Journal: Biological Chemistry

Article Title: Peritumoral administration of GPI-anchored TIMP-1 inhibits colon carcinoma growth in Rag-2 γ chain-deficient mice

doi: 10.1515/bc.2009.098

Figure Lengend Snippet: Figure 2 The GPI-anchored TIMP-1 suppresses proliferation of SW480 cells and inhibits tumor invasion. The effect of cell surface engineering with TIMP-1-GPI on SW480 invasion through the Matrigel basement membrane model was assessed. Optimal invasion of the SW480 cells in response to 10% FCS, 4 ng/ml EGF, or 4 ng/ml VEGF were set to ‘zero’, and the 100% inhibition value was set to the migration level observed with untreated SW480 cells (Djafarzadeh et al., 2004). 5=103 SW480 cells per 100 ml medium were cultured in 96-well microtiter plates for 24 h under standard conditions to yield firmly attached and stably growing cells (Djafarzadeh et al., 2006). The SW480 cells were then pretreated with 2, 4, 6, 8, 10, 12, and 14 ng/ml of TIMP-1-GPI or 14 ng/ml of rhTIMP-1 as described previously (Djafarzadeh et al., 2006). After 1 h the cells were washed. (A) The effect of increasing levels of TIMP-1-GPI or rhTIMP-1 control protein on the proliferation of SW480 was measured using an MTT assay (Djafarzadeh et al., 2006). MTT was added after 24 h, 48 h, or 72 h as indicated. (B) The effect on the ability of SW480 to invade Matrigel was measured using a modified Boyden chamber as described earlier (Djafarzadeh et al., 2004, 2006).

Article Snippet: The effect of TIMP-1-GPI vs. rhTIMP-1 treatment on the ability of SW480 cells to invade Matrigel was measured using induced migration to fetal calf serum (FCS) (Biocrom, Berlin, Germany), vascular endothelial growth factor (VEGF) (R&D systems, Minneapolis, USA), and EGF (Biochrom, Berlin, Germany) using a modified Boyden chamber (Djafarzadeh et al., 2004, 2006).

Techniques: Membrane, Inhibition, Migration, Cell Culture, Stable Transfection, Control, MTT Assay, Modification

Journal: Communications Biology

Article Title: Functional characterization of a bioengineered liver after heterotopic implantation in pigs

doi: 10.1038/s42003-021-02665-2

Figure Lengend Snippet: a Overview of vessel cannulation and detergent perfusion methods for whole liver decellularization. b , c Photographs of representative porcine ( b ) native and ( c ) decellularized whole livers. d , e Hematoxylin and eosin staining of histological sections from ( d ) native and ( e ) decellularized porcine liver tissue demonstrating the efficient removal of cellular material while preserving the native tissue architecture. f – i Representative immunofluorescence micrographs from native ( f , h ) and decellularized ( g , i ) liver tissue demonstrating retention of Collagen I and Collagen IV, respectively, in cell-free scaffolds. The absence of DAPI staining in the decellularized scaffolds demonstrates removal of cellular DNA. j Schematic of perfusion bioreactor design. BELs are suspended in a vessel with culture media. An extended coil of silicone tubing (GEC) is included in the perfusion circuit to facilitate gas exchange. A bubble trap is positioned directly upstream of the BEL perfusion inlet. During media perfusion, a pressure transducer provides real-time feedback to a controller which in turn adjusts the flow rate on a peristaltic pump to maintain a constant perfusion pressure. k Schematic depicting bioreactor culture and cell seeding events. Decellularized scaffolds were pre-qualified in antibiotic-free media for 3 days prior to HUVEC seedings. HUVEC-seeded liver constructs were cultured in endothelial cell growth media until a 24 h average glucose consumption rate of 50–90 mg/h was reached (typically 13–16 days), at which point hepatocytes were seeded into the scaffold. BELs were maintained in co-culture media formulated for simultaneous culture of endothelial cells and hepatocytes for the remainder of the experiment (typically 1–3 additional days). l Overview of HUVEC culture and seeding approach to generate a reendothelialized liver construct. Cells were infused first through the cannulated iIVC followed by a second cell infusion through the PV 24 h later. m Schematic of porcine donor liver hepatocyte isolation and seeding of bioengineered liver constructs. Whole porcine livers are enzymatically digested, and dissociated cells were filtered through a series of mesh sieves to remove large debris and cell aggregates. Hepatocytes were enriched through multiple low speed (70 × g ) centrifugation and washing steps. DAPI—4′,6′-diamidino-2-phenylindole; BEL—bioengineered liver; GEC—gas exchange coil; BT—bubble trap; iIVC—infrahepatic inferior vena cava; PV—portal vein.

Article Snippet: Human umbilical vein endothelial cells (HUVECs) (Lonza, C2517A) were cultured at 37 °C and 5% CO 2 in antibiotic-free Endothelial Cell Growth Media (R&D Systems, CCM027) supplemented with 2% fetal bovine serum (Corning), 50 mg/L ascorbic acid (Sigma), 1 mg/L hydrocortisone (Sigma), 20 μg/L FGF (R&D Systems), 5 μg/L VEGF (R&D Systems), 5 μg/L EGF (R&D Systems), 15 μg/L R 3 IGF (Sigma), 1000 U/L heparin (Sigma), and 1.5 μM acetic acid (Sigma).

Techniques: Staining, Preserving, Immunofluorescence, Construct, Cell Culture, Co-Culture Assay, Isolation, Centrifugation

Journal: Communications Biology

Article Title: Functional characterization of a bioengineered liver after heterotopic implantation in pigs

doi: 10.1038/s42003-021-02665-2

Figure Lengend Snippet: a Representative photograph of a BEL seeded with HUVECs and porcine hepatocytes. b Hematoxylin and eosin staining of representative co-culture BEL tissue sections fixed 48 h after seeding hepatocytes. c – e Immunofluorescent staining of cell lineage markers in non-serial tissue sections 48 h after seeding hepatocytes: ( c ) CD31 & albumin; ( d ) CD31 & FAH; ( e ) CD31 and LYVE1. f Schematic depicting seeding and culture timeline for HUVEC only, hepatocyte only, and co-culture BEL constructs used in ( g , h , j , k ). g vWF production in grafts before and after hepatocyte seeding. Data from independent HUVEC only ( n = 5), hepatocyte only ( n = 7), and co-culture ( n = 7) BEL constructs are shown. Error bars denote the mean and standard deviation at each time point. h 24-h average albumin production in co-culture grafts 48 h following hepatocyte seeding. Data from independent HUVEC only ( n = 5), hepatocyte only ( n = 7), and co-culture ( n = 7) BELs are shown. Error bars denote the mean and standard deviation. i Schematic of in vitro ammonia clearance and urea production assay. Ammonium chloride is added to the bioreactor media at a concentration of 0.8 mM. Ammonia and urea levels are measured in media samples taken at t = 0, 1, 2 7, and 23 h following the addition of ammonium chloride. Error bars denote the mean and standard deviation each time point. j , k Ammonia clearance ( j ) and urea production kinetics ( k ) following the addition of ammonium chloride to the bioreactor perfusion media. Data from independent HUVEC only ( n = 5), hepatocyte only ( n = 7), and co-culture ( n = 7) BELs, media only controls ( n = 4) are shown. Error bars denote the mean and standard deviation each time point. BEL—bioengineered liver; FAH—fumarylacetoacetate hydrolase; CD31—cluster of differentiation 31; LYVE1—lymphatic vessel endothelial hyaluronan receptor 1; vWF—von Willebrand factor.

Article Snippet: Human umbilical vein endothelial cells (HUVECs) (Lonza, C2517A) were cultured at 37 °C and 5% CO 2 in antibiotic-free Endothelial Cell Growth Media (R&D Systems, CCM027) supplemented with 2% fetal bovine serum (Corning), 50 mg/L ascorbic acid (Sigma), 1 mg/L hydrocortisone (Sigma), 20 μg/L FGF (R&D Systems), 5 μg/L VEGF (R&D Systems), 5 μg/L EGF (R&D Systems), 15 μg/L R 3 IGF (Sigma), 1000 U/L heparin (Sigma), and 1.5 μM acetic acid (Sigma).

Techniques: Staining, Co-Culture Assay, Construct, Standard Deviation, In Vitro, Concentration Assay

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Skeletal colonization by breast cancer cells is stimulated by an osteoblast and β2AR-dependent neo-angiogenic switch

doi: 10.1002/jbmr.3133

Figure Lengend Snippet: A) Representative 40× H&E images of hind limb bone sections from athymic nude mice treated with PBS, 3mg/kg Isoproterenol (ISO), or Chronic Immobilization Stress (CIS). Blood vessels can be recognized by their shape and staining of red blood cells (red). Bar: 100 μm. B) Quantification of vessel area (VsA) and vessel number normalized by tissue area (TA) in mice that received PBS (N=12) or ISO (N=7) (p=.009 and p=.037), as well as mice subjected to control (N=5) or CIS (N=6) (p=.296 and p=.004). C) Quantification of primary mouse BMEC tube length (*=p<.05, N=3). D) Quantification of HUVECs tube length (*p=<.05, ***=p<.001, N=6). E) Schematic of the metatarsal assay and representative 4× Images of metatarsals according to each treatment. After 2 weeks of culture, mouse explants were stained for the endothelial cell marker CD31. F) Quantification of the number of CD31+ parental vessels sprouting from the metatarsal bones (*P=<.05, ****P=<.0001, N≥3).

Article Snippet: Twenty-four well plates were coated with 200 μL of growth factor reduced matrigel (Corning #354230) for 45 mins. at 37°C before 30,000 HUVECs (Gibco #C0035C), or BMECs from wild-type C57BL/6 mice were plated and cultured in 500 μL of Basal Endothelial Cell Growth Media (ScienCell #1001).

Techniques: Staining, Control, Marker