Separation of Living Cells - American Chemical Society

Chapter 1

Separation of Living Cells 1

2

Paul Todd and Thomas G. Pretlow l

Center for Chemical Technology, National Institute of Standards and Technology, 325 Broadway 831.02, Boulder, CO 80303-3328 Department of Pathology, Case Western Reserve University, Cleveland, OH 44106

Downloaded by ADAMS STATE UNIV on February 18, 2015 | http://pubs.acs.org Publication Date: June 10, 1991 | doi: 10.1021/bk-1991-0464.ch001

2

There are several reasons for isolating subpopulations of living cells, including biochemical study, product analysis in non-clonogenic cells, selection of fused cells, isolation of rare cell types for cloning, preparation of pure cells for transplantation, and bioreactor maintenance. Methods for separating cells include flow sorting, sedimentation, biphasic extraction, field-flow fractionation, affinity methods, magnetically enhanced affinity methods, and electrophoresis. Each method can be considered in terms of its resolution, purity, gentleness, convenience and cost. Recent advances in each of these methods improves its utility in routine cell-separation practice and its potential for scale-up.

The c h a p t e r s t h a t f o l l o w c o v e r , i n one o r more forms, n e a r l y a l l o f the methods u s e d i n t h e s e p a r a t i o n o f l i v i n g c e l l s . This chapter introduces the r a t i o n a l e s f o r s e p a r a t i n g l i v i n g c e l l s , the parameters w i t h w h i c h s e p a r a t i o n methods a r e e v a l u a t e d , and some p h y s i c a l c h a r a c t e r i s t i c s o f each o f t h e methods r e p o r t e d i n t h e subsequent chapters. Books c o n t a i n i n g r e v i e w a r t i c l e s (1-4) appear from time t o time on t h i s s u b j e c t . To date, most a p p l i c a t i o n s o f l i v i n g c e l l s e p a r a t i o n have o c c u r r e d i n t h e b i o m e d i c a l r e s e a r c h f i e l d and i n t h e f i e l d o f c e l l and m o l e c u l a r b i o l o g y . Applications to bioprocessing a r e s t i l l emerging.

Why s e p a r a t e

living

cells?

N o v e l p r o d u c t s o f b i o t e c h n o l o g y i n c l u d e p a r t i c u l a t e m a t e r i a l s . These t a k e t h e form o f s u b c e l l u l a r p a r t i c l e s , b a c t e r i a l i n c l u s i o n b o d i e s , whole c e l l s , and i n s o l u b l e m a c r o m o l e c u l a r a g g r e g a t e s . T h e o r e t i c a l l y ,

0097-6156/91/0464-0001$07.00/0 © 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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the l o g i c a l way t o p u r i f y t h e s e p r o d u c t s i s b y c e n t r i f u g a t i o n . I n an i n c r e a s i n g number o f c a s e s o f s u b c e l l u l a r p a r t i c l e s t h i s a p p r o a c h fails. Some v e r y s m a l l p a r t i c l e s have v e r y low s e d i m e n t a t i o n r a t e s and hence r e q u i r e u l t r a c e n t r i f u g a t i o n - - a s a t i s f a c t o r y method o n l y a t bench s c a l e . Some p a r t i c l e s a r e c o n t a m i n a t e d w i t h unwanted p a r t i c l e s t h a t have i d e n t i c a l s e d i m e n t a t i o n c o e f f i c i e n t s . P r o c e s s i n g methods t h a t c a n d e a l w i t h t h e s e troublesome s e p a r a t i o n problems include two-phase extraction, free electrophoresis, affinity a d s o r p t i o n and f i e l d - f l o w f r a c t i o n a t i o n . The r e a s o n s f o r p u r i f y i n g suspended a n i m a l and p l a n t c e l l s a r e numerous, and t h e a c t i v i t y i t s e l f i s amply j u s t i f i e d by t h e demands f o r pure c e l l p o p u l a t i o n s as objects o f chemical research, l i v i n g m a t e r i a l f o r t r a n s p l a n t a t i o n , and s o u r c e s o f u n c o n t a m i n a t e d b i o p r o d u c t s . Although h i s t o c h e m i c a l s t u d i e s a r e c a p a b l e o f d e m o n s t r a t i n g w h i c h a n t i g e n s o r w h i c h enzyme activities a r e made by c e r t a i n c e l l s , they show v e r y little i n f o r m a t i o n about p r o c e s s i n g , m o l e c u l a r w e i g h t forms, i n h i b i t o r s o r growth f a c t o r s . Any c e l l type w i t h l i m i t e d o r no p r o l i f e r a t i v e c a p a b i l i t i e s t h a t i s needed i n pure form must be s e p a r a t e d b y a t e c h n i q u e t h a t p r o v i d e s adequate p u r i t y , adequate y i e l d , adequate r e l a t i o n s h i p t o c e l l f u n c t i o n , and adequate f u n c t i o n a f t e r s e p a r a t i o n (Todd e t a l . , 1986). L i g h t - a c t i v a t e d f l o w s o r t i n g , w h i c h may p r o c e s s a few h u n d r e d i n i t i a l c e l l s p e r second, i s i n c a p a b l e o f p r o d u c i n g adequate numbers o f r a r e c e l l t y p e s f o r b i o c h e m i c a l a n a l y s i s . Cell e l e c t r o p h o r e s i s ( 6 ) , h i g h g r a d i e n t m a g n e t i c f i l t r a t i o n ( 7 ) , two-phase partitioning (8, 9 ) , and a f f i n i t y adsorption (10) have been identified as s u i t a b l e processes for cell separation. These processes, although not e s p e c i a l l y new (11, 12.), a r e i n t h e a d o l e s c e n t phase o f development, and p r o c e s s e s y e t t o be d i s c o v e r e d may be a p p l i c a b l e t o t h e p a r t i c l e p u r i f i c a t i o n problem. Relating Cell S t r u c t u r e and F u n c t i o n . S i z e and d e n s i t y a r e d i s t r i b u t e d v a l u e s i n any l i v i n g c e l l p o p u l a t i o n . (See F i g u r e 1 ) . These may o r may n o t be r e l a t e d t o c e l l f u n c t i o n . F o r example, mast c e l l s and somatotrophs a r e e x c e p t i o n a l l y dense owing t o t h e i r d e n s e l y packed granules o f s e c r e t o r y m a t e r i a l s . Sedimentation h e l p s separate such cells from o t h e r s w i t h which they are n a t u r a l l y mixed. H e t e r o g e n e i t y o f sedimentation i n a pure s u b p o p u l a t i o n i n t e r f e r e s w i t h p u r i t y and y i e l d i n most p u r i f i c a t i o n p r o c e s s e s , including c e r t a i n t y p e s o f c e l l e l e c t r o p h o r e s i s (13). In density gradient e l e c t r o p h o r e s i s i t h a s been shown t h a t human embryonic k i d n e y c e l l s m i g r a t e i n d e p e n d e n t l y o f s i z e , d e n s i t y , and p o s i t i o n i n t h e c e l l c y c l e (5) ; and a d u l t mammalian k i d n e y c e l l s c a n be s e p a r a t e d by e l e c t r o p h o r e s i s into subpopulations that vary with respect to r e n i n p r o d u c t i o n and o t h e r f u n c t i o n s (14, 1 5 ) . Two-phase e x t r a c t i o n , w h i c h depends on c e l l s p a r t i t i o n i n g i n t o upper and lower i m m i s c i b l e aqueous s o l u t i o n s o f polymers, has been a p p l i e d t o c e l l s e p a r a t i o n problems i n hematology and immunology ( 4 ) . W h i l e h i s t o c h e m i s t s r o u t i n e l y r e l a t e c e l l s t r u c t u r e and f u n c t i o n , h i s t o c h e m i s t r y a l o n e c a n n o t r e v e a l t h e s u b t l e ( a n d sometimes substantial) differences that have been discovered by cell purification, such as i m m u n o l o g i c a l l y similar molecules with d i f f e r e n t b i o l o g i c a l f u n c t i o n p r o d u c e d by s e p a r a b l e , m o r p h o l o g i c a l l y



TODD & PRETLOW

Separation of Living CeUs

10 20 30 CELL DIAMETER,μΜ

40

F i g u r e 1. S i z e h e t e r o g e n e i t y o f human k i d n e y c e l l s i n f i r s t p a s s a g e c u l t u r e , measured m i c r o s c o p i c a l l y . Dashed v e r t i c a l l i n e s indicate c a l i b r a t i o n points. The range o f d i a m e t e r s i s n e a r l y a f a c t o r o f 3.



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s i m i l a r , c e l l s (16, 17). H i s t o c h e m i s t r y cannot measure the m o l e c u l a r w e i g h t s o f c e l l p r o d u c t s o r determine gene sequences i n s u c h c e l l s ; c e l l p u r i f i c a t i o n has made i t p o s s i b l e t o do t h e s e . Owing t o e l e c t r o p h o r e t i c p u r i f i c a t i o n , we now know, f o r example, t h a t two p i t u i t a r y c e l l t y p e s make two t y p e s o f growth hormone and that different electrophoretic subpopulations of kidney cells make plasminogen a c t i v a t o r molecules having d i f f e r e n t p r o p e r t i e s . The f i n d i n g s r e q u i r e d p r o c e d u r e s f o r r e l i a b l y i s o l a t i n g s e v e r a l s p e c i f i c c e l l t y p e s from mammalian s o l i d t i s s u e s (13,17). The accomplishment o f m e t h o d o l o g i c a l g o a l s s u c h as t h e s e p r o v i d e s new means t o h i g h l y s i g n i f i c a n t ends: a way t o s t u d y the f u n c t i o n s o f specific cells o f t i s s u e s and e f f e c t i v e methods o f b u l k cell s e p a r a t i o n . The p h a r m a c o l o g i c a l s t u d y o f s e p a r a t e d c e l l p o p u l a t i o n s a l s o has the p o t e n t i a l o f r e d u c i n g the amount o f c o s t l y (and, t o some, o b j e c t i o n a b l e ) a n i m a l r e s e a r c h . C l o n o g e n i c but Rare P l a n t and A n i m a l C e l l s . Most c l o n o g e n i c c e l l s from a l l l i v i n g kingdoms c a n be i s o l a t e d by c l a s s i c a l methods c o n s i s t i n g o f s e l e c t i n g a whole c o l o n y o f c e l l s h a v i n g the d e s i r e d p r o p e r t i e s and p r o p a g a t i n g c e l l s from i t i n c o n t i n u o u s o r b a t c h c u l t u r e . Such t e c h n i q u e s as r e p l i c a p l a t i n g , c o l o n y " p i c k i n g " ( l o o p , t o o t h p i c k , p i p e t t e , s e l e c t i o n r i n g , e t c . ) , and d i l u t i o n i n m i c r o w e l l s are used t o i s o l a t e whole c o l o n i e s t h a t a r e presumed t o have d e s c e n d e d from a s i n g l e c e l l . S e l e c t i n g one c e l l w i t h a m i c r o p i p e t t e under a m i c r o s c o p e a l s o e x p l o i t s the c l o n o g e n i c p o t e n t i a l o f a d e s i r e d c e l l type. These c l a s s i c a l methods a r e s t i l l the most e f f i c i e n t f o r most g e n e t i c c l o n i n g g o a l s ; however, when the d e s i r e d c e l l t y p e i s l e s s t h a n 0.01% o f the c l o n o g e n i c p o p u l a t i o n , t h e n more t h a n 10,000 c o l o n i e s must be s c r e e n e d t o o b t a i n each c l o n e u n l e s s a g e n e t i c s e l e c t i o n t e c h n i q u e i s used. Genetic s e l e c t i o n techniques ( k i l l i n g a l l b u t the d e s i r e d c l o n e s ) can be c o n t r i v e d f o r most m i c r o b i a l c e l l s , t h e r e b y s o l v i n g t h i s problem, b u t few p l a n t and a n i m a l c e l l i s o l a t i o n problems have ready-made s e l e c t i v e markers. V i a b l e - c e l l s e p a r a t i o n p r o c e s s e s a p p l i e d t o t h i s same p r o b l e m c a n i s o l a t e 1,000 d e s i r e d c e l l s i n a s i n g l e s t e p s t a r t i n g w i t h 10 c e l l s . F o r example, i f an o p t i c a l marker i s a v a i l a b l e , c l o n o g e n i c c e l l s c a n be c o l l e c t e d by s o r t i n g each d e s i r e d c e l l i n t o a s i n g l e w e l l w i t h a c e l l s o r t e r , r e s u l t i n g i n 10 m i c r o w e l l p l a t e s w i t h a s e l e c t e d , c l o n o g e n i c c e l l i n each w e l l . S i m i l a r l y , i f s u r f a c e markers e x i s t , s e l e c t i o n w i t h an a f f i n i t y l i g a n d i s p o s s i b l e ; i f s u r f a c e c h a r g e differences exist, electrophoretic i s o l a t i o n w i l l y i e l d a bulk s u s p e n s i o n o f s e l e c t e d c e l l s i n a few h u n d r e d μL o f l i q u i d ; e t c . 7

Hybrid (Electrofused) C e l l s . I n a r i g o r o u s s t a t i s t i c a l s t u d y o f the rescue of desired hybridoma cells d u r i n g monoclonal antibody p r o d u c t i o n , Adamus e t a l . ( 1 8 ) , found i t n e c e s s a r y t o p e r f o r m two s e r i a l c l o n a l s e l e c t i o n s by p e r f o r m i n g s e r i a l d i l u t i o n s i n 4 9 6 - w e l l m i c r o t i t e r p l a t e s a t each s t e p u s i n g 0.2 - 10 c e l l s p e r w e l l . A p h y s i c a l p r o c e s s t h a t can a c c o m p l i s h the same g o a l s a u t o m a t i c a l l y would o b v i o u s l y be h e l p f u l as hybridoma t e c h n o l o g y i s one o f the most intense users of c e l l selection.


1.

T O D D & PRETLOW

Separation of Living Cells

P r e l i m i n a r y r e s e a r c h has i n d i c a t e d t h a t p a i r s o f c e l l t y p e s can be chosen with differing electrophoretic mobility and that h e t e r o d i k a r y o n s o f such c e l l s have an i n t e r m e d i a t e e l e c t r o p h o r e t i c m o b i l i t y (19). The use o f a p h y s i c a l p r o p e r t y a v e r t s the need f o r a s e l e c t a b l e g e n e t i c marker i n the h e t e r o k a r y o n s . Thus i t i s no l o n g e r n e c e s s a r y t o f u s e o n l y c e l l s t h a t have been g e n e t i c a l l y m a n i p u l a t e d t o p o s s e s s markers t h a t a r e l e t h a l i n h y b r i d s e l e c t i o n medium.


Non-clonogenic C e l l s . D i s p e r s e d c e l l s from p l a n t and a n i m a l t i s s u e s c o n t a i n v e r y few c e l l s c a p a b l e o f p r o l i f e r a t i o n , b u t e v e r y t i s s u e c o n t a i n s numerous t y p e s o f c e l l s . P h y s i c a l processes that separate c e l l t y p e s from one a n o t h e r a r e r e q u i r e d f o r the p e r f o r m a n c e o f f u n c t i o n a l s t u d i e s or s t u d i e s o f composition. Biochemical study. One o f the major g o a l s of c e l l separation t e c h n o l o g y i s the i s o l a t i o n o f adequate numbers o f c e l l s for biochemical study. The g o a l s o f b i o c h e m i c a l a n a l y s i s o f i s o l a t e d c e l l s u b p o p u l a t i o n s are too numerous t o l i s t , b u t a few examples w i l l i l l u s t r a t e the importance o f t h i s g o a l . C e l l subsets f o r t r a n s p l a n t a t i o n . C e l l subsets f o r t r a n s p l a n t a t i o n c a n be i s o l a t e d by p h y s i c a l methods, p r o v i d i n g p o p u l a t i o n s t h a t can be t r a n s p l a n t e d i n the absence o f a n t i g e n - p r e s e n t i n g c e l l s , which enhance the h o s t ' s immune r e j e c t i o n r e s p o n s e , f o r example. Other types of undesired cells can be eliminated from transplant p o p u l a t i o n s , s u c h as p r o l a c t i n - p r o d u c i n g c e l l s o f the p i t u i t a r y . In a c l a s s i c a l example, g r a f t - v s . - h o s t c e l l s have been removed from bone marrow c e l l p o p u l a t i o n s , t h e r e b y e n h a n c i n g h o s t s u r v i v a l (20.21). Productive from Non-productive Bioreactor Cells. In modern b i o r e a c t o r technology c e l l s are o f t e n c u l t u r e d to produce products s p e c i f i e d by f o r e i g n genes. C e l l s w i t h f o r e i g n genes s u f f e r from an a d d i t i o n a l metabolic burden that u s u a l l y slows their rate of m u l t i p l i c a t i o n r e l a t i v e to t h a t o f t h e i r c o u n t e r p a r t c e l l s w i t h o u t f o r e i g n genes. T h e r e f o r e , c e l l s t h a t have l o s t f o r e i g n genes can t a k e o v e r a b i o r e a c t o r c u l t u r e i n a few g e n e r a t i o n s , so s t r a t e g i e s must be u s e d t o r i d b i o r e a c t o r s o f n o n - p r o d u c t i v e c e l l s . H i g h - c e l l - d e n s i t y b i o r e a c t o r s a l s o t e n d to accumulate dead c e l l s , which are a l s o non-productive. A procedure f o r continuously or r e g u l a r l y r i d d i n g b i o r e a c t o r c u l t u r e s o f dead c e l l s i s a l s o u s e f u l , as dead cells can have various adverse affects on reactor p r o d u c t i v i t y , s u c h as s e c r e t i n g p r o d u c t s t h a t p o i s o n the l i v e c e l l s o r t h e i r p r o d u c t s , consuming n u t r i e n t s from the medium u s e l e s s l y , etc. P r e p a r a t i o n o f Pure M a t e r i a l s . When an i n t r a c e l l u l a r p r o d u c t i s made by a s m a l l f r a c t i o n o f the t o t a l c e l l s i n a p o p u l a t i o n , s u c h as an a n i m a l t i s s u e , i t i s n o t always u s e f u l t o i s o l a t e the p r o d u c t from the t o t a l t i s s u e , b u t i n s t e a d , from a s u b p o p u l a t i o n o f c e l l s from that tissue.


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Study o f the p r o c e s s i n g o f m e t a b o l i t e s i n o r g a n s . D i f f e r e n t c e l l types i n a given t i s s u e p o s s e s s d i f f e r e n t m e t a b o l i c pathways, i n c l u d i n g pathways o f hormone and v i t a m i n transformation. The r e l a t i o n s h i p between m e t a b o l i c s t e p s and f u n c t i o n a l c e l l t y p e s c a n n o t always be e s t a b l i s h e d by h i s t o c h e m i s t r y . While h i s t o c h e m i s t r y can o f t e n e s t a b l i s h the p r e s e n c e o r absence o f c e r t a i n enzymes, i n c a n n o t p r o v i d e enzyme k i n e t i c d a t a o r enzyme c h a r a c t e r i z a t i o n d a t a .

Evaluation

of separation

methods


Resolution. The q u a n t i t a t i o n o f r e s o l u t i o n tends t o f o l l o w the paradigm o f m u l t i - s t a g e e x t r a c t i o n or a d s o r p t i o n (chromatography). R -

(x

2

- Χι)/2(σ! + σ )

(1)

2

where R - " r e s o l u t i o n " x and x are the m i g r a t i o n d i s t a n c e s o f any two separands, and σ and σ are their respective standard deviations. The i n v e r s e v i e w i s a l s o u s e f u l . In sedimentation, i n which resolution depends on the standard d e v i a t i o n of the distance s e d i m e n t e d and e l e c t r o p h o r e s i s , i n w h i c h i t depends on the s t a n d a r d d e v i a t i o n o f m o b i l i t y , and i n f l o w s o r t i n g , where i t depends on the s t a n d a r d d e v i a t i o n o f o p t i c a l f l u o r e s c e n c e , f o r example - - a l l c a s e s i n w h i c h the moments o f the a p p r o p r i a t e measurement a r e known -- the i n v e r s e o f the " T h e o r e t i c a l S t a g e s " i s a u s e f u l v a r i a b l e . This i s the c o e f f i c i e n t o f v a r i a t i o n ("CV") o r " r e l a t i v e s t a n d a r d d e v i a t i o n " , i n any c a s e the r a t i o o f s t a n d a r d d e v i a t i o n t o the mean. For example, 2

1

x

2

CV

- αχ/Xi.

(2)

T h e r e a r e u s u a l l y p h y s i c a l c o n s t r a i n t s on r e s o l u t i o n . The r e c i p r o c a l o f maximum number o f f r a c t i o n s t h a t can be c o l l e c t e d , h e r e termed number o f s e p a r a t i o n u n i t s , NSU, cannot e x c e e d the CV. T h i s measure of "geometrical r e s o l u t i o n " i s d e f i n e d s t r i c t l y i n terms o f the geometry o f the system. In a t y p i c a l c e l l h a r v e s t i n g process, t h i s would be d e t e r m i n e d from NSU

= volume o f s e p a r a t o r / v o l u m e p e r

fraction.

(3)

I n f l o w s o r t i n g , i t works out t h a t NSU = number o f c e l l s s o r t e d and can be very large (more than 1 million). In some cell e l e c t r o p h o r e s i s p r o c e d u r e s , NSU can be as s m a l l as 12. Sometimes t h e s e f r a c t i o n s a r e f u r t h e r p o o l e d i n t o 4-6 s u s p e n s i o n s , r e d u c i n g NSU to 4-6. These numbers s h o u l d be c o n s i d e r e d i n comparison w i t h chromatography, f o r example, where NSU = 10,000 i s common ( t h i s number r e l a t e s t o the p r o c e d u r e f o r f r a c t i o n c o l l e c t i o n and i s n o t the same as the number o f t h e o r e t i c a l p l a t e s ) .


1.

T O D D & PRETLOW

Separation of Living Cetts

Purity. P u r i t y i s d e f i n e d as t h e p r o p o r t i o n that c o n s i s t s o f the d e s i r e d product ( c e l l types, i : Purity -

κ /Σχ 1

of a separated f r a c t i o n type 1) among a l l c e l l

,

ι

7

(4)

i


where x ^ s a r e c e l l c o n c e n t r a t i o n s . In a t y p i c a l separation a trade i s made between p u r i t y and r e c o v e r y . The h i g h e s t p u r i t y i s f o u n d i n one ( p o s s i b l y two) f r a c t i o n ( s ) . I f t h e s e c o n t a i n o n l y 1/3 o r l e s s o f the p r o d u c t , a d j a c e n t f r a c t i o n s c a n be p o o l e d w i t h t h e c e n t r a l f r a c t i o n , thereby i n c r e a s i n g r e c o v e r y b u t r e d u c i n g the average p u r i t y to Purity -

(EiXi/ZxJVjl/iZVj) J

for a l l c e l l

i

(5)

j

types i i n j pooled f r a c t i o n s .

One c a n n o t a s s e s s " p u r i f i c a t i o n " u n l e s s one knows t h e p u r i t i e s o f b o t h t h e s e p a r a t e d f r a c t i o n s and t h e i n i t i a l s u s p e n s i o n o f c e l l s . When r e p o r t s s t a t e t h a t c e r t a i n s e p a r a t e d f r a c t i o n s c o n t a i n a c t i v i t y or "purified" cells without giving the l e v e l o f a c t i v i t y o r c o n c e n t r a t i o n o f c e l l s i n the s t a r t i n g suspension, i t i s impossible t o know whether o r n o t a p u r i f i c a t i o n t o o k p l a c e . The f a c t t h a t different fractions from a separation contain different concentrations o f separands i s almost inevitable and i s n o t n e c e s s a r i l y e v i d e n c e f o r any degree o f p u r i f i c a t i o n . T h i s p o i n t may n o t be a p p r e c i a t e d when y i e l d s a r e n o t a l s o c a l c u l a t e d . T h e r e a r e many means o f a s s e s s i n g p u r i t y . One c a n a s s e s s a f u n c t i o n a l p a r a m e t e r and e x p r e s s p u r i t y as a f u n c t i o n a l u n i t , i . e . , s e c r e t i o n o f a hormone, a c t i v i t y o f an enzyme, a b i l i t y t o k i l l neoplastic c e l l s etc., per " p u r i f i e d " c e l l . I t i s i m p o r t a n t t o be aware t h a t p u r i t y , as a s s e s s e d m o r p h o l o g i c a l l y o r as e x p r e s s e d as proportions o f c e l l s with given phenotypic c h a r a c t e r i s t i c s ( l a b e l l i n g w i t h a monoclonal antibody, c o n t a i n i n g phagocytosed b a c t e r i a , or w h a t e v e r ) i s a l m o s t n e v e r d i r e c t l y r e l a t e d t o p u r i t y e x p r e s s e d as a v e r a g e amount o f f u n c t i o n / c e l l . L e t us c o n s i d e r a n example t h a t d e m o n s t r a t e s t h e importance o f m i c r o s c o p i c e x a m i n a t i o n . One might have a f r a c t i o n number 12 t h a t c o n t a i n s 100% macrophages w i t h one o r two i n g e s t e d b a c t e r i a p e r c e l l . By t h i s s t a n d a r d , t h e p u r i f i c a t i o n has been a b s o l u t e , and t h e macrophages t h a t c o u l d n o t i n g e s t t h e t e s t e d b a c t e r i u m have been p a r t i t i o n e d i n t o d i f f e r e n t f r a c t i o n s from the f r a c t i o n w i t h 100% macrophages t h a t c o n t a i n b a c t e r i a . Itis p o s s i b l e t h a t t h e same p r o c e d u r e f o r f r a c t i o n a t i o n c o u l d y i e l d a f r a c t i o n 6 w i t h (a) 25% macrophages t h a t c o n t a i n an a v e r a g e o f 50 b a c t e r i a / macrophage and (b) 75% macrophages t h a t were u n a b l e t o ingest bacteria. L e t us f u r t h e r assume t h a t t h e b a c t e r i a have n o t been k i l l e d and t h a t t h e a s s a y o f p u r i t y i s t o l y s e t h e macrophages, c u l t u r e t h e b a c t e r i a , and c o u n t t h e number o f b a c t e r i a l colonies obtained. I f we i n v e n t a s i t u a t i o n i n w h i c h a l l b a c t e r i a grow, one would e x p e c t t o g e t 12.5 b a c t e r i a l c o l o n i e s / c e l l from f r a c t i o n 6 and



8

CELL

SEPARATION

SCIENCE AND

TECHNOLOGY

1.2 b a c t e r i a l c o l o n i e s / c e l l from f r a c t i o n 12. I f no attempt were made t o a s s e s s each c e l l i n d i v i d u a l l y , e i t h e r m o r p h o l o g i c a l l y or e l e c t r o n i c a l l y w i t h the a p p r o p r i a t e l a b e l s , one might e r r o n e o u s l y c o n c l u d e t h a t macrophages w i t h the c a p a c i t y t o i n g e s t b a c t e r i a were most h i g h l y p u r i f i e d i n f r a c t i o n 6. I f the i n v e s t i g a t o r were n o t t o examine the p u r i f i e d c e l l s i n t h i s model, i t i s a l s o conceivable that the described model would show p u r i f i c a t i o n of presumed macrophages t h a t c o n t a i n b a c t e r i a i n f r a c t i o n s t h a t c o n t a i n o n l y masses o f b a c t e r i a a g g r e g a t e d w i t h l a r g e fragments o f a c e l l u l a r debris. I t i s d i f f i c u l t t o o v e r s t a t e the v a l u e o f p h o t o m i c r o g r a p h s o f purified cells. Photomicrographs are v a l u a b l e data i n t h a t they t e l l the r e a d e r (a) what k i n d o f p u r i f i c a t i o n was a c c o m p l i s h e d and (b) how satisfactory the criteria were for the evaluation of the purification. The l a t t e r p o i n t may seem t r i v i a l ; however, on occasion, unknown to the i n v e s t i g a t o r , the quality of the p r e p a r a t i o n s were q u i t e i n a d e q u a t e to j u s t i f y the c o n c l u s i o n s t h a t were drawn. Most d e s c r i p t i o n s o f p r e v i o u s l y u n r e p o r t e d methods f o r c e l l s e p a r a t i o n s s h o u l d i n c l u d e p h o t o m i c r o g r a p h s (22 ,.23). These p h o t o m i c r o g r a p h s s h o u l d be t a k e n a t a m a g n i f i c a t i o n t h a t p e r m i t s the i n c l u s i o n o f enough c e l l s t o c o n v i n c e the r e a d e r t h a t a homogeneous f r a c t i o n was o b t a i n e d . A p h o t o m i c r o g r a p h o f one c e l l w i l l always show 100% p u r i t y . I t i s n o t d i f f i c u l t t o f i n d f i e l d s w i t h a few c e l l s o f the same type i n most s u s p e n s i o n s o f c e l l s p r i o r t o the separation of c e l l s . Bands t h a t can be v i s u a l i z e d o r p h o t o g r a p h e d i n d e n s i t y g r a d i e n t s t e l l s one n o t h i n g about p u r i f i c a t i o n and little about relative concentration. The a b i l i t y o f p a r t i c l e s t o s c a t t e r l i g h t and the r e s u l t a n t t u r b i d i t y c a u s e d by a p a r t i c l e i s a f u n c t i o n o f s i z e . The p r e s e n c e o f v i s i b l e bands can o f t e n be t a k e n as e v i d e n c e f o r the aggregation of p a r t i c l e s (organelles) or c e l l s . V i s i b l e , bands may o c c u r a t the l o c a t i o n i n a g r a d i e n t where c e l l s a r e p r e s e n t a t the h i g h e s t c o n c e n t r a t i o n s , s i n c e c e l l s t e n d t o a g g r e g a t e more a t h i g h e r c o n c e n t r a t i o n s ; however, c o n c e n t r a t i o n i s n o t the o n l y d e t e r m i n a n t o f the p r o p e n s i t y o f c e l l s t o a g g r e g a t e , and the h i g h e s t c o n c e n t r a t i o n o f c e l l s and/or the modal l o c a t i o n o f a p a r t i c u l a r p o p u l a t i o n o f c e l l s i n a g r a d i e n t may be p h y s i c a l l y remote from the g r o s s l y v i s i b l e bands c a u s e d by the a g g r e g a t i o n o f c e l l s t h a t have a g g r e g a t e d . If h e t e r o g e n e o u s groups o f c e l l s were a g g r e g a t e d b e f o r e t h e y were l a y e r e d o v e r 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 , (a) one w i l l r e l i a b l y g e t g r o s s l y v i s i b l e "bands" i n the g r a d i e n t and (b) the heterogeneous nature of the aggregates precludes significant p u r i f i c a t i o n o f homogeneous s u b p o p u l a t i o n s from c e l l s that are aggregated. I n the p u r i f i c a t i o n o f m a t e r i a l s o t h e r t h a n c e l l s i t has been c o n v e n t i o n a l f o r y e a r s f o r i n v e s t i g a t o r s to keep a b a l a n c e s h e e t t h a t shows where a l l s e p a r a t e d m a t e r i a l s went. I t i s i m p o r t a n t to p r e s e n t some form o f q u a n t i f i c a t i o n o f r e c o v e r y and a graph t h a t shows the l o c a t i o n s o f a l l c e l l s a t the end o f the s e p a r a t i o n . It is i m p o s s i b l e to a s s e s s c r i t i c a l l y the v a l u e o f a c e l l s e p a r a t i o n i n the absence o f such d a t a .



1.

TODD & PRETLOW

9


Viability. I t i s usually possible to contrive separation conditions t h a t do n o t k i l l l i v i n g c e l l s . I n some c a s e s , i n g e n u i t y i s r e q u i r e d to minimize shear forces, eliminate t o x i c chemicals (including c e r t a i n a f f i n i t y ligands), incorporate p h y s i o l o g i c a l l y acceptable b u f f e r i o n s , m a i n t a i n o s m o l a r i t y , c o n t r o l the temperature, e t c . N e a r l y e v e r y c e l l s e p a r a t i o n p r o c e s s i s i n danger o f i n t r o d u c i n g c o n d i t i o n s t h a t k i l l c e l l s , and a d d i t i v e s such as g l y c i n e , a l b u m i n , serum, and n e u t r a l polymers a r e f r e q u e n t l y u s e d i n c e l l s e p a r a t i o n systems. The measurement and d e f i n i t i o n o f " v i a b i l i t y " may be complex, d i f f i c u l t , and c o n t r o v e r s i a l . The f a c t t h a t c e l l s a r e j u d g e d t o be v i a b l e by one o r more c r i t e r i a does n o t guarantee t h a t t h e y (a) were n o t i n j u r e d by t h e p r o c e d u r e f o r c e l l s e p a r a t i o n o r (b) c a n p e r f o r m a l l o f the f u n c t i o n s t h a t are g e n e r a l l y a s s o c i a t e d w i t h v i a b i l i t y . F o r example, some forms o f z o n a l r o t o r s a r e e q u i p p e d w i t h r o t a t i n g seals. I n t h e case o f one type o f t h e s e r o t o r s , t h e s h e a r f o r c e s e x p e r i e n c e d by even v e r y "tough" c e l l s i n p a s s i n g t h r o u g h a r o t a t i n g s e a l were l e t h a l . A f t e r p a s s i n g t h r o u g h one o f t h e r o t a t i n g s e a l s t h a t i s s t a n d a r d equipment w i t h a p a r t i c u l a r z o n a l r o t o r , E h r l i c h a s c i t e s tumor c e l l s appeared m o r p h o l o g i c a l l y i n t a c t , e x c l u d e d t r y p a n b l u e , and e x h i b i t e d t i m e - l a p s e m o t i l i t y ; however, t h e y ware n o t t u m o r i g e n i c i n s y n g e n e i c mice even when t e s t e d w i t h a t h o u s a n d - f o l d m u l t i p l e o f t h e u s u a l t u m o r i g e n i c dose ( P r e t l o w e t a l . , u n p u b l i s h e d ) . Capacity. The c a p a c i t y o f a c e l l s e p a r a t i o n method c a n be measured i n number o f c e l l s p r o c e s s e d p e r hour. S i g n i f i c a n t s c a l e - u p above 1 0 c e l l s / h o u r i s seldom p r a c t i c e d , p a r t l y owing t o t h e a v a i l a b i l i t y o f c e l l s and p a r t l y owing t o t h e c o s t o f p r o d u c i n g l a r g e r numbers o f c e r t a i n c e l l t y p e s t h a t need t o be s e p a r a t e d . C e l l s with i n f i n i t e r e p r o d u c t i v e c a p a c i t y , such as y e a s t and b a c t e r i a , c a n u s u a l l y be maintained and c u l t i v a t e d as pure p o p u l a t i o n s , w i t h t h e major e x c e p t i o n b e i n g c u l t u r e s w i t h a h i g h r a t e o f m u t a g e n e s i s c a u s e d by the l o s s o f recombinant p l a s m i d ( 2 4 ) . 7

Relationship to B i o l o g i c a l Function. Only i n t h e c a s e s o f a f f i n i t y methods and f l o w s o r t i n g a r e c e l l s e p a r a t i o n p r o c e d u r e s b a s e d on t h e b i o l o g i c a l f u n c t i o n f o r which t h e c e l l s a r e b e i n g s e p a r a t e d . I n the cases o f sedimentation, field-flow and e l e c t r o k i n e t i c methods, physical properties must be fortuitously linked to physiological/biological function. Convenience. I n the l a b o r - i n t e n s i v e arena o f b i o m e d i c a l r e s e a r c h c o n v e n i e n c e may come i n one o f two forms: a s i m p l e p r o c e s s t h a t requires very l i t t l e engineering s k i l l or physical manipulation or a complex p r o c e s s t h a t has been h i g h l y automated by an e x p e n s i v e machine. These two extremes a r e perhaps r e p r e s e n t e d by 1-g s e d i m e n t a t i o n on t h e one hand and by h i g h - s p e e d f l o w s o r t i n g on t h e o t h e r . G e n e r a l l y , b i o m e d i c a l r e s e a r c h e r s who do n o t w i s h t o d e d i c a t e e x c e s s i v e amounts o f manpower t o s e p a r a t i o n p r o c e s s development seek convenience i n a s e p a r a t i o n process, p o s s i b l y t o the e x c l u s i o n o f other a t t r i b u t e s .


10

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SEPARATION

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TECHNOLOGY

Cost. The c o s t o f a s e p a r a t i o n p r o c e s s i n c l u d e s c a p i t a l equipment (apparatus), r e a g e n t s and l a b o r . The economics o f i n v e s t i n g i n s e p a r a t i o n t e c h n o l o g y a r e h i g h l y dependent upon g o a l s . A frequently r e p e a t e d p r o c e s s , f o r example, i s b e t t e r done by a n automated system and i s capital-intensive. A r a r e l y performed, intellectually demanding p r o c e d u r e would be l a b o r - i n t e n s i v e . And a p r o c e s s t h a t r e q u i r e s l a r g e q u a n t i t i e s o f e x p e n s i v e a f f i n i t y r e a g e n t s ( s u c h as antibodies) i s expendables-intensive and would n o t be u s e d t o separate large quantities o f separand without provisions f o r r e c y c l i n g ligands or reducing t h e i r cost.


Methods o f c e l l

separation

O p t i c a l and e l e c t r o n i c s o r t i n g General characteristics. Flow cytometry (FCM) i s t h e p e r f o r m a n c e o f measurements on c e l l s as t h e y f l o w p a s t a s e n s o r , t h e s i g n a l o f w h i c h c a n be t r a n s l a t e d i n t o a n e l e c t r i c a l impulse upon w h i c h q u a n t i t a t i v e measurements ( h e i g h t , a r e a , w i d t h , r i s e time, etc.) c a n be made and t a b u l a t e d . Flow s o r t i n g c o n s i s t s o f t r a n s p o r t i n g c e l l s , s i n g l e f i l e , through a n o z z l e i n t o a stream o f s o l v e n t ( u s u a l l y p h y s i o l o g i c a l s a l i n e ) so t h e c e l l s c a n be m o n i t o r e d e l e c t r o n i c a l l y ( " C o u l t e r volume") o r o p t i c a l l y one a t a t i m e . The r e s u l t i n g e l e c t r o n i c s i g n a l i s used t o charge o r n o t charge a d r o p l e t containing a specific c e l l . T h i s drop, i f c h a r g e d , i s d e f l e c t e d b y a DC e l e c t r i c f i e l d i n t o a c o l l e c t i o n v e s s e l . See F i g u r e 1 i n c h a p t e r 2. I t i s p o s s i b l e t o c o l l e c t up t o 5 s u b p o p u l a t i o n s t h i s way. I n a d d i t i o n t o d e f l e c t i n g charged drops c o n t a i n i n g c e l l s i t has r e c e n t l y become p o s s i b l e t o m a n i p u l a t e s i n g l e c e l l s i n s u s p e n s i o n b y o p t i c a l pressure or o p t i c a l trapping. T h i s e x c i t i n g new development i s d e s c r i b e d i n t h e c h a p t e r b y T. B u i c a n ( 2 5 ) . Flow s o r t i n g s e p a r a t e s c e l l s one a t a t i m e . Thus r e s o l u t i o n depends on t h e i n t r i n s i c o p t i c a l p r o p e r t y o f the c e l l that i s measured as selection criterion. This property i s always d i s t r i b u t e d , and CV - 0.20 c a n o c c u r f r e q u e n t l y . I n the case o f n a r r o w l y d i s t r i b u t e d p r o p e r t i e s , such as DNA c o n t e n t , CV as low as 0.01 h a s been a c h i e v e d . Thus t h e r e s o l u t i o n o f t h e s o r t i n g p r o c e s s depends on t h e CV o f a c e l l p r o p e r t y i n t h e s t a r t i n g p o p u l a t i o n . Applications. Flow c y t o m e t r y (FCM) h a s f o u n d e x t e n s i v e u s e i n c l i n i c a l immunology, and i t s m a t u r i t y as a r o u t i n e t e c h n o l o g y ( v s . a b a s i c r e s e a r c h t o o l ) has been p r o v e n i n t h i s f i e l d . A l i m i t e d number o f a p p l i c a t i o n s i n tumor p a t h o l o g y and c l i n i c a l hematology have a l s o become r o u t i n e and a c c e p t e d i n t h e r e s p e c t i v e p r o f e s s i o n s . The f l o w c y t o m e t e r has become a b a s i c r e s e a r c h t o o l i n s e v e r a l a r e a s o f c e l l b i o l o g y , p a t h o b i o l o g y , and t o x i c o l o g y , t o name a few examples, and a few t h o u s a n d i n s t r u m e n t s a r e now i n o p e r a t i o n a r o u n d t h e w o r l d . M e t a b o l i c c h a r a c t e r i z a t i o n o f c e l l s suspended from s o l i d t i s s u e s can be p e r f o r m e d u s i n g b l u e autofluorescence (an i n d i c a t o r o f NADH/NADPH l e v e l s ) , rhodamine 123 s t a i n i n g ( m i t o c h o n d r i a l a c t i v i t y ) and p y r o n i n Y s t a i n i n g (RNA c o n t e n t ) . These markers have been e x p l o i t e d (26) i n t h e c h a r a c t e r i z a t i o n o f a i r w a y e p i t h e l i a l c e l l s


1.

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i s o l a t e d by c e n t r i f u g a t i o n and n o n - i n v a s i v e flow s o r t i n g . The combination o f these techniques made i t possible to follow d i f f e r e n t i a t i o n pathways i n h e t e r o t o p i c t r a c h e a l g r a f t s o f p u r e p o p u l a t i o n s o f b a s a l and s e c r e t o r y c e l l s . The c h a r a c t e r i z a t i o n o f messenger RNA b e i n g made b y c e l l s i n v i v o i s p o s s i b l e through i n s i t u h y b r i d i z a t i o n u s i n g c l o n e d n u c l e i c a c i d p r o b e s complementary t o s p e c i f i c mRNA. H i g h l y r a d i o a c t i v e probes, either synthesized or n i c k - t r a n s l a t e d using P - l a b e l e d nucleotides, can be u s e d i n " N o r t h e r n " b l o t s and c e l l r a d i o a u t o g r a p h y . Nucleic a c i d p r o b e s c a n a l s o be tagged w i t h f l u o r e s c e n t o r immunoenzymatic l a b e l s o r w i t h b i o t i n , and methods have been d e v e l o p e d f o r a p p l y i n g such l a b e l e d p r o b e s t o whole c e l l s i n s u s p e n s i o n . These c e l l s c a n t h e n be e v a l u a t e d b y f l o w o r image c y t o m e t r y . Non-cellular a p p l i c a t i o n s a r e a l s o p o s s i b l e ; beaded media, d r o p l e t s u n d e r g o i n g phase s e p a r a t i o n , p r e c i p i t a t e s , s t a n d a r d t e s t p a r t i c l e s , and o t h e r n o n - l i v i n g components o f b i o p r o c e s s t e c h n o l o g y can be a s s e s s e d by FCM.


3 2

Flow S o r t i n g o f C e l l s and Chromosomes. Most e a r l y flow c y t o m e t r y e x p e r i m e n t s were c o n f i n e d t o t h e s t u d y o f c e l l s grown i n vitro, immunological and h e m a t o l o g i c a l c e l l s , and c e r t a i n tumor cells. Improved cell dispersal methods have broadened the a p p l i c a b i l i t y o f flow cytometry, even t o i n c l u d e r e t r o s p e c t i v e a n a l y s i s o f DNA d i s t r i b u t i o n s i n n u c l e i p r e p a r e d from t i s s u e c e l l s embedded i n p a r a f f i n ( 2 7 ) . Flow k a r y o t y p i n g , t h e s t u d y o f i s o l a t e d metaphase chromosomes i n s u s p e n s i o n by f l o w c y t o m e t r y , h a s advanced t o a h i g h l e v e l o f s o p h i s t i c a t i o n , and t h e e f f e c t s o f g e n o t o x i c a g e n t s a r e d e t e c t e d i n t h e form o f m o d i f i e d f l u o r e s c e n c e i n t e n s i t y d i s t r i b u t i o n s . I f t h e r e have been t r a n s l o c a t i o n s t h a t have been r e p l i c a t e d , f o r example, t h e s e w i l l appear as new peaks i n t h e f l o w karyogram. I f t h e r e has been e x t e n s i v e f r a g m e n t a t i o n , t h e " b a s e l i n e " between chromosome peaks w i l l r i s e . Mutant gene p r o d u c t s c a n a l s o be sought b y f l o w c y t o m e t r y . The p r e p a r a t i o n o f chromosomes by combined s e d i m e n t a t i o n and f l o w s o r t i n g methods t o produce enough m a t e r i a l f o r g e n e t i c a n a l y s i s i s d e t a i l e d by A l b r i g h t and M a r t i n i n t h i s volume (28). By i n s t r u m e n t i n g t h e f l o w c y t o m e t e r t o d e t e c t " r a r e e v e n t s " and by u s i n g a h i g h l y s p e c i f i c f l u o r e s c e n t a n t i b o d y s t a i n i t i s p o s s i b l e t o d e t e c t 1 c e l l w i t h a mutated s u r f a c e p r o t e i n p e r few m i l l i o n c i r c u l a t i n g erythrocytes. T h i s a p p r o a c h was a p p l i e d b y B i g b e e , Langlois and J e n s e n (29) t o demonstrate that v a r i a n t human e r y t h r o c y t e s e x p r e s s i n g n e i t h e r t h e M n o r Ν g l y c o p h o r i n A gene o c c u r more f r e q u e n t l y i n i n d i v i d u a l s b e a r i n g d e f e c t i v e r e p a i r genes o r exposed t o genotoxic agents. The r o u t i n e d e t e c t i o n o f 1 c e l l i n 10,000 i s p r e s e n t e d by L e a r y e t a l . i n t h i s volume ( 3 0 ) . Bioprocessing applications. To d a t e , t h i s method has f o u n d i m p o r t a n t b u t l i m i t e d a p p l i c a t i o n t o b i o p r o c e s s e n g i n e e r i n g and b i o p r o c e s s i n g r e s e a r c h . Many b i o p r o c e s s i n g r e s e a r c h p r o j e c t s e n t a i l t h e s t u d y o f l i v i n g c e l l s : b a c t e r i a , y e a s t , o t h e r f u n g i , p l a n t c e l l s , and animal c e l l s . The s t u d y o f each from t h e b i o p r o c e s s i n g p e r s p e c t i v e has i t s own problems r e l a t i v e t o s e n s i t i v i t y , w a v e l e n g t h , p u l s e



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a n a l y s i s , and d a t a a n a l y s i s . The f u l l range o f c e l l t y p e s t o w h i c h FCM c a n be a p p l i e d i n b i o p r o c e s s i n g has n o t been e x p l o r e d . For example, the m o n i t o r i n g o f v i a b i l i t y , k i n e t i c s , m e t a b o l i s m , and production by single cells i n a suspended-cell r e a c t e r can be e f f e c t i v e l y p e r f o r m e d by FCM. I t has become a c h o s e n m o n i t o r o f gene expression i n e u k a r y o t e s i n m o l e c u l a r b i o l o g y and biotechnology. B a i l e y , O l l i s , and o t h e r s have u s e d FCM i n the v e r i f i c a t i o n o f biomass models i n c e l l b i o r e a c t o r s ( 3 1 ) . However, the number o f b i o p r o c e s s r e s e a r c h e r s u s i n g FCM t o d a y i s s t i l l m i n u s c u l e . This i s s u r p r i s i n g i n v i e w o f the a b i l i t y o f the f l o w c y t o m e t e r t o measure n e a r l y a l l o f the s i n g l e - c e l l p r o p e r t i e s t h a t a r e c r i t i c a l t o c e l l bioreactor function. C e l l growth can be measured by the d i r e c t e n u m e r a t i o n o f c e l l s by e l e c t r o n i c o r o p t i c a l c o u n t i n g o f c e l l s i n a s p e c i f i e d volume o f medium. C e l l v i a b i l i t y can be e v a l u a t e d on the b a s i s o f f l u o r e s c e i n diacetate staining; fluorescein diacetate enters cells and is d e - a c y l a t e d t o become f l u o r e s c e n t . Dead c e l l s do n o t r e t a i n the dye w h i l e v i a b l e ones do ( 3 2 ) . S i m i l a r l y , dead c e l l s admit p r o p i d i u m i o d i d e t h r o u g h d e f e c t i v e membranes, and t h i s s t a i n s a l l n u c l e i c a c i d s i n the c e l l ( 3 3 ) . Thus two o r more methods a r e a v a i l a b l e t o d e t e r m i n e v i a b l e c e l l count, and e i t h e r l i v e o r dead c e l l s c a n be s t a i n e d . F l u o r e s c e n t a n t i b o d i e s r e a c t i n g w i t h mouse I g a r e c o m m e r c i a l l y a v a i l a b l e , and t h e s e can be u s e d t o measure s u r f a c e o r i n t e r n a l I g , d e p e n d i n g on the method o f c e l l p r e p a r a t i o n . Brief s t a i n i n g of l i v e c e l l s with fluorescent a n t i - I g stains only surface Ig, while i n t e r n a l Ig i s a l s o s t a i n e d i n a l c o h o l - f o r m a l i n - t r e a t e d cells. C e l l s i z e can be measured on the b a s i s o f the r e s i s t i v e i m p u l s e volume ( " e l e c t r o n i c " , o r " C o u l t e r " v o l u m e ) , f o r w a r d a n g l e l i g h t s c a t t e r (0-2 d e g r e e s ) , and p u l s e - h e i g h t independent o p t i c a l ( s c a t t e r o r f l u o r e s c e n c e ) p u l s e w i d t h measurement ( 3 4 ) . Position in the c e l l c y c l e can be d e t e r m i n e d on the b a s i s o f DNA c o n t e n t , w h i c h is measured using fluorescent s t a i n i n g with propidium iodide ( f o l l o w i n g a l c o h o l f i x a t i o n and RNAase t r e a t m e n t ) o r w i t h the DNA-specific dye H o e c h s t 33258 (UV i l l u m i n a t i o n ) . The l e v e l of " r e d u c e d n u c l e o t i d e " (NADH + NADPH) can be measured i n each c e l l on the b a s i s o f " a u t o f l u o r e s c e n c e " (35) s t i m u l a t e d a t 365 nm. This i s a v e r y i m p o r t a n t i n d e x o f the m e t a b o l i c ( r e d o x ) s t a t e o f the c e l l and c a n be c o r r e l a t e d w i t h c e l l k i n e t i c s , p r o d u c t r e l e a s e , and redox e l e c t r o d e measurements i n the b r o t h . The a b i l i t y o f f l o w c y t o m e t e r s to make measurements on i n d i v i d u a l b a c t e r i a has improved o v e r the l a s t s e v e r a l y e a r s ( 3 6 ) . With a modest mercury a r c lamp, adequate n u m e r i c a l a p e r t u r e , and a p p r o p r i a t e staining (mithramycin p l u s ethidium bromide) i t i s p o s s i b l e to measure DNA in bacterial cells, t h e r e b y a s s e s s i n g the s t a t e o f bioreactor cultures. B a c t e r i a l c e l l s , being small, s c a t t e r large amounts o f l i g h t a t 90 d e g r e e s . Adequate s e n s i t i v i t y u s i n g a n t i b o d y s t a i n i n g r e q u i r e s the use o f s p e c i a l a m p l i f i c a t i o n t e c h n i q u e s , such as enzyme-coupled avidin and biotin derivatives of primary a n t i b o d i e s . Most dye l i g a n d s u s e d i n f l u o r e s c e n c e f l o w c y t o m e t r y a r e compounds of traditional aromatic dyes, such as fluorescein, rhodamine, H o e c h s t compounds o r carbocyanines, but e f f o r t s at a c h i e v i n g i n c r e a s e d s e n s i t i v i t y and r e d u c e d s p e c t r a l o v e r l a p by the


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use o f r a r e - e a r t h t h i s volume (37.) .

13

Separation of Living Celk compounds a r e

d e t a i l e d by

V a l l a r i n o and

Leif

in


Sedimentation. L i k e most p h y s i c a l methods, s e d i m e n t a t i o n separates c e l l s on the b a s i s o f p h y s i c a l p r o p e r t i e s i r r e s p e c t i v e o f t h e i r b i o l o g i c a l function. However, the d e n s i t y and r a d i u s o f numerous e u k a r y o t i c c e l l t y p e s a r e consequences o f t h e i r f u n c t i o n . For example, h e a v i l y g r a n u l a t e d c e l l s , such as g r a n u l o c y t e s and hormones e c r e t i n g c e l l s o f the a n t e r i o r p i t u i t a r y a r e l a r g e and dense, owing to t h e i r cytoplasms being f i l l e d with h i g h - d e n s i t y granules that s h a r e many p r o p e r t i e s w i t h p r o t e i n c r y s t a l s . The g e n e r a l e q u a t i o n o f m o t i o n t h a t i s e x p l o i t e d i n s e d i m e n t a t i o n is ν - dx/dt - 2(p

-

2

)α α/9η

(6)

Ρο

where ν i s the t e r m i n a l v e l o c i t y a c h i e v e d when the S t o k e s d r a g f o r c e on a s p h e r e , 6ttrçav, e x a c t l y b a l a n c e s the a c c e l e r a t i o n , a, and buoyancy, (p - p ), forces. The p a r t i c l e ( c e l l ) r a d i u s i s a, i t s d e n s i t y i s p, and η and p a r e v i s c o s i t y and d e n s i t y o f the medium, respectively. I n 1-g s e d i m e n t a t i o n the a c c e l e r a t i o n i s g = 9.8 m/s , and t h i s has been u s e d i n bone marrow and p e r i p h e r a l b l o o d c e l l s e p a r a t i o n s (38)· I n t y p i c a l 1-g a p p l i c a t i o n s , a d e n s i t y g r a d i e n t p ( x ) i s used, so d x / d t i s a f u n c t i o n o f χ and t h e r e f o r e o f t i m e . In simple c e n t r i f u g a t i o n the a c c e l e r a t i o n v e c t o r i s ω χ , where χ - d i s t a n c e o f the s e p a r a n d p a r t i c l e from the c e n t e r o f r o t a t i o n . C l e a r l y d x / d t i s a f u n c t i o n of χ i n c e n t r i f u g a t i o n with or without a d e n s i t y gradient. C e n t r i f u g a t i o n i n the absence o f a d e n s i t y g r a d i e n t d i s t r i b u t e s c e l l s r a d i a l l y a c c o r d i n g t o t h e i r r a d i u s and d i f f e r e n t i a l d e n s i t y . This p r o c e s s has been d e f i n e d as " d i f f e r e n t i a l c e n t r i f u g a t i o n " , meaning "the s e p a r a t i o n o f homogeneous m i x t u r e s o f h e t e r o g e n e o u s p a r t i c l e s by c e n t r i f u g a t i o n i n the absence o f a d e n s i t y g r a d i e n t w i t h just s u f f i c i e n t f o r c e t o p e r m i t a c r u d e p u r i f i c a t i o n o f the most r a p i d l y s e d i m e n t i n g p a r t i c l e s on the bottom o f the c e n t r i f u g e tube and o f the most s l o w l y s e d i m e n t i n g p a r t i c l e s t h a t r e m a i n i n s u s p e n s i o n (.39) . Q

Q

2

Q

2

T h r e e v a r i a n t s can be u s e d t o e x t e n d the s e p a r a t i o n p r o c e s s : one i s t o c r e a t e a d e n s i t y g r a d i e n t t h a t r e s u l t s i n (ρ - ρ )χ/η - const. , which gives ν - constant. Such a d e n s i t y g r a d i e n t i s an " i s o k i n e t i c g r a d i e n t " , as d i s c u s s e d i n the c h a p t e r by P r e t l o w and P r e t l o w ( 4 0 ) . A l s o , f l u i d can be pumped inward a t v e l o c i t y d x / d t f o r a p a r t i c u l a r s e p a r a n d , so t h a t s l o w l y s e p a r a t i n g separands a r e pumped b a c k out the t o p , and r a p i d l y s e d i m e n t i n g components c o n t i n u e out the "bottom" ( o u t e r r a d i u s ) o f the c e n t r i f u g e t h i s i s e l u t r i a t i o n and i s d e t a i l e d i n the c h a p t e r by Keng ( 4 1 ) . I s o p y c n i c s e d i m e n t a t i o n i s an equilibrium process. P a r t i c l e s sediment, e i t h e r a t u n i t g r a v i t y o r i n a c e n t r i f u g a l f i e l d , i n a density gradient u n t i l ρ - p , a t which time s e d i m e n t a t i o n (buoyancy) s t o p s . F r a c t i o n s a r e t h e n c o l l e c t e d on the b a s i s o f p a r t i c l e d e n s i t y . 0

Q



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Affinity Adsorption. From the standpoint of separation thermodynamics, a f f i n i t y a d s o r p t i o n i s a n e q u i l i b r i u m p r o c e s s . It has t r a d i t i o n a l l y been p r a c t i c e d i n t h r e e ways: s i n g l e - s t a g e b a t c h d e s o r p t i o n , m u l t i - s t a g e a d s o r p t i o n and d e s o r p t i o n , and c o n t i n u o u s d e s o r p t i o n , as i n chromatography. Most a p p l i c a t i o n s t o c e l l s a r e single-stage batch separations. The s e l e c t i o n and e v a l u a t i o n o f a d s o r p t i o n media f o r c e l l s i s d i s c u s s e d i n t h e c h a p t e r b y K a t a o k a (42). Few p r o c e s s e s s e p a r a t e c e l l s on t h e b a s i s o f t h e i r i n t r i n s i c magnetic p r o p e r t i e s . However, h i g h l y s e l e c t i v e , high-capacity hydrodynamic c a p t u r e methods have been d e v e l o p e d u s i n g magnetic f i e l d s , such as magnetic f i l t r a t i o n , h i g h - g r a d i e n t f i e l d s e p a r a t i o n , and m a g n e t i c f l o t a t i o n ( 4 3 ) . A f f i n i t y a d s o r p t i o n o f magnetic p a r t i c l e s by c e l l s i s t h e s o u r c e o f s p e c i f i c i t y i n m a g n e t i c c e l l separations. M a g n e t i c a l l y enhanced a f f i n i t y methods a r e becoming p o p u l a r i n c a s e s i n which t h e attachment o f c e l l s t o a m a c r o s c o p i c s u r f a c e may be u n d e s i r a b l e due t o i n t e r f e r e n c e b y n o n - s p e c i f i c adhesion or untimely a c t i v a t i o n o f c e l l u l a r processes s u c h as b l a s t o g e n e s i s o r c y t o k i n e p r o d u c t i o n . An example o f p r o g r e s s i n c e l l i s o l a t i o n by m a g n e t i c a l l y enhanced a f f i n i t y methods i s p r e s e n t e d i n the chapter by Powers and Heath ( 4 4 ) , and i t s p o t e n t i a l c o m m e r c i a l i z a t i o n i s d i s c u s s e d by L i b e r t i ( 4 5 ) . One o f t h e major problems i n magnetic c a p t u r e i s t h e o c c u r r e n c e o f u n d e s i r e d a d v e n t i t i o u s i n t e r a c t i o n s o f m a g n e t i c p a r t i c l e s owing t o an a t t r a c t i v e - o n l y f o r c e t h a t cannot be s w i t c h e d o f f . The r e s u l t i s c e l l a g g r e g a t i o n and t h e a g g r e g a t i o n o f m i c r o b e a d s i n t h e absence o f c e l l s o r a magnetic f i e l d . The g e n e r a l a p p r o a c h t o s o l v i n g t h i s p r o b l e m h a s been t h e development o f p a r a m a g n e t i c o r s u p e r p a r a m a g n e t i c microspheres with a f f i n i t y l i g a n d s . Superparamagnetic p a r t i c l e s possess no m a g n e t i c moment u n l e s s t h e y a r e i n a n inhomogeneous magnetic f i e l d o f h i g h g r a d i e n t . Such p a r t i c l e s have been made o f f e r r i t i n and d e x t r a n o r o t h e r beaded media (46, 47) . Magnetic capture and a f f i n i t y b i n d i n g a r e b o t h e q u i l i b r i u m processes. Equations o f motion t h e r e f o r e apply only t o the r a t e a t which e q u i l i b r i u m i s achieved. Free energies o f these e q u i l i b r i a depend on t h e m a g n e t i c d i p o l e moment o f each p a r t i c l e , t h e number o f p a r t i c l e s adsorbed p e r c e l l , s t r e n g t h o f the magnetic f i e l d g r a d i e n t , and t h e l i g a n d a f f i n i t y c o n s t a n t .

B i p h a s i c E x t r a c t i o n . B i p h a s i c e x t r a c t i o n i s one o f t h e most p o p u l a r p u r i f i c a t i o n methods u s e d i n t h e c h e m i c a l i n d u s t r y today. I t has f o u n d l i m i t e d p o p u l a r i t y i n b i o p r o c e s s i n g owing t o t h e damaging e f f e c t s o f o r g a n i c s o l v e n t s on b i o m o l e c u l e s and c e l l s , whereas aqueous two-phase systems, due t o t h e i r h i g h w a t e r c o n t e n t , a r e b i o c o m p a t i b l e (48,49). Moreover, these systems a r e r e p o r t e d t o have p r o v i d e d s t a b i l i t y t o b i o l o g i c a l l y a c t i v e s u b s t a n c e s , s u c h as enzymes (50). Due t o t h e i r s i m i l a r p h y s i c a l p r o p e r t i e s , i m m i s c i b l e aqueous phases do n o t s e p a r a t e r a p i d l y i n l a r g e volumes, as i n p r o d u c t i o n scale purifications. D e s p i t e some 800 p a p e r s on t h i s s u b j e c t ( 4 ) , l a r g e - s c a l e commercial a p p l i c a t i o n s a r e n o t w i d e s p r e a d . The h i g h c o s t o f lower-phase polymers i s a n o t h e r d e t e r r e n t t o i t s w i d e s p r e a d


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u s e . C o s t c o n t a i n m e n t h a s been r e c e n t l y a f f e c t e d b y t h e i n t r o d u c t i o n o f l o w - c o s t polymer aqueous phase systems ( 5 1 ) . In p r a c t i c e , multistage e x t r a c t i o n s a r e performed t o achieve h i g h - r e s o l u t i o n s e p a r a t i o n s ( 5 2 ) . The p h y s i c a l problems a s s o c i a t e d w i t h b i p h a s i c e x t r a c t i o n r e s e a r c h c a n be d i v i d e d i n t o two major c a t e g o r i e s , a l t h o u g h t h e y b e a r c e r t a i n thermodynamic s i m i l a r i t i e s : phase s e p a r a t i o n - - t h e f o r m a t i o n o f two phases from a d i s p e r s i o n , and p a r t i t i o n i n g -- t h e p r e f e r e n t i a l t r a n s f e r o f a s e p a r a n d i n t o one phase. Phase s e p a r a t i o n . When two polymers A and Β a r e d i s s o l v e d i n aqueous s o l u t i o n a t c o n c e n t r a t i o n s t h a t cause phase s e p a r a t i o n a n u p p e r phase forms t h a t i s r i c h i n A and p o o r i n B, and a l o w e r phase forms t h a t i s r i c h i n Β and p o o r i n A. T y p i c a l l y A i s p o l y e t h y l e n e g l y c o l (PEG), c o n s i d e r e d a r e l a t i v e l y h y d r o p h o b i c s o l u t e , a n d Β i s dextran or a s i m i l a r polysaccharide. Β c a n a l s o be a s a l t a t h i g h concentration. The phase s e p a r a t i o n p r o c e s s i s d e s c r i b e d by? a twod i m e n s i o n a l phase diagram, such as i n F i g u r e 2, i n w h i c h h i g h c o n c e n t r a t i o n s o f A and Β cause t h e f o r m a t i o n o f top-phase s o l u t i o n s w i t h c o m p o s i t i o n s g i v e n b y p o i n t s i n t h e upper l e f t and bottom-phase s o l u t i o n s w i t h compositions g i v e n by p o i n t s i n the lower r i g h t . Each c o m b i n a t i o n o f A and Β f a l l s on a " t i e l i n e " c o n n e c t i n g t h e r e s u l t i n g top a n d bottom phase compositions at equilibrium. Higher c o n c e n t r a t i o n s o f polymers r e s u l t i n l o n g e r t i e l i n e s on t h e phase diagram. Any c o m p o s i t i o n t h a t l i e s on a t i e l i n e w i l l r e s u l t i n t h e same e q u i l i b r i u m c o m p o s i t i o n s , and t h e r a t i o o f t h e volumes o f t h e two phases i s r e l a t e d t o t h e p o s i t i o n o f t h e i n i t i a l c o m p o s i t i o n on the t i e l i n e . The c u r v e t h a t forms t h e e n v e l o p e c o n n e c t i n g t h e ends o f t h e t i e l i n e s , t h e " b i n o d i a l " , shown i n F i g u r e 2, s e p a r a t e s t h e 1phase a n d 2-phase r e g i o n s on t h e diagram. As a n example, one twophase e x t r a c t i o n system i s d e s c r i b e d b y t h e l o c a t i o n s o f the e n c i r c l e d p o i n t s on t h e phase d i a g r a m o f t h e PEG/dextran/water system a t 25°C shown i n F i g u r e 2. The t o p and bottom phase d e n s i t i e s o f t h i s system a r e 1.0164 and 1.1059 g/cm , respectively, and t h e c o r r e s p o n d i n g v i s c o s i t i e s a r e 0.0569 and 4.60 P o i s e . 3

Such phase diagrams a r e s t r i c t l y e x p e r i m e n t a l ; however, t h e y r e p r e s e n t thermodynamic e q u i l i b r i a , and t h e y s h o u l d be p r e d i c t a b l e on the b a s i s o f thermodynamic p r i n c i p l e s . Cabezas e t a l . (.53), chose t o a p p l y s t a t i s t i c a l mechanics v i a t h e s o l u t i o n t h e o r y o f T e r r e l l H i l l (54). A t e q u i l i b r i u m the chemical p o t e n t i a l o f each s u b s t i t u e n t ( t y p i c a l l y d e x t r a n , PEG and w a t e r ) i s t h e same i n t h e t o p and bottom phase, and t h e c h e m i c a l p o t e n t i a l o f e a c h c a n be d e t e r m i n e d from t h e i r f r a c t i o n a l m o l a l i t i e s mi and t h e i r o s m o t i c v i r i a l c o e f f i c i e n t s Cij b y Δμ

2

- -RT[ I n m

+ 2C m

2

+ 2C m ]

(7a)

Δμ

3

- -RT[ln m

+ 2C m

2

+ 2C m ]

(7b)

2

3

22

23

-RT[m + m 2

3

+ C m 22

23

33

2 2

3

3

+ 2C m m 23

2

2

3

+ C m ] 33

3


(7c)


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Wt % DEXTRAN 480,000 Figure 2. Phase diagram of the dextran-water-polyethylene glycol system showing tie lines and binodial curve. The vertical limb of the binodial curve gives compositions of upper phases and the horizontal limb gives compositions of lower phases at equilibrium. (Reproduced with permission from reference 61. Copyright 1990 by Marcel Dekker.)


1.


T O D D & PRETLOW

17

where s u b s c r i p t s 1, 2 and 3 c o r r e s p o n d t o water, PEG and d e x t r a n , respectively. The same r e l a t i o n s h i p s a p p l y t o any p a i r o f p o l y m e r s , but n o t to polymer-salt c o m b i n a t i o n s t h a t form two phases, as e l e c t r o s t a t i c s must be added t o a c c o u n t f o r t h e c h e m i c a l p o t e n t i a l s of s a l t s . Thermodynamic a n a l y s e s t h a t a c c o u n t f o r t h e i n t e r a c t i o n s o f i o n s have been r e c e n t l y a c h i e v e d ( 5 5 ) . The osmotic v i r i a l coefficients a r e f o r polymers and c o n s t i t u t e a d d i t i o n a l unknowns i n e q u a t i o n s ( 7 ) . These c a n be d e r i v e d from group r e n o r m a l i z a t i o n t h e o r y as a p p l i e d t o polymer s o l u t i o n s (56) b y u s i n g monomer-monomer i n t e r a c t i o n c o e f f i c i e n t s b j : A

C

3

2

- b N * [l

+ (2/9)ln(H„ /M )J

(8a)

C33 = b N " [ l

+ (2/9)^(^3/^3)]

(8b)

2 2

2 2

2


3

3 3

C

3

3

3

2 3

2

n2

2

3

3

» b {N * [l + (2/9)lnOWH2)] [N " [l + 2 3

2

(8c)

3

( 2 / 9 ) 1 ^ ^ / ^ 3 ) J} ' 1

2

where C i s b a s e d on an e m p i r i c a l a p p l i c a t i o n o f t h e g e o m e t r i c mean rule. A t e q u i l i b r i u m t h e c h e m i c a l p o t e n t i a l o f each c o n s t i t u e n t i n the t o p phase i s e q u a l t o i t s c h e m i c a l p o t e n t i a l i n t h e bottom phase. These e q u a l i t i e s r e s u l t i n a system o f e q u a t i o n s w i t h t h e same number o f e q u a t i o n s as unknowns which c a n be s o l v e d f o r m and m t o o b t a i n e q u i l i b r i u m c o n c e n t r a t i o n s i n b o t h phases. These c o n c e n t r a t i o n s have been f o u n d t o s u c c e s s f u l l y p r e d i c t phase diagrams, i n c l u d i n g t h e one shown i n F i g u r e 2 ( 5 3 ) . I n a d d i t i o n t o t h e s e e q u i l i b r i u m phenomena, r a t e s o f phase s e p a r a t i o n ("demixing") a r e o f t e c h n i c a l i n t e r e s t ( 5 7 ) . When two polymers a r e d i s s o l v e d i n aqueous s o l u t i o n a t c o n c e n t r a t i o n s that cause phase s e p a r a t i o n , c e r t a i n d i s s o l v e d i o n s s u c h as phosphate a r e u n e q u a l l y p a r t i t i o n e d between t h e phases (.58) l e a d i n g t o a Donnan p o t e n t i a l a c r o s s t h e i n t e r f a c e (.59) and an e l e c t r o k i n e t i c ( z e t a ) p o t e n t i a l a t t h e i n t e r f a c e ( 6 0 ) . As a consequence o f t h e l a t t e r , phase d e m i x i n g c a n be h a s t e n e d by t h e a p p l i c a t i o n o f an e l e c t r i c f i e l d (61). 2 3

2

3

P a r t i t i o n i n g . P a r t i t i o n i n g o f m o l e c u l e s and c e l l s between phases d u r i n g d e m i x i n g i s a l s o c o n s i d e r e d a thermodynamic p r o c e s s and n o t a rate process. T h i s means t h a t s c a l e up under r e l a t i v e l y n o n - h o s t i l e c o n d i t i o n s s h o u l d be f e a s i b l e , and t h i s i s t h e main p r o m i s e o f b i p h a s i c aqueous e x t r a c t i o n as a p u r i f i c a t i o n method. P a r t i t i o n i n g was o r i g i n a l l y m o d e l l e d by B r o n s t e d (62), who n o t e d t h a t , a t t h e v e r y l e a s t , p a r t i t i o n c o e f f i c i e n t , K, s h o u l d depend on t h e m o l e c u l a r weight o f the separand Κ - exp(ÀM/k T) B

(9)

but there a r e a t l e a s t 4 p r o p e r t i e s that determine a molecule's p a r t i t i o n c o e f f i c i e n t : molecular weight, h y d r o p h o b i c i t y , charge d e n s i t y , and b i n d i n g a f f i n i t y . Brooks and o t h e r s (49, 63, 64» 65) consider the surface area, (which does depend on m o l e c u l a r w e i g h t )


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o f a p a r t i t i o n i n g m o l e c u l e and i t s i n t e r f a c i a l f r e e e n e r g y p e r u n i t area Δ7, as the s i g n i f i c a n t measurable v a r i a b l e s i n d e t e r m i n i n g partition coefficient: Κ -

(10)

exp(A7AM/k T). B

W h i l e s u c h a r e l a t i o n s h i p seems v a l i d f o r s o l u t e m o l e c u l e s , i t does n o t s a t i s f a c t o r i l y d e s c r i b e the p a r t i t i o n i n g o f p a r t i c l e s , s u c h as cells. I n one view, e q u a t i o n (10) would o n l y be s a t i s f a c t o r y i f Τ c o u l d be s e t t o 10 - 10 °K. Such a h i g h k T i m p l i e s r a n d o m i z i n g effects due to non-thermal forces, such as gravity (63-65). Furthermore i t i s necessary to account f o r e l e c t r o k i n e t i c t r a n s p o r t as a means o f r e a c h i n g e q u i l i b r i u m when an e l e c t r i c a l p o t e n t i a l , ΔΦ, between the two phases e x i s t s ( 4 8 ) . A p a r t i c l e can i n t e r a c t w i t h b o t h phases s i m u l t a n e o u s l y , so the d i f f e r e n c e between i t s i n t e r f a c i a l f r e e e n e r g i e s w i t h r e s p e c t t o the top phase, 7 and w i t h r e s p e c t t o the b o t t o m phase, 7 , which i s Δ 7 , i s a determinant of p a r t i t i o n coefficient. The i n t e r f a c i a l f r e e e n e r g y between top and b o t t o m phase 7 p l a y s a r o l e i n e x c l u d i n g p a r t i c l e s from the i n t e r f a c e . The s u g g e s t e d o v e r a l l thermodynamic r e l a t i o n s h i p f o r p a r t i t i o n i n g out o f the i n t e r f a c e i s 4

5


B

PT

PB

TB

Κ - Bexp{-

7 t b

A[l

- (Δ7

+ α ΔΨ/ β

2

7 ι Λ

] /4k T} , B

(11)

where Β i s a c o n s t a n t o f p r o p o r t i o n a l i t y . When s a l t s , e s p e c i a l l y salts of s u l f a t e and phosphate, are dissolved in PEG-dextran s o l u t i o n s , an e l e c t r o c h e m i c a l p o t e n t i a l i s d e v e l o p e d between the phases (58,59). Arguments d e r i v e d from e q u i l i b r i u m thermodynamics s u g g e s t t h a t t h i s c o u l d be a Donnan p o t e n t i a l d e v e l o p e d by the u n e q u a l p a r t i t i o n i n g o f i o n s between the two phases ( 5 9 ) , and t h i s p o t e n t i a l can d r i v e c e l l s i n t o the upper (more p o s i t i v e ) phase on the b a s i s o f c e l l s u r f a c e charge d e n s i t y ("zeta p o t e n t i a l " ) . F i e l d Flow F r a c t i o n a t i o n . T h i s t e c h n i q u e , i n v e s t i g a t e d and d e v e l o p e d m a i n l y by J . C. G i d d i n g s and co-workers (see c h a p t e r s 9 and 10 by G i d d i n g s (66) and by B i g e l o w e t a l . (67)) and a b b r e v i a t e d FFF, i s d e f i n e d as any s e p a r a t i o n method i n w h i c h a t r a n s v e r s e field is imposed on d i s s o l v e d o r suspended separands as t h e y f l o w t h r o u g h a chamber. The t r a n s v e r s e f i e l d may be a p r e s s u r e drop imposed t h r o u g h p o r o u s chamber w a l l s ( f l o w f i e l d ) , an e l e c t r i c f i e l d , a c e n t r i f u g a l a c c e l e r a t i o n f i e l d ( s t e r i c FFF) an a d h e s i o n f o r c e a t the chamber w a l l ( 6 7 ) , o r s i m p l y the s h e a r r a t e due t o the v e l o c i t y g r a d i e n t imposed by P o i s e u i l l e l a m i n a r f l o w . T y p i c a l l y , i n a p p l i c a t i o n s t o c e l l s , a s u s p e n s i o n o f mixed c e l l s f l o w s i n t o a chamber t h a t i s 100 - 300 μια t h i c k , a few cm wide and s e v e r a l cm l o n g . The t h i c k n e s s o f the chamber e s t a b l i s h e s the s t r e n g t h o f t r a n s v e r s e f i e l d t h a t c a n be a p p l i e d ( u s u a l l y f l o w f i e l d ) , the w i d t h o f the chamber e s t a b l i s h e s i t s c a p a c i t y , and the l e n g t h o f the chamber e s t a b l i s h e s , up t o some maximum l e n g t h , the r e s o l u t i o n o f the s e p a r a t i o n . A p p l i c a t i o n s o f f i e l d - f l o w s e p a r a t i o n s t o c e l l s have n o t b e e n numerous. I t i s p r i m a r i l y a chemical separation technique. Recent s t u d i e s have shown q u i t e c l e a r l y , n e v e r t h e l e s s , that p a r t i c l e s of



1.

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19

d i f f e r e n t s i z e s , d i f f e r i n g by p e r h a p s 1 μιη i n d i a m e t e r , c a n be s e p a r a t e d from one a n o t h e r w i t h u s e f u l r e s o l u t i o n . I n a n o t h e r r e c e n t development, the s e p a r a t i o n o f c e l l s on the b a s i s o f t h e i r d i f f e r e n t a d h e s i o n s t r e n g t h s t o the chamber w a l l s has been a c c o m p l i s h e d (67.) . I n a sense, t h i s i s a c l a s s i c a l c h r o m a t o g r a p h i c t e c h n i q u e , i n w h i c h adsorption i s the thermodynamic v a r i a b l e e x p l o i t e d t o e f f e c t a separation. T h i s method a l s o has a n a l y t i c a l as w e l l as s e p a r a t i v e v a l u e ; by knowing the s h e a r r a t e o f the f l o w i n g b u f f e r r e q u i r e d t o remove a d h e r e n t c e l l s , the magnitude o f the a d h e s i o n f o r c e c a n be evaluated. F i e l d - f l o w t e c h n o l o g y has not e n j o y e d w i d e s p r e a d use i n e i t h e r l a b o r a t o r y or i n d u s t r i a l c e l l separations. T h i s i s the c a s e w i t h several biophysical separation methods i n which p r a c t i t i o n e r s u n f a m i l i a r w i t h the p h y s i c a l p r i n c i p l e s u n d e r l y i n g a separation p r o c e s s a r e r e l u c t a n t t o e x p l o i t i t s e f f i c i e n c y owing t o a p h o b i a f o r physical/mechanical things. There i s a w i d e s p r e a d f e e l i n g t h a t c e n t r i f u g e s and chromatographs are f o r b i o l o g i s t s and biochemists while free electrophoresis, e l u t r i a t i o n , f i e l d - f l o w f r a c t i o n a t i o n and, t o some e x t e n t , o p t i c a l s o r t i n g methods a r e f o r p h y s i c i s t s and engineers. E l e c t r o k i n e t i c Methods. E l e c t r o p h o r e s i s i s the m o t i o n o f p a r t i c l e s (molecules, s m a l l p a r t i c l e s and whole b i o l o g i c a l cells) in an electric field and i s one of several e l e c t r o k i n e t i c transport processes. The v e l o c i t y o f a p a r t i c l e p e r u n i t a p p l i e d e l e c t r i c f i e l d i s i t s e l e c t r o p h o r e t i c m o b i l i t y , μ; t h i s i s a c h a r a c t e r i s t i c o f i n d i v i d u a l p a r t i c l e s and can be u s e d as a b a s i s o f s e p a r a t i o n and purification. T h i s s e p a r a t i o n method i s a r a t e ( o r transport) process. The four p r i n c i p a l e l e c t r o k i n e t i c processes of i n t e r e s t are electrophoresis (motion o f a particle i n an electric field), s t r e a m i n g p o t e n t i a l (the c r e a t i o n o f a p o t e n t i a l by f l u i d f l o w ) , sedimentation p o t e n t i a l (the c r e a t i o n o f a p o t e n t i a l by p a r t i c l e m o t i o n ) , and e l e c t r o e n d o s m o s i s (the i n d u c t i o n o f f l o w a t a c h a r g e d s u r f a c e by an e l e c t r i c f i e l d , a l s o c a l l e d e l e c t r o o s m o s i s ) . These phenomena always o c c u r , and t h e i r r e l a t i v e magnitudes d e t e r m i n e the p r a c t i c a l i t y o f an e l e c t r o p h o r e t i c s e p a r a t i o n o r an e l e c t r o p h o r e t i c measurement. I t i s g e n e r a l l y d e s i r a b l e , f o r example, t o m i n i m i z e m o t i o n due t o e l e c t r o e n d o s m o s i s in practical applications. A brief d i s c u s s i o n o f the g e n e r a l e l e c t r o k i n e t i c r e l a t i o n s h i p s f o l l o w s . The surface charge of suspended particles prevents their c o a g u l a t i o n and l e a d s t o s t a b i l i t y o f l y o p h o b i c c o l l o i d s . This s t a b i l i t y d e t e r m i n e s the s u c c e s s e s o f p a i n t s and c o a t i n g s , p u l p and p a p e r , sewage and f e r m e n t a t i o n , and numerous o t h e r m a t e r i a l s and processes. The s u r f a c e charge a l s o l e a d s t o m o t i o n when such p a r t i c l e s a r e suspended i n an e l e c t r i c f i e l d . The p a r t i c l e s u r f a c e has an e l e c t r o k i n e t i c ("zeta") p o t e n t i a l , ζ, p r o p o r t i o n a l t o σ , i t s s u r f a c e c h a r g e d e n s i t y - a few mV a t the hydrodynamic s u r f a c e o f s t a b l e , non-conducting p a r t i c l e s , i n c l u d i n g b i o l o g i c a l c e l l s , in aqueous s u s p e n s i o n . I f the s o l u t i o n has e l e c t r i c a l p e r m i t t i v i t y e (= 7 χ 10" F/m i n w a t e r ) , the e l e c t r o p h o r e t i c v e l o c i t y i s β

9


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(12)

f o r s m a l l p a r t i c l e s , such as m o l e c u l e s , whose r a d i u s o f c u r v a t u r e i s s i m i l a r t o t h a t o f a d i s s o l v e d i o n (Debye-Huckel p a r t i c l e s ) , and


ν - ^ -

Ε

(13)

for large ("von Smoluchowski") p a r t i c l e s , such as cells and organelles, η i s the v i s c o s i t y o f the b u l k medium. A t t y p i c a l i o n i c s t r e n g t h s (0.01 - 0.2 g - i o n s / L ) p a r t i c l e s i n the nanometer s i z e range u s u a l l y have m o b i l i t i e s (v/E) i n t e r m e d i a t e between t h o s e s p e c i f i e d by e q u a t i o n s (11) and (12) ( 6 8 ) . Analytical electrophoresis of proteins and other solutes is p e r f o r m e d i n a g e l m a t r i x , b e c a u s e c o n v e c t i o n i s s u p p r e s s e d ; however, h i g h sample l o a d s cannot be u s e d owing t o the l i m i t e d volume o f g e l t h a t can be c o o l e d s u f f i c i e n t l y t o p r o v i d e a u n i f o r m e l e c t r i c f i e l d , and p a r t i c u l a t e separands as l a r g e as c e l l s do n o t m i g r a t e t h r o u g h gels. C a p i l l a r y zone e l e c t r o p h o r e s i s , a p o w e r f u l , h i g h - r e s o l u t i o n a n a l y t i c a l t o o l ( 6 9 ) , depends on p r o c e s s e s a t the m i c r o m e t e r s c a l e and i s n o t a p p l i c a b l e t o p r e p a r a t i v e c e l l e l e c t r o p h o r e s i s . Therefore p r e p a r a t i v e e l e c t r o p h o r e s i s must be p e r f o r m e d i n f r e e f l u i d . The twa most f r e q u e n t l y u s e d f r e e - f l u i d methods a r e zone e l e c t r o p h o r e s i s i n a d e n s i t y g r a d i e n t and f r e e - f l o w ( o r c o n t i n u o u s f l o w ) e l e c t r o p h o r e s i s (FFE o r CFE). O t h e r p r e p a r a t i v e methods, more s u i t a b l e f o r m o l e c u l a r s e p a r a t i o n s , a r e d e s c r i b e d i n r e v i e w s by I v o r y (70) and by Mosher e t l - (21)· The c o m b i n a t i o n o f CFE w i t h c o m p l i m e n t a r y methods i s shown t o be a p o w e r f u l a p p r o a c h t o the a n a l y s i s o f c e l l s o f the immune system i n c h a p t e r s 13 and 14 by C r a w f o r d e t a l . (72) and by Bauer (22). A n a l y t i c a l and p r e p a r a t i v e c e l l e l e c t r o p h o r e t i c methods were compared c r i t i c a l l y i n a r e v i e w by P r e t l o w and P r e t l o w i n 1979 (74) . The number o f p r e p a r a t i v e methods a l o n e has s i n c e grown t o a t l e a s t 13, and t h e s e a r e i n t r o d u c e d , r e v i e w e d and compared q u a n t i t a t i v e l y i n C h a p t e r 15 ( 7 5 ) . a

The e l e c t r o p h o r e s i s o f l i v i n g c e l l s imposes p h y s i c a l c o n s t r a i n t s on s o l u t i o n s t h a t can be u s e d f o r e l e c t r o p h o r e s i s b u f f e r s . While maintaining low conductivity i t i s also necessary to maintain i s o t o n i c c o n d i t i o n s f o r the c e l l s . T h i s i s u s u a l l y a c h i e v e d by the a d d i t i o n o f n e u t r a l s o l u t e s t h a t are n o t h a r m f u l t o c e l l s , s u c h as s u g a r s . W i t h a few n o t a b l e e x c e p t i o n s , l i v i n g c e l l s do n o t t o l e r a t e t e m p e r a t u r e s above 40°C, so, t h e r m o r e g u l a t i o n d e s i g n e d t o p r e v e n t n a t u r a l c o n v e c t i o n a l s o must a c c o u n t f o r the t e m p e r a t u r e s e n s i t i v i t y o f the s e p a r a n d s . These and o t h e r c o n s t r a i n t s a r e a d d r e s s e d i n C h a p t e r s 12-15.


1.

T O D D & PRETLOW

Separation ofLiving CeUs

21

Summary The c h a p t e r s t h a t f o l l o w i n d i c a t e t h a t t h e s c i e n c e and t e c h n o l o g y o f c e l l s e p a r a t i o n i s becoming more d i v e r s e , t h a t f o u r p r i n c i p a l methods o f c e l l s e p a r a t i o n a r e emerging and b e i n g a p p l i e d t o i n c r e a s i n g l y diverse separation problems, that cell separation s c i e n c e and t e c h n o l o g y c o n t i n u e s t o be a r e s e a r c h a r e a o f i t s own as a p p l i c a t i o n s increase, that each technology i s increasing i n scientific sophistication, and t h a t c o n t i n u i n g dialogue between u s e r s and d e v e l o p e r s o f c e l l s e p a r a t i o n methods c o n t r i b u t e s t o p r o g r e s s i n t h i s important f i e l d .


Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

Cell Separation Methods Bloemendal, H. Ed. Elsevier/North-Holland Biomedical Press: Amsterdam, 1977. Cell Separation. Methods and Selected Applications. Pretlow, T.G.,II; Pretlow, T.P., Eds.; Academic Press: New York, NY, 1978-1987, Vol. 1-Vol. 5. Methods of Cell Separation: Catsimpoolas, Ν., Ed.; Plenum Press: New York, NY, 1977, 1979; Vol. 1, Vol 2. Partitioning in Aqueous Two-Phase Systems. Walter, H.; Brooks, D.E.; Fisher, D., Eds.; Academic Press: New York, NY, 1985. Todd,P.; Plank, L.D.; Kunze, M.E.; Lewis, M.L.; Morrison, D.R.; Barlow, G.H.; Lanham, J.W.; Cleveland, C. J. Chromatography 1986, 364, 11-24. Hannig, K. Electrophoresis 1982, 3, 235-243. Takayasu, M.; Maxwell, E.; Kelland, D.R. IEEE Transactions on Magnetics 1984, 10, 1186-1188. Walter, H.; Coyle, R.P. Biochim. Biophys. Acta 1968, 165, 540543. Walter, H.; Krob, E.J.; Brooks, D.E. Biochemistry 1976, 15, 2959-2964. Schlossman, S.; Hudson, L. J. Immunol. 1973, 110, 313-315. Lillie, R.S. Amer. J. Physiol. 1902, 8, 273-283. Beijerinck, M.W. Zeitz. für Chemie u. Industrie der Koll. 1910, 7, 16-20. Plank, L.D.; Hymer, W.C.; Kunze, M.E.; Todd, P. J. Biochem. Biophys. Meth. 1983, 8, 273-289. Heidrich, H.-G.; and Dew, M.E. J. Cell Biol. 1983, 74, 780-788. Kreisberg, J.I.; Sachs, G.; Pretlow, T.G.,II; andMcGuire, R.A. J. Cell Physiol. 1977, 93, 169-172. Hymer, W.C.; Hatfield, J. In Methods in Enzymology; Colowick, S.J.; Kaplan, N.O., Eds.; Academic Press: New York, NY, 1983, Vol. 103; pp. 257-287. Hymer, W.C.; Barlow, G.H.; Cleveland, C.; Farrington, M.; Grindeland, R.; Hatfield, J.M.; Lanham, J.W.; Lewis, M.L.; Morrison, D.R.; P. H. Rhodes, P.H.; Richman, D.; Rose, J . ; Snyder, R.S.; Todd, P.; Wilfinger, W. Cell Biophysics 1987, 10, 61-85.


22

18. 19. 20. 21. 22.


23. 24. 25. 26.

27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47.

CELL

SEPARATION SCIENCE AND

TECHNOLOGY

Adamus, G.; Zam, Z.S.; Emerson, S.S.; Hargrave, P.A.. In Vitro Cell Dev. Biol. 1988, 25, 1141-1146. Saunders, J.A.; Beretta, D.; Todd, P.; Matthews, B.S.; Singer, D.W. International School of Biomembrane and Receptor Mechanisms, Cannizzaro, Italy, Sep-Oct, 1985. Nordling, S.; Anderson, L.C.; Häyry, P. Science 1972, 178, 1001-1002. Zeiller, K.; Hannig, K.; Pascher, G. Hoppe-Seyler's Z. Physiol. Chem. 1971, 352, 1168-1170. Pretlow, T.G., II; Weir, E.E.; Zettergren, J.G. Int. Rev. Exp. Pathol. 1975, 14, 91-204. Pretlow, T.G.; Jones, C.M.; Pretlow, T.P. Biophys. Chem. 1976, 5, 99-106. Davis, R.H.; Lee, C.Y.; Batt, B.C.; Kompala, D.S. This volume. Buican, T. This volume. Johnson, N.F.; Hubbs, A.F.; Thomassen, D.G. In Multilevel Health Effects Research: from Molecules to Man. Park, J. F.; Pelroy, R.A., Eds.; Battelle Press: Columbus, OH, 1989, pp. 135-138. Kute, T.D.; Gregory, B.; Galleshaw, J . ; Hopkins, M.; Buss, D.; Case, D. Cytometry 1988, 9, 494-498. Albright, K.; Martin, J. This volume. Bigbee, W.L.; Langlois, R.G.; Jensen, R.H. In Multilevel Health Effects Research: from Molecules to Man. Park, J.F.; Pelroy, R.A., Eds.; Battelle Press: Columbus, OH, 1989, pp. 139-148. Leary, J.F. This volume. Srienc, F.; Campbell, J.L.; Bailey, J.E. Biotech. Ltrs. 1983, 5, 43. Rotman, B.; Papermaster, B.W. Proc. Natl. Acad. Sci. U.S.A. 1966, 55, 134-141. Wallen, C.A.; Higashikubo, R.; Roti Roti, J.L. Cell Tissue Kinet. 1983, 16, 357-365. Leary, J.F.; Todd, P.; Wood, J.C.S.; Jett, J.H. J. Histochem. Cytochem. 1979, 27, 315-320. Thorell, B. Cytometry 1983, 4, 61. Steen, H.B.; Skarstad, K.; Boye, E. Ann. Ν. Y. Acad. Sci. 1988, 468, 329-338. Leif, R.C.; Vallarino, L.M. This volume. Miller, R.C.; Phillips, R.A. J. Cell Physiol. 1969 73, 191-201. Anderson, N.G. National Cancer Institute Monograph 1966, 21, 940. Pretlow, T.G.,II; Pretlow, T.P. This volume. Keng, P.C. This volume. Kataoka, K. This volume. Owen, C.S. Cell Biophys. 1986, 8, 287-296. Powers, F.; Heath, C.A.; Ball E. D.; Vredenburg, J . ; Converse, A. O. This volume. Liberti, P.A.; Feeley, B.P. This volume. Owen, C.S. IEEE Trans. Magn. 1982, 18, 1514-1516. Kronick, P.L.; Campbell, G.L.M.; Joseph, K. Science 1978, 200, 1074-1076.


1.

T O D D & PRETLOW

48. 49. 50. 51. 52.


53.

54. 55. 56. 57. 58. 59. 60. 61. 62. 63.

65.

66. 67. 68. 69. 70.


23

P.-Å. Albertsson. Partition of Cell Particles and Macromolecules. 2nd Ed. Wiley-Interscience, New York, NY, 1971. P.-Å. Albertsson. Partition of Cell Particles and Macromolecules. Third Ed., John Wiley and Sons, New York, NY, 1986. Shanbhag, V.P. Biochim. Biophys. Acta 1973, 320, 517-527. Szlag, D.C.; Giuliano, K.A. Biotechnol. Techniques 1988, 2, 277-282. Van Alstine, J.M.; Snyder, R.S.; Karr, L.J.; Harris, J.M. J. Liquid. Chromatog. 1985, 8, 2293-2313. Cabezas, H. Jr.; Evans, J . ; Szlag, D.C. In Downstream Processing and Bioseparation, Recovery and Purification of Biological Products. Hamel, J-F. P.; Hunter J.B.; Sikdar, S.K., Eds.; ACS Symposium Series 419, American Chemical Society: Washington, DC, 1990, pp. 38-52. T. L. Hill, T.L. J. Chem. Phys. 1959, 30, 93-97. Cabezas, Η., Jr.; Kabiri-Badr, M.; Snyder, S.M.; Szlag, D.C. In Frontiers in Bioprocessing II: Sikdar, S.K.; Bier, M.; Todd, P., Eds.; American Chemical Society, Washington, DC, 1991. Schafer, L.; Kappeler, C. J. de Phys. 1985, 46, 1853. Baird, J.K. In Proc. 5th Europ. Symp. on Materials Science under Microgravity. European Space Agency: Paris, Series ESA SP-222, 1984, pp. 319-324. Johansson, G. Biochim. Biophys. Acta 1970, 221, 387-390. Bamberger, S.; Seaman, G.V.F.; Brown, J.A.; Brooks, D.E. J. Colloid. Interface Sci. 1984, 99, 187-193. Brooks, D.E.; Sharp, K.A.; Bamberger, S.; Tamblyn, C.H.; Seaman, G.V.F.; Walter, H. J. Colloid. Interface Sci. 1984, 102, 1-13. Raghava Rao, K.S.M.S.; Stewart, R.M.; Todd, P. Sep. Sci. Technol. 1990, 25, 985-996. Brønsted, J.N. Z. Phys. Chem. A. Bodenstein Festband, 1931, 257-266. Brooks, D.E.; Bamberger, S.; Harris, J.M.; Van Alstine, J. Proc. 5th European Symp. Material Sciences under Microgravity 1984, ESA SP-222, 315-318. 64. Brooks, D.E.; Sharp, K.A.; Fisher, D. In Partitioning in Aqueous Two-Phase Systems, Walter, H.; Brooks, D.E.; Fisher D., Eds.; Academic Press, New York, NY, 1985, pp. 11-84. Van Alstine, J.M.; Karr, L.J.; Harris, J.M.; Snyder, R.S.; Bamberger, S.B.; Matsos, H.C.; Curreri, P.A.; Boyce, J . ; Brooks, D.E. In Immunobiology of Proteins and Peptides IV. Atassi, M.Z., Ed.; Plenum Publishing Corp., New York, NY, 1987, pp. 305-326. Giddings, J.C.; Barman, B.N.; Liu, M.-K. This volume. Bigelow, J.C.; Nabeshima, Y.; Kataoka, K.; Giddings, J.C. This volume. R. W. O'Brien, R.W.; and L. R. White, L.R. J. Chem. Soc. Faraday II 1978, 74, 1607-1626. Jorgenson, J.W.; Lukacs, K.D. Science 1983 222, 266-272. Ivory, C.F. Sep. Sci. and Technol. 1988, 23, 875-912.


24

71.

72. 73. 74. 75.

CELL

SEPARATION SCIENCE AND

TECHNOLOGY

Mosher, R.; Thormann, W.; Egen, N.B.; Couasnon, P.; Sammons, D.W. In New Directions in Electrophoretic Methods, J.W. Jorgenson, J.W.; Phillips M. Eds.; American Chemical Society, Washington, DC, 1987, pp. 247-262. Crawford, N.; Eggleton, P.; Fisher, D. This volume. Bauer, J. This volume. Pretlow, T.G.,II; Pretlow, T.P. Int. Rev. Cytol., 1979, 61, 85-128. Todd, P. This volume.


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Separation of Living Cells - American Chemical Society

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