Applied Chemistry at Protein Interfaces

14 Demonstration of the Involvement of Adsorbed Proteins in Cell Adhesion and

Downloaded by UNIV OF ROCHESTER on January 19, 2018 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch014

Cell Growth on Solid Surfaces R. E. BAIER and L. WEISS Department of Biophysics, Roswell Park Memorial Institute, Buffalo, Ν. Y.

Experiments with solids immersed in cell culture media, with and without living cells present, convincingly demon strate the absolute condition that protein-dominated films accumulate at the solid-liquid boundaries prior to cell ad hesion. The reality and speed of this spontaneous, adsorptive event are documented by using infrared-transmitting, mul tiple internal reflection plates as the immersed solid with care to prevent film transfer from gas—liquid interfaces.

*"phis brief report highlights the involvement of adsorbed ( conditioning ) films of proteins in the behavior of cells at solid surfaces, particularly during routine experiments designed to investigate the cell adhesion, cell migration, cell growth, or cell aggregation process. Adsorbable macromolecules, used in supplements to otherwise well-defined culture media, affect such experiments greatly. All our methods have been reported (1,2,3). The cells were derived from Ehrlich-Lettre hyperdiploid ascites tumors (EAT) kept in culture at the Roswell Park Memorial Institute. For details of the culture method, cultured cells, and culture media, see Refs. 4 and 5. ,

Typical

Results

A n I R s p e c t r u m of f r e s h l y p r e p a r e d R P M I 1630 c e l l c u l t u r e m e d i u m s u p p l e m e n t e d w i t h 5 % c a l f s e r u m , the m e d i u m u s e d r o u t i n e l y to c u l t u r e v i a b l e cells, is p r e s e n t e d i n F i g u r e 1.

F i g u r e 1 is a n I R s p e c t r u m of

c e n t r i f u g a l l y c o n c e n t r a t e d a n d w a s h e d E A T cells w h i c h w e r e c u l t u r e d i n this m e d i u m .

T h e I R s p e c t r u m of t h e salt-free r e s i d u e f r o m

the

s u p e r n a t a n t l i q u i d o b t a i n e d after c e n t r i f u g i n g the cells constitutes F i g u r e 300 Baier; Applied Chemistry at Protein Interfaces Advances in Chemistry; American Chemical Society: Washington, DC, 1975.

14.

B A I E R A N D WEISS


3600

Figure 1.

3.

3200

Adsorbed

?300

2400

301

Proteins FREQUENCY (CM') 2000 1800

1600

1400

1200

IR spectrum of freshly prepared RPMI 1630 cell culture (+ 5% calf serum supplement) with no cells present

1000

medium

C e l l lines u s u a l l y cannot b e successfully p r o p a g a t e d i n v a r i o u s s y n -

thetic media without supplemental acterized)

s e r u m components.

(and, unfortunately, poorly

char-

This requirement for supplemental pro-

t e i n m a t e r i a l s p r o b a b l y reflects—at least i n i t i a l l y — t h e n e e d for a d s o r b a b l e proteinaceous constituents w h i c h w i l l spontaneously a c c u m u l a t e at a n d favorably

modify

(for cell

of t h e c u l t u r e containers.

adhesion

a n d propagation)

t h e surfaces

T h e r e w a s essentially n o spontaneous

film

a d s o r p t i o n f r o m f r e s h l y p r e p a r e d R P M I 1630 c u l t u r e m e d i u m w i t h o u t

FREQUENCY (CM') 2000 1800

Figure 2. IR spectrum of centrifugally concentrated and washed cells which had been cultured in RPMI 1630 medium ( + 5 % calf serum supplement)

Baier; Applied Chemistry at Protein Interfaces Advances in Chemistry; American Chemical Society: Washington, DC, 1975.


302

APPLIED CHEMISTRY AT PROTEIN INTERFACES

a d d e d c a l f s e r u m d u r i n g a 1 0 - m i n i m m e r s i o n of a n i n t e r n a l reflection p r i s m w i t h w h i c h e v e n m o n o l a y e r a m o u n t s of o r g a n i c c o n t a m i n a t i o n c a n b e d e t e c t e d , a n d there w e r e n o significant a b s o r p t i o n b a n d s i n t h e I R spectrum ( F i g u r e 4).

B y contrast, a n o r g a n i c film w a s s p o n t a n e o u s l y

a d s o r b e d onto another p r i s m d u r i n g a 1 0 - m i n i m m e r s i o n i n a f r e s h l y p r e p a r e d c e l l c u l t u r e m e d i u m w i t h a 5%

calf serum supplement ( F i g u r e 5 ) .

T h i s s p o n t a n e o u s l y a d s o r b e d film w a s d o m i n a t e d b y a g l y c o p r o t e i n c o m -

FREQUENCY (CM) 2000 1800

1400

1200

1000

Figure 4. IR spectrum demonstrating essential absence of spontaneous film adsorption from freshly prepared RPMI 1630 cell culture medium (without added calf serum) during 10-min immersion with no cells present Technique:

MA1R,

sensitive to even a monolayer of organic

contamination


14.


Adsorbed

303

Proteins

p o n e n t . O n c e a d s o r b e d , i t w a s c o m p l e t e l y i n s o l u b l e i n w a t e r a n d i n saline m e d i a ; thus a d s o r p t i o n h a d d e n a t u r e d t h e film sufficiently so t h a t its o r i g i n a l s o l u b i l i t y characteristics h a d b e e n lost. W a t e r o r s a l i n e e x t r a c t i o n u s u a l l y removes g l y c o p r o t e i n o r p r o t e o g l y c a n films w h i c h are not s t r o n g l y b o u n d to the s o l i d - l i q u i d b o u n d a r y ( 3 ) . S i m i l a r l y a film w a s s p o n t a n e ously adsorbed

during a 10-min prism immersion i n used

s i d e r e d e x h a u s t e d ) c u l t u r e m e d i u m i n w h i c h cells h a d b e e n

(often

con-

propagated

for some t i m e ( F i g u r e 6 ) ; g l y c o p r o t e i n a d s o r p t i o n , e v e n f r o m t h e exhausted m e d i u m , was r a p i d a n d irreversible.


4000

3600

3200

2800

2400

FREQUENCY (CM ') 2000 1800

1600

1400

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1000

100

Figure 5. IR spectrum of film spontaneously adsorbed during 10-min immersion in freshly prepared cell culture medium (RPMI 1630 + 5 % calf serum supplement) with no cells present T h e presence o f these s p o n t a n e o u s l y a d s o r b e d (i.e. a d s o r b e d d u r i n g m i n i m u m exposure

t i m e s ) , t h i n films o n p o l y m e r i c substrates s u c h as

p o l y s t y r e n e c u l t u r e dishes a n d glass plates u s u a l l y c a n n o t b e d e m o n strated b y other spectroscopic

methods.

F o r example, the modified i n -

t e r n a l reflection spectroscopic t e c h n i q u e , i n w h i c h a n a u x i l i a r y salt p r i s m ( u s u a l l y the m a l l e a b l e salt K R S - 5 ) is pressed against t h e d e m o n s t r a b l y ( b y other t e c h n i q u e s )

p r o t e i n - c o a t e d substrates, a l w a y s f a i l s ; this t e c h -

n i q u e is not sensitive e n o u g h f o r the near m o n o l a y e r ranges r e q u i r e d f o r this d e m o n s t r a t i o n .

T h e r e q u i r e d s e n s i t i v i t y is a c h i e v e d o n l y w h e n a d -

s o r p t i o n occurs d i r e c t l y o n the face of a c l e a n , o r t h i n

film-coated,

internal

reflection element. A c u r r e n t g o a l is to establish l o n g t e r m b i o c o m p a t i b i l i t y o f engineeri n g a n d s t r u c t u r a l m a t e r i a l s , e s p e c i a l l y f o r p r o s t h e t i c devices, b y p r o m o t i n g the r a p i d a t t a c h m e n t a n d p r o l i f e r a t i o n o f l i v i n g cells c o m p a t i b l e w i t h t h e site o f u l t i m a t e i m p l a n t a t i o n . F i n e f a b r i c meshes a r e of great u t i l i t y i n this a p p l i c a t i o n .

T h e great

sensitivity ( a n d simplicity) of



304

APPLIED CHEMISTRY

Figure 7.

AT PROTEIN

IR spectrum of cell culture on 5-μ thick, microfabric substrate

INTERFACES

polypropylene-based,

m o n i t o r i n g c e l l adhesive events w i t h i n a m i c r o f a b r i c l a y e r b y i n t e r n a l reflection spectroscopic m e t h o d s (1,2,3)

is i l l u s t r a t e d b y F i g u r e s 7 a n d 8.

F i g u r e 7 is t h e I R s p e c t r u m after 2 hrs o f c e l l c u l t u r e w i t h i n a 5-μ t h i c k , p o l y p r o p y l e n e fiber, m i c r o f a b r i c substrate ( a p p l i e d to a g e r m a n i u m i n t e r n a l reflection p r i s m a t U n i o n C a r b i d e C o r p . t h r o u g h t h e courtesy of J o s e p h B y c k ) . L i g h t m i c r o s c o p y r e v e a l e d that t h e large p r o t e i n s p e c t r a l b a n d s r e s u l t e d f r o m extensive c e l l u l a r coverage;

thus, even d u r i n g this

t i m e , c e l l a t t a c h m e n t a n d g r o w t h ( a s i n d i c a t e d b y t h e a b u n d a n c e of c e l l u l a r m a t e r i a l ) w e r e excellent. I 4000

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Figure 6. IR spectrum of film spontaneously adsorbed during 10-min immersion in used culture medium (RPMI 1630) in which cell growth had occurred after removal of cells by centrifugation


14.


Adsorbed

305

Proteins

d e t a c h m e n t e v e n b y l e n g t h y a n d v i g o r o u s r i n s i n g i n saline m e d i a a n d w a t e r . B y contrast, i n experiments to c u l t u r e t h e same c e l l l i n e o n b a r e g e r m a n i u m p r i s m s , there w a s l i t t l e p e r m a n e n t c e l l a t t a c h m e n t i n t w o hours. D e s p i t e a p p a r e n t success i n u s i n g m a n y m i c r o f a b r i c s to p r o m o t e c e l l a t t a c h m e n t a n d c e l l g r o w t h , o u r experiments also d e m o n s t r a t e d some w o r r i s o m e features of this m a t e r i a l . F o r e x a m p l e , s i m p l e s o a k i n g i n d i s t i l l e d w a t e r seriously l e a c h e d r e s i d u a l m i c r o f a b r i c c o n t a m i n a n t s f r o m t h e polypropylene mesh ( F i g u r e 8 ) ; depending o n their composition, such c o m p o n e n t s l e a c h e d i n t o a d e l i c a t e c e l l c u l t u r e m e d i u m m i g h t seriously


i n t e r f e r e w i t h a t t a c h m e n t o r p r o p a g a t i o n o f less v i a b l e c e l l lines.

Figure

8.

IR spectra illustrating severe leaching taminants

of microfabric

con

L o w e r trace, 5-μ thick polypropylene-based microfabric as prepared; and u p p e r trace, same fabric after soaking in distilled water

Discussion O n l y t h e m e t h o d s u s e d i n this s t u d y ( J , 2, 3) c a n detect a n d f o l l o w the i n i t i a l events at t h e b o u n d a r y b e t w e e n biological milieu.

a s o l i d substrate a n d t h e

T h i s e x t r a o r d i n a r y s e n s i t i v i t y derives f r o m t h e a b i l i t y

to m o n i t o r events f r o m w i t h i n t h e substrate itself b y m a k i n g t h e substrate c a p a b l e o f s u p p o r t i n g m u l t i p l e a t t e n u a t e d i n t e r n a l reflection ( M A I R ) at a v a r i e t y of u s e f u l s p e c t r o s c o p i c w a v e l e n g t h s .

I n other studies, i t w a s

c o n c l u d e d that a d s o r b e d p r o t e i n o n s o l i d substrates is of essentially n a t i v e (i.e.

s o l u t i o n or v o l u m e p h a s e ) c o n f i g u r a t i o n a n d present i n significant

thickness (6).

T h o s e studies u s e d a different v e r s i o n o f t h e i n t e r n a l

reflection s p e c t r o s c o p i c m e t h o d w h e r e i n a p r i s m element w a s f o r c e f u l l y


306

APPLIED CHEMISTRY

AT PROTEIN

INTERFACES

p r e s s e d against a p l a s t i c or f o i l surface w h i c h h a d a c c u m u l a t e d , after v e r y l o n g i m m e r s i o n p e r i o d s , e q u i l i b r i u m amounts of a d s o r b e d p r o t e i n . ther, those

studies w e r e

generally conducted

i n the

Fur-

absence of

the

n o r m a l l y a t t a c h i n g cells w h i c h w o u l d h a v e m o d i f i e d the process of

film

b u i l d u p b e f o r e i t r e a c h e d e q u i l i b r i u m . S u c h studies d e t e c t e d o n l y the n a t u r e of the p r o t e i n a d s o r b e d onto other layers of p r o t e i n a l r e a d y at the s o l i d - s o l u t i o n interface.

T h e p r i m a r y , i n t e r f a c i a l l y l o c a l i z e d layers c a n -

not b e d e t e c t e d b y m e t h o d s other t h a n those u s e d here. relevance

to s p e c u l a t i o n a b o u t

the effect of

these

T h e r e is l i t t l e

secondary

protein

layers w h i c h are p r o b a b l y not e v e n present w h e n cells first a r r i v e a n d Downloaded by UNIV OF ROCHESTER on January 19, 2018 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch014

a t t a c h to s o l i d surfaces.

Figure 9.

Phase contrast micrographs

Culture medium, RPMI 1640;

of live cells adherent to glass

left, with no added protein; and right, with 20% calf serum supplement

fetal

W o r k is just n o w b e g i n n i n g o n the effect of substrate c h e m i s t r y o n p r o t e i n a d s o r p t i o n f r o m s u p p l e m e n t e d c u l t u r e m e d i a a n d o n the seco n d a r y effect of these layers o n c e l l a d h e s i o n processes (7).

In preliminary

w o r k , w e d e t e r m i n e d that a m a r k e d d i f f e r e n t i a t i o n i n c e l l

morphology

reflects the presence of a d s o r b e d proteinaceous c o m p o n e n t s at a s u b s t r a t e s o l u t i o n interface.

F o r e x a m p l e , w h e n l i v i n g cells settle to c l e a n glass

surfaces i n the absence of s u p p l e m e n t a l p r o t e i n , the cells r e m a i n r o u n d e d a n d p o o r l y a d h e s i v e , a n d t h e y are u n a b l e to d e v e l o p g o o d c e l l m o n o l a y e r c o v e r a g e of the surface (see

Figure 9).

O n the other h a n d , the presence

of s u p p l e m e n t a l p r o t e i n i n the m e d i u m i n d u c e s greater c e l l a d h e s i o n ; i n c r e a s e d a d h e s i o n is a p p a r e n t l y m e d i a t e d b y extensive

morphological

changes i n the c e l l ( m a n y e l o n g a t e d f o r m s ) , a n d e v e n t u a l l y t h e cells c o m p l e t e l y cover the s o l i d surface.


14.


Adsorbed

Proteins

307


Literature Cited 1. Baier, R. E., Loeb, G. I., "Multiple Parameters Characterizing Interfacial Films of a Protein Analogue," in "Polymer Characterization: Interdisci plinary Approaches," C. D. Craver, Ed., pp. 79-96, Plenum, New York, 1971. 2. Baier, R. E., Dutton, R. C., "Initial Events in Interactions of Blood with a Foreign Surface," J. Biomed. Mater. Res. (1969) 3, 191-206. 3. Baier, R. E., "Applied Chemistry at Protein Interfaces," ADVAN. CHEM. SER. (1975) 145, 1. 4. Moore, G. E., Sandburg, Α. Α., Ulrich, Κ., "Suspension Cell Culture and In Vivo and In Vitro Chromosome Constitution of Mouse Leukemia L-1210,"J. Nat. Cancer Inst. (1966) 36, 405-413. 5. Mayhew, E., "Electrophoretic Mobility of Ehrlich Ascites Carcinoma Cells Grown In Vitro and In Vivo," Cancer Res. (1968) 28, 1590-1595. 6. Lyman, D. J., Brash, J. L., Chaikin, S. W., Klein, K. G., Carini, M., "Protein and Platelet Interaction with Polymer Surfaces," Trans. Amer. Soc. Artif. Intern. Organs (1968) 14, 250-255. 7. Wilkins, J., "Adhesion of Ehrlich Ascites Tumor Cells to Surface-Modified Glass," Ph.D. Dissertation, State University of New York at Buffalo, 1974. RECEIVED

June 7, 1974.


Applied Chemistry at Protein Interfaces

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