Changes in the properties of cell-substrate adhesion during cultivation of chicken fibroblasts in vitro in a serum-free medium

Experimental Cell Research 68 (I 971) 88-96

CHANGES IN THE PROPERTIES OF CELL-SUBSTRATE DURING

CULTIVATION

OF CHICKEN

IN VITRO IN A SERUM-FREE

ADHESION

FIBROBLASTS MEDIUM

M. TAKEICHI Laboratory

for Cell Diffrventiation

ami Morphogenesis, Institute for Biophysics, Faculty of Science, Uniwrsity of Kyoto, Kyoto, Japan

SUMMARY Fibroblasts from a culture of chick embryo sclera were trypsinized and plated in serum-free Eagle MEM in Falcon plastic Petri dishes at 37°C. The inculated cells rapidly attached to the plate. The once-attached cells were not detached from the plate by treatment with either crude trypsin or EDTA within 60-180 min of inoculation, but after 24 h many cells became easily detachable by these treatments. This change did not occur in the cold. When the cells were plated in dishes which had been coated with conditioned medium (CM) harvested from a culture incubated for 24 h, the attached cells were detachable by trypsin or EDTA even within 180 min of inoculation. Divalent cations were unnecessary for cell attachment to the clean plastic, but their presence was necessary for cell attachment to the CM-treated plate. This effect of CM was not detected when the CM-coated plate was previously treated with crystallized trypsin. These observations suggest that the cells-adhering tb substrate coated with the material(s) contained in CM become sensitive to trypsin and EDTA. The material(s), presumably extracellular, may be released from the cells into both medium and interface between cells and plastic during culture in vitro in serum-free media.

Most cells can adhere to non-cellular substrates. The surface properties of the substrates are, however, important for cell adhesion. By coating a substrate with serum [15] or chemicals such as fatty acid [IO, 1I] or metal [3], cell adhesion is greatly affected. It has been suggested that some cellular products also affect cell adhesion to the substrate. Rosenberg [9] reported the deposition of a material exuded from cells on glass. Weiss [ 161 indicated that a glass surface was altered, so that cells were more adhesive to it, by the presence of a material that was left on the glass after cells cultured on it had been mechanically removed. In spite of these findings, the existence of extracellular material(s) concerned with cell adhesion to subExptl Cell Res 68

strates does not yet seem to be well confirmed, and our knowledge concerning the role of such material(s) is still meager. In order to analyse the mechanism of cell adhesion to substrates, it seems important, first of all, to clarify the issue of whether cells actually produce some material(s) relating to cell adhesion. The use of embryonic cells in the study of cell adhesion will be particularly valuable, because here will be an opportunity to provide some basic knowledge for the analysis of morphogenetic phenomena in animal development. In such a study, the use of serum-free culture media seems to be essential,

because

the

constitute a “noise”

presence

of

serum

may

that will interfere with

Changes of cell-substrate adhesion the exact detection of the predicted active substance(s) produced by the cell itself. Freshly isolated embryonic cells, however, are often difficult to maintain for a long period in serum-free media, as pointed out by Taylor [I 51. We have succeeded in maintaining fibroblasts derived from the sclera of chick embryos in a serum-free medium for a few weeks in a healthy condition. By using a subculture system it was found that a gradual change occurs in the characteristics of cell-substrate adhesion, in relation with the production of a material(s) presumably extracellularly released from living cells. MATERIAL

AND METHODS

Material Sclera removed from chicken days of incubation was used.

embryos

at

12-13

Solutions for cell dissociation and culture media Balanced salt solution (BSS) was prepared by modifying Hanks solution to be used in the air phase. Its composition is NaCl 8.0 g, KCI 0.4 g, KH,PO, 0.06 g, Na,HPO, 12H,O 0.87 g, CaCI, 0.11 g, MgSO, 7H,O 0.247 g and glucose 1.0 g in 1000 ml double-glass-distilled water. Ca2+ and Mg2+-free balanced salt solution (CMF) was prepared from BSS. 0.1-0.5 “” crude trypsin (Difco, 1 :250) in CMF was used for dissociation, and 0.05 u0 crystallized trypsin (Sigma, 2) in CMF was used for digestion of a material in conditioned medium. The pH of the trypsin solutions was adjusted to about 7.6 with 1 N NaOH. 2 mM EDTA in CMF, pH 8.0, adjusted with I N NaOH, was used to detach cells from plates. Eagle MEM (Nissui, Tokyo) supplemented with 6 % fetal calf serum (GIBCO. New York) was used for the primary culture. Serum-free MEM was used for the secondary culture. These media contained respectively 0.24 g and 0.20 g of NaHCO,, in 100 ml. Phospate-buffered serum-free MEM with or without Ca”‘~ and Mg’+ was prepared by dissolving powder of salt-free MEM (Nissui) in BSS or CMF, respectively. The pH of both media was adjusted to 7.4 with I N NaOH. CMF was autocalved. The other solutions were filtered through a Millipore HA filter, which was washed before use according to Cahn et al. [2]. Trypsin solutions and sera were stored at -2o”C, the others, at 4’C.

Cell culture Eyeballs were removed from chick embryos in BSS and the neural retina and pigment cpithelium were

89

removed. Layers of sclera were peeled from the scleral cartilage with forceps under a dissecting microscope. Sclerae isolated from seven embryos were transferred to a centrifuge tube with about 5 ml of 0.5 :b trypsin, and incubated for 30 min at 37 “C. As much as nossible of the trvnsin solution was removed without disturbing the softened tissues, and 4 ml of cold MEM with serum (culture medium) was added. The tissues were gently pipetted until the cells were dispersed. After the cell suspension was filtered through 4-layered gauze, it was centrifuged at 120 g for 4 min, and the supernatant was discarded. The cells were resuspended in about 8 ml culture medium and cell-counting was done with a FuchsRosenthal hemocytometer. About I IO” cells were plated into 3 ml culture medium in a 60-mm Falcon plastic Petri dish. The incubation was carried out in a water-saturated atmosnhere of 5 o,, CO, in air. The medium was changed every second day-for the first 6 days, and every day thereafter. The ceils multiplied to 6-10 i IO” per dish after 8-10 davs. although their growth rate varied somewhat with the batch of serum in the culture medium. These cells were sub-cultured. After removing the culture medium, the cells were washed once with CMF and 2 ml of 0.25”,, trvosin was added to incubate for I5 min at 37 ‘C. Thk’cells thus harvested from three dishes, after two washings with CMF by centrifugation, were suspended in 9 ml phosphatcbuffered serum-free MEM with Ca”’ and Mg”&, and incubated for IO-15 min at 37’C for complete removal of the trvnsin or serum proteins oresumablv remaining on the-cell surface. After another centrifugation, the cells were finally suspended in 8 ml of the serum-free MEM. The cells obtained by this method appeared round with smooth outlines, and were fully dispersed. I IO’ cells were usually plated with 3 ml culture medium into a 60-mm Falcon plastic Petri dish to incubate at 37-C in a watersaturated atmosphere of 51’,, CO, in air. Sometimes phosphate-buffered serum-free Eagle MEM was used for short-term cultures of up to 3 h, since bicarbonate-CO, buffer required a somewhat longer time to be equilibrated. The pH of these media was carefully maintained at approximately 7.4 throughout the period of culture.

Measurement of cell attachment and detachment The secondary culture described above was an assaying system for studying the properties of cell adhesion to the plastic surface. After an appronriate period of incubation of the secondary culture, all floating cells were collected by gentle washing of the culture with about 4 ml CMF twice, with care taken not to disturb the cells adhering to the plate. The cells were transferred to a 12-ml conical centrifuge tube, into which about 0.1 ml serum had previously been dropped to prevent cell adhesion to the wall of the tube. After centrifugation the pellet was flushed to obtain 1.0 ml suspension in CMF with serum for cell counting with the Fuchs-Rosenthal hemocytometer. After reinoving the floating cells, 2.5 ml of 0.1 “,) E.xptl Cell Rrs 68

90

M. Takeichi

trypsin or 2 mM EDTA was gently poured into the dish. The dishes were placed on a gvratorv shaker (New Brunswick Scientific Co., Modei G-2;) which was rotated at 90 rpm for an appropriate time at 37°C. The detached cells were collected as described above and counted. In order to calculate the percentage of detached cells, the total number of cells adhering to the plate must be obtained. This was estimated by counting the number of unattached cells in the culture within 3 h of inoculation. However, the possibility of cell loss by lysis may not be neglected in the culture after a long-term incubation. In this case, the total number of adhering cells was directly counted by detaching all cells from the plate with vigorous pipetting after a 30-min incubation in 2 ml of 0.5 “;, trypsin. The data in each experiment show an average of values obtained from 2 or 3 dishes.

RESULTS Attachment of cells to the plate The rate of cell attachment to the plastic plate was measured in the secondary cultures in the serum-free- medium. It was observed that by 30 min after inoculation, 90-95 “;, of the cells attached, while the cells left unattached by this time no longer attached to the plate (fig. 1). The number of floating cells tended to increase during incubation as the result of spontaneous detachment of cells from the plate (fig. 2). This fact indicates that a small fraction of the attached cells lose adhesiveness to the plate, perhaps partly due to cell death. Most cells attached to the plate, however, started to spread within 60 min after inoculation and exhibited a fibroblastic appearance at 180 min (fig. 3). Detachment of cells by treatment Mlith trypsin and EDTA The sensitivity of the adhering cells to trypsin and EDTA was tested. The cells at 60 min and 24 h after inoculation were incubated in trypsin and EDTA for various periods, and the numbers of detached cells were counted (fig. 4). BSS was used as a control of the enzymatic or chelating activity of the reagents. Attachment of the cells Exptl Cell Res 68

I:;I /f--60

4 Oo

10

20

30

40,

50

60t

Fig. 1. Abscissa: time (min); ordinate:

‘I<)cells attached. Rate of cell attachment to the plastic in serum-free Eagle MEM.

cultured for 60 mm was hardly affected by either trypsin or EDTA; usually less than IOC1,,of the adhering cells were detached. On the other hand, high percentages of the cells cultured for 24 h were detached with the same treatment, their number reaching a maximum after 30-min. Although the percentages of detached cells varied from experiment to experiment, usually 60-90 “;, of thecells adhered to theplateweredetached with trypsin, and 30-60 o. with EDTA. The failure of these reagents to detach all the adhering cells was not due to a loss of their activity, since these trypsin or EDTA solutions used to detach the cells were still capable of detaching cells in other plates. The number of cells detachable with trypsin increased with the time of culturing; sometimes almost 100 O. of the cells became detachable with trypsin after 24-72 h. The number of cells detachable with EDTA remained much lower, and often rather decreased with longer culturing. Fig. 5 shows a comparison of the changes with time in trypsin-(or EDTA-)sensitive cell4l

Fig. 2. Abscissu: time (hours); ordinate: cells/dish IO 5. Number of cells remaining unattached to plate at various times after inoculation. Data from three experiments are shown separately.

Changes of’ cell-substrate adhesion

Fig. 3. Photomicrographs of cells attached to the 1Jlastic, 180 min after inoculation: 180. to CM-treated plate.

substrate adhesion during culture in serumfree medium. In order to test whether such a change in the adhesive properties of the cells, from trypsin-insensitive to trypsin-sensitive, is temperature-dependent, the culture plates were placed at OC for 24 h after an initial 60 min at 37’C for cell attachment. As shown in table 1, the number of cells detachable with trypsin and EDTA hardly increased after 24 h culture under cold. &ffect of conditioned medium on attachment and detachment of cells In some preliminary tests, it was experienced that cell density in a plate affected the percentage of cells detachable with trypsin or EDTA. This fact suggests the possibility that “conditioning” of the medium by cells may influence their adhesive property. To investigate this, cells were plated in condi-

(a) to normal

plate;

9I

(b)

tioned medium (CM) collected from cultures incubated For 24 h. A considerable number of cells attached to the plate with CM were easily detached by treatment with trypsin even at 60 min after inoculation (table 2). The effects of CM on the attachment and detachment of cells were, therefore, investigated further. CM was prepared as follows; 2 1: 10” cells were inoculated per plate with 3 ml serumfree MEM. After incubation for 24 h at 37°C the medium was harvested and centrifuged at 230 g for 10 min to remove floating cells. The resultant supernatant, CM, was stored at 4’C until use. A test was first made to determine whether material(s) in the CM capable of affecting the adhesive property of the cells might adsorb onto the plastic surface. 2 ml of CM was spread evently on the surface of a plate. The plate was placed in a CO,-incubator for 60 min or 24 h at Exptl Cell Res 68

92

M. Takeichi

Table 1. Effect of incubation at 0°C on cellular sensitivity

to trypsin or EDTA

,:o_

‘Z

~~ the ‘“““‘“‘per after inoculation :ho:vr

a-

+<

2..:: 1

LoI

x/\ x .

20

15

cent age detached with Trypsin

EDTA

;;;

;;

xjx

0 0

for detachment

30

7

9

45

60

Fig. 4. Abscissa: time (min); ordinate: o,, cells detached. Percentage of cells detached after incubation for various periods in 0.1 96 crude trvkn (o--o). 2 mM EdTA ( %). or BSS (iLo). Top: &s at 60 min after inoculation; bottom: cells at 24 h after inoculation. In the lower figure, data from two experiments are shown separately.

37°C. After incubation, the CM was removed, and the dish was washed three times with 5 ml of CMF and 5 ml of CMF was left in it until use. After the CMF was removed, cells were plated into such a treated plate with serum-free MEM. Attachment of the cells was somewhat retarded in the CM-treated plates, particularly in those treated for 24

h (fig. 6). At 120-180 min after inoculation, the cells adhering to the treated surface extended much longer pseudopodia than those of cells in untreated plates (fig. 3), suggesting a difference in the affinity of cells to the CM-treated and untreated substrate. When the cells were treated with trypsin or EDTA at 180 min after inoculation, many of them were detached from the CM-treated surface and only a few from the untreated plate (table 3). These results show that CM alters the adhesiveness of cells to the plate, perhaps by adsorbing onto its surface. The fact that the cells are detachable from the CM-treated plate with EDTA suggests that divalent cation(s) are required for attachment to the treated plate. A test was then made by plating the cells, using Ca”+- and Mg2+-free MEM, in CM-treated and untreated plates. The cells attached rapidly to Table 2. Effect of CM 011 cellular to trypsin for clftachnwnt

Fig. 5. Abscissa: time (hours); ordirme: ‘lo cells detached. Change of the percentage of cells detached. Incubation for 30 min in 0.1 ‘111crude trypsin (C-C;) and 2 mM EDNA ( I - / ). Data from two experiments are shown separately. Exptl Cell Res 68

sensitil>ity front the substrate

Expt

(I~,detached

1. Control CM

3.0 28.0

2. Control CM

3.5 24.0

3. Control CM

9.5 61.5

Changes of cell-substrate adhesion 8 .

100 -

.-80

-

CI

60 -

&----A

93

Table 4. Effect of trypsinization of CM-treated plates on cellular sensitivity to trypsin or EDTA for detachment from the substrate Percentage

/

detached with

40 -

Expt

20 -

Untreated plate Untreated plate + trypsin CM-treated plate CM-treated plate ~- trypsin

Trypsin 2.9 6.0 44.8 7.1

EDTA 4.1 1.2 27.4 3.3

60

Plates treated with CM for 60 min.

180

OOk-kkhC?

Fin. 6. Abscissa: time (min); ordinate: % cells attached. Comparisons of the iate of cell attachment to plates treated with CM in normal (O-O) or in Ca”+- and Mg”+-free (J-L) media, and to untreated plates in normal (O-G) or in Cazf- and Mg2+-free ( i. - j ) media. Top: plates treated for 60 min; bottom: plates treated for 24 h.

the untreated plate in the absence of divalent cations, but attachment to the CM-treated plate took place very slowly and a large number of cells remained unattached for at Table 3. Effect of treating plates with CM on cellular sensitivity to trypsin or EDTA for detachment from the substrate Percentage detached with Expt

Trypsin

1. Untreated Treated’

3.4 55.5

4.4 25.0

2. Untreated Treateda

3.6 37.8

4.0 1 I.0

3. Untreated Treateda

3.1 22.3

1.8 16.6

EDTA

Plates treated with CM for 60 min, and cells assayed at 180 min after inoculation. a Counts of cells detached in BSS were subtracted from the original values.

least 3 h (fig. 6). If they adhered, their spreading was inhibited in Ca2+- and Mg2+-free medium in the treated plate. To test the possibility that detachment of cells from the CM-treated plate with trypsin was due to enzymatic digestion of a material(s) coating the plate surface, 2 ml of 0.05 96 crystallized trypsin was placed in a CM-treated plate and incubated for 60 min at 37°C. Since the trypsin molecule itself may be adsorbed onto the plastic surface and affect the adhesion of cells, a plate untreated with CM was also incubated with the enzyme as a control. After incubation, the trypsin was removed and the dishes were washed three times with 5 ml CMF and used immediately. The cells were plated into four kinds of dishes: CM-treated plates incubated with or without trypsin, and untreated plates incubated with or without trypsin. When the cells were tested at 180 min after inoculation, the result showed that pretreatment of CMtreated plates with crystallized trypsin evidently reduced the number of cells detached with crude trypsin (table 4). Takahashi & Okada [13] found that some molecules which were contained in CM collected from a monolayer culture of chicken fibroblasts raised the plating efficiency of Exptl Cell Res 68

94

40 c o+Ll 0

M. Tukeichi

EDTA. The result summarized in table 5 indicates that CM treatment of cells before plating hardly influenced their further attachment, in contrast to the positive influence of previously treating the substrate with CM.

d IO

20

30

40

I 50

I 60

Fig. 7. Abscissa: time (min); o/&zafe: 0Ocells attached. Comparison of the rate of attachment of treated (O-O) and untreated V--c) cells to the plates.

cells on plastic plates. They suggested that these molecules adsorb onto the cell surface to display their effect. A test was therefore made to see whether the present effect of CM also might depend on the adsorption or absorption of effective substance(s) to or by cells. 6 1 lo6 cells dissociated and washed as described above were suspended in 9 ml CM in a centrifuge tube and incubated for 60 min at 37°C. After incubation, the cells were centrifuged, washed two times with BSS, and suspended in fresh culture medium. The rate of attachment to the plate of these CM-treated cells was compared with that of untreated cells. The CM-treated cells attached to the plate within 30 min similarly to the untreated cells (fig. 7). At 60 min after inoculation, the cells were treated with trypsin and

Table 5. Effect of treating

cells Hxith CM on cellular sensitivity to trypsin OP EDTA for detachment from the substrate Percentage detached with Expt

Trypsin

EDTA

1. Untreated Treated

2.0 3.3

2.6 2.9

2. Untreated Treated

6.5 1.5

5.5 9.0

3. Untreated Treated

2.1 3.8

2.8 3.4

Exptl

Cell Res 68

DISCUSSION The rapid attachment of cells to glass in strum-free media has already been reported by several investigators [4. 9, 151. The same phenomenon was observed in the present experiment, in which fibroblasts from sclerae of chick embryos in a serum-free medium arc found to adhere to the plastic surface of Falcon Petri dishes. In both cases, attachment occurs too rapidly to assume the synthesis of any interfacial material between cells and substrates as a prerequisite for the initial step of cell adhesion. It is possible that the two surfaces of cell and substrate may be attracted directly by physico-chemical force(s). Such a state of adhesion. however, cannot persist for long in the cultivation of the present cells, because the property of cell adhesion to plastic changes with the length of cultivation, as revealed by changes in the sensitivity of adhesion to trypsin or EDTA. The occurrence of such change can be mainly attributed to the appearance of a material in the interface between cells and plastic, as suggested by the observation that when the plastic surface was treated with CM, the cell adhesion was sensitive to trypsin or EDTA even after a brief cultivation period. This fact should indicate that some material(s) contained in the CM adsorbed onto the plastic surface to alter its property. Since the CM was actually collected from a culture in which many cells could be detached with trypsin and EDTA, it seems reasonable to consider that the adsorption of the same material(s) onto the plastic surface must

Changes

occur in the secondary culture of the present assay system. It is therefore concluded that the material(s) is released by the cells into the medium as well as into the interspace between cells and plastic, and, as the result of its adsorption onto the substrate, the cells can establish trypsin-(or EDTA-)sensitive adhesion to the plastic. Since the trypsinor EDTA-sensitive cell adhesion did not occur in cultures maintained at 0°C such material(s) must be released through an energy-dependent process. There have been several reports concerning extra-cellular material(s) which are believed to play a crucial role in cell adhesion. Rosenberg [9] described that cells cultured in vitro produced a material, which he called “microexudate”, within a few hours of contact with the glass. There are some differences between his and our results. In his experiment, cells were not detachable with trypsin and EDTA even at the time when the microexudate became detectable on the glass, unless the cell or glass was washed with serum. The material(s) postulated in the present work, on the other hand. if adsorbed onto the plastic, facilitated the detachment of cells with trypsin and EDTA even in a serum-free condition. Its activity therefore seems to correspond rather to the property of serum indicated by Rosenberg. The production of Rosenberg’s microexudate was independent of temperature. Taken altogether, his material(s), which was detected on the glass after mechanical removal of cells cultured for a short period, seems to be a different molecule from the one found in the present experiment. This notion is also supported by another observation of Rosenberg’s, that the material adsorbable to glass was not released into the medium by the cells. Of course, this is not the case in our experiment, although we did not use glass as the substrate. It should be noted, however,

of‘ cell-substrate

adhesion

9.5

that Rosenberg used a different cell type from that of the present experiment. His cell was a strain cell of human conjunctiva origin. It is conceivable that some types of strain cells may not release macromolecular material into the medium, as suggested by Oellermann & Miller [8], whereas embryonic cells cultured in vitro do release macromolecules [5, 6, 7, 8, 12, 13, 141, some of which are shown to be effective for cell adhesion. It is necessary to determine whether the material(s) in the CM described here is a true extracellular molecule produced through normal cellular metabolic processes. or a substance which leaked from the cells following their lysis or other pathological condition. In fact, a fraction of the inoculated cells remained unattached or were detached spontaneously from the plate during the culture. and some of them tended to be cytolysed. Therefore, cytoplasmic materials exuding from these cells must be included in the CM to some extent. It has sometimes been observed, however, that CM taken from quite healthy cultures in which relatively little cell debris appeared floating in the medium. is similarly effective in rendering cells trypsin(or EDTA-)sensitive after a very brief period of cultivation. This suggests that the factor essential for modifying the property of cell adhesion is not a cytoplasmic constituent abnormally leaking from cells, but is actually secreted by the living cells. The material which is contained in CM and present between cells and plastic, probably does not consist of a single kind of molecule, because the number of cells detached with EDTA varied independently of the number detached with trypsin from experiment to experiment, indicating the presence of at least two kinds of molecules mediating cell adhesion to the plastic. Cells can adhere to a clean plastic surface in the absence of divalent cations. The same Exptl Cell Res 68

96

M. Takeichi

is true in the case of cell adhesion to glass in serum-free media [I, 4, 9, 151. On the other hand, once the plastic surface was treated with CM, the presence of divalent cations became necessary for cell attachment. This suggests that adsorption of the material(s) to the plastic leads to an alteration in the physico-chemical properties of its surface. Ilf such material(s) can be adsorbed onto the cell surface itself or the intercellular matrix in tissue, some effect would be expected also on the cell-to-cell adhesion or cell-to-matrix adhesion in vivo. It is still not clear, however, whether the material(s) can actually adsorb onto the cell surface.

The author is indebted to Professor T. S. Okada for encouragement and critical reading of the manuscript. This work was partly supported by a grant for basic cancer research from the Ministry of Education of Japan.

Exptl Cell Res 68

REFERENCES 1. Berwick, L & Coman, D R, Cancer res 22 (1962) 982. 2. Cahn, R D, Coon, H G & Cahn, M B, Methods in developmental biology (ed F H Wilt & N K Wessells), p. 493. Thomas Y. Crowell. New York ( 1967). 3. Carter. S B. Nature 208 (1965) 1183. 4. Easty,‘G C; Easty, D M’ & Ambrose, E J, Exptl cell res 19 (1960) 539. 5. Halpern, M & Rubin, H, Exptl cell res 60 (1970) 86. 6. - Ibid 60 (1970) 96. 7. Lillien. J E, Develou biol 17 (1968) 657. 8. Oellermann, R A & Miller, E M; J cell physiol 74 (1969) 299. 9. Rosenberg, M D, Biophys j I (1960) 137. 10. - Proc natl acad sci US 48 (1962) 1342. ’ 1I. - Science 139 (1963) 411. i 12. Rubin. H. The suecificitv of cell surfaces (ed B D Davis &’ L Warren) - p. 181. Prentice-Hall, Englewood Cliffs. N J (1967). 13. Takahashi, K & bkada, T S, Develop growth & differ 12 (1970) 65. 14. - Ibid. In press. 15. Taylor, A C, Exptl cell res, suppl. 8 (1961) 154. 16. Weiss, L, Exptl cell res 25 (1961) 504. Received March 15, 1971