Use of microcarriers in Mobius® CellReady bioreactors to support growth of adherent cells

The Mobius CellReady bioreactor product platform incorporates novel disposable technologies that provide optimal performance for suspension mammalian cell culture. Here we show the utility of EMD Millipore’s 3L and 50L CellReady single use bioreactors for the cultivation of adherent mammalian cells on microcarriers. Cytodex and Solohill collagen microcarriers were first tested in a mixing study to assess feasibility. We evaluated the normalized mixing speed required in the 3L and 50L to achieve a suspension of the microcarriers and enable growth of the cells. Mixing Manufacturer specifications show Cytodex and Solohill microcarriers to be similar in density and size. Working with this assumption, mixing studies where performed using the Cytodex microcarriers in 3L Mobius CellReady and Solohill Collagen coated in 50L single use bioreactor to determine the slowest agitation speed or the just suspended mixing power inputs (P/V)js, required to fully suspend the microcarriers so that the beads are equally distributed in the bioreactor. Microcarrier distribution was assessed by sampling the bioreactor at varying depths. Then the dry weight of the microcarrier was used to determine the% relative sample weight to the target weight. Mixing Results Data show the (P/V)js to be ~0.6W/m 3 in both the 3L and 50L single use bioreactors 100% distribution corresponds to the theoretical concentration of microcarriers, which is 3g/L Cytodex in 3L bioreactor and 15g/L Solohill Collagen microcarriers in 50L bioreactor Cell Growth Initial cell culture runs were performed with MDCK and Human Mesenchymal Stem Cells (hMSCs) to evaluate the bioreactor agitation to support cell growth in the 3L Mobius CellReady single use bioreactor. The conditions that showed the best performance could then scaled to the 50L Mobius bioreactor. 1. Cultured MDCK cells on Cytodex microcarriers grew to a peak cell density of ~1e6cells/mL using a power input of 0.6W/m with a 2L working volume after 3 days. 2. Cultured hMSCs on Solohill microcarriers grew to a maximum total cell number of 6e6 cells using power input of 0.6-0.8W/m with a 2.4L working volume after 12 days. Conclusions 1. Data from the mixing experiments demonstrate the just suspended mixing power input was determined to be ~0.6W/m. 2. Cell growth experiments with hMSCs demonstrate comparable cell growth in the 3L and 50L Mobius CellReady bioreactor with total number of hMSCs * Correspondence: [email protected] EMD Millipore Corporation, 80 Ashby Rd, Bedford MA 01730, USA McGlothlen et al. BMC Proceedings 2013, 7(Suppl 6):P95 http://www.biomedcentral.com/1753-6561/7/S6/P95 © 2013 McGlothlen et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Table 1 Physical Characteristics of Microcarriers Microcarrier Cytodex 3 ® Solohill Collagen Coated Density (g/ml) 1.04 1.03 Hydrated Size (μm) 141-211 125-212 Concentration (g/ml) 3 15 Table 2 MDCK/ Cytodex 3 ® Microcarriers Process Table Variable Value Cells MDCK hMSCs Inoculation Density 4e5 cells/mL 5e3 cells/mL Substrate Cytodex 3 ® Solohill Growth Media DMEM w/ 4.5g/L Glucose, 2% FBS, 1% NEAA and 2mM L-Glutamine DMEM low glucose, 10% FBS, 8ng/ml bFGF, 2mM Glutamine, 1X Pen/Strep pH 7 NA DO (% Saturation) 45 NA Feed 1 Day 1: 100% Growth Media Day 6: 1000ml low glucose fresh medium Feed 2 Day 3: Drain 50% of the working volume and reefed with equal volume of Growth Media Day 9: 400ml high glucose fresh medium Batch Duration 7 days 12 days Figure 1 Illustrates the attachment of MDCK and hMSCs to Cytodex and Solohill microcarriers Figure 2 compares the viable cell density of MDCK cells at increasing power/volume impeller inputs and different bioreactors McGlothlen et al. BMC Proceedings 2013, 7(Suppl 6):P95 http://www.biomedcentral.com/1753-6561/7/S6/P95 Page 2 of 3