Development and Characterisation of a Human Chronic Skin Wound Cell Line-Towards an Alternative for Animal Experimentation

Abstract

Background: Chronic skin wounds are a growing financial burden for healthcare providers, causing discomfort/immobility to patients. Whilst animal chronic wound models have been developed to allow for mechanistic studies and to develop/test potential therapies, such systems are not good representations of the human chronic wound state. As an alternative, human chronic wound fibroblasts (CWFs) have permitted an insight into the dysfunctional cellular mechanisms that are associated with these wounds. However, such cells strains have a limited replicative lifespan and therefore a limited reproducibility/usefulness. Objectives: To develop/characterise immortalised cell lines of CWF and patient-matched normal fibroblasts (NFs). Methods and Results: Immortalisation with human telomerase resulted in both CWF and NF proliferating well beyond their replicative senescence end-point (respective cell strains senesced as normal). Gene expression analysis demonstrated that, whilst proliferation-associated genes were up-regulated in the cell lines (as would be expected), the immortalisation process did not significantly affect the disease-specific genotype. Immortalised CWF (as compared to NF) also retained a distinct impairment in their wound repopulation potential (in line with CWF cell strains). Conclusions: These novel CWF cell lines are a credible animal alternative and could be a valuable research tool for understanding both the aetiology of chronic skin wounds and for therapeutic pre-screening.

Keywords: animal alternative; chronic wound; fibroblasts; microarray; wound healing.

Figure 1

Cumulative population doublings for normal skin (NF) (A,C,E) and chronic wound fibroblasts (CWF) (B,D,F) primary cells (▲), mock-infected cells (■) and catalytic component (hTERT)-infected cells (●) from Patient 1 (A,B), Patient 2 (C,D) and Patient 3 (E,F).

Figure 2

Immortalised NF and CWF cell lines express hTERT and template component (hTR) as demonstrated by RT-PCR (A) and have active telomerase as demonstrated by the appearance of a 6 bp ladder (B). CWF immortalisation and maintenance in culture (C) changes the appearance of the chronic cells from flattened and spread out to more bi-polar (and thereby more like the appearance of the NF cells). Scale bar = 20 µm.

Figure 3

Utilising a monolayer wound scratch assay and time lapse microscopic analysis immortalised NF, like the primary cells from which they were established, clearly repopulated the wound space over a period of 24 h whereas the patient-matched CWF cells (both primary and immortalised) failed to do so. Scale bar = 100 µm.