首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 31 毫秒
1.
For preparing cell sheets effectively for cell sheet-based regenerative medicine, cell-adhesion strength to thermoresponsive cell culture surfaces need to be controlled precisely. To design new thermoresponsive surfaces via a terminal modification method, thermoresponsive polymer brush surfaces were fabricated through the surface-initiated reversible addition-fragmentation chain transfer (RAFT) radical polymerization of N-isopropylacrylamide (IPAAm) on glass substrates. The RAFT-mediated grafting method gave dithiobenzoate (DTB) groups to grafted PIPAAm termini, which can be converted to various functional groups. In this study, the terminal carboxylation of PIPAAm chains provided high cell adhesive property to thermoresponsive surfaces. Although cell adhesion is generally promoted by a decrease in the grafted PIPAAm amount, the decrease also decelerated thermally-induced cell detachment, whereas the influence of terminal modification was negligible on the cell detachment. Consequently, the terminally modified PIPAAm brush surfaces allowed smooth muscle cells (SMCs) to simultaneously adhere strongly and detach themselves rapidly. In this study, SMCs were unable to reach a confluent monolayer on as-prepared PIPAAm brush surfaces (grafted amount: 0.41 μg/cm(2)) without terminal carboxylation due to their insufficient cell-adhesion strength. On the other hand, though a decrease in the PIPAAm amount allowed SMCs to form a confluent cell monolayer on the PIPAAm brush surface, the SMCs were unable to be harvested as a monolithic cell sheet by low-temperature culture at 20 °C. Because of their unique property, only terminal-carboxylated PIPAAm brush surfaces achieved rapid harvesting of complete cell sheets by low-temperature culturing.  相似文献   

2.
Cell sorting of specific target cells from a mixture of different cell types is a prerequisite for development of functional engineered tissues based on stem-cell and tissue engineering. This paper presents a new method of cell sorting that uses a mixture of thermoresponsive cell-adhesive and non-cell-adhesive substances. The former substance is poly(N-isopropylacrylamide)-grafted gelatin (PNIPAM-gelatin) and the latter is PNIPAM. Graded cell adhesion, produced by mixed coating of these thermoresponsive substances at an appropriate mixing ratio, clearly differentiated the adhesive potentials of two bovine vascular cell types (endothelial cell and smooth muscle cell). The sequential procedures of detachment at room temperature and subsequent replating at 37 degrees C on dishes coated with a mixed coating with the same composition as that employed previously yielded remarkably pure target cells, as determined using confocal laser scanning fluorescence microscopy. This method, leading to harvesting of target cells, is characteristic of simple manipulation with no cell damage. Such advantages are expected to facilitate stem-cell and tissue engineering.  相似文献   

3.
Temperature-dependent regulation of affinity binding between bioactive ligands and their cell membrane receptors is an attractive approach for the dynamic control of cellular adhesion, proliferation, migration, differentiation, and signal transduction. Covalent conjugation of bioactive ligands onto thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-grafted surfaces facilitates the modulation of one-on-one affinity binding between bioactive ligands and cellular receptors by changing temperature. For the dynamic control of the multivalent affinity binding between heparin and heparin-binding proteins, thermoresponsive cell culture surface modified with heparin, which interacts with heparin-binding proteins such as basic fibroblast growth factor (bFGF), has been proposed. Heparin-functionalized thermoresponsive cell culture surface induces (1) the multivalent affinity binding of bFGF in active form and (2) accelerating cell sheet formation in the state of shrunken PIPAAm chains at 37°C. By lowering temperature to 20°C, the affinity binding between bFGF and immobilized heparin is reduced with increasing the mobility of heparin and the swollen PIPAAm chains, leading to the detachment of cultured cells. Therefore, heparin-functionalized thermoresponsive cell culture surface was able to enhance cell proliferation and detach confluent cells as a contiguous cell sheet by changing temperature. A cell cultivation system using heparin-functionalized thermoresponsive cell culture surface is versatile for immobilizing other heparin-binding proteins such as vascular endothelial growth factor, fibronectin, antithrombin III, and hepatocyte growth factor, etc. for tuning the adhesion, growth, and differentiation of various cell species.  相似文献   

4.
The smart thermoresponsive coatings and surfaces that have been explicitly designed for cell culture are mostly based on poly(N-isopropylacrylamide) (PNIPAAm). This polymer is characterized by a sudden precipitation on heating, switching from a hydrophilic to a hydrophobic state. Mammalian cells cultured on such thermoresponsive substrates can be recovered as confluent cell sheets, while keeping the newly deposited extracellular matrix intact, simply by lowering the temperature and thereby avoiding the use of deleterious proteases. Thermoresponsive materials and surfaces are powerful tools for creating tissue-like constructs that imitate native tissue geometry and mimic its spatial cellular organization. Here we review and compare the most representative methods of producing thermoresponsive substrates for cell sheet engineering.  相似文献   

5.
Cell-based therapeutics are promising routes for the regeneration of damaged cells and organs. The recovery of cells cultured in vitro for such applications requires the use of proteolytic enzymes which deteriorate its property by disruption of cell–cell and cell–matrix interactions. Intact cell sheets can be retrieved with the use of thermo responsive polymer grafted on to the culture plates. Our study presents the use of photo-polymerization as a simple and inexpensive way to create thermo-responsive culture surfaces for the detachment of intact cell sheet. Poly (N-isopropyl acrylamide) (PNIPAAm) was synthesized by photo-polymerization and characterized by NMR spectroscopy, differential scanning calorimetry and gel permeation chromatography. Thermo-responsive culture dishes were prepared by the coating method and characterized for its thermo-responsive efficacy using FTIR spectroscopy and water contact angle measurements. Atomic force microscopy depicted the thin coating achieved with this method is similar to the conventional grafting method. Suitability for cell culture and cell sheet retrieval was assessed by culturing rat aortic smooth muscle cells in the PNIPAAm coated tissue culture plates. The cells remained viable as evident from the live dead assay and the cell sheet was detached by low temperature treatment. The results demonstrate a versatile method for creating thermo responsive culture surfaces while eliminating the use of expensive radiation sources for the conventional grafting method.  相似文献   

6.
Cell sheet engineering has been progressing rapidly during the past few years and has emerged as a novel approach for cell based therapy. Cell sheet harvest technology enables fabrication of viable, transplantable cell sheets for various tissue engineering applications. Currently, the majority of cell sheet studies use thermo-responsive systems for cell sheet detachment. However, other responsive systems began showing their potentials for cell sheet harvest. This review provides an overview of current techniques in creating cell sheets using different types of responsive systems including thermo-responsive, electro-responsive, photo-responsive, pH-responsive and magnetic systems. Their mechanism, approach, as well as applications for cell detachment have been introduced. Further development of these responsive systems will allow efficient cell sheet harvesting and patterning of cells to reconstruct complex tissue for broad clinical applications.  相似文献   

7.
Cell sheets (CS) from c-kit+ cardiac stem cell (CSC) hold a potential for application in regenerative medicine. However, manufacture of CS may require thermoresponsive dishes, which increases cost and puts one in dependence on specific materials. Alternative approaches were established recently and we conducted a short study to compare approaches for detachment of CS from c-kit+ CSC. Our in-house developed method using chelation by Versene solution was compared to UpCell? thermoresponsive plates in terms of CSC proliferation, viability, gap junction formation and engraftment in a model of myocardial infarction. Use of Versene solution instead of thermoresponsive dishes resulted in comparable CS thickness (approximately 100 mcm), cell proliferation rate and no signs of apoptosis detected in both types of constructs. However, we observed a minor reduction of gap junction count in Versene-treated CS. At day 30 after delivery to infarcted myocardium both types of CS retained at the site of transplantation and contained comparable amounts of proliferating cells indicating engraftment. Thus, we may conclude that detachment of CS from c-kit+ CSC using Versene solution followed by mechanical treatment is an alternative to thermoresponsive plates allowing use of routinely available materials to generate constructs for cardiac repair.  相似文献   

8.
聚N-异丙基丙烯酰胺(poly(N-isopropylacrylamide),PNIPAAm),温敏性聚合物,可利用其温敏特性替代酶类物质或细胞刮刀用于贴壁细胞的收获,从而有效避免酶解和机械损伤,可为生物医药领域提供品质优良的种子细胞。重点阐述促进细胞有效粘附和快速脱附的温敏性PNIPAAm二维平面的研发情况,包括选取特殊基材、引入亲水基团、调节反应物比率、控制聚合物厚度/密度、提供适宜外力等方式,从而有效改善细胞对温敏性平面的适应性并降低染菌风险以及减少低温处理对细胞的影响。同时介绍PNIPAAm微载体、支架和凝胶等温敏性三维培养介质的研究进展,此方式不仅增加细胞生长面积,更可以模拟体内微环境,从而保持细胞原始生理特征,同时实现大规模扩增和非酶解收获细胞以及组织器官修复和重构的目标。最后简单说明PNIPAAm培养平台的应用,PNIPAAm的研发为再生医学的发展提供了崭新思路。  相似文献   

9.
Newly developed fabrication technique of thermoresponsive surface using RAFT-mediated block copolymerization and photolithography achieved stripe-like micropatterning of poly(N-isopropylacrylamide) (PIPAAm) brush domains and poly(N-isopropylacrylamide)-b-poly(N-acryloylmorpholine) domains. Normal human dermal fibroblasts were aligned on the physicochemically patterned surfaces simply by one-pot cell seeding. Fluorescence images showed the well-controlled orientation of actin fibers and fibronectin in the confluent cell layers with associated extracellular matrix (ECM) on the surfaces. Furthermore, the aligned cells were harvested as a tissue-like cellular monolayer, called "cell sheet" only by reducing temperature below PIPAAm's lower critical solution temperature (LCST) to 20 °C. The cell sheet harvested from the micropatterned surface possessed a different shrinking rate between vertical and parallel sides of the cell alignment (approximately 3:1 of aspect ratio). This indicates that the cell sheet maintains the alignment of cells and related ECM proteins, promising to show the mechanical and biological aspects of cell sheets harvested from the functionalized thermoresponsive surfaces.  相似文献   

10.
Thermoresponsive surfaces are prepared via a spin-coating method with a block copolymer consisting of poly(N-isopropylacrylamide) (PIPAAm) and poly(butyl methacrylate) (PBMA) on polystyrene surfaces. The PBMA block suppresses the removal of deposited PIPAAm-based polymers from the surface. The polymer coating affects the temperature-dependent cellular behavior of the surfaces with respect to protein adsorption. By adjusting layer thicknesses, PBMA-b-PIPAAm-coated surfaces are optimized to regulate the adhesion/detachment of cells by temperature changes. Thus, thermoresponsive polymer-coated surfaces are able to harvest contiguous cell sheets with their basal extracellular matrix proteins.  相似文献   

11.
Summary Highly contractile skeletal myotubes differentiated in tissue culture are normally difficult to maintain on collagen-coated tissue culture dishes for extended periods because of their propensity to detach as a sheet of cells from their substratum. This detachment results in the release of mechanical tension in the growing cell “sheet” and, consequently, loss of cellular protein. We developed a simple method of culturing high density contractile primary avian myotubes embedded in a collagen gel matrix (collagel) attached to either a stainless steel mesh or nylon support structure. With this system the cells are maintained in a highly contractile state for extended periods in vitro under tension. Structural integrity of the myotubes can be maintained for up to 10 d in basal medium without serum or embryo extract. Total cellular protein and myosin heavy chain accumulation in the cells can be maintained for weeks at levels which are two to three times those found in timematched controls that are under little tension. Morphologically, the myotubes are well differentiated with structural characteristics of neonatal myofibers. This new collagel culture system should prove useful in the analysis of in vitro gene expression during myotube to myofiber differentiation and its regulation by various environmental factors such as medium growth factors, innervation, and mechanical activity. This work was supported by grant AM 36266 from the National Institutes of Health, Bethesda, MD, and grant NAG2-414 from the National Aeronautics and Space Administration, Washington, D.C. Parts of this work have appeared in abstract form, In Vitro 23:24a; 1987.  相似文献   

12.
Retraction is a major rate-limiting step in cell motility, particularly in slow moving cell types that form large stable adhesions. Myosin II dependent contractile forces are thought to facilitate detachment by physically pulling up the rear edge. However, retraction can occur in the absence of myosin II activity in cell types that form small labile adhesions. To investigate the role of contractile force generation in retraction, we performed traction force microscopy during the movement of fish epithelial keratocytes. By correlating changes in local traction stress at the rear with the area retracted, we identified four distinct modes of retraction. “Recoil” retractions are preceded by a rise in local traction stress, while rear edge is temporarily stuck, followed by a sharp drop in traction stress upon detachment. This retraction type was most common in cells generating high average traction stress. In “pull” type retractions local traction stress and area retracted increase concomitantly. This was the predominant type of retraction in keratocytes and was observed mostly in cells generating low average traction stress. “Continuous” type retractions occur without any detectable change in traction stress, and are seen in cells generating low average traction stress. In contrast, to many other cell types, “release” type retractions occur in keratocytes following a decrease in local traction stress. Our identification of distinct modes of retraction suggests that contractile forces may play different roles in detachment that are related to rear adhesion strength. To determine how the regulation of contractility via MLCK or Rho kinase contributes to the mechanics of detachment, inhibitors were used to block or augment these pathways. Modulation of MLCK activity led to the most rapid change in local traction stress suggesting its importance in regulating attachment strength. Surprisingly, Rho kinase was not required for detachment, but was essential for localizing retraction to the rear. We suggest that in keratocytes MLCK and Rho kinase play distinct, complementary roles in the respective temporal and spatial control of rear detachment that is essential for maintaining rapid motility.  相似文献   

13.
Human mesenchymal stem cells (hMSCs) are colony‐forming unit fibroblasts (CFU‐F) derived from adult bone marrow and have significant potential for many cell‐based tissue‐engineering applications. Their therapeutic potential, however, is restricted by their diminishing plasticity as they are expanded in culture. In this study, we used N‐isopropylacrylamide (NIPAM)‐based thermoresponsive polyelectrolyte multilayer (N‐PEMU) films as culture substrates to support hMSC expansion and evaluated their effects on cell properties. The N‐PEMU films were made via layer‐by‐layer adsorption of thermoresponsive monomers copolymerized with charged monomers, positively charged allylamine hydrochloride (PAH), or negatively charged styrene sulfonic acid (PSS) and compared to fetal bovine serum (FBS) coated surfaces. Surface charges were shown to alter the extracellular matrix (ECM) structure and subsequently regulate hMSC responses including adhesion, proliferation, integrin expression, detachment, and colony forming ability. The positively charged thermal responsive surfaces improved cell adhesion and growth in a range comparable to control surfaces while maintaining significantly higher CFU‐F forming ability. Immunostaining and Western blot results indicate that the improved cell adhesion and growth on the positively charged surfaces resulted from the elevated adhesion of ECM proteins such as fibronectin on the positively charge surfaces. These results demonstrate that the layer‐by‐layer approach is an efficient way to form PNIPAM‐based thermal responsive surfaces for hMSC growth and removal without enzymatic treatment. The results also show that surface charge regulates ECM adhesion, which in turn influences not only cell adhesion but also CFU‐forming ability and their multi‐lineage differentiation potential. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010  相似文献   

14.
15.
In contrast to living cells, glycerin extracted mouse embryo fibroblasts do not round up after detachment from the substrate. The addition of ATP makes these fibroblasts round up. Thus, the rounding of the detached cell occurs in result of active, ATP-requiring contractile forces rather than due to the action of elastic forces or of surface tension. The ATP-induced contraction of the glycerinated cell is accompanied with the loss of the parallel orientation of 50-70 A microfilaments. The loss is suggested to result from the attachment of different microfilaments of the same bundle to different points of the cell surface. Microtubules are not essential for the contraction: the rounding of living or glycerin-treated cells is not colcemide affected. Living cells treated with cytochalasine B (CH) reversibly lose their ability to round up after detachment. ATP is able to induce no contraction of glycerin-extracted cells treated with CH before extraction. In contrast, the addition of CH to the ATP-containing solution does not inhibit the contraction of glycerin-extracter normal cells. These results give reason to suggest that CH may inactivate contractile structures of the cell. It may be thought that some unknown additional factors, available in the living cell and not available in the glycerin-extracted one, are essential for this inactivation.  相似文献   

16.
Here we propose a bio-MEMS device designed to evaluate contractile force and conduction velocity of cell sheets in response to mechanical and electrical stimulation of the cell source as it grows to form a cellular sheet. Moreover, the design allows for the incorporation of patient-specific data and cell sources. An optimized device would allow cell sheets to be cultured, characterized, and conditioned to be compatible with a specific patient's cardiac environment in vitro, before implantation. This design draws upon existing methods in the literature but makes an important advance by combining the mechanical and electrical stimulation into a single system for optimized cell sheet growth. The device has been designed to achieve cellular alignment, electrical stimulation, mechanical stimulation, conduction velocity readout, contraction force readout, and eventually cell sheet release. The platform is a set of comb electrical contacts consisting of three-dimensional walls made of polydimethylsiloxane and coated with electrically conductive metals on the tops of the walls. Not only do the walls serve as a method for stimulating cells that are attached to the top, but their geometry is tailored such that they are flexible enough to be bent by the cells and used to measure force. The platform can be stretched via a linear actuator setup, allowing for simultaneous electrical and mechanical stimulation that can be derived from patient-specific clinical data.  相似文献   

17.
Tissue engineering aims to regenerate tissues that can successfully take over the functions of the native tissue when it is damaged or diseased. In most tissues, collagen makes up the bulk component of the extracellular matrix, thus, there is great emphasis on its accurate quantification in tissue engineering. It has already been reported that pepsin digestion is able to solubilize the collagen deposited within the cell layer for accurate quantification of collagen content in cultures, but this method has drawbacks when cultured cells are hyperconfluent. In this condition, Pepsin digestion will result in fragments of the cell layers that cannot be completely resolved. These fragments of the undigested cell sheet are visible to the naked eye, which can bias the final results. To the best of our knowledge, there has been no reported method to accurately quantify the collagen content in hyperconfluent cell sheet. Therefore, this study aims to illustrate that sonication is able to aid pepsin digestion of hyperconfluent cell layers of fibroblasts and bone marrow mesenchymal stem cells, to solubilize all the collagen for accurate quantification purposes.  相似文献   

18.
Cell transfer printing from patterned poly(ethylene glycol)-oleyl surfaces onto biological hydrogel sheets is investigated herein, as a new cell stamping method for both cell microarray and tissue engineering. By overlaying a hydrogel sheet on the cells immobilized on the poly(ethylene glycol)-oleyl surface and successively peeling it off, the immobilized cells were transferred onto a hydrogel sheet because the adhesive interaction between the cells and the hydrogel was stronger than that between the cells and the poly(ethylene glycol)-oleyl surface. Four types of human cell could be efficiently transferred onto a rigid collagen sheet. The transfer printing ratios, for all cells, were above 80% and achieved within 90 min. A cell microarray was successfully prepared on a collagen gel sheet using the present stamping method. We have also demonstrated that the transferred pattern of endothelial cells is transformed to the patterned tube-like structure on the reconstituted basement membrane matrix. Finally, the patterns of two types of endothelial cell are shown to be easily prepared on the matrix, and the desired tube-like structures, including the orderly pattern of the two different cells, were formed spontaneously. Thus, the present poly(ethylene glycol)-oleyl coated substrates are useful for rapid and efficient cell stamping, in the preparation of multi-cellular pattern on extracellular matrices.  相似文献   

19.
A novel method to prepare multicellular spheroids from varied cell types   总被引:1,自引:0,他引:1  
A simple method for preparing multicellular spheroids from varied cell types has been successfully developed by using a stepwise gradient surface in cell attachability or detachability. The surface was composed of poly-N-isopropylacrylamide (PNIPAAm), a temperature responsive polymer, as a cell detaching component, and collagen as a cell attaching component. The surface functions as a culture substratum at 37 degrees C; then, when lowering the temperature of culture medium, the cells attached to it detach as a self-supporting sheet. This is because PNIPAAm dissolves into the culture medium below the lower critical solution temperature (LCST; about 30 degrees C), but it is insoluble above the LCST. The detached cell sheet forms a multicellular spheroid. The stepwise gradient surface which consisted of six different sectors was prepared by exposing a surface of the PNIPAAm-collagen mixture to ultraviolet (UV) irradiation six times using a photomask, sliding the hole position in the photomask, and changing the energy of UV irradiation. This was because crosslinking of collagen depended on the energy of UV irradiation, then, cell attachability to and detachability from the surface were tightly controlled by changing the energy.The stepwise gradient surface allowed us to easily determine optimal surface conditions to obtain good cell attachment and detachment as a self-supporting sheet from the surface to prepare multicellular spheroids. According to the evaluation of the attachability and detachability of 23 cell types, the optimal surface condition remarkably depended on each cell type. The detached cells under optimal surface conditions, including fibroblasts, osteoblastic cells, smooth muscle cells, and measangial cells, which were very difficult to form spherioids using conventional methods, were able to form multicellular spheroids. The results clearly demonstrate that the above-described method for preparing multicellular spheroids can be applied to varied cell types. (c) 1995 John Wiley & Sons, Inc.  相似文献   

20.
Mandal K  Balland M  Bureau L 《PloS one》2012,7(5):e37548
We describe the design of micropatterned surfaces for single cell studies, based on thermoresponsive polymer brushes. We show that brushes made of poly(N-isopropylacrylamide) grafted at high surface density display excellent protein and cell anti-adhesive properties. Such brushes are readily patterned at the micron scale via deep UV photolithography. A proper choice of the adhesive pattern shapes, combined with the temperature-dependent swelling properties of PNIPAM, allow us to use the polymer brush as a microactuator which induces cell detachment when the temperature is reduced below [Formula: see text]C.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号