Tissue Culture Laboratory
Unit Head: Joanne Paddle-Ledinek AM
Skin culture
For burns grafting
The keratinocyte cell is the building block of the top layer of skin, the epidermis. Burns are traditionally treated by taking a thin layer of skin from an undamaged area (donor site) and placing it onto the burn wound. In severely burnt patients, the donor sites are few and the potential to re-harvest the area is limited. Skin allografts (harvested from one person and grafted onto another) can be used as temporary dressings, but are rejected by the body within a short time.
The Tissue Culture laboratory grows keratinocyte cells into epithelial grafts for burn patients in hospitals around Australia. From a small piece (2 x 2cm) of the patient’s own skin, we can grow enough epithelial grafts to cover a whole person in 3 weeks. The individual grafts are typically 10 x 7cm in size and are multi-layered, very much like normal epidermis. At the base of the graft is the basal cell layer. As the cells move through each layer of the skin, they become increasingly differentiated. Once the epithelial graft is placed on the patient and exposed to air, the top layer takes on the protective role of the skin by becoming cornified.
Figure 1. Normal epidermis
Figure 2. Cultured epithelium
(H&E stain, magnification approx. x350)
To date more than 200 patients with burns up to 96% Total Body Surface Area (TBSA) have been grafted with a total of almost 10,000 grafts.
Figure 3. Detached cultured epithelial graft
For chronic skin ulcer treatment
We, and others, have shown that cultured epithelial autografts or allografts can be used to successfully treat chronic leg ulcers.
Keratinocytes secrete a number of growth factors (e.g. TGF-a ) that are essential for their own proliferation. Even in deep ulcers, patches of keratinocytes belonging to hair follicles or sweat glands may remain. These cells are stimulated to migrate and proliferate by such autocrine factors. A similar response in keratinocytes bordering the wound shrinks the perimeter of the ulcer.
We have established a keratinocyte cell line from neonatal foreskin. These cells are free of contamination by HIV, Hepatitis B & C and CMV and have been used to produce cultured epithelial allografts for the successful treatment of chronic leg ulcers.
Cryo-preserved allografts are available as biological dressings for immediate use on request.
Chondrocyte culture
For cartilage repair
Chondrocytes are the cells responsible for setting up and maintaining the structure of hyaline cartilage that is the load-bearing surface in articular joints (eg. knees). In situ, chondrocytes do not proliferate and so are unable to repair structural defects, however they can be grown in vitro. To do this, a very small cartilage biopsy (100mg) is harvested arthroscopically from a low weight-bearing area of the patient’s knee.
Within two weeks, we can grow sufficient autologous chondrocytes to repair such defects.
The repair is achieved by injecting a suspension of chondrocytes back into the defect area beneath a fixed periosteal patch. Over 200 patients with knee joint defects have been treated in this way. The resultant repairs exhibit excellent macroscopic characteristics. In those cases where core biopsies have been obtained, the replacement cartilage has been predominantly hyaline in type.
Arthroscopic appearance.
Figure 4. Original cartilage defect (20x18mm)
Figure 5. Healed cartilage defect after 9 months.
Cross-sections of 2mm diameter core biopsy from the healed defect at 9 months post-implantation
(Magnification approx. x50)
Figure 6. H&E stain
Figure 7. Masson stain.
Figure 8. Alcian /Van Gieson stain
The three stained sections of core biopsy reveal chondracytic cells and mature hyaline cartilage abutting the cortical bone. Although superficially the cells are more spindle in outline, the extensive mucin accumulation indicates on-going cartilage maturation.
Further research in this area will be directed to the possibility of using cultured chondrocyte seeded polymeric implants.
Other cell research
During the course of previous and current projects we have isolated other primary cell lines (e.g. Desmoid tumor cells, gastric epithelial cells, mammary gland epithelial cells, urothelial cells). We have also developed models to study the possible cytotoxic or growth-promoting substances on keratinocytes and other cells.
Research Group
Further information
You can contact Joanne on (03) 9903 0615 or (03) 9903 0616,
Or email her at Joanne.Paddle-Ledinek@med.monash.edu.au
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