Preliminary ground based experiments strongly support the validity of a proposed space experiment in the separation of biological cells by counter current distribution (CCD). It was shown that a low level applied selective field can accelerate the separation of the phases by a very large factor. An electric field therefore should be very effective in space in lieu of gravity for the phase separation step. This appears to solve a central problem in implementing CCD in space and opens the technique to many types of large cell mixtures for which it has not been applicable before due to rapid settling effects. It was determined that electrophoretic migration of the cells, as a possibly disturbing factor, will be of negligible effect. Attenuation of a light beam by scattering generated a signal indicating the course of the phase separation

Brooks, D. E.

English

NASA Technical Reports Server

General Disclaimer 
One or more of the Following Statements may affect this Document 
 
 This document has been reproduced from the best copy furnished by the 
organizational source. It is being released in the interest of making available as 
much information as possible. 
 
 This document may contain data, which exceeds the sheet parameters. It was 
furnished in this condition by the organizational source and is the best copy 
available. 
 
 This document may contain tone-on-tone or color graphs, charts and/or pictures, 
which have been reproduced in black and white. 
 
 This document is paginated as submitted by the original source. 
 
 Portions of this document are not fully legible due to the historical nature of some 
of the material. However, it is the best reproduction available from the original 
submission. 
 
 
 
 
 
 
 
Produced by the NASA Center for Aerospace Information (CASI) 
https://ntrs.nasa.gov/search.jsp?R=19780021239 2020-03-22T03:36:45+00:00Z
{
( !N A S A-4R- 150766) SPAR PROJECT: COUNTER
	 N78-29182CUR;.ENT DISTRIBUTION OF BIntO6V-ALS Final
Report, i Dec. 1976 - 31 Dec. 1977 (Oregon
Uftiv. Health Sciences Center) 12 p HC
	 Unclas	 _	 3802/t!F 801
	 CSCL 07D G3/25 28519
FINAL REPORT
on
SPAR PROJECT: COUNTER CURRENT
DISTRIBUTION OF BIOLOGICALS
December 1, 197;,-December 31, 1977
Contract No. NAS8 -32353
Principal Investigator: D.E. Brooks
Prepared for:
National Aeronautics and Space Administration
George C. Marshall Space Flight Center
Marshall Space Flight Center, Alabama 35812
By:
Department of Neurology
University of Oregon Health Sciences Center
3181 S.W. Sam Jackson Park Road
Portland, Oregon 97201
X11. M^
4 _
COUNTERCGRREN.T DISTRIBCTION
PRELIMINARY REPORT
SV'MARY
Preliminary ground-based experiments strongly support the validity of a proposed
space experiment in the separation of biological cells by counter current dis-
tribution (CCD). It is shown that a low-level applied selective field (=or ex-
ample as 5 V/cm) can accelerate the separation of the phases by a very lave
factor (for example 35-fold). An electric field therefore shouted be vat- effec-
tive in space in lieu of gravity for the phase separation step. This ap pears to
solve a central problem in implementing CCD in space and opens the technique to
many types of large cell mixtures for which it has not been applicable before
due to rapid settling effects. It is shown that electrophoretic migration of
the cells, as a possibly disturbing factor, will be of negligible effect. The
phase forming polymers used in the preliminary experiments are not, per se, com-
patible with biological cells. However, other suitable polymers are already
under development, and are predicted to give similar rapid phase separation.
The present experiments were implemented with a prototype chamber similar to
that proposed for space. Attenuation of a light beam by scattering generated
a signal indicating the course of the phase separation. The highly prcmd sing
results-of these experiments now appear clearly to justify a SPAR expe:iWent as
a next logical phase of investigation.
tINTROXTCTIO'X
Following the submission of our proposal "Counter Current Distributica of Bio-
logicals" in response to A.O. No. OA-76-02 of the Space Processing Rocket Ex-
periment Project, a contract (NAS8-32353) was awarded to the University of
Oregon Health Sciences Center to allow further definition and deve lop-:e^.t of
the flight experiment. The Statement of Work in that contract included the
following tasks:
1. Verify that there is an aqueous polymer system that is significantly
separated by an applied electric field. Within the limits imposed
by 1 g conditions, analyze field-induced phase separation and the
influence the field and separating phases have on the biological
cells of interest. Design ground experiments to assess experiment
operation in space.
2. Do a detailed analysis of the equipment proposed for the SPAR to show
that the proposed components can accomplish the planned objectives.
In particular, analyze (a) the removal of bubbles, and (b) optical
devices proposed or otherwise available to measure the progression
of phase separation.
3. Supply general operating parameters for an electric field-driven CCD
apparatus as determined by 1 g experiments.
The principal results obtained during this study are summarized below. They
verify that the approach we have proposed is a valid and promising one and that
a SPAR flight experiment is indeed an appropriate next phase of experimentation.
ELECTRIC FIELD-DRIVEN PHASE SEPARATION
The central problem to be solved if phase-separated aqueous polymer systems are
to be used as cell partition media in space is that of finding a ethod for
5	 -1-
separating the emulsified phases in the effective absence of gravity-driven
settling. We proposed to use an applied electric field as the drivic; -force
since droplets of one phase suspended in the other are known to exhibit electro-
phoretic mobilities which increased linearly with drop radius. 	 b deconstrate
that field-driven separation can in fact occur, even in the presence of gravity,
phase systems with very high droplet electrophoretic mobilities had to be de-
veloped. Suitable systems were found consisting of Sodium Dextran Sulfate
(NaDS, Mw--500,000) and Pluronic O P-104 (a block co-polymer of poly (ethylene
glycol) and poly (propylene glycol) in a weight ratio of 2:3, M W - 5,400) with
potassium citrate as the supporting electrolyte. Two phase compositions were
utilized: 8% w/w NaDS, 8% w/w P-104, 0.22H K3 citrate (8/8/.2) ; and 5: •./•,:
NaDS, 10% w/w P-104. O AM K3 citrate (5/10/.1). Typical droplet mobilities for
these systems were as shown in the table below for 6.5u diameter drops.
Mobility (cm2iv-5)
Bottom Phase Drops	 Top Phase Drops
System	 in Top Phase	 in Bottom Phase
5/10/.1	 +8.8x10-4	 -8.4x10-4
8/8/.2	 +17.6x10-4	 -15.3x10-4
In these systems the top phase is rich in P-104 and the bottom is predominantly
NaDS. To drive phase separation in coincidence with gravity, then, t=e electric
field is applied with the anode at the top and the cathode at the bot:or of the
sample chamber.
A prototype phase separation chamber was constructed similar to that described
in Section 4.2.1 of our original proposal. A cross-sectional vier of the chamber
is shown in Figure 1. The stirring screen was omitted, since mixed pease systems
could be introduced directly into the chamber.
The apparatus consists of two electrode chambers containing brig hat
 Pt electrodes
separated from a phase chamber (5em L x 0.5 em W x 0.2 cm deep) by twc a- ►i:on
.CM-100 membranes. The phase chamber is split in half horizontally so that the
two halves can be displaced laterally relative to one another, al_owi-g is_la-
tion of the contents of each half. Feeder ports give access to tae ca er for
-2-
U
O
m
H
'S
C..
a
a,a
c
0
a
u
a
.ry
s7XU
O
Z
:J
J
1-^
1
JU
QL
N_
v
Fy
N
Q Q C.
O e^7 4J
r.w ^.. I,: d
tj
F- ...1 
0 <
to C. Cn 1
W
Co
Lu
Qxc.^
J O
^ d m
-3-
filling and drainage in either the separated or contiguous configuration.
Electrode rinse buffer is circulated through the upper and lcwer electrode
chambers to remove electrode reaction products. The chamber and electrode
assembly is made of poly (methyl methacrylate) lapped and polished, to provide
a good sliding seal between the upper and lower halves. The optical system
used to follow phase separation turbidimetrically consists of a s^all rubf
laser whose beam diameter is limited to — 0.03 cm by'an entrance aperture.
P
	 The beam traverses the width of the phase chamber at a vertical position
which can be adjusted relative to the midline split. The beam irtensity is
measured with a solid state detector and amplifier after traversing t::e
chamber and an 0.03 cm diameter exit aperture. The beam and apertures are
aligned with water in the phase chamber so that when a turbid mixed prase
system is present only non-scattered light is detected.
A typical trace of detector voltage as a function of time with no field applied
is shown in Figure 2 for the 8/8/.2 system made up on a phase volume ratio of
9 parts top phase:l part bottom phase. The beam samples all of the bcttom
phase and a portion of the top phase near the interface. At this phase volume
ratio, the 1 g settling time is prolonged and the kinetics are easy to follow.
The signal initially increases linearly in the early stages of phase coalescence,
then fluctuates more seve-ely as larger droplets form and the system separates
supralinearly with time. At this amplifier gain setting for this run, full
scale deflection of 100 mV corresponds to an essentially separated system. The
degree of separation sufficient for all partition work corresponds to ,cwething
less than this level (-50 mV) since residual drops smaller than cell dimensions
have little effect on cell partition behavior yet still scatter a considerable
amount of light.
The effect of applying an electric field of 5.1 v/cm to this system is saoun, in
Figure 3. An immediate increase in the rate of separation is seen. The siape
increases by a factor of about 35 and the intensity increases to an output of
20 mV (an arbitrary but convenient measure of separation ti=me) in 1.1 min. as
opposed to 10.5 min. in Figure 2.
-4-
- 0 ______ ..... 
I! 
t : 
- -.~ ~--'----.,~.- .~ .. -- -. i ~ 
,:: ~\. ~ _:_~ _: _·-.:.:-=-=L~_~~~~ j,~ 
~~ .. -,--~-t:--~--;· ...• i·:~--! -._~f:j 
I I f j i I 
-. ~ f --- ; ---i~-· -~ - . . .. ~; 
~ •. _--~.- -.-~------ .--_ .. _--- ,&~. 
, , 
- -- ~ --L_ .... __ . ______ ! ~ 
: . .J 
! ! 
!. ., ___ ~_._il ... _+ -.. L 
I • 
, L_ . ; .. _. 
p; 
i j : i I' 
.. -:--1-~ -- -+-! --+:....;.;--;---
i I I . I' i ~ ri .. ·-t--i---1-·-i--r-1-·'~ 
~~.-,.-. -.. -. ~-'--'-t-'-~--~t-,~-. ~-~-~;-: .-_---~;-l---'.·-_-:j-.. - .. -:-~.-~-·--·-"'i:~-.-,---~;---.. """' H 
.rr; . I ;' .-
~ 
r--' 
L,-, 
,I 'j 
11 I ,i.. 
E;==:~-:~~-:-~;-~f~F~~·~_] 
4-1----:.~··--.··--t_l-_i.·-·-l_.;, --1-
~" :·;--i--i .. j .-1._ .. , ~- -.; ··-H
I # i' I ' I I . ~.--.~ - ---;---I-j--4 i I 
L. _ : .. 1.---1 __ 1- -. I,-~-L--+ •. -.-L. I : I Ii' ~--~~.;--=t-.l... ,-'-l--.c..t-' 1-4-' _! _i~,---
I : i. I 'i • " I .' j !. 
-5-
I 
~ 
I. 
~ 
~ 
I 
i 
i 
f 
I 
Fig::::.::: J. System 8/8/.2. Ch.-u't. Speed .:lnd mV S.:"'l.~ as in Fig:.::;! 2_ 
Showing FielJ T!'.1!1sitior: from E=i) to !::=./.S :','..:-.-::. 
-6-
Figure 4 gives another example of the field effect at ten times higher gain.
As the field is increased from 0 to 2.8 v/cm, the separation ra_e increases
until the field is turned off, at which point the rate slows. R_-application
of 2.4 v/cm, then 3.4 v/cm rapidly clears the system.
Effects such as these were repeatedly seen in both the 8/8/.2 and 3/10/.1
systems, although the former always exhibited larger field effec_s (initial
slopes 3 to 30 times greater) than the system with the lower droplet ;nobility.
The effects could not have been due to unstable convection in the chamber b'nce
the Rayleigh number for these experiments was between 1 and 10 and the maximum
temperature rise in the sample was less than 3°C. Moreover, the effect depended
on the direction of the electric field; reversing its polarity induced mixing
in a partially separated system, reducing the signal to zero. These results,
then, are consistent with the presence of field-driven phase separation in these
systems and represent, we believe, the first demonstration of this effect.
FIELD EFFECTS ON CELL SEPARATIONS
Since biological cells are known to exhibit characteristic electrophoresic
mobilities, the possibility should be considered that an electric field applied
to drive the phase separation could affect the cell partitioning process. Cell
electrophoresis might, for instance, pull cells out of the interface and thus
change their position in the phase system. We have as yet been unable to test
this possibility directly since the NaDS/P-104/E3 citrate system is highly non-
physiological and damaging to cells. However, it seems likely that such effects
will be absent or insignificant for the following reason: All biological cells
under physiological conditions bear a negative surface charge and hence, will
migrate upward in our apparatus.
In virtually all phase systems used in cell separation work, partition occurs
between the top phase and the interface with no cells remaining in the bottom
phase. With the electric field applied, then, cells which have partitioned into
the top phase will only tend to move closer to the top electrode. Cells adsorbed
in the interface will also tend to move in this direction, but the interfacial
tension is sufficient to held the cells in place. We have examined this point
- i-
k
rte_ Lam— _	 ± OR IN
rz
---^-- -_
	
7 -::
---	 ,—^
E=1.83 Vilcm
-^ ..— - i ._..._, —_.^_ 
+	
_	 —• -- .. _	 - ..	 E-2.8 V/ cm —^—..—	 =1; c, y
_ —	
!	 y
	
_	 _	
s^	
S, y
Qj
c 44
fA
Sk
Ci
bv observinc the electro phoresis of ph;:se drops to which cells art ac_orbed.
At field sc~engths egvzl to those used here, the cells remains-' 	 t,e inter-
face as th:- droplets mov24 at velocities one to two orders of :^a °.iCurz -reater
than those at which the cells along would ,.ova in free suspension.. T.a latter
:velocities are very small compared to droplet velocities because o t*-e rela-
tively high phase viscoss,ies aid low cell surface potential. T'a istarfacial
tension (which is under experimental control) is apparently stror.S en_u;h to
maintain the relative po_ition of the cell and interface under condit'.ons
similar to those present during field-driven phase spearation.
F1 7T D-DPI'EN PHASE SEPA3ATION TIME
At the phase volume ratios used in the present experiments, the NaDS/P-104/
citrate system separated in 1 to 2 min. at field strengths of 5 vjen or less.
Although we did not investigate the dependence of separation rate on phase
volu•se ratio specifically, it was clear that systems having more ,early a 1:1
top:bottom ratio separated considerably faster, as would be expected. We could
not operate at a 1:1 ratio with the NaDS/P-104 system because of a field-depen-
dent instability associated with high contents of NaDS-rich botto= phase, which
caused mixing. Presumably the instability is caused by the opposite mobilities
of droplets and molecules of NaDS, a negatively charged polyelectrolyte. ::ole-
cules of NaDS in the separated bottom phase will move toward the anode while
bottom phase droplets, which have a positive surface potential, -.'grate ca_hodi-
cally. No mixing was observed at smaller bottom-phase volume fractions, hc:ever.
It is pot anticipated that the observed instability will occur ': •hen both polymers
are neutral which will be the case in the cell partition systems to be used in
the flight experiments. Even if similar mixing is observed, it will still be
possible to eliminate it by varying the phase volume ratio since caly one sulk
phase and the interface are actually occupied by cells after partition. occurs.
Since we could not operate the demonstration system with equal top an= bottom
phase volumes, it was not passible to correlate directly the drog_2t -cbi1:_-as
with separation rates for systems corresponding to those ro be us--j in the flig-.t
experiments. We can,.therefore, not directly calculate the separation times to
z e::pe:'C2d in a Zero g e%periment. However, ail est mate may be -.adz ;s fZ-11c%s:
-9-
Khen the phases separate after sha•ring, the interface z y r.:s faiziy rapidl, as
the majority of the droplets coalesce into bulk phases. A haze of residual
droplets is 12ft in each phase thi gh then settles and com?letes the separation.
If these resilu al droplets are large enough (greater than cell dimen,ions),
they can contain or adsorb cells and contribute to the final cell distribution.
If they are about the size of or smaller than tba cells beir .3 partitianed, they
will not contribute and need not be allowea • o settle before the cell distribu-
tion is deter-ined or a transfer made in a countercurrent distribution experi-
ment. An estimate of the effective separation time may then be obtained from
the time required for droplets sore^,hat larger than cells to migrate thro•.:gh
half the depth of the chamber. For gravitational settling, the settl _ng time,
t5ep , will therefore be given approximately by:
t	 -	 d	 d	 r 271m +sep
	 2vsed	 4 'ap ' g ' r2 l Tlm +'7d
where d - depth of chamber - 0.2 cm
dp - density difference between phases - 4 x 10-2g/cm3
g - 980 cm/s2
r - drop radius
77m - medium viscosity - 4 x 10-2p
7Id - droplet viscosity - 0.3p
For r - 10;: (about 2x cell radius) this expression gives t 5ep -
 
7.5 m=a., which
corresponds closely to the observed time of about 5 min. in the CCD a;paratus.
For field-driven separation, the appropriate velocity to use in (1) is the
electrophoretic velocity v - uE, where u is the droplet mobility and E the
applied electric field. Phase systems with high mobilities useful in cell
partition work are currently under deve lopment but a conservative estimate
for the mobility of such systems is u - 0.5 rs
-1 . The corresponding separation
tine in a 5 v/cn field for r - 10:: is t Sep - d/2uE - 33 s. Accor-jingly, pease
separation should occur very rapidly in the flight experiment. T-e separation
period should therefore be readily compatible with the duration o' redaced
;cavity available on a SPAR flight.
EQL'IP'`;T PERFOP4CE
It is clear from the results presented above that the apparatus performs sat.s.
factorily. With a phase system in the sample cha:aber and an electrode rinse
flow of 400 to 600 ml/nin., a curr-.!nt of 0.5A-*5% (corresponding to an electric
field of abcut 13 vlcm in the sample chamber) can be maintained continuously
for at least 15 minutes. There is occasional difficulty associated with foam
in the electrode rinse after the buffer has re-cycled szireral times. The P-104
is quite surface active, and the small  mount that leaches through the membranes
can form stable foams during electrolysis that are sonewhat difficult to remove.
This problem► should not occur in flight experiments, however, where poly (ethy-
lene glycol), which has little surface activity, will replace the P-104.
The optical system employed also behaves satisfactorily. The read-out of detector
voltage versus time provides an ecellent record of the progression of phase separa-
tion, and it should prove feasible to follow the kinetics of separation even in
the rresence of high concentrations of cells.
CO` C:	 7y
We believe that the .results cited above provide excellent evidence that the ap-
proach described in Beckman Proposal CS76-328, "Countercurrent Distribution of
Biologicals", is valid. It would appear appropriate as a next step to test the
cell separation under micro-gravity conditions. The. experimental protocol we
have outlined fits the requirements of a SPAR flight experiment particularly
well. I We feel that such an experiment is justified by the potential importance
of this cell separation technique and the highly promising results now achieved
in our areliminary experiments.


SPAR project: Counter current distribution of biologicals

Abstract

Similar works

Full text

Available Versions

NASA Technical Reports Server