Novel bioprinted 3D model to human fibrosis investigation

Petrachi, Tiziana; Portone, Alberto; Arnaud, Gaelle Françoise; Ganzerli, Francesco; Bergamini, Valentina; Resca, Elisa; Accorsi, Luca; Ferrari, Alberto; Delnevo, Annalisa; Rovati, Luigi; Marra, Caterina; Chiavelli, Chiara; Dominici, Massimo; Veronesi, Elena

doi:10.1016/j.biopha.2023.115146

Cited by 3 publications

(1 citation statement)

References 23 publications

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“…Individual ECM proteins have long been used by different authors to elucidate their functions in the mentioned processes [20, 21]. However, the requirements for recreating the complexity of the system lead to the emergence of models in which the authors combine several types of ECM components [22] or three-dimensional models to recreate the three-dimensional architecture of tissues [23]. For maximum accuracy in recreating the profibrotic matrix, we have developed a method for its production by human fibroblasts with the addition of ascorbic acid, which is a cofactor of prolyl hydroxylase required for the maturation of collagen fibers.…”

Section: Discussionmentioning

confidence: 99%

Modeling the profibrotic microenvironmentin vitro: model validation

Grigorieva,

Basalova,

Dyachkova

et al. 2023

Preprint

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Establishing the molecular and cellular mechanisms of fibrosis requires the development of validated and reproducible models. The complexity ofin vivomodels challenges the monitoring of an individual cell fate, in some cases making it impossible. However, the set of factors affecting cells inin vitroculture systems differ significantly fromin vivoconditions, insufficiently reproducing living systems. Thus, to model profibrotic conditionsin vitro, usually the key profibrotic factor, transforming growth factor beta (TGFβ-1) is used as a single factor. TGFβ-1 stimulates the differentiation of fibroblasts into myofibroblasts, the main effector cells promoting the development and progression of fibrosis. However, except for soluble factors, the rigidity and composition of the extracellular matrix (ECM) play a critical role in the differentiation process. To develop the model of more complex profibrotic microenvironmentin vitro, we used a combination of factors: decellularized ECM synthesized by human dermal fibroblasts in the presence of ascorbic acid if cultured as cell sheets and recombinant TGFβ-1 as a supplement. When culturing human mesenchymal stromal cells derived from adipose tissue (MSCs) under described conditions, we observed differentiation of MSCs into myofibroblasts due to increased number of cells with stress fibrils with alpha-smooth muscle actin (αSMA), and increased expression of myofibroblast marker genes such as collagen I, EDA-fibronectin and αSMA. Importantly, secretome of MSCs changed in these profibrotic microenvironment: the secretion of the profibrotic proteins SPARC and fibulin-2 increased, while the secretion of the antifibrotic hepatocyte growth factor (HGF) decreased. Analysis of transciptomic pattern of regulatory microRNAs in MSCs revealed 49 miRNAs with increased expression and 3 miRNAs with decreased expression under profibrotic stimuli. Bioinformatics analysis confirmed that at least 184 gene targets of the differently expressed miRNAs genes were associated with fibrosis. To further validate the developed model of profibrotic microenvironment, we cultured human dermal fibroblasts in these conditions and observed increased expression of fibroblast activation protein (FAPa) after 12 hours of cultivation as well as increased level of αSMA and higher number of αSMA+ stress fibrils after 72 hours. The data obtained allow us to conclude that the conditions formed by the combination of profibrotic ECM and TGFβ-1 provide a complex profibrotic microenvironmentin vitro. Thus, this model can be applicable in studying the mechanism of fibrosis development, as well as for the development of antifibrotic therapy.

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Section: Discussionmentioning

confidence: 99%

Modeling the profibrotic microenvironmentin vitro: model validation

Grigorieva,

Basalova,

Dyachkova

et al. 2023

Preprint

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3D Humanized Bioprinted Tubulointerstitium Model to Emulate Renal Fibrosis In Vitro

Addario,

Fernández‐Pérez,

Formica

et al. 2024

Adv Healthcare Materials

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Chronic kidney disease (CKD) leads to a gradual loss of kidney function, with fibrosis as pathological endpoint, which is characterized by extracellular matrix (ECM) deposition and remodeling. Traditionally, in vivo models are used to study interstitial fibrosis, through histological characterization of biopsy tissue. However, ethical considerations and the 3Rs (replacement, reduction, and refinement) regulations emphasizes the need for humanized 3D in vitro models. This study introduces a bioprinted in vitro model which combines primary human cells and decellularized and partially digested extracellular matrix (ddECM). A protocol was established to decellularize kidney pig tissue and the ddECM was used to encapsulate human renal cells. To investigate fibrosis progression, cells were treated with transforming growth factor beta 1 (TGF‐β1), and the mechanical properties of the ddECM hydrogel were modulated using vitamin B2 crosslinking. The bioprinting perfusable model replicates the renal tubulointerstitium. Results show an increased Young's modulus over time, together with the increase of ECM components and cell dedifferentiation toward myofibroblasts. Multiple fibrotic genes resulted upregulated, and the model closely resembled fibrotic human tissue in terms of collagen deposition. This 3D bioprinted model offers a more physiologically relevant platform for studying kidney fibrosis, potentially improving disease progression research and high‐throughput drug screening.

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Hybrid biofabricated blood vessel for medical devices testing

Portone,

Ganzerli,

Petrachi

et al. 2024

Science and Technology of Advanced Materials

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Current in vitro and in vivo tests applied to assess the safety of medical devices retain several limitations, such as an incomplete ability to faithfully recapitulate human features, and to predict the response of human tissues together with non-trivial ethical aspects. We here challenged a new hybrid biofabrication technique that combines bioprinting and Fast Diffusion-induced Gelation strategy to generate a vessel-like structure with the attempt to spatially organize fibroblasts, smooth-muscle cells, and endothelial cells. The introduction of Fast Diffusion-induced Gelation minimizes the endothelial cell mortality during biofabrication and produce a thin endothelial layer with tunable thickness. Cell viability, Von Willebrand factor, and CD31 expression were evaluated on biofabricated tissues, showing how bioprinting and Fast Diffusion-induced Gelation can replicate human vessels architecture and complexity. We then applied biofabricated tissue to study the cytotoxicity of a carbothane catheter under static condition, and to better recapitulate the effect of blood flow, a novel bioreactor named CuBiBox (Customized Biological Box) was developed and introduced in a dynamic modality. Collectively, we propose a novel bioprinted platform for human in vitro biocompatibility testing, predicting the impact of medical devices and their materials on vascular systems, reducing animal experimentation and, ultimately, accelerating time to market.

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Novel bioprinted 3D model to human fibrosis investigation

Cited by 3 publications

References 23 publications

Modeling the profibrotic microenvironmentin vitro: model validation

Modeling the profibrotic microenvironmentin vitro: model validation

3D Humanized Bioprinted Tubulointerstitium Model to Emulate Renal Fibrosis In Vitro

Hybrid biofabricated blood vessel for medical devices testing

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