| 1. |
- Malakpour Permlid, Atena, et al.
(författare)
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A Novel 3D Polycaprolactone High-Throughput System for Evaluation of Toxicity in Normoxia and Hypoxia
- 2021
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Ingår i: Toxicology Reports. - : Elsevier BV. - 2214-7500. ; 8, s. 627-635
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Tidskriftsartikel (refereegranskat)abstract
- Two-dimensional (2D) culturing of cancer cells has been indispensable for the development of anti-cancer drugs. Drug development, however, is lengthy and costly with a high attrition rate, calling to mind that 2D culturing does not mimic the three-dimensional (3D) tumour microenvironment in vivo. Thus, began the development of 3D culture models for cancer research. We have constructed a 3D 96-well plate using electrospun fibres made of biocompatible polycaprolactone (PCL). Finely-cut PCL fibre pieces in water/ethanol solution was pipetted to the wells of hydrophobic 96-well plates. A fibrous network of approximately 200 µm thickness and high porosity was formed after crosslinking and drying. Human JIMT-1 breast cancer cells and fibroblasts were seeded into the network. Confocal microscopy shows that the cells grow throughout the fibre network. The toxicity of paclitaxel and an experimental salinomycin analogue was assessed and compared in 2D and 3D cultures incubated under conditions of normoxia and hypoxia often found in tumours. The toxicity of both compounds is lower when the cells are cultured in 3D compared to 2D when cultured in either normoxia or hypoxia. We conclude that our 96-well assay is a cost-efficient tool that may be used for high-throughput pre-clinical screening of potential anti-cancer compounds.
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| 2. |
- Malakpour Permlid, Atena
(författare)
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From a Road Less Travelled to a Worn Path: Three-Dimensional Tumour Models for Cancer Research and Therapeutics
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cancer is the second leading cause of death among both men and women worldwide. During therecent years, three-dimensional (3D) tumour models have gained increasing interest as a pre-clinicalplatform for screening of compounds for potential use in cancer therapy. It is becoming recognizedthat two-dimensional (2D) cell culturing, in which cells are grown on physiologically irrelevant flatsurfaces, are not reliable tools for investigating chemo sensitivity of anti-cancer drug candidates.Therefore, the emerging miniaturized 3D tumour models in vitro are better representatives of thehuman tumour growing in the in vivo microenvironment. We have established a complex 3D humantumour outside the body using randomly oriented highly porous electrospun polycaprolactone (PCL)fibres, which mimic the collagen structure of the extracellular matrix (ECM). The data show that monoculturesof cancer cells grow as dense multicellular spheroids in the biocompatible 3D scaffolds,while normal cells show spread-out and elongated morphology. When co-cultured, JIMT-1 breastcancer cells and human dermal fibroblasts (HDFs) show a growth pattern similar to what is found ina tumour with the cancer cells growing in tight clusters surrounded by the fibroblasts. When grown inmono-culture or co-culture, the cells grow in the entire depth of the 3D PCL network. In addition, wecharacterized the proteins deposited by the cells in the 3D scaffolds incubated in the absence orpresence of transforming growth factor-β1 (TGF-β1), a tumour promoting cytokine. The data showthat the fibrous ECM proteins fibronectin, collagen I, and laminin are deposited throughout the depthof 3D structure. TGF-β1 treatment did not have a significant effect on protein deposition butsignificantly modulated the activity of matrix metalloproteinases and the level of interleukine-6cytokines in the medium of our 3D culture. In TGF-β1-treated co-cultures, the cancer cells changedthe growth pattern from tight clusters to be spread out along elongated HDFs. The 3D human tumourin vitro was utilized for evaluation of efficacy of two anti-cancer compounds; a well-known anti-cancerdrug paclitaxel and an experimental salinomycin analogue (SAEC). The experiments were performedin hypoxia and normoxia. Paclitaxel treatment was more toxic to the cancer cells while the SAEC wasmore toxic to the HDFs in normoxia and hypoxia. Furthermore, we fabricated and validated a 96-wellplates with 3D PCL fibre network as a high-throughput screening (HTS)-based assay. We comparedthe toxicity of paclitaxel and SAEC in 2D and 3D under normoxic and hypoxic condition. The datashow that the 96-well 3D system is a cost-efficient tool that can be used for assessing of new potentialchemotherapeutic drugs in an HTS manner. Thus, we have “proof of principle” that our tailor-designedhuman tumour outside the body has structure similar to a tumour in the body and that it can be usedfor investigation of chemical toxicity.
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| 3. |
- Malakpour Permlid, Atena, et al.
(författare)
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Identification of Extracellular Matrix Proteins Secreted by Human Dermal Fibroblasts Cultured in 3D Electrospun Scaffolds
- 2021
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 11
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Tidskriftsartikel (refereegranskat)abstract
- The appreciation that cell interactions in tissues is dependent on their three dimensional (3D) distribution has stimulated the development of 3D cell culture models. We constructed an artificial 3D tumour by culturing human breast cancer JIMT-1 cells and human dermal fibroblasts (HDFs) in a 3D network of electrospun polycaprolactone fibres. Here, we investigate ECM components produced by the cells in the artificial 3D tumour, which is an important step in validating the model. Immunostaining and confocal fluorescence microscopy show that the ECM proteins fibronectin, collagen I, and laminin are deposited throughout the entire 3D structure. Secreted soluble factors including matrix metalloproteinases (MMPs) and interleukine-6 (IL-6) were analysed in collected medium and were found to be mainly derived from the HDFs. Treatment with transforming growth factor-β1 (TGF-β1), a major cytokine found in a tumour, significantly alters the MMP activity and IL-6 concentration. In addition, TGF-β1 treatment, changes the morphology of the HDFs to become more elongated and with increased linearized actin filaments compared to non-treated HDFs. Collectively, these novel findings suggest that the artificial 3D tumour displays a clear cell distribution and ECM deposition that resembles a tumour environment in vivo, suggesting an innovative biological model to study a human tumour.
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| 4. |
- Malakpour-Permlid, Atena, et al.
(författare)
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Two-dimensional cell culturing on glass and plastic : the past, the present, and the future
- 2022
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Ingår i: 3D Lung Models for Regenerating Lung Tissue. - 9780323908719 - 9780323908726 ; , s. 21-35
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Bokkapitel (refereegranskat)abstract
- Two-dimensional (2D) cell culturing implies the cultivation of cells, isolated from an organism, to permit growth in 2D. Two-dimensional tissue culture surfaces are made of different kinds of glass and plastics with various degrees of hydrophilicity. The hydrophilicity is determined by the surface chemistry, which determines the adsorption of proteins required for cell attachment. Cellular physiology is highly affected by the surface chemistry due to the preadsorption of proteins. The relevance of 2D cell culturing must be considered in the context of how the cells grow in the organism. The use of 2D cell culturing for cancer cells is questionable, since a tumor is a disorganized three-dimensional (3D) structure. However, epithelial cells grow in 2D although on a highly folded 2D basement membrane, which is supported by a 3D environment. To increase reproducibility of all cell culturing, standardization of surfaces and medium composition is required.
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| 5. |
- Malakpour Permlid, Atena, et al.
(författare)
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Unique animal friendly 3D culturing of human cancer and normal cells
- 2019
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Ingår i: Toxicology in Vitro. - : Elsevier BV. - 1879-3177 .- 0887-2333. ; 60, s. 51-60
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Tidskriftsartikel (refereegranskat)abstract
- Two-dimensional cell culturing has proven inadequate as a reliable preclinical tumour model due to many inherent limitations. Hence, novel three-dimensional (3D) cell culture models are needed, which in many aspects can mimic a native tumour with 3D extracellular matrix. Here, we present a 3D electrospun polycaprolactone (PCL) mesh mimicking the collagen network of tissue. The naturally hydrophobic PCL mesh was subjected to O2 plasma treatment to obtain hydrophilic fibres. Biocompatibility tests performed using L929 fibroblasts show that the 3D PCL fibre unit compartments were non-toxic. The human breast cancer cell lines MCF-7 and JIMT-1, the normal-like human breast cell line MCF-10A, and human adult fibroblast were cultured in PCL meshes and cell proliferation was monitored using the AlamarBlue® assay. Confocal microscopy and cryosectioning show that the cells penetrated deep into the fibre mesh within 1 week of cell culturing. The cancer cells form spheroids with the cells attaching mainly to each other and partly to the fibres. The human adult fibroblasts stretch out along the fibres while the MCF-10A cells stretch between fibres. Overall, we show that normal and cancer cells thrive in the 3D meshes cultured in fetal bovine free medium which eliminates the use of collagen as an extracellular matrix support.
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| 6. |
- Rafnsdóttir, Ólöf Birna, et al.
(författare)
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A new animal product free defined medium for 2D and 3D culturing of normal and cancer cells to study cell proliferation and migration as well as dose response to chemical treatment
- 2023
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Ingår i: Toxicology Reports. - 2214-7500. ; 10, s. 509-520
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Tidskriftsartikel (refereegranskat)abstract
- Cell culturing methods are increasingly used to reduce and replace the use of live animals in biomedical research and chemical toxicity testing. Although live animals are avoided when using cell culturing methods, they often contain animal-derived components of which one of the most commonly used is foetal bovine serum (FBS). FBS is added to cell culture media among other supplements to support cell attachment/spreading and cell proliferation. The safety, batch-to-batch variation, and ethical problems with FBS are acknowledged and therefore world-wide efforts are ongoing to produce FBS free media. Here, we present the composition of a new defined medium with only human proteins either recombinant or derived from human tissues. This defined medium supports long-term culturing/routine culturing of normal cells and of cancer cells, and can be used for freezing and thawing of cells, i.e. for cell banking. Here, we show for our defined medium, growth curves and dose response curves of cells grown in two and three dimensions, and applications such as cell migration. Cell morphology was studied in real time by phase contrast and phase holographic microscopy time-lapse imaging. The cell lines used are human cancer-associated fibroblasts, keratinocytes, breast cancer JIMT-1 and MDA-MB-231 cells, colon cancer CaCo-2 cells, and pancreatic cancer MiaPaCa-2 cells as well as the mouse L929 cell line. In conclusion, we present the composition of a defined medium without animal-derived products which can be used for routine culturing and in experimental settings for normal cells and for cancer cells, i.e. our defined medium provides a leap towards a universal animal product free cell culture medium.
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