Development of 3D drug-testing platform for chronic lymphocytic leukemia

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F65269705%3A_____%2F19%3A00071807" target="_blank" >RIV/65269705:_____/19:00071807 - isvavai.cz</a>

Výsledek na webu

<a href="https://library.ehaweb.org/eha/2019/24th/266759/hana.svozilova.development.of.3d.drug-testing.platform.for.chronic.lymphocytic.html" target="_blank" >https://library.ehaweb.org/eha/2019/24th/266759/hana.svozilova.development.of.3d.drug-testing.platform.for.chronic.lymphocytic.html</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1097/01.HS9.0000562852.16850.75" target="_blank" >10.1097/01.HS9.0000562852.16850.75</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Development of 3D drug-testing platform for chronic lymphocytic leukemia

Popis výsledku v původním jazyce

Background: Conventionally, the efficacy of anti-cancer drugs in vitro has been tested in the traditional two-dimensional (2D) cell culture, mostly with plastic platforms. However, in the last few decades, studies have introduced a novel experimental design - a three-dimensional (3D) cell culture on the so-called scaffolds. These models have been found to represent a more suitable and accurate approach than the 2D systems (Gurski et al., 2010). Additionally, a co-culture with other cell types, which are normally present in the tissue, leads to an even closer approximation of the real in vivo situation. Developing an appropriate 3D co-culture in vitro drug-testing platform for chronic lymphocytic leukemia (CLL) is of utmost importance, given the microenvironment-dependency of CLL cells, which normally shortly undergo apoptosis in the conventional 2D cultures, even without any treatment. Aims: To develop an in vitro CLL model which enables a long-term culture of primary CLL cells and a subsequent drug-treatment of the cells. Methods: Primary CLL cells were cultured on two types of synthetic materials: i) 0.5% collagen scaffolds and ii) porous hydrogels made of poly(2-hydroxyl methacrylate-co-2-aminoethyl-methacrylate) modified with the cell adhesion peptide RGDS [p(HEMA-co-AEMA)-RGDS]. Cell distribution, proliferation, and viability were quantified by AlamarBlue assay or confocal microscopy. Results: We optimized a cell-seeding method which resulted in a homogenous cell distribution in the pores throughout the whole material. This method required the scaffold to be rotated in the tube with cell suspension with a concentration of 50 million cells per ml. Although overloaded in the first place, the scaffolds later tended to accommodate only hundreds of thousands of cells per piece. The number of living cells on the scaffolds was then maintained by co-culture with bone marrow stromal cells, which are normally present in the microenvironment of primary CLL cells in vivo. Supplementing the medium with recombinant interleukin 4 (10 ng/μl) and CD40 ligand (1 μg/μl), which were previously found to inhibit apoptosis of CLL cells (Mertens et al., 2012), also contributed to prolonged survival of the cells in vitro. Furthermore, we aimed to support media exchange in the pores of the materials by the gentle motion on a lab rocker. However, this motion was repeatedly proven to be insufficient, since it had no impact on the overall cell survival. Conclusion: We introduced first steps in creating an appropriate CLL 3D scaffold-based drug-testing platform, which mimics the real in vivo CLL microenvironment. The co-culture on the porous synthetic materials was found to possesses many important advantages, such as larger surface resulting in more frequent cell-cell and cell-surface contacts. In the future, we plan to implement either a dynamic growth in a bioreactor with microgravity (Pavesi et al., EHA 2018) or culture in a perfusion reactor (Bourgine et al., 2018).

Název v anglickém jazyce

Development of 3D drug-testing platform for chronic lymphocytic leukemia

Popis výsledku anglicky

Background: Conventionally, the efficacy of anti-cancer drugs in vitro has been tested in the traditional two-dimensional (2D) cell culture, mostly with plastic platforms. However, in the last few decades, studies have introduced a novel experimental design - a three-dimensional (3D) cell culture on the so-called scaffolds. These models have been found to represent a more suitable and accurate approach than the 2D systems (Gurski et al., 2010). Additionally, a co-culture with other cell types, which are normally present in the tissue, leads to an even closer approximation of the real in vivo situation. Developing an appropriate 3D co-culture in vitro drug-testing platform for chronic lymphocytic leukemia (CLL) is of utmost importance, given the microenvironment-dependency of CLL cells, which normally shortly undergo apoptosis in the conventional 2D cultures, even without any treatment. Aims: To develop an in vitro CLL model which enables a long-term culture of primary CLL cells and a subsequent drug-treatment of the cells. Methods: Primary CLL cells were cultured on two types of synthetic materials: i) 0.5% collagen scaffolds and ii) porous hydrogels made of poly(2-hydroxyl methacrylate-co-2-aminoethyl-methacrylate) modified with the cell adhesion peptide RGDS [p(HEMA-co-AEMA)-RGDS]. Cell distribution, proliferation, and viability were quantified by AlamarBlue assay or confocal microscopy. Results: We optimized a cell-seeding method which resulted in a homogenous cell distribution in the pores throughout the whole material. This method required the scaffold to be rotated in the tube with cell suspension with a concentration of 50 million cells per ml. Although overloaded in the first place, the scaffolds later tended to accommodate only hundreds of thousands of cells per piece. The number of living cells on the scaffolds was then maintained by co-culture with bone marrow stromal cells, which are normally present in the microenvironment of primary CLL cells in vivo. Supplementing the medium with recombinant interleukin 4 (10 ng/μl) and CD40 ligand (1 μg/μl), which were previously found to inhibit apoptosis of CLL cells (Mertens et al., 2012), also contributed to prolonged survival of the cells in vitro. Furthermore, we aimed to support media exchange in the pores of the materials by the gentle motion on a lab rocker. However, this motion was repeatedly proven to be insufficient, since it had no impact on the overall cell survival. Conclusion: We introduced first steps in creating an appropriate CLL 3D scaffold-based drug-testing platform, which mimics the real in vivo CLL microenvironment. The co-culture on the porous synthetic materials was found to possesses many important advantages, such as larger surface resulting in more frequent cell-cell and cell-surface contacts. In the future, we plan to implement either a dynamic growth in a bioreactor with microgravity (Pavesi et al., EHA 2018) or culture in a perfusion reactor (Bourgine et al., 2018).

Druh

O - Ostatní výsledky

CEP obor

—

OECD FORD obor

30205 - Hematology

Projekt

<a href="/cs/project/NV15-31834A" target="_blank" >NV15-31834A: Vliv selekce genomických poškození na průběh chronické lymfocytární leukémie</a><br>

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)<br>I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2019

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů