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Tracing human keratinocytes and melanocytes with carboxyfluorescein hydroxysuccinimidyl ester (CFSE) staining
Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Medicine and Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Anaesthetics, Operations and Specialty Surgery Center, Department of Hand and Plastic Surgery.
2015 (English)Manuscript (preprint) (Other academic)
Abstract [en]

Burn treatment and conditions of hypopigmentation may require autologous transplantation of keratinocytes and melanocytes. The tracing of transplanted cells presents a challenge. We report a methodology based on passive staining with carboxyfluorescein hydroxysuccinimidyl ester (CFSE) that enables localising cells in tissue sections to investigate the fate of transplanted cells in wound re-epithelialisation. CFSE-stained keratinocytes and CFSE-stained melanocytes were transplanted to human full thickness in vitro wounds either as cell suspension for keratinocytes, or with the aid of  macroporous gelatin microcarriers for both cells types in single and co-culture. Viability and migration of CFSE-stained keratinocytes and melanocytes were investigated, and proliferation of the cells cultured on microcarriers was measured with flow cytometry. Wounds with transplanted cells were harvested after seven, 14 and 21 days in culture, cryosectioned and investigated using fluorescence microscopy. Sections from wounds with transplanted co-cultured keratinocytes and melanocytes were stained for pancytokeratin to distinguish double stained keratinocytes. The CFSE-staining of keratinocytes and melanocytes did not affect the viability, migration or proliferation of the cells. Transplanted cells were traced in tissue sections after 21 days and wound re-epithelialisation was not affected. We propose a novel application of CFSE-staining in transplantation studies here presented with primary human keratinocytes and melanocytes.

Place, publisher, year, edition, pages
2015.
National Category
Clinical Medicine
Identifiers
URN: urn:nbn:se:liu:diva-123310OAI: oai:DiVA.org:liu-123310DiVA: diva2:881355
Available from: 2015-12-10 Created: 2015-12-10 Last updated: 2015-12-10Bibliographically approved
In thesis
1. Applications of human skin in vitro
Open this publication in new window or tab >>Applications of human skin in vitro
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Chronic wounds are a substantial problem in today’s health care and place significant strains on the patient. Successful modelling of the wound healing process is pivotal for the advancement of wound treatment research. Wound healing is a dynamic and multifactorial process involving all constituents of the skin. The progression from haemostasis and inflammation to proliferation of epidermal  keratinocytes and dermal fibroblasts, and final scar maturation can be halted and result in a chronic wound that fails to re-epithelialise. The wound healing process constitutes an example of dynamic reciprocity in tissue where cellular changes take place on cues from the extracellular matrix and vice versa when tissue homeostasis is disturbed. The extracellular matrix provides a structural context for the resident cells and the epidermal keratinocytes, and a functioning interplay between the two tissue compartments is crucial for successful wound healing to take place. Work included in this thesis has applied viable human full thickness skin in vitro to investigate the re-epithelialisation process and barrier function of intact skin.The use of full thickness skin in vitro can take into account the contextual aspect of the process where the epidermal keratinocytes are activated and obtain a migratory phenotype, and are continuously dependent on the cues from the extracellular matrix and support of the dermis. When utilising skin for studies on re-epithelialisation, circular standardised full thickness wounds were created and cultured  for up to four weeks in tissue culture. In paper I, the organisation of a thick neoepidermis was investigated in the in vitro wound healing model when resident cells were provided with a porous suspended three dimensional gelatin scaffold. In paper II we investigated the use of a fluorescent staining conventionally used for proliferation studies to facilitate the tracing of transplanted epidermal cells in in vitro  wounds, in order to improve and expand the use of the model. In paper III the model was utilised to investigate the treatment approach of acidification of wounds to evaluate the suitability of such intervention in regards to keratinocyte function and re-epithelialisation. Studies on re-epithelialisation with the aid of the in vitro wound healing model provided insight in neoepidermal structure with porous gelatin scaffolding in the wound, a novel methodological approach to tracing cells and response to constrained wound healing environment. In paper IV, intact human skin was evaluated for modelling the cytotoxic response after exposure to a known irritant compound. To study barrier function, intact skin was exposed to irritants by restricting exposure topically, and full thickness skin in vitro was found suitable for modelling cytotoxicity responses. Employing human full thickness skin in vitro makes use of the actual target tissue of interest with epidermal and dermal cells, and full barrier function.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2016. 99 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1493
National Category
Clinical Medicine Clinical Science
Identifiers
urn:nbn:se:liu:diva-123313 (URN)10.3384/diss.diva-123313 (DOI)978-91-7685-895-0 (print) (ISBN)
Public defence
2016-01-29, Berzeliussalen, Campus US, Linköping, 13:00 (English)
Opponent
Supervisors
Available from: 2015-12-10 Created: 2015-12-10 Last updated: 2015-12-18Bibliographically approved

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Lönnqvist, SusannaKratz, Gunnar
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Division of Clinical SciencesFaculty of Medicine and Health SciencesDepartment of Clinical and Experimental MedicineDepartment of Hand and Plastic Surgery
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