Cell Separation Science and Technology - ACS Publications

Chapter 6

Separation of Cells by Sedimentation

Downloaded by STANFORD UNIV GREEN LIBR on August 2, 2012 | http://pubs.acs.org Publication Date: June 10, 1991 | doi: 10.1021/bk-1991-0464.ch006

Thomas G. Pretlow and Theresa P. Pretlow Department of Pathology, Case Western Reserve University, Cleveland, OH 44106

Isopycnic and velocity sedimentation have proved to be broadly applicable techniques for the separation of mammalian cells according to their physical properties, i.e., density and effective diameter. Isopycnic sedimentation has been useful for the determination of the densities of cells; however, isopycnic sedimentation has been less widely applicable than velocity sedimentation for the purification of most kinds of mammalian cells. There are four techniques for velocity sedimentation that have been used by many: elutriation, sedimentation at unit gravity, sedimentation in an isokinetic gradient of Ficoll in tissue culture medium, and sedimentation in a reorienting gradient zonal rotor. All four of these techniques for velocity sedimentation are used with shallow (gm/ml/cm) gradients and are capable of high-resolution separations of cells based on their rates of sedimentation. The kinds of data required for the evaluation of cell separations are discussed. Because of the broad scope of this subject, we have attempted to supply references that will provide specific details that will not fit in this brief review. V e l o c i t y s e d i m e n t a t i o n and i s o p y c n i c c e n t r i f u g a t i o n i n c o n t i n u o u s g r a d i e n t s a r e w i d e l y used t e c h n i q u e s f o r t h e p r e p a r a t i v e and a n a l y t i c a l separation of c e l l s . H i s t o r i c a l l y , the f i r s t s e r i o u s e f f o r t s to p u r i f y c e l l s by means o f s e d i m e n t a t i o n were i n t h e l a b o r a t o r y o f L i n d a h l (_1). L i n d a h l has p r e s e n t e d a c r i t i c a l a n a l y s i s o f t h e t e c h n i q u e t h a t he i n v e n t e d ( 2 ) and c a l l e d c o u n t e r - s t r e a m i n g c e n t r i f u g a tion. T h i s t e c h n o l o g y was t r a n s f e r r e d t o o t h e r l a b o r a t o r i e s s l o w l y because o f t h e requirement f o r r e l a t i v e l y e x p e n s i v e equipment t h a t was n o t r e a d i l y a v a i l a b l e c o m m e r c i a l l y ; however, t h e Beckman I n s t r u ment Company has manufactured a s l i g h t l y m o d i f i e d form o f L i n d a h l ' s i n s t r u m e n t and has c a l l e d i t an " e l u t r i a t o r . " I n t h e 1960s and

0097-6156/91/0464-0090$06.00/0 © 1991 American Chemical Society

In Cell Separation Science and Technology; Kompala, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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1970s, o t h e r approaches t o c e l l s e p a r a t i o n by s e d i m e n t a t i o n were developed r a p i d l y . These i n c l u d e s e d i m e n t a t i o n a t u n i t g r a v i t y , s e d i m e n t a t i o n i n an i s o k i n e t i c g r a d i e n t of F i c o l l i n t i s s u e c u l t u r e medium, c e l l s e p a r a t i o n i n a r e o r i e n t i n g g r a d i e n t z o n a l r o t o r , and i s o p y c n i c sedimentation i n continuous g r a d i e n t s .


Theory In the d e s i g n o f experiments f o r the s e p a r a t i o n o f c e l l s , perhaps the most u s e f u l and most commonly n e g l e c t e d f a c t i s t h a t t h e r e a r e o n l y two p h y s i c a l p r o p e r t i e s of c e l l s t h a t determine t h e i r r a t e s o f s e d i m e n t a t i o n : d i a m e t e r and d e n s i t y . I f i n v e s t i g a t o r s were more w i d e l y c o g n i z a n t of t h i s f a c t , methods f o r the s e p a r a t i o n of c e l l s c o u l d be d e s i g n e d much more e f f i c i e n t l y ; and the r e s u l t a n t p u r i f i c a t i o n s of c e l l s would be o f much h i g h e r q u a l i t y . The s e d i m e n t a t i o n of c e l l s i s governed by the Stokes e q u a t i o n which i s g i v e n below. ν = dr/dt =

2

[2a (£p -

2

^m)6) r]/9^

In t h i s e q u a t i o n , £ r e p r e s e n t s the d i s t a n c e of the c e l l from the c e n t e r of r e v o l u t i o n ; _t, time; d r / d t , the v e l o c i t y o f the c e l l ; a, the r a d i u s o f the c e l l ; £j>, the d e n s i t y o f the c e l l ; Pm, the d e n s i t y of medium i n which the c e l l i s suspended a t the l o c a t i o n o f the c e l l ; £), the a n g u l a r v e l o c i t y of the c e n t r i f u g e ; and tj_ the v i s c o s i t y o f the medium i n which the c e l l i s suspended a t the l o c a t i o n o f the c e l l . F o r c e l l s t h a t a r e not s p h e r i c a l , we can determine an e f f e c t i v e v a l u e f o r the r a d i u s , i . e . , a i n the Stokes e q u a t i o n . We s h o u l d emphasize the f a c t t h a t the i n t e g r a t i o n o f the Stokes e q u a t i o n can be used e f f e c t i v e l y f o r c e l l s such as neurons and o s t e o c l a s t s t h a t a r e not s p h e r i c a l even i n s u s p e n s i o n . One can use e x p e r i m e n t a l l y d e t e r m i n e d v e l o c i t i e s of s e d i m e n t a t i o n and o t h e r measured o r known parameters i n the Stokes e q u a t i o n t o s o l v e f o r what we (_3) have termed the " e f f e c t i v e d i a m e t e r . " The e f f e c t i v e d i a m e t e r cannot be proved t o d e s c r i b e a n y t h i n g more about a p a r t i c u l a r c e l l than the f a c t o r (a) t h a t d e s c r i b e s the b e h a v i o r o f t h a t c e l l d u r i n g sedimen t a t i o n ; however, w i t h a p e r f e c t l y c i r c u l a r e r r o r and g r e a t p r a c t i c a l u t i l i t y , t h i s v a l u e f o r " e f f e c t i v e d i a m e t e r " can be used w i t h the i n t e g r a t e d Stokes e q u a t i o n f o r the s i m u l a t i o n o f the s e d i m e n t a t i o n b e h a v i o r of the c e l l under c o n s i d e r a t i o n and f o r the d e s i g n o f c o n d i t i o n s and g r a d i e n t s t h a t w i l l p e r m i t the o p t i m a l p u r i f i c a t i o n o f that c e l l . y

Boone e t a l . (4) f i r s t proposed t h a t the i n t e g r a t i o n o f the Stokes e q u a t i o n might p r o v i d e a u s e f u l approach t o the d e s i g n o f experiments i n the s e p a r a t i o n of c e l l s by v e l o c i t y s e d i m e n t a t i o n . S u b s e q u e n t l y , we (_5) demonstrated f o r the f i r s t time t h a t the Stokes e q u a t i o n , i n t e g r a t e d by a t r a p e z o i d a l computer i n t e g r a t i o n , a c c u r a t e l y d e s c r i b e s c e l l s e p a r a t i o n s as o b s e r v e d d u r i n g g r a d i e n t c e n t r i f u g a t i o n i n the l a b o r a t o r y . Both t r a p e z o i d a l (5) and c l o s e d (6) i n t e g r a t i o n s of the Stokes e q u a t i o n f o r the p u r i f i c a t i o n o f c e l l s have been d e s c r i b e d .


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Isopycnic Centrifugation In the " i s o p y c n i c c e n t r i f u g a t i o n " of c e l l s , one s e p a r a t e s c e l l s a c c o r d i n g to t h e i r buoyant d e n s i t i e s and uses a s u f f i c i e n t p r o d u c t of c e n t r i f u g a l f o r c e and time,(J r t , t o sediment c e l l s t o the l o c a t i o n s i n the g r a d i e n t where the d e n s i t i e s of the g r a d i e n t and the c e l l s a r e e q u a l . From the Stokes e q u a t i o n i t becomes apparent t h a t , when the c e l l s a p p r o a c h t h e i r d e n s i t i e s i n the g r a d i e n t , t h e i r v e l o c i t i e s a p p r o a c h z e r o ; and a d d i t i o n a l time or c e n t r i f u g a l f o r c e w i l l not change t h e i r p o s i t i o n s i n the g r a d i e n t . L e i f and V i n o g r a d (7^) were the f i r s t t o r e a l i z e the importance of c o n t i n u o u s g r a d i e n t s f o r the i s o p y c n i c c e n t r i f u g a t i o n of c e l l s and were the f i r s t t o s e p a r a t e c e l l s by i s o p y c n i c c e n t r i f u g a t i o n i n c o n t i n u o u s g r a d i e n t s . They used t h i s t e c h n i q u e f o r the p u r i f i c a t i o n of s u b p o p u l a t i o n s o f e r y t h r o c y t e s ( 7 ) . The two groups t h a t p i o n e e r e d the a p p l i c a t i o n of i s o p y c n i c c e n t r i f u g a t i o n i n c o n t i n u o u s g r a d i e n t s f o r the s e p a r a t i o n of c e l l s were L e i f * s group and Shortman's group. Both o f these i n v e s t i g a t o r s have reviewed c e l l s e p a r a t i o n by s e d i m e n t a t i o n (8,9.)· The most w i d e l y used p o t e n t i a l l y i s o t o n i c media f o r the i s o p y c n i c c e n t r i f u g a t i o n o f c e l l s have been albumin ( 7 , 1 0 ) , c o l l o i d a l s i l i c a (11, 12), and c o l l o i d a l s i l i c a c o a t e d w i t h p o l y v i n y l p y r r o l i d o n e and g i v e n the name " P e r c o l l " ( 1 3 ) . I n a d d i t i o n to t h e s e , many o t h e r media have been used f o r the i s o p y c n i c c e n t r i f u g a t i o n of c e l l s i n c l u d i n g F i c o l l (4_,_5,14-24), M e t r i z a m i d e o r 2-(3-acetamido-5-Nmethylacetamido-2,4,6-triiodobenzaraido)-2-deoxyglucose (25-27), and many o t h e r s (3,28,29). Many o f these media have s p e c i f i c d i s a d v a n tages. F o r example, F i c o l l i s more v i s c o u s than most g r a d i e n t media and r e q u i r e s v e r y h i g h c e n t r i f u g a l f o r c e s f o r i s o p y c n i c c e n t r i f u g a tion. Albumin and P e r c o l l a r e p r o b a b l y the b e s t media f o r the i s o p y c n i c sedimentation of c e l l s . P e r c o l l o f f e r s the advantage t h a t i t i s c o m m e r c i a l l y r e a d i l y a v a i l a b l e i n a form t h a t r e q u i r e s l i t t l e f u r t h e r p r e p a r a t i o n . Note t h a t our u n d e r s t a n d i n g o f the o s m o l a r i t y of P e r c o l l i s d i f f e r e n t i n r e c e n t y e a r s (13,30). In n a t u r e , most r e c o g n i z a b l e s i n g l e t y p e s o f c e l l s e x h i b i t broad and o v e r l a p p i n g ranges of d e n s i t y (3_). As a r e s u l t of t h i s broad range of d e n s i t i e s observed f o r most mammalian c e l l s , i s o p y c n i c s e d i m e n t a t i o n has been r e l a t i v e l y l i m i t e d i n i t s a p p l i c a t i o n s f o r the p u r i f i c a t i o n of c e l l s . I t has been p a r t i c u l a r l y u s e f u l f o r the s e p a r a t i o n of r e d b l o o d c e l l s and s u b p o p u l a t i o n s o f r e d b l o o d c e l l s (7^), v a r i o u s c l a s s e s o f mast c e l l s ( 1 6 ) , c a r d i a c myocytes ( 1 9 ) , p a n c r e a t i c a c i n a r c e l l s ( 2 3 ) , p a r o t i d a c i n a r c e l l s ( 3 1 ) , and rat proximal tubule c e l l s (32). Velocity

Sedimentation

" V e l o c i t y " o r " r a t e - z o n a l " s e d i m e n t a t i o n r e f e r s t o the use o f e x p e r i m e n t a l c o n d i t i o n s under which s e d i m e n t a t i o n i s stopped b e f o r e c e l l s a r r i v e a t t h e i r i s o p y c n i c d e n s i t i e s i n the media i n which they a r e sedimented. Most w e l l d e s i g n e d systems f o r v e l o c i t y s e d i m e n t a t i o n employ g r a d i e n t s i n which the a c t u a l d e n s i t i e s o f the c e l l s b e i n g s e p a r a t e d a r e not even r e p r e s e n t e d . C e l l s of e q u a l d e n s i t y but d i f f e r e n t d i a m e t e r s can be s e p a r a t e d by v e l o c i t y s e d i m e n t a t i o n . Cells of e q u a l d i a m e t e r but d i f f e r e n t d e n s i t i e s can be s e p a r a t e d i n d e p e n d e n t l y by v e l o c i t y or by i s o p y c n i c s e d i m e n t a t i o n . Since c e l l s are



6.

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s e p a r a t e d d u r i n g i s o p y c n i c c e n t r i f u g a t i o n based on d i f f e r e n c e s i n the d e n s i t i e s o f c e l l s and d u r i n g v e l o c i t y s e d i m e n t a t i o n based on d i f f e r e n c e s i n r a t e s of s e d i m e n t a t i o n , a p r o p e r t y t h a t i s dependent upon b o t h d i a m e t e r and d e n s i t y , i t i s o c c a s i o n a l l y u s e f u l t o employ a two-step p r o c e d u r e i n which v e l o c i t y s e d i m e n t a t i o n and i s o p y c n i c c e n t r i f u g a t i o n a r e used. When t h i s a p p r o a c h i s t a k e n , i s o p y c n i c c e n t r i f u g a t i o n i s o f t e n most u s e f u l as the second s t e p , s i n c e v e r y l a r g e sample volumes composed of f r a c t i o n s c o l l e c t e d from v e l o c i t y s e d i m e n t a t i o n o r from o t h e r p r o c e d u r e s f o r p u r i f i c a t i o n can be l a y ered d i r e c t l y over g r a d i e n t s f o r i s o p y c n i c c e n t r i f u g a t i o n . Starting samples f o r v e l o c i t y s e d i m e n t a t i o n must be s m a l l e r and of low density. T h i s two-step approach t o the s e p a r a t i o n of c e l l s by sediment a t i o n , i . e . , v e l o c i t y s e d i m e n t a t i o n f o l l o w e d by i s o p y c n i c sediment a t i o n , was f i r s t employed i n 1970 (16,33) and has been used f o r s e v e r a l d i f f e r e n t problems i n the s e p a r a t i o n o f c e l l s s i n c e 1970. V e l o c i t y s e d i m e n t a t i o n i n an i s o k i n e t i c g r a d i e n t of F i c o l l i n t i s s u e c u l t u r e medium has been a p p l i c a b l e f o r the s e p a r a t i o n o f many d i f f e r e n t kinds of c e l l s . We have reviewed t h i s method as used i n our l a b o r a t o r y and i n the l a b o r a t o r i e s o f o t h e r s r e c e n t l y ( 3 4 ) . The uses o f o t h e r methods f o r the v e l o c i t y s e d i m e n t a t i o n of c e l l s have a l s o been r e v i e w e d . These approaches have i n c l u d e d e l u t r i a t i o n (35, 3 6 ) , s e d i m e n t a t i o n i n a r e o r i e n t i n g g r a d i e n t z o n a l r o t o r (37-41), and s e d i m e n t a t i o n a t u n i t g r a v i t y (42-46). Sedimentation

at Unit Gravity

S e d i m e n t a t i o n a t u n i t g r a v i t y was f i r s t used by Mel and h i s c o l l a b o r a t o r s (47-50). I n i t s most w i d e l y used form, s e d i m e n t a t i o n a t u n i t g r a v i t y has been c a r r i e d out w i t h s l i g h t m o d i f i c a t i o n s of a t e c h n i q u e d e s c r i b e d by P e t e r s o n and Evans ( 4 5 ) . The t h e o r y o f v e l o c i t y s e d i m e n t a t i o n as used a t u n i t g r a v i t y has been d e s c r i b e d (46,51). The method f o r s e p a r a t i n g c e l l s a t u n i t g r a v i t y has the advantage t h a t the r e q u i r e d equipment i s i n e x p e n s i v e . W h i l e t h i s method has been employed f o r the p u r i f i c a t i o n of l a r g e numbgrs of c e l l s , i t i s b e s t s u i t e d f o r the p u r i f i c a t i o n of l e s s than 10 cells. We (3) and o t h e r s (42-44,46) have r e v i e w e d a p p l i c a t i o n s o f c e l l s e p a r a t i o n a t unit gravity previously. The time r e q u i r e d f o r s e d i m e n t a t i o n o f c e l l s a t u n i t g r a v i t y can be s h o r t e n e d c o n s i d e r a b l y w i t h the use of a r e o r i e n t i n g g r a d i e n t i n a d e v i c e i n v e n t e d by Bont and H i l g e r s ( 5 2 ) . T h i s a p p a r a t u s i s a v a i l a b l e c o m m e r c i a l l y as m o d i f i e d by W e l l s ( 5 3 ) . The major d i s a d vantage i n the use of u n i t g r a v i t y s e d i m e n t a t i o n r e s u l t s from the f a c t t h a t o n l y the e a r t h ' s g r a v i t a t i o n a l f i e l d i s a v a i l a b l e . This r e s u l t s i n a slow s e p a r a t i o n t h a t g e n e r a l l y r e q u i r e s a t l e a s t two hours and may r e q u i r e s e d i m e n t a t i o n o v e r n i g h t . I f the c e l l s tend t o a g g r e g a t e , as the m a j o r i t y o f v i a b l e c e l l s do, a g g r e g a t e s may form as c e l l s a r e t o g e t h e r i n the same zone o f the g r a d i e n t f o r h o u r s . I f a g g r e g a t e s form, they sediment w i t h l a r g e r e f f e c t i v e d i a m e t e r s ( d e f i n e d i n _3) and a r e found i n zones o f the g r a d i e n t which c o n t a i n c e l l s t h a t a r e l a r g e r than the monomers o f the c e l l s t h a t form the a g g r e g a t e ; i . e . , a g g r e g a t i o n w i l l a l t e r the s e d i m e n t a t i o n o f c e l l s and g e n e r a l l y cause a d e c r e a s e d p u r i t y and/or r e c o v e r y of c e l l s a f t e r sedimentation. I f aggregation r e s u l t s i n aggregates that are heterogeneous w i t h r e s p e c t t o the k i n d s o f c e l l s t h a t a r e i n c o r p o -


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r a t e d as i s g e n e r a l l y the c a s e , t h i s p r e c l u d e s a h i g h degree o f p u r i f i c a t i o n of the c e l l s i n v o l v e d i n these aggregates.


Isokinetic

Sedimentation

I s o k i n e t i c sedimentation i n a gradient of F i c o l l i n t i s s u e c u l t u r e medium, l i k e s e d i m e n t a t i o n a t u n i t g r a v i t y , i s b e s t s u i t e d f o r the p u r i f i c a t i o n o f r e l a t i v e l y s m a l l numbers o f c e l l s . For i s o k i n e t i c s e d i m e n t a t i o n , i n a d d i t i o n t o any r e f r i g e r a t e d , s w i n g i n g bucket c e n t r i f u g e , o n l y commonly a v a i l a b l e , i n e x p e n s i v e l a b o r a t o r y equipment i s r e q u i r e d . One c a n s e p a r a t e up t o a p p r o x i m a t e l y 35 m i l l i o n c e l l s per g r a d i e n t w i t h the t e c h n i q u e t h a t we have d e s c r i b e d (29,34,54, 55). I t i s p o s s i b l e t o r u n t h r e e o r f o u r g r a d i e n t s so t h a t the range ο g c e l l s t o be p u r i f i e d may go as h i g h as o r s l i g h t l y h i g h e r than 10 c e l l s . As a p r a c t i c a l a p p r o a c h t o t h e p u r i f i c a t i o n o f c e l l s , i s o k i n e t i c s e d i m e n t a t i o n has been employed f o r t h e p u r i f i c a t i o n o f a wide range o f types o f c e l l s i n many d i f f e r e n t l a b o r a tories (34). Elutriation The most commonly employed a p p r o a c h t o v e l o c i t y s e d i m e n t a t i o n f o r the p u r i f i c a t i o n o f l a r g e numbers o f c e l l s i s e l u t r i a t i o n . There have been many r e v i e w s o f the p u r i f i c a t i o n o f c e l l s by e l u t r i a t i o n (2,35,56,57). E l u t r i a t i o n has been used e x t e n s i v e l y and s u c c e s s f u l l y by many i n v e s t i g a t o r s e x p e r i e n c e d i n t h e use o f t h i s t e c h n i q u e (36,58-63). I t i s o u r i m p r e s s i o n from d i s c u s s i o n s w i t h many who use and who have t r i e d t o use t h e e l u t r i a t o r t h a t t h i s t e c h n i q u e has r e s o l u t i o n t h a t c l o s e l y approaches the r e s o l u t i o n t h a t one o b t a i n s w i t h s e d i m e n t a t i o n a t u n i t g r a v i t y o r s e d i m e n t a t i o n i n the i s o k i n e t i c g r a d i e n t ; however, i t appears t h a t t h e most e f f e c t i v e use o f the e l u t r i a t o r r e q u i r e s c o n s i d e r a b l y more e x p e r i e n c e and e x p e r t i s e than t h e use o f e i t h e r o f t h e s e o t h e r two methods f o r v e l o c i t y s e d i mentation. A d d i t i o n a l a s p e c t s o f e l u t r i a t i o n a r e d e t a i l e d i n the next c h a p t e r . Reorienting Gradient Zonal

Rotor

A r e l a t i v e l y s i m p l e method f o r the p u r i f i c a t i o n o f a p p r o x i m a t e l y t h e same number o f c e l l s as c a n be p u r i f i e d by e l u t r i a t i o n i s c e n t r i f u gation i n a reorienting gradient zonal rotor. L i k e e l u t r i a t i o n , the use o f t h i s t e c h n i q u e r e q u i r e s a s p e c i a l c e n t r i f u g e head. Both 8 9 t e c h n i q u e s a r e c a p a b l e o f p u r i f y i n g 10 -10 c e l l s . Neither tech nique works w e l l f o r v e r y s m a l l numbers o f c e l l s . The use o f t h e r e o r i e n t i n g gradient zonal rotor requires very l i t t l e experience; however, i t has n o t been promoted e f f e c t i v e l y , and most o f the s c i e n t i f i c community seems t o be unaware o f t h i s a p p r o a c h . Separa t i o n o f c e l l s i n t h e r e o r i e n t i n g g r a d i e n t z o n a l r o t o r has been r e viewed and has been employed f o r the p u r i f i c a t i o n o f many k i n d s o f c e l l s (37-41,64).


6.

PRETLOW & PRETLOW



Comparison o f I s o p y c n i c and V e l o c i t y

95

Sedimentation

Comparisons o f v e l o c i t y and i s o p y c n i c s e d i m e n t a t i o n have been made and d i s c u s s e d i n d e t a i l p r e v i o u s l y (3,28,29,65). I s o p y c n i c sediment a t i o n i s w e l l s u i t e d f o r the p u r i f i c a t i o n of s e v e r a l kinds of c e l l s as d e s c r i b e d above; however, as a means o f p u r i f y i n g c e l l s , i s o p y c n i c s e d i m e n t a t i o n has more l i m i t a t i o n s than does v e l o c i t y s e d i m e n t a tion. We a r e aware o f o n l y a few s p e c i f i c a p p l i c a t i o n s i n which m o r p h o l o g i c a l l y r e c o g n i z a b l e k i n d s o f c e l l s c a n be b e t t e r p u r i f i e d by i s o p y c n i c s e d i m e n t a t i o n than by v e l o c i t y s e d i m e n t a t i o n . These a p p l i c a t i o n s i n c l u d e t h e p u r i f i c a t i o n o f v a r i o u s k i n d s o f mast c e l l s ( 1 6 ) , t h e s e p a r a t i o n o f r e d b l o o d c e l l s from some v a r i e t i e s o f nucl e a t e d c e l l s , and t h e p u r i f i c a t i o n o f r a t p r o x i m a l t u b u l e c e l l s (32). I n t e r e s t i n g l y , w h i l e one c a n p u r i f y r a t p r o x i m a l t u b u l e c e l l s r e l a t i v e l y e f f e c t i v e l y by i s o p y c n i c s e d i m e n t a t i o n ( 3 2 ) , hamster p r o x i m a l t u b u l e c e l l s a r e more e f f e c t i v e l y p u r i f i e d by i s o k i n e t i c s e d i m e n t a t i o n than by i s o p y c n i c s e d i m e n t a t i o n ( 6 6 ) . I f one i s a b l e t o o b t a i n a p p r o x i m a t e l y comparable p u r i f i c a t i o n by v e l o c i t y sediment a t i o n and by i s o p y c n i c s e d i m e n t a t i o n f o r a p a r t i c u l a r k i n d o f c e l l , v e l o c i t y s e d i m e n t a t i o n o f f e r s the advantage t h a t t h e c e l l w i l l be exposed t o many-fold l e s s c e n t r i f u g a l f o r c e when s e p a r a t e d by v e l o c i t y s e d i m e n t a t i o n than when s e p a r a t e d by i s o p y c n i c s e d i m e n t a t i o n . While c e r t a i n c e l l f u n c t i o n s have been shown t o be c a p a b l e o f s u r v i v i n g f o r c e s as h i g h as 20,000g ( 6 7 ) , most c e l l s a r e s e v e r e l y i n j u r e d o r k i l l e d by t h e s e v e r y l a r g e c e n t r i f u g a l f o r c e s . Discontinuous

Gradients

D i s c o n t i n u o u s g r a d i e n t s have been w i d e l y employed f o r the p u r i f i c a t i o n of blood c e l l s . Red b l o o d c e l l s a r e p r e s e n t i n human p e r i p h e r a l b l o o d a t a p p r o x i m a t e l y 500 t o 1 , 0 0 0 - f o l d h i g h e r c o n c e n t r a t i o n s than a r e n u c l e a t e d c e l l s . I f one wishes t o p u r i f y a p a r t i c u l a r k i n d o f n u c l e a t e d c e l l from p e r i p h e r a l b l o o d , i t i s not f e a s i b l e to do t h i s under i d e a l c i r c u m s t a n c e s because o f t h i s l a r g e i n i t i a l r a t i o of red blood c e l l s to nucleated c e l l s . I n o r d e r t o g e t back a u s e f u l number o f n u c l e a t e d c e l l s , i t i s n e c e s s a r y t o l o a d a much l a r g e r number o f c e l l s on a g r a d i e n t than i s o p t i m a l . As d i s c u s s e d i n d e t a i l p r e v i o u s l y (3,28) each g r a d i e n t has a maximum c a p a c i t y known as a band c a p a c i t y . When t h i s i s exceeded, n o n i d e a l sediment a t i o n o c c u r s ; and g e n e r a l l y lower degrees o f p u r i f i c a t i o n a r e achieved. D i s c o n t i n u o u s g r a d i e n t s have been n e c e s s a r y f o r the separ a t i o n o f n u c l e a t e d c e l l s from e r y t h r o c y t e s because o f t h i s enormous problem r e l a t e d t o the r a t i o o f r e d b l o o d c e l l s t o n u c l e a t e d c e l l s . Boyum d e v e l o p e d methods t h a t a r e commonly used f o r t h i s a p p r o a c h (68-74). I t s h o u l d be noted t h a t Boyum p o i n t e d out t h a t c e n t r i f u g a l f o r c e i s n o t r e q u i r e d f o r t h e s e p a r a t i o n s t h a t he a c h i e v e d . The p r o c e s s i s somewhat a c c e l e r a t e d by the use o f a c e n t r i f u g e . Boyum used a g r a d i e n t medium which causes r e d b l o o d c e l l a g g l u t i n a t i o n , and t h i s i s an i m p o r t a n t c h a r a c t e r i s t i c o f t h e medium which Boyum explored f o r blood c e l l s . The l i m i t a t i o n s o f t h i s v a l u a b l e method have been d i s c u s s e d i n d e t a i l ( 7 5 ) . The use o f d i s c o n t i n u o u s g r a d i e n t s f o r most purposes i s n o t a d v a n t a g e o u s . When one uses a d i s c o n t i n u o u s g r a d i e n t , i t i s n e c e s s a r y f o r t h e c e l l s t o pass through a s u c c e s s i o n o f i n t e r f a c e s where they


96

CELL

SEPARATION

SCIENCE AND

TECHNOLOGY


a r e c o n c e n t r a t e d and o f t e n a g g r e g a t e Another disadvantage r e s u l t s from the f a c t t h a t c e l l s cannot be m a x i m a l l y r e s o l v e d i n d i s c o n t i n uous g r a d i e n t s s i n c e the c o n t i n u o u s range o f d e n s i t i e s and v i s c o s i t i e s cannot be r e p r e s e n t e d i n d i s c o n t i n u o u s g r a d i e n t s . The absence o f g r a d i e n t s i n the i n t e r v e n i n g d i s t a n c e s between s u c c e s s i v e i n t e r f a c e s r e s u l t s i n a l o s s o f the s t a b i l i z a t i o n of the column of f l u i d t h a t i s the major advantage of c o n t i n u o u s g r a d i e n t s . Thus, i n the absence o f c o n t i n u o u s g r a d i e n t s , s e v e r a l a r t i f a c t s a r e enhanced as has been d i s c u s s e d by many a u t h o r s (3,8,9,28,76). F o r r e a s o n s t h a t seem l e s s than j u s t i f i e d to us, some i n v e s t i g a t o r s have been a t t r a c t e d to the " s h a r p " bands t h a t a r e o b t a i n e d at i n t e r f a c e s i n discontinuous g r a d i e n t s . I t i s p o s s i b l e to combine f r a c t i o n s of d i f f e r e n t k i n d s of c e l l s s e p a r a t e d i n c o n t i n u o u s g r a d i ents. I t i s i m p o s s i b l e to s e p a r a t e the d i f f e r e n t k i n d s o f c e l l s t h a t would have been s e p a r a t e d i n a c o n t i n u o u s g r a d i e n t by c e n t r i f u g i n g them a g a i n s t the b a r r i e r s of i n c r e a s e d d e n s i t y and v i s c o s i t y t h a t a r e e n c o u n t e r e d a t the i n t e r f a c e s i n d i s c o n t i n u o u s g r a d i e n t s . As s t a t e d by de Duve ( 7 6 ) : "The d i s c o n t i n u o u s g r a d i e n t i s e s s e n t i a l l y a d e v i c e f o r g e n e r a t i n g a r t i f i c i a l bands. T h i s may be a c o n v e n i ent way o f compressing t o g e t h e r f o r p r e p a r a t i v e purposes c e r t a i n segments of the d i s t r i b u t i o n s o b s e r v e d i n c o n t i n u o u s g r a d i e n t s . But i t i s a l s o a v e r y dangerous p r o c e d u r e , i n t h a t i t c r e a t e s the i l l u s i o n of c l e a r - c u t s e p a r a t i o n . . . D e n s i t y - g r a d i e n t c e n t r i f u g a t i o n i n a c o n t i n u o u s g r a d i e n t i s the a n a l y t i c a l method 'par e x c e l l e n c e . It l e n d s i t s e l f to an e n t i r e l y o b j e c t i v e assessment of the f r e q u e n c y d i s t r i b u t i o n c u r v e s of c e r t a i n p h y s i c a l p r o p e r t i e s , such as d e n s i t y or s e d i m e n t a t i o n c o e f f i c i e n t , from which i n t u r n o t h e r c h a r a c t e r i s t i c s o f a p o p u l a t i o n , i n c l u d i n g i t s s i z e d i s t r i b u t i o n , can be derived." 1

Design of

Gradients

The d e s i g n of g r a d i e n t s f o r i s o p y c n i c c e n t r i f u g a t i o n has been d i s c u s s e d ( 3 ) . S i n c e the purpose of these g r a d i e n t s i s to p r o v i d e a means of s e p a r a t i n g c e l l s a c c o r d i n g to t h e i r d e n s i t i e s , the o n l y necessary property of a g r a d i e n t f o r i s o p y c n i c c e n t r i f u g a t i o n i s t h a t i t c o n t a i n a range o f d e n s i t i e s t h a t i n c l u d e s the d e n s i t i e s of the c e l l s to be s e p a r a t e d . I n the name o f b r e v i t y , we s h a l l emphas i z e o n l y one, o f t e n o v e r l o o k e d f e a t u r e o f i s o p y c n i c g r a d i e n t s . If t h e r e a r e l a r g e d i f f e r e n c e s , w i t h r e s p e c t t o d e n s i t y and v i s c o s i t y , between the sample to be l a y e r e d over the g r a d i e n t and t h a t p a r t o f the g r a d i e n t a t which the c e l l s e n t e r the g r a d i e n t , c e l l s w i l l be compressed and c o n c e n t r a t e d a g a i n s t t h i s i n t e r f a c e j u s t as they a r e a g a i n s t the i n t e r f a c e s i n d i s c o n t i n u o u s g r a d i e n t s . While the r e s u l t a n t a g g r e g a t i o n of c e l l s has been m i n i m i z e d by some i n v e s t i g a t o r s by s t i r r i n g the i n t e r f a c e w i t h a p i p e t t e p r i o r to c e n t r i f u g a t i o n , t h i s i s not n e c e s s a r y when the g r a d i e n t i s w e l l d e s i g n e d . The d e s i g n o f g r a d i e n t s f o r v e l o c i t y s e d i m e n t a t i o n has been discussed i n d e t a i l (77). I t i s worthy of note t h a t the d i s t a n c e o f the g r a d i e n t from the c e n t e r of the c e n t r i f u g e w i l l i n f l u e n c e the degree of s e p a r a t i o n of c e l l s t h a t i s a c h i e v e d ( 7 7 , 7 8 ) . As f o r a l l g r a d i e n t s f o r the s e p a r a t i o n of c e l l s , the o s m o l a r i t y of the g r a d i ent i s i m p o r t a n t . As d i s c u s s e d by W i l l i a m s ( 7 9 ) , the importance of d i a m e t e r i n d e t e r m i n i n g the r a t e s of s e d i m e n t a t i o n o f c e l l s has o f -



6.

PRETLOW & PRETLOW


97

t e n been emphasized t o an i n a p p r o p r i a t e d e g r e e . D e s p i t e t h i s f a c t , v e l o c i t y s e d i m e n t a t i o n i s used most commonly f o r t h e s e p a r a t i o n o f c e l l s t h a t d i f f e r w i t h r e s p e c t t o d i a m e t e r . When t h i s i s t h e g o a l , the e f f e c t o f t h e d i a m e t e r o f t h e c e l l c a n be maximized and t h e e f f e c t o f t h e d e n s i t y o f t h e c e l l c a n be m i n i m i z e d by t h e d e s i g n o f a g r a d i e n t o f low d e n s i t y . T h i s has been d i s c u s s e d w i t h some p r a c t i c a l examples ( 2 8 ) . R e s o l u t i o n w i l l be maximized when the s l o p e (gm/ml/cm) o f t h e g r a d i e n t i s as s m a l l as i s c o n s i s t e n t w i t h t h e s t a b i l i t y o f t h e column o f f l u i d ( 7 7 ) . When t h e s l o p e o f t h e g r a d i ent i s l a r g e , r a p i d l y s e d i m e n t i n g c e l l s t h a t have become s e p a r a t e d from l e s s r a p i d l y s e d i m e n t i n g c e l l s a r e r e t a r d e d by a b a r r i e r o f i n c r e a s i n g v i s c o s i t y and d e n s i t y ; t h i s d e c r e a s e s t h e f i n a l d i s t a n c e between c e l l s . The i s o k i n e t i c g r a d i e n t o f F i c o l l i n t i s s u e c u l t u r e medium t h a t we d e s c r i b e d (34,54) combines a low d e n s i t y w i t h a s l o p e t h a t i s s u f f i c i e n t l y s m a l l as t o a p p r o a c h t h e lower l i m i t o f s l o p e t h a t i s c o m p a t i b l e w i t h t h e s t a b i l i t y o f the g r a d i e n t . Parenthetic a l l y , t h e low d e n s i t y and low v i s c o s i t y o f t h i s g r a d i e n t makes i t p o s s i b l e t o s e p a r a t e o r g a n e l l e s i n t h i s g r a d i e n t by v e l o c i t y s e d i m e n t a t i o n w i t h h i g h e r r e s o l u t i o n and much lower c e n t r i f u g a l f o r c e s t h a n have been used t r a d i t i o n a l l y f o r t h e p u r i f i c a t i o n o f o r g a n e l l e s by v e l o c i t y s e d i m e n t a t i o n ( 7 7 ) . S e d i m e n t a t i o n A r t i f a c t s and E v a l u a t i o n o f Data The f a i l u r e o f many i n v e s t i g a t o r s t o be aware o f common a r t i f a c t s i n s e d i m e n t a t i o n and t h e l a c k o f a r i g o r o u s e v a l u a t i o n o f d a t a f o l l o w i n g s e d i m e n t a t i o n have r e s u l t e d i n t h e m i s i n t e r p r e t a t i o n o f a v e r y l a r g e p r o p o r t i o n o f e x p e r i m e n t s on t h e s e d i m e n t a t i o n o f c e l l s . We have stepped i n most o f t h e a v a i l a b l e p o t h o l e s a t l e a s t once and cannot emphasize t o o s t r o n g l y t h e importance o f a r t i f a c t s t h a t may occur during sedimentation (3,28). I t i s v e r y i m p o r t a n t f o r anyone who p l a n s t o use v e l o c i t y s e d i m e n t a t i o n f o r t h e p u r i f i c a t i o n o f c e l l s t o be aware o f the concept o f band c a p a c i t y and t h e s l i g h t l y d i f f e r e n t concept o f g r a d i e n t c a p a c i t y . R e l e v a n t t o the k i n d s o f d a t a t h a t a r e r e q u i r e d f o r t h e e v a l u a t i o n o f e x p e r i m e n t s i n c e l l s e p a r a t i o n , we (80) w i s h t o emphasize p a r t i c u l a r d a t a t h a t a r e o f t e n o m i t t e d from p u b l i s h e d papers t h a t s h o u l d be i n c l u d e d . S p e c i f i c a l l y , we b e l i e v e t h a t i t i s v e r y import a n t t o q u a n t i f y t h e markers o f f u n c t i o n and p u r i t y i n the s e p a r a t e d f r a c t i o n s and i n t h e s t a r t i n g s u s p e n s i o n o f c e l l s . F o r most c e l l s e p a r a t i o n s , we would l i k e t o see p h o t o m i c r o g r a p h s o f permanent preparations of p u r i f i e d c e l l s with s u f f i c i e n t c e l l s being present t o g i v e a s e m i q u a n t i t a t i v e message about p u r i t y and, more import a n t l y , a c l e a r i n d i c a t i o n of the q u a l i t y of the (a) preparations examined and ( b ) c e l l s r e c o v e r e d . W h i l e morphology o f h i g h q u a l i t y does not g u a r a n t e e t h a t t h e c e l l s a r e f u n c t i o n a l l y i n t a c t o r even a l i v e , i t i s o f t e n p o s s i b l e t o t e l l t h a t c e l l s a r e damaged o r h i g h l y s e l e c t e d based on t h e morphology o f c e l l s i n t h e s t a r t i n g sample as compared w i t h t h e q u a l i t y o f t h e p u r i f i e d c e l l s . Permanent p r e p a r a t i o n s ( a ) always g i v e a l e s s o p t i m i s t i c e s t i m a t e o f t h e degree o f p u r i f i c a t i o n than wet p r e p a r a t i o n s and (b) p r o v i d e a permanent r e c o r d t h a t c a n be examined and compared w i t h o t h e r e x p e r i m e n t s i n future years.



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Some i n v e s t i g a t o r s e x p r e s s contempt f o r those who a r e i n t e r e s t e d i n s e p a r a t i n g c e l l s o f type A from c e l l s of type Β w h i l e emphasizing the v a l u e o f t e c h n i q u e s f o r c e l l s e p a r a t i o n t o s e p a r a t e f u n c t i o n a l l y h e t e r o g e n e o u s s u b p o p u l a t i o n s of s i n g l e k i n d s o f c e l l s such as g r a n u l o c y t e s o r macrophages. While i t i s v a l u a b l e t o sepa r a t e heterogeneous s u b p o p u l a t i o n s of p a r t i c u l a r k i n d s of c e l l s t h a t d i f f e r w i t h r e s p e c t t o f u n c t i o n , i t i s of l i t t l e use t o show t h a t c e l l o f type A i s d i s t r i b u t e d i n f r a c t i o n s 6 through 10 and t h a t t h e s e f r a c t i o n s c o n t a i n d i f f e r e n t average amounts of f u n c t i o n / c e l l . I f the c e l l s i n f r a c t i o n 6 c o n t a i n o n l y h a l f as much average f u n c t i o n / c e l l as c e l l s i n f r a c t i o n 10, does t h i s r e p r e s e n t t h e i r c h a r a c t e r i s t i c h e t e r o g e n e i t y i n f u n c t i o n t h a t was p r e s e n t i n v i v o , o r were c e l l s i n f r a c t i o n 6 p a r t i t i o n e d t o f r a c t i o n 6 because they were i n j u r e d o r p o s s e s s e d an u n r e l a t e d p h y s i c a l p r o p e r t y ? How does the c o l l e c t i v e f u n c t i o n o f c e l l s i n a l l f r a c t i o n s compare w i t h the f u n c t i o n a v a i l a b l e b e f o r e the p r o c e d u r e f o r p u r i f i c a t i o n was performed? Other s i m i l a r q u e s t i o n s have been d i s c u s s e d i n d e t a i l ( 8 0 ) . D i r e c t i o n s f o r the

Future

I t has become i n c r e a s i n g l y apparent t h a t the o p t i m a l p u r i f i c a t i o n of most k i n d s of c e l l s r e q u i r e s the s u c c e s s i v e use o f more than one k i n d of t e c h n i q u e f o r the s e p a r a t i o n of c e l l s . F o r example, i n p u r i f y i n g the p u t a t i v e p r e c u r s o r s of h e p a t o c e l l u l a r carcinoma from the l i v e r s of r a t s t h a t had been t r e a t e d w i t h c a r c i n o g e n , c o n s i d e r a b l e advantage was found i n the s e q u e n t i a l c o m b i n a t i o n of v e l o c i t y s e d i m e n t a t i o n and f r e e - f l o w e l e c t r o p h o r e s i s ( 8 1 ) . While e l e c t r o p h o r e s i s has not been used as e x t e n s i v e l y as many o t h e r approaches t o the s e p a r a t i o n of c e l l s , e l e c t r o p h o r e s i s i s an important approach t h a t ( a ) t a k e s advantage of a q u a l i t a t i v e l y d i f f e r e n t s e t of c h a r a c t e r i s t i c s o f c e l l s from those t h a t a r e important f o r o t h e r c e l l s e p a r a t i o n s and (b) i s g e n e r a l l y a p p l i c a b l e t o a l l c e l l s (82-84). The de velopment of e l e c t r o p h o r e t i c b u f f e r s o f low i o n i c s t r e n g t h and t h e i r a p p l i c a t i o n by the l a t e K l a u s Z e i l l e r i n c o l l a b o r a t i o n w i t h K u r t Hannig d i d much t o e s t a b l i s h the u s e f u l n e s s o f e l e c t r o p h o r e s i s f o r the s e p a r a t i o n of l a r g e numbers of c e l l s (85-87). The number o f m o n o c l o n a l a n t i b o d i e s t h a t a r e s p e c i f i c f o r the s u r f a c e components of p a r t i c u l a r k i n d s of c e l l s i s i n c r e a s i n g r a p i d l y , and the a v a i l a b l e t e c h n o l o g y f o r the c r e a t i o n o f new, h i g h l y s p e c i f i c monoclonal a n t i b o d i e s a g a i n s t s p e c i f i c k i n d s of c e l l s i s g r e a t l y improved ( 8 8 ) . These a n t i b o d i e s w i l l be used i n c r e a s i n g l y i n p r o v i d i n g a d d i t i o n a l , o f t e n v e r y s p e c i f i c means f o r the p u r i f i c a t i o n of c e l l s . They may be used t o f a c i l i t a t e the e l e c t r o n i c s o r t i n g o f c e l l s (89) o r may be used a t t a c h e d t o m i c r o s p h e r e s (90,91) t h a t w i l l r e s u l t i n changes i n c h a r g e , d e n s i t y , o r s u s c e p t i b i l i t y t o magnetic f o r c e s t h a t can be e x p l o i t e d f o r the p u r i f i c a t i o n of the c e l l - m i c r o s p h e r e complex as d e s c r i b e d i n l a t e r c h a p t e r s . Particles t h a t can be a t t r a c t e d w i t h magnets have been used f o r y e a r s w i t h hand-held magnets. More r e c e n t l y , a r e l a t i v e l y i n e x p e n s i v e and much more p o w e r f u l d e v i c e (92) has been f a b r i c a t e d t h a t p e r m i t s one t o s e p a r a t e c e l l s t h a t f l o w t h r o u g h a column from which they can be r e c o v e r e d when the e l e c t r o m a g n e t i s s w i t c h e d o f f . We a n t i c i p a t e t h a t s e d i m e n t a t i o n w i l l c o n t i n u e t o p r o v i d e an i m p o r t a n t a p p r o a c h to c e l l s e p a r a t i o n t h a t w i l l be combined w i t h




99

many o t h e r q u a l i t a t i v e l y d i f f e r e n t approaches t o g e n e r a t e strategies f o r m u l t i - s t e p p u r i f i c a t i o n s f o r the successive e x p l o i t a t i o n of q u a l i t a t i v e l y d i f f e r e n t c h a r a c t e r i s t i c s o f t h e c e l l s t o be s e p a r a t e d . Acknowledgment s T h i s work was s u p p o r t e d by P u b l i c H e a l t h S e r v i c e g r a n t s ROI CA36467, ROI CA38727, ROI CA48032, and P30 CA43703 from t h e N a t i o n a l Cancer I n s t i t u t e and by g r a n t //89B48 from t h e American I n s t i t u t e f o r Cancer Research.


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