Three Dimensional Monolayer Culture of Epithelial and Endothelial Cells on Poly(vinyl alcohol) Nanofibrous Membrane Containing Integrin-binding Ligands 1 Min-Ho Choi , Yong-Su Kim , Jung-In Shin , Ji-Hyun Lee , Perry Ayn Mayson A. Maza , Minsook Ryu , Jong-Yong Kwak 1,4,5 1,2 1,2 3 1,2 1,2 1 Department of Pharmacology, School of Medicine, Ajou University Suwon 16499, South Korea, 2 Department of Biomedical Sciences, Graduate school, Ajou University Suwon 16499, South Korea, 3 Department of Allergy, Ajou University School of Medicine, Suwon 16499, South Korea, 4 3D Immune System Imaging Core
Center, Ajou University, Suwon 16499, South Korea Korea, 5 Immune Network Pioneer Researcg Center, Ajou University, Suwon 16499, South Korea, 6 Nanofaentech Inc. Kimhae 50969, South Korea BACKGROUND AIM Adherence of epithelial cells to traditional two-dimensional (2D) culture dish may induce aberrant In this study, we developed a three dimensional (3D) culture system of epithelial and cell functions, including epithelial-mesenchymal transition. Poly(vinyl alcohol) (PVA) is one of the endothelial cells with PVA nanofibrous membrane which contains integrin-binding most prevalent and versatile
synthetic polymers abundantly used in tissue engineering as peptides to improve cell adhesion, growth, and functions. biomaterials, but high hydrophilicity of PVA results in poor cell adhesion, leading to aggregation and spheroid formation of cultured cells on PVA nanofibrous membrane. METHODS 1. Electrospinning - Electrospun nanofibers were prepared from blends of PVA, integrin-binding peptides, and chemical cross linkers. The electrospun PVA nanofibrous membranes were then further crosslinked by exposure of the membranes to HCl vapor and dimethylformamide solution. 2. Cell culture – MLE-12
lung epithelial cells, bEND.3 endothelial cells, mouse primary hepatocytes, CT26 colon cancer cells, HepG2 hepatoma cells, and fibroblasts were plated on 2D culture dish, PVA nanofibrous membrane, and PVA nanofibrous membrane containing peptide of fibronectin and laminin. 3. Scanning electron microscopy (SEM) - The ultrastructure of nanofibrous membrane and cultured cells were analysed by SEM. 4. Image analysis - Cell morphology was determined by laser scanning confocal microscopy. Cells were labelled with DAPI, FITC-conjugated phalloidin, PE-conjugated anti-zona occluding 1 (ZO-1) and
anti-E-cadherin. Morphology of fluorescence-labelled cells were observed using a confocal microscope. Images was analysed using the ImageJ software. RESULTS The novel combination of PVA/polyacrylic acid/glutaraldehyde crosslinked with combination of heat, HCl, and dimethylformamide was used for the production of water-stable and transparent electrospun PVA membrane in culture media. When mouse primary hepatocytes and lung epithelial cell line, MLE-12 cells, and other various cells were cultured on PVA nanofiber membrane, the cells adhered to the cross-linked PVA nanofiber membrane but formed
cell aggregate and discoidal shaped spheroid. RGD and YIGSR peptides, which are derived from domains of cell binding in fibronectin and laminin, respectively, were blended in PVA nanofibers. Formation of cell aggregates was increased on RGD-PVA membrane due to release of blended but slightly bound peptides which inhibited the binding of cells to nanofibers. The release of peptides was enhanced by treatment of peptide-PVA membrane with NaOH. However, peptides in NaOH- treated membrane still existed and were not further released in culture media. When hepatocytes were cultured on NAOH-treated
RGD-PVA membrane, the cells adhered to and formed monolayer on the membrane rather than formation of spheroid. Although MLE-12 lung epithelial and bEND.3 endothelial cells adhered to the surface of PVA membrane and formed cell aggregates, endothelial and epithelial cells on YIGSR-PVA membranes showed tube-like and sheet-like cell growth, respectively. The expression of E-cadherin and ZO-1 in culture epithelial and endothelial cells were maintained during culture time. CONCLUSION REFERENCES These results suggest that PVA nanofibrous membrane Kapp TG, Rechenmacher F, Neubauer S, Maltsev OV,
Cavalcanti- Adam EA, Zarka R, et al. A comprehensive evaluation of the activity containing integrin-binding peptides provides similar and selectivity profile of ligands for RGD-binding integrins. Sci. Rep. environment as extracellular matrix in tissue and can support for 2017;7:39805. attachment of various cells including epithelial cells. Epithelial and endothelial cells cultured on cell adhesion ligand-containing Severgnini M, Sherman J, Sehgal A, Jayaprakash NK, Aubin J, Wang G, et al. A rapid two-step method for isolation of functional primary PVA nanofibrous membrane grow three
dimensionally in mouse hepatocytes: cell characterization and asialoglycoprotein monolayer and maintain their functions for long time. receptor based assay development. Cytotechnol. 2012;64:187-95. [E. Cell adhesion and cytoskeletal dynamics-2] Three dimensional monolayer culture of epithelial and endothelial cells on poly(vinyl alcohol) nanofibrous membrane containing integrin-binding ligands Min-Ho Choi¹˙⁵, Yong-Su Kim¹˙², Jung-In Shin¹˙², Sang-Won Lee¹˙⁵, Perry Ayn Mayson A Maza¹˙², Min-Sook Ryu³, Jong-Young Kwak¹˙⁴˙⁵˙* ¹Department of Pharmacology, School of Medicine, Ajou University, Suwon
16499, South Korea, ²Department of Biomedical Sciences, Graduate school, Ajou University , Suwon 16499, South Korea, ³Department of Allergy, Ajou University School of Medicine, Suwon 16499, South Korea, ⁴Department of 3D Immune System Imaging Core Center, Ajou University, Suwon 16499, South Korea, ⁵Department of Immune Network Pioneer Researcg Center, Ajou University, Suwon 16499, South Korea, ⁶Department of Fabrication engineering, Nanofaentech Inc, Kimhae 50969, South Korea Adherence of epithelial cells to two-dimensional (2D) culture dish may induce aberrant cell functions, including
epithelial-mesenchymal transition. Poly(vinyl alcohol) (PVA) is one of the versatile synthetic polymers abundantly used in tissue engineering as biomaterials. In this study, we developed a three dimensional (3D) culture system of epithelial and endothelial cells with PVA nanofibrous membrane which contains integrin-binding peptides (RGD and YIGSR) to improve cell adhesion, growth, and functions. Primary hepatocytes and lung epithelial cell line, MLE-12 cells were cultured on PVA nanofiber membrane, the cells adhered to the PVA nanofiber membrane but formed cell aggregate and discoidal shaped
spheroid. Hepatocytes cultured on RGD-PVA membrane adhered to and formed monolayer on the membrane rather than formation of spheroid. Although MLE-12 lung epithelial and bEND.3 endothelial cells adhered to the surface of PVA membrane and formed cell aggregates, endothelial and epithelial cells on YIGSR-PVA membranes showed tube-like and sheet-like cell growth, respectively. These results suggest that PVA nanofibrous membrane containing integrin-binding peptides provides similar environment as extracellular matrix in tissue and can support for attachment of various cells. Cytoskeleton Dependent
Activation of Tentonin 3/TMEM150C, an Essential Component of Mechanosensitive Channel Sungmin Pak , Gyu-Sang Hong , Hyungsup Kim , Jaehyouk Choi , Huanjun Lu , Kyungmin Kim , Hyesu Kim , 1 1 1 1 1 1 1 Luan Nguyen Thien , Taewoong Ha , Sujin Lim , Mi-Sun Kim , and Uhtaek Oh 1 1 1 1 1 1 Sensory Research Center, CRI, Brain Science Institute, Korea Institute of Science and Technology, Seoul 02792, Korea Abstract Introduction-1 Tentonin3/TMEM150C (TTN3) is a mechanosensitive ion channel that has characteristics of slowly inactivating kinetics by mechanical stimulation. Recent studies have proposed
whether TTN3 is an ion DRG Sensory neurons Mechanosensitive channel channel and its independent channel activity is validated. The opening mechanism of mechanosensitive ion channels have been suggested into two theories. One theory is regarding the activation through ? direct cell plasma membrane stretch, for example, the Piezo family. Another theory pertains the influence of tethering proteins near the plasma membrane activating mechanosensitive channels, for example, MEC complex in C. elegans. Focal Adhesion (FA) Complex is a multi-protein assembly of transmembrane proteins and intracellular
proteins that provides connection between the cell and the extracellular matrix. We report that the FA Complex of Piezo1-Knockout (P1KO) HEK293T cells is hampered and selective knockdown of FA related proteins, Talin1, Vinculin and Integrin, diminished TTN3 mechanical current in Ttn3-transfected HEK293T cells. Therefore, we propose the disturbed FA Piezo2 Complex in P1KO HEK293T cells is responsible for the absence of TTN3 mechanical current in ttn3- (Delmas, 2011, Nature Review) transfected P1KO-HEK293T cells. There are mainly three types of MS currents in isolated DRG neurons, determined by
inactivation time Introduction-2 kinetics under whole-cell patch mode: rapidly adapting (RA) currents (~10 ms), intermediately adapting (IA) currents (10 ms–30 ms) and slowly adapting (SA) currents (>30 ms) (Huand Lewin, 2006). Although Piezo2 has been TTN3 is a novel mechanosensitive (MS) ion discovered as a RA MS channel (Coste, 2010), molecular identity of SA MS channel is not known yet. Introduction-3 channel Candidate Is TTN3 a Modulator of MS channels? No activation of TTN3 in HEK-P1KO Kinetics of TREK1 & PiezosTTN3 modulates inactivation (Dubin et al., Neuron, 2017) (Hong et al.,
Neuron, 2016) Independent of TTN3 from Piezo1 1. Activated by mechanical stimuli with slow-inactivation kinetics. 2. Required for SA MS currents in DRG neurons. 3. Present in muscle spindles and contributes to proprioception. 1 A B HEK HEK- (Hong et al., Neuron, (Anderson et al., Cell reports, 2018) △P1 3 2017) Z- 4 3 4 3 A B C D Figure 3. Focal Adhesion (FA) Stacked 3 6 3 6 TTN3/S 600 TTN3/VCLsi 600 TTN3/TL1si dependency of TTN3 MA current F-actin ddCt (to GAPDH) H 500 pA 100 pA 100 pA Vinculin siRNA or Ttn3 + Talin-1 siRNA (C) C 600 ms crbl ms ms ** *** (A–C) Representative traces of
whole-cell MA currents in Ttn3 + scrambled siRNA, ttn3 + HEK- transfected HEK293T cells. E (D-F) Summary of average peak amplitude (D), △P1 K (G) Peak current amplitudes of Ttn3 co- on transfected with Vcl, Tl1, and Kk1. Figure 1. Defects of intracellular cytoskeletal structure in HEK-P1KO cells. E *** ** *** F G Co-expressi threshold (E) and peak distance (F). (A) Combined Z-stack images of HEK293T and P1KO-HEK293T cells. ANOVA one-way ANOVA with Dunnett’s (B) Summary of number and intensity of foci counted between HEK293T and P1KO-HEK293T cells. correction, **p < 0.005, ***p < 0.001). Data
(C) mRNA expression levels of hPiezo1 (hP1), TTN3, Vinculin (VCL), Talin1 (TL1), and Kank1 (KK1) in shown as mean ± SEM. HEK293T and P1KO-HEK293T cells. 2 4 5 A Tal F-actin M Figure 5. Generation in- e of TTN3 MS current in 1 r g HEK-P1KO cells. H 10 e μm HEK- (A) Immunofluorescent B E △P1 images, stained with Talin- K Vincu M Merg Talin Vinc 1 lin e Phalloidin(F-actin) in ulin er HEK-P1KO cells after 2 g hours treatment of DMSO H e HEK- (i) E (ii) △P1 or 250nM Jasplakinolide. K (B–D) Representative traces of whole-cell MS currents in response to 2 μm 2 μm mechanical indentation Figure 2.
Difference in cytoskeletal structure steps from 6 to 8 μm in between HEK293T and P1KO-HEK293T cells. TTN3 (B and D) or GFP (A) and (B) Immunofluorescence images of cells stained (C) overexpressing HEK- with Talin1 antibody and Phalloidin (A) and Talin1 and P1KO cells. 250 nM Vinculin antibody (B) in HEK293T and P1KO-HEK293T Figure 4. TTN3 dependent MS currents in HEK cells Jasplakinolide (JAS) or cells. DMSO was applied to the Conclusion (A) Representative traces of maximal MS currents in mTTN3 cultured medium for 2 expressing or non-expressing HEK cells. Blue arrowhead represents hours before
the mechanical indentation starting point of 600 m
