BACKGROUND: Chronic lymphocytic leukemia (CLL) is a common adult leukemia characterized by the accumulation of neoplastic mature B cells in blood, bone marrow, lymph nodes, and spleen. The disease biology remains unresolved in many aspects, including the processes underlying the disease progression and relapses. However, studying CLL in vitro poses a considerable challenge due to its complexity and dependency on the microenvironment. Several approaches are utilized to overcome this issue, such as co-culture of CLL cells with other cell types, supplementing culture media with growth factors, or setting up a three-dimensional (3D) culture. Previous studies have shown that 3D cultures, compared to conventional ones, can lead to enhanced cell survival and altered gene expression. 3D cultures can also give valuable information while testing treatment response in vitro since they mimic the cell spatial organization more accurately than conventional culture. METHODS: In our study, we investigated the behavior of CLL cells in two types of material: (i) solid porous collagen scaffolds and (ii) gel composed of carboxymethyl cellulose and polyethylene glycol (CMC-PEG). We studied CLL cells' distribution, morphology, and viability in these materials by a transmitted-light and confocal microscopy. We also measured the metabolic activity of cultured cells. Additionally, the expression levels of MYC, VCAM1, MCL1, CXCR4, and CCL4 genes in CLL cells were studied by qPCR to observe whether our novel culture approaches lead to increased adhesion, lower apoptotic rates, or activation of cell signaling in relation to the enhanced contact with co-cultured cells. RESULTS: Both materials were biocompatible, translucent, and permeable, as assessed by metabolic assays, cell staining, and microscopy. While collagen scaffolds featured easy manipulation, washability, transferability, and biodegradability, CMC-PEG was advantageous for its easy preparation process and low variability in the number of accommodated cells. Both materials promoted cell-to-cell and cell-to-matrix interactions due to the scaffold structure and generation of cell aggregates. The metabolic activity of CLL cells cultured in CMC-PEG gel was similar to or higher than in conventional culture. Compared to the conventional culture, there was (i) a lower expression of VCAM1 in both materials, (ii) a higher expression of CCL4 in collagen scaffolds, and (iii) a lower expression of CXCR4 and MCL1 (transcript variant 2) in collagen scaffolds, while it was higher in a CMC-PEG gel. Hence, culture in the material can suppress the expression of a pro-apoptotic gene (MCL1 in collagen scaffolds) or replicate certain gene expression patterns attributed to CLL cells in lymphoid organs (low CXCR4, high CCL4 in collagen scaffolds) or blood (high CXCR4 in CMC-PEG).

Advanced Biomaterials Central European Institute of Technology Brno University of Technology Brno Czech Republic

Center of Molecular Medicine Central European Institute of Technology Masaryk University Brno Czech Republic

Department of Internal Medicine Hematology and Oncology University Hospital Brno and Faculty of Medicine Masaryk University Brno Czech Republic

Institute of Medical Genetics and Genomics University Hospital Brno and Faculty of Medicine Masaryk University Brno Czech Republic

Zobrazit více v PubMed

Agathangelidis A, Scarfò L, Barbaglio F, Apollonio B, Bertilaccio MTS, Ranghetti P, Ponzoni M, Leone G, De Pascali V, Pecciarini L, Ghia P, Caligaris-Cappio F, Scielzo C. Establishment and characterization of PCL12, a novel CD5+ chronic lymphocytic leukaemia cell line. PLOS ONE. 2015;10(6):e0130195. doi: 10.1371/journal.pone.0130195. PubMed DOI PMC

Agis H, Beirer B, Watzek G, Gruber R. Effects of carboxymethylcellulose and hydroxypropylmethylcellulose on the differentiation and activity of osteoclasts and osteoblasts. Journal of Biomedical Materials Research. Part A. 2010;95(2):504–509. doi: 10.1002/jbm.a.32842. PubMed DOI

Aljitawi OS, Li D, Xiao Y, Zhang D, Ramachandran K, Stehno-Bittel L, Van Veldhuizen P, Lin TL, Kambhampati S, Garimella R. A novel three-dimensional stromal-based model for in vitro chemotherapy sensitivity testing of leukemia cells. Leukemia & Lymphoma. 2014;55(2):378–391. doi: 10.3109/10428194.2013.793323. PubMed DOI PMC

Aravamudhan A, Ramos DM, Nada AA, Kumbar SG. Chapter 4-natural polymers: polysaccharides and their derivatives for biomedical applications. In: Kumbar SG, Laurencin CT, Deng M, editors. Natural and Synthetic Biomedical Polymers. Oxford: Elsevier; 2014. pp. 67–89. DOI

Ariyoshi W, Usui M, Sano K, Kawano A, Okinaga T, Nakashima K, Nakazawa K, Nishihara T. 3D spheroid culture models for chondrocytes using polyethylene glycol-coated microfabricated chip. Biomedical Research. 2020;41(4):187–197. doi: 10.2220/biomedres.41.187. PubMed DOI

Babrnáková J, Pavliňáková V, Brtníková J, Sedláček P, Prosecká E, Rampichová M, Filová E, Hearnden V, Vojtová L. Synergistic effect of bovine platelet lysate and various polysaccharides on the biological properties of collagen-based scaffolds for tissue engineering: Scaffold preparation, chemo-physical characterization, in vitro and ex ovo evaluation. Materials Science and Engineering: C. 2019;100(1–2):236–246. doi: 10.1016/j.msec.2019.02.092. PubMed DOI

Badekila AK, Rai P, Kini S. Identification and evaluation of an appropriate housekeeping gene for real time gene profiling of hepatocellular carcinoma cells cultured in three dimensional scaffold. Molecular Biology Reports. 2022;49(1):797–804. doi: 10.1007/s11033-021-06830-y. PubMed DOI

Bae J, Leo CP, Hsu SY, Hsueh AJ. MCL-1S, a splicing variant of the antiapoptotic BCL-2 family member MCL-1, encodes a proapoptotic protein possessing only the BH3 domain. The Journal of Biological Chemistry. 2000;275(33):25255–25261. doi: 10.1074/jbc.M909826199. PubMed DOI

Barbaglio F, Belloni D, Scarfò L, Sbrana FV, Ponzoni M, Bongiovanni L, Pavesi L, Zambroni D, Stamatopoulos K, Caiolfa VR, Ferrero E, Ghia P, Scielzo C. 3D co-culture model of chronic lymphocytic leukemia bone marrow microenvironment predicts patient-specific response to mobilizing agents. Haematologica. 2020;106(9):2334–2344. doi: 10.3324/haematol.2020.248112. PubMed DOI PMC

Barisione G, Fabbi M, Cutrona G, De Cecco L, Zupo S, Leitinger B, Gentile M, Manzoni M, Neri A, Morabito F, Ferrarini M, Ferrini S. Heterogeneous expression of the collagen receptor DDR1 in chronic lymphocytic leukaemia and correlation with progression. Blood Cancer Journal. 2017;7(1):e513. doi: 10.1038/bcj.2016.121. PubMed DOI PMC

Belloni D, Ferrarini M, Ferrero E, Guzzeloni V, Barbaglio F, Ghia P, Scielzo C. Protocol for generation of 3D bone marrow surrogate microenvironments in a rotary cell culture system. STAR Protocols. 2022;3(3):101601. doi: 10.1016/j.xpro.2022.101601. PubMed DOI PMC

Belloni D, Heltai S, Ponzoni M, Villa A, Vergani B, Pecciarini L, Marcatti M, Girlanda S, Tonon G, Ciceri F, Caligaris-Cappio F, Ferrarini M, Ferrero E. Modeling multiple myeloma-bone marrow interactions and response to drugs in a 3D surrogate microenvironment. Haematologica. 2018;103(4):707–716. doi: 10.3324/haematol.2017.167486. PubMed DOI PMC

Blanco TM, Mantalaris A, Bismarck A, Panoskaltsis N. The development of a three-dimensional scaffold for ex vivo biomimicry of human acute myeloid leukaemia. Biomaterials. 2010;31(8):2243–2251. doi: 10.1016/j.biomaterials.2009.11.094. PubMed DOI

Bray LJ, Binner M, Körner Y, von Bonin M, Bornhäuser M, Werner C. A three-dimensional ex vivo tri-culture model mimics cell-cell interactions between acute myeloid leukemia and the vascular niche. Haematologica. 2017;102(7):1215–1226. doi: 10.3324/haematol.2016.157883. PubMed DOI PMC

Bruce A, Evans R, Mezan R, Shi L, Moses BS, Martin KH, Gibson LF, Yang Y. Three-dimensional microfluidic tri-culture model of the bone marrow microenvironment for study of acute lymphoblastic leukemia. PLOS ONE. 2015;10(10):e0140506. doi: 10.1371/journal.pone.0140506. PubMed DOI PMC

Burger JA, Burger M, Kipps TJ. Chronic lymphocytic leukemia B cells express functional CXCR4 chemokine receptors that mediate spontaneous migration beneath bone marrow stromal cells. Blood. 1999;94(11):3658–3667. doi: 10.1182/blood.V94.11.3658. PubMed DOI

Burgess M, Cheung C, Chambers L, Ravindranath K, Minhas G, Knop L, Mollee P, McMillan NAJ, Gill D. CCL2 and CXCL2 enhance survival of primary chronic lymphocytic leukemia cells in vitro. Leukemia & Lymphoma. 2012;53(10):1988–1998. doi: 10.3109/10428194.2012.672735. PubMed DOI

Cao L, Zhao H, Qian M, Shao C, Zhang Y, Yang J. Construction of polysaccharide scaffold-based perfusion bioreactor supporting liver cell aggregates for drug screening. Journal of Biomaterials Science, Polymer Edition. 2022;33(17):2249–2269. doi: 10.1080/09205063.2022.2102715. PubMed DOI

Chiaraviglio L, Kirby JE. Evaluation of impermeant, DNA-binding dye fluorescence as a real-time readout of eukaryotic cell toxicity in a high throughput screening format. Assay and Drug Development Technologies. 2014;12(4):219–228. doi: 10.1089/adt.2014.577. PubMed DOI PMC

Clarke SA, Hoskins NL, Jordan GR, Henderson SA, Marsh DR. In vitro testing of Advanced JAXTM Bone Void Filler System: species differences in the response of bone marrow stromal cells to β tri-calcium phosphate and carboxymethylcellulose gel. Journal of Materials Science: Materials in Medicine. 2007;18(12):2283–2290. doi: 10.1007/s10856-007-3099-1. PubMed DOI

Clift MJD, Doak SH. Advanced in vitro models for replacement of animal experiments. Small. 2021;17(15):2101474. doi: 10.1002/smll.202101474. PubMed DOI

Crassini K, Shen Y, Mulligan S, Giles Best O. Modeling the chronic lymphocytic leukemia microenvironment in vitro. Leukemia & Lymphoma. 2017;58(2):266–279. doi: 10.1080/10428194.2016.1204654. PubMed DOI

Decrock E, De Vuyst E, Vinken M, Van Moorhem M, Vranckx K, Wang N, Van Laeken L, De Bock M, D’Herde K, Lai CP, Rogiers V, Evans WH, Naus CC, Leybaert L. Connexin 43 hemichannels contribute to the propagation of apoptotic cell death in a rat C6 glioma cell model. Cell Death & Differentiation. 2009;16(1):151–163. doi: 10.1038/cdd.2008.138. PubMed DOI

Eichhorst B, Robak T, Montserrat E, Ghia P, Niemann CU, Kater AP, Gregor M, Cymbalista F, Buske C, Hillmen P, Hallek M, Mey U. Chronic lymphocytic leukaemia: ESMO clinical practice guidelines for diagnosis, treatment and follow-up. Annals of Oncology. 2021;32(1):23–33. doi: 10.1016/j.annonc.2020.09.019. PubMed DOI

Fabbri G, Holmes AB, Viganotti M, Scuoppo C, Belver L, Herranz D, Yan XJ, Kieso Y, Rossi D, Gaidano G, Chiorazzi N, Ferrando AA, Dalla-Favera R. Common nonmutational NOTCH1 activation in chronic lymphocytic leukemia. Proceedings of the National Academy of Sciences of the United States of America. 2017;114(14):E2911–E2919. doi: 10.1073/pnas.1702564114. PubMed DOI PMC

Ferrarini M, Steimberg N, Ponzoni M, Belloni D, Berenzi A, Girlanda S, Caligaris-Cappio F, Mazzoleni G, Ferrero E. Ex-vivo dynamic 3-D culture of human tissues in the RCCSTM bioreactor allows the study of multiple myeloma biology and response to therapy. PLOS ONE. 2013;8(8):e71613. doi: 10.1371/journal.pone.0071613. PubMed DOI PMC

Hallek M, Al-Sawaf O. Chronic lymphocytic leukemia: 2022 update on diagnostic and therapeutic procedures. American Journal of Hematology. 2021;96(12):1679–1705. doi: 10.1002/ajh.26367. PubMed DOI

Haselager M, Perelaer E, Kater AP, Eldering E. Development of a novel lymph node-based 3D culture system promoting chronic lymphocytic leukemia proliferation and survival. Blood. 2020;136:47–48. doi: 10.1182/blood-2020-141962. DOI

Haselager MV, van Driel BF, Perelaer E, de Rooij D, Lashgari D, Loos R, Kater AP, Moerland PD, Eldering E. In vitro 3D spheroid culture system displays sustained T cell-dependent CLL proliferation and survival. HemaSphere. 2023;7(9):e938. doi: 10.1097/HS9.0000000000000938. PubMed DOI PMC

Herman SEM, Wiestner A. Preclinical modeling of novel therapeutics in chronic lymphocytic leukemia: the tools of the trade. Seminars in Oncology. 2016;43(2):222–232. doi: 10.1053/j.seminoncol.2016.02.007. PubMed DOI PMC

Hoferkova E, Kadakova S, Mraz M. In vitro and in vivo models of CLL-T cell interactions: implications for drug testing. Cancers. 2022;14(13):3087. doi: 10.3390/cancers14133087. PubMed DOI PMC

Karimpoor M, IIlangakoon E, Reid AG, Claudiani S, Edirisinghe M, Khorashad JS. Development of artificial bone marrow fibre scaffolds to study resistance to anti-leukaemia agents. British Journal of Haematology. 2018;182(6):924–927. doi: 10.1111/bjh.14883. PubMed DOI

Kitada S, Zapata JM, Andreeff M, Reed JC. Bryostatin and CD40-ligand enhance apoptosis resistance and induce expression of cell survival genes in B-cell chronic lymphocytic leukaemia. British Journal of Haematology. 1999;106(4):995–1004. doi: 10.1046/j.1365-2141.1999.01642.x. PubMed DOI

Kong X, Tang Q, Chen X, Tu Y, Sun S, Sun Z. Polyethylene glycol as a promising synthetic material for repair of spinal cord injury. Neural Regeneration Research. 2017;12(6):1003–1008. doi: 10.4103/1673-5374.208597. PubMed DOI PMC

Kurtova AV, Balakrishnan K, Chen R, Ding W, Schnabl S, Quiroga MP, Sivina M, Wierda WG, Estrov Z, Keating MJ, Shehata M, Jäger U, Gandhi V, Kay NE, Plunkett W, Burger JA. Diverse marrow stromal cells protect CLL cells from spontaneous and drug-induced apoptosis: development of a reliable and reproducible system to assess stromal cell adhesion-mediated drug resistance. Blood. 2009;114(20):4441–4450. doi: 10.1182/blood-2009-07-233718. PubMed DOI PMC

Lanemo Myhrinder A, Hellqvist E, Bergh A-C, Jansson M, Nilsson K, Hultman P, Jonasson J, Buhl AM, Bredo Pedersen L, Jurlander J, Klein E, Weit N, Herling M, Rosenquist R, Rosén A. Molecular characterization of neoplastic and normal “sister” lymphoblastoid B-cell lines from chronic lymphocytic leukemia. Leukemia & Lymphoma. 2013;54(8):1769–1779. doi: 10.3109/10428194.2013.764418. PubMed DOI

Lee SY, Bang S, Kim S, Jo SY, Kim BC, Hwang Y, Noh I. Synthesis and in vitro characterizations of porous carboxymethyl cellulose-poly(ethylene oxide) hydrogel film. Biomaterials Research. 2015;19(1):12. doi: 10.1186/s40824-015-0033-3. PubMed DOI PMC

Lezina L, Spriggs RV, Beck D, Jones C, Dudek KM, Bzura A, Jones GDD, Packham G, Willis AE, Wagner SD. CD40L/IL-4-stimulated CLL demonstrates variation in translational regulation of DNA damage response genes including ATM. Blood Advances. 2018;2(15):1869–1881. doi: 10.1182/bloodadvances.2017015560. PubMed DOI PMC

Metzger W, Sossong D, Bächle A, Pütz N, Wennemuth G, Pohlemann T, Oberringer M. The liquid overlay technique is the key to formation of co-culture spheroids consisting of primary osteoblasts, fibroblasts and endothelial cells. Cytotherapy. 2011;13(8):1000–1012. doi: 10.3109/14653249.2011.583233. PubMed DOI

Okkenhaug K, Burger JA. PI3K signaling in normal B cells and chronic lymphocytic leukemia (CLL) In: Kurosaki T, Wienands J, editors. B Cell Receptor Signaling. Current Topics in Microbiology and Immunology. Cham: Springer International Publishing; 2016. pp. 123–142. PubMed DOI PMC

Palma M, Krstic A, Peña Perez L, Berglöf A, Meinke S, Wang Q, Blomberg KEM, Kamali-Moghaddam M, Shen Q, Jaremko G, Lundin J, De Paepe A, Höglund P, Kimby E, Österborg A, Månsson R, Smith CIE. Ibrutinib induces rapid down-regulation of inflammatory markers and altered transcription of chronic lymphocytic leukaemia-related genes in blood and lymph nodes. British Journal of Haematology. 2018;183(2):212–224. doi: 10.1111/bjh.15516. PubMed DOI

Palomero T, Lim WK, Odom DT, Sulis ML, Real PJ, Margolin A, Barnes KC, O’Neil J, Neuberg D, Weng AP, Aster JC, Sigaux F, Soulier J, Look AT, Young RA, Califano A, Ferrando AA. NOTCH1 directly regulates c-MYC and activates a feed-forward-loop transcriptional network promoting leukemic cell growth. Proceedings of the National Academy of Sciences of the United States of America. 2006;103(48):18261–18266. doi: 10.1073/pnas.0606108103. PubMed DOI PMC

Panayiotidis P, Ganeshaguru K, Jabbar SA, Hoffbrand AV. Interleukin-4 inhibits apoptotic cell death and loss of the bcl-2 protein in B-chronic lymphocytic leukaemia cells in vitro. British Journal of Haematology. 1993;85(3):439–445. doi: 10.1111/j.1365-2141.1993.tb03330.x. PubMed DOI

Panayiotidis P, Jones D, Ganeshaguru K, Foroni L, Hoffbrand AV. Human bone marrow stromal cells prevent apoptosis and support the survival of chronic lymphocytic leukaemia cells in vitro. British Journal of Haematology. 1996;92(1):97–103. doi: 10.1046/j.1365-2141.1996.00305.x. PubMed DOI

Pascutti MF, Jak M, Tromp JM, Derks IAM, Remmerswaal EBM, Thijssen R, van Attekum MHA, van Bochove GG, Luijks DM, Pals ST, van Lier RAW, Kater AP, van Oers MHJ, Eldering E. IL-21 and CD40L signals from autologous T cells can induce antigen-independent proliferation of CLL cells. Blood. 2013;122(17):3010–3019. doi: 10.1182/blood-2012-11-467670. PubMed DOI

Passaro D, Abarrategi A, Foster K, Ariza-McNaughton L, Bonnet D. Bioengineering of humanized bone marrow microenvironments in mouse and their visualization by live imaging. JoVE. 2017;2017(126):e55914. doi: 10.3791/55914-v. PubMed DOI PMC

Philipp-Abbrederis K, Herrmann K, Knop S, Schottelius M, Eiber M, Lückerath K, Pietschmann E, Habringer S, Gerngroß C, Franke K, Rudelius M, Schirbel A, Lapa C, Schwamborn K, Steidle S, Hartmann E, Rosenwald A, Kropf S, Beer AJ, Peschel C, Einsele H, Buck AK, Schwaiger M, Götze K, Wester HJ, Keller U. In vivo molecular imaging of chemokine receptor CXCR4 expression in patients with advanced multiple myeloma. EMBO Molecular Medicine. 2015;7(4):477–487. doi: 10.15252/emmm.201404698. PubMed DOI PMC

Pozzo F, Bittolo T, Vendramini E, Bomben R, Bulian P, Rossi FM, Zucchetto A, Tissino E, Degan M, D’Arena G, Di Raimondo F, Zaja F, Pozzato G, Rossi D, Gaidano G, Del Poeta G, Gattei V, Dal Bo M. NOTCH1-mutated chronic lymphocytic leukemia cells are characterized by a MYC-related overexpression of nucleophosmin 1 and ribosome-associated components. Leukemia. 2017;31(11):2407–2415. doi: 10.1038/leu.2017.90. PubMed DOI

Priya G, Madhan B, Narendrakumar U, Suresh Kumar RV, Manjubala I. In vitro and in vivo evaluation of carboxymethyl cellulose scaffolds for bone tissue engineering applications. ACS Omega. 2021;6(2):1246–1253. doi: 10.1021/acsomega.0c04551. PubMed DOI PMC

Prosecká E, Rampichová M, Litvinec A, Tonar Z, Králíčková M, Vojtová L, Kochová P, Plencner M, Buzgo M, Míčková A, Jančář J, Amler E. Collagen/hydroxyapatite scaffold enriched with polycaprolactone nanofibers, thrombocyte-rich solution and mesenchymal stem cells promotes regeneration in large bone defect in vivo. Journal of Biomedical Materials Research. Part A. 2015;103(2):671–682. doi: 10.1002/jbm.a.35216. PubMed DOI

Pérez del Río E, Santos F, Rodriguez Rodriguez X, Martínez-Miguel M, Roca-Pinilla R, Arís A, Garcia-Fruitós E, Veciana J, Spatz JP, Ratera I, Guasch J. CCL21-loaded 3D hydrogels for T cell expansion and differentiation. Biomaterials. 2020;259:120313. doi: 10.1016/j.biomaterials.2020.120313. PubMed DOI

Ran Y, Dong Y, Li Y, Xie J, Zeng S, Liang C, Dai W, Tang W, Wu Y, Yu S. Mesenchymal stem cell aggregation mediated by integrin α4/VCAM-1 after intrathecal transplantation in MCAO rats. Stem Cell Research & Therapy. 2022;13(1):507. doi: 10.1186/s13287-022-03189-0. PubMed DOI PMC

Reuss-Borst MA, Ning Y, Klein G, Müller CA. The vascular cell adhesion molecule (VCAM-1) is expressed on a subset of lymphoid and myeloid leukaemias. British Journal of Haematology. 1995;89(2):299–305. doi: 10.1111/j.1365-2141.1995.tb03304.x. PubMed DOI

Rezvani Ghomi E, Nourbakhsh N, Akbari Kenari M, Zare M, Ramakrishna S. Collagen-based biomaterials for biomedical applications. Journal of Biomedical Materials Research. Part B, Applied Biomaterials. 2021;109(12):1986–1999. doi: 10.1002/jbm.b.34881. PubMed DOI

Ribezzi D, Pinos R, Bonetti L, Cellani M, Barbaglio F, Scielzo C, Farè S. Design of a novel bioink suitable for the 3D printing of lymphoid cells. Frontiers in Biomaterials Science. 2023;2:2102. doi: 10.3389/fbiom.2023.1081065. DOI

Rosén A, Bergh AC, Gogok P, Evaldsson C, Myhrinder AL, Hellqvist E, Rasul A, Björkholm M, Jansson M, Mansouri L, Liu A, Teh BT, Rosenquist R, Klein E. Lymphoblastoid cell line with B1 cell characteristics established from a chronic lymphocytic leukemia clone by in vitro EBV infection. Oncoimmunology. 2012;1(1):18–27. doi: 10.4161/onci.1.1.18400. PubMed DOI PMC

Santos F, Valderas-Gutiérrez J, Pérez Del Río E, Castellote-Borrell M, Rodriguez XR, Veciana J, Ratera I, Guasch J. Enhanced human T cell expansion with inverse opal hydrogels. Biomaterials Science. 2022;10(14):3730–3738. doi: 10.1039/D2BM00486K. PubMed DOI

Sbrana FV, Pinos R, Barbaglio F, Ribezzi D, Scagnoli F, Scarfò L, Redwan IN, Martinez H, Farè S, Ghia P, Scielzo C. 3D bioprinting allows the establishment of long-term 3D culture model for chronic lymphocytic leukemia cells. Frontiers in Immunology. 2021;12:38. doi: 10.3389/fimmu.2021.639572. PubMed DOI PMC

Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez J-Y, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A. Fiji: an open-source platform for biological-image analysis. Nature Methods. 2012;9(7):676–682. doi: 10.1038/nmeth.2019. PubMed DOI PMC

Scielzo C, Ghia P. Modeling the leukemia microenviroment in vitro. Frontiers in Oncology. 2020;10:615. doi: 10.3389/fonc.2020.607608. PubMed DOI PMC

Sen A, Kallos MS, Behie LA. Expansion of mammalian neural stem cells in bioreactors: effect of power input and medium viscosity. Developmental Brain Research. 2002;134(1–2):103–113. doi: 10.1016/S0165-3806(01)00328-5. PubMed DOI

Shen ZH, Zeng DF, Wang XY, Ma YY, Zhang X, Kong PY. Targeting of the leukemia microenvironment by c(RGDfV) overcomes the resistance to chemotherapy in acute myeloid leukemia in biomimetic polystyrene scaffolds. Oncology Letters. 2016;12(5):3278–3284. doi: 10.3892/ol.2016.5042. PubMed DOI PMC

Svozilová H, Plichta Z, Proks V, Studená R, Baloun J, Doubek M, Pospíšilová Š, Horák D. RGDS-modified superporous poly(2-Hydroxyethyl Methacrylate)-based scaffolds as 3D in vitro leukemia model. International Journal of Molecular Sciences. 2021;22(5):2376. doi: 10.3390/ijms22052376. PubMed DOI PMC

Tavakol DN, Bonini F, Tratwal J, Genta M, Brefie-Guth J, Braschler T, Naveiras O. Cryogel-based injectable 3D microcarrier co-culture for support of hematopoietic progenitor niches. Current Protocols. 2021;1(11):e275. doi: 10.1002/cpz1.275. PubMed DOI

Trimarco V, Ave E, Facco M, Chiodin G, Frezzato F, Martini V, Gattazzo C, Lessi F, Giorgi CA, Visentin A, Castelli M, Severin F, Zambello R, Piazza F, Semenzato G, Trentin L. Cross-talk between chronic lymphocytic leukemia (CLL) tumor B cells and mesenchymal stromal cells (MSCs): implications for neoplastic cell survival. Oncotarget. 2015;6(39):42130–42149. doi: 10.18632/oncotarget.6239. PubMed DOI PMC

Unnikrishnan K, Thomas LV, Ram Kumar RM. Advancement of scaffold-based 3D cellular models in cancer tissue engineering: an update. Frontiers in Oncology. 2021;11:733652. doi: 10.3389/fonc.2021.733652. PubMed DOI PMC

Velasco-Mallorquí F, Rodríguez-Comas J, Ramón-Azcón J. Cellulose-based scaffolds enhance pseudoislets formation and functionality. Biofabrication. 2021;13(3):035044. doi: 10.1088/1758-5090/ac00c3. PubMed DOI

Vojtová L, Zikmund T, Pavliňáková V, Šalplachta J, Kalasová D, Prosecká E, Brtníková J, Žídek J, Pavliňák D, Kaiser J. The 3D imaging of mesenchymal stem cells on porous scaffolds using high-contrasted x-ray computed nanotomography. Journal of Microscopy. 2019;273(3):169–177. doi: 10.1111/jmi.12771. PubMed DOI

Wu D, Wang Z, Li J, Song Y, Perez MEM, Wang Z, Cao X, Cao C, Maharjan S, Anderson KC, Chauhan D, Zhang YS. A 3D-bioprinted multiple myeloma model. Advanced Healthcare Materials. 2022;11(7):e2100884. doi: 10.1002/adhm.202100884. PubMed DOI PMC

Xiong GF, Xu R. Function of cancer cell-derived extracellular matrix in tumor progression. Journal of Cancer Metastasis and Treatment. 2016;2(9):357–364. doi: 10.20517/2394-4722.2016.08. DOI