Introduction: Numerous clinical trials in which MSC cells were used have lead to different results, not always as positive as expected. According to our hypothesis MSC can be replaced in some therapies by active factors they produce. Therefore, we decided to replace the primary MSC cultures with immortalized line derived from human adipose tissue (HATMSC1) to produce biologically active factors for basic and application study.
Methodology: The MSCs phenotype of immortalized cell line HATMSC1was confirmed using the flow cytometry method. The content of biologically active factors in conditioned media (CM) were assessed in standard culture conditions (however, without fetal bovine serum) and in CM stimulated by hypoxia and immunomodulatory factors (TNF-α or INF-γ or LPS) using RayBio® C-Series Human Cytokine Antibody Array C1000. The level of five selected cytokines was confirmed using the Multiplex ELISA method, Human Cytokine/Chemokine Magnetic Bead Panel MILLIPLEX® MAP Kit. Pro-proliferative activity of CM was evaluated using MTT assay and target cell lines involved in wound healing: fibroblasts (MSU-1.1), keratinocytes (HaCaT) and endothelial cells (HSkMEC.2). Pro-angiogenic activity was evaluated using Capillary-like Structures Formation Assay in the Matrigel.
Results: Rapidly proliferating immortalized cell line (HATMSC1) with the phenotype of MSCs produced above 100 out of 120 analyzed cytokines. Hypoxia trigger augmentation in the production of certain pro-angiogenic growth factors e.g. Angiogenin, IL-6, MCP-1, GRO, IL-8, TPO and VEGF by 20% - 30%. The production level of selected factors as assessed using the Multiplex ELISA was highest in the case of IL-8 and VEGF-A and reached a value of 2–2.5µg/mL while angiogenin was produced at the lowest concentration of 250 pg/mL.
The immunomodulatory factors like TNF-α, INF-γ or LPS caused an increase in production of many bioactive factors such as: GM-CSF, IL-5, IL-6, MCP-1, RANTES or IL-8 from 100 to 1200%!, compared to CM from standard culture conditions. Interestingly, the concentration of cytokines depends also on the cell density at the time of setting up the culture. There were cytokines produced more efficiently in dense culture (MCP-1 or IL-8) and those that were produced more efficiently in culture with low density (IL-6 or Osteoprotegerin). The in vitro pro-proliferative and pro-angiogenic activity of the CM produced by the HATMSC1 line was confirmed. Proliferation of target skin-origin cell lines increased up to 300% of control after 72h of culture when 50% CM was used. The highest pro-angiogenic effect was observed by increasing average mesh size by 200% over the control and was achieved after 14h with the use of 10% CM.
Conclusion(s): The results showed that immortalized cell line HATMSC1 cultured in the presence of TNF-α or INF-γ or LPS increased their ability to produce biologically active factors in terms of both their quantity and concentration. The data suggest that the factors produced under different culture conditions could potentially be used in different clinical conditions in regenerative medicine e.g. when increased concentration of pro-angiogenic factors is needed or in inflammatory diseases when high concentration of immunomodulatory factors is desired.