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Assessing the Layer-by-Layer Assembly of Cellulose Nanofibrils and Polyelectrolytes in Pancreatic Tumor Spheroid Formation
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Fibre Technology.ORCID iD: 0000-0002-6544-9955
KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.ORCID iD: 0009-0005-3637-2513
Department of Genetics, Genomics and Experimental Pathology, The Oncology Institute “Prof. Dr. I. Chiricuta”, 400015 Cluj-Napoca, Romania.ORCID iD: 0000-0001-5082-9341
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
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2023 (English)In: Biomedicines, E-ISSN 2227-9059, Vol. 11, no 11Article in journal (Refereed) Published
Abstract [en]

Three-dimensional (3D) tumor spheroids are regarded as promising models for utilization as preclinical assessments of chemo-sensitivity. However, the creation of these tumor spheroids presents challenges, given that not all tumor cell lines are able to form consistent and regular spheroids. In this context, we have developed a novel layer-by-layer coating of cellulose nanofibril–polyelectrolyte bilayers for the generation of spheroids. This technique builds bilayers of cellulose nanofibrils and polyelectrolytes and is used here to coat two distinct 96-well plate types: nontreated/non-sterilized and Nunclon Delta. In this work, we optimized the protocol aimed at generating and characterizing spheroids on difficult-to-grow pancreatic tumor cell lines. Here, diverse parameters were explored, encompassing the bilayer count (five and ten) and multiple cell-seeding concentrations (10, 100, 200, 500, and 1000 cells per well), using four pancreatic tumor cell lines—KPCT, PANC-1, MiaPaCa-2, and CFPAC-I. The evaluation includes the quantification (number of spheroids, size, and morphology) and proliferation of the produced spheroids, as well as an assessment of their viability. Notably, our findings reveal a significant influence from both the number of bilayers and the plate type used on the successful formation of spheroids. The novel and simple layer-by-layer-based coating method has the potential to offer the large-scale production of spheroids across a spectrum of tumor cell lines.

Place, publisher, year, edition, pages
MDPI AG , 2023. Vol. 11, no 11
Keywords [en]
pancreatic ductal adenocarcinoma, three-dimensional tumor model, layer-by-layer, cellulose nanofibrils
National Category
Biomaterials Science
Identifiers
URN: urn:nbn:se:kth:diva-339943DOI: 10.3390/biomedicines11113061ISI: 001107899000001Scopus ID: 2-s2.0-85178372456OAI: oai:DiVA.org:kth-339943DiVA, id: diva2:1814068
Note

QC 20231215

Available from: 2023-11-22 Created: 2023-11-22 Last updated: 2023-12-29Bibliographically approved
In thesis
1. Utilizing Biopolymers in 3D Tumor Modeling and Tumor Diagnosis
Open this publication in new window or tab >>Utilizing Biopolymers in 3D Tumor Modeling and Tumor Diagnosis
2023 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cancer represents a significant global public health challenge and ranks as the second mostcommon cause of death in the United States. The onset of cancer entails an initial phasewhere cells lose their polarity and disconnect from the normal basement membrane, allowingthem to form distinct three-dimensional (3D) configurations that interact with adjacent cellsand the surrounding microenvironment. Cells grown in 2D monolayers demonstrate differentgene expression patterns and different activation of signaling pathways compared to cellscultivated within the natural structure of tumor tissue of the same cell type. Multicellulartumor spheroids (MCTS) are extensively investigated as a well-studied model of organotypiccancer. These spheroids are formed by tumor cells, either alone or in combination with othercell types, and they can be created with or without the application of supportive scaffolds.The MCTSs are also considered promising models for preclinical assessments of chemosensitivity.However, the creation of these tumor spheroids presents challenges, as not alltumor cell lines can consistently form regular spheroids.Cellulose nanofibrils (CNF) have become essential as a sustainable and environmentallyfriendly material. For example, thin films, with inherent mechanical properties, and flexibility,offer versatility across various applications. Also known for its biocompatibility and non-toxicnature, native CNF is a natural option to use. Its fibrous structure closely mimics the collagenmatrix in human tissue, showing potential as an effective scaffold for 3D cell culture. In thisregard, an innovative Layer-by-Layer (LbL) coating technique using CNF-polyelectrolytebilayers was investigated to generate spheroids. This method constructs bilayers of CNFand polyelectrolytes and can coat various surfaces. In this thesis, the first focus was ondemonstrating the spheroid formation capability using low molecular weight polyelectrolytesin LbL assembly. Secondly, an investigation was conducted involving embedding of LbLgrownspheroids in a decellularized extracellular matrix (ECM) aiming to determine howECM, possessing suitable mechanical characteristics, could influence the cancer stem celltraits in spheroids. Thirdly, the thesis demonstrated the utilization of LbL for capturing andreleasing of circulating tumor cells. Lastly, the shift from using low molecular weightpolyelectrolytes in the LbL assembly to high molecular weight counterparts and analyzingthe differences in spheroid formation abilities to assess the underlying differences inmolecular weights of the polyelectrolytes was explored. All-in-all, employing the CNF-basedLbL surface coating strategy explored in the thesis has proven to be promising for thedevelopment of spheroid models closely resembling in vivo conditions and holds significantpotential for applications in drug development.

Abstract [sv]

Cancer utgör en betydande global utmaning inom folkhälsan och rankas som den nästvanligaste dödsorsaken i USA. Cancer börjar med en initial fas där celler förlorar sin polaritetoch lossnar från basalmembranet, vilket tillåter dem att bilda distinkta tredimensionella (3D)kluster som interagerar med intilliggande celler och den omgivande mikromiljön. Celler somodlas i 2D-monolager visar olika genuttryck och olika signalvägar jämfört med celler somodlas som mer naturlig 3D struktur likt tumörvävnad. Multicellulära tumörsfärer (MCTS) ärväl studerade som modell för organotypisk cancer, dessa sfärer bildas av tumörceller,antingen av samma typ eller i kombination med andra celltyper, och de kan skapas medeller utan användning av underliggande stödjande strukturer. MCTS betraktas även somlovande modell för preklinisk bedömning av cellernas kemokänslighet. Dock är skapandetav dessa tumörsfärer utmanande, eftersom alla tumörcellinjer inte verkar kunna bildaregelbundna sfärer.Cellulosananofibriller (CNF) är ett alternativ som hållbart och miljövänligt material. Sombland annat kan forma till tunna filmer, med inbyggda mekaniska egenskaper, flexibilitet ,erbjuder mångsidighet över olika tillämpningsområden, till följd av dess flexibilitet och dessinbyggda mekaniska egenskaper. Känd för sin biokompatibilitet och ofarliga natur fungerarCNF som ett bra alternativ för användning även inom biomedicinska tillämpningar. CNFstrukturen liknar kollagenmatrisen i mänsklig vävnad och visar potential som ett effektivtunderlag för 3D-cellodling. Inom detta område undersöktes en innovativ lager-på-lager (LbL)beläggningsmetod innehållande CNF och polyelektrolyt bilager för att skapa sfärer. Metodbygger bilager av CNF och polyelektrolyter som kan belägga ytor av olika material. Dennaavhandling fokuseradar först på att demonstrera sfärformationsförmågan vid användning avpolyelektrolyter med lågmolekylvikt i de tillverkade LbL-modifieringarna. Däreftergenomfördes en undersökning som innefattar inbäddning av sfärerna som bildades pådessa LbL-ytor i extracellulärmatris (ECM) dels i form av collagen samt decellulariseradextracellulärmatris. För att undersöka om ECMs mekaniska egenskaper kan påverkacancercellernas egenskaper i sfärerna samt om EMC med liknande mekaniska egenskaperlikt naturlig vävnad är lämplig. Därefter demonstrerar avhandlingen användningen av LbLför att fånga cirkulerande tumörceller som sedan kunde släppas i en liten volym. Till sistutforskades övergången från användning av polyelektrolyter med låg molekylvikt vid LbLuppbyggnadentill samma typ av polyelektrolyter men med högre molekylvikt, samtanalysera skillnaderna i möjligheten att bilda sfärer. Sammantaget har användningen avCNF-baserad LbL-ytbeläggningsstrategi som utforskats i avhandlingen visat sig varalovande för utvecklingen av multicellulära sfäroidmodeller och som kan ha en betydandepotential för tillämpningar inom läkemedelsutveckling.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2023. p. 64
Series
TRITA-CBH-FOU ; 2023:57
Keywords
3D tumor modeling, Spheroids, Cellulose Nano Fibrils, Layer by Layer, Extracellular matrix, Circulating Tumor Capturing, Pancreatic ductal adenocarcinoma (PDAC), 3D-tumörmodellering, Sfärer, Cellulosa, Lager-på-lager, Extracellulärmatris, Infångning av cirkulerande tumörceller, Mänskliga Duktala Adenocarcinom-Hepatocyter
National Category
Biomaterials Science
Research subject
Fibre and Polymer Science
Identifiers
urn:nbn:se:kth:diva-339964 (URN)978-91-8040-788-5 (ISBN)
Public defence
2023-12-15, F3 (Flodis), Lindstedtsvägen 26, Stockholm, 13:00 (English)
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Supervisors
Note

QC 20231124

Available from: 2023-11-24 Created: 2023-11-23 Last updated: 2024-01-08Bibliographically approved

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Abbasi Aval, NegarLahchaichi, EkeramFayazbakhsh, FarzanehPettersson, TorbjörnRussom, Aman

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