The objective of this study was to investigate the utility of gelatin hydrogel nonwoven fabrics (GHNFs) as a carrier membrane in preparing multilayered cell sheets. The GHNFs were fabricated by a blow method of gelatin solution. When the cell sheet of human mesenchymal stromal cells was piled up to formulate three-layered cell sheets, the GHNFs were used to allow the cell sheet to detach from the cell culture dish and transfer. The cell sheet harvest and transfer processes were performed simpler and faster than those without using the GHNF. The lactate/glucose ratio of a metabolic activity was significantly lower, and the ATP production was higher for the three-layered cell sheets formulated with the GHNF than that obtained without the GHNF. During the culture, the cell migration from cell sheets into the GHNF was observed. The GHNF was promising to simply and fast fabricate three-layered cell sheets with the activity remaining.
Impact Statement
This study introduces the utility of gelatin hydrogel nonwoven fabrics (GHNFs) for cell sheet engineering. The GHNF had the mechanical property strong enough to hold by forceps even in the swollen condition. The cell sheet harvest and transfer processes were performed simpler and faster than those without using the GHNF. The GHNF facilitates the metabolic activity of three-layered cell sheets, and the cell migration from cell sheets into the GHNF was observed. The GHNF is a promising material used to support cell sheets during the process of assemble formulation and contributes to the improved biological functions of tissue-like cell constructs.
Get full access to this article
View all access options for this article.
References
1.
MatsuuraK., UtohR., NagaseK., et al.Cell sheet approach for tissue engineering and regenerative medicine. J Control Release, 190, 228, 2014.
2.
KirbyG.T.S., MichelmoreA., SmithL.E., WhittleJ.D., and ShortR.D.Cell sheets in cell therapies. Cytotherapy, 20, 169, 2017.
3.
KaweckiF., ClafshenkelW.P., FortinM., AugerF.A., and FradetteJ.Biomimetic tissue-engineered bone substitutes for maxillofacial and craniofacial repair: the potential of cell sheet technologies. Adv Healthc Mater, 7, 1, 2018.
SakaiY., KoikeM., YamanouchiK., et al.Time-dependent structural and functional characterization of subcutaneous human liver tissue. J Tissue Eng Regen Med, 12, 2287, 2018.
6.
MasumotoH., and YamashitaJ.K.Human iPS cell-engineered three-dimensional cardiac tissues perfused by capillary networks between host and graft. Inflamm Regen, 38, 26, 2018.
7.
HaraguchiY., ShimizuT., SasagawaT., et al.Fabrication of functional three-dimensional tissues by stacking cell sheets in vitro. Nat Protoc, 7, 850, 2012.
8.
SasagawaT., ShimizuT., SekiyaS., et al.Design of prevascularized three-dimensional cell-dense tissues using a cell sheet stacking manipulation technology. Biomaterials, 31, 1646, 2010.
9.
KikuchiT., ShimizuT., WadaM., YamatoM., and OkanoT.Automatic fabrication of 3-dimensional tissues using cell sheet manipulator technique. Biomaterials, 35, 2428, 2014.
10.
TadakumaK., TanakaN., HaraguchiY., et al.A device for the rapid transfer/transplantation of living cell sheets with the absence of cell damage. Biomaterials, 34, 9018, 2013.
11.
ShimizuH., OhashiK., UtohR., et al.Bioengineering of a functional sheet of islet cells for the treatment of diabetes mellitus. Biomaterials, 30, 5943, 2009.
12.
AmagaiY., KarasawaK., KyungsookJ., et al.Development of a novel carrier optimized for cell sheet transplantation. Biomatter, 5, e1027846, 2015.
13.
MouthuyP.-A., El-SherbiniY., CuiZ., and YeH.Layering PLGA-based electrospun membranes and cell sheets for engineering cartilage-bone transition. J Tissue Eng Regen Med, 10, E263, 2016.
14.
HaraguchiY., KagawaY., HasegawaA., KuboH., and ShimizuT.Rapid fabrication of detachable three-dimensional tissues by layering of cell sheets with heating centrifuge. Biotechnol Prog, 34, 692, 2018.
15.
ShimizuT., SekineH., YangJ., et al.Polysurgery of cell sheet grafts overcomes diffusion limits to produce thick, vascularized myocardial tissues. FASEB J, 20, 708, 2006.
16.
SekiyaS., and ShimizuT.Introduction of vasculature in engineered three-dimensional tissue. Inflamm Regen, 37, 25, 2017.
17.
SekineW., HaraguchiY., ShimizuT., UmezawaA., and OkanoT.Thickness limitation and cell viability of multi-layered cell sheets and overcoming the diffusion limit by a porous-membrane culture insert. J Biochips Tissue Chips, S1, 007, 2011.
18.
MatsuoT., MasumotoH., TajimaS., et al.Efficient long-term survival of cell grafts after myocardial infarction with thick viable cardiac tissue entirely from pluripotent stem cells. Sci Rep, 5, 16842, 2015.
19.
MedeirosE.S., GlennG.M., KlamczynskiA.P., OrtsW.J., and MattosoL.H.C.Solution blow spinning: a new method to produce micro- and nanofibers from polymer solutions. J Appl Polym Sci, 113, 2322, 2009.
20.
OzekiM., and TabataY.In vivo degradability of hydrogels prepared from different gelatins by various cross-linking methods. J Biomater Sci Polym Ed, 16, 549, 2005.
21.
OkamotoT., AoyamaT., NakayamaT., et al.Clonal heterogeneity in differentiation potential of immortalized human mesenchymal stem cells. Biochem Biophys Res Commun, 295, 354, 2002.
22.
WeiF., QuC., SongT., et al.Vitamin C treatment promotes mesenchymal stem cell sheet formation and tissue regeneration by elevating telomerase activity. J Cell Physiol, 227, 3216, 2012.
23.
HasegawaA., HaraguchiY., ShimizuT., and OkanoT.Rapid fabrication system for three-dimensional tissues using cell sheet engineering and centrifugation. J Biomed Mater Res Part A, 103, 3825, 2015.
24.
NamJ.H., ErmonvalM., and SharfsteinS.T.Cell attachment to microcarriers affects growth, metabolic activity, and culture productivity in bioreactor culture. Biotechnol Prog, 23, 652, 2007.
25.
SekineH., ShimizuT., SakaguchiK., et al.In vitro fabrication of functional three-dimensional tissues with perfusable blood vessels. Nat Commun, 4, 1310, 2013.
26.
SaitoK., LeeS., ShiuchiT., et al.An enzymatic photometric assay for 2-deoxyglucose uptake in insulin-responsive tissues and 3T3-L1 adipocytes. Anal Biochem, 412, 9, 2011.
27.
TabataY., NaganoA., and IkadaY.Biodegradation of hydrogel carrier incorporating fibroblast growth factor. Tissue Eng, 5, 127, 1999.
28.
YoungS., WongM., TabataY., and MikosA.G.Gelatin as a delivery vehicle for the controlled release of bioactive molecules. J Control Release, 109, 256, 2005.
29.
HayashiK., and TabataY.Preparation of stem cell aggregates with gelatin microspheres to enhance biological functions. Acta Biomater, 7, 2797, 2011.
30.
TabataY.Biomaterials design of culture substrates for cell research. Inflamm Regen, 31, 137, 2011.
31.
LiuX., and MaP.X.Phase separation, pore structure, and properties of nanofibrous gelatin scaffolds. Biomaterials, 30, 4094, 2009.
32.
MatsudaN., ShimizuT., YamatoM., and OkanoT.Tissue engineering based on cell sheet technology. Adv Mater, 19, 3089, 2007.
33.
PangestyI.A., ArahiraT., and TodoM.Characterization of tensile mechanical behavior of MSCs/PLCL hybrid layered sheet. J Funct Biomater, 7, 14, 2016.
34.
TabataY., YamadaK., MiyamotoS., et al.Bone regeneration by basic fibroblast growth factor complexed with biodegradable hydrogels. Biomaterials, 19, 807, 1998.
35.
LienS.M., KoL.Y., and HuangT.J.Effect of pore size on ECM secretion and cell growth in gelatin scaffold for articular cartilage tissue engineering. Acta Biomater, 5, 670, 2009.
36.
StephanouA., and JayasingheS.N.Cell electrospining: a unique biotechnique for encapsulating living organisms for generating active biological microthreads/scaffolds. Biomacromolecules, 7, 3364, 2006.
37.
JayasingheS.N.Cell electrospinning: a novel tool for functionalising fibres, scaffolds and membranes with living cells and other advanced materials for regenerative biology and medicine. Analyst, 138, 2215, 2013.
