| For the past centenary,glass flasks,plastic culture dishes,and microplates have been used for cell culture.In those culture settings,cells grow on two-dimensional(2D)substrates as a monolayer,which significantly differs from real in vivo environments,in which cells are surrounded by other cells and the extracellular matrix in a 3D fashion.It has been postulated that the reason in vitro assays for selected compounds have not been successfully translated to clinical practice is because cells grown in 2D monolayers cannot reproduce the complicated signals that occur between cells and the matrix in vivo.Thus,growing attention has been paid to the development of in vitro culture systems that mimic the in vivo environment.Paper,one of the most ancient inventions,has been successfully used to culture cells and assemble tissue-like structures in vitro because the flexibility in stacking and de-stacking paper allows evaluation of the metabolic profile of cells at different layers.Although distinct stacking and destocking procedure to build 3D culture model demonstrated the potential of paper-based devices for cell-based studies,it is of importance to see that the paper of choice is limited to certain types of paper—the most popular are Whatman brand 114 chromatography filter paper(CFP-114)and lens paper(LP).Whatman CFP-114 paper is quite thick(~200 ?m),and this will affect microscopy studies.Lens paper is ~40 ?m thick.Because of its looser cellulose matrix,two pieces of lens paper are normally laminated to one paper layer to avoid disassembling the fiber networks.In addition,to provide a 3D environment for cell growth,the hydrogel is loaded onto filter paper.The cellulose fiber network of filter paper in this cells-in-gel-in-paper(CiGiP)model functions as a framework to ensure the gelling and shaping of the hydrogel,while the hydrogel not only increases the water intake but also provides a 3D environment for cell growth.However,one of the concerns is that the convenience of the CiGiP platform has been compromised because the hydrogel has to be prepared and loaded onto the paper-like substrate before usage.After several hours of gelling,the substrate can be used for subsequent cell assays.For the above concerns,this study focuses on following part: 1.To explore the potential of Chinese calligraphy rice paper as an economical and cytocompatible substrate for cell biological assaysFor the first time,we show the potential of rice paper(an organic paper specifically used in Chinese calligraphy)for building cell analysis platforms.Rice paper's solution wicking and surface characterization proves that it has a similar chemical configuration as standard Whatman filter paper.Moreover,lactate dehydrogenase(LDH)release assay and WST-1 cell growth assay show that rice paper has better cell-compatibility features and improved cell distribution.The cell anchors and spreads along the cellulose fiber of the rice paper,while the porous rice paper matrix provides a sufficient surface area for cell growth.The cell-based immunohistochemistry was conducted to measure the expression of O-linked N-acetylglucosamine(O-GlcNAc)protein on prostate cancer cell DU145.An enhanced colorimetric signal can be observed from rice paper-based cell culture platform than that from standard 2D culture dish.The feasibility of fabricating rice paper with both direct crafting and wax printing—as well as on-paper cell immunoassays for on-demand applications—confirms the potential of rice paper as a new substrate for building paper devices for cell biology studies.2.Freeze-drying prepared gelatin aerogel@polypropylene nonwoven sheet as a ready-to-use substrate for 3D cell cultureInspired by the CiGiP model,we proposed a free-standing,ready-to-use gelatin aerogel impregnated nonwoven scaffold(gelatin aerogel@nonwoven)for cell culture.Polypropylene(PP)is one of the most commonly used polymers and has good mechanical properties,heat resistance,low cost,ease of processing,and full recyclability.The PP nonwoven fabric serves as a skeleton or scaffold that ensures the stability of thin sheet structures,which are the base for stacking and de-stacking and assembling tissue-like structures.The impregnated gelatine aerogel functions as an interconnected hydrogel base for solution and nutrient storage and niches for cell growth.In this study,gelatin aerogel@PP nonwoven hybrids were prepared by the freeze-drying approach,after which the porosity and swelling property of the hybrid scaffolds were characterized.The cell adhesion and cell proliferation assays showed that the gelatin aerogel@PP nonwoven hybrid is a ready-to-use substrate for cell-based assays.The ready-to-use convenience of the gelatin aerogel@PP nonwoven hybrid was ensured,as the physicochemical properties(chemical group,pore ratio,and swelling rate)and cell compatibility were well preserved after 90 days of storage.3.Cytokine-functionalized gelatin aerogel@polypropylene nonwoven sheet for mimicking microenvironment of cellsCytokines are functional proteins secreted by immune cells and stromal cells.To improve the biological function of paper-like materials,especially to mimics the microenvironment for cell growth,we proposed to introduce cytokines onto gelatin aerogel@PP nonwoven hybrid.First,Human vascular endothelial growth factor(hrVEGF)was loaded on gelatin aerogel@PP nonwoven hybrid through physical adsorption,EDC/NHS mediated covalent conjugation,and pre-mixing.The effects of VEGF functionalization on cell adhesion and proliferation were studied.The results showed that cytokines coating not only promotes the adhesion of human vascular endothelial cells on gelatin aerogel@PP nonwoven hybrid,but also enhances the proliferation of attached cells.Moreover,a multi-layer gelatin @ polypropylene hybrids composed cell migration system was assembled.The results showed that cells attended to grow into VEGF-coated gelatin @PP nonwoven hybrid,demonstrating the potential of cytokines-functionalized paper-like materials in assembling of 3D system for cell study. |
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