The Experts below are selected from a list of 300 Experts worldwide ranked by ideXlab platform
Swathi Sundarraj - One of the best experts on this subject based on the ideXlab platform.
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Explant Culture a simple reproducible efficient and economic technique for isolation of mesenchymal stromal cells from human adipose tissue and lipoaspirate
Journal of Tissue Engineering and Regenerative Medicine, 2014Co-Authors: Nancy Priya, Shilpita Sarcar, Anish Sen Majumdar, Swathi SundarrajAbstract:Adipose tissue has emerged as a preferred source of mesenchymal stem/stromal cells (MSC), due to its easy accessibility and high MSC content. The conventional method of isolation of adipose tissue-derived stromal cells (ASC) involves enzymatic digestion and centrifugation, which is a costly and time-consuming process. Mechanical stress during isolation, use of bacterial-derived products and potential contamination with endotoxins and xenoantigens are other disadvantages of this method. In this study, we propose Explant Culture as a simple and efficient process to isolate ASC from human adipose tissue. This technique can be used to reproducibly isolate ASC from fat tissue obtained by liposuction as well as surgical resection, and yields an enriched ASC population free from contaminating haematopoietic cells. We show that Explanting adipose tissue results in a substantially higher yield of ASC at P0 per gram of initial fat tissue processed, as compared to that obtained by enzymatic digestion. We demonstrate that ASC isolated by Explant Culture are phenotypically and functionally equivalent to those obtained by enzymatic digestion. Further, the Explant-derived ASC share the immune privileged status and immunosuppressive properties implicit to MSC, suggesting that they are competent to be tested and applied in allogeneic clinical settings. As Explant Culture is a simple, inexpensive and gentle method, it may be preferred over the enzymatic technique for obtaining adipose tissue-derived stem/stromal cells for tissue engineering and regenerative medicine, especially in cases of limited starting material. Copyright © 2012 John Wiley & Sons, Ltd.
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Explant Culture a simple reproducible efficient and economic technique for isolation of mesenchymal stromal cells from human adipose tissue and lipoaspirate
Journal of Tissue Engineering and Regenerative Medicine, 2014Co-Authors: Nancy Priya, Shilpita Sarcar, Anish Sen Majumdar, Swathi SundarrajAbstract:Adipose tissue has emerged as a preferred source of mesenchymal stem/stromal cells (MSC), due to its easy accessibility and high MSC content. The conventional method of isolation of adipose tissue-derived stromal cells (ASC) involves enzymatic digestion and centrifugation, which is a costly and time-consuming process. Mechanical stress during isolation, use of bacterial-derived products and potential contamination with endotoxins and xenoantigens are other disadvantages of this method. In this study, we propose Explant Culture as a simple and efficient process to isolate ASC from human adipose tissue. This technique can be used to reproducibly isolate ASC from fat tissue obtained by liposuction as well as surgical resection, and yields an enriched ASC population free from contaminating haematopoietic cells. We show that Explanting adipose tissue results in a substantially higher yield of ASC at P0 per gram of initial fat tissue processed, as compared to that obtained by enzymatic digestion. We demonstrate that ASC isolated by Explant Culture are phenotypically and functionally equivalent to those obtained by enzymatic digestion. Further, the Explant-derived ASC share the immune privileged status and immunosuppressive properties implicit to MSC, suggesting that they are competent to be tested and applied in allogeneic clinical settings. As Explant Culture is a simple, inexpensive and gentle method, it may be preferred over the enzymatic technique for obtaining adipose tissue-derived stem/stromal cells for tissue engineering and regenerative medicine, especially in cases of limited starting material.
Aldo C Zamudio - One of the best experts on this subject based on the ideXlab platform.
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inhibition of tgfβ cell signaling for limbal Explant Culture in serumless defined xeno free conditions
Experimental Eye Research, 2016Co-Authors: Aldo C Zamudio, Zheng Wang, Sohyang Chung, Mario J WolosinAbstract:Outgrowths of limbal epithelium by Explant Culture are used to treat limbal stem cell deficiency (LSCD). The Explant Culture medium is always complemented with serum, a complex solution which includes TGFβ. Since TGFβ is a cytostatic effector for epithelial proliferation we examined its effect on these Cultures. Limbal biopsies were set on Explant Culture in DMEM/F12 with 5 ng/ml EGF and cholera toxin (ChT), ITS, and 5% FBS, henceforth SHEM or a) SHEMSB=SHEM plus SB431542 an inhibitor of TGFβ signaling; b) sfSHEM = SHEM with FBS replaced by 0.05% Albumax II; and c) sfSHEMSB and sfSHEMA83 = sfSHEM plus, respectively, SB431542 or A-83-01, another TGFβ inhibitor. After the initial outgrowths reached 3 cm in diameter, the limbal biopsies were serially transferred up to six times onto new inserts. Biopsy Explant outgrowths were trypsinized and cell yield, morphology and stem-cell related JC-1 exclusion (IOVS, 52:4330) were determined by flow cytometry. Cells we plated at low density seeding to compare relative clonal proliferative activity. The expression of three proteins whose levels are associated with growth and differentiation states, Krt3, connexin 43 and p63 were determined by immunohistology and/or Western blot. Cell yield in rabbit, relative to SHEM (in %) were, SHEMSB, 104 ± 13 (p > 0.95); sfSHEM: 5 ± 3; and sfSHEMSB, 94 ± 18 (p > 0.95). Cell size and morphology, JC1 dye exclusion, Krt3, p63 and connexin 43 content, proliferation efficiency and the preservation of extended proliferative potential of the serially Cultured biopsies were similar for SHEM, SHEMSB and sfSHEMSB. The only differences observed where reduced expression of Krt3 and increased preservation of p63 in the FBS-free medium. Removal of EGF from sfSHEMSB reduced yield by 92 ± 6% (p < 0.05). Removal of Albumax and ChT to establish a xeno-free medium caused a small, non-statistical decrease in growth rates. Equivalent results were observed in a preliminary experiment in human. These results suggest that in the absence serum endogenously generated TGFβ act as an autocrine cytostatic agent and that TGFβ inhibitors allow Explant Culture in xeno-free, chemically defined medium. Furthermore, the pro-growth effect of serum in limbal Explant Cultures may result exclusively from neutralization of the TGFβ cytostatic effect.
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Inhibition of TGFβ cell signaling for limbal Explant Culture in serumless, defined xeno-free conditions
Experimental eye research, 2015Co-Authors: Aldo C Zamudio, Zheng Wang, Sohyang Chung, J. Mario WolosinAbstract:Outgrowths of limbal epithelium by Explant Culture are used to treat limbal stem cell deficiency (LSCD). The Explant Culture medium is always complemented with serum, a complex solution which includes TGFβ. Since TGFβ is a cytostatic effector for epithelial proliferation we examined its effect on these Cultures. Limbal biopsies were set on Explant Culture in DMEM/F12 with 5 ng/ml EGF and cholera toxin (ChT), ITS, and 5% FBS, henceforth SHEM or a) SHEMSB=SHEM plus SB431542 an inhibitor of TGFβ signaling; b) sfSHEM = SHEM with FBS replaced by 0.05% Albumax II; and c) sfSHEMSB and sfSHEMA83 = sfSHEM plus, respectively, SB431542 or A-83-01, another TGFβ inhibitor. After the initial outgrowths reached 3 cm in diameter, the limbal biopsies were serially transferred up to six times onto new inserts. Biopsy Explant outgrowths were trypsinized and cell yield, morphology and stem-cell related JC-1 exclusion (IOVS, 52:4330) were determined by flow cytometry. Cells we plated at low density seeding to compare relative clonal proliferative activity. The expression of three proteins whose levels are associated with growth and differentiation states, Krt3, connexin 43 and p63 were determined by immunohistology and/or Western blot. Cell yield in rabbit, relative to SHEM (in %) were, SHEMSB, 104 ± 13 (p > 0.95); sfSHEM: 5 ± 3; and sfSHEMSB, 94 ± 18 (p > 0.95). Cell size and morphology, JC1 dye exclusion, Krt3, p63 and connexin 43 content, proliferation efficiency and the preservation of extended proliferative potential of the serially Cultured biopsies were similar for SHEM, SHEMSB and sfSHEMSB. The only differences observed where reduced expression of Krt3 and increased preservation of p63 in the FBS-free medium. Removal of EGF from sfSHEMSB reduced yield by 92 ± 6% (p
Mario J Wolosin - One of the best experts on this subject based on the ideXlab platform.
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inhibition of tgfβ cell signaling for limbal Explant Culture in serumless defined xeno free conditions
Experimental Eye Research, 2016Co-Authors: Aldo C Zamudio, Zheng Wang, Sohyang Chung, Mario J WolosinAbstract:Outgrowths of limbal epithelium by Explant Culture are used to treat limbal stem cell deficiency (LSCD). The Explant Culture medium is always complemented with serum, a complex solution which includes TGFβ. Since TGFβ is a cytostatic effector for epithelial proliferation we examined its effect on these Cultures. Limbal biopsies were set on Explant Culture in DMEM/F12 with 5 ng/ml EGF and cholera toxin (ChT), ITS, and 5% FBS, henceforth SHEM or a) SHEMSB=SHEM plus SB431542 an inhibitor of TGFβ signaling; b) sfSHEM = SHEM with FBS replaced by 0.05% Albumax II; and c) sfSHEMSB and sfSHEMA83 = sfSHEM plus, respectively, SB431542 or A-83-01, another TGFβ inhibitor. After the initial outgrowths reached 3 cm in diameter, the limbal biopsies were serially transferred up to six times onto new inserts. Biopsy Explant outgrowths were trypsinized and cell yield, morphology and stem-cell related JC-1 exclusion (IOVS, 52:4330) were determined by flow cytometry. Cells we plated at low density seeding to compare relative clonal proliferative activity. The expression of three proteins whose levels are associated with growth and differentiation states, Krt3, connexin 43 and p63 were determined by immunohistology and/or Western blot. Cell yield in rabbit, relative to SHEM (in %) were, SHEMSB, 104 ± 13 (p > 0.95); sfSHEM: 5 ± 3; and sfSHEMSB, 94 ± 18 (p > 0.95). Cell size and morphology, JC1 dye exclusion, Krt3, p63 and connexin 43 content, proliferation efficiency and the preservation of extended proliferative potential of the serially Cultured biopsies were similar for SHEM, SHEMSB and sfSHEMSB. The only differences observed where reduced expression of Krt3 and increased preservation of p63 in the FBS-free medium. Removal of EGF from sfSHEMSB reduced yield by 92 ± 6% (p < 0.05). Removal of Albumax and ChT to establish a xeno-free medium caused a small, non-statistical decrease in growth rates. Equivalent results were observed in a preliminary experiment in human. These results suggest that in the absence serum endogenously generated TGFβ act as an autocrine cytostatic agent and that TGFβ inhibitors allow Explant Culture in xeno-free, chemically defined medium. Furthermore, the pro-growth effect of serum in limbal Explant Cultures may result exclusively from neutralization of the TGFβ cytostatic effect.
Nancy Priya - One of the best experts on this subject based on the ideXlab platform.
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Explant Culture a simple reproducible efficient and economic technique for isolation of mesenchymal stromal cells from human adipose tissue and lipoaspirate
Journal of Tissue Engineering and Regenerative Medicine, 2014Co-Authors: Nancy Priya, Shilpita Sarcar, Anish Sen Majumdar, Swathi SundarrajAbstract:Adipose tissue has emerged as a preferred source of mesenchymal stem/stromal cells (MSC), due to its easy accessibility and high MSC content. The conventional method of isolation of adipose tissue-derived stromal cells (ASC) involves enzymatic digestion and centrifugation, which is a costly and time-consuming process. Mechanical stress during isolation, use of bacterial-derived products and potential contamination with endotoxins and xenoantigens are other disadvantages of this method. In this study, we propose Explant Culture as a simple and efficient process to isolate ASC from human adipose tissue. This technique can be used to reproducibly isolate ASC from fat tissue obtained by liposuction as well as surgical resection, and yields an enriched ASC population free from contaminating haematopoietic cells. We show that Explanting adipose tissue results in a substantially higher yield of ASC at P0 per gram of initial fat tissue processed, as compared to that obtained by enzymatic digestion. We demonstrate that ASC isolated by Explant Culture are phenotypically and functionally equivalent to those obtained by enzymatic digestion. Further, the Explant-derived ASC share the immune privileged status and immunosuppressive properties implicit to MSC, suggesting that they are competent to be tested and applied in allogeneic clinical settings. As Explant Culture is a simple, inexpensive and gentle method, it may be preferred over the enzymatic technique for obtaining adipose tissue-derived stem/stromal cells for tissue engineering and regenerative medicine, especially in cases of limited starting material. Copyright © 2012 John Wiley & Sons, Ltd.
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Explant Culture a simple reproducible efficient and economic technique for isolation of mesenchymal stromal cells from human adipose tissue and lipoaspirate
Journal of Tissue Engineering and Regenerative Medicine, 2014Co-Authors: Nancy Priya, Shilpita Sarcar, Anish Sen Majumdar, Swathi SundarrajAbstract:Adipose tissue has emerged as a preferred source of mesenchymal stem/stromal cells (MSC), due to its easy accessibility and high MSC content. The conventional method of isolation of adipose tissue-derived stromal cells (ASC) involves enzymatic digestion and centrifugation, which is a costly and time-consuming process. Mechanical stress during isolation, use of bacterial-derived products and potential contamination with endotoxins and xenoantigens are other disadvantages of this method. In this study, we propose Explant Culture as a simple and efficient process to isolate ASC from human adipose tissue. This technique can be used to reproducibly isolate ASC from fat tissue obtained by liposuction as well as surgical resection, and yields an enriched ASC population free from contaminating haematopoietic cells. We show that Explanting adipose tissue results in a substantially higher yield of ASC at P0 per gram of initial fat tissue processed, as compared to that obtained by enzymatic digestion. We demonstrate that ASC isolated by Explant Culture are phenotypically and functionally equivalent to those obtained by enzymatic digestion. Further, the Explant-derived ASC share the immune privileged status and immunosuppressive properties implicit to MSC, suggesting that they are competent to be tested and applied in allogeneic clinical settings. As Explant Culture is a simple, inexpensive and gentle method, it may be preferred over the enzymatic technique for obtaining adipose tissue-derived stem/stromal cells for tissue engineering and regenerative medicine, especially in cases of limited starting material.
Pranab K Das - One of the best experts on this subject based on the ideXlab platform.
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subtoxic concentrations of allergenic haptens induce lc migration and maturation in a human organotypic skin Explant Culture model a novel method for identifying potential contact allergens
Experimental Dermatology, 2006Co-Authors: Cynthia L Lehe, John J L Jacobs, Graham R Elliott, Chi Mei Hua, P Courtellemont, Pranab K DasAbstract:The accelerated migration of Langerhans cells (LCs) out of the epidermis and up-regulation of maturation markers, upon treatment with subtoxic concentrations of chemicals, were used as the criteria to determine the potential of allergenic chemicals capable of inducing a hapten-specific delayed-type hypersensitivity reaction. Here we report the findings of a study in which seven chemicals, coded and tested in a blind fashion, were classified as contact allergens or non-allergens using the human organotypic skin Explant Culture (hOSEC) model. All chemicals that were identified as a contact sensitizer on decoding induced a definite decrease in the number of CD1a and HLA-DR-positive epidermal LCs in the epidermis of the skin Explants, as determined by both semiquantitative immunohistochemistry and quantitative flow cytometric analysis. A significant increase in the number of CD83+ cells was accompanied by up-regulation of activation molecules in the epidermis of hOSEC exposed specifically to contact allergens. In contrast, there were only minor alterations in epidermal LC numbers, expression of CD83 and other activation markers by LCs when the biopsies were treated with non-toxic concentrations of non-allergenic irritants and vehicles. The data suggest that an increased epidermal LC migration and maturation accompanied by increased expression of activation markers could be used as end-point determinants to screen allergens in a non-animal alternative hOSEC model. © Blackwell Munksgaard, 2006.
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a two centre evaluation of the human organotypic skin Explant Culture model for screening contact allergens
Atla-alternatives To Laboratory Animals, 2003Co-Authors: Cynthia L Lehe, John J L Jacobs, Graham R Elliott, Pranab K DasAbstract:Animal models are considered to be the "gold standard" for determining the potential contact allergenicity of low molecular weight chemicals. However, governmental regulations and ethical considerations limit the use of animals for such purposes. There is therefore a need for in vitro alternative models. The human organotypic skin Explant Culture (HOSEC) model is reported to be a promising alternative method for the predictive testing of contact allergens. The accelerated migration of Langerhans cells from the epidermis upon exposure to contact allergens is used to identify chemicals that are potentially capable of inducing a delayed-type hypersensitivity. In the study described in this paper, the model was further refined, and used, in two independent laboratories, to screen 23 low molecular weight compounds of known classification for their allergenicity. Each laboratory was able to accurately detect the contact allergens, despite small variations in the protocols used. However, the classification of dermal irritants, which have often been falsely classified as allergens, varied between the two laboratories. Despite the current limitations of the HOSEC model, the accuracy of the predictions made (sensitiser or non-sensitiser) compare favourably with classifications obtained with commonly used animal models. The HOSEC model has the potential to be developed further as an in vitro alternative to animal models for screening for contact allergens.
