Table 2 3D in vitro models of CAVD
From: Models for calcific aortic valve disease in vivo and in vitro
Classification | Type | Molding methods | Results | Advantages | Disadvantages |
|---|---|---|---|---|---|
Ex vivo | valve leaflets (Weber et al. 2021) | AV leaflets from healthy 6–9 months Ovis; stretched with needles on silicon rubber rings; under pro-degenerative conditions for 14d-56d | At 14 d begins to form and at 56 d massive calcium accumulation in three layers by histologic staining; Col1A1, Col3A1, VIM, ACTA-2, OPN↑ | easily applicable, reproducible, and cost effective; native valvular ECM and realistic VIC–VEC interactions | processed cultured under passive tension; regardless of cell types and factors in the blood circulation |
perfusion heart ( Kruithof et al. 2021) | whole mouse hearts (2–6 months) in MTCS; perfusion with osteogenic medium (OSM) or inorganic phosphates + Dex for 1 weeks | PI + Dex but not OSM induces valve leaflets calcification indicated by alizarin red-stainning; ALP and RUNX1/2/3 immunostaining; Endochondral differentiation staining | culture of mouse valves in their natural position in the heart and under specific hemodynamic conditions; exposing the leaflets to pro-calcific enviroment (relative native); shorten culture time | retrograde flow created a mechanical environment favoring calcification; regardless of cell types and factors in the blood circulation | |
Hydrogel-based 3D culture | Scaffold-based coculture (Hof et al. 2016) | Sheep aortic valves were decellularized and treated with trypsin or laser perforation, then reseeded with sheep VICs | low activation of repopulating VIC after 7 days of culture;MMP2,MMP9,αSMA↑ | using fsL-mediated photodisruption to increase dECM permeability; short enzymatic treatment facilitate the migration of seeded VIC into the ECM | Not for CAVD modeling; Relatively limited interstital repopulation; hard to identify exact photodisrupted regions |
Scaffold-based coculture (Stadelmann et al. 2022) | A bilayer cryo-electrostatically spun scaffold, 6-y porcine VEC and VIC seeded onto FN-functionalized scaffold, cultured for 4 weeks in CM or OM | cell adherence, homogenous migration and proliferation↑; VICs interact with VECs↑; Runx2,SPP1↑; | a promising platform material to study calcification on a soft substrate; | could be integrated into perfusion or dynamic culture systems for studying disease progression | |
3D stacked paper-based culture (Sapp et al. 2015) | 6-m pVICs, filter paper layer printed with a wax-well plate template, implanted with a mixture of VICs and collagen | VIC migration↑; αSMA↑ | Allows customization of the ECM and incorporates the ability to stack individual layers to control the thickness of the total culture | the position of cells in each layer cannot be controlled | |
Hydrogel-based culture plateform (Porras et al. 2018) | pVICs seeded on either GelMA only or GelMA/GAG hydrogels (HA, CS), treated with 25 μg/mL human LDL or oxLDL for 72 h | GAGs enriched ECM leads to inflammatory↓, angiogenesis↑, deposition of oxidized lipoproteins↑ | mimics enriched GAGs, quiescent VICs, and presence of lipoproteins in early CAVD | It does not include studies of the factors that regulate the onset of GAG enrichment or the importance of early features in fibrosis and calcification | |
Micropatterning hydrogel based plateform (Duan et al. 2019) | 12-year(Normal) or 75-y(CAVD) individual origin HAVIC were seeded on 3D micropatterned bioactive hydrogels consisting of Me-HA/Me-Gel, with a customized mask-guided photocrosslinking method;in OGM | αSMA↑, MMP-1↑, ALP↑; osteogenic differentiation↑in diseased HAVIC with patterning, | bioactive Me-HA/Me-Gel hydrogels with VIC ECM-like components and similar stiffness to the ventricularis and fibrosa layers of aortic heart valve leaflets | The width and space of the micropattern, and different degrees of alignment, were not studied | |
Bioreactor model (Gould et al. 2012) | compression springs with gel inoculation, solidified for 60 min and seeded with porcine valve mesenchymal stromal cells | proliferation and apoptosis↑; F-actin↑; ACTA2↑ | Implemented a novel bioreactor to investigate the relationship between anisotropic strain, cell differentiation, and matrix remodeling in 3D culture | It is unclear how cells interpret time- and direction-varying anisotropic strain fields in defined three-dimensional matrix structures | |
Bioreactor culture (Ferdous et al. 2011) | HASMC and HAVIC obtained from non-sclerotic patients, molded in tubular collagen-cell mixture and cultured in pneumatic bioreactor with osteogenic media for 9 or 21 d | collagen I,MMP-2↑; calcium deposition↑; Runx2,ALP,αSMA↑; HASMCs expresses higher osteogenic markers and matrix remodeling than HAVICs | Comparing vascular versus valvular calcification with tissue-engineered collagen gels | Could not interprete regional differences due to mechanical force variation | |
VICs 3D culture (Lim et al. 2016) | mVICs were encapsulated in 2 mg/mL collagen, treated with 5 mmol/L β-GP, and 50 μg/mL of ascorbic acid in α-MEM | thickness, calcification↑; fibronectin, a-SMA, collagen receptor, discoidin domain tyrosine kinase receptor 2↑; F-actin, NF-κB, JNK↑ | linking inflammation with the clinical features of aortic stenosis: valvular retraction, stiffening, and formation of calcified nodules | Inflammatory factors are limited to TNF-α | |
VICs 3D culture (Hjortnaes et al., 2015) | 10-m pVICs cultured in 1% HAMA-5% GelMA, treated by TGFβ | vimentin, αSMA, MMP9, Col1A1↑ | enlable to maintain a quiescent VIC phenotype before stimulation | Hydrogel platforms are not in an environment of repetitive strain and pressure | |
VECs and VICs co-culture (Gee et al. 2021) | pVECs and pVICs seeded in a mechanically constrained collagen alone or in co-culture configurations | cell and matrix aggregates↑; αSMA,pSAMD2,ACTA2↑; SOX9↓ | The model supports its use to test mechanisms of intercellular communication in valves and their pharmacological control | mechanical characterization of collagen gel is not feasible | |
VECs and VICs co-culture (Bramsen et al. 2022) | 6–8 m pAVICs were seeded into and pAVEC were seeded on top of hydrogels with different collagen composition (Con:1.5 mg/mL; stiff: 2.2 mg/mL; with CS: 1.5 mg/mL collagen + 20 mg/mL CS; + HA:1.5 mg/mL collagen + 20 mg/mL HA) for 2w | αSMA,ALP↑; cellular invasion rates↑; proliferation activity↑ | GAGs (CS and HA) mimic altered ECM (matrix mineralization situation) to study EndMT-derived aVIC activity | Gender was not addressed | |
VECs and VICs co-culture (Vadana et al. 2020) | VICs and VECs from calcified human, Gelatin-based 3D constructs with VIC encapsulated in hydrogel and VEC seeded on top, exposed to osteogenic medium (10 mmol/L β-GP, 10 ng/mL ascorbic acid and 10−8 mol/L dexamethasone) | αSMA↓; vimentin-; MMP1,MMP13,MMP2, MMP9↑; Runx2, OCN,OPN↑; HG trigger BMP and TGF-β signaling further | 3D model with human valvular cells plus high glucose imply the increased risk of degenerative aortic valve disease and calcification found in diabetic patients | Lack of flow-induced shear stress and hemodynamic forces | |
3D-bioprinting (Immohr et al. 2022) | oVICs dissolved in a hydrogel composed of 2% alginate and 8% gelatin, 3D bioprinting and incubated for 28d | cell viability↑ | reduce associated VIC damage and increase long-term cell viability | cannot establish 3D-bioprinting of even more vulnerable aortic valvular endothelial cells | |
3D-bioprinting (Immohr et al. 2023) | oVICs and VECs, dissolved in a hydrogel consisting of 2% alginate and 8% gelatin, 3D bioprinting with architectures | cell viability↑ | first 3D-bioprinted AV model combining both VIC and VEC in a single multicellular construct | unintentional concomitant induction of endothelial-to-mesenchymal transition |