A novel honeycomb cell assay kit designed for evaluating horizontal cell migration in response to functionalized self-assembling peptide hydrogels

doi:10.1007/s11706-017-0369-9

Front. Mater. Sci.

2017, Vol. 11

Issue (1) : 13-21 https://doi.org/10.1007/s11706-017-0369-9

RESEARCH ARTICLE

A novel honeycomb cell assay kit designed for evaluating horizontal cell migration in response to functionalized self-assembling peptide hydrogels

Fengyi GUAN,Jiaju LU,Xiumei WANG(

)

Key Laboratory of Advanced Materials of Ministry of Education, School of Materials Science and Engineering, Tsinghua University, Beijing 100084, China

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Abstract

A clear understanding on cell migration behaviors contributes to designing novel biomaterials in tissue engineering and elucidating related tissue regeneration processes. Many traditional evaluation methods on cell migration including scratch assay and transwell migration assay possess all kinds of limitations. In this study, a novel honeycomb cell assay kit was designed and made of photosensitive resin by 3D printing. This kit has seven hexagonal culture chambers so that it can evaluate the horizontal cell migration behavior in response to six surrounding environments simultaneously, eliminating the effect of gravity on cells. Here this cell assay kit was successfully applied to evaluate endothelial cell migration cultured on self-assembling peptide (SAP) RADA (AcN-RADARADARADARADA-CONH₂) nanofiber hydrogel toward different functionalized SAP hydrogels. Our results indicated that the functionalized RADA hydrogels with different concentration of bioactive motifs of KLT or PRG could induce cell migration in a dose-dependent manner. The total number and migration distance of endothelial cells on functionalized SAP hydrogels significantly increased with increasing concentration of bioactive motif PRG or KLT. Therefore, the honeycomb cell assay kit provides a simple, efficient and convenient tool to investigate cell migration behavior in response to multi-environments simultaneously.

Keywords honeycomb cell assay kit cell migration self-assembling peptide hydrogels endothelial?cells

Corresponding Author(s): Xiumei WANG

Online First Date: 12 January 2017 Issue Date: 22 January 2017

Cite this article:

Fengyi GUAN,Jiaju LU,Xiumei WANG. A novel honeycomb cell assay kit designed for evaluating horizontal cell migration in response to functionalized self-assembling peptide hydrogels[J]. Front. Mater. Sci., 2017, 11(1): 13-21.

URL:

https://academic.hep.com.cn/foms/EN/10.1007/s11706-017-0369-9
https://academic.hep.com.cn/foms/EN/Y2017/V11/I1/13

Fig.1 Schematic illustration of the honeycomb cell assay kit: (a) The three orthographic views of the top part of the kit; (b) The stereo rendering of the top part of the kit; (c) The three orthographic views of the bottom part of the kit; (d) The stereo rendering of the bottom part of the kit; (e) A top perspective view of the filter membrane (Membrane I: microporous water permeable?membrane with a pore size of 0.2 μm, and Membrane II: microporous polycarbonate?membrane with a pore size of 10 μm); (f) A kit sample in 60-mm tissue culture dish.

Tab.1 Designer functionalized SAPs used in this work

Fig.2 Molecular models of designer SAPs and schematic illustrations of SAP nanofiber scaffolds. (a) Molecular models of designer peptides RADA and functionalized RADA that represent a β-sheet secondary structure. Hydrophobic alanine side groups are present on one side of SAP RADA and the other side is populated with alternating positive and negative charges due to the arginine and aspartic acid residues, respectively. (b) Schematic illustrations of SAP nanofibers formation after mixing RADA with functionalized peptide through ionic complementary and hydrophobic interactions. The functional motifs extrude from nanofiber backbones. (c) Typical gross morphology of the functionalized peptides nanofiber hydrogel. (d) Typical SEM morphology of the functionalized RADA nanofiber hydrogel.

Fig.3 The design of cell migration assay in?SAP hydrogels. (a) The top view of the distribution of various peptide solution (RAD: RADA; Gel 1: RAD/KLT-20; Gel 2: RAD/KLT-40; Gel 3: RAD/KLT-60; Gel 4: RAD/PRG-20; Gel 5: RAD/PRG-40; Gel 6: RAD/PRG-60). (b) Side view of the migration assay kit along the dotted line in (a) indicating the cell migration in SAP hydrogels. The yellow arrow represents the direction of horizontal cell migration. (c) A gross image of the kit sample with SAP hydrogels incubated in a 60-mm tissue culture dish.

Fig.4 The fluorescence imaging results of HUVECs migration on various concentrations of KLT-functionalized SAP hydrogels after 7 d of culture by using a confocal microscope: (a) RAD/KLT-20; (b) RAD/KLT-20; (c) RAD/KLT-60. Fluorescent staining with rhodamin-phalloidin for F-actin (red) showed the cell attachments and viabilities.

Fig.5 The fluorescence imaging results of HUVECs migration on various concentrations of PRG-functionalized SAP hydrogels after 7 d of culture by using a confocal microscope: (a) RAD/PRG-20; (b) RAD/PRG-20; (c) RAD/PRG-60. Fluorescent staining with rhodamin-phalloidin for F-actin (red) and DAPI for nuclei (blue) showed the cell attachments and viabilities.

Fig.6 The typical morphology of HUVECs growing on various concentration of SAP hydrogels at a given migration distance away from the starting point of the cell migration at day 7: (a) RAD/KLT-20, 5 mm; (b) RAD/KLT-40, 5 mm; (c) RAD/KLT-60, 5 mm; (d) RAD/PRG-20, 2 mm; (e) RAD/PRG-40, 2 mm; (f) RAD/PRG-60, 2 mm. Fluorescent staining with rhodamin-phalloidin for F-actin (red) and DAPI for nuclei (blue) showed the cell attachments and viabilities.

Fig.7 The proliferation profile of HUVECs evaluated with cell counting kit-8 (CCK-8) on the pure RADA or functionalized SAP hydrogels (RAD/KLT-20, RAD/KLT-40, RAD/KLT-60, RAD/PRG-20, RAD/PRG-40, and RAD/PRG-60) after 1, 4 and 7 d of culture (* P<0.05; ** P>0.05).

1	Lauffenburger D A, Horwitz A F. Cell migration: a physically integrated molecular process. Cell, 1996, 84(3): 359–369 https://doi.org/10.1016/S0092-8674(00)81280-5 pmid: 8608589
2	Reig G, Pulgar E, Concha M L. Cell migration: from tissue culture to embryos. Development, 2014, 141(10): 1999–2013 https://doi.org/10.1242/dev.101451 pmid: 24803649
3	Van Nieuwenhuysen T, Vleminckx K. Cell migration during development. eLS, 2014, https://doi.org/10.1002/9780470015902.a0020864.pub2
4	Shaw T J, Martin P. Wound repair at a glance. Journal of Cell Science, 2009, 122(18): 3209–3213 https://doi.org/10.1242/jcs.031187 pmid: 19726630
5	Gurtner G C, Werner S, Barrandon Y, . Wound repair and regeneration. Nature, 2008, 453(7193): 314–321 https://doi.org/10.1038/nature07039 pmid: 18480812
6	Yamaguchi H, Wyckoff J, Condeelis J. Cell migration in tumors. Current Opinion in Cell Biology, 2005, 17(5): 559–564 https://doi.org/10.1016/j.ceb.2005.08.002 pmid: 16098726
7	Hollister S J. Porous scaffold design for tissue engineering. Nature Materials, 2005, 4(7): 518–524 https://doi.org/10.1038/nmat1421 pmid: 16003400
8	Griffith L G, Naughton G. Tissue engineering — current challenges and expanding opportunities. Science, 2002, 295(5557): 1009–1014 https://doi.org/10.1126/science.1069210 pmid: 11834815
9	Petrie R J, Doyle A D, Yamada K M. Random versus directionally persistent cell migration. Nature Reviews: Molecular Cell Biology, 2009, 10(8): 538–549 https://doi.org/10.1038/nrm2729 pmid: 19603038
10	Pankov R, Endo Y, Even-Ram S, . A Rac switch regulates random versus directionally persistent cell migration. The Journal of Cell Biology, 2005, 170(5): 793–802 https://doi.org/10.1083/jcb.200503152 pmid: 16129786
11	Haeger A, Wolf K, Zegers M M, . Collective cell migration: guidance principles and hierarchies. Trends in Cell Biology, 2015, 25(9): 556–566 https://doi.org/10.1016/j.tcb.2015.06.003 pmid: 26137890
12	Rørth P. Whence directionality: guidance mechanisms in solitary and collective cell migration. Developmental Cell, 2011, 20(1): 9–18 https://doi.org/10.1016/j.devcel.2010.12.014 pmid: 21238921
13	Lutolf M P, Hubbell J A. Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering. Nature Biotechnology, 2005, 23(1): 47–55 https://doi.org/10.1038/nbt1055 pmid: 15637621
14	Kramer N, Walzl A, Unger C, . In vitro cell migration and invasion assays. Mutation Research/Reviews in Mutation Research, 2013, 752(1): 10–24 https://doi.org/10.1016/j.mrrev.2012.08.001 pmid: 22940039
15	Hulkower K I, Herber R L. Cell migration and invasion assays as tools for drug discovery. Pharmaceutics, 2011, 3(1): 107–124 https://doi.org/10.3390/pharmaceutics3010107 pmid: 24310428
16	Lei K F, Tseng H P, Lee C Y, . Quantitative study of cell invasion process under extracellular stimulation of cytokine in a microfluidic device. Scientific Reports, 2016, 6: 25557 https://doi.org/10.1038/srep25557 pmid: 27150137
17	Meng Q, Yao S, Wang X, . RADA16: A self-assembly peptide hydrogel for the application in tissue regeneration. Journal of Biomaterials and Tissue Engineering, 2014, 4(12): 1019–1029 https://doi.org/10.1166/jbt.2014.1246
18	Liu X, Wang X, Wang X, . Functionalized self-assembling peptide nanofiber hydrogels mimic stem cell niche to control human adipose stem cell behavior in vitro. Acta Biomaterialia, 2013, 9(6): 6798–6805 https://doi.org/10.1016/j.actbio.2013.01.027 pmid: 23380207
19	Wang X, Qiao L, Horii A. Screening of functionalized self-assembling peptide nanofiber scaffolds with angiogenic activity for endothelial cell growth. Progress in Natural Science: Materials International, 2011, 21(2): 111–116 https://doi.org/10.1016/S1002-0071(12)60043-4
20	Wang X, Horii A, Zhang S. Designer functionalized self-assembling peptide nanofiber scaffolds for growth, migration, and tubulogenesis of human umbilical vein endothelial cells. Soft Matter, 2008, 4(12): 2388–2395 https://doi.org/10.1039/b807155a
21	Liu X, Wang X, Horii A, . In vivo studies on angiogenic activity of two designer self-assembling peptide scaffold hydrogels in the chicken embryo chorioallantoic membrane. Nanoscale, 2012, 4(8): 2720–2727 https://doi.org/10.1039/c2nr00001f pmid: 22430460
22	D’Andrea L D, Iaccarino G, Fattorusso R, . Targeting angiogenesis: structural characterization and biological properties of a de novo engineered VEGF mimicking peptide. Proceedings of the National Academy of Sciences of the United States of America, 2005, 102(40): 14215–14220 https://doi.org/10.1073/pnas.0505047102 pmid: 16186493
23	Kouvroukoglou S, Dee K C, Bizios R, . Endothelial cell migration on surfaces modified with immobilized adhesive peptides. Biomaterials, 2000, 21(17): 1725–1733 https://doi.org/10.1016/S0142-9612(99)00205-7 pmid: 10905454
24	Aguzzi M S, Giampietri C, De Marchis F, . RGDS peptide induces caspase 8 and caspase 9 activation in human endothelial cells. Blood, 2004, 103(11): 4180–4187 https://doi.org/10.1182/blood-2003-06-2144 pmid: 14982875
25	Form D M, Pratt B M, Madri J A. Endothelial cell proliferation during angiogenesis. In vitro modulation by basement membrane components. Laboratory Investigation, 1986, 55(5): 521–530 pmid: 2430138

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