Applications of Adsorbed Proteins at Solid and Liquid Substrates

Chapter 36

Applications of Adsorbed Proteins at Solid and Liquid Substrates Ivar Giaever and Charles R. Keese

Downloaded by UNIV OF CALIFORNIA SANTA CRUZ on October 10, 2014 | http://pubs.acs.org Publication Date: July 13, 1987 | doi: 10.1021/bk-1987-0343.ch036

Corporate Research and Development, General Electric Company, Schenectady, NY 12301

The understanding of protein adsorption onto nonbiological substrates is an important problem in biotechnology that may lead to many practical advances. For example, solid-phase immunology tests such as enzyme-linked immunosorbant assays rely on a preadsorbed layer of either antigen or antibody on a plastic or glass surface. Protein-covered interfaces are also very important in tissue culture research, as the cells attach to a protein film adsorbed at the plastic surface of the tissue culture dish and not directly to the dish itself. In addition, because of the increasing use of artificial organs, understanding of the adsorption of protein from body fluids and the search for a nonthrombogenic surface has intensified. If successful, such surfaces will be of major importance in medicine. This paper presents a summary of work in our laboratory to understand the phenomenon of protein adsorption and to apply this understanding to biotechnological problems. Adsorption of Protein on Solid Surfaces It is generally agreed that proteins adsorb to most artificial surfaces in a monolayer; however, much confusion exists with regard to the desorption and replacement of protein. One reason for this is the variety of different buffers used in studying this phenomenon. In this laboratory we have confirmed earlier findings (1) that phosphate and borate-based buffers under certain conditions interfere with the adsorption process, and can also cause proteins to desorb. Thus to avoid this complication, these buffers have been generally avoided in our work, and Tris is used in most experiments requiring a buffer. If protein is adsorbed from a saline solution onto a solid surface, we believe that the protein binds in a random orientation at the site of the molecule's first encounter with the surface. We simulated this process on a computer, approximating the proteins with a disk, and found that the final 0097-6156/87/0343-0582$06.25/0 © 1987 American Chemical Society

In Proteins at Interfaces; Brash, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

36.

GIAEVER AND KEESE

Adsorbed Proteins at Solid and Liquid Substrates

f r a c t i o n a l c o v e r a g e w a s o n l y 0.547 c o m p a r e d t o 0.907 f o r a c l o s e packed surface. I n Figure 1, o u r c o m p u t e r calculation i s contrasted with an experimental observation o f the adsorption o f ferritin onto a carbon surface. The agreement between fractional coverage measurement for t h e model and t h e experimental s i t u a t i o n i s e x c e l l e n t (2).


Detection

o f Adsorbed P r o t e i n

Layers

Many ingenious methods have been introduced t o study p r o t e i n adsorption. I f the kinetics of the adsorption process are i m p o r t a n t , t h e e l l i p s o m e t r i c method i n t r o d u c e d by Rothen (3) is probably the best. I n t h i s method p r o t e i n a d s o r p t i o n c a n be studied i nsitu from a solution. The method h a s b e e n u s e d to study t h e k i n e t i c s o f b o t h the adsorption of protein i n single layers and i n double layers that can occur i n the immune-reaction. When p r o t e i n s u c h as bovine serum albumin (BSA) was a d s o r b e d f r o m a d i l u t e s o l u t i o n o n t o a s u r f a c e , a f t e r a delay o f a few seconds, s t e a d y - s t a t e d i f f u s i o n c o n t r o l l e d t h e adsorption process a n d , c o n s e q u e n t l y , t h e amount b o u n d t o t h e surface increased l i n e a r l y w i t h time. However, as t h e s u r f a c e became covered, adsorption slowed down, b e c a u s e i t was now l i m i t e d b y t h e number o f a v a i l a b l e s i t e s on t h e s u r f a c e . The f i n a l l a y e r o f BSA was r o u g h l y 2 n a n o m e t e r t h i c k . Figure 2 i s a good illustration o f t h e power o f t h e e l l i p s o m e t r i c technique. C u r v e ( a ) o n t h e f i g u r e shows t h e specific attachment o f antibody t o a p r e a d s o r b e d BSA l a y e r . Curve (b) i s a r e p e a t o f t h e experiment, except r o u g h l y 90 s e c . after the s t a r t o f t h e r u n , a d d i t i o n a l B S A was a d d e d t o t h e s o l u t i o n thus e f f e c t i v e l y n e u t r a l i z i n g t h e s p e c i f i c a n t i b o d i e s . F o r s t a t i c m e a s u r e m e n t o f p r o t e i n f i l m s we h a v e d e v e l o p e d a method that relies on l i g h t s c a t t e r i n g ; the technique i s r e f e r r e d t o a s t h e I n d i u m S l i d e M e t h o d (4.5.6^. When i n d i u m i s evaporated onto a transparent surface such as g l a s s o r p l a s t i c i n a vacuum, t h e i n d i u m atoms w i l l condense upon t h e s u r f a c e i n small particles. The p h y s i c a l s i z e o f the indium p a r t i c l e s depends m a i n l y o n t h e amount o f i n d i u m e v a p o r a t e d , b u t a l s o on the temperature of the substrate. The optimum s i z e o f t h e p a r t i c l e s f o r t h i s method i s r o u g h l y e q u a l t o t h e w a v e l e n g t h o f light, i . e . a few hundred nanometers i n diameter. The t e s t r e l i e s on t h e f a c t t h a t v i s i b l e l i g h t s c a t t e r e d by p a r t i c l e s i n this size range i s markedly increased i f the particles are covered w i t h t h i n d i e l e c t r i c l a y e r s . Adsorbed p r o t e i n acts as t h i s d i e l e c t r i c l a y e r a n d , i n g e n e r a l , t h e more p r o t e i n a d s o r b e d the more t h e s c a t t e r i n g i n c r e a s e s . Thus i t i s possible to quantify t h e amount o f p r o t e i n a d s o r b e d b y m e a s u r i n g t h e amount of l i g h t t r a n s m i t t e d through the s l i d e w i t h the help o f a simple densitometer, o r one c a n s i m p l y e s t i m a t e t h e amount o f p r o t e i n by v i s u a l i n s p e c t i o n . A p p l i c a t i o n o f Adsorbed P r o t e i n Layers

i n Immunology

We h a v e u s e d t h e s l i d e s e x t e n s i v e l y f o r m e a s u r i n g v a r i o u s forms o f t h e immune r e a c t i o n , f r o m s c r e e n i n g f o r m o n o c l o n a l a n t i b o d i e s


583


584

PROTEINS AT INTERFACES

Figure la. Monte Carlo simulation of protein adsorption. At the jamming limit for disks, the final coverage is 0.547 of the available area.



36.

GIAEVER

A N D KEESE


Figure lb. Ferritin (horse spleen) adsorbed on carbon and stained with uranyl acetate.


585


586


TIME ( s e c )

Figure 2 (a) E l l i p s o m e t r i c detection o f the adsorption of r a b b i t a n t i s e r u m t o BSA onto a monolayer o f BSA a t a gold surface. (b) Same c o n d i t i o n s as i n c u r v e ( a ) , b u t a t the i n d i c a t e d time, e x c e s s BSA was added t o the a n t i s e r u m dilution.



36.

GIAEVER AND

KEESE


(R. Rej , I. Giaever and C.R. Keese, A Screening Technique for Monoclonal Antibody Production: Application of an Indium Slide Immunoassay, submitted for publication) to detecting h e p a t i t i s or schistosomiasis infections (4). In a t y p i c a l test, antigen i s f i r s t adsorbed onto the s l i d e as a small spot from a s o l u t i o n of a r e l a t i v e l y pure protein. The protein adsorbs i n a monolayer, and i f the s l i d e i s rinsed and dried, this layer can e a s i l y be detected by a change i n the amount of transmitted l i g h t . Next, i f desired, the s l i d e i s "masked" by adsorbing an inert protein over the remaining surface of the s l i d e that has the same l i g h t scattering e f f e c t as the antigen. The slide is now exposed to a s o l u t i o n that may or may not contain antibodies to the adsorbed antigen. I f antibody i s absent, no a d d i t i o n a l protein w i l l attach to any portion of the s l i d e , and the l i g h t scattering w i l l not change. On the other hand, i f s p e c i f i c antibody i s present, some of the antibody w i l l bind to the preadsorbed antigen causing a d i s t i n c t change i n the transmitted l i g h t i n that region. There are several v a r i a t i o n s of this procedure. For example, i t i s possible to enhance the e f f e c t by using a second antibody. I f i t i s desired to detect antigen, i t i s necessary to do an i n h i b i t i o n test (5), as only a small f r a c t i o n of the antibodies remain active i f they are adsorbed on a surface. Figure 3 shows a photograph of the indium s l i d e applied to detection of rheumatoid factor (6). Figure 3a i s a photograph of a naked indium s l i d e . Figure 3b i s a photograph of the s l i d e following the adsorption of antigen spots, on the l e f t i s human IgG and on the right, rabbit IgG. Figure 3c shows the s l i d e a f t e r i t has been dipped into a s o l u t i o n of aldolase. The aldolase, which alters the light scattering with approximately the same intensity as the IgG molecules, adsorbs around the antigen "masking" them from view. Figure 3d i s the appearance of a s l i d e after i t has been incubated i n a serum that does not contain rheumatoid factor. Figure 3e, on the other hand, shows the s l i d e after i t has been incubated with a serum containing rheumatoid factors against both the human and rabbit antigen. The "antigen spots" are now v i s i b l e because the monomolecular layer of adsorbed IgG i s now covered with a layer of rheumatoid factor. F i n a l l y Figure 3f shows the r e s u l t of incubation with a serum whose rheumatoid factor only reacted with the human IgG, a much more common occurrence. Adsorbed Protein Layers and C e l l s i n Tissue Culture Since the 1950's i t has been possible to grow mammalian c e l l s i s o l a t e d from a v a r i e t y of d i f f e r e n t tissues and organisms i n the laboratory. In tissue culture, c e l l s divide and carry on a v a r i e t y of b i o l o g i c a l a c t i v i t i e s while feeding on a r i c h nutrient medium that supplies a l l of the necessary molecules for their survival. Unlike b a c t e r i a l cultures, most commonly cultured normal mammalian c e l l s , such as f i b r o b l a s t s , w i l l not grow i n suspension but require attachment to a r i g i d surface i n order to undergo mitosis. T r a d i t i o n a l l y , this substrate has been glass or polystyrene that has been treated to render i t


587



588

F i g u r e 3 D e t e c t i o n o f r h e u m a t o i d f a c t o r i n human sera using t h e i n d i u m s l i d e immunoassay. (a) Indium s l i d e before spotting. (b) A n t i g e n s p o t s a p p l i e d . ( c ) Spots masked. (d) S l i d e exposed t o c o n t r o l serum. (e,f) Slide exposed t o two d i f f e r e n t p o s i t i v e s e r a .



36.

GIAEVER AND KEESE


hydrophilic. In most tissue culture work, the medium contains large amounts (e.g. 10%) of plasma or serum i n addition to defined components such as s a l t s , sugars, vitamins, etc. Consequently, c e l l s i n tissue culture are grown i n the presence of protein molecules, and the surfaces with which they interact are covered with a monolayer of protein that spontaneously adsorbs on the s o l i d interface. Our laboratory has been engaged i n studies of the interactions of f i b r o b l a s t i c c e l l s with these adsorbed protein layers. There are many facets to this study. When surfaces are i n i t i a l l y inoculated, the c e l l s are introduced as a monodisperse suspension with roughly a spherical morphology. Upon s e t t l i n g to the substrate, a complex series of events occur where the c e l l s attach to the surface, spread into highly flattened, i r r e g u l a r shapes, and then crawl about (Figure 4a). Although the attachment and subsequent spreading and locomotion involve making and breaking contact and exerting forces upon the adsorbed proteins, as a general rule, we have found that the type of protein adsorbed at the surface has only subtle e f f e c t s on c e l l behavior. One exception to t h i s generalization was observed when c e l l s were grown on substrates covered with IgG molecules. In t h i s case the a b i l i t y of the c e l l s to attach and spread upon the substrate was noticeably impaired. An even more pronounced e f f e c t was observed when the substrate was coated with a bimolecular protein layer consisting of IgG molecules s p e c i f i c a l l y bound to an adsorbed antigen layer. In t h i s s i t u a t i o n , no c e l l attachment or spreading was detected for a wide v a r i e t y of both normal and transformed c e l l l i n e s (7). Figure 4b demonstrates t h i s e f f e c t and emphasizes the fact that c e l l s i n culture interact with i n t e r f a c i a l protein layers and not d i r e c t l y with the s o l i d substrate. To produce the e f f e c t shown, d i f f e r e n t protein layers were placed on a glass coverslip i n defined regions using a UV lithographic technique (8). The substrate was then inoculated using standard tissue culture protocol with WI-38 human embryoic lung f i b r o b l a s t s . Following overnight incubation the coverslip was gently rinsed with tissue culture medium to remove unattached c e l l s . The remaining c e l l s were f i x e d and stained to reveal t h e i r location. The background showing normal c e l l - s u b s t r a t e i n t e r a c t i o n i s covered with a layer of BSA while the pattern (GE100) consists of a base layer of BSA covered with s p e c i f i c a l l y attached IgG molecules and, consequently, i s void of c e l l s . Monitoring

C e l l Attachment and Spreading E l e c t r i c a l l y

I t i s possible to detect small differences i n c e l l - s u b s t r a t e interactions using weak e l e c t r i c f i e l d s and i n t h i s manner to q u a n t i t a t i v e l y measure differences i n the dynamics of c e l l attachment and spreading to defined protein monolayers. The d e t a i l s of the system have been previously described (9-10). In b r i e f , c e l l s were cultured on gold electrodes under standard tissue culture conditions. To minimize the e f f e c t of


589


590


SPREADING FIBROBLAST ADSORBED PROTEIN MONOLAYER ~ 20 Â THICK

ADHESION PLAQUE

Figure 4a. The illustration depicts a spread Fibroblast on a layer of adsorbed protein; the arrows represent forces generated by the microfilaments of the cell as an action-reaction pair. These forces are involved in the process of spreading as well as locomotion of the cells on the substrate.



36.

GIAEVER AND KEESE


Figure 4b. Haptotactic behavior of WI-38 cells on a specially prepared glass coverslip. The background, showing normal cell-substrate interaction, is covered with a layer of adsorbed BSA, while the pattern (GE100) is covered with IgG molecules specifically attached to a base layer of BSA.


591


592

solution resistance, the system was designed with one small electrode (=* 10 " c m ) and one large electrode; under these conditions the measurement i s d o m i n a t e d by the e l e c t r o l y t i c i n t e r f a c e between the small electrode and the solution. An a p p l i e d a l t e r n a t i n g e l e c t r i c f i e l d ( n o r m a l l y 4000 Hz) p r o d u c e d a v o l t a g e d r o p o f a few m i l l i v o l t a t the boundary o f t h e s o l u t i o n a n d t h e e l e c t r o d e , a n d t h e c u r r e n t d e n s i t y was a few milliamperes/cm . Under these c o n d i t i o n s , t h e r e were no detectable e f f e c t s o f t h e e l e c t r i c f i e l d s on c e l l s as j u d g e d by c e l l m o r p h o l o g y , l e n g t h o f g e n e r a t i o n t i m e , e t c . As fibroblasts attached and s p r e a d on t h i s s u r f a c e , however, t h e impedance o f t h e e l e c t r o d e was o b s e r v e d t o i n c r e a s e , r e a c h i n g a maximum v a l u e about two h o u r s a f t e r i n o c u l a t i o n ( u s i n g a s u f f i c i e n t n u m b e r o f c e l l s for a confluent layer). At this point, the impedance decreased slightly, and a f t e r a p p r o x i m a t e l y another hour the average value stabilized. At this time, the impedance fluctuated about the mean a s the f i b r o b l a s t s crawled about, a l t e r i n g t h e i r c o n t a c t w i t h the e l e c t r o d e surface. Figure 5a illustrates these e v e n t s w i t h WI-38 c e l l s i n w h i c h t h e d a t a i s p r e s e n t e d as the m e a s u r e d i n - and o u t - o f - p h a s e p o t e n t i a l across t h e s m a l l e l e c t r o d e as a f u n c t i o n o f t i m e . 4

2


2

We h a v e a p p l i e d t h i s new means o f m o n i t o r i n g c e l l behavior t o s t u d y t h e i n t e r a c t i o n o f c e l l s i n c u l t u r e medium w i t h d e f i n e d layers of adsorbed p r o t e i n . Before the a d d i t i o n of the t i s s u e c u l t u r e m e d i u m c o n t a i n i n g s e r u m , t h e s m a l l e l e c t r o d e was exposed for 15 m i n . to a 100 Mg/ml s o l u t i o n of a selected protein. F o l l o w i n g a d s o r p t i o n , t h e e l e c t r o d e was t h o r o u g h l y rinsed free of unadsorbed protein and inoculated with a fibroblast suspension. F i g u r e 5b p r e s e n t s d a t a obtained when electrodes coated w i t h adsorbed l a y e r s of plasma f i b r o n e c t i n , g e l a t i n , BSA a n d f e t u i n w e r e i n o c u l a t e d w i t h WI-38/VA 13 c e l l s , a transformed ( c a n c e r o u s ) c e l l l i n e d e r i v e d f r o m WI-38. As c a n be s e e n , t h e r e was a p r o n o u n c e d d i f f e r e n c e i n t h e r e s p o n s e o f t h e c e l l s t o the different protein layers. A l t h o u g h the r a t e o f change i n the r e s i s t i v e c o m p o n e n t o f t h e i m p e d a n c e was g r e a t l y r e d u c e d f o r BSA and f e t u i n , e v e n t u a l l y the f i n a l change i n impedance, and hence i n c e l l - s u b s t r a t e i n t e r a c t i o n , a p p e a r e d t o be equivalent (data not shown i n f i g u r e . ) When d i f f e r e n t c e l l l i n e s w e r e c o m p a r e d , the o r d e r i n g of the p r o t e i n l a y e r s w i t h r e g a r d to the rate of impedance increase varied, but i n a l l cases examined, r a p i d i n i t i a l change i n impedance o c c u r r e d when t h e p r o t e i n coat was fibronectin; this p r o t e i n has l o n g b e e n the l e a d i n g c a n d i d a t e for the "glue" that connects c e l l s to a s u r f a c e . In a d d i t i o n to studies i n v o l v i n g the dynamics of cell attachment and spreading on protein-coated substrates, the s y s t e m has a l s o been employed to study cell locomotion as revealed by oscillations in the impedance o b s e r v e d f o l l o w i n g c e l l attachment and spreading. The belief that these are related to cell motion is supported by d r u g s t u d i e s where compounds known t o i n t e r f e r e w i t h c e l l motion greatly reduced the amplitude of these f l u c t u a t i o n s (9). An e x t e n s i v e search was u n d e r t a k e n t o d i s c o v e r i f d o m i n a n t f r e q u e n c i e s w e r e present by digitally processing the signals. So f a r t h i s s e a r c h has been negative, but the power density spectrum of the




GIAEVER AND KEESE

Figure 5 (a) Monitoring c e l l - s u b s t r a t e interactions i n tissue culture using weak e l e c t r i c f i e l d s . (b) The e f f e c t of d i f f e r e n t adsorbed protein layers on WI-38/VA13 c e l l s monitored using weak e l e c t r i c f i e l d s .


594


cell-induced fluctuations has been obtained from this study. In general, the magnitude of the spectrum varies inversely with the square of the frequency of the f l u c t u a t i o n (Brownian noise) and i s much larger for cancer c e l l s than normal c e l l s . Because there i s a large amount of scatter i n the data, we have not yet been able to r e l a t e the noise to the various proteins used as substrates.


Oil-water Interfaces as Substrates

for C e l l Growth

In 1964, Rosenberg introduced the use of a f l u i d substrate for the growth of both transformed and anchorage-dependent c e l l s (11). In this method, a c e l l suspension was introduced over a hydrophobic l i q u i d having a density greater than that of the aqueous medium, and then the c e l l s , being of intermediate density, s e t t l e d to the interface where they were observed. Using fluorocarbon f l u i d s , Rosenberg found that such interfaces could serve as supports for attachment, spreading, and growth of a v a r i e t y of c e l l l i n e s . Again, as i n the case of s o l i d substrates, the interface was coated with a monolayer of adsorbed protein from the culture medium. Since this i n i t i a l observation, we have demonstrated that the adsorbed proteins on highly p u r i f i e d fluorocarbon f l u i d s do not form adequate i n t e r f a c i a l substrates unless the o i l contains small amounts of s p e c i f i c surface active compound (12). The compound we have found to be most e f f e c t i v e i n this capacity i s pentafluorobenzoyl chloride (PFBC). To produce an i n t e r f a c i a l substrate that i s adequate for the growth of most f i b r o b l a s t i c mammalian c e l l s , t h i s compound i s added to the oil-phase to y i e l d a f i n a l surface concentration of at least 0.25 μg per square centimeter (Figure 6a). The necessity for this (or s i m i l a r compound) for c e l l growth has been thoroughly investigated i n our laboratory because i t a f f e c t s the mechanical strength of the adsorbed protein. In order to achieve a spread morphology and to move about on a surface, c e l l s i n culture exert forces at t h e i r points of attachment. These forces are generated by an i n t r a c e l l u l a r system of muscle-like f i b e r s referred to as microfilaments and composed mainly of the muscle protein a c t i n . I f the adsorbed protein layer at the oil-water interface i s unable to support such forces, i t w i l l yield causing the c e l l s to r e t r a c t to a rounded state. Hence, i f one i s to use fluorocarbon fluid-water interfaces as tissue culture supports, they must s a t i s f y the minimal mechanical properties required to sustain the forces involved i n c e l l spreading. We have investigated the alteration in mechanical properties of the protein layer caused by the PFBC using a modified surface viscometer. The protein f i l m was placed under a shearing stress by the a p p l i c a t i o n of a small torque to a t e f l o n paddle wheel inserted into the i n t e r f a c i a l boundary, and the angular deformation of the f i l m was measured. From this data i t was possible to obtain s t r e s s - s t r a i n curves and to determine the surface shear modulus and surface fracture point for the protein layer. In most studies protein was adsorbed to the interface of perfluorotributylamine from either a buffered



36.

GIAEVER AND KEESE


BSA s o l u t i o n or culture medium containing 10% serum. Following a 24-hour period, to allow d i f f u s i o n of PFBC to the interface, measurements were c a r r i e d out. The r e s u l t s are shown i n Figure 6b where the conversion of an e s s e n t i a l l y f l u i d layer of adsorbed protein to an e l a s t i c f i l m by the presence of increasing amounts of the acid chloride i s shown. We believe there are two possible mechanisms to account for this alteration. Following adsorption of the interfacial protein layer, the PFBC molecules d i f f u s i n g to the interface w i l l react with the acid chloride moiety either with the water or, more importantly, with functional groups on the surface of the adsorbed protein by a condensation reaction. In this manner what were formerly the most hydrophilic residue are chemically modified into groups that now are l i k e l y to be inserted into the oil-phase. This a l t e r a t i o n could r e s u l t i n severe denaturation of the protein molecules, such that adjacent molecules would tangle to form an e l a s t i c f i l m . I t i s also possible that the f l u o r i n e i n the para p o s i t i o n could undergo a n u c l e o p h i l i c replacement reaction. In this manner the PFBC could be acting as a b i f u n c t i o n a l c r o s s l i n k i n g compound, j o i n i n g adjacent proteins by covalent bonds. We are now using i n t e r f a c i a l protein layers to characterize some of the mechanical properties required of a c e l l substrate. To carry out these studies two d i f f e r e n t measurements are being made. F i r s t , the surface shear modulus and surface fracture point of 24-hour o l d adsorbed protein layers are measured as a function of the surface concentration of PFBC as described above. Next, identical interfaces are inoculated with f i b r o b l a s t s and incubated for 16 hours. Following this period, the c e l l s at the interface are f i x e d and stained, and the projection area of the c e l l s i s measured with a Zeiss IBAS image analysis system. From this data, the r e l a t i v e amount of c e l l spreading i s calculated. By c o r r e l a t i n g the r e s u l t s of these two types of experiments, we expect to determine the minimal mechanical proparties required of a substrate for d i f f e r e n t c e l l lines. Conversely, these values should also allow us to i n f e r the magnitude of the forces exerted by d i f f e r e n t c e l l l i n e s upon the substrate. In vivo these forces are thought to be associated with normal c e l l migration i n development and wound healing. They have also been implicated i n the process of metastasis whereby a cancer c e l l i s able to leave i t s primary l o c a t i o n and e s t a b l i s h secondary tumors throughout the body. Applications of Oil-water

I n t e r f a c i a l Protein Layers

By u t i l i z i n g fluorocarbon f l u i d s containing pentafluorobenzoyl chloride, a l i q u i d microcarrier system has been developed capable of use with a v a r i e t y of c e l l types including normal human f i b r o b l a s t . In this configuration, c e l l s on the surface of a coarse oil dispersion (- 150 μτα. diameter) exhibit exponential growth (Figure 7). In addition, a microcarrier based on s i l i c o n e o i l has been formed and used to culture mouse f i b r o b l a s t s (12-13). These novel i n t e r f a c i a l substrates may allow manipulation


595


Figure 6a. 3T3-L1 mouse Fibroblasts are shown atfluorocarbonoil-water interfaces 16 h after inoculation. In this photograph, where the cells have not spread but have clustered together in spheroids, the oil received no additive. Continued on next page.



Figure 6a.—Continued. 3T3-L1 mouse Fibroblasts are shown atfluorocarbonoil-water inter faces 16 h after inoculation. The photograph shows a confluent layer of spread Fibroblasts. PFBC was present in the oil phase at a surface concentration of 1 /xg/cm .




598

BSA

FC-43

LOG S U R F A C E CONC.

2 4 hrs

(pgm/cm ) 2

Figure 6b. Mechanical properties of adsorbed BSA layers at a fluorocarbon oil-water interface as a function of the log of the surface concentration of PFBC (Ο» surface fracture point; ·, surface shear modulus).



36.


GIAEVER AND KEESE

MRC-5 CELLS

1000

I

C F, t

e

WITH PENTAFLU0R0BENZ0YL

CHLORI0E (SOO^/ta!)

I

100 b-

/

i.

10 0

40

t

t

80

120

1

160

200

I t TIME (hr)

F i g u r e 7a. Growth c u r v e f o r the human f i b r o b l a s t MRC-5 a t the i n t e r f a c e of o i l droplets (microcarriers).


599


600 PROTEINS AT INTERFACES



36. GIAEVER AND KEESE

Adsorbed Proteins at Solid and Liquid Subst

of cells in culture in ways previously not possible using solid-substrate culturing. The liquid microcarrier system permits mass culturing cells in arrangements commonly used with solid microcarriers but also allows one to mechanically harvest the cells by breaking the dispersion into its two component phases. Such an arrangement may prove to be of particular value in studies involving purification of large quantities of receptors and other surface molecules, where chemical methods of cell harvesting could damage the components of interest. Another interesting property of this arrangement is its capacity for delivery of water insoluble compounds to cells. By first dissolving such compounds in the oil-phase they could then continuously partition from the oil-phase into the cells membranes. Adsorbed protein on fluorocarbon oil-water interfaces have also been used in our laboratory to develop a variation of the latex agglutination assay to detect immunological molecules. Fluorocarbon oil was emulsified by sonication in the presence of an antigenic protein that also serves as the emulsifying agent. Average particles had a diameter of the order of 1 μπι and were highly stable without the addition of other emulsifiers or the additive, PFBC. When these droplets were combined with specific antiserum and allowed to slide by each other with a gentle rocking motion, agglutination could be observed and quantitated using a image analysis system. The system had a sensitivity of 1 /ig/ml of antibody for a 15-min. reaction time. Interestingly, the most sensitive results were obtained when an impure antigen was used to stabilize the emulsion (14) Acknowledgments This work was carried out in part pursuant to a contract with the National Foundation for Cancer Research. Literature Cited 1. Trurnit, H.J. Science III 1950, 1. 2. Feder, J.; Giaever, I. J. Colloid & Interface Science 1980, 78, 144. 3. Rothen, Α.; Mathot, C. Hel. Chim. Acta 1971, 54, 1208. 4. Giaever, I.; Laffin, R.J. Proc. Natl. Acad.Sci.,USA 1974, 71, 4533. 5. Rej, R.; Keese, C.R.; Giaever, I. Clinical Chemistry 1981, 27, 1597. 6. Giaever, I.; Keese, C.R.; Rynes, R.I. Clinical Chemistry 1984, 30, 880. 7. Giaever, I.; Ward, E. Proc. Natl. Acad. Sci., USA 1978, 75, 1366. 8. Panitz, J.Α.; Giaever, I. Surface Science 1980, 97, 25. 9. Giaever, I.; Keese, C.R. Proc. Natl. Acad.Sci.,USA 1984, 81, 3761. 10. Giaever, I.; Keese, C.R. IEEE Trans. Biomed. Engrg. 1986, 33, 242.



602 11. 12.


13. 14.

Rosenberg, M.D. Cellular Emmelot, P. and Muhlbock, 1964, pp 146-164. Keese, C.R.; Giaever, I. 1983, 80, 5622. Keese, C.R.; Giaever, I. Prather, T.L.; Grande, J.; J. Immunol. Methods 1986,

RECEIVED January

Control Mechanisms and Cancer; O., Eds.; Elsevier: Amsterdam, Proc. Natl. Acad. Sci., USA Science 1983, 219, 1448. Keese, C.R.; Giaever, I. 87, 211.

3, 1987


Applications of Adsorbed Proteins at Solid and Liquid Substrates

Recommend Documents