OBJECTIVE: Mesenchymal Stromal Cells (MSCs) obtained from different tissues (eg: bone marrow, adipose tissue, gingival papilla) can be loaded in vitro with anti-cancer drugs by using simple standardized procedure. This provides a new tool to be used for drug delivery. However, the restricted lifespan of the MSCs represents a significant limitation for producing them in large amounts or long time studies. For that reason our aim was to establish immortalized MSCs that can contribute to improve both basic in vitro and preclinical studies providing high amounts of cells with stable characters and facilitate procedures for preparing large amounts of secretome containing microvesicles. Furthermore, immortalized-GFP (Green Fluorescent Protein) transfected cells could be also of help in cell-tracking and homing studies in in vivo models. MATERIALS AND METHODS: Immortalized cell line hASCs-TS were obtained by co-transducing cells with lentiviral vectors expressing the telomerase reverse transcriptase gene in combination with the SV40 viral gene (pLenti-hTERT and pLenti-III-SV40). hASCs-TS expressing the GFP (GFP-hASCs-TS) were obtained by lentiviral transduction. The line was characterized for expression of the MSCs markers and chondro-osteo-adipo differentiation ability. hASCs-TS/GFP+ were studied for their ability to uptake and release paclitaxel (PTX) and gemcitabine (GCB). From PTX treated cells (hASCs-TS/GFP+/PTX) were isolates, by ultracentrifugation, microvesicles associated to paclitaxel (PTX/MVs) that were analysed by TEM, counted by qNano Gold apparatus and tested on a standard pancreatic carcinoma cell line (CFPAC-1) proliferation by using a MTT in vitro assay. RESULTS: hASCs-TS/GFP+ maintained mesenchymal characters and were able to uptake both PTX and GCB and to release them in the CM after cell subculture. The amount of the drug releasing was enough to dramatically affect the in vitro proliferation of CFPAC-1 cells. This means that also immortalized cells hASCs-TS/GFP+ (like MSCs) were able to internalize and then release active molecule and no inactivation of GCB (a prodrug) was seen. From hASCs-TS/GFP+/PTX we isolated MVs in amounts enough to inhibit in vitro CFPAC-1 cells proliferation. By comparing PTX/MVs anticancer activity with that of pure PTX we approximately estimated that to each MV is associated an amount of about 1.90 fg of PTX. This suggest that by using immortalized cell lines, that can be expanded to obtain very high number of cells, it is possible to enter a large scale production of PTX/MVs. CONCLUSION: Human immortalized MSCs provides an interesting tool for a large scale production of cells to use for a new approach of cell mediated drug-delivery, that could be considered as an implement of surgical, radiological and traditional chemotherapy. The secretion of drug-associated microvesicles could represents a way to produce new drugs by biogenesis with reduced work and time. This could also help to simplify GMP-procedures requested by regulatory agency to carry out advanced cell-therapy.
Fluorescent-immortalized human adipose-derived stromal cells (hASCs-TS/GFP+) for studying cell drug delivery mediated by microvesicles / V.Coccè, L.Balducci, M.L. Falchetti, L.Pascucci, E. Ciusani, A.T. Brini, F. Sisto, G. Piovani, G. Alessandri, E. Parati, L. Cabeza, A. Pessina. ((Intervento presentato al convegno GISM tenutosi a Brescia nel 2016.
|Titolo:||Fluorescent-immortalized human adipose-derived stromal cells (hASCs-TS/GFP+) for studying cell drug delivery mediated by microvesicles|
|Data di pubblicazione:||2016|
|Settore Scientifico Disciplinare:||Settore MED/07 - Microbiologia e Microbiologia Clinica|
Settore BIO/14 - Farmacologia
|Citazione:||Fluorescent-immortalized human adipose-derived stromal cells (hASCs-TS/GFP+) for studying cell drug delivery mediated by microvesicles / V.Coccè, L.Balducci, M.L. Falchetti, L.Pascucci, E. Ciusani, A.T. Brini, F. Sisto, G. Piovani, G. Alessandri, E. Parati, L. Cabeza, A. Pessina. ((Intervento presentato al convegno GISM tenutosi a Brescia nel 2016.|
|Appare nelle tipologie:||14 - Intervento a convegno non pubblicato|