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Office Method for Tissue Culture
Preliminary and Short Report OFFICE METHOD FOR TISSUE CULTURE R. NEAL SCENEIDEEMAN, M.D.*
After having performed a large number of obtained from blood of healthy patients on whom dermabrasions, it occurred to me that large quan- routine blood counts were being performed. The tities of living cells were being wastefully dis- composite material was poured into plastic culcarded onto my walls, ceilings and assistants.
ture plates. The plates were coated with the
It became apparent that the successful culture of explants as described, and then placed into an such small explants might be of some practical incubator at 37° C. The plates were observed use in the future. A search of the modest tissue under the microscope at regular intervals. No
culture literature as regards the skin revealed attempt was made to control oxygen-carbon no similar attempt to culture skin by the use of dioxide tension, pH of the media or disposal of such a method. The equipment available was waste materials. minimal, but sufficient for my purposes. RESULTS
MATERIALS AND METHODS
Immediately after preparation of a plate for
The subjects available were young men and women who were undergoing the dermabrasion procedure for the improvement of scarring from acne vulgaris. The instrument used for the abrasion was a Kurtin plastic planer with a maximum speed of 15,000 RPM. The abrasive brush was of a rough diamond fraize type. All of the patients were prepared for the procedure with a ten minute
phisohex scrub and 100 mgm Demeral I. M.
Frigiderm was the local refrigerant. The explants
were taken from normal, unrefrigerated skin. Small areas were abraded as superficially as
culture, inspection under the microscope revealed numerous discs of tissue about six to ten cells in
diameter, thick in the central portions and thin
at the periphery. These discs, as mentioned, were separated by distances varying from two to three disc diameters. In the initial group of six cultures
the following were average descriptions of the culture: Day 3—early budding Day 7—moderate budding Day 10—marked budding and bridging
Day 13—colonies stopped growing and media became dessicated. possible but deep enough to produce a minimum In the second group of cultures, half of the of bleeding to insure presence of basal cells. This procedure produced a minimum of discomfort to plates were as in the first series and half of the plates had 20 units of ACTH added to each plate. the patients. The method of collecting the explants was The ACTH plates were prepared at the same time simple. The direction of spray from the rapidly and under the same conditions as were the nonrotating wheel was determined to be approxi- ACTH plates. In the plain plates the results were mately parallel to the surface of skin being as in the first series. In the ACTH group, 4 of the abraded. The plastic culture plate was held by an 6 plates became quickly overgrown with bacteria, assistant about 6 inches from the wheel and when fungi, or both. The remaining two ACTH plates a fairly even distribution of the explants was grew for 11 days until contaminents obscured the obtained on the plate, the collection was complete. field. In these two plates, the rate of growth was With a little practice the same quantity was markedly accelerated. By the fifth day marked approximated so that the explants were separated budding had occurred and by the tenth day many by a distance equal to that of about two to three bridges had formed. In one section of one of the ACTH plates a thin sheet of tissue seemed to be times their diameter. The greatest problem was the search for .a forming under the microscope. On day eleven an suitable medium and this still, of course, is the attempt was made to lift the apparent sheet off culture plate but this was unsuccessful and major problem. After attempting a variety of the media, the following is the one that was used for only bits of tissue came off. After day eleven all the ACTH plates were overgrown with conthis first study: taminents. Mitoses were not observed, but the 500 cc. Nutrient Agar density of the culture medium made such observa30 mg. Chloromycetin tion difficult. 10 cc. Human Serum Originally, the serum of the patient whose skin CONCLUSIONS was being abraded was used, but because of the difficulties in preparing the medium in small The above data is self-explanatory. Without quantities and in time for the procedure, it was great speculation, no conclusions may be drawn
decided to use pooled "normal" human serum at this time. It may be stated, however, that hmited viability of epidermal cells was maintained by this method. More and better controlled experiments are being planned.
* Dallas, Texas.
Received for publication July 3, 1961.
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4
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
DIAGRAMATIC REPRESENTATION OF CULTURE PLATES THROUGH CESSATION OF GROWTH.
oo 00 C 0O DAY
0
DAY 7
0
OO 90 DAY
3
DAY 10
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
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linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.
24. Wynn, C. H. and Iqbal, M.: Isolation of rat
skin lysosomes and a comparison with liver Path., 80: 91, 1965. and spleen lysosomes. Biochem. J., 98: lOP, 37. Nicolaides, N.: Lipids, membranes, and the 1966.
human epidermis, p. 511, The Epidermis
Eds., Montagna, W. and Lobitz, W. C. Acascopic localization of acid phosphatase in demic Press, New York. human epidermis. J. Invest. Derm., 46: 431, 38. Wills, E. D. and Wilkinson, A. E.: Release of 1966. enzymes from lysosomes by irradiation and 26. Rowden, C.: Ultrastructural studies of kerathe relation of lipid peroxide formation to tinized epithelia of the mouse. I. Combined enzyme release. Biochem. J., 99: 657, 1966. electron microscope and cytochemical study 39. Lane, N. I. and Novikoff, A. B.: Effects of of lysosomes in mouse epidermis and esoarginine deprivation, ultraviolet radiation and X-radiation on cultured KB cells. J. phageal epithelium. J. Invest. Derm., 49: 181, 25. Olson, R. L. and Nordquist, R. E.: Ultramicro-
No warranty is given about the accuracy of the copy.
Users should refer to the original published dermal cells. Nature, 216: 1031, 1967. version of1965. the material. vest. Derm., 45: 448, 28. Hall, J. H., Smith, J. G., Jr. and Burnett, S. 41. Daniels, F., Jr. and Johnson, B. E.: In prepa1967.
Cell Biol., 27: 603, 1965.
27. Prose, P. H., Sedlis, E. and Bigelow, M.: The 40. Fukuyama, K., Epstein, W. L. and Epstein, demonstration of lysosomes in the diseased J. H.: Effect of ultraviolet light on RNA skin of infants with infantile eczema. J. Inand protein synthesis in differentiated epi-
C.: The lysosome in contact dermatitis: A ration. histochemical study. J. Invest. Derm., 49: 42. Ito, M.: Histochemical investigations of Unna's oxygen and reduction areas by means of 590, 1967. 29. Pearse, A. C. E.: p. 882, Histochemistry Theoultraviolet irradiation, Studies on Melanin, retical and Applied, 2nd ed., Churchill, London, 1960.
30. Pearse, A. C. E.: p. 910, Histacheini.stry Thearetscal and Applied, 2nd ed., Churchill, London, 1960.
31. Daniels, F., Jr., Brophy, D. and Lobitz, W. C.: Histochemical responses of human skin fol-
lowing ultraviolet irradiation. J. Invest. Derm.,37: 351, 1961.
32. Bitensky, L.: The demonstration of lysosomes by the controlled temperature freezing section method. Quart. J. Micr. Sci., 103: 205, 1952.
33. Diengdoh, J. V.: The demonstration of lysosomes in mouse skin. Quart. J. Micr. Sci., 105: 73, 1964.
34. Jarret, A., Spearman, R. I. C. and Hardy, J. A.:
Tohoku, J. Exp. Med., 65: Supplement V, 10, 1957.
43. Bitcnsky, L.: Lysosomes in normal and pathological cells, pp. 362—375, Lysasames Eds., de Reuck, A. V. S. and Cameron, M. Churchill, London, 1953.
44. Janoff, A. and Zweifach, B. W.: Production of inflammatory changes in the microcirculation by cationic proteins extracted from lysosomes. J. Exp. Med., 120: 747, 1964.
45. Herion, J. C., Spitznagel, J. K., Walker, R. I. and Zeya, H. I.: Pyrogenicity of granulocyte lysosomes. Amer. J. Physiol., 211: 693, 1966.
46. Baden, H. P. and Pearlman, C.: The effect of ultraviolet light on protein and nucleic acid synthesis in the epidermis. J. Invest. Derm.,
Histochemistry of keratinization. Brit. J. 43: 71, 1964. Derm., 71: 277, 1959. 35. De Duve, C. and Wattiaux, R.: Functions of 47. Bullough, W. S. and Laurence, E. B.: Mitotic control by internal secretion: the role of lysosomes. Ann. Rev. Physiol., 28: 435, 1966. the chalone-adrenalin complex. Exp. Cell. 36. Waravdekar, V. S., Saclaw, L. D., Jones, W. A. and Kuhns, J. C.: Skin changes induced by
Res., 33: 176, 1964.
After having performed a large number of obtained from blood of healthy patients on whom dermabrasions, it occurred to me that large quan- routine blood counts were being performed. The tities of living cells were being wastefully dis- composite material was poured into plastic culcarded onto my walls, ceilings and assistants.
ture plates. The plates were coated with the
It became apparent that the successful culture of explants as described, and then placed into an such small explants might be of some practical incubator at 37° C. The plates were observed use in the future. A search of the modest tissue under the microscope at regular intervals. No
culture literature as regards the skin revealed attempt was made to control oxygen-carbon no similar attempt to culture skin by the use of dioxide tension, pH of the media or disposal of such a method. The equipment available was waste materials. minimal, but sufficient for my purposes. RESULTS
MATERIALS AND METHODS
Immediately after preparation of a plate for
The subjects available were young men and women who were undergoing the dermabrasion procedure for the improvement of scarring from acne vulgaris. The instrument used for the abrasion was a Kurtin plastic planer with a maximum speed of 15,000 RPM. The abrasive brush was of a rough diamond fraize type. All of the patients were prepared for the procedure with a ten minute
phisohex scrub and 100 mgm Demeral I. M.
Frigiderm was the local refrigerant. The explants
were taken from normal, unrefrigerated skin. Small areas were abraded as superficially as
culture, inspection under the microscope revealed numerous discs of tissue about six to ten cells in
diameter, thick in the central portions and thin
at the periphery. These discs, as mentioned, were separated by distances varying from two to three disc diameters. In the initial group of six cultures
the following were average descriptions of the culture: Day 3—early budding Day 7—moderate budding Day 10—marked budding and bridging
Day 13—colonies stopped growing and media became dessicated. possible but deep enough to produce a minimum In the second group of cultures, half of the of bleeding to insure presence of basal cells. This procedure produced a minimum of discomfort to plates were as in the first series and half of the plates had 20 units of ACTH added to each plate. the patients. The method of collecting the explants was The ACTH plates were prepared at the same time simple. The direction of spray from the rapidly and under the same conditions as were the nonrotating wheel was determined to be approxi- ACTH plates. In the plain plates the results were mately parallel to the surface of skin being as in the first series. In the ACTH group, 4 of the abraded. The plastic culture plate was held by an 6 plates became quickly overgrown with bacteria, assistant about 6 inches from the wheel and when fungi, or both. The remaining two ACTH plates a fairly even distribution of the explants was grew for 11 days until contaminents obscured the obtained on the plate, the collection was complete. field. In these two plates, the rate of growth was With a little practice the same quantity was markedly accelerated. By the fifth day marked approximated so that the explants were separated budding had occurred and by the tenth day many by a distance equal to that of about two to three bridges had formed. In one section of one of the ACTH plates a thin sheet of tissue seemed to be times their diameter. The greatest problem was the search for .a forming under the microscope. On day eleven an suitable medium and this still, of course, is the attempt was made to lift the apparent sheet off culture plate but this was unsuccessful and major problem. After attempting a variety of the media, the following is the one that was used for only bits of tissue came off. After day eleven all the ACTH plates were overgrown with conthis first study: taminents. Mitoses were not observed, but the 500 cc. Nutrient Agar density of the culture medium made such observa30 mg. Chloromycetin tion difficult. 10 cc. Human Serum Originally, the serum of the patient whose skin CONCLUSIONS was being abraded was used, but because of the difficulties in preparing the medium in small The above data is self-explanatory. Without quantities and in time for the procedure, it was great speculation, no conclusions may be drawn
decided to use pooled "normal" human serum at this time. It may be stated, however, that hmited viability of epidermal cells was maintained by this method. More and better controlled experiments are being planned.
* Dallas, Texas.
Received for publication July 3, 1961.
3
4
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
DIAGRAMATIC REPRESENTATION OF CULTURE PLATES THROUGH CESSATION OF GROWTH.
oo 00 C 0O DAY
0
DAY 7
0
OO 90 DAY
3
DAY 10
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
94
linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.
24. Wynn, C. H. and Iqbal, M.: Isolation of rat
skin lysosomes and a comparison with liver Path., 80: 91, 1965. and spleen lysosomes. Biochem. J., 98: lOP, 37. Nicolaides, N.: Lipids, membranes, and the 1966.
human epidermis, p. 511, The Epidermis
Eds., Montagna, W. and Lobitz, W. C. Acascopic localization of acid phosphatase in demic Press, New York. human epidermis. J. Invest. Derm., 46: 431, 38. Wills, E. D. and Wilkinson, A. E.: Release of 1966. enzymes from lysosomes by irradiation and 26. Rowden, C.: Ultrastructural studies of kerathe relation of lipid peroxide formation to tinized epithelia of the mouse. I. Combined enzyme release. Biochem. J., 99: 657, 1966. electron microscope and cytochemical study 39. Lane, N. I. and Novikoff, A. B.: Effects of of lysosomes in mouse epidermis and esoarginine deprivation, ultraviolet radiation and X-radiation on cultured KB cells. J. phageal epithelium. J. Invest. Derm., 49: 181, 25. Olson, R. L. and Nordquist, R. E.: Ultramicro-
No warranty is given about the accuracy of the copy.
Users should refer to the original published dermal cells. Nature, 216: 1031, 1967. version of1965. the material. vest. Derm., 45: 448, 28. Hall, J. H., Smith, J. G., Jr. and Burnett, S. 41. Daniels, F., Jr. and Johnson, B. E.: In prepa1967.
Cell Biol., 27: 603, 1965.
27. Prose, P. H., Sedlis, E. and Bigelow, M.: The 40. Fukuyama, K., Epstein, W. L. and Epstein, demonstration of lysosomes in the diseased J. H.: Effect of ultraviolet light on RNA skin of infants with infantile eczema. J. Inand protein synthesis in differentiated epi-
C.: The lysosome in contact dermatitis: A ration. histochemical study. J. Invest. Derm., 49: 42. Ito, M.: Histochemical investigations of Unna's oxygen and reduction areas by means of 590, 1967. 29. Pearse, A. C. E.: p. 882, Histochemistry Theoultraviolet irradiation, Studies on Melanin, retical and Applied, 2nd ed., Churchill, London, 1960.
30. Pearse, A. C. E.: p. 910, Histacheini.stry Thearetscal and Applied, 2nd ed., Churchill, London, 1960.
31. Daniels, F., Jr., Brophy, D. and Lobitz, W. C.: Histochemical responses of human skin fol-
lowing ultraviolet irradiation. J. Invest. Derm.,37: 351, 1961.
32. Bitensky, L.: The demonstration of lysosomes by the controlled temperature freezing section method. Quart. J. Micr. Sci., 103: 205, 1952.
33. Diengdoh, J. V.: The demonstration of lysosomes in mouse skin. Quart. J. Micr. Sci., 105: 73, 1964.
34. Jarret, A., Spearman, R. I. C. and Hardy, J. A.:
Tohoku, J. Exp. Med., 65: Supplement V, 10, 1957.
43. Bitcnsky, L.: Lysosomes in normal and pathological cells, pp. 362—375, Lysasames Eds., de Reuck, A. V. S. and Cameron, M. Churchill, London, 1953.
44. Janoff, A. and Zweifach, B. W.: Production of inflammatory changes in the microcirculation by cationic proteins extracted from lysosomes. J. Exp. Med., 120: 747, 1964.
45. Herion, J. C., Spitznagel, J. K., Walker, R. I. and Zeya, H. I.: Pyrogenicity of granulocyte lysosomes. Amer. J. Physiol., 211: 693, 1966.
46. Baden, H. P. and Pearlman, C.: The effect of ultraviolet light on protein and nucleic acid synthesis in the epidermis. J. Invest. Derm.,
Histochemistry of keratinization. Brit. J. 43: 71, 1964. Derm., 71: 277, 1959. 35. De Duve, C. and Wattiaux, R.: Functions of 47. Bullough, W. S. and Laurence, E. B.: Mitotic control by internal secretion: the role of lysosomes. Ann. Rev. Physiol., 28: 435, 1966. the chalone-adrenalin complex. Exp. Cell. 36. Waravdekar, V. S., Saclaw, L. D., Jones, W. A. and Kuhns, J. C.: Skin changes induced by
Res., 33: 176, 1964.