Adipose-derived stem cells (ASCs) culture in spinner flask: improving the parameters of culture in a microcarrier-based system.

Prior to clinical use, extensive in vitro proliferation of human adipose-derived stem cells (ASCs) is required. Among the current options, spinner-type stirred flasks, which use microcarriers to increase the yield of adherent cells, are recommended. Here, we propose a methodology for ASCs proliferation through cell suspension culture using Cultispher-S® microcarriers (MC) under agitation in a spinner flask, with the aim of establishing a system that reconciles the efficiency of cell yield with high viability of the culture during two distinct phases: seeding and proliferation. The results showed that cell adhesion was potentiated under intermittent stirring at 70 rpm in the presence of 10% FBS for an initial cell concentration of 2.4 × 104 cells/mL in the initial 24 h of cultivation. In the proliferation phase, kinetic analysis showed that cell growth was higher under continuous agitation at 50 rpm with a culture medium renewal regime of 50% every 72 h, which was sufficient to maintain the culture at optimal levels of nutrients and metabolites for up to nine days of cultivation, representing an 11.1-fold increase and a maximum cell productivity of 422 cells/mL/h (1.0 × 105 viable cells/mL). ASCs maintained the immunophenotypic characteristics and mesodermal differentiation potential of both cell lines from different donors. The established protocol represents a more efficient and cost-effective method to obtain a high proliferation rate of ASCs in a microcarrier-based system, which is necessary for large-scale use in cell therapy, highlighting that the manipulation of critical parameters optimizes the ASCs production process.

Basophil activation test with indomethacin to assess hypersensitivity to non-steroidal anti-inflammatory drugs: a preliminary study.

BACKGROUND: The basophil activation test has been investigated for diagnosing hypersensitivity to non-steroidal anti-inflammatory drugs (NSAIDs). This has not yet been done in relation to indomethacin. OBJECTIVE: First seek to establish the viable concentrations of indomethacin and the diluent propylene glycol (PPG) in relation to basophils then test this in patients with hypersensitivity to NSAIDs. MATERIALS & METHODS: The ideal concentrations of PPG and indomethacin were assessed by incubating them with basophils from an atopic donor and evaluating the intensity of expression of CD63 molecules by means of flow cytometry. We also evaluated the cell viability directly using the trypan blue in seven controls. Then indomethacin was tested in ten patients with hypersensitivity to NSAIDs compared with eight persons in control group. RESULTS: In relation to the toxicity of propylene glycol, concentrations less than or equal to 0.5% are safe. There was no cytotoxicity or nonspecific stimulation from using indomethacin at concentrations of 10 mcg/mL, 1 mcg/mL and 0.1 mcg/mL. Then indomethacin was tested at concentration of 10 mcg/mL diluted in 0.5% propylene glycol in both groups. There was no statistical difference in the intensity of activation of basophils comparing the group of patients with hypersensitivity to NSAIDs and the control group. CONCLUSIONS: As a diluent for indomethacin, PPG should be used at concentrations less than or equal to 0.5%. The indomethacin at concentration of 10 mcg/mL was not able to differentiate patients with and without hypersensitivity to NSAIDs.