Construction of novel temperature-responsive hydrogel culture system based on the biomimetic method for stem cell sheet harvest

2019 ◽  
Vol 34 (3) ◽  
pp. 229-245 ◽  
Author(s):  
Zengjie Fan ◽  
Yingying Nie ◽  
Zizi Chen ◽  
Xuzhuzi Xie ◽  
Xiaozhu Liao ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Stem Cell ◽  
Culture System ◽  
Cell Sheet ◽  
Sheet Thickness ◽  
Temperature Sensitive ◽  
Preparation Methods ◽  
Tissue Culture Polystyrene ◽  
Temperature Responsive ◽  
Biomimetic Method

Temperature-responsive hydrogel culture system is considered as an ideal platform for cell sheet harvest, but its complex preparation methods and harsh reaction conditions limit its application. Inspired by the marine mussels, a biomimetic method presented here is to construct a novel temperature-responsive hydrogel culture system for stem cell sheet harvest. The tissue culture polystyrene is first modified with polydopamine coating, and then amine-terminated poly(N-isopropylacrylamide) is grafted onto the coating via the Schiff base or Michael addition reaction to construct the temperature-sensitive hydrogel culture system. Then, bone marrow stromal cells are cultured on the culture system to construct cell sheets. The prepared culture system shows significant temperature-sensitive property with the grafted concentrations of poly(N-isopropylacrylamide) ranging from 0.5 to 1 g/L. Meanwhile, the constructed culture system has low cytotoxicity and facilitates the stem cell adhesion, proliferation, and cell sheet formation at 37°C. When the culture system is placed in a 20°C environment, the cell sheet can be completely detached from the surface of tissue culture polystyrene without being treated with any enzymes. More importantly, the cell morphology, cell sheet thickness, and the fibril structure of the associated proteins are similar to the cells cultured on the tissue culture polystyrene without modification. The biomimetic, simple, inexpensive, and environmentally friendly preparation of the culture system enables it to be used for the harvest of cell sheet and even applied to tissue engineering for tissue regeneration.

scholarly journals Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

10.3791/58624 ◽  
2018 ◽  
Author(s):  
Yingwei Wang ◽  
Cheng Lu ◽  
Chengzhi He ◽  
Baoxin Chen ◽  
Youling Zheng ◽  
...  
Keyword(s):  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Culture System ◽  
Cell Sheet ◽  
Dynamic Culture ◽  
Mesenchymal Stem Cell Sheet

scholarly journals Phenotypic traits of mesenchymal stem cell sheets fabricated by temperature-responsive cell culture plate: structural characteristics of MSC sheets

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Mitsuyoshi Nakao ◽  
Kyungsook Kim ◽  
Kenichi Nagase ◽  
David W. Grainger ◽  
Hideko Kanazawa ◽  
...  
Keyword(s):  
Cell Culture ◽  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Cell Sheet ◽  
Cellular Structures ◽  
Phenotypic Traits ◽  
Cell Sheets ◽  
Temperature Responsive ◽  
Cell Sheet Technology ◽  
Responsive Cell

Abstract Background In most stem cell therapy strategies reported to date, stem cells are introduced to damaged tissue sites to repair and regenerate the original tissue structure and function. MSC therapeutic efficacies are inconsistent, largely attributed to transplanted MSC difficulties both in engrafting at tissue sites and in retaining their therapeutic functions from suspension formulations. MSC functional components, including cell adhesion and cell–cell junction proteins, and ECM that contribute to essential cellular therapeutic effects, are damaged or removed by proteolytic enzymes used in stem cell harvesting strategies from culture. To overcome these limitations, methods to harvest and transplant cells without disrupting critical stem cell functions are required. Cell sheet technology, exploiting temperature-responsive cell culture surfaces, permits cell harvest without cell protein damage. This study is focused on phenotypic traits of MSC sheets structurally and functionally to understand therapeutic benefits of cell sheets. Methods/results This study verified cleaved cellular proteins (vinculin, fibronectin, laminin, integrin β-1, and connexin 43) and increased apoptotic cell death produced under standard trypsin harvesting treatment in a time-dependent manner. However, MSC sheets produced without trypsin using only temperature-controlled sheet harvest from culture plastic exhibited intact cellular structures. Also, MSCs harvested using enzymatic treatment (i.e., chemical disruption) showed higher pYAP expression compared to MSC sheets. Conclusion Retention of cellular structures such as ECM, cell–cell junctions, and cell–ECM junctions is correlated with human umbilical cord mesenchymal stem cell (hUC-MSC) survival after detachment from cell culture surfaces. Retaining these proteins intact in MSC cultures using cell sheet technology is proposed to enhance stem cell survival and their function in stem cell-based therapy.

scholarly journals Laminin-521 Promotes Rat Bone Marrow Mesenchymal Stem Cell Sheet Formation on Light-Induced Cell Sheet Technology

2017 ◽  
Vol 2017 ◽  
pp. 1-11 ◽  
Author(s):  
Zhiwei Jiang ◽  
Yue Xi ◽  
Kaichen Lai ◽  
Ying Wang ◽  
Huiming Wang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Cell Sheet ◽  
Sheet Thickness ◽  
Collagen Type I ◽  
Type I ◽  
Cell Sheets ◽  
Cell Sheet Technology ◽  
Rat Bone

Rat bone marrow mesenchymal stem cell sheets (rBMSC sheets) are attractive for cell-based tissue engineering. However, methods of culturing rBMSC sheets are critically limited. In order to obtain intact rBMSC sheets, a light-induced cell sheet method was used in this study. TiO2 nanodot films were coated with (TL) or without (TN) laminin-521. We investigated the effects of laminin-521 on rBMSCs during cell sheet culturing. The fabricated rBMSC sheets were subsequently assessed to study cell sheet viability, reattachment ability, cell sheet thickness, collagen type I deposition, and multilineage potential. The results showed that laminin-521 could promote the formation of rBMSC sheets with good viability under hyperconfluent conditions. Cell sheet thickness increased from an initial 26.7 ± 1.5 μm (day 5) up to 47.7 ± 3.0 μm (day 10). Moreover, rBMSC sheets maintained their potential of osteogenic, adipogenic, and chondrogenic differentiation. This study provides a new strategy to obtain rBMSC sheets using light-induced cell sheet technology.

scholarly journals In vitromethod for safety testing of foot-and-mouth disease vaccines

1970 ◽  
Vol 68 (2) ◽  
pp. 159-172 ◽  
Author(s):  
E. C. Anderson ◽  
P. B. Capstick ◽  
G. N. Mowat ◽  
F. B. Leech
Keyword(s):  
Tissue Culture ◽  
Culture System ◽  
Cell Sheet ◽  
Foot And Mouth Disease ◽  
Kidney Cells ◽  
Live Virus ◽  
Safety Testing ◽  
Safety Test ◽  
Antigen Present ◽  
Baby Hamster Kidney Cells

SummaryThe susceptibility of the tissue culture system to small amounts of residual live virus was not influenced by the inactivated antigen present. The depth of inoculum over the cell sheet did not affect results. Negative cultures frequently gave positive first (but not second or later) sub-cultures.Baby hamster kidney cells were always more sensitive than cattle tongues to infection with any of the strains used.Confidence in the safety test depends on the number of vaccination doses used; the tissue culture test can be made much more reliable than the cattle test because it is not limited to the 15 ml. of inoculum that restricts the cattle test.

scholarly journals The Porcine Aortic Tissue Culture System in vitro for Stem Cell Research

2011 ◽  
Vol 4 (2) ◽  
pp. 116-122
Author(s):  
Dong-Eun Kim ◽  
Keun-Hee Oh ◽  
Ji-Hye Yang ◽  
Sun-Keun Kwon ◽  
Tae-Jun Cho ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Stem Cell ◽  
Stem Cell Research ◽  
Culture System ◽  
Aortic Tissue ◽  
Tissue Culture System

Towards the Development of a Closed, Nanofiber-Based Culture System for Clinical Expansion of Cord Blood-Derived CD34+ Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4411-4411
Author(s):  
Stephen E Fischer ◽  
Yiwei Ma ◽  
Caitlin Smith ◽  
Anirudhasingh Sodha ◽  
Yukang Zhao
Keyword(s):  
Bone Marrow ◽  
Tissue Culture ◽  
Stem Cell ◽  
Cord Blood ◽  
Culture System ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Cell Phenotype ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 4411 Interest in ex vivo hematopoietic stem and progenitor cell (HSPC) expansion has increased in recent years due to the growing importance of these cells in the treatment of a variety of both malignant and non-malignant diseases. Ex vivo expansion of cord blood-derived cells has been particularly investigated because cord is a valuable and readily available source of HSPCs, yet contains limited numbers of cells in each unit. Despite these efforts, most attempts to use expanded cord blood HSPCs in the clinic have been unsuccessful due to the generation of insufficient numbers of cells with the appropriate phenotype and the ability to function in vivo. In many ex vivo culture systems, HSPCs are cultured as a suspension cells and cultured in the presence of various media additives that act to enhance cell proliferation while reducing differentiation. An often-overlooked factor influencing fate decisions is the interaction of HSPCs with a substrate. In the natural bone marrow microenvironment, HSPCs maintain close contact with a complex network of stromal cells and extracellular matrix, likely indicating that cell-cell and cell-matrix interactions play an important role in maintaining their stem cell phenotype. With the goal of mimicking the bone marrow stem cell niche, Arteriocyte, Inc. has developed a 3-D nanofiber-based cell culture substrate (NANEX™). The functionalized NANEX™ substrate is designed to provide topographical and substrate-immobilized biochemical cues that act in synergy with media additives to enhance HSPC proliferation while minimizing differentiation. Here, we present our recent work towards developing a closed, NANEX™-based platform for large-scale clinical expansions of cord blood-derived CD34+ cells. We demonstrate that NANEX™ expands CD34+ cells from cord an average of more than 150-fold in 10 day culture, which is at least 2-fold higher than that obtained in standard tissue culture plates. Additionally, we show an approximately 1.5-fold higher proliferation of colony forming cells and a significantly higher engraftment rate in NSG mice for NANEX™-expanded cells compared to cells cultured in tissue culture plates. Furthermore, we demonstrate that the NANEX™ scaffold maintains its HSPC growth promoting characteristics after processing into a closed culture system and offers significant advantages over other culture platforms typically used for HSPC expansions in the clinic (culture bags and T-flasks). Our data indicates that NANEX™ technology provides a robust ex vivo expansion of cord blood HSPCs and, with further development, offers great potential for clinical applications requiring large numbers of functional cells. Disclosures: No relevant conflicts of interest to declare.

A Method for Electron Microscopic Study of Tissue Culture Cell Monolayers Grown in Tubes

1967 ◽  
Vol 25 ◽  
pp. 132-133
Author(s):  
R. Stephens ◽  
G. Schidlovsky ◽  
S. Kuzmic ◽  
P. Gaudreau
Keyword(s):  
Electron Microscopy ◽  
Tissue Culture ◽  
Light Microscopy ◽  
Cell Sheet ◽  
Culture Cell ◽  
Tissue Culture Cell ◽  
Electron Microscopic ◽  
Cell Sheets ◽  
Morphological Alterations ◽  
Culture Cells

The usual method of scraping or trypsinization to detach tissue culture cell sheets from their glass substrate for further pelletization and processing for electron microscopy introduces objectionable morphological alterations. It is also impossible under these conditions to study a particular area or individual cell which have been preselected by light microscopy in the living state.Several schemes which obviate centrifugation and allow the embedding of nondetached tissue culture cells have been proposed. However, they all preserve only a small part of the cell sheet and make use of inverted gelatin capsules which are in this case difficult to handle.We have evolved and used over a period of several years a technique which allows the embedding of a complete cell sheet growing at the inner surface of a tissue culture roller tube. Observation of the same cell by light microscopy in the living and embedded states followed by electron microscopy is performed conveniently.

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