The University of Southampton
University of Southampton Institutional Repository

Development of multi-layered models of the airway mucosa

Development of multi-layered models of the airway mucosa
Development of multi-layered models of the airway mucosa
Introduction: Asthma is a global burden, leading to around 180,000 deaths worldwide annually. Translation of therapies from animal models is limited, suggesting these models do not recapitulate adequately genetic and environmental aspects of asthma. Existing human cell culture models are usually simplistic and do not mimic the in vivo tissue architecture; therefore there is a need to improve three-dimensional human tissue culture models. We hypothesised that use of cell sheet engineering would help overcome this problem.

Aims: Thermoresponsive polymers or ultrasound standing waves (Sonotweezers) will be tested for their compatibility for the formation of cell sheets which can be used for tissue engineering.

Methods: Poly (2-alkyl-2-oxazolines) are polymers with thermoresponsive properties and were tested for biocompatibility using cell motility and adhesion assays, immunofluorescent staining, and measurement of p38 phosphorylation. Multiple cell types were seeded onto thermoresponsive polymers (either NiPAAm, poly(2-alkyl-2-oxazolines) or thermo-gels of poly(2-isopropyl-2-oxazoline)-carboxymethylcellulose)) and cell sheet release assessed after incubation at 20ºC. Cell sheets were also created using ultrasound standing waves (Sonotweezers) or incubation on a thermo-gel. Adhesion junctions in cell sheets were assessed by staining for E-cadherin, ZO-1 and the actin cytoskeleton; cell viability was monitored using 7-Aminoactinomycin D. Subsequently, epithelial cell sheets were used to created co-cultures with fibroblasts. Cytokine release (IL-6 and IP-10) following Poly (I:C) stimulation from cell-sheet co-cultures was compared to conventional models by ELISA.

Results: Using conventional cell culture techniques a multi-layered cell structure by the addition of epithelial cells onto a confluent fibroblast layer was not possible as it resulted in redistribution of the cells to form a single layer comprising islands of epithelial cells surrounded by fibroblasts. Consequently alternative methods were explored using thermoresponsive polymers or Sonotweezers. Cells could be lifted from a commercial NiPAAm coated dish (UpCell) after attachment to a membrane, but during the release process cells either rounded up and lost contact (fibroblasts, HeLa cells) or epithelial cell sheets were damaged and incompletely released. Sonotweezers also could not generate sufficient force to release and levitate the cells. As alternatives, cell sheets were created by levitation in the Sonotweezers device or overlaying the cells on a thermo-gel to allow cell sheet formation followed by sedimentation onto an underlying layer of fibroblasts. Levitation of single cells resulted in the formation of a cell sheet within 2 hours, which gradually contracted becoming three-dimensional by 24 hours. Contraction could be inhibited by removal of Ca2+ to prevent adherens junction formation or by adding cytochalasin D to prevent actin filaments or an E-cadherin neutralising antibody to prevent adherens junction formation. After 2 hours of levitation, the cell sheet could be placed onto confluent MRC5 fibroblasts and epithelial cells used plithotaxis to spread across the fibroblasts to create two distinct layers. Oxazoline polymers with a range of hydrophobicities covalently attached to glass were biocompatible, but not thermoresponsive. A gel of poly(2-isopropyl-2-oxazoline-co-2-butyl2-oxazoline)-carboxymethylcellulose was thermoresponsive, enabling formation of epithelial cell sheets which were used to form cell-sheet co-cultures. The cell-sheet co-cultures achieved an electrically tight barrier and when challenged with the viral mimic Poly(I:C), showed increased IL-6 and IP-10 release. IL-6 release was predominantly apical, whereas IP-10 was basolateral, suggesting polarised mediator release.

Conclusions: Multi-layered cell culture models can be created using either the Sonotweezers device or gelling polymers. The latter offers potential for formation of multi-layered structures in a high through-put manner.
University of Southampton
Tait, Angela
91d3e403-54d5-4efd-94c5-8b476e6af283
Tait, Angela
91d3e403-54d5-4efd-94c5-8b476e6af283
Davies, Donna
7de8fdc7-3640-4e3a-aa91-d0e03f990c38
Hill, Martyn
d65df777-edc9-47f3-9a7d-7593e5ac5d90

Tait, Angela (2014) Development of multi-layered models of the airway mucosa. University of Southampton, Doctoral Thesis, 207pp.

Record type: Thesis (Doctoral)

Abstract

Introduction: Asthma is a global burden, leading to around 180,000 deaths worldwide annually. Translation of therapies from animal models is limited, suggesting these models do not recapitulate adequately genetic and environmental aspects of asthma. Existing human cell culture models are usually simplistic and do not mimic the in vivo tissue architecture; therefore there is a need to improve three-dimensional human tissue culture models. We hypothesised that use of cell sheet engineering would help overcome this problem.

Aims: Thermoresponsive polymers or ultrasound standing waves (Sonotweezers) will be tested for their compatibility for the formation of cell sheets which can be used for tissue engineering.

Methods: Poly (2-alkyl-2-oxazolines) are polymers with thermoresponsive properties and were tested for biocompatibility using cell motility and adhesion assays, immunofluorescent staining, and measurement of p38 phosphorylation. Multiple cell types were seeded onto thermoresponsive polymers (either NiPAAm, poly(2-alkyl-2-oxazolines) or thermo-gels of poly(2-isopropyl-2-oxazoline)-carboxymethylcellulose)) and cell sheet release assessed after incubation at 20ºC. Cell sheets were also created using ultrasound standing waves (Sonotweezers) or incubation on a thermo-gel. Adhesion junctions in cell sheets were assessed by staining for E-cadherin, ZO-1 and the actin cytoskeleton; cell viability was monitored using 7-Aminoactinomycin D. Subsequently, epithelial cell sheets were used to created co-cultures with fibroblasts. Cytokine release (IL-6 and IP-10) following Poly (I:C) stimulation from cell-sheet co-cultures was compared to conventional models by ELISA.

Results: Using conventional cell culture techniques a multi-layered cell structure by the addition of epithelial cells onto a confluent fibroblast layer was not possible as it resulted in redistribution of the cells to form a single layer comprising islands of epithelial cells surrounded by fibroblasts. Consequently alternative methods were explored using thermoresponsive polymers or Sonotweezers. Cells could be lifted from a commercial NiPAAm coated dish (UpCell) after attachment to a membrane, but during the release process cells either rounded up and lost contact (fibroblasts, HeLa cells) or epithelial cell sheets were damaged and incompletely released. Sonotweezers also could not generate sufficient force to release and levitate the cells. As alternatives, cell sheets were created by levitation in the Sonotweezers device or overlaying the cells on a thermo-gel to allow cell sheet formation followed by sedimentation onto an underlying layer of fibroblasts. Levitation of single cells resulted in the formation of a cell sheet within 2 hours, which gradually contracted becoming three-dimensional by 24 hours. Contraction could be inhibited by removal of Ca2+ to prevent adherens junction formation or by adding cytochalasin D to prevent actin filaments or an E-cadherin neutralising antibody to prevent adherens junction formation. After 2 hours of levitation, the cell sheet could be placed onto confluent MRC5 fibroblasts and epithelial cells used plithotaxis to spread across the fibroblasts to create two distinct layers. Oxazoline polymers with a range of hydrophobicities covalently attached to glass were biocompatible, but not thermoresponsive. A gel of poly(2-isopropyl-2-oxazoline-co-2-butyl2-oxazoline)-carboxymethylcellulose was thermoresponsive, enabling formation of epithelial cell sheets which were used to form cell-sheet co-cultures. The cell-sheet co-cultures achieved an electrically tight barrier and when challenged with the viral mimic Poly(I:C), showed increased IL-6 and IP-10 release. IL-6 release was predominantly apical, whereas IP-10 was basolateral, suggesting polarised mediator release.

Conclusions: Multi-layered cell culture models can be created using either the Sonotweezers device or gelling polymers. The latter offers potential for formation of multi-layered structures in a high through-put manner.

Text
Angela Tait Final Thesis 2014 - Version of Record
Available under License University of Southampton Thesis Licence.
Download (9MB)

More information

Published date: April 2014

Identifiers

Local EPrints ID: 435481
URI: https://eprints.soton.ac.uk/id/eprint/435481
PURE UUID: 65ebca10-ea8d-4050-a5a9-845d842f9bff
ORCID for Donna Davies: ORCID iD orcid.org/0000-0002-5117-2991

Catalogue record

Date deposited: 07 Nov 2019 17:30
Last modified: 08 Nov 2019 01:39

Export record

Download statistics

Downloads from ePrints over the past year. Other digital versions may also be available to download e.g. from the publisher's website.

View more statistics

Atom RSS 1.0 RSS 2.0

Contact ePrints Soton: eprints@soton.ac.uk

ePrints Soton supports OAI 2.0 with a base URL of https://eprints.soton.ac.uk/cgi/oai2

This repository has been built using EPrints software, developed at the University of Southampton, but available to everyone to use.

We use cookies to ensure that we give you the best experience on our website. If you continue without changing your settings, we will assume that you are happy to receive cookies on the University of Southampton website.

×