Keratinocyte culture – short notes

Types of keratinocyte culture –

  1. Autologous
  2. Allografts

 

Autologous keratinocyte culture –

Patient selection –

Usually patient with >50% TBSA burn with most being 3rd degree

Contraindications – Cutaneous tuberculosis, AIDS, Hepatitis B

Age and inhalational injury are not contraindications.

Biopsy –

Minimum delay from biopsy to culture plating.

Less than 48 hours.

If >48 hours, antibiotics needs to be added to culture medium.

Biopsy – A SSG of 1-5 cm2 is harvested.

 

Keratinocytes isolation –

Sample is washed

Differential trypsinization is done

Separated epidermal component is centrifuged and cell suspension obtained

 

Technique of culture –

Described by Green

Keratinocytes are now routinely cultured on feeder layers

Feeder layer consists of fibroblasts (locally irradiated or treated with mitomycin)

Murine fibroblast line or human fibroblasts are used.

Isolated keratinocytes are seeded on petridish/culture flask containing mitomycin C treated mouse fibroblast 3T3 cells.

Keratinocytes are cultured in 3:1 mixture of Dulbeco’s modified Eagle’s medium supplemented with –

Hams F12 medium

Hydrocortisone (0.4 mg/ml)

Insulin

Transferrin (5mg/ml)

Adenine (8 x 10-4 mmol/l)

Triiodothyronine (2 x 10-2 mmol/l)

Cholera toxin (10-10 mmol/l)

Fetal calf serum (10%)

Antibiotics

Culture is incubated @ 37°C in humidified atmosphere with 5% CO2

After 3 days, 10 ng/ml of human epidermal growth factor  is added.

Primary culture is now established and can be passed ‘n’ number of times depending on requirement.

Keratinocytes adhere to matrix synthesized by fibroblasts and produce 3 types of clones –

Holoclones

Meroclones

Paraclones

Basal cell produce holoclone (with maximum growth potential allowing 5-6 subculture)

Suprabasal cell produce paraclones (with least growth potential).

Holoclones gradually develop into meroclones during culture.

So, only 2nd and 3rd passage are used for grafting.

Each colony grows from periphery and become confluent by 8-10 days.

Cultures are seeded/reseeded with attenuated 3T3 cells.

Once a confluent and multicellular epithelium is obtained, it is detached enzymatic from culture disk/flask by dispose and rinsed in PBS.

Epidermis is then taken on paraffin gauze.

Cultured epithelial autografts usually takes 2-3 weeks.

Detached epithelium is assessed for viability.

The cultured graft is transported in aseptic conditions in petri-dishes containing DMEM under 5% CO2 to OT.

 

Patient preparation –

Meanwhile patient burn wounds in serially debrided.

Patient condition in stabilized.

Excision is done as required.

 

Grafting

Grafting is done as soon as cultured graft arrives in OT

Carrier gauze is directly applied to the prepared bed.

Carrier gauze is then covered with saline gauze f/b thick dry dressing.

1st dressing is done after 5 days.

Then alternate day.

Carrier gauze is removed at 6-8 days.

 

Drawbacks of cultured keratinocytes –

(Most of the drawback is due to absence of dermal layer.)

Healed wound are thin and stiff

Lacks durability

Scarring and wound contraction are often

Keratinocytes also lack a well formed BM (or forms gradually) making grafts to shear off easily.

Fragile graft – leads to ‘delayed losses’.

 

Future –

Lack of dermal component makes cultured keratinocytes less than desirable – using dermal equivalent is a option to increase uptake (but, makes it costly.)

Using acellular fibrin gel as biological support media – does not require enzymatic detachment –

Improves adhesion potential of cells – increases attachment to the bed.

Also reduces culture time to 15 days to produce sheet graft.

 

Epidermal allograft culture –

Easily available

But, only a temporary measure (it gets rejected after some time).

 

 

 

 

 

 

 

 

 

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