Pharmaceutical Production Research Facility, Schulich School of Engineering, University of Calgary, Calgary, Alberta, Canada.
Cytotherapy. 2013 Jul;15(7):805-19. doi: 10.1016/j.jcyt.2013.01.011. Epub 2013 Mar 9.
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.
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.
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.
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.
滑膜来源的间充质基质细胞（S-MSCs）在临床关节修复应用中具有潜在的应用价值。然而，由于组织中 S-MSCs 的稀缺性，它们不能大量分离，需要在培养中进行扩增。由于体内滑膜组织暴露于比培养介质中通常发现的更高的钙（Ca（2+））水平，因此本研究检查了 Ca（2+）补充对培养中 S-MSC 增殖速度的影响。
S-MSCs 连续培养有或没有 Ca（2+）补充。通过使用 Ca（2+）通道阻滞剂评估抑制 Ca（2+）摄取的效果。在长时间暴露于高浓度 Ca（2+）后，通过评估表面标记物谱、进行逆转录定量聚合酶链反应和进行三系分化测定来对 S-MSCs 进行表征。
升高的 Ca（2+）浓度导致 S-MSC 增殖增强。在 5.0mmol/L Ca（2+）时达到峰值生长，与未补充培养基相比，在 8 个传代中平均每个传代的增长率为 4.52±0.65。Ca（2+）通道阻滞剂抑制增殖。补充 Ca（2+）的细胞表现出增强的成骨能力（17.82±4.21μg Ca（2+）/样品与 12.70±2.11μg Ca（2+）/样品）和成脂能力（0.47±0.04mg 油红 O/样品与 0.352±0.005mg 油红 O/样品），并保持其软骨形成能力（1.37±0.07μg 糖胺聚糖/小球与 1.33±0.17μg 糖胺聚糖/小球）。在高 Ca（2+）中培养的 S-MSCs 表达增强的 SOX-9 和过氧化物酶体增殖物激活受体γ的信使 RNA 水平，并降低胶原 I 的水平。
以前没有报道过 S-MSC 对 Ca（2+）的敏感性。这些发现表明，通过 Ca（2+）补充可以上调 S-MSC 群体扩增率，而不会损害其特征细胞特性。本研究举例说明了在培养干细胞时需要考虑培养基组成的必要性。
