Skin donation, skin banking, skin culture

1. Skin donation, skin banking,
skin culture

2. History
• 1871 -First skin autograft was described by
Reverdin
• 1874- Thiersch used small thin grafts, pinch
grafts, epidermis grafts, or razor grafts
• 1881- first successful use of allogeneic skin for
burn wound coverage by Girdner
• Early 1900- Webster and Matthews described
healing of skin autografts stored for 3 weeks
at 4–7°C

3. • 1949- establishment of United States Navy
tissue bank
• 1952- Billingham and Medawar demonstrated
that skin could be effectively cryopreserved
using glycerol
• Brown and Jackson popularize use of
allogeneic human skin grafts as biologic
dressings for extensive burns and denuded
tissue

4. • 1966- Cochrane reported first successful use
of frozen autologous skin grafts following
controlled-rate freezing in 15% glycerol and
rapid rewarming prior to implantation
• 1971- Bondoc and Burke are credited with
establishment of the first functional skin bank.

5. Concept
• The Skin Bank is a service, not a product
• Central service, available to the Plastic Surgery
Department and especially to the Burns Unit

6. Clinical uses of allograft skin

9. Appearance of cryopreserved
and fresh, refrigerated
allograft skin on postoperative
day 5

10. Meshed allograft overlay technique
as described by Alexander et al.

11. Disadvantages
• Infection
– Bacterial contamination
– Transmission of HIV, hepatitis, CMV
• Rejection
– Vascularized allogeneic skin grafts typically remain
intact on the wound of a burn patient for 2–3
weeks
– Survive up to 67 days due to the inherent
immunosuppression of extensive burn injury
– Langerhans cells which express class II antigens on
their surface- Acute rejection

12. Technical aspects of skin banking
• Donor screening
• Skin recovery
• Skin processing
• Refrigeration
• Cryopreservation
• Lyophilisation
• Transport
• Rewarming

13. Donor screening
• Ethical/legal consideration
• Donor Medical History and Behavioral Risk
Assessment Questionnaire
• Serologic screening tests for transmissible viral
diseases (HIV-1/2 antibody, hepatitis B surface
antigen and B core antibody, and hepatitis C
and HTLV-1 antibodies, treponemal antibody)
• Microbiology reports and cause of'death on
the death certificate.

15. • Sepsis or bacteraemia
• Dermatitis or other infection of the skin
• Pneumonia or other respiratory infection
• Addiction to controlled substances (due to risk of hepatitis)
• Toxic or viral hepatitis
• Antibody HLA
• Treponemal antigen or antibody
• Malignancy (including sarcoma, carcinoma, lymphoma and
leukaemia)
• Recent carcinoma chemotherapy or radiation therapy
• Auto lmmune disease affecting integrity of skin
• Collagen disease afecting integrity of skin
• Homosexuality, or heterosexual donors who have been
involved in prostitution.

16. Skin recovery
• In an appropriate facility (i.e. hospital morgue
or operating room, medical examiner’s office,
or the tissue bank)
• Within 24 hours of death if the donor was
refrigerated within 12 hours of asystole or
within 15 hours if refrigeration did not occur

17. • Aseptic precautions
• Limited to the torso, hips, thighs, and upper
calves
• Dermatome at a thickness of 0.012–0.018
inches
• skin is placed in tissue medium and
maintained at 1–10°C during transport

19. Skin processing
• Processing environment:
– Aseptic precautions

20. • Microbiologic testing:
– 1 cm2 biopsy sample for each 10% of the body surface
area
– microorganisms:
• • coagulase-positive staphylococci,
• • group A, beta-hemolytic streptococci,
• • enterococci,
• • Gram-negative organisms,
• • Clostridium sp., or
• • yeast or fungi
– Culture results be reported after 7 days of incubation

21. • Maintenance of viability:
– Nutrient tissue culture medium- Eagle’s MEM and
RPMI-1640 continue to be generally accepted
– University of Wisconsin (UW) solution
– Cryoprotectants- Glycerol (10–20%) and
dimethylsulfoxide (10–15%)

23. Information sheet:
1 . The generic name of the product
2. Instructions for thawing and use
3. Donor blood type, if known
4. Cryoprotective fluid composition, including
antibiotic and other supplements
5. Name and adress of the collecting skin bank
6. Dimensions of the skin strip contained
7. Required storage temperature
8. A space for recipient identification

25. Refrigeration
• stored at 4°C in tissue culture medium with or
without antibiotics
• free-floating in an aseptic container with
approximately 300 mL of medium per square
foot of skin
• skin viability can be maintained for up to 2
weeks at 4°C if the nutrient medium is
changed every 3 days

26. Cryopreservation
• Within 10 days of procurement if nutrient media
is changed every 3 days otherwise within 96
hours of recovery
• Incubated in cryoprotectant solution for 30
minutes at 4°C
• Slow-rate cooling at a rate of approximately −1°C
per minute
• Skin stored in mechanical freezer (−70 to −100°C)
can be maintained for 3–6 months, if stored in
liquid nitrogen (−150 to −196°C) viable up to 10
years

27. Lyophilization
• Skin can also be lyophilized by freeze drying or
incubation in glycerol
• decrease biologic degradation and antigenicity
• epidermal cell destruction and the loss of
barrier function
• poor adherence to excised wound bed

28. Request for information
• 1. Recipient patient's name
2. Patient's hospital registration number or
similar identification
3. Name of requesting physician
4. Anticipated time and date of use
5. Amount of skin required
6. Part of body to be covered

29. Transport
• Refrigerated skin: in tissue culture medium at
wet ice temperatures (1–10°C) in an insulated
container
• Frozen allograft skin: on dry ice in an insulated
container to prevent the skin temperature
from rising to greater than −50°C

30. Rewarming
• performed in 2–4 minutes or less at a
temperature of 10–37°C (127–470°C/min).

31. • National Burns Centre (a tertiary burn care
centre) along with Rotary International and
Euro Skin Bank joined hands to plan and
develop a sustainable skin banking model in
Mumbai

32. 1. the finance of setting-up and running a skin bank
was supported by Rotary
2. the technical assistance was provided by Euro
Skin Bank
3.the procurement, processing, preservation and
distribution were looked after by National Burns
Centre
4. the continuous large scale awareness campaign
was supported and executed by a group of
Nongovernmental Organizations (NGOs) led by
Rotary along with National Burns Centre

35. The future of skin banking
• Technological advances may include
modifications to reduce immunogenicity and/or
the potential for disease transmission
• Deepidermized allograft dermis could become:
• a source of growth factors and antimicrobial agents,
• a permanent full-thickness wound cover seeded with
the patient’s autologous keratinocytes and fibroblasts,
• a bilayer membrane system for epidermal
autografting,
• a readily-available cover preseeded with non-antigenic
allogeneic keratinocytes, fibroblasts, and melanocytes

36. Skin donation

43. Skin culture

44. • 1981- Reconstituted human epidermal tissues
were used to treat the burns (O'Connor et al.,
1981)
• Dermis: matrix + fibroblasts

46. • Fibroblast functionality
– Capacity of fibroblasts to grow, remodel the
matrix, secrete and degrade proteins, and produce
numerous growth factors or cytokines is crucial for
the regulation of the tissue structure and its
cellular microenvironment.

47. 1. Cell passage number
For a skin graft, sub-culturing of fibroblasts for 8
passages, corresponding to at least 25 doubling
populations where one isolated cell will generate
8.4 million cells
2. Age and sex of the donor
– No Variations in the doubling time with donor
age or sex

48. 3. Location of the biopsy
– No difference in doubling time
– cells were isolated from the scalp or abdomen,
the dermis was thicker than when fibroblasts
were isolated from the breast or forearm

49. Reconstruction of a tissue-engineered
endothelialized skin
• Dermal fibroblasts are cultured on petri dishes
with the fibroblast medium supplemented
with ascorbic acid (50µg/ml).
• Cells secrete and remodel matrix that form a
manipulable sheet after 4 weeks.
• Endothelial cells are then seeded on the sheet
and cultured for an additional week in EGM-2
medium with ascorbic acid

50. • Two of these sheets are then superimposed to
form an endothelialized tissue-engineered
dermal substitute.
• Keratinocytes can then be added onto the
dermis to form an epidermis after
differentiation at the air-liquid interface

51. 1. Human embryonic stem cell (HESC) model-
shown an integrated basement membrane
2. Organotypic culture (OTC) with human
keratinocytes and fibroblasts

52. Three-Dimensional Organotypic
Human Skin Culture
• basic 3D skin culture system is composed of an
upper chamber and lower chamber that are
separated by collagen gel or dermal matrices,
such as split thickness human dermis derived
from cadaver or surgically discarded skin

54. • media on the top chamber is removed,
creating an air–liquid interface
• bottom chamber will be replenished with
fresh media every 2–3 days.

55. Other possible improvements
• Autologous hypodermis using human adipose-
derived stem/stromal cells and
• Wound stimulus and wnt pathway activation
trigger de novo hair follicle formation from
epidermal stem cells in reconstructed skin
• Langerhans and dendritic cells, immunological
cells and nerves to improve skin sensation

56. References
• David N Hernon. Total burn care. 3rd edn
• Keswani S. M. (2018). Skin banking at a
regional burns centre—The way forward.
Burns, 44(4)
• Pousa real F. Skin bank organization. Annals
oj'the MBC - vol. 3