11.8 ± 0.8 × 106 cells/cm2; schedule (iii) 12.9 ± 0.6 × 106 cells/cm2 (n = 3)). However, cultures that received all 10 ml/cm2 of medium up-front (schedule (iii)) grew exponentially and reached their maximum cell density by day 9–10 of culture, whereas addition of medium in a staggered fashion resulted in an interrupted growth pattern where the cells fluctuated between log and lag phase growth, prolonging the time until maximal cell output was achieved. Thus, we have demonstrated that 10 ml of medium per cm2 administered at culture setup results in the shortest time required to achieve maximum cell numbers.

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.