Effect of calcium on the proliferation kinetics of synovium-derived mesenchymal stromal cells.

BACKGROUND AIMS: Synovium-derived mesenchymal stromal cells (S-MSCs) have potential utility in clinical joint repair applications. However, their scarcity in tissues means S-MSCs cannot be isolated in large quantities and need to be expanded in culture. Because synovial tissues in vivo are exposed to higher calcium (Ca(2+)) levels than typically found in culture media, this study examined the impact of Ca(2+) supplementation on the rate of S-MSC proliferation in culture. METHODS: S-MSCs were serially cultured with or without Ca(2+) supplementation. The effect of inhibiting Ca(2+) uptake was assessed using Ca(2+) channel blockers. After extended exposure to elevated Ca(2+) concentrations, S-MSCs were characterized by evaluating surface marker profiles, performing reverse transcriptase quantitative polymerase chain reaction and carrying out tri-lineage differentiation assays. RESULTS: Elevated Ca(2+) concentrations resulted in enhanced S-MSC proliferation. Peak growth occurred at 5.0 mmol/L Ca(2+), with an average fold increase of 4.52 ± 0.65 per passage over 8 passages compared with 2.03 ± 0.46 in un-supplemented medium. Proliferation was inhibited by Ca(2+) channel blockers. Ca(2+)-supplemented cells showed enhanced capacity toward osteogenesis (17.82 ± 4.21 µg Ca(2+) deposited/sample vs. 12.70 ± 2.11 µg Ca(2+) deposited/sample) and adipogenesis (0.47 ± 0.04 mg oil red O/sample vs. 0.352 ± 0.005 mg oil red O/sample) and retained their capacity to undergo chondrogenesis (1.37 ± 0.07 µg glycosaminoglycan/pellet vs. 1.33 ± 0.17 µg glycosaminoglycan/pellet). S-MSCs cultured in elevated Ca(2+) expressed enhanced messenger RNA levels for SOX-9 and peroxisome proliferator activated receptor gamma and depressed levels for collagen I. CONCLUSIONS: S-MSC sensitivity to Ca(2+) has not been reported previously. These findings indicate that S-MSC population expansion rates may be up-regulated by Ca(2+) supplementation without compromising defining cell characteristics. This study exemplifies the need to consider medium composition when culturing stem cells.

Enhanced bioproduction of carvone in a two-liquid-phase partitioning bioreactor with a highly hydrophobic biocatalyst.

The microbial biotransformation of (-)-trans-carveol to the flavor and fragrance compound (R)-(-)-carvone by Rhodococcus erythropolis DCL14 was carried out in a 3 L two phase partitioning bioreactor with an immiscible liquid second phase in an effort to improve upon the reactor performance achieved in a single aqueous phase system. The purpose of employing the liquid second phase is to minimize biotransformation rate inhibition due to the accumulation of the toxic substrate (cis-carveol) and product (carvone) in the aqueous phase. 1-Dodecene was chosen as the solvent for this application because it is biocompatible, non-biodegradable and has a superior affinity for the target product (carvone) relative to the other solvents tested. However, when 1-dodecene was used in the biotransformation, the extremely hydrophobic R. erythropolis DCL14 created an emulsion with the organic solvent with significant sequestering of the cells into the organic phase and negligible substrate conversion. To overcome these operational difficulties, silicone oil, which is considered a liquid polymer, was used with the aim of preventing emulsification and sequestration of cells in the non-aqueous phase. Although some emulsification of the water-silicone oil was again created by the cells, operability was improved and, in fed-batch mode, the system was able to convert approximately 2(1/2) times more carveol than a benchmark single aqueous phase system before substrate/product toxicity caused the biotransformation to stop. This study has demonstrated enhancement of a microbial biotransformation for the production of a high value nutraceutical compound via the use of a second partitioning phase, along with operational challenges arising from the use of a highly hydrophobic organism in such systems.