The lymph node microenvironment is composed of different cell types, as fibroblasts reticular cells (FRCs), follicular dendritic cells (FDCs), and it is essential in providing signals that promote activation, proliferation and differentiation of normal hematopoietic cells and malignant cells, as leukemias and lymphomas. In my thesis work I have initially characterized the morphology, proliferation rate and gene expression of primary human lymph node stromal cells recently established in our laboratory. The morphological analysis of the cell line (named SLN3) revealed heterogeneity, indicating the presence of different cellular subpopulations. Based on this observation, we performed cell cloning by serial dilutions and isolated 10 clones with different proliferation rates and gene expression profiles. We then tested the growth under tridimensional (3D) culture conditions of SLN3 cells, by seeding them on porous scaffolds consisting of natural fibers of Collagen type I inserted inside a miniaturized optically accessible bioreactor (Moab). This bioreactor provides the opportunity of performing long-term dynamic in vitro cultures/co-cultures, allowing to grow human and mouse cells to perform drug testing, with the goal of minimizing the use of animal models. After 1-2 weeks of culture under perfusion, we performed immunofluorescence analyses and observed that the parental stromal line SLN3 and clone number 10 grew efficiently on scaffolds, recreating a lymph node-like microenvironment. Based on these results, we proceeded to perform 3D co-cultures of the stromal cells with primary human chronic lymphocytic leukemia cells from different patients. With these experiments, we showed that the 3D lymph node-like microenvironment created in vitro by human stromal cells inside our millifluidic system was able to support survival of leukemic cells, as shown by immunofluorescence analysis. The development of a tissue-like 3D system that mimics the spatial and cellular organization of the microenvironment in which chronic lymphocytic leukemia is activated and expanded is important, as it could allow to perform screening of antitumoral drugs, under conditions that mimic more closely the native lymphoid tissues. In conclusion, with our work we have developed a three-dimensional in vitro model that could also be used to study the biology of stroma-leukemia interactions.
Il microambiente linfonodale è composto da diversi tipi di cellule, come i fibroblasti reticolari (FRC) e le cellule dendritiche follicolari (FDC), ed è essenziale nel fornire segnali che promuovono l'attivazione, la proliferazione e la differenziazione delle cellule ematopoietiche normali, e di quelle tumorali, come leucemie e linfomi. Nel mio lavoro di tesi ho inizialmente caratterizzato la morfologia, il tasso di proliferazione e l'espressione genica di cellule stromali linfonodali umane primarie recentemente create nel nostro laboratorio. L’analisi morfologica della linea cellulare (denominata SLN3) ha mostrato eterogeneità, ad indicare la presenza di diverse sottopopolazioni cellulari. Alla luce di questa osservazione abbiamo eseguito il clonaggio delle cellule mediante diluizioni seriali e isolato 10 cloni con un tasso di proliferazione ed espressione genica diverso. Abbiamo poi testato la crescita in condizioni di coltura tridimensionale (3D) delle cellule SLN3, seminandole su supporti porosi costituiti da fibre naturali di Collagene di tipo I, inseriti all'interno di un bioreattore millifluidico (Moab). Quest’ultimo offre la possibilità di eseguire colture/co-culture dinamiche in vitro a lungo termine, consentendo di coltivare cellule umane e di topo per eseguire test con farmaci, con l'obiettivo di ridurre l'uso di modelli animali. Dopo colture di 1-2 settimane in perfusione, abbiamo eseguito analisi di immunofluorescenza, ed osservato che la linea stromale parentale SLN3 e il clone numero 10 crescevano in modo efficiente sugli scaffold, ricreando un microambiente simile a quello dei linfonodi. Sulla base di questi risultati, abbiamo proceduto ad eseguire co-culture 3D delle cellule stromali con cellule umane primarie di leucemia linfatica cronica provenienti da diversi pazienti. Con questi esperimenti abbiamo dimostrato che il microambiente 3D simile a un linfonodo, creato in vitro dalle cellule stromali umane all'interno del nostro sistema millifluidico, era in grado di supportare la sopravvivenza delle cellule leucemiche, come dimostrato dall'analisi di immunofluorescenza. Lo sviluppo di un sistema 3D simile a un tessuto che imita l'organizzazione spaziale e cellulare del microambiente in cui la leucemia linfatica cronica si attiva ed espande è importante, in quanto potrebbe consentire di eseguire lo screening di farmaci antitumorali in condizioni che imitano più da vicino i tessuti linfoidi nativi. In conclusione, con il nostro lavoro abbiamo sviluppato un modello tridimensionale in vitro che potrebbe essere utilizzato anche per studiare la biologia delle interazioni stroma-leucemia.
Sviluppo di un microambiente linfonodale umanizzato 3D per studiare le interazioni stroma-leucemia
FRANCHINO, MARTINA
2021/2022
Abstract
The lymph node microenvironment is composed of different cell types, as fibroblasts reticular cells (FRCs), follicular dendritic cells (FDCs), and it is essential in providing signals that promote activation, proliferation and differentiation of normal hematopoietic cells and malignant cells, as leukemias and lymphomas. In my thesis work I have initially characterized the morphology, proliferation rate and gene expression of primary human lymph node stromal cells recently established in our laboratory. The morphological analysis of the cell line (named SLN3) revealed heterogeneity, indicating the presence of different cellular subpopulations. Based on this observation, we performed cell cloning by serial dilutions and isolated 10 clones with different proliferation rates and gene expression profiles. We then tested the growth under tridimensional (3D) culture conditions of SLN3 cells, by seeding them on porous scaffolds consisting of natural fibers of Collagen type I inserted inside a miniaturized optically accessible bioreactor (Moab). This bioreactor provides the opportunity of performing long-term dynamic in vitro cultures/co-cultures, allowing to grow human and mouse cells to perform drug testing, with the goal of minimizing the use of animal models. After 1-2 weeks of culture under perfusion, we performed immunofluorescence analyses and observed that the parental stromal line SLN3 and clone number 10 grew efficiently on scaffolds, recreating a lymph node-like microenvironment. Based on these results, we proceeded to perform 3D co-cultures of the stromal cells with primary human chronic lymphocytic leukemia cells from different patients. With these experiments, we showed that the 3D lymph node-like microenvironment created in vitro by human stromal cells inside our millifluidic system was able to support survival of leukemic cells, as shown by immunofluorescence analysis. The development of a tissue-like 3D system that mimics the spatial and cellular organization of the microenvironment in which chronic lymphocytic leukemia is activated and expanded is important, as it could allow to perform screening of antitumoral drugs, under conditions that mimic more closely the native lymphoid tissues. In conclusion, with our work we have developed a three-dimensional in vitro model that could also be used to study the biology of stroma-leukemia interactions.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/15053