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Dermal-epidermal Separation, Part II: Enzymatic Digestion

April 27, 2017 | Contact Author | By: Ying Zou, Shanghai Skin Disease Hospital, Shanghai and University of California, San Francisco, and Howard I. Maibach, M.D., University of California, San Francisco
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Keywords: enzyme | skin | research | digestion | DEJ | trypsin | collagenase | pancreatin | pronase | thermolysin

Abstract: Readers have expressed interest in understanding the most efficient and reliable methods to separate the epidermis and dermis as an important technique for basic skin investigation. This second installment in a three-part series reviews separation via enzyme digestion.

The first column in this skin care and cosmetic research series on dermal-epidermal separation methods reviewed advantages and disadvantages of acid, alkalis and neutral salts for this purpose. This second column reviews optimum dermal-epidermal separation methods utilizing enzyme digestion options.

Several enzymes can separate the epidermis from the dermis. They do so at the dermal-epidermal junction (DEJ) by digesting specific structures.

Trypsin: Basement membrane elastic fibers play an important part in anchoring the epidermis to the underlying tissue. Medawer observed human skin incubated in trypsin and found it possible to disengage the epidermis in the form of an intact sheet. Klein also separated mouse epidermis using trypsin and elastase.

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Editor's Note: This article has been adapted with permission from Arch Derm Res (in review).

The first column in this skin care and cosmetic research series on dermal-epidermal separation methods reviewed advantages and disadvantages of acid, alkalis and neutral salts for this purpose. This second column reviews optimum dermal-epidermal separation methods utilizing enzyme digestion options.

Enzymatic Digestion

Several enzymes can separate the epidermis from the dermis (see Table 2). They do so at the dermal-epidermal junction (DEJ) by digesting specific structures.

Trypsin: Basement membrane elastic fibers play an important part in anchoring the epidermis to the underlying tissue. Medawer7 observed human skin incubated in trypsin and found it possible to disengage the epidermis in the form of an intact sheet. Klein8 also separated mouse epidermis using trypsin and elastase.

Shorter incubation was required for elastase than trypsin, and the ease or difficulty of separation was directly related to the total amount and dimensions of skin samples incubated. Trypsin separation remains widely used and is reported to be less damaging to keratinocytes than other physical and chemical procedures.9

Separation predominantly occurs between basal and suprabasal cells by disruption of desmosomes, which may cause basal cells to remain loosely attached to the basement membrane.10 Trypsinization causes loosening of the keratinocytes intercellular connections, and vigorous washing of the isolated epidermal sheet removes contaminating dermal fibroblasts and dissociates keratinocytes. Slight modifications of trypsinization can also separate the suprabasal layer.

Pancreatin: Becker et al.11 described the pancreatic enzyme method; results resembled those of Medawer7 obtained with trypsin. Fan,12 however, showed crystalline and purified trypsin-separated epidermis using equal or smaller amounts than pancreatin. Purified trypsin freed the epidermis the most efficiently.

Pronase: Einbinde et al.13 compared pronase, a broad-spectrum proteinase, and the relative digestive action with five other proteolytic enzymes: trypsin, collagenase, pancreatic elastase, fungal elastase and keratinase (see Table 2). The endpoint of digestion was similar. Optimal enzyme concentration, pH and time varied.

Pronase was extremely active although its proteolytic and elastolytic activity was not significantly different from the others. Separation at the DEJ was followed by acantholysis and subsequently, the loss of dermal appendages and fibrous elements. Mature keratin structures or basement membrane structures did not appear altered.

Dispase: Dispase is a bacterial neutral protease obtained from Bacillus polymyxa culture filtrate, whose cytotoxicity is low. Kitano et al. isolated epidermal sheets in vivo without dissociating cells.14 After 24 hr, epidermal sheets from human skin treated with 500 and 1,000 U/mL of dispase were easily peeled from the dermis, and its undersurface retained rete ridges.

Electron microscopy showed the basal surface composed of cells with numerous slender villi and cytoplasmic projections. Although intercellular spaces of spinous as well as basal layers were wide, desmosomes were intact with their accompanying tonofilaments.

Einbinder et al.13 compared the digestive action on skin of six proteolytic enzymes. Optimal enzyme concentration, pH and time varied. They ascertained that skin must be incubated in enzyme solutions for consistent separation, since topical application in vivo or in vitro, as well as intracutaneous injection in vivo, did not produce consistent results.

Takahashi et al.15 compared trypsin with dispase isolation using rat skin. Quantitative estimations were made of the yield, viability and number of attached epidermal cells. Dispase was superior to trypsin, especially for cell yield, which was four times higher; 1.1 × 107 vs. 0.24 × 107 cells/10 cm2 of skin, respectively. Viability and rate of attachment of recovered epidermal cells were also higher with dispase than trypsin.

Thermolysin caused detachment in the basal membrane zone while trypsin caused chaotic segmentation and digestion of the keratinocytes.

Thermolysin: Thermolysin is isolated from filtrates of Bacillus thermoproteolyticus cultures. Walzer et al.16 reported the human epidermis could easily be separated from the dermis following incubation at 4ºC for 1 hr in a solution containing 250-500 ug/mL thermolysin. Light and electron microscopy revealed dermal-epidermal separation occurred at the basement membrane between sites of bullous pemphigoid antigen and laminin, and the hemidesmosomes were selectively disrupted. They concluded thermolysin treatment appears to be a useful alternative to trypsin for dermal-epidermal separation.

Gragnani et al. also compared keratinocyte isolation methods using trypsin and thermolysin.17 In histological evaluations, thermolysin caused detachment in the basal membrane zone, while trypsin caused chaotic segmentation and the digestion of keratinocytes. Contamination was not present with fibroblasts in the thermolysin group but it was in the trypsin group. The number of keratinocyte colonies in the thermolysin group was also significantly greater than with trypsin.

The final column in this series will focus on heat and mechanical separation methods—and offer recommendations for choosing the appropriate method.

References

  1. PB Medawar, Sheets of pure epidermal epithelium from human skin, Nature 148 783 (1941)
  2. M Klein and LR Fitzgerald, Enzymatic separation of intact epidermal sheets from mouse skin, J Invest Dermatol 39 111-114 (1962)
  3. K Ma, Biochemistry and Physiology of the Skin, Oxford University Press, New York (1983)
  4. SC Liu and M Karasek, Isolation and growth of adult human epidermal keratinocytes in cell-culture, J Invest Dermatol 71 157-162 (1978)
  5. SW Becker, TB Fitzpatrick and H Montgomery, Human melanogenesis: Cytology and histology of pigment cells (melanodendrocytes), Arch Dermatol and Syphilol 65 511-523 (1952)
  6. J Fan, Epidermal separation with purified trypsin, J Invest Dermatol 30 271 (1958)
  7. JM Einbinde, RA Walzer and I Mandl, Epidermal-dermal separation with proteolytic enzymes, J Invest Dermatol 46 492-504 (1966)
  8. Y Kitano and N Okada, Separation of the epidermal sheet by dispase, Brit J Derm 108 555-560 (1983)
  9. H Takahashi, K Sano, K Yoshizato, N Shioya and K Sasaki, Comparative studies on methods of isolating rat epidermal-cells, Ann Plas Surg 14 258-266 (1985)
  10. C Walzer, M Benathan and E Frenk, Thermolysin treatment: A new method for dermo-epidermal separation, J Invest Derm 92 78-81 (1989)
  11. A Gragnani, CS Sobral and LM Ferreira, Thermolysin in human cultured keratinocyte isolation, Braz J Biol 67 105-109 (2007)
 

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Table 2. Enzymatic Separation

Table 2. Enzymatic Separation

Several enzymes can separate the epidermis from the dermis. They do so at the dermal-epidermal junction (DEJ) by digesting specific structures.

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