Increasing efficiency of human mesenchymal stromal cell culture by optimization of microcarrier concentration and design of medium feed.

BACKGROUND AIMS: Large amounts of human mesenchymal stromal cells (MSCs) are needed for clinical cellular therapy. In a previous publication, we described a microcarrier-based process for expansion of MSCs. The present study optimized this process by selecting suitable basal media, microcarrier concentration and feeding regime to achieve higher cell yields and more efficient medium utilization. METHODS: MSCs were expanded in stirred cultures on Cytodex 3 microcarriers with media containing 10% fetal bovine serum. Process optimization was carried out in spinner flasks. A 2-L bioreactor with an automated feeding system was used to validate the optimized parameters explored in spinner flask cultures. RESULTS: Minimum essential medium-α-based medium supported faster MSC growth on microcarriers than did Dulbecco's modified Eagle's medium (doubling time, 31.6 ± 1.4 vs 42 ± 1.7 h) and shortened the process time. At microcarrier concentration of 8 mg/mL, a high cell concentration of 1.08 × 10(6) cells/mL with confluent cell concentration of 4.7 × 10(4)cells/cm(2) was achieved. Instead of 50% medium exchange every 2 days, we have designed a full medium feed that is based on glucose consumption rate. The optimal medium feed that consisted of 1.5 g/L glucose supported MSC growth to full confluency while achieving the low medium usage efficiency of 3.29 mL/10(6)cells. Finally, a controlled bioreactor with the optimized parameters achieved maximal confluent cell concentration with 16-fold expansion and a further improved medium usage efficiency of 1.68 mL/10(6)cells. CONCLUSIONS: We have optimized the microcarrier-based platform for expansion of MSCs that generated high cell yields in a more efficient and cost-effective manner. This study highlighted the critical parameters in the optimization of MSC production process.

Evolution and diversity of community-associated methicillin-resistant Staphylococcus aureus in a geographical region.

BACKGROUND: Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) was first reported in remote regions of Western Australia and is now the predominant MRSA isolated in the state. The objective of this study is to determine the genetic relatedness of Western Australian CA-MRSA clones within different multilocus sequence type (MLST) clonal clusters providing an insight into the frequency of S. aureus SCCmec acquisition within a region. RESULTS: The CA-MRSA population in Western Australia is genetically diverse consisting of 83 unique pulsed-field gel electrophoresis strains from which 46 MLSTs have been characterised. Forty five of these sequence types are from 18 MLST clonal clusters and two singletons. While SCCmec IV and V are the predominant SCCmec elements, SCCmec VIII and several novel and composite SCCmec elements are present. The emergence of MRSA in diverse S. aureus clonal clusters suggests horizontal transmission of the SCCmec element has occurred on multiple occasions. Furthermore DNA microarray and spa typing suggests horizontal transfer of SCCmec elements has also occurred within the same CC. For many single and double locus variant CA-MRSA clones only a few isolates have been detected. CONCLUSIONS: Although multiple CA-MRSA clones have evolved in the Western Australian community only three clones have successfully adapted to the Western Australian community environment. These data suggest the successful evolution of a CA-MRSA clone may not only depend on the mobility of the SCCmec element but also on other genetic determinants.