recent update in vitiligo

1. Cultured Melanocyte
Transplantation In Vitiligo
Presenter: Dr. Ankita
Moderator: Dr Aakash Singhal

2. Introduction
• Various treatment modalities for re-pigmentation of vitiliginous skin
• Medical and surgical techniques
• Lack of response to non invasive (medical) treatment esp. in hand & feet and
segmental vitiligo
• Tissue grafts has several limitation (inability to cover large areas, textural changes)
• Melanocyte transplantation: Autologous melanocytes are transplanted to
depigmented area
Cultured pure melanocytes suspension
Non cultured epidermal cellular suspension

3. Rationale for cultured melanocyte transplantation
• Cover large areas
• Less number of settings required
• Greater patient comfort
• Uniform pigmentation
• Less textural changes of donor skin
• Cells can cryopreserved and reculture in future

4. History
• Funan Hu et al (1956)
First studied melanocytes in benign pigmented naevi and foreskin of white and black infants
• Bellingham et al (1960)
Cultivated epithelial cell and fibroblasts
• Wilkins and Szabo (1981)
Able to grow melanocytes purely (95% purity)
• Gilchrist et al (1984):
Selective cultivation and long term maintenance in culture (serial passage up to 6 times)
• Eisinger and Nielson (1984)
Able to grow more pure melanocytes for >43 weeks (10 passages)

5. • 1992 : Use of non-cultured epidermal cellular grafting for treatment of stable
vitiligo. Donor sample -hair scalp
• 1998 : Olsson and Juhlin reported comparable but improved technique. The donor
skin – gluteal region.
• 2001 : Van Geel et al introduced use of hyaluronic acid - biodegradable cell carrier -
increase viscosity of suspension

6. Separation of epidermal cells from sample
• Splitting along the basement membrane
• Decrease the possibility of epidermal stem cell dissipation
• Various enzymes used
Trypsin
Collagenase
Dispases
• Best enzyme: Trypsin
High purity and viability of melanocytes
Fibroblast contamination was minimum

7. Medium and factors for melanocyte culture
ACS Textbook on Cutaneous and Aesthetic Surgery (2012)2nd ed. Jaypee Brothers New Delhi chapter 24 pg 327-384

8. Common components of melanocyte culture
• Keratinocyte inhibitors:
• Increased calcium
• PMA (Phorbol 12- myristate 13- acetate)
• Fibroblasts inhibitors:
• Cholera toxin
• Geneticin (commonly used)
• Melanocyte growth stimulators
• Potent enhancer of growth of melanocytes
• High yield of melanocyte in culture

9. Melanocyte growth stimulators
Chemical mitogens having tumor
promoting activity
(Not preferred due to mutagenicity)
Biological mitogens (without tumor
promoting activity)
(Preferred)
• Phorbol esters
• 12-tetradecanoyl phorbol 12
acetate (TPA)
• Phorbol 12- myrisate 13- acetate
(PMA)
Epidermal growth factor
Basic fibroblast growth factor
Endothelin-1
Alpha melanocyte stimulating hormone
Keratinocyte growth factor
Autologous human serum
Human placental extract
• Telecidin
• Aplysiataxin
Zokaei S, Farhud DD, Keykhaei M, Zarif Yeganeh M, Rahimi H, Moravvej H. Cultured Epidermal Melanocyte
Transplantation in Vitiligo: A Review Article. Iran J Public Health. 2019 March

10. Medium used for melanocyte culture
• Foetal bovine serum
• Ham’s nutrient mixture F 12 medium
• MCDB 153 medium (modification of F12 medium)
• Eagle’s minimal essential medium (EMEM)
• Hank’s salt with eagle’s minimal essential medium (HMEM)
• Dulbecco’s modified eagle’s medium (DMEM)
• DMEM and F12 medium
• Melanocyte growth medium (MGM)
• M2 medium

11. Fetal bovine serum
• Universal growth supplement of cell and tissue culture medium
• Natural cocktail of growth factors for cell attachment, growth and
proliferation
• Biosafety aspects: may contain adverse factors such as
Endotoxins
Mycoplasma
Viral components
Prion protein
• Ethical issues (collection from bovine fetuses)
• Serum free mediums were designed later

12. Ham’s nutrient mixture F 12 medium
• Serum free synthetic culture medium
• Components:
• Zinc
• Putrescine
• Hypoxanthine and Thymidine
• Choline and Inositol
• Amino acids
• Disadvantage
• No proteins and growth factors
• Requires supplementation with fetal bovine serum for optimal growth
• Soda bicarbonate buffer requires 5-10% CO2 to maintain physiological Ph

13. MCDB 153 medium
• Modification of Ham’s nutrient mixture F12
• Buffer changed
• HEPES (4-2 hydroxy ethyl 1- piperazine ethane sulfonic acid)
• Extensive trace elements
• Selenium
• Transferrin
• Insulin
• Tri-iodothyronine
• Testosterone
• Hydrocortisone
• Proteins and growth factors lacking

14. Eagle’s minimal essential medium (EMEM)
• Components
• 6 salts (CaCl2, KCL, MgSO4, NaCl, sodium phosphate and sodium
bicarbonate)
• 13 Amino acids
• 8 Vitamins (B1, B2, B3, B5, B6, B9)
• Modifications:
• HMEM: Nucleic acid precursors were also added
• DMEM: Four fold increase in concentration of amino acids and
vitamins, higher glucose
• DMEM with F12 Medium
Disadvantages:
• No growth supplements, protein, lipid and hormones
• Sodium bicarbonate buffer

15. • Melanocyte growth medium
• Used PMA
• Bovine pituitary extract
• M2 medium
• Used commonly
• PMA free (hence non mutagenic)
• Bovine serum free
• No antibiotics or antimycotics
• Contained bFGF, recombinant human insulin
Zokaei S, Farhud DD, Keykhaei M, Zarif Yeganeh M, Rahimi H, Moravvej H. Cultured Epidermal Melanocyte
Transplantation in Vitiligo: A Review Article. Iran J Public Health. 2019 Mar

16. Melanocyte Growth Medium M2 :
• serum and Phorbol Myristate Acetate free.
• The medium suitable - human pigment epithelial cells, neuronal cells,
and melanoma cells.
• available in two formats: Medium (ready-to-use) and Medium Kit.
• Medium (ready-to-use) : 500 mL bottle of Basal Medium and 1 vial of
supplement mix.
• Medium Kit : 500 mL bottle of Basal Medium and supplement pack
individual vials with pre-measured supplements.
• Includes components : optimal growth of human melanocytes.
• Not contain antibiotics or antimycotics
• formulated for use in incubator with 5% atmospheric CO2.

17. concentrations in Melanocyte Growth Medium (after addition)
Component
Melanocyte
Growth Medium
Melanocyte
Growth Medium
M2
Bovine Pituitary
Extract
0.004 mL / mL
Proprietary serum-
free, PMA-free
formulation
Basic Fibroblast
Growth Factor
(recombinant
human)
1 ng / mL
Insulin
(recombinant
human)
5 μg / mL
Hydrocortisone 0.5 μg / mL
Phorbol Myristate
Acetate
10 ng / mL

19. Co-culturing
• Keratinocyte- melanocyte co-culturing:
• Few keratinocytes aids in growth and differentiation of melanocytes
• Release of growth factors by keratinocytes
• Attachment of dendrites of melanocytes to keratinocytes enhancing the
growth
• Melanocyte co-culture with adipose derived stem cells
• Cell to cell interaction between melanocytes and stem cells
• Induction of melanocyte proliferation

20. Transplantation of melanocyte culture
Preparation of recipient sites
• Dermabrasion
• Blister formation
• Suction method
• Using liquid nitrogen gas
• CO2 laser
• Erbium Yag laser
• Direct injection using a needle
• Timed surgery: programmed diathermy device by Guerra et al
• Various scaffolds can be used for transplantation
Guerra L, Capurro S, Melchi F, et al. Treatment of “stable” vitiligo by Timedsurgery and transplantation of cultured epidermal autografts. Arch
Dermatol. 2000;136:1380-9

21. Various scaffold models
• Extracellular matrix components
Hyaluronic acid membrane
Andreassi et al observed less intense repigmentation in most of the
treated lesions
Collagen coated membranes
FACIT collagen (Fibril associated collagen with interrupted triple
helices)
• Degradable polymers
• Polylactic acid
• Polyglycolic acid
• Amniotic membrane

22. Amniotic membrane
• Native human or bio-engineered
• Angiogenic, anti-inflammatory and antimicrobial properties
• Lack antigenicity
• Facilitate re-epithelisation of skin
• Reduces scarring
• In a pilot study on four patients by Redondo et al
• Replated cultured melanocytes onto the basement membrane side of amniotic
membrane
• Allowed the melanocytes to grow for another 3–4 days
• Amniotic membranes placed on dermabraded recipient site
• Achieve excellent re-pigmentation in all patients
Redondo P, Giménez de Azcarate A, Marqués L, et al. Amniotic Membrane as a Scaffold for Melanocyte Transplantation in Patients with
Stable Vitiligo. Dermatol Res Pract. 2011;2011:532139. Epub 2011 Aug 18

23. Technique of melanocyte culture

24. Cryopreservation
• Cultured melanocytes preservation for prolonged periods
Procedure:
• Suspension of cultured cells centrifuged to form a pellet
• Resuspended in cryoprotectant (8% dimethyl sulfoxide in undiluted newborn calf serum).
• 1 ml of cryoprotectant for 10 million cells
• Transfer to cryotube, kept on ice for 10 min and placed in a freezer (-70° to -85°C).
• Whenever needed, these cells can be thawed by placing in a 37°c water bath
• About 70% of cells retain viability after 1 year

25. Multi-lineage differentiating stress-enduring
(Muse) cells
• Source of pluripotent cells naturally present within mesenchymal tissues
including dermis
• Muse cells do not undergo tumorigenesis
• Help in tissue regeneration and functional recovery as described in different
animal disease models including vitiligo
• These cells have immunomodulatory properties.
• Can be employed in surgically treating vitiligo lesions with minimal stability
• Shorten the waiting period for patients willing for vitiligo surgery
• Decrease the chances of graft failure with cellular transplantation
• Prevents the occurrence of peri-graft halo phenomenon.
Zhu MC, Ma HY, Zhi Zhan et al (2017). Detection of auto antibodies and transplantation of cultured autologous melanocytes for
the treatment of vitiligo. Exp Ther Med, 13

26. Factor affecting the results of cultured
melanocyte transplantation
• Age: results in children and adolescents were better but no significant difference
• Clinical stability of vitiligo lesion: stable lesion have better outcomes
• Short duration of disease
• Site of lesions: acral sites have poor outcomes
• Heavily cornified skin: poor outcome
• Hypothyroidism: less response
• Number of melanocytes transplanted: >1500 cells/mm3 required for good
results
Rao A, Gupta S, Dinda AK et al (2012). Study of clinical, biochemical and immunological factors determining stability of disease in patients
with generalized vitiligo undergoing melanocyte transplantation. Br J Dermatol, 166(6):1230-1236.

27. Studies comparing cultured vs non- cultured
melanocyte transfer

28. No. of patients Method Recipient site
preparation
Medium used No. of
melanocytes
transferred
Results
100 sites 50 sites for
cultured
melanocyte
transfer and 50
sites for non
cultured
Dermabrasion
of recipient
site
DMEM/ F12
medium used
with bFGF +
isobutyl
methyl
xanthine+
cholera toxin+
glutamine+
fetal bovine
serum
1000-2000
melanocytes
per mm2
Excellent
response seen
in 62.17%
cases with non
cultured
melanocyte
suspension
and 52% with
melanocyte
culture
Verma, R., Grewal, R. S., Chatterjee, M., Pragasam, V., Vasudevan, B., & Mitra, D. (2014). A comparative study of efficacy of cultured versus
non cultured melanocyte transfer in the management of stable vitiligo. Medical journal, Armed Forces India, 70(1), 26–31.

29. Studies comparing cultured vs non- cultured
melanocyte transfer ( continue)
• Period : 1 year
• Conclusion : No statistically significant difference between two
groups
• NCMT method : show slightly better results
1. The cumbersomeness method
2. Delay in transplantation : 3 weeks after the donor graft

30. Pandya V, Parmar KS, Shah BJ, Bilimoria F E. A study of autologous melanocyte transfer in treatment of stable vitiligo. Indian J Dermatol Venereol
Leprol 2005;71:393-397
No. of patients Method Recipient site
preparation
Medium used No. of
melanocytes
transferred
Results
27 patients 23 patients:
autologous
melanocyte
suspension
4 patients:
melanocyte
culture
- - 1000-2000
melanocytes
per mm2
Excellent
response seen
in 52.17%
cases with non
cultured
melanocyte
suspension
and 50% with
melanocyte
culture

31. No. of
patients
Method Recipient site
preparation
Medium
used
No. of
melanocytes
transferred
Results
30 cases 30 cases each
with 2 patches
each treated
with either non
cultured
epidermal
suspension or
cultured
melanocytes
- - - • Higher number of sites
resulted in excellent and good
pigmentation with cultured
method
• All segmental cases and 25%
of vitiligo vulgaris cases
achieved >90% pigmentation
with cultured
• Only fair to good pigmentation
on elbow and feet with CMT
Verma G, Varkhande SR, Kar HK, Rani R. Evaluation of Repigmentation with Cultured Melanocyte Transplantation (CMT) Compared with Non-Cultured
Epidermal Cell Transplantation in Vitiligo at 12th Week Reveals Better Repigmentation with CMT. J Invest Dermatol. 2015 Oct;135(10):2533-2535

32. Studies (using only melanocyte culture
method) with successful repigmentation
Kaufmann R et al
(1998)1
Used erbium yag laser for recipient preparation for irregular lesions and delicate sites
Lin SJ et al (2006)2 Chitosan based scaffold was used for melanocyte culture and successful
repigmentation
Wu K J et al (2017)3 > 50% repigmentation achieved in 79% patient with facial segmental vitiligo
Flabella et al (2009)4 Used fetal bovine serum, >80% repigmentation
Flabella et al (1989)5 Used liquid nitrogen for recipient preparation with permanent pigmentation of
refractory areas
1.Kaufmann R, Greiner D, Kippenberger S, et al (1998). Grafting of in vitro cultured melanocytes onto laser-ablated lesions in vitiligo. Acta Derm Venereol,
78(2): 136-138.
2. Lin SJ, Jee SH, Hsiao WC, et al (2006). Enhanced cell survival of melanocyte spheroids in serum starvation condition. Biomaterials, 27(8):1462- 1469
3. Wu KJ, Tang LY, Li J, et al (2017). Modified Technique of Cultured Epithelial Cells Transplantation on Facial Segmental Vitiligo. J Craniofac Surg, 28(6): 1462-
1467.
5. Falabella R, Escobar C, Borrero I (1989). Transplantation of in vitro cultured epidermis bearing melanocytes for repigmenting vitiligo. J Am Acad Dermatol,
21(2 Pt 1):257-264.
4. Falabella, R. (2009) Vitiligo and the melanocyte reservoir. Indian J Dermatol 54, 313- 318

33. Thank you

34. References
• Venkataram M. & Association of Cutaneous Surgeons of India. (2017). Acs(i) textbook of cutaneous and
aesthetic surgery (Second edition)
• Zokaei S, Farhud DD, Keykhaei M, Zarif Yeganeh M, Rahimi H, Moravvej H. Cultured Epidermal Melanocyte
Transplantation in Vitiligo: A Review Article. Iran J Public Health. 2019 Mar;48(3):388-399
• Matin R. Vitiligo in adults and children: surgical interventions. BMJ Clin Evid. 2015 Mar 20;2015:1717
• Im S, Hann SK, Park YK, Kim HI. Culture of melanocytes obtained from normal and vitiligo subjects. Yonsei
Med J. 1992 Dec;33(4):344-50.
• Rao A, Gupta S, Dinda AK et al (2012). Study of clinical, biochemical and immunological factors determining
stability of disease in patients with generalized vitiligo undergoing melanocyte transplantation. Br J
Dermatol, 166(6):1230-1236.
• Olsson MJ, Juhlin L (2002). Longterm followup of leucoderma patients treated with transplants of autologous
cultured melanocytes, ultrathin epidermal sheets and basal cell layer suspension. Br J Dermatol,147(5):893-
904.
• Zhu MC, Ma HY, Zhi Zhan et al (2017). Detection of auto antibodies and transplantation of cultured
autologous melanocytes for the treatment of vitiligo. Exp Ther Med, 13(1):23-28.
• Verma G, Varkhande SR, Kar HK et al (2015). Evaluation of repigmentation with cultured melanocyte
transplantation (CMT) compared with non-cultured epidermal cell transplantation in vitiligo at 12th week
reveals better repigmentation with CMT. J Invest Dermatol,135(10):2533-2535