Primary cultured fibroblasts derived from patients with chronic wounds: a methodology to produce human cell lines and test putative growth factor therapy such as GMCSF

Abstract

Background: Multiple physiologic impairments are responsible for chronic wounds. A cell line grown which retains its phenotype from patient wounds would provide means of testing new therapies. Clinical information on patients from whom cells were grown can provide insights into mechanisms of specific disease such as diabetes or biological processes such as aging. The objective of this study was 1) To culture human cells derived from patients with chronic wounds and to test the effects of putative therapies, Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) on these cells. 2) To describe a methodology to create fibroblast cell lines from patients with chronic wounds.

Methods: Patient biopsies were obtained from 3 distinct locations on venous ulcers. Fibroblasts derived from different wound locations were tested for their migration capacities without stimulators and in response to GM-CSF. Another portion of the patient biopsy was used to develop primary fibroblast cultures after rigorous passage and antimicrobial testing.

Results: Fibroblasts from the non-healing edge had almost no migration capacity, wound base fibroblasts were intermediate, and fibroblasts derived from the healing edge had a capacity to migrate similar to healthy, normal, primary dermal fibroblasts. Non-healing edge fibroblasts did not respond to GM-CSF. Six fibroblast cell lines are currently available at the National Institute on Aging (NIA) Cell Repository.

Conclusion: We conclude that primary cells from chronic ulcers can be established in culture and that they maintain their in vivo phenotype. These cells can be utilized for evaluating the effects of wound healing stimulators in vitro.

Figure 1

Fibroblast deriving from different location of the wound exhibit different morphology. The picture of the wound is shown in the center. Circles indicate origin of specific locations from which biopsies were taken. Fibroblasts deriving from each location are shown. Cells from location B exhibit different phenotype (larger in size; clumped) whereas cells from Locations C and A exhibit phenotype similar to normal healthy fibroblasts.

Figure 2

Cells from different wound locations exhibit distinct migration capacity. Wound scratch assay is shown. Cells from Location C migrated equally to the healthy control whereas cells from Location B have the slowest rate.

Figure 3

Human Recombinant GM-CSF Accelerate Migration of Fibroblasts deriving from Location C. Full lines indicate initial wound area; dotted lines demarcate migrating front of cells. GM – CSF treatment of fibroblasts deriving from location A (A) and location B (B). GM – CSF treatment of fibroblasts deriving from location C stimulated migration the most. (D) Surface area not covered by fibroblasts from scratch wounds are shown. GM-CSF markedly reduced wound area of fibroblasts from location C.