- Email: [email protected]
Novel method of skin substitution in plastic surgery
Burns (1990), 16, 71-72 Printed in Great Britain
71
letters to the Editor
Novel method of skin substitution plastic surgery
in
Dear Sir, In common with other laboratories in the UK, we have for some years been engaged in the culture of human skin epithelial cells using the technique of Rheinwald and Green. As is well known, this method requires the presence of feeder layers of mouse embryo fibroblast (3T3) cells and a complex medium with the addition of foetal bovine serum (FBS) together with hormones and other additives. We were, therefore, surprised to read the paper of Khalfan et al. (Bums, 15,335-7) in which it is reported that confluent epithelial cultures may be obtained in 7 days from collagenase-treated skin biopsies without the use of feeder cell layers, and in Minimal Essential Medium (MEM) supplemented with 10% FBS alone. The method is also reported to require a seeding density of cells of only one fifth or less of that commonly employed in the Rheinwald and Green system. Furthermore such cultures appear to be obtainable from an 80 year-old patient whose bums healed as a result, it is claimed, of the application of a suspension of the cultured cells directly onto the wound. In our experience it has not proved possible to prepare confluent sheets of keratinocytes suitable for grafting in less than 12 days from very young children. In patients over 65 years of age it is often very difficult to obtain confluent cultures at all. De Luca et al. in the same issue of Burns also make this observation. It is not clear from the paper what steps the authors have taken to prevent the growth of fibroblasts under their culture conditions. Perhaps the authors would furnish exact details of their methods and cell preparations, including photographs of the cells so that other investigators may seek to confirm the apparent advantages of their culture systems. V. A. Shakespeare,P. G. Shakespeare,Laiig Laboratory for Bum Injury Investigation, Odstock Hospital, Salisbury, Wilts, UK.
Dear Sir, I would like to comment on the paper by Khalfan et al. (Bums 1989 15, 335-7). These authors apparently made no effort to avoid fibroblast contamination of their ‘epithelial cells from split thickness skin. Indeed their use of collagenase would more efficiently release fibroblasts and endothelial cells from the dermis, than keratinocytes from the epidermis. Unless very high numbers 2 x 105/cm2) of keratinocytes (Eisinger et al. 1979) are seeded into flasks without feeder layers the cells either fail to proliferate or are outgrown by contaminating fibroblasts. These authors seeded low nurn0 19% ButteMrorth & Co (Publishers) Ltd 0305-4179/90/010071~2
bers of cells (2 x 102/crn”). Also I cannot understand how such rapid keratinocyte growth is achieved without using medium supplements such as cholera toxin, and why the cells exhibited monolayer growth without stratification in the absence of calcium modulation. The authors did not report using methods to confirm the epithelial origin of the cultured cells. The cell growth characteristics observed and methods used would suggest that the majority of cells cultured were not keratinocytes, but possibly fibroblasts. If that is the case, the stimulation of autologous reepithelialisation by cultured fibroblasts may be an interesting phenomenon, however, double blind controlled trials on more than two patients would be required to confirm this effect. J. N. Keamey, Director Yorkshire Regional Tissue Bank, Pinderfields General Hospital, Wakefield WF 1 4DG, UK.
Dear Sir, It was with some interest that we read ‘Novel method of skin substituion in plastic surgery’ by H. A. Khalfan et al. (Bums, 1989,15,335-7). However we feel that the methods described require further explanation. In our experience an average cell yield of 2 x 106/crnz of tissue is obtained when extracting epidermal cells from split thickness skin grafts with trypsin. We have no experience with collagenase but find the 2 x 10’ cells/cm2 yield stated to be very low. Epidermal cells (keratinocytes) have been estimated to have a cell doubling time of 26-30 h in vih-0 (Kitano et al., (1983), Langdon and McGuire (1986)). For the 2 x lo5 cells obtained to produce a confluent 75 cm’ flask within 7-8 days, every cell would have to be capable of growth i.e. a plating efficiency of 100%. This is unlikely as any population of keratinocytes must include differentiating cells which loose their ability to divide. Green et al., (1975, 1979), showed that when using simple media, similar to that described, the plating efficiency of cells derived from biopsies was always less than 10%. and less than 1% in elderly patients. A more refined and complex medium (O’Connor et al., (1984)) is used by ourselves and other groups for the production of cultured epidermal grafts. Using this we have successfully produced flasks of confluent keratinocytes within 6 days. This required an inoculum of 4 x lo6 cells from a 10 month old patient. In elderly patients, such a good rate of growth is seldom seen. From a 71 year-old, cells with a plating efficiency of 2% required 13 days to reach confluence from an inoculum of 3.4 x lo6 cells. Furthermore, irradiated fibroblasts are required to suppress overgrowth of the keratinocytes by autologous fibroblasts, which will grow readily even in simple media. Human fibroblasts are
71
letters to the Editor
Novel method of skin substitution plastic surgery
in
Dear Sir, In common with other laboratories in the UK, we have for some years been engaged in the culture of human skin epithelial cells using the technique of Rheinwald and Green. As is well known, this method requires the presence of feeder layers of mouse embryo fibroblast (3T3) cells and a complex medium with the addition of foetal bovine serum (FBS) together with hormones and other additives. We were, therefore, surprised to read the paper of Khalfan et al. (Bums, 15,335-7) in which it is reported that confluent epithelial cultures may be obtained in 7 days from collagenase-treated skin biopsies without the use of feeder cell layers, and in Minimal Essential Medium (MEM) supplemented with 10% FBS alone. The method is also reported to require a seeding density of cells of only one fifth or less of that commonly employed in the Rheinwald and Green system. Furthermore such cultures appear to be obtainable from an 80 year-old patient whose bums healed as a result, it is claimed, of the application of a suspension of the cultured cells directly onto the wound. In our experience it has not proved possible to prepare confluent sheets of keratinocytes suitable for grafting in less than 12 days from very young children. In patients over 65 years of age it is often very difficult to obtain confluent cultures at all. De Luca et al. in the same issue of Burns also make this observation. It is not clear from the paper what steps the authors have taken to prevent the growth of fibroblasts under their culture conditions. Perhaps the authors would furnish exact details of their methods and cell preparations, including photographs of the cells so that other investigators may seek to confirm the apparent advantages of their culture systems. V. A. Shakespeare,P. G. Shakespeare,Laiig Laboratory for Bum Injury Investigation, Odstock Hospital, Salisbury, Wilts, UK.
Dear Sir, I would like to comment on the paper by Khalfan et al. (Bums 1989 15, 335-7). These authors apparently made no effort to avoid fibroblast contamination of their ‘epithelial cells from split thickness skin. Indeed their use of collagenase would more efficiently release fibroblasts and endothelial cells from the dermis, than keratinocytes from the epidermis. Unless very high numbers 2 x 105/cm2) of keratinocytes (Eisinger et al. 1979) are seeded into flasks without feeder layers the cells either fail to proliferate or are outgrown by contaminating fibroblasts. These authors seeded low nurn0 19% ButteMrorth & Co (Publishers) Ltd 0305-4179/90/010071~2
bers of cells (2 x 102/crn”). Also I cannot understand how such rapid keratinocyte growth is achieved without using medium supplements such as cholera toxin, and why the cells exhibited monolayer growth without stratification in the absence of calcium modulation. The authors did not report using methods to confirm the epithelial origin of the cultured cells. The cell growth characteristics observed and methods used would suggest that the majority of cells cultured were not keratinocytes, but possibly fibroblasts. If that is the case, the stimulation of autologous reepithelialisation by cultured fibroblasts may be an interesting phenomenon, however, double blind controlled trials on more than two patients would be required to confirm this effect. J. N. Keamey, Director Yorkshire Regional Tissue Bank, Pinderfields General Hospital, Wakefield WF 1 4DG, UK.
Dear Sir, It was with some interest that we read ‘Novel method of skin substituion in plastic surgery’ by H. A. Khalfan et al. (Bums, 1989,15,335-7). However we feel that the methods described require further explanation. In our experience an average cell yield of 2 x 106/crnz of tissue is obtained when extracting epidermal cells from split thickness skin grafts with trypsin. We have no experience with collagenase but find the 2 x 10’ cells/cm2 yield stated to be very low. Epidermal cells (keratinocytes) have been estimated to have a cell doubling time of 26-30 h in vih-0 (Kitano et al., (1983), Langdon and McGuire (1986)). For the 2 x lo5 cells obtained to produce a confluent 75 cm’ flask within 7-8 days, every cell would have to be capable of growth i.e. a plating efficiency of 100%. This is unlikely as any population of keratinocytes must include differentiating cells which loose their ability to divide. Green et al., (1975, 1979), showed that when using simple media, similar to that described, the plating efficiency of cells derived from biopsies was always less than 10%. and less than 1% in elderly patients. A more refined and complex medium (O’Connor et al., (1984)) is used by ourselves and other groups for the production of cultured epidermal grafts. Using this we have successfully produced flasks of confluent keratinocytes within 6 days. This required an inoculum of 4 x lo6 cells from a 10 month old patient. In elderly patients, such a good rate of growth is seldom seen. From a 71 year-old, cells with a plating efficiency of 2% required 13 days to reach confluence from an inoculum of 3.4 x lo6 cells. Furthermore, irradiated fibroblasts are required to suppress overgrowth of the keratinocytes by autologous fibroblasts, which will grow readily even in simple media. Human fibroblasts are