thermodynamic aspects of plant cell adhesion to polymer surfaces

Appl Microbiol Biotechnol (1988) 29:346--355 Applied Microbiology

Biotechnology © Springer-Verlag 1988

Thermodynamic aspects of plant cell adhesion to polymer surfaces

P. J. Facchini 1, A. W. Neumann TM, and F. DiCosmo 1'3

1 Centre for Plant Biotechnology, Department of Botany, 2 Department of Mechanical Engineering, and 3 Institute of Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada MSS 1A1 4 Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada MSG 1X8

Summary. A thermodynamic model of particle adhesion from a suspension onto a solid surface is used to predict the extent of adhesion of suspension-cultured C a t h a r a n t h u s r o s e u s cells to the following polymer substrates: fluorinated ethylene-propylene (FEP), polystyrene (PS), polyethylene terephthalate (PET), sulphonated polystyrene (SPS), and glass. According to this model, the extent of adhesion is determined by the surface tensions of the plant cells, the polymer substrates, and the suspending liquid medium. Experimen- tally, adhesion of the washed plant cells was found to decrease with increasing substrate surface tension, following the sequence FEP > PS > PET > SPS > glass, when the surface tension of the liquid was greater than that of the plant cells, in agreement with the model. Howev- er, adhesion increased with increasing substrate surface tension when the liquid surface tension was lower than the cellular surface tension, also in agreement with the model. When the liquid and cellular tensions were equal the extent of adhesion was independent of the substrate surface tension. This also agrees with model predictions and leads to a value for the surface tension of C. ro-

s e u s cells of approximately 54 ergs/cm 2 which is in agreement with a value obtained from contact angle measurements on layers of cells and sedimentation volume analysis. The cellular surface tension determined by the sedimentation volume method showed a biphasic alteration during growth cycles of C. r o s e u s cell cultures. These var- iations (between 55 and 58 ergs/cm 2) agree with the pattern of adhesion previously described.

Offprint requests to: F. DiCosmo, Department of Botany, Uni- versity of Toronto, Toronto, Ontario, M5S 1A4, Canada

Introduction

The adhesion of cultured plant cells to a support matrix effectively immobilizes the cells and pro- motes a cellular aggregation that may simulate or- ganized plant tissue. As with other methods of im- mobilization the aim is to improve the perform- ance of the classical plant cell bioreactor process for the production of secondary metabolites. Im- mobilized plant cells demonstrate greater meta- bolic activity than freely-suspended cells (Furuya et al. 1984; Lindsey and Yeoman 1985), and pro- vide an alternative in fermentation applications because of the possibility of re-using the biocata- lyst. Also, there is no need to separate the product from the catalyst in continuous flow-through cul- turing and extraction procedures. The potential advantages (for product isolation) of immobilized plant cell reactors relative to conventional plant cell culture methods have been extensively re- viewed (Tanaka 1981; Brodelius and Mosbach 1982).

The large scale cost-effectiveness of an immo- bilization process is enhanced by using low-cost polymer material that can be manufactured in any geometry as the support substrate, and by exploit- ing the spontaneous adhesive behavior of the cultured plant cells. For this purpose, the attachment of cells to surfaces is recognized as mediated by two independent processes: 1) the initial physico- chemical events occurring when the cell surface interacts with the support substrate, and 2) the physiological response of the organism in producing extracellular adhesive secretions (Fletcher 1980). Indeed, the firm retention of plant cells on polyurethane and polyvinyl supports has been shown to involve extracellular mucilaginous films (Robins et al. 1986). However, the initial interactions of cultured plant cells with surfaces are gov-

P. J. Facchin i et al.: P lant cell adhes i on 347

erned by surface forces acting at the plant cell- substrate interface (Facchini et al. 1988), and remain to be fully characterized. An understanding of the fundamental mechanisms governing the initial process of plant cell adhesion to polymer materials is essential in order to control and modify the factors which promote cell adhesion and increase the strength of the adhesive bond between the cells and the substrate.

Cell adhesion phenomena have been investi- gated in relation to surface-induced thrombosis and embolism (Absolom et al. 1979), infection of various tissue (Woods et al. 1980), ship-fouling (Rosen et al. 1981), fermentation (Marshall et al. 1971), decay (Atkinson and Fowler 1974), and wastewater and sewage treatment (Characklis 1973). This literature demonstrates that the process is too complex to be studied in toto, therefore, we will initially examine individual aspects. The extent of adhesion of suspension-cultured Catha- ranthus roseus cells to polymer substrates is re- lated to the substrate surface tension as predicted by thermodynamic considerations (Facchini et al. 1988). In this paper we investigate more fully the role of surface thermodynamics in the process of plant cell adhesion.

Theoretical considerations

A fundamental question that a thermodynamic approach can answer is the directionality of a nat- ural process. This implies that a properly defined thermodynamic potential, the free energy function (F), will be minimized at equilibrium. There- fore, plant cell adhesion will be favoured if it causes the thermodynamic function F to decrease; if F increases then adhesion will not occur. When the effect of electric charges and specific bio- chemical interactions (e. g. receptor-ligand) can be neglected, the change in the free energy function (AF adh) per unit surface area is:

A F a a h = ?/cs - Y c t - ?/~t (1)

where F adh is the free energy of adhesion, ?/cs is the plant cell-substrate interfacial tension, ?/d is the plant cell-liquid interfacial tension, and ?'st is the substrate-liquid interfacial tension. The crucial point in the application of this simple free energy balance is the determination of values for the interfacial tensions ?/cs, ?/d, and ?/s~. These data may be obtained from an equation of state approach (Neumann et al. 1974), which allows the calculation of the surface tension ?/sv of a solid (S) in con-

tact with its vapour (V), from a measured liquid surface tension ?/t~ and the contact angle O which a drop of the liquid makes with the solid. Briefly, this technique uses Young's equation:

?/sv - -? /s l=?/ lv c O S O (2)

where )'so, ?'st, and ?/to are, respectively, the interfacial tension between a solid substrate S and the vapour phase V, between S and the liquid L, and between L and V; 0 represents the contact angle. Of these four quantities only ?/t~ and 0 are readily determined experimentally. Therefore, a further relationship between these and the other two quantities is necessary. It has been demonstrated thermodynamically that an equation of state of the form:

)'st =f(?/sv, ?%) (3)

must exist (Ward and Neumann 1974). Using experimental data for contact angles and liquid vapour interfacial tensions, equation 3 has been ex- plicitly formulated (Neumann et al. 1974) as:

(?/sv - - ?/lv) 2

)'st = 1 - 0 . 0 1 5 ~ (4)

Applying Young's equation to this relation yields:

cos O = (0.015 ?/s~-2.00) ?/1 y/~ ?/t o +?/to ¢ 7 2 ?/,o - 11

(5)

The surface tension of the substrate 7% may be calculated using equation 5. Accordingly, any interfacial tension ?/12 can be calculated from given or predetermined interfacial tensions ?'13 and ?/23 where the subscripts 1, 2, and 3 refer to the different phases. This allows all interfacial tensions in equation 1 to be calculated, making explicit thermodynamic predictions of the relative extent of plant cell adhesion to various substrates possible. Certain purely mathematical limitations of equa- tions 4 and 5 can be readily circumvented by the use of tables (Neumann et al. 1980a) and com- puter programs (Neumann et al. 1980b). These are available upon request.

The thermodynamic model predicts a complex pattern of adhesion. For example, a theoretical calculation of AP ~dh for the adhesion of cultured plant cells from suspension to various substrates as a function of ?/sv is illustrated in Fig. 1. This graph can be generated with data for the surface

348 P.J. Facchini et al.: Plant cell adhesion

- 1 2

E 0

¢- " 0

EL

- 8 -

- 4 -

O -

+4

~'lv > ')'cv

I I I I I I I :lO 20 30 40 50 60 70

~s,, (ergs/cm 2)

Fig. 1. Dependence of the change in free energy of adhesion (AF adh) of particles as a function of substrate surface tension Ycv. The plant cells are assumed to have a surface tension of 7/c, = 55 ergs/cm 2. For Ytv > 7/cv, Ylo = 72.5 ergs/cm 2 was chosen, while for y~v <y~, yzv=44.5 ergs/cm 2 was assumed

tensions of the three interacting components; y~v, y~o, and Yl~. The theoretical calculation demonstrates two distinct situations. When

Yl. < Yc~ (6)

A-b "udh decreases with increasing ys~, predicting increasing plant cell adhesion with increasing surface tension of the substrate over a wide range of Y~v values. Alternatively, when

ytv > y ~ (7)

the opposite pattern of adhesion prevails. In the case of equality

r,.=rco (8)

A f t ah becomes equal to zero independently of the value of Ysv. This limiting situation implies that adhesion does not depend on the substrate surface tension and should be zero if no other forces, such as electrostatic interactions, are active in the system (Neumann et al. 1980a).

The purpose of this paper, therefore, is to study the effect of the surface tension of the substrate (Ysv), of the suspending liquid medium (YI~), and of the cultured plant cells (Yc~) on the relative extent of plant cell adhesion to a range of polymer surfaces. We shall demonstrate below that plant cell adhesion can be predicted using the thermodynamic model outlined.

Materials and methods

Plant cell cultures

Cell suspensions of Catharanthus roseus (cell line LBE-1) have been maintained for one year in Murashige and Skoog medium (Murashige and Skoog 1962) containing 0.5 mg/1 alpha naphthalenacetic acid, 0.1 mg/1 kinetin, and 3% (w/v) sucrose. Cells were propagated as 75 ml cultures in 250 ml Erlenmeyer flasks in the dark at 27°C on a gyratory shaker (120 rpm). Stock cultures were subcultured every 14 days using a one to four dilution of cells to medium. Biomass was evaluated as fresh weight (Wilson et al. 1971) by determining the mean value of three flasks of cell suspension harvested each day during the 14 day growth cycle.

Polymer substrates and suspendin9 liquid medium

Plant cell static adhesion experiments were performed with the substrate materials listed in Table 1. Preparation of the surfaces was performed as described earlier (Facchini et al. 1988) and as indicated in Table 1. The advancing contact angles were measured by means of the conventional sessile drop technique using double distilled water (Neumann and Good 1979). The substrate surface tensions were determined using the equation of state approach (Neumann et al. 1974).

The suspending liquid medium used for adhesion and sedimentation volume experiments was distilled water containing known amounts of 1-propanol to a maximum of 6% (v/v) resulting in a liquid surface tension range from 72.5 to 44.5 erg/cm 2. The effect of various concentrations of 1-propanol

Table 1. Solid substrates used in plant cell adhesion experiments

Material Source Preparation Contact angle Surface with water tension (OHio [°]) (ergs/cm ~)

Glass Coming Glass Works, Coming, New York Used as received 0 Sulphonated polystyrene (SPS) Central Research Lab, Dow Chemical Co. Used as received 24+3 Polyethylene terephthalate (PET) Celanese, Toronto, Canada Used as received 60___ 2 Polystyrene (PS) Central Research Lab, Dow Chemical Co. Used as received 95 +2 Fluorinated ethylene-propylene (FEP) Commercial Plastics, Toronto, Canada Heat press 110___ 3

>72.5 66.7 47.0 25.6 16.4

P. J. Facchini et al.: Plant cell adhesion 349

"E

=.

t 60- °~o

5 0

40 , I , 0 1

O~o~ ° ~ o

I : ~ : I : I : I : ~ 2 3 4 5 6 7

Percent p r o p a n o l / w a t e r (v/v)

Fig. 2. Reduction in the surface tension Ytv of distilled water by the additon of 1-propanol. Error bars (standard deviation are within the radius of the symbols

on the surface tension (Yzv) of distilled water is presented in Fig. 2; the specific 1-propanol-water mixtures used for the adhesion experiments are shown in Table 2. For sedimentation volume experiments a 2.5% (v/v) 1-propanol-water mixture re- suiting in a 7~v value of 55.5 ergs/cm z was also used. Liquid surface tensions were measured using the Wilhelmy technique (Neumann et al. 1973). The pH of the suspending liquid was maintained at pH 7.0 with the addition of 1.0 M NaOH.

Cell preparation

The cell suspensions used for all experiments were prepared by diluting a 75 ml cell culture with six volumes of distilled water and filtering the dilute suspension through a 500, 350, and 210 txm nylon mesh filter series under gentle vacuum. The resulting suspension was centrifuged at 600 xg for three minutes, and the pellet resuspended in fresh distilled water. For sedimentation volume and contact angle measurement experiments this process was repeated 3 times. For the adhesion experiments the process was repeated twice with the third resus- pension and centrifugation performed in a 100 ~tM EGTA solution rather than distilled water. These procedures produce cell suspensions consisting of greater than 97% small aggre- gates (2-5 cells) with low levels of extracellular polysacchar- ides and proteins in the suspending liquid (Facchini et al. 1988). For adhesion experiments the plant cells were centri-

Table 2. Surface tension of suspending medium at 25 o C

Medium Concentration of Surface tension 1-propanol Ytv ergs/cm 2 (%, v/v)

Distilled water 0 72.5 Water-l-propanol I 1 63.0 Water-l-propanol 2 2 58.0 Water- 1-propanol 3 3 53.0 Water- 1-propanol 6 6 44.5

fuged again at 600 xg for 3 min then resuspended in the appro- priate liquid (Table 2) as determined by te experimental protocol. The final suspensions were adjusted to a 1% packed cell volume (PVC). Cell viability was determined to be a greater than 98% in all 1-propanol-water mixtures (from 0 to 7% 1- propanol) over a one hour period. Viability tests were performed by dye exclusion of a 0.5% (w/v) solution of Evan's Blue stain (Taylor and West 1980) and by using the fluorescein diacetate method (Widholm 1972).

Adhesion protocol

Static adhesion experiments to the various substrate materials were performed as described previously (Facchini et al. 1988). Briefly, 1 ml of the 1% PCV plant cell suspension in the appro- priate test medium was placed into 1 ml capacity wells formed in Teflon blocks secured to the polymer surface retained on a glass microscope slide. Silastic gaskets were used between the Teflon blocks and the polymers to prevent leakage. Six replicate wells for each test liquid and polymer surface were tested. After a 20-min incubation period at 25°C the Teflon blocks with surfaces still attached were submerged and subsequently inverted in a distilled water bath at 25 °C for 10 rain to remove non-adherent cells. The blocks were removed from the bath and allowed to stand at room temperature for 20 minutes. Sub- sequently, the Teflon blocks were removed and the substrates were air dried.

The percent area of the substrate surface covered by adherent plant cells was determined using an automated image analysis system (Omnicon 3000; Bauch and Lomb, Rochester, NY) as described previously (Facchini et al. 1988). Ten area fields were required to analyse the entire surface of each well. Each of the reported values consists of the mean of all the fields from the wells of each test liquid-polymer surface combination on the specified sample day during the growth cycle. Plant cell adhesion experiments were performed in triplicate for day 4 cell cultures and every second day during one complete growth cycle.

Contact angle measurements on layers of cells

The surface tension of the suspension-cultured C. roseus cells was determined by measuring the contact angle formed by drops of distilled water on layers of the cells (van Oss et al. 1975). Smooth layers were produced by collecting cells from a 100 ml sample of a 1% PCV filter and washed suspension on a 0.45 ~tm HAWP Millipore filter under gentle vacuum. The re- suiting cellular layers were approximately 1-2 mm in thick- ness. A 20 ~tl volume of double-distilled water at 25 ° C and pH 7.0 was used to determine the advancing contact angle of the liquid on the cellular material at specific times after the forma- tion of the plant cell layer (van Oss et al. 1975). The contact angle of 10 different drops was determined, each on a previously unused area of the cell layer. Reported values represent the mean of these measurements. The experiment was repeated in triplicate at 2 days intervals during one complete growth cycle.

Sedimentation volume experiments

An alternative technique used to determine cellular surface tension uses the correlation of sedimentation volume, particle surface tension, and liquid surface tension (Vargha-Butler et al. 1985a). For these experiments it is essential to produce

350 P.J. Facchini et al.: Plant cell adhesion

plant cell suspensions of constant concentration in the various test liquids (Table 2, and 2.5% (v /v) 1-propanol-water). The filtered and washed plant cells were collected on a 105 lxm pore nylon filter under gentle vacuum. The vacuum was allowed to suction off water from the layer of cells for 60 s, and the cells were air dried for 3 min. Subsequently, 1.00 g samples were resuspended in 5 ml of the various test liquids. The final PCV concentration of these suspensions was approximately 17%. Cells manipulated in this way were greater than 95% viable as assessed by the Evan's Blue staining method (Taylor and West 1980). The final suspensions in the various test liquids were transferred quantitatively into 1 ml capacity Wintrobe micro test tubes (100 mm high with an inner diameter of 3 mm) using 9 inch long Pasteur pipettes. The tubes were maintained verti- cally and the cells were allowed to sediment under the influence of gravity at 25 ° C. The sedimentation volume was determined after 20 min. Three tubes were tested for each test liquid. These experiments were performed in triplicate during three consecutive growth cycles. Reported values represent the mean of combined data from the three growth cyles tested.

Results and discussion

The linear relationship between the extent of adhesion of Catharan thus roseus cells suspended in distilled water and the substrate hydrophobicity (surface tension) is consistent with the predictions of the thermodynamic model (Facchini et al. 1988). Three parameters control the extent of adhesion as described by this model: the surface tensions of the substrate (7/sv), the suspending liquid (76v), and the plant cells (7/c~). In order to use the thermodynamic model to predict the extent of plant cell adhesion in any given system values are required for all of these factors. The surface tensions 7s,and 76~ can be readily determined as pre- visously described. Measurement of the surface tension of the cultured C. roseus ceils is critical for a functional description of the surface thermodynamic adhesion process.

Previously, we suggested that changes in the surface tension of C. roseus cells could explain the consistent pattern of adhesion observed during growth cycles of the cell cultures (Facchini et al. 1988). Cell suspension cultures of C. roseus

proceed through an initial lag phase (day 0 to 2), an early (day 2 to 6) and late (day 6 to 10) exponential phase, and a stationary phase (day 10 to 14) as shown in Fig. 3. Filtered and washed C. roseus

cells demonstrate a biphasic adhesion pattern with maxima occurring at the early exponential and stationary phases of growth (Facchini et al. 1988).

In the present study, the cellular surface tension was initially determined from contact angle measurements on layers of cells (Fig. 4). Initially the contact angle (69) which the water drop makes

E-

@

E

25-

20-

15-

t 0 -

5 -

./ /

2 4 ; ~ I , I , I , I

6 8 10 12 14

T i m e ( d a y s )

Fig. 3. Growth cycle of C. roseus cells in suspension culture. The error bars (standard deviation) for some points are within the dot size

with the layer is close to zero degrees but, due to water evaporation, increases to reach a stable plateau. The contact angles of the plateau represent Young's contact angles, i.e., angles that are thermodynamically characteristic of the substrate (viz. the plant cells) and are not influenced by factors such as surface roughness (Neumann et al. 1974). For C. roseus cells these angles are usually stable for approximately 60 min, then the cells become desiccated and 69 rapidly increases. Using the plateau contact angle of 51 degrees and the surface

90.

o~

80.

70-

60-

50.

40-

30-

20-

10-

±

90 ,0o 14o 1B0', o'2 o'2 o' ' 240

T ime (m ins )

Fig. 4. Contact angle of double distilled water on layers of suspension-cultured day 4 C. roseus cells as a function of water evaporation from the wet plant cell substrate, measured in terms of time. Each value represents the average of 10 contact angle measurements on different drops placed at approximately the same time, on fresh areas of the substrate. Error bars are standard deviation

P. J. Facchini et al.: Plant cell adhes ion 351

tension ytv=72.5 ergs/cm 2 for double distilled water at 25 ° C, the surface tension (Yc~) of the day 4 C. roseus cells was calculated to be 52.5 ergs/ cm 2. Values of y~ obtained with this method were consistent for each replicate on every day of the growth cycle tested (i.e., at two-day intervals). The technique has been successfully applied to numerous microorganisms including human lym- phocytes (van Oss et al. 1975), granulocytes (Ab- solom et al. 1979), macrophages (Thrasher et al. 1973), erythrocytes (van Oss et al. 1975), porcine platelets (Absolom et al. 1982), serum proteins (van Oss et al. 1981), and bacteria (Absolom et al. 1983). However, the relatively large size of C. roseus cell particles causes the cellular layer to be highly absorbent. Consequently, the water drop remains visible on the surface of the cell layer for only about one scond. Although precise measurements are difficult, the contact angle was estimated by making repeated measurements.

The results for one typical adhesion experiment, using the various 1-propanol-water mixtures and performed on day 4 of the growth cycle, are presented in Fig. 5. Data from all adhesion experiments could not be combined because, although adhesion patterns observed were consistent for all experiments on each day tested, the absolute magnitude of adhesion varied from day to day during the growth cycle as well as among

35-

co 30-

25-

8 t5-

5-

: : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : 0 ,0 20 30 40 50 60 70

"~sv (e rgs / cm 2)

Fig. 5. Adhes ion of suspens ion cultured day 4 Catharanthus roseus cells as a funct ion o f surface tens ion Y,v for the various 1-propanol concentra t ions . Lines were p lo t ted using a least squares estimate. For graphical reasons error bars (95% confi- dence limits) are omit ted in some cases but are similar for all values. The poin ts for glass (y,v > 72.5 e rg s / cm 2) were not used to fit lines because its precise surface tens ion was unknown. The liquid surface tens ions used were: ( O ) - - 72.5 ergs /cm2; ( A ) - - 63.0 e rgs /cm2; ( ~ ) - - 58.0 e rgs /cm2; ( 0 ) - - 53.0 e rgs / cm2; and ( A ) _ 44.5 e r g s / c m 2

growth cycles as reported previously (Facchini et al. 1988). The theoretical predictions in Fig. 1 (i.e., whether or not the extent of adhesion will increase or decrease with increasing substrate surface tension) developed from free energy calcula- tions using the relevant interfacial tensions described in Eq. (1), are substantiated by our experimental observations (Fig. 5). When the surface tension Ytv of the suspending liquid is greatest, plant cell adhesion decreases with increasing Ysv values of the substrate. As the surface tension Yto is lowered, the change in the extent of adhesion of the C. roseus cells with increasing ys~ becomes less pronounced. At a certain intermediate Y1~ value between 58 and 53 ergs/cm 2, adhesion becomes independent of 7~. At still lower values of 700, the trend reverses and adhesion increases with increasing y~v. Thus with the direct measurement of the liquid surface tension and the calculation of the substrate and cellular surface tensions using Eq. (5), the relative values of AP ~ah under various conditions may be determined using Eq. (1). The combination of liquid, substrate and cellular surface tensions that resulted in the most negative value of A/~ah produced the greatest level of adhesion in these experiments as predicted by Eq. (1).

The influence of the substrate surface tension 7~ on the extent of plant cell adhesion is illustrated in Fig. 6. In Figs. 6a and b the liquid surface tension 7tv was 72.5 ergs/cm 2, while the substrates FEP and SPS had y~ values of 16.4 and 66.7 ergs/cm 2, respectively. In Fig. 6c and d the extent of adhesion of C. roseus cells to FEP and SPS is shown when y~ has been substantially lowered to 44.5 ergs/cm 2. The results presented in Fig. 5 are clearly illustrated by comparing Figs. 6a and b with Figs. 6c and d. When ytv is lowered from 72.5 to 44.5 ergs/cm 2 the extent of adhesion of C. roseus cells per unit surface area decreases in the case of FEP but increases for SPS under otherwise identical conditions.

It is important to note several points about these experiments: (1) the plant cells were iso- lated and resuspended, after repeated centrifugation washes, in an extracellular protein-and car- bohydrate-free liquid medium ensuring that the polymer surfaces were not pre-coated with these substances prior to cell adhesion; (2) the plant cells were washed in a solution containing the chelating agent EGTA to reduce the effect of possible divalent cationic bridging in cell-cell and cell-surface interactions; and (3) the washed plant cells were resuspended in protein-and carbohy- drate-free liquids of various surface tensions (Yt~).

352 P.J. Facchini et al. : Plant cell adhesion

Fig. 6. Photomicrographs of the adhesion of suspension-cultured day 4 C. roseus cells under varing conditions. (a) FEP, Ysv = 16.4 ergs/cm2; Yt~ =72.5 ergs/cm 2. (b) SPS, )%=66.7 ergs/cm2; yl~ =72.5 ergs/cm 2. (c) FEP, y~v = 16.4 ergs/cm2; yt~ =44.5 ergs/cm 2. (d) SPS, Ytv = 66.7 ergs/cmZ; y~o = 44.5 ergs/cm 2. Photomicrographs were taken by rehydrating the air dried substrates with Toluid- ine Blue (TBO) stain from the adhesion experiment of Fig. 5

Thus, the role of electrostatic and specific bio- chemical interactions in producing the observed effects in Figs. 5 and 6 were minimized and were neglected in this study. The thermodynamic model (Equation 1), together with the equation of state approach for interfacial tensions (Neumann et al. 1974) predicts the initial plant cell adhesion process well under conditions used herein.

The pattern of plant cell adhesion shown in Fig. 5 is divided into three regions: (1) y~v>Yc~, where adhesion decreases with increasing Ys~; (2) 7/~ < y~v, where adhesion increases with increasing ys,: and (3) Yl~ = Y~o, where the extent of adhesion is independent of Ysv. Investigating this further, the slopes of the straight lines in Fig. 5 were plotted against Y1~ in Fig. 7. Since the slope becomes equal to zero when Yl~ = Yc~, the intercept with Yl~ axis, according to the thermodynamic model, is equal to the surface tension of the suspension-cultured C. roseus cells. From Fig. 7, the slope becomes equal to zero when y~v = 54 ergs/cm 2 imply- ing that this is the surface tension of the C. roseus cells at 25 o C. This agrees well with the value of

53 ergs/cm 2 estimated from contact angle measurements. All adhesion experiments performed (10 in total) produced Ycv values of 56___ 3 ergs/cm 2.

.6

"5

i i I l l n 0 J CO

.5-

. 4 -

. 2 -

. ! -

o

- . ~ t -

- . 2 -

- . 3 -

- . 4 -

I I I I I I I 40 45 50 55 60 6 5 70 75

"~lv (ergs/cm 2) Fig. 7. Slopes of the straight lines in Fig. 5 vs. Ytv. The slope is equal to zero for )'to = Yc, = 54 ergs/cm 2. Line was plotted by means of a second order polynomial computerized curve fit


However, it was decided that this technique, like the contact angle method, was not sensitive enough to detect y~v changes during the growth cycle.

Resolution of the cellular surface tension with a high degree of sensitivity was achieved using a third alternate method for determining the surface tension of small particles. The principle of this technique, known as the sedimentation volume method, has been described in detail previously (Vargha-Butler et al. 1985a) and thus will be dis- cussed here only briefly. This technique has been used to determine the surface tension of various polymer particles (Vargha-Butler et al. 1985a), protein-coated polymer particles (Absolom et al. 1987), and numerous coal fractions (Vargha- Butler et al. 1985b; Vargha-Butler et al. 1985c). When an equal mass of these particles was suspended in liquids of known surface tension it was found that the sedimentation volume (V~ed) of the particles was altered in response to the liquid surface tension y~. An extremum in V~d occurs when the surface tension of the suspending liquid ?'lo is equal to the surface tension of the particles as predicted on a theoretical basis (Vargha-Butler et al. 1985a).

The measured sedimentation volumes of the C. roseus cells at two day intervals during the growth cycle of the cultures is shown in Fig. 8. The maximum in V, ee was consistently observed in the range of 55 to 58 ergs/cm 2 (viz. at maximum Vsed Yr, = Y~ = 55-58 ergs/cm2). These values are similar to y~v values determined using the techniques described above. However, we found the sedimentation volume technique to discrimi- nate between relatively small y~v differences. This is presumably due to the small number of factors involved in producing the experimental effects; i.e., specific y~, values, precise cell concentrations in each liquid, and the variable y~ values. Results were consistent for all three growth cycles tested.

The sedimentation volume experiments demonstrate that ?% changes during the growth cycle. The y~ values on different days of the growth cycle determined from Fig. 8 are plotted in Fig. 9b. In Fig. 9a, the extent of adhesion of the C. roseus cells suspended in distilled water (Yt, = 72.5 ergs/cm 2) to three polymers (FEP, PS, and PET) is also plotted for different days of the growth cycle. The consistent biphasic pattern illustrated by Fig. 9a is reflected to a large extent by the biphasic pattern of y~ changes through the growth cycle (Fig. 9b). Although the first maximum in the adhesion pattern occurs at day 6 and the corre- sponding y~, maximum occurs at day 4, it has

×

5~

E

60' -a 5 8 ' 5 6 '

54- b 52 50

64 c 62 ~ 6O ~

64- d 6 2 ' 6O ~

60- e 58- 5 6 " -

5 2 ~ - f

48

62

,4O

~1 I I I

I

45 50 55 60 65 70

"Ylv ( e r g s / c m 2)

Fig. 8. Sedimentation volumes of suspension-cultured C. ro-

s e u s cells at different days of the growth cycle plotted as a function of the surface tension Yt~ of the suspending liquid. Days of the growth cycle tested: a - - day 2; b - - day 4; c - - day 6 ; d - - day 8 ; e - - day 10; f - - day12; g - - day 14

been previously demonstrated that while the trends are consistent, the magnitudes and precise times of adhesion maxima and minima are variable (Facchini et al. 1988).

The patterns demonstrated in Fig. 9 are in agreement with predictions of the thermodynamic model. When values of 7tv and Y~v remain constant the change in the free energy of adhesion (AP dh)

0

0 0

o

o

v

8

a 30-

25-

20-

t5-

10-

5-

0 b

60-

59-

58-

57-

56-

55-

54

:J.j'J I I I I I I I

0 / 0 ~ .

o/ \ o / ( I I I I I I

2 4 6 8 t0 12 ~4

T ime (days)

Fig. 9. (a) The extent of adhesion of suspension-cultured C. r o s e u s cells suspended in distilled water (Y~v =72.5 ergs/cm 2) to polymers (Zx - - FEP; A -- PS; • - - PET) as a function of the growth cycle measured in terms of time. (b) The surface tension Ycv of the C. r o s e u s cells as determined from the maximum sedimentation volumes in Fig. 8 as a function of the growth cycle

354 P.j. Facchini et al.: Plant cell adhesion

becomes more negative, and accordingly , adhe- s ion becomes more extensive, when the cel lular surface tens ion Ycv decreases. Therefore , it is ap- pa ren t tha t the b iphas ic pa t t e rn o f adhes ion can be ascr ibed to changes in the surface tens ion 7c~ of the p lan t cells.

Acknowledgements. We are grateful to J. Young for technical assistance with the photomicrography. This research was sup- ported by a Medical Research Council of Canada grant (No. MA-9857) to F. DiCosmo.

References

Conclusion

The theoret ica l predic t ions of the t h e r m o d y n a m i c mode l are bo rne out by the exper imenta l results o f this invest igat ion. We have demons t r a t ed that the f u n d a m e n t a l m e c h a n i s m s govern ing the initial in teract ion o f Catharan thus roseus cells with polymer substra tes involve substrate, suspending liquid, and cel lular surface tensions. Values for these pa rame te r s are essential i f we are to use the pre- dictive capac i ty of the model . The relative values o f these fac tors de te rmine the extent o f adhes ion of the C. roseus cells at var ious stages o f their growth cycle. Our d a t a suggests tha t the the rmo- dynamic mode l is the p r o p e r start ing po in t to s tudy the initial phases o f p lant cell adhes ion phenomena . This po in t is re inforced by the fact tha t pro te in adso rp t ion (van Oss et al. 1981), and bacterial (Abso lom et al. 1983) and m a m m a l i a n cell ( N e u m a n n et al. 1979) adhes ion fol low similar pa t te rns descr ibed for cul tured p lan t cells.

The mos t difficult surface tens ion measure - men t is a lways tha t o f the microorgan ism. The ag reemen t o f three i ndependen t me thods o f cell surface tens ion measu remen t , as well as the ther- m o d y n a m i c cons is tency with mode l predic t ions , suppor t ou r results indicat ing changing cellular surface tens ion be tween 55 and 58 e r g s / c m 2 th rough the g rowth cycle o f the cell cultures. Plant cell surface tensions a p p e a r to be consistent ly lower ( indicat ing that the cells are more hy- d rophob ic ) t han mos t bacter ia l or m a m m a l i a n cells. For example , the xy lem cells o f Pseudo t suga menziesii , Western hem l ock and English O a k have been measu red as 59.0, 56.5 and 49.8 e rgs / cm 2, respect ive ly (Nguyen and John ' s 1970). Mos t bacter ial , and all m a m m a l i a n cells, tes ted have surface tens ions grea ter than 63 e r g s / c m 2. Con- tact angle m e a s u r e m e n t s on layers of hemicel lu- lose, a m a j o r c o m p o n e n t o f the p lant cell wall, in- dicate tha t the surface tens ion of this po lymer i c po lysacchar ide is 56 e r g s / c m 2 (Abso lom et al. 1986). This suggests tha t c o m p o n e n t s o f the p lan t cell wall are respons ib le for p roduc ing the observed cel lular surface tens ion and poss ib ly the pa t te rn of cell surface tens ion change th rough the growth cycle.

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Received January 13, 1988/Accepted May 24, 1988

thermodynamic aspects of plant cell adhesion to polymer surfaces

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