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Studies on cellular adhesion in tissue culture
Experimental
Cell
STUDIES
Research
17, 499-507
ON CELLULAR I. THE
499
(1959)
ADHESION EFFECT
IN TISSUE
CULTURE
OF SERUM
L. WEISS The
John
Burford
Carlill
Pathological
Laboratories, England
Westminster
School
of
Medicine,
London,
Received November 25, 1958
adhesion is logically studied by a tripartite analysis of the cell surface, the cellular environment, and the substratum to which the cell adheres. The present communication deals with the adhesion of cells in culture to glass. Experiments are described which show the effects of some modifications of cell surfaces and cellular environment (media) upon adhesion, with particular reference to the part played by serum. CELLULAR
Tissue Cultures The cells used in the experiments described were all of human origin and were grown in Pyrex glass “baby bottles”. HeLa and Sta (Carcinoma of thyroid) cells had undergone transformation in tissue-culture before use. Par (thyroid), Col. (thyroid) and H.Ki (kidney) cells were in the untransformed, fibroblast-like state. The method of preparation of cultures of thyroid cells and the significance of transformation in them is discussed by Pulvertaft, Weiss, Davies and Wilkinson [17]. No difference in the behaviour of these various cells was detected in the experiments to be described. The trypsin solution used throughout was “Difco” 1: 250, made up as a 0.25 per cent solution in the phosphate buffered saline of Dulbecco and Vogt [3]. EXPERIMENTS
The Efect of Whole Serum Material
and
Methods
Cells spread out in monolayers were detached from the surfaces of glass bottles with a stream of culture fluid gently delivered from a bulb-pipette. A cellular deposit was obtained by slow centrifugation; this was resuspended in Hanks’ solution and Experimental
Cell
Research
17
L. Weiss
500
divided into three parts. The first part was left untreated, the second washed three times in Hanks’ solution, and the third batch was washed three times in Hanks’ solution and then incubated in trypsin solution for 30 minutes at 37°C. After incubation, these cells were again washed and resuspended in Hanks’ solution. At the conclusion of these procedures the three suspensions were diluted to contain about 250,000 cells per ml. Media had been previously made up in 1.8 ml quantities in culture bottles as shown in Table I. Each series of media contained serial ten-fold dilutions of pooled human serum, which had previously been inactivated at 56°C for 35 minutes. The actual tenfold dilutions were made with separate pipettes, as the “carry-over” on a single pipette proved sufficient to invalidate the results.
I. The effect of serum concentration upon the adhesion of HeLa cells to glass.
TABLE
Cells
Media Lactalbumen hydrolysate 0.5 % in Hanks’ solution Bactoyeast 0.2 % in Hanks’ solution Lactalbumen hydrolysate 0.5 % + Bactoyeast 0.2 %
10
Serum concentration, % 1 0.1 0.01 0.001 0.0001
Unwashed Washed x 3 Trypsinised
+ + +
+ + -
+
Unwashed Washed x 3
+ +
+ +
+ +
Unwashed Washed x 3 Trypsinised
+ + +
+ + -
+ -
-
.-
.-
+ -
= -
= -
-
-
-
To each bottle containing medium was added 0.2 ml of the cell suspension in Hanks’ fluid. The bottles were incubated at 37°C overnight and examined with an inverted microscope for the presence of adherent viable cells. Very occasional adherent forms were ignored, as this was taken to mean that the preinoculation treatment of the particular batch of cells was uneven.
Results The results are shown in Table I in which the presence or absence of adherent cells is indicated. It is seen that for adhesion to glass to occur, trypsintreated cells require approximately ten times as much serum as do thrice washed cells. In turn, thrice washed cells require ten times as much serum as do the unwashed cells. Experimental
Cell Research
17
Cellular
501
adhesion and serum
The E$ect of Serum Fractions Materials
and Methods
Specimens of human serum fractions were used throughout. Serum albumin, y-globulin, and the crude lipoprotein-rich fraction G2 of Kekwick and MacKay [lo] were obtained from ether-precipitated samples.A fraction containing both albumin and p-globulin was obtained by salting out with ammonium sulphate and subsequent dialysis. The constituents of all the fractions were checked by paper electrophoresis. The fractions were made in solutions containing twice the normal physiological amounts. Culture media were then prepared as follows: 80 parts 0.5 per cent lactalbumin hydrolysate in Hanks’ solution and 20 parts solution of fraction, in Hanks’ solution. Human serum and phosphate buffered saIine were used in the positive and negative control media respectively. Cellswere trypsinised as previously described and resuspendedin Hanks’ solution. 0.2 ml of cell suspensionwere added to culture bottles containing 1.8 ml of culture fluid. After overnight incubation at 37°C the cultures were examined for the presence of adherent cells. The media were numbered as shown in Table II. Results
The results are tabulated in Table II. In this table, where mixtures of media are indicated, equal parts of all the constituent media were used in a total volume of 1.8 ml per bottle. It is seen that for the adhesion of trypsin-treated cells to glass, fraction G2 may replace serum. None of the other fractions tested would do this.
The Assay of Fraction Materials
62 for Adhesive Activity
and Methods
Fraction G2 was reconstituted with phosphate buffered saline to the volume of the original plasma samplefrom which it was obtained. Ten-fold dilutions of “normal” G2 were made in 0.1 per cent bacto yeast in Hanks’ fluid. Similar dilutions were made of whole serum. 0.2 ml of trypsin-treated cells suspendedin Hanks’ solution were added to 1.8 ml of media, as in the previous experiments. Results
After overnight incubation the bottles were examined for adherent cells. The results shown in Table III indicate that in its capacity for enabling cells to adhere to glass, fraction G2 is as effective in the same order of concentration as whole serum. Experimental
Cell Research 17
502
L. Weiss
The Efect
of Species Diference
and Inactivation Cellular
Materials
of Serum
on the Strength
of
Adhesion
and Methods
Sera were obtained from a number of different species. One half of each specimen was inactivated at 56°C for 30 minutes. Media were made up containing 90 per cent of 0.1 per cent bactoyeast in Hanks’ solution, together with 10 per cent of the “test” serum. Trypsinised cells were cultured in the various test media in bottles. After overnight culture, the bottles were gently rocked, and the culture fluid and any unattached cells were poured off. The cultures were then allowed to equilibrate at room temperature, at which all further manipulations were carried out. One ml of Hanks’ fluid was added to each bottle, and groups of four bottles were clamped into a “Microid” wrist-action shaker (Griffin & Tatlock, Ltd.) and shaken at a set speed as previously determined by experiment, for 2 minutes. The bottles were then removed and emptied. The residual cells adhering to the bottle were removed and dispersed by incubation at 37°C for 30 minutes with a measured quantity of 0.25 per cent trypsin containing 0.025 per cent ethylenediamine tetra-acetic acid, disodium salt. The cells which had been poured off after shaking were washed, and then also dispersed with the trypinTABLE
II.
The
effect
of’ serum
fractiom on glass.
the adhesion
Adherent Fractions
Medium
present
,!-Globulin -I- albumin y-Globulin Albumin “G2” Whole serum Phosphate-buffered saline (P.B.S.) These effects control 1+4 2 -t4 3+4 6+4
HeLa
-I+
of cultured
viable
cells
Par
sta
+ -1.
+ +
-~
results could have been due to toxic of media 1, 2, 3, and 6. The following mixtures were then used:
B + y-Globulins + G2 y-Globulin + G2 Albumin + G2 P.R..‘% + G2
+ I-
+
-t-
i +
+
+ + + +
The positive results are thus due to G2 and the previous negative results are not due to the toxic effects of media 1, 2, 3, and 6.
Experimental
Cell
Research
17
cells
to
Cellular
adhesion
503
and serum
versene mixture. The cell densities in both suspensions were estimat.ed from cell counts (in duplicate) on 4 cm samples in a Fuchs Rosenthal chamber and the volumes of the suspensions were measured to 0.1 ml; the product of these two measurements gave the total number of cells. The total of both cell counts gave the original total population in any one bottle. The numbers of cells freed from the glass were expressed as a percentage of their total-bottle population. Thus, the higher the percentage, the weaker the cellular adhesion to glass.
III.
TABLE
The comparison of fbaction G2 with human serum fbr adhesive activity.
CdlS
Media
10
HeLa
Whole Serum G2 Whole Serum G2 Whole Serum G2
+ + + + + +
co1 H.Ki.
TABLE
-
i+ + + + +
--
-
-
.-
IV. The effect ofspecies and inaction ofserum in media, upon the strength of adhesion of cells to glass.
Serum Human: Dre Joh Nit Tib Wei Dog Rat Pig Rabbit Guinea pig a Arithmetic 33
Concentration, % 1 0.1 0.01
- 593706
Percentage’ of cells liberated by standard agitation Inactivated serum Fresh serum
14 8 18 15 11
29 31 36 33 33
79 13 62
85 75 91 57 79
44 25
mean of 6 cultures. The lower the number,
the greater the strength of adhesion. Experimental
Cell
Research
17
L. Weiss Results The results are given in Table IV. A species difference is shown ability of serum to promote cellular adhesion to glass. In all species, possibly “dog”, inactivation of serum is associated with impairment “adhesive activities”.
in the except of its
DISCUSSION
In an earlier paper [21] a loss of cellular dry mass following trypsin treatment was described. This loss of dry mass was associated with the appearance of an amorphous mutinous protein. In the present communication, trypsin treatment is shown to be associated with a loss of adhesive properties which may be restored by the addition of serum to the culture fluid. More serum is required to restore the adhesiveness of trypsin-treated cells than of washed cells. One explanation of these results would be that an adsorbed extramembranous protein layer is incompletely removed by washing and more completely removed by enzyme treatment. An alternative explanation is that two extramembranous layers exist, the more superficial one consisting of protein adsorbed from the medium and the deeper one being attached by enzymedissociable links to the cell membrane. In favour of this is the fact that protein is removed from tissue cells by up to three washings, but not after this. More material however may subsequently be removed by trypsin-treatment. Easty and Lowick [4] measured the electrophoretic mobilities of murine sarcoma cells before and after treatment with papain and noted that the papain treated cells behaved as if their surface charges had been constantly reduced. This observation is taken to support the hypothesis that something is removed from the cell surface by enzymatic digestion. Against this hypothesis is the failure to identify an extramembranous layer by histochemical methods. Hale’s staining method [S] which utilises the formation of a complex between dialysed iron and certain mucoproteins has been unsuccessful. According to lmmers [9] this technique would also show up polynucleotides, phosphoThe combination of Alcian blue and chlorantinc proteins and phospholipids. fast red stain for muco-polysaccharides as described by Lison [13] was also negative, as were the periodic acid Schiff and metachromatic toluidine blue stains. These negative results could he explained by assuming the coat to be so thin as to be beyond the resolving power of the ordinary microscope. coat could be discarded in Alternatively, the idea of a complete “adhesive” favour of “adhesive” patches occurring on the plasma membrane. This concept fits in with the observations of 13irbeck and Mercer [2] and Mercer [ 141 who note the electron micrographic appearance of small amounts of “cement” Esperimenfal
Cdl
Research
17
Cellular
505
adhesion and serum
external to the plasma membrane. These patches are not well defined owing to their low density and their failure to react well with the osmium-containing fixatives used [ 151. It is shown that serum plays a definite role in permitting trypsin-treated cells to adhere to glass. These results are in agreement with those of Rinaldini and Lucy [19] who found that trypsin-dispersed embryonic chick cells when resuspended in synthetic media would adhere to glass in the presence of serum. These workers grew their trypsin-dispersed cells in media containing 20 per cent of serum, before subculturing them in synthetic media. This ability of cells to carry over serum probably accounts for the different results obtained with the cultivation of various cells in the many synthetic media now available. It is suggested that synthetic media should not be considered complete unless extramembranous material is first removed from the cells by enzyme treatment. Puck, Marcus and Ciecuira [16] measured the surface areas of cells cultured on flat glass surfaces. They found that HeLa cells grown in media containing human serum, presented four times as much surface area as those grown in porcine or bovine serum. This variation in surface area was not associated with variation of cell volume. The greater strength of cellular adhesion to glass in the presence of human serum in the presen.t studies, is possibly explained on the basis of greater areas of contact between the cells and their substratum, but the problem of why cells should spread over a greater area in some sera than in others remains unsolved. It is well known that sera from some animals appear to enhance cellular adhesion to glass, and growth, more than sera from others and also, that sera appear to be adversely affected by inactivation as far as enhancement of growth is concerned. In the experiments described, these phe:nomena are given a quantitative basis. Unfortunately the results obtainable by this method have no absolute values, and are comparable numerically only with results obtained with the same apparatus. Within these limitations it has been shown that some sera cause firmer adhesions to glass than others. With the possible exception of dog serum, inactivation by heating at 56°C for 30 minutes impairs the adhesive activity of serum. This diminution of adhesive activity by heat inactivation suggested that complement might be involved, but complement titrations on media containing guinea pig serum, after cells cultured in them have adhered to glass failed to show significant change of titre when compared with controls. As so far only four separate sera have been tested, these preliminary results are not considered decisive. To restore adhesive properties to trypsin-treated cells, serum may be reExperimentnl
Cell Research
17
L. Weiss placed by fraction G2 of Kekwick and Mackay [lo]. This fraction is prepared from human plasma by ether precipitation at critical concentrations and temperatures. It has no clinical importance, and very little work appears to have been done to determine its chemical constitution. It is rich in B-lipoproteins which give it a characteristic turbid appearance, and also contains a large part of the serum a-globulins. Bachtold and Gebhardt [ 1 ] using quantitative electrophoretic analysis, determined the patterns of serum utilisation during the outgrowth in vitro of monkey kidney cells. They noted that the main protein utilisation occurred during the first three days of their cultures, when monolayers had become well established but were not confluent. The main protein loss was observed in the a,-globulin fractions. As one of the main activities of cells during the establishment of a monolayer on glass appears to be connected with adhesion, this observation on the selective loss of the &,-fraction is regarded as corroboratory evidence of the activity of the fraction G2. It must be noted that this fraction is only interchangeable with whole serum for adhesion of cells to glass and not for nutrition. Lieberman and Ove [ll] obtained a purified serum fraction which caused cells to adhere to glass surfaces. This material was composed mainly of glycoprotein, and its electrophoretic mobility was that of the cc-globulins. It was heat-labile, which would account for the effects of heat inactivation of serum described in the present communication. a-globulins were also associated with cellular adhesion to glass by Fisher, Puck and Sato [7], who noted that the active component appeared to resemble fetuin. This is an a,-globulin comprising 45 per cent of foetal calf serum protein. However, Lieberman an 1 Ove [la] have shown that fetuin can be separated from the “adhesion-active” protein factor by column chromatography. Strain 929 clone L (mouse Iibroblast) cells can grow and adhere to glass chemically defined media as described by Evans and her in protein-free, associates [5, 61 and Waymouth [20]. However, it is difficult to grow most cell lines adherent to glass in such media, immediately following trypsin-treatment (see above). This suggests that the substances involved in the “protein-mechanism” of adhesion can be produced immediately by some cells, but have to be provided in the medium for others. Rappaport [18] has shown that trypsinised monkey kidney cells will spread on glass in the absence of serum, in the presence of high concentrations of calcium ions (physiological x 4). The calcium-dependent mechanism is considered to coexist with the protein mechanisms of adhesion and will be discussed in a future paper. Experimental
Cell Research
17
Cellular
507
adhesion and serum SUMMARY
Some effects of serum on the adhesion of tissue cells to glass in culture, are described. 1. The amount of serum required for adhesion is greater in trypsin-treated than in washed cells. 2. From the aspect of cellular adhesion, the lipoprotein-rich serum fraction G2 may replace whole serum. 9. A method is described for the quantitative assessment of the effects on cellular adhesion of species difference and heat inactivation of serum. The results are discussed in terms of cell-membrane structure and the composition of culture media. I have received much help and encouragement from Professor R. J. \‘. Pulvertaft throughout these experiments. I am indebted to Dr. H. B. Fell for giving me considerable advice on the presentation of this paper. My thanks are also due to Miss E. R. Blake and Miss. H. Youle for technical assistance. This work was made possible by a generous grant from the British Empire Cancer Campaign. The various serum fractions were kindly provided by the Lister Institute. REFERENCES J. G. and GEBHARDT, L. P., Expfl. Cell Research 13, 432 (1957). 2. BIRBECK, M. S. and MERCER, E. H., Nature 178, 985 (1956). 3. DULBECCO, R. and VOGT, M. J., J. Exptl. Med. 99, 167 (1954). 4. EASTY, G. and LOWICK, J., unpublished data (1957). 5. EVANS, V. J., BRYANT, J. C., FIORAMONTI, M. C., MCQUILKIS, W. T., S.ZNDFOEID, K. K. and EARLE, W. R., Cancer Research 16, 77 (1956). 6. -Cancer Research 17, 315 (1957). 7. FISHER, H. W., PUCK, T. T. and SATO, G., Proc. Satl. Acad. Sci. 44, 4 (1958). 8. HALE, E. W., JVature 157, 802 (1946). 9. IMMERS, J., Exptl. Cell Research 6, 127 (1954). 10. KEKWICK, R. A. and MACKAY, M. E., Ned. Research Council Spec. Rep. Ser., No. 286 (1954). 11. LIEBERMAN, I. and OVE, P., Biochim. et Biophys. Acta 25, 449 (1957). 12. ~ J. Biol. Chem. 233, 637 (1958). 13. LISON, L., Stain Technol. 29, 131 (1954). 14. MERCER, E. H., Nature 180, 831 (1957). 15. MERCER, E. H., personal communication (1958). 16. PUCK, T. T., MARCUS, P. I. and CIECIURA, S. J., J. Exptl. Med. 103, 273 (1956). R. .J. V., WEISS, L., DAVIES, J. R. and WILKIXSOX, .J. H.. J. Pathol. Bacferiot. 17. PULVERTAFT, (in press 1959). 18. RAPPAPORT, C., Proc. Sot. Exptl. Bio/. Med. 91, 464 (1956). 19. RINALDINI, L. M. and LUCY, J. A., B.E.C.C. Ann. Rep. 244 (1956). 20. WAYMOUTH, C., J. Nafl. Cancer Inst. 17, 315 (1956). 21. WEISS, L., Exptl. Cell Research 14, 80 (1958).
1. BACHTOLD,
Erperimrnfal
Cer’l Rrsearch
17
Cell
STUDIES
Research
17, 499-507
ON CELLULAR I. THE
499
(1959)
ADHESION EFFECT
IN TISSUE
CULTURE
OF SERUM
L. WEISS The
John
Burford
Carlill
Pathological
Laboratories, England
Westminster
School
of
Medicine,
London,
Received November 25, 1958
adhesion is logically studied by a tripartite analysis of the cell surface, the cellular environment, and the substratum to which the cell adheres. The present communication deals with the adhesion of cells in culture to glass. Experiments are described which show the effects of some modifications of cell surfaces and cellular environment (media) upon adhesion, with particular reference to the part played by serum. CELLULAR
Tissue Cultures The cells used in the experiments described were all of human origin and were grown in Pyrex glass “baby bottles”. HeLa and Sta (Carcinoma of thyroid) cells had undergone transformation in tissue-culture before use. Par (thyroid), Col. (thyroid) and H.Ki (kidney) cells were in the untransformed, fibroblast-like state. The method of preparation of cultures of thyroid cells and the significance of transformation in them is discussed by Pulvertaft, Weiss, Davies and Wilkinson [17]. No difference in the behaviour of these various cells was detected in the experiments to be described. The trypsin solution used throughout was “Difco” 1: 250, made up as a 0.25 per cent solution in the phosphate buffered saline of Dulbecco and Vogt [3]. EXPERIMENTS
The Efect of Whole Serum Material
and
Methods
Cells spread out in monolayers were detached from the surfaces of glass bottles with a stream of culture fluid gently delivered from a bulb-pipette. A cellular deposit was obtained by slow centrifugation; this was resuspended in Hanks’ solution and Experimental
Cell
Research
17
L. Weiss
500
divided into three parts. The first part was left untreated, the second washed three times in Hanks’ solution, and the third batch was washed three times in Hanks’ solution and then incubated in trypsin solution for 30 minutes at 37°C. After incubation, these cells were again washed and resuspended in Hanks’ solution. At the conclusion of these procedures the three suspensions were diluted to contain about 250,000 cells per ml. Media had been previously made up in 1.8 ml quantities in culture bottles as shown in Table I. Each series of media contained serial ten-fold dilutions of pooled human serum, which had previously been inactivated at 56°C for 35 minutes. The actual tenfold dilutions were made with separate pipettes, as the “carry-over” on a single pipette proved sufficient to invalidate the results.
I. The effect of serum concentration upon the adhesion of HeLa cells to glass.
TABLE
Cells
Media Lactalbumen hydrolysate 0.5 % in Hanks’ solution Bactoyeast 0.2 % in Hanks’ solution Lactalbumen hydrolysate 0.5 % + Bactoyeast 0.2 %
10
Serum concentration, % 1 0.1 0.01 0.001 0.0001
Unwashed Washed x 3 Trypsinised
+ + +
+ + -
+
Unwashed Washed x 3
+ +
+ +
+ +
Unwashed Washed x 3 Trypsinised
+ + +
+ + -
+ -
-
.-
.-
+ -
= -
= -
-
-
-
To each bottle containing medium was added 0.2 ml of the cell suspension in Hanks’ fluid. The bottles were incubated at 37°C overnight and examined with an inverted microscope for the presence of adherent viable cells. Very occasional adherent forms were ignored, as this was taken to mean that the preinoculation treatment of the particular batch of cells was uneven.
Results The results are shown in Table I in which the presence or absence of adherent cells is indicated. It is seen that for adhesion to glass to occur, trypsintreated cells require approximately ten times as much serum as do thrice washed cells. In turn, thrice washed cells require ten times as much serum as do the unwashed cells. Experimental
Cell Research
17
Cellular
501
adhesion and serum
The E$ect of Serum Fractions Materials
and Methods
Specimens of human serum fractions were used throughout. Serum albumin, y-globulin, and the crude lipoprotein-rich fraction G2 of Kekwick and MacKay [lo] were obtained from ether-precipitated samples.A fraction containing both albumin and p-globulin was obtained by salting out with ammonium sulphate and subsequent dialysis. The constituents of all the fractions were checked by paper electrophoresis. The fractions were made in solutions containing twice the normal physiological amounts. Culture media were then prepared as follows: 80 parts 0.5 per cent lactalbumin hydrolysate in Hanks’ solution and 20 parts solution of fraction, in Hanks’ solution. Human serum and phosphate buffered saIine were used in the positive and negative control media respectively. Cellswere trypsinised as previously described and resuspendedin Hanks’ solution. 0.2 ml of cell suspensionwere added to culture bottles containing 1.8 ml of culture fluid. After overnight incubation at 37°C the cultures were examined for the presence of adherent cells. The media were numbered as shown in Table II. Results
The results are tabulated in Table II. In this table, where mixtures of media are indicated, equal parts of all the constituent media were used in a total volume of 1.8 ml per bottle. It is seen that for the adhesion of trypsin-treated cells to glass, fraction G2 may replace serum. None of the other fractions tested would do this.
The Assay of Fraction Materials
62 for Adhesive Activity
and Methods
Fraction G2 was reconstituted with phosphate buffered saline to the volume of the original plasma samplefrom which it was obtained. Ten-fold dilutions of “normal” G2 were made in 0.1 per cent bacto yeast in Hanks’ fluid. Similar dilutions were made of whole serum. 0.2 ml of trypsin-treated cells suspendedin Hanks’ solution were added to 1.8 ml of media, as in the previous experiments. Results
After overnight incubation the bottles were examined for adherent cells. The results shown in Table III indicate that in its capacity for enabling cells to adhere to glass, fraction G2 is as effective in the same order of concentration as whole serum. Experimental
Cell Research 17
502
L. Weiss
The Efect
of Species Diference
and Inactivation Cellular
Materials
of Serum
on the Strength
of
Adhesion
and Methods
Sera were obtained from a number of different species. One half of each specimen was inactivated at 56°C for 30 minutes. Media were made up containing 90 per cent of 0.1 per cent bactoyeast in Hanks’ solution, together with 10 per cent of the “test” serum. Trypsinised cells were cultured in the various test media in bottles. After overnight culture, the bottles were gently rocked, and the culture fluid and any unattached cells were poured off. The cultures were then allowed to equilibrate at room temperature, at which all further manipulations were carried out. One ml of Hanks’ fluid was added to each bottle, and groups of four bottles were clamped into a “Microid” wrist-action shaker (Griffin & Tatlock, Ltd.) and shaken at a set speed as previously determined by experiment, for 2 minutes. The bottles were then removed and emptied. The residual cells adhering to the bottle were removed and dispersed by incubation at 37°C for 30 minutes with a measured quantity of 0.25 per cent trypsin containing 0.025 per cent ethylenediamine tetra-acetic acid, disodium salt. The cells which had been poured off after shaking were washed, and then also dispersed with the trypinTABLE
II.
The
effect
of’ serum
fractiom on glass.
the adhesion
Adherent Fractions
Medium
present
,!-Globulin -I- albumin y-Globulin Albumin “G2” Whole serum Phosphate-buffered saline (P.B.S.) These effects control 1+4 2 -t4 3+4 6+4
HeLa
-I+
of cultured
viable
cells
Par
sta
+ -1.
+ +
-~
results could have been due to toxic of media 1, 2, 3, and 6. The following mixtures were then used:
B + y-Globulins + G2 y-Globulin + G2 Albumin + G2 P.R..‘% + G2
+ I-
+
-t-
i +
+
+ + + +
The positive results are thus due to G2 and the previous negative results are not due to the toxic effects of media 1, 2, 3, and 6.
Experimental
Cell
Research
17
cells
to
Cellular
adhesion
503
and serum
versene mixture. The cell densities in both suspensions were estimat.ed from cell counts (in duplicate) on 4 cm samples in a Fuchs Rosenthal chamber and the volumes of the suspensions were measured to 0.1 ml; the product of these two measurements gave the total number of cells. The total of both cell counts gave the original total population in any one bottle. The numbers of cells freed from the glass were expressed as a percentage of their total-bottle population. Thus, the higher the percentage, the weaker the cellular adhesion to glass.
III.
TABLE
The comparison of fbaction G2 with human serum fbr adhesive activity.
CdlS
Media
10
HeLa
Whole Serum G2 Whole Serum G2 Whole Serum G2
+ + + + + +
co1 H.Ki.
TABLE
-
i+ + + + +
--
-
-
.-
IV. The effect ofspecies and inaction ofserum in media, upon the strength of adhesion of cells to glass.
Serum Human: Dre Joh Nit Tib Wei Dog Rat Pig Rabbit Guinea pig a Arithmetic 33
Concentration, % 1 0.1 0.01
- 593706
Percentage’ of cells liberated by standard agitation Inactivated serum Fresh serum
14 8 18 15 11
29 31 36 33 33
79 13 62
85 75 91 57 79
44 25
mean of 6 cultures. The lower the number,
the greater the strength of adhesion. Experimental
Cell
Research
17
L. Weiss Results The results are given in Table IV. A species difference is shown ability of serum to promote cellular adhesion to glass. In all species, possibly “dog”, inactivation of serum is associated with impairment “adhesive activities”.
in the except of its
DISCUSSION
In an earlier paper [21] a loss of cellular dry mass following trypsin treatment was described. This loss of dry mass was associated with the appearance of an amorphous mutinous protein. In the present communication, trypsin treatment is shown to be associated with a loss of adhesive properties which may be restored by the addition of serum to the culture fluid. More serum is required to restore the adhesiveness of trypsin-treated cells than of washed cells. One explanation of these results would be that an adsorbed extramembranous protein layer is incompletely removed by washing and more completely removed by enzyme treatment. An alternative explanation is that two extramembranous layers exist, the more superficial one consisting of protein adsorbed from the medium and the deeper one being attached by enzymedissociable links to the cell membrane. In favour of this is the fact that protein is removed from tissue cells by up to three washings, but not after this. More material however may subsequently be removed by trypsin-treatment. Easty and Lowick [4] measured the electrophoretic mobilities of murine sarcoma cells before and after treatment with papain and noted that the papain treated cells behaved as if their surface charges had been constantly reduced. This observation is taken to support the hypothesis that something is removed from the cell surface by enzymatic digestion. Against this hypothesis is the failure to identify an extramembranous layer by histochemical methods. Hale’s staining method [S] which utilises the formation of a complex between dialysed iron and certain mucoproteins has been unsuccessful. According to lmmers [9] this technique would also show up polynucleotides, phosphoThe combination of Alcian blue and chlorantinc proteins and phospholipids. fast red stain for muco-polysaccharides as described by Lison [13] was also negative, as were the periodic acid Schiff and metachromatic toluidine blue stains. These negative results could he explained by assuming the coat to be so thin as to be beyond the resolving power of the ordinary microscope. coat could be discarded in Alternatively, the idea of a complete “adhesive” favour of “adhesive” patches occurring on the plasma membrane. This concept fits in with the observations of 13irbeck and Mercer [2] and Mercer [ 141 who note the electron micrographic appearance of small amounts of “cement” Esperimenfal
Cdl
Research
17
Cellular
505
adhesion and serum
external to the plasma membrane. These patches are not well defined owing to their low density and their failure to react well with the osmium-containing fixatives used [ 151. It is shown that serum plays a definite role in permitting trypsin-treated cells to adhere to glass. These results are in agreement with those of Rinaldini and Lucy [19] who found that trypsin-dispersed embryonic chick cells when resuspended in synthetic media would adhere to glass in the presence of serum. These workers grew their trypsin-dispersed cells in media containing 20 per cent of serum, before subculturing them in synthetic media. This ability of cells to carry over serum probably accounts for the different results obtained with the cultivation of various cells in the many synthetic media now available. It is suggested that synthetic media should not be considered complete unless extramembranous material is first removed from the cells by enzyme treatment. Puck, Marcus and Ciecuira [16] measured the surface areas of cells cultured on flat glass surfaces. They found that HeLa cells grown in media containing human serum, presented four times as much surface area as those grown in porcine or bovine serum. This variation in surface area was not associated with variation of cell volume. The greater strength of cellular adhesion to glass in the presence of human serum in the presen.t studies, is possibly explained on the basis of greater areas of contact between the cells and their substratum, but the problem of why cells should spread over a greater area in some sera than in others remains unsolved. It is well known that sera from some animals appear to enhance cellular adhesion to glass, and growth, more than sera from others and also, that sera appear to be adversely affected by inactivation as far as enhancement of growth is concerned. In the experiments described, these phe:nomena are given a quantitative basis. Unfortunately the results obtainable by this method have no absolute values, and are comparable numerically only with results obtained with the same apparatus. Within these limitations it has been shown that some sera cause firmer adhesions to glass than others. With the possible exception of dog serum, inactivation by heating at 56°C for 30 minutes impairs the adhesive activity of serum. This diminution of adhesive activity by heat inactivation suggested that complement might be involved, but complement titrations on media containing guinea pig serum, after cells cultured in them have adhered to glass failed to show significant change of titre when compared with controls. As so far only four separate sera have been tested, these preliminary results are not considered decisive. To restore adhesive properties to trypsin-treated cells, serum may be reExperimentnl
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L. Weiss placed by fraction G2 of Kekwick and Mackay [lo]. This fraction is prepared from human plasma by ether precipitation at critical concentrations and temperatures. It has no clinical importance, and very little work appears to have been done to determine its chemical constitution. It is rich in B-lipoproteins which give it a characteristic turbid appearance, and also contains a large part of the serum a-globulins. Bachtold and Gebhardt [ 1 ] using quantitative electrophoretic analysis, determined the patterns of serum utilisation during the outgrowth in vitro of monkey kidney cells. They noted that the main protein utilisation occurred during the first three days of their cultures, when monolayers had become well established but were not confluent. The main protein loss was observed in the a,-globulin fractions. As one of the main activities of cells during the establishment of a monolayer on glass appears to be connected with adhesion, this observation on the selective loss of the &,-fraction is regarded as corroboratory evidence of the activity of the fraction G2. It must be noted that this fraction is only interchangeable with whole serum for adhesion of cells to glass and not for nutrition. Lieberman and Ove [ll] obtained a purified serum fraction which caused cells to adhere to glass surfaces. This material was composed mainly of glycoprotein, and its electrophoretic mobility was that of the cc-globulins. It was heat-labile, which would account for the effects of heat inactivation of serum described in the present communication. a-globulins were also associated with cellular adhesion to glass by Fisher, Puck and Sato [7], who noted that the active component appeared to resemble fetuin. This is an a,-globulin comprising 45 per cent of foetal calf serum protein. However, Lieberman an 1 Ove [la] have shown that fetuin can be separated from the “adhesion-active” protein factor by column chromatography. Strain 929 clone L (mouse Iibroblast) cells can grow and adhere to glass chemically defined media as described by Evans and her in protein-free, associates [5, 61 and Waymouth [20]. However, it is difficult to grow most cell lines adherent to glass in such media, immediately following trypsin-treatment (see above). This suggests that the substances involved in the “protein-mechanism” of adhesion can be produced immediately by some cells, but have to be provided in the medium for others. Rappaport [18] has shown that trypsinised monkey kidney cells will spread on glass in the absence of serum, in the presence of high concentrations of calcium ions (physiological x 4). The calcium-dependent mechanism is considered to coexist with the protein mechanisms of adhesion and will be discussed in a future paper. Experimental
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Cellular
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adhesion and serum SUMMARY
Some effects of serum on the adhesion of tissue cells to glass in culture, are described. 1. The amount of serum required for adhesion is greater in trypsin-treated than in washed cells. 2. From the aspect of cellular adhesion, the lipoprotein-rich serum fraction G2 may replace whole serum. 9. A method is described for the quantitative assessment of the effects on cellular adhesion of species difference and heat inactivation of serum. The results are discussed in terms of cell-membrane structure and the composition of culture media. I have received much help and encouragement from Professor R. J. \‘. Pulvertaft throughout these experiments. I am indebted to Dr. H. B. Fell for giving me considerable advice on the presentation of this paper. My thanks are also due to Miss E. R. Blake and Miss. H. Youle for technical assistance. This work was made possible by a generous grant from the British Empire Cancer Campaign. The various serum fractions were kindly provided by the Lister Institute. REFERENCES J. G. and GEBHARDT, L. P., Expfl. Cell Research 13, 432 (1957). 2. BIRBECK, M. S. and MERCER, E. H., Nature 178, 985 (1956). 3. DULBECCO, R. and VOGT, M. J., J. Exptl. Med. 99, 167 (1954). 4. EASTY, G. and LOWICK, J., unpublished data (1957). 5. EVANS, V. J., BRYANT, J. C., FIORAMONTI, M. C., MCQUILKIS, W. T., S.ZNDFOEID, K. K. and EARLE, W. R., Cancer Research 16, 77 (1956). 6. -Cancer Research 17, 315 (1957). 7. FISHER, H. W., PUCK, T. T. and SATO, G., Proc. Satl. Acad. Sci. 44, 4 (1958). 8. HALE, E. W., JVature 157, 802 (1946). 9. IMMERS, J., Exptl. Cell Research 6, 127 (1954). 10. KEKWICK, R. A. and MACKAY, M. E., Ned. Research Council Spec. Rep. Ser., No. 286 (1954). 11. LIEBERMAN, I. and OVE, P., Biochim. et Biophys. Acta 25, 449 (1957). 12. ~ J. Biol. Chem. 233, 637 (1958). 13. LISON, L., Stain Technol. 29, 131 (1954). 14. MERCER, E. H., Nature 180, 831 (1957). 15. MERCER, E. H., personal communication (1958). 16. PUCK, T. T., MARCUS, P. I. and CIECIURA, S. J., J. Exptl. Med. 103, 273 (1956). R. .J. V., WEISS, L., DAVIES, J. R. and WILKIXSOX, .J. H.. J. Pathol. Bacferiot. 17. PULVERTAFT, (in press 1959). 18. RAPPAPORT, C., Proc. Sot. Exptl. Bio/. Med. 91, 464 (1956). 19. RINALDINI, L. M. and LUCY, J. A., B.E.C.C. Ann. Rep. 244 (1956). 20. WAYMOUTH, C., J. Nafl. Cancer Inst. 17, 315 (1956). 21. WEISS, L., Exptl. Cell Research 14, 80 (1958).
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