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The biotechnology slant is the culture and application of human dermal keratinocytes. Autologous (the patient's own) skin keratinocytes can be utilized to restore epithelial coverage of small burns. To culture sheets of keratinocytes is time consuming and labour intensive. The cells can be delivered to the wound by aerosol spray or direct instillation. A recent article in the medical journal, Burns (2005) show that cultivated keratinocytes are particularly useful in selected children afflicted with partial thickness scald burns and renders constantly reliable results compared to skin grafting. The advantages of keratinocyte treatment is that painful skin donor grafting can be reduced as well as the amount of blood transfusions. Less scarring may well be observed after keratinocyte treatment. Cultured keratinocytes may well be of value in varicose ulceration of the lower leg provided venous hypertension dynamics and underlying causes have been corrected. BOLAND CELL has specialized technologies in this area for clinical application in persons with burns and leg ulceration. This biological therapy is also promising in persons with chronic diabetic neuropathic foot ulcers. The treatment is well tolerated, can be combined with standard care of diabetes and has a good safety profile. FDA approval in the United States has been given for the biological treatment of burns and wounds with keratinocytes under controlled conditions. See contact details in South Africa .


Patients with extensive burns are associated with an appreciable morbidity and mortality. Inhalation burns and extensive burns of the head and neck region in children are potentially lethal. Death may follow super infection with Pseudomonas organisms, other nosocomial infections, renal failure and septicaemia. Cadaveric skin is valuable in patients with extensive burns and could be life-saving. Cultured skin keratinocytes can be sprayed onto the burn provided the right local conditions prevail and local conditions have been optimalized in the burn unit. Academic centres can provide these services in addition to conventional auto grafting and cadaveric allogeneic transplantation. Obviously during this time optimal nursing, nutritional support, prevention of cross-infection, physio and occupational therapy, and enhancement of the immune system is critical for a positive outcome. Top academic units can proliferate keratinocytes as an adjunct to conventional burn therapy. There is proof-of-science for the application thereof and is an important research focus. Drawbacks include cost and time of cell proliferation. See resources: Burns 2006,31:578-86; J Trauma 2006, 60:821-9; Burns 2006, 32: 395-401; Biomed Mater Eng 2006, 16: S63-71.


BOLANDCELL in this upgraded overview on keratinocyte-derived cell the rapy provides details on:

  • Potential application of cultured keratinocytes
  • Potential application of non-cultured keratinocytes
  • Technology for investors and venture capitalists

Definition: Dorland's Medical Dictionary defines the keratinocyte as follows: " The epidermal cell which synthesizes keratin; constituting 95% of the epidermal cells and, with the melanocyte, forming the binary cell system of the epidermis. In its various successive stages it is known as basal cell, prickle cell, and granular cell. Called also malpighian cell ".

BASIC HISTOLOGY : BOLANDCELL provides a comprehensive updated overview on the biology and application of autologous keratinocyte transplantation in the clinic.

The epidermis is composed chiefly of stratified squamous keratinized epithelium (Junqueira et al 2003), but is also to a much lesser degree endowed with melanocytes, Langerhan's cells, and Merkel's cells. The layers of the epidermis are the strata corneum, lucidum, granulosum, spinosum and basale.

For the moment there are two types of keratinocyte suspensions available in the clinic. One is cultured autologous keratinocytes ex vivo in a commercial lab and takes about 6 weeks to multiply sufficient numbers of cells. This approach has been available for the past 20 years, and has been predominantly applied in persons with burns. The second preparation is spray on non-cultured keratinocytes. A small skin graft is obtained and the keratinocytes are dispersed by enzymatic digestion in the operating suite. The cell suspension so obtained is then concentrated in a syringe and sprayed or dripped onto the chosen recipient site. This approach has been marketed by ReCell® in England and Europe .

Regarding the epidermis, the predominant cell type is the keratinocyte, which produce keratin (Kierszenbaum 2002). As previously described, keratinocytes are arranged in 5 strata ( Kieszenbaum 2002). Keratinocytes in the strata lucidum and corneum are difficult to culture as the cells are being shed. During wound healing keratinocyte migration is enhanced by epidermal growth factor (EGF) and keratinocyte growth factor. Especially on histological assessment are the presence of membrane coated granules (also referred to as lamellar bodies), and tonofibrils that extend into the cytoplasmic spinous-like processes. Keratohyalin granules occur in the keratinocytes derived from the stratum granulosum ( Kierszenbaum 2002). The expression of keratinocytes is important to master if one is involved with cell culture of keratinocytes. The lamellar product and glycolipid acylglucosylceramide is released by the cells into the intercellular spaces. This is alleged to waterproof the cells and epidermis. Keratins are classified as Keratin 2e and 9, 1 and 10, 5 and 14. Desmosomes support adjacent cells and hemidesmosomes anchor the cells to the basal lamina. The prominent product of keratinocytes in the stratum granulosum is filaggrin. In the stratum basale ( Malpighian layer) are found mitotically dividing stem cells. Langerhan's cells ( dendritic cells) are associated with keratinocytes through E-cadherin. All mitosis is confined to the Malpighian layer and this of critical importance to the cell biologist involved in the proliferation and multiplication of keratinocytes, ex vivo. During proteolytic cell separation, trypsin is used to lyse the cells and in particular the keratin filaments (tonofibrils) that attach the cells ( Junqueira et al 2003).


Gilchrest et al 1994, from the Boston University School of Medicine, Mass, showed that both aging and photoaging alter the expression of selected genes that are implicated in growth, differententiation, immunomodulation, and UV response in human epidermis. This has bearing on photocarcinogenesis in persons with solar damage. Habitual sun exposure ( photoaging), results in alterations in signal transduction and differentiation state in cultured human keratinocytes. Epidermal cell-derived thymocyte-activating factor ( ATAF) is reduced with age ( Sauder et al 1988). Others have shown that the telomere length in keratinocytes is shortened with aging ( Matsui et al 2000).


  • Rapid and effective closure of full-thickness burn wounds remains a challenge in persons with greater than 50% ( TBSA) burns
  • Cultured skin substitutes consisting of autologous cultured keratinocytes and fibroblast is an option that can be considered. This approach may reduce requirements for donor skin harvesting, reduce time to wound closure, morbidity and mortality
  • Competition for the keratinocyte approach is the application of skin banking and the use of cadaveric skin allografts
  • In children, the use of cultivated keratinocytes in partial thickness scald burns is a procedure that renders constantly reliable results ( Rab et al 2005). Use of allogeneic keratinocytes can also result in epithelisation and a reduction in secondary skin grafting procedures. In children some units have reported constantly reliable results with the use of cultivated allogeneic keratinocytes
  • Wood ( 1996) showed the efficacy of cultured epithelial autograft for the treatment of burn wounds, but pointed out that the resulting scar was subject to breakdown with minimal trauma, necessitating early aggressive pressure therapy. These healed burn surfaces are very fragile and tend to break down, probably because it takes time to mature the dermal-epidermal junction. That group showed that the cultured keratinocytes have a fragile hold on the underlying wound bed in the initial stages . Because of this the wounds are susceptible to shearing forces. They have pointed out that until the keratinocytes have differentiated sufficiently to form an established layer of keratin, the surface is vulnerable to maceration ( Wood et al 1996)
  • Do cultured epithelial autografts " Take" ? Stoner et al 1999 have answered the question and shown unequivocally that marked cultured cells of sole origin do engraft ( cell attachment) as indicated by cytokeratin 9
  • Is it possible to treat hypopigmented lesions with cultured epithelial autograft? Stoner et al 2000 have answered that question and confirmed that the hypopigmentation in burn patients can be repigmented with CEA.
  • Can melanocyte repopulation in full-thickness wounds be affected by using a cell spray apparatus? Navarro et al 2001 have indicated that it is possible but that more studies are needed to control the expression of melanin
  • Is there any experimental data to back the use of spray-on keratinocyte suspensions? Nevarro et al 2000, from Western Australia has shown that seeded keratinocytes can epithelialise a denuded surface in a porcine model
  • A benchmark article was produced by Wood, also from Western Australia ( 2003). She explains the use of epidermal reconstruction using cellular autologous epithelial suspension in combination with traditional skin grafting techniques in burn subjects. Their group were able to minimize skin scarring, improve scar quality and, a reduced need for pressure garments
  • Steinlechner et al 1993 of London demonstrated that low-level laser therapy could stimulate keratinocyte proliferation in culture, but this work is not focused on burn therapy. Nonetheless the results are impressive
  • Currie et al 2003 ( see Burns,2003 29[677-685] showed that cultured and suspended autologous keratinocytes could be suspended in Tisseel fibrin glue
  • An important paper appeared from the U.S. Army Institute of Surgical Research in 1993, in which this group failed to identify the positive impact of cultured autologous keratinocytes, on wound closure in extensively burned patients. In their study they were also able to conclude that CAK exerted no demonstrable effect on the outcome of extensively burned patients


Basic science relevant to understanding autologous skin cell therapy for vitiligo.

Vitiligo is a clinically relevant dermatological condition and characterized by skin depigmentation (1,2,3). Persons from dark-skinned races may experience significant emotional stress as a result of progressive depigmentation (2). Histologically, there is a complete absence of melanocytes and therefore differs from albinism in which melanocytes are present, but no melanin is formed because of defective tyrosinase (2). New developments in biotechnology, autologous spray-on cell therapy, including keratinocytes and light phototherapy have strengthened the physician's armamentarium in the treatment of vitiligo and leucoderma (3). Anecdotal reports and small case studies show promising results in the hands of experts (3). The long term efficacy and safety of non cultured melanocyte-keratinocyte cell transplantation for the treatment of segmental and stable vitiligo are now apparent, but further controlled trials are desirable (3). To understand, the biotechnological cell therapy approach to vitiligo needs a comprehensive grounding in basic medical science and clear understanding of skin histology regarding the morphology of the integument (4). For instance, it is important to have theoretical and practical knowledge of the different layers and cell types of the epidermis: stratum corneum, lucidum, granulosum, spinosum and basale. Langerhan's cells typically occur in the stratum granulosum and melanocytes in the stratum basale (4). Therefore, if a thin split-thickness skin biopsy is taken to generate an ex-vivo melanocyte- keratinocyte cell preparation, then the inclusion of the stratum basale must be ensured (3,5,6). Keratinocytes contain abundant amounts of the protein, termed keratin (2,4). The deeper parts of the skin contain living keratinocytes, but the outer layer consists of skeletons of cells called corneocytes (2,4). Relevant to wound healing and cell transplantation is the fact that keratinocytes are associated with the release of secretory proteins such as, acylglucosylceramide, keratohyalin, thymulin or thymopoietin- like substance, IL-1, epidermal growth factor and transforming growth factor alpha (TGF-a)(4). In the basal strata of the epidermis, living keratinocytes express integrins that determine cellular adhesive properties (4). Knowledge of melanocytes is mandatory, if successful cell therapy outcome is to be predicted for the treatment of selected patients with vitiligo (3,5,6). Melanocytes, of neural crest origin, interdigitate with keratinocytes in the basal layer of the skin. Melanocytes produce melanin, an important endogenous integument pigment and tyrosinase plays a pivotal role in the formation of melanin (2,4). An important cell-to-cell transfer occurs in the skin when melanin granules are phagocytosed from the dendritic processes of melanosomes by surrounding keratinocytes (4).


Clinical data show that skin repigmentation in selected patients with focal and segmental vitiligo is achievable by melanocyte-keratinocyte cell therapy (7-15). Mulekar and others have demonstrated that utilization of a small targeted skin biopsy, together with trypsin digestion to produce a cellular suspension of keratinocytes and melanocytes, can induce repigmentation in leucoderma (3,6,11,12). A five-year follow-up period was recorded by his team using cell therapy for vitiligo, and very favourable results were documented (5). Positive efficacy and safety profiles are documented (12). Patients with segmental and focal vitiligo, showed good results in 84% and 73%, after cell therapy, respectively (11). New technology provides for the generation of a non-cultured, autologous cell preparation of melanocytes and keratinocytes to a wound surface to promote enhanced wound healing ( ReCell®). A split thickness skin biopsy (0.2- 0.3 mm) skillfully taken, is needed and procurred under local anaesthesia using either a Zimmer® dermatome, silver knife, Humby knife or Dermablade® (3). A skin biopsy size of 2 x 2cm and 1 x 1 cm can treat designated areas of 320cm 2 and 80 cm 2 , by cell suspension application, respectively, if the ReCell® technology is used. This cell therapy technology is thus highly suitable, practical and affordable for treatment of select burns, leucoderma, and stable vitiligo (3,5,6). Because the patients' own skin is used to generate the autologous cell suspension, the chances of transmission of infections is completely excluded (3). Depigmented lesions sited in the genital region may benefit from cell therapy (5). Enhanced results in patients with vitiligo vulgaris, may be anticipated in those receiving combination low-dose oral pulse betamethasone and autologous, non-cultured melanocyte-keratinocyte cell transplantation (6). Other workers from Belgium , using subjective and objective measurements, have demonstrated the potential benefits of non-cultured epidermal cell transplantation for rapid treatment, in patients with stabile vitiligo (7,9,10). Engraftment, " take", and survival of transplanted autologous keratinocytes has been demonstrated by immunocytochemistry. Interesting work from Taiwan , suggest that PUVA treatment can be considered as treatment modality in persons with the active stage of vitilligo (8). This approach can theoretically slow down the destruction of melanocytes. Cell therapy for skin repair is thus another treatment option in the armamentarium of the dermatologist and plastic surgeon regarding vitiligo (3,5,7,9,11,13,14,15). This approach can supplement conventional treatment: topical steroids, betamethasone, tacrolimus 0.1% ointment or pimecrolimus 1% cream, XeCI excimer laser phototherapy, and camouflaging make-up.


Harvested autologous epithelial cells (ReCell®) from a dermatological and plastic surgery practice perspective, has proven useful in the following clinical scenario:

  • Revision of scar tissue (hypopigmented facial scars: post irradiation of basal cell carcinoma).
  • Amelioration of selected patients with stable vitiligo (3,5,6).
  • Amelioration of persistent hypopigmentation (hypopigmented scarring, post laser treatment and hypopigmentation); by reintroduction of pigment producing cells and autologous epithelial spray.
  • Amelioration of post-chemical peel hypopigmentation, keloid formation or induction of hypertrophic scarring.
  • Amelioration of small partial thickness burns on the dorsum of the hand and other anatomical areas.
  • Amelioration of vitiligo in conjunction with conventional laser-assisted treatment.
  • Facilitation of burn wound healing, reduction in scar formation and altered pigmentation.
  • Facilitation of facial rejuvenation and other related aesthetic procedures to enhance skin resurfacing.


    • ReCell® provides accessible, non-invasive and simple spray-on technology for the clinician. The procedure can be completed in 90 minutes. ReCell® provides a medical device ( kit) for harvesting and processing of autologous skin cells. Repigmentation induced by melanocyte repopulation is expected to be permanent in persons with stable vitiligo. Informed consent is important to ensure realistic expectations and treatment outcome. Marked improvement, rather than perfection should be explained to the patient.
    • The technology relies on the characteristics of basal cells from the epidermal-dermal junction. A maximum area of 320cm 2 can be treated with ReCell®. Indications for ReCell® treatment include: stable vitiligo, vitiligo refractory to standard medical treatments, segmental and focal vitiligo, persons with leucoderma ( piebaldism, postburn leucoderma, chemical leucoderma, nevus depigmentus, and halo nevus). Contraindications to the use of ReCell© are few, but include, underlying infections, bleeding disorders, history of hypertrophic scarring or keloids, and history of the Köbner phenomenon.
    • Wounds meeting inclusion criteria and sprayed with autologous keratinocytes, show faster and better quality of epitheliasation. Scarring is potentially reduced.
    • Reintroduction of autologous melanocytes enables repigmentation of stable vitiligo and other forms of leucoderma. Epidermal derived cells can be sprayed in suspension onto areas of the patient's epidermis where they are deficient.
    • There are three critical factors that impact on the outcome of autologous cell therapy: thickness of donor skingraft, preparation of keratinocyte-melanocyte cell suspension, preparation and optimal debridement of the recipient site. Recipient site preparation may include, mechanical dermabrasion, use of CO 2 and Erbium lasers, blistering with suction pump or liquid nitrogen, and rarely ultrasound and UVA treatment. The skin biopsy must be ultrathin and transparent to facilitate the epidermal-junction split by trypsin.
    • Optimal dressings are important and patient follow-up is mandatory, to document outcomes and to reassure the recipient.
    • Expectant outcomes include, epithelialisation within 5-8 days and repigmentation within 1 month. Continued improvement can be anticipated over the next 6 months, at a rate depending on the type of vitiligo and skin type of the individual. Temporary and reversible hyperpigmentaion has been observed in dark skin races. A failure of cell therapy is predicable in persons with active vitiligo. In patient's displaying partial and a static response, can be reconsidered for retreatment 6 months after the initial ReCell© therapy.


  1. Ortonne J. P. Vitiligo and other hypomelanoses of skin and hair. New York , Plenuim Press, 1982.
  2. Robbins S. L, Cotran R. S, Kumar V (eds). Pathological Basis of Disease. 3 rd edition, 1984, W. B. Saunders., Philadelphia .
  3. Mulekar S. V. Long-term follow-up study of segmental and focal vitiligo treatment by autologous, non-cultured melanocyte-keratinocyte cell transplantation. Arch Dermatol 2004, 140:1211-5.
  4. Kessel R. G. Basic Medical Histology. The Biology of Cells, Tissues, and Organs. Oxford University Press, Oxford , 1998:338-353.
  5. Mulekar S. V, Al Issa A, Al Elisa A, Asaad M. Genital vitiligo treated by autologous, non-cultured melanocyte-keratinocyte cell transplantation. Dermatol Surg, 2005, 31:1737-9.
  6. Mulekar S. V. Stable vitiligo treated by a combination of low-dose oral pulse betamethasone and autologous, non-cultured melanocyte-keratinocyte cell transplantation. Dermatol Surg. 2006, 32:536-41.
  7. Van Geel N, Ongenae K, Van der Haegen Y, Verveaet C, Naeyaert J. M. Subjective and objective evaluations of non-cultured epidermal cellular grafting for repigmentating vitiligo. Dermatol 2006, 213:23-9.
  8. Wu C. S, Lan C. C, Wang L. F, Chen G. S, Yu H. S. Effects of psoralen plus ultraviolet A irradiation on cultured epidermal cells in vitro and patients with vitilligo in vivo. Br. J. Dermat, 2007, 156:122-9.
  9. Van Geel N, Ongene K, De Mil M, Naeyaert J. M. Modified technique of autologous non cultured epidermal cell transplantation for repigmentating vitiligo. A pilot study. Dermatol Surg, 2001:27:873-6.
  10. Van Geel N, Ongene K, Naeyaert J. M. Surgical techniques for vitilligo: a review. Dermatol, 2001:202:162-6.
  11. Mulekar S. V. Long-term follow-up of 142 patients with vitiligo vulgaris treated by autologous non-cultured melanocyte-keratinocyte cell transplantation. Int J Dermatol, 2005, 44, 841-5.
  12. Mulekar S. V. Melanocyte-keratinocyte cell transplantation for stable vitiligo. Int J Dermatol 2003, 42:132-6.
  13. Olsson M. J, Juhlin L. Long-term follow-up of leucoderma patients treated with transplants of autologous cultured melanocytes, ultrathin epidermal sheets and basal cell layer suspension. Br. J Dermatol 2002, 147:893-904.
  14. Olsson M. J, Juhlin L. Leucoderma treated by transplantation of a basal cell layer enriched suspension. Br J Dermatol 1998, 138:644-8.
  15. Van Geel N. A, Ongenae K, van der Haegen Y. M, Naeyert J. M. Autologous transplantation techniques for vitiligo: how to evaluate treatment outcome. Eur J Dermatol 2004, 14:46-51.
  16. Kessel RG. Basic Medical Histology: The biology of cells, tissues and organs, 1998, Oxford University Press.

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Details on technology is available for investors and venture capitalists

Disclaimer:Bolandcell only provides information of general interest, and is not descriptive or advisory of treatment choice or diagnosis. Patient's are advised to visit their own health care provider for consultation and guidance to enable them to understand their rights and to enable them to make an informed decision and consent to intervention, if any. Go to top of page

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Boland Cell - Cell Technology - Aesthetic Biotechnology