Choice of an aqueous polymer two-phase system for cell partition

101

Biochimica et Biophysica Acta, 542 (1978) 101--106

© Elsevier/North-Holland Biomedical Press

BBA 28592 CHOICE OF AN AQUEOUS POLYMER TWO-PHASE SYSTEM FOR CELL PARTITION

LARISA M. MIHEEVA, BORIS YU. ZASLAVSKY and SERGEI V. ROGOZHIN Institute of Elementoorganic Compounds, Academy of Sciences of the U.S.S.R., MoscQw V-312 (U.S.S.R.)

(Received November 29th, 1977)

Summary Partition of h u m a n erythrocytes in aqueous two-phase polymer systems produced by Ficoll and different molecular weight fractions of dextran and polyethylene glycol and the influence of the ionic composition on the cells' partition in the systems was studied. It is found that the Ficoll-dextran-40 system is characterized by a number of advantages as compared with the c o m m o n dextran-polyethylene glycol system or the others systems under study. The main advantage of the system appears to be that it is possible to concentrate the red cells in the top phase or in the b o t t o m phase of the system, depending on the system ionic composition. The influence of the nature and the concentration of salt additives on this two-phase system formation is examined.

Introduction The partition in aqueous two-phase polymer system is widely used at present for separation of various biological materials [1]. The two-phase dextran500-poly(ethylene glycol)-6000 system and its modifications c o m m o n l y used in the particles and cells partition [1--9] have several disadvantages: (1) the cell partition is restricted by one of the phases and the interface; (2) the phase polymers are not completely inert to cells distributed in the system [10,11]. It was the purpose of this study to find an aqueous two-phase polymer system for cells partition w i t h o u t the above disadvantages. We have studied h u m a n erythrocytes behaviour in systems formed by Ficoll and different molecular weight fractions of dextran and polyethylene glycol and its dependence on the ionic composition in these systems. As a result the Ficoll-dextran-40 system for cells partition is suggested and the ionic composition influence on the system formation is established.

102 Materials and Methods Materials. Dextran with molecular weight 20 000 (Ferak, West Berlin), dextran fractions with molecular weight 40 000, 80 000 (Polfa, Poland) and 150 000, 250 000, 500 000 and 2000 000 (Pharmacia Fine Chemicals, Sweden), Ficoll-400 (Pharmacia Fine Chemicals, Sweden) and polyethylene glycol fractions with molecular weight 600, 2000, 3000 (LOBA, Austria}, 4000 and 6000 (Merck, G.F.R.) were used in this work. All inorganic salts and sucrose used were of analytical reagent grade. Erythrocytes from outdated bank blood were obtained from Moscow Transfusion Center and were stored at 4 °C. Before use, the cells were washed three times with the phosphate-buffered isotonic saline (pH 7.4). The washed and packed cells were then suspended in an appropriate isotonic buffer to give a suspension of the concentration about 5 • 10 s cells per ml. Phase systems. The phase systems used in the present experiments are given below in captions to tables. The systems containing phosphate buffer and NaC1 (as indicated in captions to tables and in the text) to provide the desired final concentrations were obtained as described elsewhere [1] by mixing the stock polymer solutions in a series of test tubes. Each tube then received from 0.2 to 1.0 ml of red cells suspension to obtain the required cell concentration, namely about 1 • 10 s cells per total 5 g of the system. The contents were then mixed by inverting the tubes 20--40 times and phase separation then allowed to take place at 20--23°C. The settling time was dependent on the system. At the end of the settling time, l-ml samples were withdrawn by pipette from both phases and diluted with 0.1 ml 1% solution of sodium dodecyl sulfate and 0.9 ml water to lyse the cells. The absorbance of the lysate was measured at 413 or 535 nm and from the extinction of the cell suspension added and the total extinction present in each of the phases (obtained with regard to the phase volumes) the percentages of the cells added which were present in each phase could be calculated, as well as that attached to the interface. The phase diagrams were constructed as described in ref. 1. Results and Discussion Since erythrocytes are known [1--4,12] to favour the upper polyethylene glycol-rich phase in the dextran-500-polyethylene glycol-6000 system, we endeavoured to replace dextran by a more hydrophobic polymer, Ficoll-400. The fact that in the case of Ficoll-400 and polyethylene glycol-6000 the concentrations of the polymers necessary to produce two-phase systems are higher than those required in the case of dextran-500 and polyethylene glycol-6000 [1] indicates that Ficoll-400 is more hydrophobic than dextran-500 as well as being more compatible with polyethylene glycol-6000. When erythroeytes are placed into the Ficoll-400-polyethylene glycol-6000 system they collect in the Ficoll,rich phase independently of the ionic composition of the system: in 0.15 M NaC1 in 0.01 M sodium phosphate buffer, pH from 6.8 to 7.4; in 0.11 M sodium phosphate buffer, pH 7.4, as well as when all phosphates are replaced with 0.29 M sucrose, The cells' behaviour appears also to be independent of polyethylene glycol molecular weight; in the

103

TABLE I THE

BEHAVIOUR

OF HUMAN

ERYTHROCYTE

6000 SYSTEMS WITH DIFFERENT

FRACTIONS

IN THE DEXTRAN

- POLYETHYLENE

GLYCOL-

OF DEXTRAN

0 . 1 5 M NaC1 w i t h 0 . 0 1 M s o d i u m p h o s p h a t e b u f f e r , p H 7 . 4 w a s e m p l o y e d .

Molecular weight of dextran

20 40 80 150 250 500 2 000

Polymer composition

000 000 000 000 000 000 000

(% w / w )

Dextran

Polyethylene glycol-6000

8.5 7.4 8.5 5.5 5.25 5.0 4.2

5.0 4.5 4.7 3.8 3.74 4.0 3.4

Partition *

B B B + I I I I T

* B, T a n d I i n d i c a t e t h a t m o r e t h a n 50% o f t h e cells in t h e s y s t e m f a v o u r t h e b o t t o m phase or the interface, respectively.

phase, the top

phase systems formed by Ficoll with polyethylene glycol-6000, -4000, -3000, -2000 or -600 the red cells collect in the Ficoll-rich phase. In view of this observation we have decided to turn back to the dextranpolyethylene glycol-6000 system and to study the erythrocytes' partition dependence upon the dextran molecular weight. From the data in Table I it follows that if dextran is replaced by a lower molecular weight fraction the affinity of erythrocytes for the b o t t o m dextran-rich please increases and when the system is formed by polyethylene glycol-6000 and dextran-80, -40 or -20 the cells collect in the b o t t o m phase. Alteration in the ionic composition of dextran-polyethylene glycol phase system markedly affects the cells' partition behaviour. An increase in the relative phosphate concentration with a concomitant decrease in the NaC1 concentration (to keep the overall salt concentration at a constant osmolarity) reduces the cells' affinity for the top phase. The data in Table II show the distribution of human red cells in the dextran-20-polyethylene glycol-6000 and the dextran500-polyethylene glycol-6000 systems at different ionic compositions. The

T A B L E II PARTITION

OF

HUMAN

SYSTEMS AS A FUNCTION

ERYTHROCYTES

IN TWO DEXTRAN-POLYETHYLENE

GLYCOL-6000

O F NaC1 C O N C E N T R A T I O N

S y s t e m 1 c o n t a i n e d 8 . 5 % ( w / w ) d e x t r a n - 2 0 a n d 5% ( w / w ) p o l y e t h y l e n e g l y c o l - 6 0 0 0 ; s y s t e m 2 c o n t a i n e d 5% ( w / w ) d e x t r a n - 5 0 0 a n d 4 % ( w / w ) p o l y e t h e l e n e g l y c o l - 6 0 0 0 ; p H 7 . 4 . Ionic composition

0.11 0.03 0.09 0.15

M M M M

sodium NaC1 + NaC1 + NaCI +

phosphate buffer 0.09 M sodium phosphate buffer 0.05 M sodium phosphate buffer 0.01 M sodium phosphate buffer

* B, T a n d I are i n d i c a t e d in c a p t i o n t o T a b l e I.

Partition * 1

2

I I I + B B

T T÷I

I I

104

TABLE III PARTITION OF HUMAN TEMS WITH DIFFERENT SALT COMPOSITION

ERYTHROCYTES IN DEXTRAN-80-POLYETHYELENE G L Y C O L SYSF R A C T I O N S O F P O L Y E T H E L E N E G L Y C O L AS A F U N C T I O N O F T H E

S y s t e m 1 c o n t a i n e d 7.4% ( w / w ) d e x t r a n - 8 0 , 4.5% ( w / w ) p o l y e t h e l e n e g l y c o l - 6 0 0 0 ; s y s t e m 2 c o n t a i n e d 28% ( w / w ) d e x t r a n - 8 0 , 23% ( w / w ) p o l y e t h e l e n c g l y c o l - 4 0 0 0 . Ionic composition

0.29 0.11 0.15 0.15

M M M M

Partition *

sucrose s o d i u m p h o s p h a t e b u f f e r , p H 7.4 NaC1 + 0 . 0 1 M s o d i u m p h o s p h a t e b u f f e r , p H 7.4 NaC1 + 0 . 0 1 M s o d i u m p h o s p h a t e b u f f e r , p H 6,8

1

2

T I I + B I + B

T T T + I I

* B, T a n d I are i n d i c a t e d in c a p t i o n to T a b l e I.

data show that an increase in the relative NaC1 concentration results in an increase of the cells' affinity for the dextran-rich phase independently of the dextran molecular weight. Table III shows the behaviour of human erythrocytes in dextran-80-polyethylene glycol systems with different molecular weight fractions of polyethylene glycol at different ionic compositions and pH. It should be noted that if the salts in the dextran-80-polyethylene glycol-6000 system are replaced completely by sucrose (to maintain isotonicity) the cells concentrate in the top phase and in the system containing 0.15 M NaC1 in 0.01 M phosphate sodium buffer (pH 6.8) the cells distribute between the b o t t o m phase and the interface of the system. Thus, it appears that a reversal of the cells' affinity for the top or the b o t t o m phase is possible when the systems' conditions are altered. In view of the above results it was suggested that to find a suitable phase system for cell partition, systems produced by low molecular weight dextrans and Ficoll-400 should be studied. In Table IV the various ways of behaviour of erythrocytes in the Ficoll-400-dextran (from 40 000 to 500 000) systems at different ionic compositions are listed. The data in Table IV show that in the presence of 0.15 M NaC1 in 0.01 M sodium phosphate buffer (pH 7.4) TABLE IV BEHAVIOUR OF HUMAN ERYTHROCYTES IN THE FICOLL-DEXTRAN SYSTEMS WITH DIFFERENT FRACTIONS OF DEXTRAN AS A FUNCTION OF THE IONIC COMPOSITION S y s t e m 1, 0 . 1 1 M s o d i u m p h o s p h a t e b u f f e r , p H 7.4; s y s t e m 2, 0 . 1 5 M N a C I + 0.01 M s o d i u m p h o s p h a t e b u f f e r , p H 7.4. Molecular weight of dextran

40 80 150 250 500

000 000 000 000 000

P o l y m e r c o m p o s i t i o n (%, w / w )

Partition *

Dextran

Ficoll

1

2

10.0 7.0 7.0 5.0 3.0

10.0 10.0 10.0 i0.0 10.0

B B B B B

T T I I B

* B, T a n d I a r e i n d i c a t e d in c a p t i o n to T a b l e I.

105 123

Ca

0

I

I

t

~__1

I0

I

I

I

I

20 Ficoll, °Io w / w

I

I

I

I

3L 30

~

Fig. 1. Phase d i a g r a m f o r t h e t w o - p h a s e Ficoll-4OO-dextran-40 s y s t e m . T e m p e r a t u r e 20 ° c . 1; w a t e r ; 2, 0 . 1 5 M NaC1 + 0.01 M s o d i u m p h o s p h a t e b u f f e r , DH 7.4; 3; 0.11 M s o d i u m p h o s p h a t e b u f f e r , p H 7.4.

the cells' affinity for the b o t t o m Ficoll-rich phase is decreased when the molecular weight of dextran is decreased. In the Ficoll-400
106 TABLE V PARTITION OF HUMAN ERYTHROCYTES FUNCTION OF THE IONIC COMPOSITION

IN

THE

FICOLL-400-DEXTRAN-40

SYSTEM

AS A

P o l y m e r c o m p o s i t i o n : 1 4 % ( w / w ) Ficoll, 10% ( w / w ) d e x t r a n - 4 0 . P a r t i t i o n is e x p r e s s e d as q u a n t i t y of cells f o u n d in t h e p h a s e i n d i c a t e d ( p e r c e n t o f t o t a l cells a d d e d ) . R e s u l t s are p r e s e n t e d as the m e a n o f five exp e r i m e n t s ~ S.D. s h o w n in p a r e n t h e s e s . Ionic c o m p o s i t i o n

0 . 1 1 M s o d i u m p h o s p h a t e b u f f e r , p H 7.4 0 . 1 5 M N a C I + 0 . 0 1 M s o d i u m p h o s p h a t e b u f f e r , p H 7.4

Partition Top phase

Bottom phase

-92 (+3)

98 (_+3) --

using the system for the partition of different cells and biological particles is under study. References 1 A l b e r t s s o n , P.-~. ( 1 9 7 1 ) P a r t i t i o n o f Cell P a r t i c l e s a n d M a e r o m o l e c u l e s , 2nd e d n . , Wiley, N e w Y o r k 2 W a l t e r , H. ( 1 9 7 5 ) in M e t h o d s in Cell R e s e a r c h ( P r e s c o t t , D.M., ed.), Vol. 9, pp. 2 5 - - 5 0 , A c a d e m i c Press, N e w Y o r k 3 Walter, H. K r o b E.J. a n d G a r z a . R. ( 1 9 6 8 ) B i o c h i m . B i o p h y s . A e t a 165, 5 0 7 - - 5 1 4 4 Walter, H. ( 1 9 6 9 ) in P r o g r e s s in S e p a r a t i o n a n d P u r i f i c a t i o n ( G e r r r i t s e n , T., ed.), Vol. 2. pp. 1 2 1 - - 1 4 5 , A c a d e m i c Press, N e w Y o r k 5 R e i t h e r m a n , R., F l a n a g a n , S.D. a n d B a r o n d e s , S.H. ( 1 9 7 3 ) B i o e h i m . B i o p h y s . A e t a 297, 193 2 0 2 6 F l a n a g a n , S.D., T a y l o r , P. a n d B a r o n d e s , S.H. ( 1 9 7 5 ) N a t u r e 2 5 4 , 4 4 1 - - 4 4 3 7 E r i k s s o n E., A l h e r t s s o n , P.-A. a n d J o h a n s s o n , G. ( 1 9 7 6 ) Mol. Cell. B i o c h e m . 10, 1 2 3 - - 1 2 8 8 Walter, H. a n d K r o b , E.J. ( 1 9 7 6 ) F E B S L e t t . 61, 2 9 0 - - 2 9 3 9 Walter, H., K r o b , E.J. a n d T u n g , R. ( 1 9 7 6 ) E x p . Cell Res. 102, 14 24 10 A h k o n g , Q . F . , F i s h e r , D., T a m p i o n , W. a n d L u c y , J . A . ( 1 9 7 5 ) N a t u r e 2 5 3 , 1 9 4 - - 1 9 5 11 S k o o g W.A. a n d B e c k , W.S. ( 1 9 5 6 ) Blood 11, 4 3 6 - - 4 3 9 12 W',dter, H., K r o b , E.J. a n d B r o o k s , D.E. ( 1 9 7 3 ) B i o c h e m i s t r y 12, 2 9 5 9 - - 2 9 6 4