the skin is often known as the largest organ of the human body and contains three primary layers : epidermis , dermis , and hypodermis . skin performs in different functions such as protection , sensation , heat regulation , water resistance , and many others . today , most burn surgeons use autologous skin graft technique for patients , but a challenge remains for large surface wounds and also the high risk of infections . since 1975 , epidermal cell culture to cultivate keratinocytes in vitro was introduced and keratinocyte sheets have been used for more than 20 years in clinical setting , but these methods had major problems . recently , scientists have been thinking about using the patient 's own epidermal cells to cover the burning aria . for burned patients , epidermal replacement is an important step in which keratinocytes , langerhans cells , and melanocytes are restored . several methods have been reported by scientists for the separation of epidermis from dermis over the past decades . that elastic fibers can be rapidly and specifically dissolved by trypsin which result in separation of epidermis from dermis . then , in 1942 , heat separation was introduced as a simple and reliable method by baumberger et al . and then this method were improved by reducing the time of heating in 1982 by kassis et al . some chemicals were reported to be effective to separate the epidermis from dermis such as sodium thiocyanate , sodium bromide , and ammonium hydroxide . fraulin et al . established the use of an aerosolized spray to transfer epithelial cells to a wound bed in pigs . in 2005 , a device was introduced known as the recell system in which a small piece of skin biopsy is used to make a suspension of epidermal cells . the cell suspension can then be sprayed onto the patients wound surface as a regenerative method to accelerate the healing , minimize the risk of scarring , and improve skin texture and color . this procedure can harvest enough skin cells from a 4-cm donor site to treat an area the size of 320 cm . in this study , several methods that were previously used for the separation of epidermis from dermis and making an epidermal cell suspension the best method was defined as a method in which the least toxicity and the most alive cells are produced . eleven discarded skin biopsies were sent to our laboratory from ghotboddin burn hospital , shiraz , iran . each sample was sliced into four small pieces ( 1 cm ) using scalpel 's blade and placed in separate petri - dishes . in this study , four different chemicals were tested : sodium bromide ( 2n ) and ( 4n ) , ammonium hydroxide ( 2n ) , and trypsin ( 0.05% ) . the epidermis was put in 5 ml of mentioned chemicals for 20 minutes at 37c to begin the intercellular detachment . trypsin was quickly neutralized in trypsinized sample using 5 ml of dulbecco 's modified eagle 's medium ( dmem ) ( usa ) supplemented with 10% fetal bovine serum ( fbs ) . epidermis and dermis were separated using forceps [ figure 1 ] and cells were scraped from the junctional surfaces with the sterile scalpels blade to form a cloud of cell . trypsin ( 5 ml ) was added to samples treated with nabr ( 2n ) , ( 4n ) , and nh 4 oh and placed in 37c for 20 minutes . afterward , epidermis in all samples was sliced into small pieces manually using scalpel 's blade . then , the suspensions were filtered using cell strainer ( bd falcon , usa ) and centrifuged at 1500 rpm for 10 minutes . the supernatant was removed and the pellet was resuspended in 1 ml of dmem . cells were counted using hemocytometer and number of dead and alive cells were determined using the vital stain , 1% evans blue counterstain ( uk ) , which was diluted 1 : 20 with distilled water . the top layer ( gray ) is the epidermis and the bottom layer ( white ) is the dermis for each method , two immunocytochemistry slides were prepared using the cell centrifuge . hmb-45 and cytokeratin antibodies were used to identify the presence of melanocytes and keratinocytes , respectively , for all methods . eleven discarded skin biopsies were sent to our laboratory from ghotboddin burn hospital , shiraz , iran . each sample was sliced into four small pieces ( 1 cm ) using scalpel 's blade and placed in separate petri - dishes . in this study , four different chemicals were tested : sodium bromide ( 2n ) and ( 4n ) , ammonium hydroxide ( 2n ) , and trypsin ( 0.05% ) . the epidermis was put in 5 ml of mentioned chemicals for 20 minutes at 37c to begin the intercellular detachment . trypsin was quickly neutralized in trypsinized sample using 5 ml of dulbecco 's modified eagle 's medium ( dmem ) ( usa ) supplemented with 10% fetal bovine serum ( fbs ) . epidermis and dermis were separated using forceps [ figure 1 ] and cells were scraped from the junctional surfaces with the sterile scalpels blade to form a cloud of cell . trypsin ( 5 ml ) was added to samples treated with nabr ( 2n ) , ( 4n ) , and nh 4 oh and placed in 37c for 20 minutes . afterward , epidermis in all samples was sliced into small pieces manually using scalpel 's blade . then , the suspensions were filtered using cell strainer ( bd falcon , usa ) and centrifuged at 1500 rpm for 10 minutes . the supernatant was removed and the pellet was resuspended in 1 ml of dmem . cells were counted using hemocytometer and number of dead and alive cells were determined using the vital stain , 1% evans blue counterstain ( uk ) , which was diluted 1 : 20 with distilled water . the top layer ( gray ) is the epidermis and the bottom layer ( white ) is the dermis for each method , two immunocytochemistry slides were prepared using the cell centrifuge . hmb-45 and cytokeratin antibodies were used to identify the presence of melanocytes and keratinocytes , respectively , for all methods . eleven discarded skin biopsies were sent to our laboratory from ghotboddin burn hospital , shiraz , iran . each sample was sliced into four small pieces ( 1 cm ) using scalpel 's blade and placed in separate petri - dishes . in this study , four different chemicals were tested : sodium bromide ( 2n ) and ( 4n ) , ammonium hydroxide ( 2n ) , and trypsin ( 0.05% ) . the epidermis was put in 5 ml of mentioned chemicals for 20 minutes at 37c to begin the intercellular detachment . trypsin was quickly neutralized in trypsinized sample using 5 ml of dulbecco 's modified eagle 's medium ( dmem ) ( usa ) supplemented with 10% fetal bovine serum ( fbs ) . epidermis and dermis were separated using forceps [ figure 1 ] and cells were scraped from the junctional surfaces with the sterile scalpels blade to form a cloud of cell . trypsin ( 5 ml ) was added to samples treated with nabr ( 2n ) , ( 4n ) , and nh 4 oh and placed in 37c for 20 minutes . afterward , epidermis in all samples was sliced into small pieces manually using scalpel 's blade . then , the suspensions were filtered using cell strainer ( bd falcon , usa ) and centrifuged at 1500 rpm for 10 minutes . the supernatant was removed and the pellet was resuspended in 1 ml of dmem . cells were counted using hemocytometer and number of dead and alive cells were determined using the vital stain , 1% evans blue counterstain ( uk ) , which was diluted 1 : 20 with distilled water . the top layer ( gray ) is the epidermis and the bottom layer ( white ) is the dermis for each method , two immunocytochemistry slides were prepared using the cell centrifuge . hmb-45 and cytokeratin antibodies were used to identify the presence of melanocytes and keratinocytes , respectively , for all methods . all 44 ( 11 skin biopsies and each were divided into four pieces ) samples were counted in four different fields of hemocytometer by two experts [ figure 2a ] and the means were used for the analysis . cells with blue cytoplasm represent dead cells as they can not exit vital stain due to lack of active pumps [ figure 2b ] . these cells contain the active pumps and the shapes of them are like an empty circle ( shown by arrow in figure 2b ) . ( a ) epidermal cell suspension cells that are stained with 1% evans blue stain with 100 magnification . ( b ) dead cells are stained blue ( arrow ) with 200 magnification the results of counting the total , dead , and alive cells are shown in histogram [ figure 3 ] . friedman two - way analysis of variance test was used to compare all methods generally and wilcoxon matched - pairs signed - ranks test was used to compare each pair of methods ( data not shown ) . histogram of the different methods to obtain the best method for preparation of epidermal cell suspension . ( d ) percentage of alive cells generally , there was no significant difference between total cell counts , dead cell counts , and percent of alive cells obtained from four different methods . but there was a significant difference between alive cell counts obtained from nabr ( 4n ) method compared with other methods , as it is shown in table 1 . while comparing nabr ( 4n ) method to each of nabr ( 2n ) , nh4oh , and trypsin methods , p values were 0.008 , 0.169 , and 0.182 , respectively , that all of them are significant . considering total cell count and alive cell count , nabr ( 4n ) yielded the most cells and trypsin method was after that . on the other hand , trypsin method produced the most dead cell counts , although the differences were insignificant . the last parameter which is alive cell percentage shows no significant difference between all methods , though nabr 4n and nh4oh had the greatest percentages , respectively . while using the nh4oh method , the epidermis color changed to dark and under microscopic field yielded a percent of large squamous cells with high cytoplasm to nucleus ratio . other cells obtained by this method were small round cells similar to the cells obtained with other methods . comparison of the p values of different used methods immunocytochemical staining showed that in all methods , some cells are stained positively for cytokeratin antibody and some for melanosome antibody . keratinocytes are the dominant cells in the epidermal cell suspension but a few melanocytes also exist in suspension , as shown in figure 4 . regarding this test , there was no difference between four different methods as all of them yielded keratinocytes as the dominant cells of suspension and melanocytes that were less than 5% of the cells in suspension . only one melanocyte is observed in this figure ( shown by arrow ) , which is positively stained for hmb-45 antibody ( brown cell ) burns are among the worst traumatic problems in our society that can be caused by heat , cold , electricity , chemicals , light , radiation , or friction . burns can be minor or can present a life - threatening emergency , depending on the heat 's intensity and length of exposure to the skin . twenty years ago , burns covering half of the body frequently ended in death , although today , patients with burns covering over 90% of their body can survive . despite all of the progresses that have been made in treatment of burn patients . however , these cells undergo phenotypic changes associated with contact inhibition and their surface proteins become disabled by enzymes applied in cell culture ; so , the epidermis formed by these sheets is unstable and vulnerable to blistering with minor trauma . in adition , making these sheets is time consuming and expensive . recell procedure uses trypsin method to make the cell suspension but in this paper , we showed that trypsin method is not the method of choice . instead , we concluded that nabr 4n can be used as the best method to make the cell suspension because it yields the most alive cells . the suspension produced by this method contains necessary cells required to form a normal epidermis , means keratinocytes and melanocytes . also , there is no obvious change in color or morphology of the cells treated with nabr 4n . so , we conclude that this method can be substituted with the previously used trypsin method in clinical conditions , but more investigations are needed for evaluation of any minor effect that nabr 4n may have on cells in molecular level . now , we are working on clinical effects of this method and the previously used trypsin method .
background : burn surgeons use autologous skin graft technique for patients , but a challenge remains for large surface wounds . 
 recently , a method was described which used a small piece of skin to cover a 70 times greater surface by spraying epidermal cells on injured skin . 
 we designed a comparative study to find the best method to make an epidermal cell suspension.materials and methods : eleven discarded skin samples were sent to our laboratory from ghotboddin burn hospital , shiraz . 
 each sample was sliced into four small pieces ( 1 cm2 ) and each piece was treated with a different chemical including sodium bromide ( 2n ) and ( 4n ) , ammonium hydroxide ( 2n ) , and trypsin ( 0.05% ) for 20 minutes . the epidermis and dermis 
 were separated using forceps . 
 trypsin was added to all samples ( except the trypsinized sample ) to begin the intercellular detachment . 
 afterward , epidermis was sliced into small pieces followed by filtration and centrifugation . 
 cells were counted using hemocytometer . 
 identification of keratinocytes and melanocytes was made through immunocytochemical staining for cytokeratin and melanosome antigens , respectively.results:there was a significant difference in alive cell counts comparing cells obtained from nabr ( 4n ) method to other methods . 
 considering total cell count and alive cell count , nabr ( 4n ) yielded the most cells . 
 immunocytochemical staining showed that in all methods , some cells are stained positively for cytokeratin antibody and some for melanosome antibody.conclusion:although recent papers had advised trypsin method to make a cell suspension to use for burn patients , we found that nabr ( 4n ) method yields more alive cells and less toxicity .