Table 4 Techniques used to evaluate the adequacy of kidney decellularization
From: Current strategies on kidney regeneration using tissue engineering approaches: a systematic review
Variable | Technique of measurement |
|---|---|
DNA content | - Quantitative: Spectrophotometer, dsDNA assay kit + Multi-Mode Microplate Reader [107] - Qualitative: DAPI + fluorescence microscope, Quant-iT PicoGreen + fluorescence dsDNA assay kit - Both: Nanodrop (DeNovix Co., USA). |
Cell membrane | - Human endothelial cells: Anti-CD31 antibody (#ab187377), Anti-human nucleoli antibody (# ab190710) [105], Anti-Gb3 antibody. - Human & porcine endothelial cells: Anti-CD31 antibody (#ab28364), Anti-VE cadherin antibody (# ab33168) [105] - Tubular structures: Anti-Acetylated tubulin - Proliferating-Cell-Nuclear-Antigen (PCNA) [20] - Apoptosis evaluation: In-situ-nick-translation (ISNT) [20] - Light microscopic evaluation using staining: H&E, Masson’s Trichrome, Alcian blue, periodic acid-Schiff (AB-PAS), elastic Verhöeff-Van Gieson (EVG), Sirius red, toluidine blue, Movat’s Pentachrome, Safranin O and Fast Green, Orcein - IHC: Anti Na–K ATPase Ab [43], biotinylated LTL Ab, Cadherin-16 Ab [35], anti-Ki67 Ab, anti-CD206 Ab [84] anti-α-SMA Ab, anti-Foxp3 Ab, anti-Pax2 Ab, and anti-TNF-α Ab, anti GATA3 Ab (tubule), anti WT1 Ab (podocyte), antiaquaporin 1 Ab, anti-NPHS2 Ab, anti-CD61 Ab, anti-tubulin Ab, anti-Keratin-16 Ab, anti vimentin Ab, anti podocin Ab, anti-SIX-2 Ab, anti-SOX-17 Ab, anti HSPG Ab, anti panCytokeratin Ab - Microstructure: SEM - Cell-Cell interaction: Computational analysis of the PDGF, NOTCH, WNT, TGFB (Transforming Growth Factor), VEGF, and BMP Families [130] |
Detergent residue | - SDS residue: methylene blue + Microplate Reader [107] - Methylene blue + Spectrophotometric (colorimetric assessment) - Quantitative Assay Stains-All Dye (MilliporeSigma) [110] |
Functionality of recellularization | - Intake of Glucose, glutamine, glutamate, LDH: Cedex Bio HT Analyzer [105] - Cell viability/ proliferation: ELISA, Viability/Cytotoxicity Kit (#L3224) + fluorescence microscope [87], Hoechst 33342) + fluorescence microscope [107], A fluorescence live/dead assay [6], CCK-8 assay [6], MTT assay, TUNEL assay, Enzylight ATP assay (BioAssay Systems, Hayward, CA, USA) that determines ATP, bromodeoxyuridine (BrdU) staining [45], Resazurin - Cell nuclei: DAPI + fluorescence microscope - MRI + Iron oxide labeling - Electrolyte reabsorption ability: Cellular sodium green (ThermoFisher Scientific) uptake + fluorescence microscope - Albumin uptake: FITC-conjugated human serum + fluorescence microscope albumin (HSA) |
Vascular integrity | - Angiography [105] - Micro-computerized tomography imaging of the vasculature [18] − 50-kDa fluorescein isothiocyanate (FITC)-dextrans) + fluorescence microscope [107] - Vascular endothelial growth factor receptor-2 (VEGFR2) - Vascular resin corrosion casting - Fluoroscopic Angiography + Discrete wavelet transform (DWT) analysis + Gray Level Co-occurrence Matrix (GLCM) analysis (141) - Polymethylmethacrylate (PMMA) vascular corrosion casts [79] - Permeability assay: Evans blue dye + spectrophotometry |
Protein | - Immunofluorescence staining (NF-H antibody, GAP-3 antibody) - Western blotting (NF-H antibody, GAP-3 antibody, GADPH antibody) [6] - Mass spectrometry - Pierce Coomassie (Bradford) Protein Assay Kit (Thermo Scientific) [56] - Micro BCA protein assay kit (ThermoFisher) |
Collagen | - Anti-Collagen I antibody (#ab34710), Anti-Collagen IV antibody (#ab6586) [105] - Hydroxyproline assay kit |
Cytokine | - Radioimmunoprecipitation assay (RIPA) buffer + analyzed using a Proteome Profiler Human XL Cytokine Array Kit (R&D systems) - ELISA |
Laminin Fibronectin | - Anti-Laminin antibody (#ab11575) - Anti-Fibronectin antibody |
GAG | - Blyscan assay [15] - Immunofluorescence staining: WGA antibody, LEA antibody, heparan sulfate specific antibody [92] − 1,9-dimethyl-methylene blue (DMMB) dye assay + Multi-Mode Microplate Reader [114] |
Mechanical properties | - Microstructure, pore size and vascular structure: SEM - Water absorption: W (%) = (m2-m1)/m1 *100% * - Compression modulus: \(\:E\left(MPa\right)=\frac{\left(x2-x1\right)AB}{\left(Z2-Z1\right)C}\) ** - Porosity: \(\:\frac{(W2-W3-Ws)}{(W1-W3)}\)*100% *** - Chemical composition of scaffold: Fourier transform infrared (FTIR) spectroscopy - Tensile properties: uniaxial stretch, load–displacement curve, biaxial stress–strain test, ultimate tensile strain, suture retention strength, compressive strength [85] - Hydrophilicity: contact angle goniometry |
Thermal analysis | - Thermo-gravimetric analysis (TGA) and differential scanning calorimetry (DSC) |