Technique to accurately quantify collagen content in hyperconfluent cell culture

See, Eugene Yong-Shun; Toh, S.L.; Goh, James Cho Hong

doi:10.1007/s10735-008-9205-y

Cited by 5 publications

(3 citation statements)

References 21 publications

(17 reference statements)

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“…Briefly, gastric mucosa pepsin (Roche Diagnostics Asia Pacific, Singapore) was reconstituted in 0.25 M HCL to a concentration of 0.25 mg/ml and incubated with the cell sheets for 2 h with gentle shaking. Sonication was then introduced at the mid-point of pepsin incubation to aid in the digestion of the cell sheets (See et al, 2008). Sircol assay (Biocolor) was carried out on the digested samples according to the manufacturer's instructions.…”

Section: Collagen Deposition Normalized To Dna Contentmentioning

confidence: 99%

Temporal profiling of the growth and multi-lineage potentiality of adipose tissue-derived mesenchymal stem cells cell-sheets

Neo

See

Toh

et al. 2013

J Tissue Eng Regen Med

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Cell-sheet tissue engineering retains the benefits of an intact extracellular matrix (ECM) and can be used to produce scaffold-free constructs. Adipose tissue-derived stem cells (ASCs) are multipotent and more easily obtainable than the commonly used bone marrow-derived stem cells (BMSCs). Although BMSC cell sheets have been previously reported to display multipotentiality, a detailed study of the development and multilineage potential of ASC cell sheets (ASC-CSs) is non-existent in the literature. The aims of this study were to temporally profile: (a) the effect of hyperconfluent culture duration on ASC-CSs development; and (b) the multipotentiality of ASC-CSs by differentiation into the osteogenic, adipogenic and chondrogenic lineages. Rabbit ASCs were first isolated and cultured until confluence (day 0). The confluent cells were then cultured in ascorbic acid-supplemented medium for 3 weeks to study cell metabolic activity, cell sheet thickness and early differentiation gene expressions at weekly time points. ASC-CSs and ASCs were then differentiated into the three lineages, using established protocols, and assessed by RT-PCR and histology at multiple time points. ASC-CSs remained healthy up to 3 weeks of hyperconfluent culture. One week-old cell sheets displayed upregulation of early differentiation gene markers (Runx2 and Sox9); however, subsequent differentiation results indicated that they did not necessarily translate to an improved phenotype. ASCs within the preformed cell sheet groups did not differentiate as efficiently as the non-hyperconfluent ASCs, which were directly differentiated. Although ASCs within the cell sheets retained their differentiation capacity and remained viable under prolonged hyperconfluent conditions, future applications of ASC-CSs in tissue engineering should be considered with care. Copyright © 2016 John Wiley & Sons, Ltd.

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Section: Collagen Deposition Normalized To Dna Contentmentioning

confidence: 99%

Temporal profiling of the growth and multi-lineage potentiality of adipose tissue-derived mesenchymal stem cells cell-sheets

Neo

See

Toh

et al. 2013

J Tissue Eng Regen Med

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“…The 4 week‐old simulated IVD‐like assembly was washed twice with 1× PBS and digested with porcine gastric mucosa pepsin (2500 U/mg; Roche Diagnostics Asia Pacific, Singapore) in a final concentration of 0.25 mg/ml. To aid digestion of the hyperconfluent cell layer for accurate collagen quantification, a sonication step was introduced and quantified as previously described (See et al ., ). Collagen bands were stained using a SilverQuest kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol.…”

Section: Methodsmentioning

confidence: 97%

Simulated intervertebral disc-like assembly using bone marrow-derived mesenchymal stem cell sheets and silk scaffolds for annulus fibrosus regeneration

See

Toh

Goh

2011

J Tissue Eng Regen Med

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Most studies on the intervertebral disc (IVD) focus on the regeneration of the nucleus pulposus (NP). However, without a proper strategy to regenerate the damaged annulus fibrosus (AF), the NP replacements are bound to fail. Therefore the objective of this study was to investigate whether the use of bone marrow-derived mesenchymal stem cells (BMSCs) to form cell sheets, and incorporating them onto silk scaffolds, has the potential to regenerate the annulus fibrosus. The BMSC cell sheets and silk scaffolds were wrapped around a silicone NP substitute to form a simulated IVD-like assembly. The simulated IVD-like assembly was cultured for 4 weeks in static conditions and it was shown that the BMSC cell sheets remained viable, with no significant change in cell numbers. Histological analysis showed that the BMSC cell sheets adhered well onto the silk scaffolds and glycosaminoglycans (GAGs) were detected within the extracellular matrix (ECM). The ratio of collagen type I to collagen type II within the ECM of the BMSC cell sheets also decreased significantly over the period of culture. The results suggested that extensive remodelling of the ECM occurred within the simulated IVD-like assembly, and it is suitable for the regeneration of the inner AF.

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“…After incubation, the samples were neutralized with 0.1 N NaOH. The samples were then analyzed by SDS-PAGE under non-reducing conditions [48]. Briefly, 0.1 mg/ml of bovine type I collagen was used as standards with every gel.…”

Section: Sds-page Analysis Of Type I Collagenmentioning

confidence: 99%

Fabrication of Functional High Density Cell Sheets for Tissue Engineering Using Spin-Coated poly(N-isopropylacrylamide)Thin Films

Dzhoyashvili¹,

Joyce²,

Thompson³

et al. 2019

Insights Stem Cell Res Ther

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The current goal of cell sheet engineering technology is to provide an improved approach for more predictive and effective generation of viable tissue-like constructs. Success requires validation and characterization of cell sheet growth and detachment. In this study, we perform qualitative and quantitative assessments of cell sheets lifted from thin spin-coated poly(N-isopropylacrylamide) films.Cell morphology, detachment time, metabolic activity and viability of mouse stromal and human corneal epithelial cell sheets were examined before and after lifting and after reattachment. The histological analysis of cell sheets and the immunofluorescence analysis of acting cytoskeleton, paxillin and cadherins were performed. The structure of extracellular matrix and the content of type I collagen in stromal and epithelial cell sheets were assessed by scanning electron microscopy and SDS-PAGE analysis, respectively.Results demonstrate that lifted cell sheets remain viable and maintain tissue-like integrity that strongly depends on reinforcement of acting cytoskeleton and cadherins at cell-cell junctions. Strong cell-cell adhesions and a high density of confluent cell sheets promote collective cell sheet detachment. Preserved extracellular matrix and focal adhesion complexes support cell sheet reattachment.This study demonstrates the potential of spin-coated poly(N-isopropylacrylamide) films in producing viable cell sheets that might be considered as a building block to create large biological tissues. Our results also describe the central cellular mechanisms involved in temperature-controlled collective cell migration. This study should promote further research in engineering three-dimensional tissues with complex organizational architecture.

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Technique to accurately quantify collagen content in hyperconfluent cell culture

Cited by 5 publications

References 21 publications

Temporal profiling of the growth and multi-lineage potentiality of adipose tissue-derived mesenchymal stem cells cell-sheets

Temporal profiling of the growth and multi-lineage potentiality of adipose tissue-derived mesenchymal stem cells cell-sheets

Simulated intervertebral disc-like assembly using bone marrow-derived mesenchymal stem cell sheets and silk scaffolds for annulus fibrosus regeneration

Fabrication of Functional High Density Cell Sheets for Tissue Engineering Using Spin-Coated poly(N-isopropylacrylamide)Thin Films

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