Electrophoretic isolation of cells results in a loss of resolution power caused by the sedimentation of the cells in the media. The results of an experiment to extract urokinase from human embryos during the Apollo Soyuz mission are presented and discussed

Allen, R. E.

Barlow, G. H.

Lazer, S. L.

Rueter, A.

English

NASA Technical Reports Server

Elec t rophore t i c  Separa t ion  of Human Kidney Ce l l s  a t  Zero Gravi ty 
Grant H.  Barlow*, S. LaVera Lazer*, Annemarie Rueter* and 
Robert A1 1 en** 
*Experimental Biology Div is ion ,  Abbott Labora tor ies ,  North Chicago, 
I1 1 .  and **Biotechnology Branch, NASA, George C.  Marshal 1 Space 
F l i g h t  Center ,  Alabama 
Work supported by Cont rac t  from NASA, No. NAS-8-30591 
https://ntrs.nasa.gov/search.jsp?R=19770010742 2020-03-22T10:36:01+00:00Z
Acute thromhoen~bolic vascular  d i s ease  remains the g r e a t e s t  s i n g l e  
cause of n ior ta l i ty  i n  the middle and e l d e r  age populat ion of the U . S .  I n  
r ecen t  years  the use of f i b r i n o l y t i c  therapy has shown the  f e a s i  bi 1 i  ty  
of c l o t  l y s i s  -- i n  vivo in  such d i seases  as  pulmonary emboli and deep vein 
thrombosis.  The UPET ( 1 )  and USPET ( 2 )  mu1 t i cen t e red  c l i n i c a l  t r i a l s  
have demonstrated the  s a f e t y  of such therapy.  
The presence in  ur ine  of a  substance capable of e f f e c t i n g  the  
t ransformat i  on of plasmi nogen t o  pl asmi n,  t he  agent  necessary t o  bring 
about f i b r i n o l y s i s  was f i r s t  described by Williams ( 3 )  i n  1951 and in  
the  fol lowing yea r  by Astrup and Sterndorf f  ( 4 ) .  Sobel ( 5 )  -- e t  a1 assigned 
t h e  name urokinase (UK)  t o  t h i s  a c t i v a t o r  i n  1952. 
I t  was apparent  from the  s t a r t  t h a t  the  l o g i s t i c s  of u r ine  supply 
and t h e  c o s t  of production made the  ur ine  d e r i v a t i v e  of the enzylne 
imprac t icable .  For the  4 mi l l i on  u n i t  dose used i n  the  c l i n i c a l  t r i a l s  
a t  l e a s t  1500 l i t e r s  of u r ine  were required and the  cos t  was p roh ib i t i ve .  
These f a c t s  motivated us t o  search f o r  another  source of t h i s  f i b r i n o l y t i c  
agent ;  and s i n c e  t h i s  agent  i s  p ro te in  i n  na tu re ,  i t  i s  not d e s i r a b l e  
f o r  imniunogenic reasons ,  t o  use o the r  animal sources a s i d e  from man. 
In 1959, Barne t t  and Baron ( 6 )  demonstrated the  production of a  
plasmi nogen a c t i v a t o r  from KB c e l l s  , derived from a human epidermoid 
carcinoma and primary monkey kidney c e l l s .  In a  l a t e r  paper ( 7 )  t h e  
same authors  showed t h a t  many continuous primary c e l l  c u l t u r e s  would 
produce e i  t h e r  o r  both plas~iiinogen and protease a c t i v a t o r s .  Pa in t e r  and 
Charles  (8)  deliionstrated the  accun~ulat ion of a  f i b r i n o l y t i c  agent  i n  
c u l t u r e s  of primary monkey kidney c e l l s  and i n  an e s t ab l i shed  l i n e  of 
canine renal  c e l l s .  This agent  was shown t o  be an a c t i v a t o r  of plasminogen 
with p rope r t i e s  s i m i l a r  t o  those of urokinase.  F i n a l l y ,  Bernik and Kwaan 
( 9 ,  10) demonstrated ( a )  f i b r i n o l y t i c  a c t i v i t y  i n  cu l tu re s  of human 
kidneys,  ( b )  t h a t  t h i s  a c t i v i t y  was imniunoloyical ly  i nd i s t i ngu i shab le  
from ur inary  urokinase and ( c )  t h a t  t h i s  f i b r i n o l y t i c  agent  was produced 
t o  the g r e a t e s t  degree i n  c u l t u r e s  of human renal c e l l s  froin a 26 - 32 
week o ld  f e t u s .  These f ind ings  and the development by Weiss and Schle icher  
(11 ,  12)  of c e l l  equipment known a s  the  Mass Tissue Culture Propagator 
(MTCP) which allows f o r  the c u l t u r i n g  of c e l l s  on a l a r g e  s c a l e  prompted 
us t o  i n i t i a t e  a program t o  produce urokinase from human embryo kidney 
c e l l s .  The bas i c  methodology has been described (13)  and the  research  
has continued f o r  improvement of t he  process.  
The observat ion by Bernik and Kwaan (10)  using a f i b r i n  s l i d e  
technique t h a t  only about 5% of t he  c e l l s  produced a c t i v a t o r  led t o  the  
design of t he  experiments described here.  A method was sought t o  i s o l a t e  
t h e  producing c e l l s  t h a t  could be used eventua l ly  on a l a rge  s c a l e .  One 
poss ib l e  way i s  e l e c t r o p h o r e t i c a l l y .  However, a drawback i n  t h e  e l ec t rophores i s  
of  c e l l s  i s  the  l o s s  of reso lv ing  power due t o  the  sedimentation of t he  
c e l l s  i n  t he  media. An e l e c t r o p h o r e t i c  separa t ion  a t  zero g r a v i t y  
should obviously negate t h i s  drawback. Thus, the experiments described 
here were performed on the  Apolly-Soyuz space mission. 
Methods 
E lec t r opho res i s  i n c l u d i n g  i ns t r u rncn ta t i on ,  e l e c t r o p h o r e t i c  cond i t i ons  
ang g e l  s l i c i n g  techniques wi 11 be descr ibed  elsewhere by NASA ( 1 4 ) .  
C e l l  V i a b i l i t y  was performed us ing  a  0.4% s tock  s o l u t i o n  o f  
e r y t h r o s i n e  B made i n  phosphate b u f f e r ,  pH 7 ( 1 5 ) .  
Urok inase A c t i v i t y  determined us i ng  a  m o d i f i c a t i o n  o f  t h e  f i b r i n  
p l a t e  techn ique  as descr ibed  by Braknlan ( 16 ) .  
HGCF A c t i v i t y  determined u s i n g  human bone marrow c e l l s  i n  a  m o d i f i c a t i o n  
o f  t h e  method descr ibed  by S tan l ey  and M e t c a l f  (17 ) .  
E l e c t r o p h o r e t i c  Mobi 1  i t y  determined by the  method descr ibed  by 
Seaman (19 ) .  
Resu l t s  
The f r ozen  f r a c t i o n s  were thawed r a p i d l y  a t  37 '~ ,  c e n t r i f u g e d  and 
resuspended i n  growth media. The f r a c t i o n s  were weighed and t a r e d  t o  
de te rmine  t h e  we igh t  o f  each f r a c t i o n .  The pH on each f r a c t i o n  was a l s o  
determined. These r e s u l t s  a re  shown i n  Table I. An a l i q u o t  was taken 
f o r  v i a b l e  c e l l  coun t  and based on t h i s  i n f o r m a t i o n  t h e  c e l l s  were 
c u l t u r e d .  The d i s t r i b u t i o n  o f  v i a b l e  c e l l s  i s  shown i n  F i gu re  1. As 
can be seen about 4 subpopulat ions o f  c e l l s  can be i d e n t i f i e d .  
A f t e r  28 days o n l y  f r a c t i o n s  between 11 and 19 had reached conf luency. 
The o t h e r  f r a c t i o n s  were removed f rom the  c u l t u r e  p l a t e s  and t e s t e d  f o r  
u rok inase  a c t i v i t y  by f i b r i n  p l a t e  method and showed no f i b r i n o l y t i c  
a c t i v i t y .  The sequence o f  events  w i t h  t h e  c o n f l u e n t  p l a t e s  a re  shown 
i n  Table 2 and o f  t h e  subcu l t u re  i n  Tables 3 and 4 .  Those p l a t e s  t h a t  
went t o  p roduc t i on  were p u t  on p roduc t i on  media and t e s t e d  f o r  u rok inase  
a c t i v i t y  a t  va r i ous  t imes.  The c e l l s  t h a t  were subcu l t u red  were 
removed f rom t h e  d ishes  w i t h  EDTA and then r e c u l  tu red .  
Table 5 shows the r e s u l t s  of the  urokinase production obtained with 
t h e  primary and subrul t u r e  1 c e l l s  a f t e r  35 days on product ion.  There i s  
an obvious enrichment of urokinase a c t i v i t y  i n  Fract ion 15 when i t  i s  
recorded a s  u n i t s  of a c t i v i t y  per 100 c e l l s .  An opt imizat ion i s  a l s o  
seen in  several  o the r  f r a c t i o n s .  Control experiment with the  same c e l l s  
a t  ground base condi t ions  gave the  value 0.28/100 c e l l s .  The subcu l tu re  
2 c e l l s  d id  not  produce urokinase when placed on production media. 
P a r t  of t he  c e l l s  from subcul ture  2 were analyzed f o r  mobi l i ty  
d i s t r i b u t i o n  and the  r e s u l t s  on t h r e e  such f r a c t i o n s  i s  shown i n  Figures 
2 ,  3 and 4 .  
The mater ia l  from SC-1 was a l s o  t e s t e d  f o r  the presence of Human 
Granulocyte Conditioning Factor  and the  r e s u l t s  a r e  shown i n  Table 6.  
Di scussion 
The r e s u l t s  show t h a t  c e l l s  can be separa ted  under s t e r i l e  condi t ions  
and re turned  from o r b i t  i n  such a manner t h a t  they r e t a ined  t h e i r  a b i l i t y  
t o  grow i n cul t u r e .  
The e l ec t rophores i s  i n  space showed good separa t ion  of the  kidney 
c e l l s  i n t o  subpopulations. The r e s u l t s  i nd i ca t ed  t h a t  t he re  were a t  
l e a s t  3 and maybe 4 subpopulat ions.  This r e s u l t  i s  i n  agreement with 
some r e s u l t s  obtained using the  endless  be1 t e i ec t rophores i s .  
Even though each f r a c t i o n  showed v i ab l e  c e l l s  by the s t a i n  technique 
and they a l l  a t tached  t o  the g l a s s  su r f ace  only the few f rac t ior i s  bctwceri 
11 and 20 grew. The reason f o r  t h i s  i s  not known. The only poss ib le  
explana t ion  i s  t h a t  t he  non growers were more s e n s i t i v e  t o  unfavorable 
condi t ions  and the re fo re  could not recover  from the  shock t o  grow i n  
c u l t u r e .  
The da ta  i n d i c a t e s  an enrichment of producing c e l l s  i n  t he  a rea  
cen t e r ing  around Frac t ion  15. The r e s u j t s  can be i n t e r p r e t e d  t o  show an 
incomplete r e s o l u t i o n  between a  producing c e l l  populat ion and a  non 
producing popula t ion .  Considering t he  f a c t  t h a t  the  condi t ions  of t he  
experiment were no t  optimized f o r  kidney c e l l s  but genera l ized  f o r  t h r e e  
d i f f e r e n t  s e p a r a t i o n s ,  t h i s  r e s u l t  i s  no t  s u r p r i s i n g .  This i s  probably 
why t h e  c e l l s  f a i l e d  t o  produce a t  subcu l tu r e  2 when normally under 
optimi zed growth condi t i o n s  they produce t o  subcu l tu r e  7 .  
I t  would appear  t h a t  t he  a rea  f o r  maxiniunl product ion of Hunian 
Granulocyte Condit ioning Fac tor  does not  co inc ide  with t h a t  f o r  urokinase.  
This i s  a  very i n t e r e s t i n g  f i nd ing  and i n d i c a t e s  t h a t  t he  two products  
a r e  most l i k e l y  no t  produced by t he  same c e l l .  
The a n a l y t i c a l  mob i l i t y  da t a  a t  t he  subcu l tu r e  2 da t a  shows each 
f r a c t i o n  a t  t h i s  s t a g e  t o  have a  r a t h e r  broad d i s t r i b u t i o n .  This i s  
r a t h e r  disappoi  nti'ng i n  t h a t  one would a n t i c i p a t e  a  r a t h e r  sharp  d i s t r i b u t i o n  
based on t he  narrow f r a c t i o n  one s t a r t s  wi th .  This i s  probably explained 
by t he  f a c t  t h a t  t he  s t a r t i n g  f r a c t i o n  i s  heterogeneous and t he  c e l l  
a t tachment  and growth p a t t e r n  i s  a  random event  which can broaden a t  
each r e c u l t u r e  l e v e l .  More ana lyses  should help prove t h i s  po in t .  
U P E T  T r i a l s ,  C i r c u l a t i o n  XLVII Suppl .  11,  (1973) .  
USPET T r i a l s ,  J .  A m .  Med. Ass. 29, 1606, ( 1 9 7 4 ) .  
Wi l l i ams ,  J .  W . ,  Baldwin, R .  L . ,  Saunders ,  \#I. M .  and S q u i r e ,  P .G. ,  
9 .  Am.  Chem. Soc. 74, 1542, ( 1 9 5 2 ) .  
A s t r u p ,  T. and S t e r n d o r f f . ,  P roc .  Soc. Exp t l .  B i o l .  Med. 81, 
675, ( 1 9 5 2 ) .  
S o b e l ,  G .  W . ,  Mohler, S .  R . ,  J o n e s ,  N .  W . ,  Dowdy, A .  B .  C . ,  and 
Gues t ,  M .  M . ,  Arner. J .  P h y s i o l .  171, 1968 (1952) .  
B a r n e t t ,  E .  V .  and Baron, S . ,  P roc .  Soc. Exp t l .  B i o l .  Med. 102, 
308, (1959) .  
Baron, S. and B a r n e t t ,  E .  V . ,  Proc.  Soc. Exp t l .  B io l .  Med. 103, 
527, (1960) .  
P a i n t e r ,  R .  H .  and C h a r l e s ,  A .  F . ,  Amer. J .  Phys io l .  202, 
1125, (1962) .  
Bern ik ,  M. B .  and Kwaan, H .  C . ,  J .  Lab. C l i n .  Med. 70, 650,  
(1  967) .  
Bern ik ,  M .  B .  and Kwaan, H .  C . ,  J .  C l i n .  I n v e s t .  48, 1740, (1969) .  
Weiss,  R .  E .  and S c h l e i c h e r ,  J .  B . ,  Biotech.  and Bioeng. 10, 
601,  (1968) .  
S c h l e i c h e r ,  J .  B. and Weiss,  R .  E . ,  Biotech.  and Bioeng. 10, 
617,  (1968) .  
Barlow, G .  H,, Ruete r ,  A.,  Tr ibby ,  I .  E . ,  P r o t e a s e s  and B i o l o g i c a l  
C o n t r o l ,  Vol. 2 ,  Cold S p r i n g s  Harbor Laboratory (1975) .  
NASA: To ve pub l i shed .  
15. P h i l l i p s ,  H .  J . ,  In Tissue  Cu l tu r e ,  Methods and Appl ica t ions .  
Ed. Kruse and P a t t e r s o n .  pg. 406, Academic Press  (1973) .  
16.  Brakman, K.  , F i b r i n o l y s i s :  A Standardized F ib r in  P l a t e  Method 
and A F i b r i n o l y t i c  Assay of Plasminogen. Scheltema and Holkema 
(1967).  
17. S t an l ey ,  E .  R .  and Metcalf ,  D., J .  Lab. Cl in .  Med. 75, 657, 
( 1  972) .  
18.  Seaman, G .  V .  F . ,  In t he  Red Blood Cel l  Vol 11, pg.  1135. 
Acadenii c  Press  (1 975) 
ORIGINAL PAGE IS 
OF POOR QULImi 
\!  ' , ,: L, I 
: 
, \, ., 
. ,., . .  
!I  . I >,! Ji, 
G , l : - -  
1 4 1  5 . i  
. . 
. . 1 I :  i '!,$ 
9 . 1 6 ~ 5 3  
ij. 133L"J 
G.lL:,:S 
0 . 1  4; L; .; iJ 
3 .  ; G ' > :  J 
L J  
0. i 7 ; : : ,  
u. 1 i,;,>
(, ,;-::*,- 
. J r45 
9. 
O . ! t ; C 2  
0. iNCJ 
(j.133.)3 
G . 1 2 *: 5 
.- . U.  ~ b * r , , L  
6. 1 " " 
, ' I ; / >  
. , 
[ J .  1'. J i L  
9 . 1 . ; > : s  
- 6 5 :  I >  u '!' - 
. . 2. i ~ l , l l>  
0.  lL2Lt.! 
!i * 1 ,:,; ',:, 
: l !  :", 
L ' .  " J . , . ,  
. .  . 
i. . I I J : ..I 
TABLE 2 
FRACTION 
11 1 d i s h  
13 1  d i s h  
1 4  1  d i s h  
15 1 d i s h  
1 6  1  d i s h  
17  2 d i s h e s  
1 9  1  d i s h  
B  C o n t r o l  1 d i s h  
D C o n t r o l  1 d i s h  
SEQUENCE OF EVENTS 
PRIMARY CULTURE 
(35 mrn). P e t r i  D i s h e s  
COriDITION 
Conf 1  uen t 
Conf 1  uen  t 
C o n f l u e n t  
Conf 1  u e n t  
Conf 1  u e n t  
C o n f l u e n t  
C o n f l u e n t  
C o n f l u e n t  
C o n f l u e n t  
DISPOSITION 
To p r o d u c t i o n  
To p r o d u c t i o n  
To s u b c u l t u r e  ( 2 )  
To p r o d u c t i o n  
To s u b c u l t u r e  
To p r o d u c t i o n  ( I ) ,  t o  s u b c u l t u r e  ( 1  1 
To s u b c u l t u r e  
To p r o d u c t i o n  
To p r o d u c t i o n  
TABLE 3 
SEQUENCE OF EVENTS 
SUBCULTURE 1 
CONDITION 
Confluent  
Confl uent  
505 Conf 1 uen t 
50% Conf 1 uent  
Conf 1 uent  
Conf 1 uen t 
Conf 1 uen t 
Conf 1 uent  
DISPOSITION 
To subcul ture-25  crn2 f l a s k  
To product ion 
To product ion 
Recul tu re -no  growth 
To subcu l tu r e  2-25 ca2  f l a s k  
To product ion 
To subcu l tu r e  2-25 c s2  f l a s k  
To product ion 
F r a c t i o n  
14-2 
14-2 
17-2 
17-2 
19-2 
19-2 
TABLE 4 
SEQUENCE OF EVENTS 
SUBCULTURE 2 
Cond i t ion  
Conf 1 uen t 
Conf luent  
Conf 1 uent  
Conf 1 uen t 
Conf luent  
Conf 1 uen t 
D i s p o s i t i o n  
To Product ion 
To M o b i l i t y  Detn. 
To Product ion 
To M o b i l i t y  Detn. 
To Product ion 
To M o b i l i t y  Detn. 
FRACTION 
1 1  
13 
15 
17 
B CONTROL 
D CONTROL 
R E S U L T S  
PRIMARY CULTURE 
UI< ASSAY VIABLE CELLS 
unitsjdish XI o5 
45 .07 
535 .696 
240 .I2 
225 .744 
61 ,068 
81 .288 
SUBCULTURE 1 
85 .60 
1 24 .I32 
205 .9 
359 .222 
TABLE 6 
RESULTS 
Human Granulocyte Conditioning Fac tor  
Subcul ture  1 
Frac t ion  
14-1 
16-1 
17-1 
19-1 
HGCF 
lColoni  es formed)* 
*Colonies formed co r r ec t ed  f o r  cont ro l  p l a t e  






Electrophoretic separation of human kidney cells at zero gravity

Abstract

Similar works

Full text

Available Versions

NASA Technical Reports Server