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Separation of epidermal cells by density gradient centrifugation on a continuous colloidal silica (Percoll) gradient
ANALYTICAL
Separation
MICHAEL
119, 2466252 ( 1Y82)
BIOCHEMISTRY
of Epidermal Cells by Density Gradient Centrifugation Continuous Colloidal Silica (Percoll) Gradient’
A. GOLDENHERSH, Dermatology
Laboratory,
ROBERT Memorial New
A. GOOD, NURUL Sloan-Kettering York, New York
Cancer 10021
on a
H. SARKAR, AND BIJAN SAFA? Center,
I275
York
Avenue,
Received April 28, 1981 The epidermis, which is composed mainly of keratinocytes, undergoes continual proliferation and differentiation. Beginning as a basal cell, the keratinocyte matures and ascends through the spinous cell, granular cell, and horny cell layers. To study keratinocyte differentiation in vitro, it is necessary to separate the keratinocytes at their successive stages of maturation. A method for separation of epidermal keratinocytes is reported here. The method utilizes centrifugation on a continuous colloidal silica (Percoll) density gradient, which separates the keratinocytes according to their inherent density. The resulting arrangement of the keratinocytes in the centrifuge tube generally corresponds to their arrangement in situ, with basal cells at the bottom and the more differentiated cells above. There is a continuous decrease in the nuclear:cytoplasm ratio of keratinocytes as the cells mature. Using this technique, it was shown that the decrease in the nuclear:cytoplasm ratio corresponds to decreasing density of keratinocytes as they mature. Using hematoxylin and eosin staining and electron microscopic studies, it was shown that relatively pure fractions of basal cells, spinous cells, and granular cells are obtained by this method. The method is simple and reproducible, and should greatly facilitate biochemical analyses of the various maturation steps of epidermal keratinocytes.
The recognized morphologic stages in keratinocyte differentiation are the basal, spinous, granular, and horny cell layers. To study the biochemical events of keratinocyte differentiation, which parallel these morphological changes, it has been necessary to develop a technique which can separate keratinocytes in suspension at different stages of differentiation. While several techniques have been reported (l-9) none has become standardized for general use. Thus, there is a need for development of a reproducible method for fractionation of keratinocytes which is practical enough to be easily adapted in any laboratory. To develop such a method we evaluated ’ Aided by the USPHS Grants CA-08747, AE-I 1843, Witty’s Fund, Chesebrough-Ponds, Handmacher Foundation, Dickson’s Fund, Robert S. Sinn’s Fund, and J. Mirsky’s Fund. * To whom correspondence should be addressed. 0003-2697/82/020246-07$02.00/O Copyright 0 1982 by Academic Press. Inc. All rights of reproduction in any form reserved.
the known features of epidermal keratinocytes. As the keratinocytes differentiate and ascend through the various layers of the epidermis, they become larger and their nuclear:cytoplasm ratio decreases (5,6,8). In general, a close relationship between the nuclear:cytoplasm ratio of a given mammalian cell and its density prevails (i.e., the higher the ratio, the higher the density) (10,ll). Accordingly, it should be possibleto separate keratinocytes at different stages of maturation using a density gradient. We report here the use of density gradient centrifugation in a continuous, sigmoidal gradient of colloidal silica polyvinylpyrrolidone (CSP) for fractionation of keratinocytes. MATERIALS
AND
METHODS
Preparation of Epidermal Cell Suspensions. Previously reported techniques ( 12,13) 246
DENSITY
GRADIENT
SEPARATION
with some modifications were used to prepare epidermal cell suspensions. Meon Chase guinea pigs of mixed sex ranging 2-4 months in age served as a source of skin. Thin split thickness sections of skin were obtained using a Davol/Simon Dermatome (Davol, Providence, R. I.). These sections were then cut into smaller pieces (approx 1.5 X 0.5 cm) and incubated dermal side down on a thin layer of 0.25% solution of lyophilized trypsin (Millipore Corp., Freehold, N. J.) in a petri dish for 90 min at 37°C. The trypsin was prepared by mixing 0.25 g trypsin in 100 ml phosphate buffered saline without Ca or Mg (Walker Laboratory, Rye, N. Y.) and adjusting pH to 7.8. Following incubation, the skin sections were removed from the trypsin and the epidermis was teased from the dermis using small forceps. The separated epidermal sheets were placed in a petri dish containing Hanks Balanced Salt Solution (HBSS) (Walker Laboratory, N. Y.) until all sections were separated, after which they were transferred to a petri dish containing 0.02% solution of DNase I (Sigma Chemical Co., St. Louis, MO.). While in the DNase solution, both sides of the epidermal sheets were vigorously stirred with a glass stirring rod for 10 min. HBSS (10 ml) was then added to the petri dish and the entire suspension of cells and epidermal sheets was pipetted onto a 0.2-mm pore size sterile wire mesh (Estey Wire Co., N. J.). The resulting suspension of epidermal cells was then washed with HBSS three times for 10 min each. Preparation of gradient media. Readymade solution of CSP (Percoll) was purchased from Pharmacia Fine Chemicals (Uppsala, Sweden). CSP (9 ml) was mixed with 1 ml HBSS 10 times (GIBCO, Grand Isiand, N. Y.) and further diluted once with the necessary amount of HBSS to achieve a density of 1.066 as measured by refractometry (Abbe-3L Refractometer; Bausch & Lomb, Rochester, N. Y.). The amount of diluent needed to achieve the appropriate
OF
EPIDERMAL
CELLS
241
starting density varies according to the initial density of CSP. Fractionation of cells. Approximately 2030 million unfractionated epidermal cells (UFEC) were suspended in 8 ml of CSP solution. The cell suspension was centrifuged in a 15-ml Corex tube (Fisher Scientific, Pittsburgh, Pa.) in a Beckman J-21 highspeed centrifuge (Beckman Instruments, Palo Alto, Calif.) equipped with a JA-20 rotor at 13,000 rpm (approx 20,OOOg) for 30 min at 5°C. After centrifugation, cells were dispersed throughout the tube. In two areas, cells were sufficiently concentrated to form bands. One band was in the upper portion of the fluid column and the other in the lower portion. Following centrifugation a thin-walled metal tube (l/16 in. o.d.) was delicately inserted to near the bottom of the centrifuge tube. Seven l-ml and one 0.5-ml fractions were collected through the metal tube using a l-ml tuberculin syringe attached to a three-way stopcock. The remaining 0.5 ml of CSP was accounted for by a small portion of CSP which remained in the bottom of the tube. In a second series of experiments, the cells were collected according to the grossly visible banding evident after centrifugation. In these experiments, three fractions were collected from the bottom. The first fractions (2 ml), containing the epidermal cells with higher density (HDEC), included the cells in and below the lower band. The second fraction, containing epidermal cells with intermediate density (IDEC), included 3 ml of CSP with unbanded cells dispersed throughout the middle of the tube. The last fraction collected, including the cells in and above the upper band, was 2.5 ml and contained epidermal cells with lower density (LDEC). Each fraction was washed three times in HBSS for 10 min and resuspended in HBSS for counting. Analysis of the CSP gradient. A tube containing CSP solution of the same starting
248
GOLDENHERSH
density was centrifuged and fractionated in the same manner, but without cells. The density of each fraction was measured by refractometry. Analysis of cell fractions. Unfractionated epidermal cells and cells in each fraction were counted. To standardize our counting method we used morphology as well as size of unstained cells. Thus, three cell types could be distinguished: ( 1) Round cells which measured iess than 13 pm had relatively large nuclei, and were found in highest proportions in the denser portion of the gradient; (2) small polygonal cells which were concentrated in the middle and upper portion of the gradient measured up to 25 I.crn, and had relatively smaller nuclei and large cytoplasm; and (3) large polygonal cells which measured more than 25 pm, and were found mostly in the upper fractions of the gradient. A similar categorization of keratinocytes has been previously noted ( 14). Cells were counted in a hemocytometer using oculomicrometry for cell measurements. Cell viability in each fraction was determined by the trypan blue dye exclusion technique. In another series of cell separations, cytocentrifuge preparations were made with cells from each of the eight fractions. The
1.025 ,
LDEC _
IDEC I.?56 - I.?75 !
HDEC r
I.110
ET AL. TABLE MEAN STRATING POLYGONAL
RESULTS
OF FOUR
1 EXPERIMENTS
DEMON-
THE PERCENTAGE OF ROUND CELLS, SMALL CELLS, AND LARGE POLYGONAL CELLS IN
EACH OF EIGHT FRACTIONS COLLECTED AFTER CENTRIFUGATION OF UNFRACTIONATED EPIDERMAL CELLS IN CSP
Round cell (%) Unfrac. 1 2 3 4 5 6 I 8
56 I 12 16 22 46 76 92 90
Small polygonal (o/o) 36 93 81 15 70 52 23 8 10
Large polygonal (%I 8 0 7 9 8 2 1 0 0
Nofe. The less dense polygonal cells predominate in the upper fractions while the more dense round cells are more numerous in the lower fractions.
cells were air-dried on glass slides and stained with hematoxylin (H) and eosin (E), and the percentage of basal, spinous, granular, and horny cells was counted. Previously described criteria were used for determination of basal, spinous, granular and horny cells (l-3,5-8). Transmission electron microscopy was also used for examination of cells from each of the eight fractions.
L-----4
RESULTS
Ii,
1.02
4
1.04
1 13
1.06
IlO
1.06
8
’
1.10
’
’
1.12
Density
FIG. 1. The continuous, nearly linear, density gradient of consecutive fractions collected after centrifugation of CSP. Fraction 1, the least dense, is from the top of the centrifuge tube; fraction 8, the most dense, is from the bottom.
A continuous sigmoidal gradient is produced following centrifugation of the CSP solution (Fig. 1). The range of density (g/ ml) is from 1.025 in fraction 1 to 1.110 in fraction 8. Repeated determinations demonstrated virtually no variation in the densities of each fraction. Four experiments were done wherein the percentage of round, small polygonal and large polygonal cells were counted in eight fractions. The mean results are shown in
DENSITY
GRADIENT
SEPARATION
Table 1. Fractions 7 and 8, which are the most dense, contained over 90% round cells. The percentage of round cells continually decreased with decreasing density, such that fraction 1 had less than 10% round cells. In contrast, fraction 1, which is the least dense, contained over 90% polygonal cells. The percentage of polygonal cells decreased with increasing density such that fraction 8 contained less than 10% polygonal cells. In addition, a transition in the cell diameter of the round cells at different densities was observed. The round cells in the lower (more dense) fractions were frequently as small as 8-10 pm, while the round cells in the upper (less dense) fractions were usually larger ( 12- 13 pm). Similarly, the large polygonal cells were found almost exclusively in the upper half of the density gradient. Analogous data for the second series of four experiments are shown in Table 2. Cells were fractionated according to the cell bands (three fractions). The trend of high percentage of round cells in the high density
TABLE
2
RESULTS OF FOUR EXPEIUMENTS IN WHICH CELLS WERE COLLECTED IN THREE FRACTIONS
MEAN
Unfrac.
Round cell (70)
Small polygonal (W)
61
32
1
I7
13
IO
61
35
4
91
3
0
OF
EPIDERMAL
CELLS
249
FIG. 2. Photomicrograph of H and E stained smear of cells from fraction 2 demonstrating the more differentiated, low density cells with relatively large cytoplasm and small or absent nucleus. This fraction, along with fraction I. contains more granular and horny cells than the other fractions. Magnification X32.
zone (lower band) and high percentage of polygonal cells in the low density zone (upper band) was maintained. The intermediate density area (IDEC) contained intermediate proportions of round and polygonal cells. The total number of cells recovered after fractionation averaged 50-65% of the initial cell load. The association of cells in the low density zone and the high density zone, each of which contained a cell band, was generally 1.5 times the proportion in the intermediate density area.
Large polygonal (o/o)
Low
density Intermediate density High density
Nore. Low-density epidermal cells (LDEC) contain the cells in and above the upper band. Intermediatedensity epidermal cells (IDEC) contain cells between the bands. High-density epidermal cells (HDEC) contain cells in and below the lower band. The preponderance of round cells in the high density zone and polygonal cells in the low-density zone is consistent with the data shown in Table 1.
FIG. 3. Photomicrograph of H and E stained smear of cells from fraction 4 showing the transitional nuclear:cytoplasm ratio of the intermediate density spinous cell layers. Original magnification X32.
250
GOLDENHERSH
FIG. 4. Photomicrograph of H and E stained smear of ceils from fraction 7 showing round, high density basal cells with large concentric nuclei. Original magnification X32.
The trypan blue technique for viability was used to demonstrate that the round cells in the high density zone were more than 90% viable. The round cells in the intermediate and lower density area were 70-80% viable. the polygonal cells were 30-40% viable. The morphologic studies of H and E stained preparations from each of the eight fractions demonstrated that fractions 1 and 2 were similar and contained 50-60% granular cells and 3-8% horny cells (Fig. 2). Fractions 4 and 5 each contained 70-80% spinous cells (Fig. 3). Fractions 7 and 8 were
ET AL.
FIG. 6. Electron micrograph of cells from fraction 4 showing larger cells of intermediate density with transitional nuclear:cytoplasm ratio. Magnification X5750.
similar and contained over 85% basal cells (Fig. 4). Though no fraction was entirely pure, “contaminating cells” were generally consistent with the cell types found in the bordering fractions. Accordingly, a basal cell was rarely seen in fractions 1 and 2; and a granular or horny cell was rarely seen in fractions 7 and 8. Fractions 3 and 6, containing mixtures of the cell types from bordering fractions, appeared to be transition fractions between the above-mentioned groupings. In addition, a progression of the nuclear:cytoplasm ratio was observed; this ratio was highest at the bottom of the centrifuge tube (fractions 7 and 8) and continuously decreased to its lowest at the top of the centrifuge tube (fractions 1 and 2). The decrease in the nuclear:cytoplasm ratio with decreasing density was also observed when the cells from each fraction were examined by electron microscopy (Figs. 5-7). DISCUSSION
FIG. 5. Electron micrograph of cells from fraction 7 showing cells with large nuclei and small cytoplasm characteristic of high density basal cells. Magnification x5750.
We have described a method for fractionation of keratinocytes based on density, an inherent physical property of the cells. As shown by the results, it appears that the den-
DENSITY
GRADIENT
SEPARATION
OF EPIDERMAL
CELLS
251
to correspond to the actual densities of the cells (16). For this reason, in the CSP gradient, keratinocytes with the lowest nuclear:cytoplasm ratio (granular and horny cells) were concentrated in the low density area (fractions 1 and 2), while the denser basal cells, having a high nuclear:cytoplasm ratio, were concentrated in the high density area (fractions 7 and 8). CSP therefore appears to be a reliable medium for fractionation of keratinocytes according to their actual density. Though classifying the cells in each fracFIG. 7. Electron micrograph of cells from fraction 2 showing a low density cell with large cytoplasm contion according to the percentage of basal, taining keratohyaline granules and a scant nucteus charspinous, granular, and horny cells is very acteristic of a highly differentiated granular cell. Magappealing, it is technically difficult because nification X5750. there are many cells which are in states of transition. Since the CSP method separates sity of the keratinocytes corresponds to their the cells on a physiologic basis (density), one nuclear:cytoplasm ratio. The morphologic may speculate that the biochemical similaranalyses of various fractions showed that ity of keratinocytes of the same density may there is an association between the density be even greater than their apparent morof keratinocytes and their degree of differphological uniformity. Preliminary experientiation. The less mature cells were found ments comparing the fractions as to thyin the denser portions of the gradient and midine uptake as well as DNA and RNA the more differentiated cells in the less dense content seem to support this contention ( 15). area of the gradient. Thus, it appears that As indicated in the results, the horny cells and the granular cells were both concenthe density and the maturity of keratinocytes trated in the same fractions. Since the unare inversely related. A similar observation has been made by Sun and Green who have fractionated cells contained only 1.5-2.5% used Ficoll-Hypaque gradient to separate horny cells, it was not surprising that this cultured epidermal cells (9). These authors small quantity of horny cells could not be have demonstrated that the cell size and cell separated from the granular cells. It is also density are inversely related. They have possible that the granular cells and horny speculated that the decrease in density of the cells are not sufficiently different in density larger cells may be due to a relative decrease to be separated on a density gradient. in the number of heavy molecules such as In conclusion, the CSP technique provides RNA or glycogen. Preliminary results of a a rapid and reproducible method for fracseries of experiments on the RNA content tionation of large quantities of epidermal of the CSP-separated epidermal cells sup- keratinocytes. The technique is practical and ports Sun and Green’s speculative view ( 15). can easily be adapted in any laboratory. In Using other cell systems it has been dem- addition, the CSP technique has also been found effective for obtaining enriched poponstrated that due to its high molecular weight, CSP cannot enter the cell to con- ulations of guinea pig Langerhans cells ( 17). tribute to the osmotic pressure; thus water REFERENCES is not removed osmotically from the cells ( 16). Because of these factors, the buoyant I. Giovanella, B. C., and Heidelberger, C. (1965) Cancer Res. 25, 161-181. densities obtained in CSP gradients are said
252 2. Gumucio, (1967) 3. Laerum, 21 1.
GOLDENHERSH J., Feldkamp, C., and Bernstein, J. Invest. Dermatol. 49, 545-551, 0. D. ( 1969) J. Invest.
4. Ugel, A. R., and Idler, matol. 55, 350-353.
Dermatol.
W. (1970)
I. A. 52,204-
J. Invest.
Der-
M. A. (1971) J. Invest. 5. Moore, J. T., and Karasek, Dermatol. 56, 3 18-324. 6. Vaughan, F. L., and Bernstein, I. A. (1971) J. Znvest. Dermatol. 56, 454-466. I. Stern, I. B., and Sekeri-Pataryas, K. H. (1972) J. Invest. Dermatol. 59, 25 1-259. 8. Sasai, Y., Kawamura, K., and Namha, K. (1979) Histochemistry 63, 265-272. 9. Sun, T., and Green, H. (1976) Cell 9, 54-521. 10. Shortman, K. (1972) Annu. Rev. Biophys. Bioeng. 1,93-130.
ET AL. 11. Miller, R. G. (1973) in New Techniques in Biophysics and Cell Biology (Pain, R. H.. and Smith, B. J., eds.), Vol. 1, pp. 87- 1 12, Wiley, New York/London. 12. Steinmuller, D., and Wunderlich, J. R. (1976) Cell. Immunol. 24, 146-163. 13. Stingl, G., Katz, S. I., Shevach, E. M., WolffSchreiner, E., and Green, 1. (1978) J. Immunol. 120, 570-578. 14. Lloyd, K. O., and Darnule, T. V. (1974) J. Immunol. 112,311-319. 15. Goldenhersh, M., Andreeff, M., Lynfield, R., Cohen, L., and Safai, B. (1981) Clin. Rex 29, 596A. 16. Pertoft, H., Rubin, K., Kjellen, L., Laurent, T. C., and Klingeborn, B. (1977) Exp. Cell. Res. 110, 449-457. 17. Safai, B., Goldenhersh, M., Sarkar, N., Gupta, S., and Good, R. A. (1980) Clin. Rex 28, 252A.
Separation
MICHAEL
119, 2466252 ( 1Y82)
BIOCHEMISTRY
of Epidermal Cells by Density Gradient Centrifugation Continuous Colloidal Silica (Percoll) Gradient’
A. GOLDENHERSH, Dermatology
Laboratory,
ROBERT Memorial New
A. GOOD, NURUL Sloan-Kettering York, New York
Cancer 10021
on a
H. SARKAR, AND BIJAN SAFA? Center,
I275
York
Avenue,
Received April 28, 1981 The epidermis, which is composed mainly of keratinocytes, undergoes continual proliferation and differentiation. Beginning as a basal cell, the keratinocyte matures and ascends through the spinous cell, granular cell, and horny cell layers. To study keratinocyte differentiation in vitro, it is necessary to separate the keratinocytes at their successive stages of maturation. A method for separation of epidermal keratinocytes is reported here. The method utilizes centrifugation on a continuous colloidal silica (Percoll) density gradient, which separates the keratinocytes according to their inherent density. The resulting arrangement of the keratinocytes in the centrifuge tube generally corresponds to their arrangement in situ, with basal cells at the bottom and the more differentiated cells above. There is a continuous decrease in the nuclear:cytoplasm ratio of keratinocytes as the cells mature. Using this technique, it was shown that the decrease in the nuclear:cytoplasm ratio corresponds to decreasing density of keratinocytes as they mature. Using hematoxylin and eosin staining and electron microscopic studies, it was shown that relatively pure fractions of basal cells, spinous cells, and granular cells are obtained by this method. The method is simple and reproducible, and should greatly facilitate biochemical analyses of the various maturation steps of epidermal keratinocytes.
The recognized morphologic stages in keratinocyte differentiation are the basal, spinous, granular, and horny cell layers. To study the biochemical events of keratinocyte differentiation, which parallel these morphological changes, it has been necessary to develop a technique which can separate keratinocytes in suspension at different stages of differentiation. While several techniques have been reported (l-9) none has become standardized for general use. Thus, there is a need for development of a reproducible method for fractionation of keratinocytes which is practical enough to be easily adapted in any laboratory. To develop such a method we evaluated ’ Aided by the USPHS Grants CA-08747, AE-I 1843, Witty’s Fund, Chesebrough-Ponds, Handmacher Foundation, Dickson’s Fund, Robert S. Sinn’s Fund, and J. Mirsky’s Fund. * To whom correspondence should be addressed. 0003-2697/82/020246-07$02.00/O Copyright 0 1982 by Academic Press. Inc. All rights of reproduction in any form reserved.
the known features of epidermal keratinocytes. As the keratinocytes differentiate and ascend through the various layers of the epidermis, they become larger and their nuclear:cytoplasm ratio decreases (5,6,8). In general, a close relationship between the nuclear:cytoplasm ratio of a given mammalian cell and its density prevails (i.e., the higher the ratio, the higher the density) (10,ll). Accordingly, it should be possibleto separate keratinocytes at different stages of maturation using a density gradient. We report here the use of density gradient centrifugation in a continuous, sigmoidal gradient of colloidal silica polyvinylpyrrolidone (CSP) for fractionation of keratinocytes. MATERIALS
AND
METHODS
Preparation of Epidermal Cell Suspensions. Previously reported techniques ( 12,13) 246
DENSITY
GRADIENT
SEPARATION
with some modifications were used to prepare epidermal cell suspensions. Meon Chase guinea pigs of mixed sex ranging 2-4 months in age served as a source of skin. Thin split thickness sections of skin were obtained using a Davol/Simon Dermatome (Davol, Providence, R. I.). These sections were then cut into smaller pieces (approx 1.5 X 0.5 cm) and incubated dermal side down on a thin layer of 0.25% solution of lyophilized trypsin (Millipore Corp., Freehold, N. J.) in a petri dish for 90 min at 37°C. The trypsin was prepared by mixing 0.25 g trypsin in 100 ml phosphate buffered saline without Ca or Mg (Walker Laboratory, Rye, N. Y.) and adjusting pH to 7.8. Following incubation, the skin sections were removed from the trypsin and the epidermis was teased from the dermis using small forceps. The separated epidermal sheets were placed in a petri dish containing Hanks Balanced Salt Solution (HBSS) (Walker Laboratory, N. Y.) until all sections were separated, after which they were transferred to a petri dish containing 0.02% solution of DNase I (Sigma Chemical Co., St. Louis, MO.). While in the DNase solution, both sides of the epidermal sheets were vigorously stirred with a glass stirring rod for 10 min. HBSS (10 ml) was then added to the petri dish and the entire suspension of cells and epidermal sheets was pipetted onto a 0.2-mm pore size sterile wire mesh (Estey Wire Co., N. J.). The resulting suspension of epidermal cells was then washed with HBSS three times for 10 min each. Preparation of gradient media. Readymade solution of CSP (Percoll) was purchased from Pharmacia Fine Chemicals (Uppsala, Sweden). CSP (9 ml) was mixed with 1 ml HBSS 10 times (GIBCO, Grand Isiand, N. Y.) and further diluted once with the necessary amount of HBSS to achieve a density of 1.066 as measured by refractometry (Abbe-3L Refractometer; Bausch & Lomb, Rochester, N. Y.). The amount of diluent needed to achieve the appropriate
OF
EPIDERMAL
CELLS
241
starting density varies according to the initial density of CSP. Fractionation of cells. Approximately 2030 million unfractionated epidermal cells (UFEC) were suspended in 8 ml of CSP solution. The cell suspension was centrifuged in a 15-ml Corex tube (Fisher Scientific, Pittsburgh, Pa.) in a Beckman J-21 highspeed centrifuge (Beckman Instruments, Palo Alto, Calif.) equipped with a JA-20 rotor at 13,000 rpm (approx 20,OOOg) for 30 min at 5°C. After centrifugation, cells were dispersed throughout the tube. In two areas, cells were sufficiently concentrated to form bands. One band was in the upper portion of the fluid column and the other in the lower portion. Following centrifugation a thin-walled metal tube (l/16 in. o.d.) was delicately inserted to near the bottom of the centrifuge tube. Seven l-ml and one 0.5-ml fractions were collected through the metal tube using a l-ml tuberculin syringe attached to a three-way stopcock. The remaining 0.5 ml of CSP was accounted for by a small portion of CSP which remained in the bottom of the tube. In a second series of experiments, the cells were collected according to the grossly visible banding evident after centrifugation. In these experiments, three fractions were collected from the bottom. The first fractions (2 ml), containing the epidermal cells with higher density (HDEC), included the cells in and below the lower band. The second fraction, containing epidermal cells with intermediate density (IDEC), included 3 ml of CSP with unbanded cells dispersed throughout the middle of the tube. The last fraction collected, including the cells in and above the upper band, was 2.5 ml and contained epidermal cells with lower density (LDEC). Each fraction was washed three times in HBSS for 10 min and resuspended in HBSS for counting. Analysis of the CSP gradient. A tube containing CSP solution of the same starting
248
GOLDENHERSH
density was centrifuged and fractionated in the same manner, but without cells. The density of each fraction was measured by refractometry. Analysis of cell fractions. Unfractionated epidermal cells and cells in each fraction were counted. To standardize our counting method we used morphology as well as size of unstained cells. Thus, three cell types could be distinguished: ( 1) Round cells which measured iess than 13 pm had relatively large nuclei, and were found in highest proportions in the denser portion of the gradient; (2) small polygonal cells which were concentrated in the middle and upper portion of the gradient measured up to 25 I.crn, and had relatively smaller nuclei and large cytoplasm; and (3) large polygonal cells which measured more than 25 pm, and were found mostly in the upper fractions of the gradient. A similar categorization of keratinocytes has been previously noted ( 14). Cells were counted in a hemocytometer using oculomicrometry for cell measurements. Cell viability in each fraction was determined by the trypan blue dye exclusion technique. In another series of cell separations, cytocentrifuge preparations were made with cells from each of the eight fractions. The
1.025 ,
LDEC _
IDEC I.?56 - I.?75 !
HDEC r
I.110
ET AL. TABLE MEAN STRATING POLYGONAL
RESULTS
OF FOUR
1 EXPERIMENTS
DEMON-
THE PERCENTAGE OF ROUND CELLS, SMALL CELLS, AND LARGE POLYGONAL CELLS IN
EACH OF EIGHT FRACTIONS COLLECTED AFTER CENTRIFUGATION OF UNFRACTIONATED EPIDERMAL CELLS IN CSP
Round cell (%) Unfrac. 1 2 3 4 5 6 I 8
56 I 12 16 22 46 76 92 90
Small polygonal (o/o) 36 93 81 15 70 52 23 8 10
Large polygonal (%I 8 0 7 9 8 2 1 0 0
Nofe. The less dense polygonal cells predominate in the upper fractions while the more dense round cells are more numerous in the lower fractions.
cells were air-dried on glass slides and stained with hematoxylin (H) and eosin (E), and the percentage of basal, spinous, granular, and horny cells was counted. Previously described criteria were used for determination of basal, spinous, granular and horny cells (l-3,5-8). Transmission electron microscopy was also used for examination of cells from each of the eight fractions.
L-----4
RESULTS
Ii,
1.02
4
1.04
1 13
1.06
IlO
1.06
8
’
1.10
’
’
1.12
Density
FIG. 1. The continuous, nearly linear, density gradient of consecutive fractions collected after centrifugation of CSP. Fraction 1, the least dense, is from the top of the centrifuge tube; fraction 8, the most dense, is from the bottom.
A continuous sigmoidal gradient is produced following centrifugation of the CSP solution (Fig. 1). The range of density (g/ ml) is from 1.025 in fraction 1 to 1.110 in fraction 8. Repeated determinations demonstrated virtually no variation in the densities of each fraction. Four experiments were done wherein the percentage of round, small polygonal and large polygonal cells were counted in eight fractions. The mean results are shown in
DENSITY
GRADIENT
SEPARATION
Table 1. Fractions 7 and 8, which are the most dense, contained over 90% round cells. The percentage of round cells continually decreased with decreasing density, such that fraction 1 had less than 10% round cells. In contrast, fraction 1, which is the least dense, contained over 90% polygonal cells. The percentage of polygonal cells decreased with increasing density such that fraction 8 contained less than 10% polygonal cells. In addition, a transition in the cell diameter of the round cells at different densities was observed. The round cells in the lower (more dense) fractions were frequently as small as 8-10 pm, while the round cells in the upper (less dense) fractions were usually larger ( 12- 13 pm). Similarly, the large polygonal cells were found almost exclusively in the upper half of the density gradient. Analogous data for the second series of four experiments are shown in Table 2. Cells were fractionated according to the cell bands (three fractions). The trend of high percentage of round cells in the high density
TABLE
2
RESULTS OF FOUR EXPEIUMENTS IN WHICH CELLS WERE COLLECTED IN THREE FRACTIONS
MEAN
Unfrac.
Round cell (70)
Small polygonal (W)
61
32
1
I7
13
IO
61
35
4
91
3
0
OF
EPIDERMAL
CELLS
249
FIG. 2. Photomicrograph of H and E stained smear of cells from fraction 2 demonstrating the more differentiated, low density cells with relatively large cytoplasm and small or absent nucleus. This fraction, along with fraction I. contains more granular and horny cells than the other fractions. Magnification X32.
zone (lower band) and high percentage of polygonal cells in the low density zone (upper band) was maintained. The intermediate density area (IDEC) contained intermediate proportions of round and polygonal cells. The total number of cells recovered after fractionation averaged 50-65% of the initial cell load. The association of cells in the low density zone and the high density zone, each of which contained a cell band, was generally 1.5 times the proportion in the intermediate density area.
Large polygonal (o/o)
Low
density Intermediate density High density
Nore. Low-density epidermal cells (LDEC) contain the cells in and above the upper band. Intermediatedensity epidermal cells (IDEC) contain cells between the bands. High-density epidermal cells (HDEC) contain cells in and below the lower band. The preponderance of round cells in the high density zone and polygonal cells in the low-density zone is consistent with the data shown in Table 1.
FIG. 3. Photomicrograph of H and E stained smear of cells from fraction 4 showing the transitional nuclear:cytoplasm ratio of the intermediate density spinous cell layers. Original magnification X32.
250
GOLDENHERSH
FIG. 4. Photomicrograph of H and E stained smear of ceils from fraction 7 showing round, high density basal cells with large concentric nuclei. Original magnification X32.
The trypan blue technique for viability was used to demonstrate that the round cells in the high density zone were more than 90% viable. The round cells in the intermediate and lower density area were 70-80% viable. the polygonal cells were 30-40% viable. The morphologic studies of H and E stained preparations from each of the eight fractions demonstrated that fractions 1 and 2 were similar and contained 50-60% granular cells and 3-8% horny cells (Fig. 2). Fractions 4 and 5 each contained 70-80% spinous cells (Fig. 3). Fractions 7 and 8 were
ET AL.
FIG. 6. Electron micrograph of cells from fraction 4 showing larger cells of intermediate density with transitional nuclear:cytoplasm ratio. Magnification X5750.
similar and contained over 85% basal cells (Fig. 4). Though no fraction was entirely pure, “contaminating cells” were generally consistent with the cell types found in the bordering fractions. Accordingly, a basal cell was rarely seen in fractions 1 and 2; and a granular or horny cell was rarely seen in fractions 7 and 8. Fractions 3 and 6, containing mixtures of the cell types from bordering fractions, appeared to be transition fractions between the above-mentioned groupings. In addition, a progression of the nuclear:cytoplasm ratio was observed; this ratio was highest at the bottom of the centrifuge tube (fractions 7 and 8) and continuously decreased to its lowest at the top of the centrifuge tube (fractions 1 and 2). The decrease in the nuclear:cytoplasm ratio with decreasing density was also observed when the cells from each fraction were examined by electron microscopy (Figs. 5-7). DISCUSSION
FIG. 5. Electron micrograph of cells from fraction 7 showing cells with large nuclei and small cytoplasm characteristic of high density basal cells. Magnification x5750.
We have described a method for fractionation of keratinocytes based on density, an inherent physical property of the cells. As shown by the results, it appears that the den-
DENSITY
GRADIENT
SEPARATION
OF EPIDERMAL
CELLS
251
to correspond to the actual densities of the cells (16). For this reason, in the CSP gradient, keratinocytes with the lowest nuclear:cytoplasm ratio (granular and horny cells) were concentrated in the low density area (fractions 1 and 2), while the denser basal cells, having a high nuclear:cytoplasm ratio, were concentrated in the high density area (fractions 7 and 8). CSP therefore appears to be a reliable medium for fractionation of keratinocytes according to their actual density. Though classifying the cells in each fracFIG. 7. Electron micrograph of cells from fraction 2 showing a low density cell with large cytoplasm contion according to the percentage of basal, taining keratohyaline granules and a scant nucteus charspinous, granular, and horny cells is very acteristic of a highly differentiated granular cell. Magappealing, it is technically difficult because nification X5750. there are many cells which are in states of transition. Since the CSP method separates sity of the keratinocytes corresponds to their the cells on a physiologic basis (density), one nuclear:cytoplasm ratio. The morphologic may speculate that the biochemical similaranalyses of various fractions showed that ity of keratinocytes of the same density may there is an association between the density be even greater than their apparent morof keratinocytes and their degree of differphological uniformity. Preliminary experientiation. The less mature cells were found ments comparing the fractions as to thyin the denser portions of the gradient and midine uptake as well as DNA and RNA the more differentiated cells in the less dense content seem to support this contention ( 15). area of the gradient. Thus, it appears that As indicated in the results, the horny cells and the granular cells were both concenthe density and the maturity of keratinocytes trated in the same fractions. Since the unare inversely related. A similar observation has been made by Sun and Green who have fractionated cells contained only 1.5-2.5% used Ficoll-Hypaque gradient to separate horny cells, it was not surprising that this cultured epidermal cells (9). These authors small quantity of horny cells could not be have demonstrated that the cell size and cell separated from the granular cells. It is also density are inversely related. They have possible that the granular cells and horny speculated that the decrease in density of the cells are not sufficiently different in density larger cells may be due to a relative decrease to be separated on a density gradient. in the number of heavy molecules such as In conclusion, the CSP technique provides RNA or glycogen. Preliminary results of a a rapid and reproducible method for fracseries of experiments on the RNA content tionation of large quantities of epidermal of the CSP-separated epidermal cells sup- keratinocytes. The technique is practical and ports Sun and Green’s speculative view ( 15). can easily be adapted in any laboratory. In Using other cell systems it has been dem- addition, the CSP technique has also been found effective for obtaining enriched poponstrated that due to its high molecular weight, CSP cannot enter the cell to con- ulations of guinea pig Langerhans cells ( 17). tribute to the osmotic pressure; thus water REFERENCES is not removed osmotically from the cells ( 16). Because of these factors, the buoyant I. Giovanella, B. C., and Heidelberger, C. (1965) Cancer Res. 25, 161-181. densities obtained in CSP gradients are said
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