Guided evaluation and standardisation of mesenchymal stem cell culture conditions to generate conditioned medium favourable to cardiac c-kit cell growth

Abstract

Mesenchymal stem cells (MSCs) are known to secrete cardioprotective paracrine factors that can potentially activate endogenous cardiac c-kit cells (CCs). This study aims to optimise MSC growth conditions and medium formulation for generating the conditioned medium (CdM) to facilitate CC growth and expansion in vitro. The quality of MSC-CdM after optimisation of seeding density during MSC stabilisation and medium formulation used during MSC stimulation including glucose, ascorbic acid, serum and oxygen levels and the effects of treatment concentration and repeated CdM harvesting were assessed based on CC viability in vitro under growth factor- and serum-deprived condition. Our data showed that functional CdM can be produced from MSCs with a density of 20,000 cells/cm, which were stimulated using high glucose (25 mM), ascorbic acid supplemented, serum-free medium under normoxic condition. The generated CdM, when applied to growth factor- and serum-deprived medium at 1:1 ratio, improved CC viability, migration and proliferation in vitro. Such an effect could further be augmented by generating CdM concentrates without compromising CC gene and protein expressions, while retaining its capability to undergo differentiation to form endothelial, smooth muscle and cardiomyocytes. Nevertheless, CdM could not be repeatedly harvested from the same MSC culture, as the protein content and its effect on CC viability deteriorated after the first harvest. In conclusion, this study provides a proof-of-concept strategy to standardise the production of CdM from MSCs based on rapid, stepwise assessment of CC viability, thus enabling production of CdM favourable to CC growth for in vitro or clinical applications.

Enhanced Anti‐inflammatory Capacity of the Conditioned Medium Derived from Periodontal Ligament Stem Cells Modified with an Iron‐Based Nanodrug

“…[5] Ng et al compared CMs collected from bone marrow MSCs under 2% FBS and serum-free conditions, revealing that CM collected under serum-free conditions has more positive effects than that collected under 2%-FBS conditions. [45] Serum deprivation is used to stimulate growth factor secretion by simulating stress conditions during tissue damage. [44] The collection procedure of CM in this study follows Xia et al, [40] using media containing 10% FBS, which are not the best conditions for preparing effective CM.…”

Mesenchymal stem cells facilitate cardiac differentiation in Sox2‐expressing cardiac C‐kit cells in coculture

“…Bone marrow–derived MSCs were isolated from the femur and tibia bones of 4‐ to 6‐week‐old C57BL/6N mice, as described previously . Both the epiphysis ends of the bone were cut open with sterile scissors and flushed through using 27 to 29 G needles with an incubation medium into a 50 mL conical tube until the bones became clear.…”

“…Bone marrow-derived MSCs were isolated from the femur and tibia bones of 4-to 6-week-old C57BL/6N mice, as described previously. 32 Both the epiphysis ends of the bone were cut open with sterile scissors and flushed through using 27 to 29 G needles with an incubation medium into a 50 mL conical tube until the bones became clear. The cells were centrifuged at 380g for 5 minutes and resuspended in fresh bone marrow mesenchymal stem cell growth medium (BMGM; see Supporting Information).…”

Vibrational Spectroscopy-Based Chemometrics Analysis of Clinacanthus nutans Extracts after Postharvest Processing and Extract Effects on Cardiac C-Kit Cells

“…Despite the availability of advanced therapeutics, it is still challenging to reverse heart remodeling caused by cardiomyocyte loss [ 25 ]. Studies have reported that the adult heart possesses endogenous cardiac c-kit cells that can regenerate dead myocardium [ 26 , 27 ], and they can be activated from the quiescent state by growth factors [ 28 ] or by factors and matrices secreted by mesenchymal stem cells [ 29 – 31 ]. However, growth factors are expensive and limited by challenges such as low plasma stability, short biological half-lives, and low specificity to target organs [ 32 ].…”