A potentially powerful technique for separating different biological cell types is based on the partitioning of these cells between the immiscible aqueous phases formed by solution of certain polymers in water. This process is gravity-limited because cells sediment rather than associate with the phase most favored on the basis of cell-phase interactions. In the present contract we have been involved in the synthesis of new polymers both to aid in understanding the partitioning process and to improve the quality of separations. The prime driving force behind the design of these polymers is to produce materials which will aid in space experiments to separate important cell types and to study the partitioning process in the absence of gravity (i.e., in an equilibrium state)

Harris, J. M.

English

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FINAL REPORT
NASAS-33978
New Polymers for Low
-Gravity Purification of Cells by Phane
Partitioning
University of Alabama in Huntsville
Dr. J. Milton Harris, Principal Investigator
September 1, 1980 - September 30, 1983
5 ABSTRACT	 A	 potentially	 powerful technique
	 for	 separating
`r different	 biological cellg^	 types is based on the partitioning of
these cells between the
	 immiscible aqueous	 phases
	 formed	 by
solution
	 of	 certain	 polymers
	
in water.	 This	 process
	 is
gravity-limited because cells
	 sediment
	 rather	 than	 associate
with	 the	 phase
	 most	 favored
	
on the	 basis	 of	 cell-phase
interactions.
	 In the present contract ve have been involved
	 in
{ the	 synthesis
	 of new polymers both to aid in understanding the
partitioning process and to improve the quality of	 separations.
The	 prime	 driving force behind the design of these polymers is
to produce materials which will
	 aid in	 space
	 experiments
	 to
separate
	 important	 cell	 types
	 and to study the partitioning
process in the absence
	 of	 gravity (i.e.,	 in	 an	 equilibrium
state).
	 Two additional tasks have been:	 ( 1) Examination of the
Ito countercurrent chromatograph ( a device on loan from NIH)
	 to
determine
	 its	 suitability	 for performing
	 automated
	 cell
separations by phase partitioning; and (2) design	 of	 apparatus
suitable
	 for	 performing
	 simple phase
	 partitioning	 space
experiments , including examination of mechanisms for separating
phases in the absence of gravity.
N o
	
-:
^
(NASA -CR- 170957) Ntit POLYMERS FJR
N84-15257
LOW-GRAVITY PURIFICATIO N OF CELLS BY PHASE
PARTITIONING final Report, 1 Sep. 1980 - 30 	 Unclas
sep. 1943 (Aldbamd Univ. , Huntsville `)
	9 i 1G3/27 18002
d.0 A02/MF A01
aw
0Background
Goals of the Present Contract
New Polymers
Crown Ethers
Hydrocarbon Ethers
Proteins
Countercurrent Chromatography
Zero-6 Apparatus
Contract-Supported Publications
Appendixes I-V
age 1
3
3
4
4
5
5
6
6
7
at
r	
- 3 -
Backaround
Modern biomedical research, whether molecular or cellular,
depends heavily upon effective techniques for separation and
purification. Separation methods for molecular biology are well
developed. However, the preparation of homogeneous,
functionally specific populations of living cells remains one of
the chief obstacles to progress in cell biology today. Phase
partitioning, a sensitive and increasingly important method for
purifying samples of biological cells, involves a relatively
simple process. The impure cell sample is added to a container
holding two immiscible liquids (phases). The container is
shaken thoroughly to disperse the cells uniformly and mix the
two phases, which are insoluble in each other. The container is
then set aside and gravity causes the two phases, which are of
different densities, to separate into a top layer and a bottom
layer. Because of its biochemical nature, each cell type has a
preference to be surrounded by one phase or the other, and thus
will tend to become concentrated in either the top or the bottom
layer. This allows separation of the cell sample into
top-preferring and bottom-preferring fractions, However, the
cells are more dense than the phase solutions, and thus tend to
sediment to the bottom of the container before reaching
equilibrium with the preferred phase. This limits the
resolution of phase partitioning, especially when the sample to
be purified contains large or dense cells.
The rationale for carrying out phase partitioning:
experiments in microgravity is threefold: (1) It will allow
theoretical examination of the complex micro-events of this
process.	 The delicate studies needed cannot be performed on 	 j
Earth because of gravity-induced movement and interaction of
phase globules during the phase separation process. (2) It will
allow partitioning of large cells and other particles that
sediment too rapidly for purifications in unit gravity. (3) It
may permit higher resolution separations of all cell
partitioning procedures, since the process can be allowed to
proceed to equilibrium only in the absence of gravity. However,
"
	
	 phase partitioning in microgravity will obviously require some
alternate method of separating the two phases from each other.
coals of the Present Contract
The goals nf the present contract as described in the
original Scope of Work were to prepare new polymers which would
provide improved separations and which would facilitate space
experiments.	 An expansion of the scope resulted from the
October 82 continuation of the contract to include: (1)
examination of the Ito countercurrent chromatograph (a device on
loan from NIH) to determine its suitability for performing
o'
I - 4 -
automated cell separations by phase partitioning; and (2) design
of apparatus suitable for performing simple phase partitioning
space experiments, including development of polymer-coated
vessels.
New Polymers (details in Appendixes I and II)
The polymer phase systems generally used in phase
partitioning are made from aqueous suiutions of the polymers
poly ethylene glycol (PEG) and dextran. The great majority of
polymers we have made have been modified PEG's, although a few
dextran derivatives have also been prepared. Our basic approach
has been to attach to the polymers ligands which have high
affinity for cells. The new polymers which we have synthesized
in this work are as follows: (1) PEG's with attached crown
ethers; (2) PEG's with attached long-chain hydrocarbons; (3)
PEG's with attached proteins (lectins in particular); and (4)
dextrans with attached long-chain hydrocarbons. A rationale for
preparing each of the polymer types follows along with a brief
summary of the work on each polymer type.
	
Crown Ethers. Crown ethers are cyclic compounds, composed	 ^e>
of varying numbers of ethylene-oxide units, whose structures
resemble crowns with oxygen atoms at the points of the crown.
They are chemically interesting because of their ability to form
complexes selectively with metal cations. This property is of
interest in the present context as it introduces the ability to
prepare PEG derivatives which will bind metal cations and
transfer them to the PEG phase of a polymer phase system. 	 Such	 -
a charged PEG phase would then be expected to interact with the
charged portions of a cell surface. Consequently it might be
expected that cell types with a larger portion of charged groups
	
on its surface would be more strongly attracted to the PEG phase
	
p
than a cell type with a lower portion of charges on its surface.
We have prepared the PEG-crowns and have found that they do
in fact produce a large interfacial potential in PEG-dextran
phase systems (details follow in Appendix I). We have not
chosen to examine the utility of these polymers for cell
purifications because some of the other polymers prepared have
proven to be very powerful and have thus received a higher
priority. An interesting spin-off of this synthetic work has
been the observation of catalytic activity for the crown
polymers and also for the PEG's with hydrocarbon "tails;" this
catalytic_ work is described in detail in Appendixes III and IV
and in general below.
Hydrocarbon Ethers. The second type of polymer prepared was
PEG with long (up to 18 carbons) hydrocarbon tails attached.
These polymers were prepared because of the known ability of
hydrocarbon tails to binr (in unknown fashion) to nonpolar
regions of the cell sur •race. These polymers proved to be very
0
(R.
I - 5 -
effective at selectively binding cells, and offer much potential
for cell separations. A major contribution of ours has been to
prepare such polymers with highly stable chemical linkages thus
making their use much more practical than would otherwise be the
case.
As noted above, the PEG's with hydrOLabons attached have
proven to be important catalysts. This work is presented in
detail in Appendix IV.
Proteins. PEG-proteins constitute our third polymer type,
and have more potential than any of our other polymers because
of the much greater selectivity for cell binding by protein
ligands. The proteins examined have been of two types: lectins
and antibodies. Lectins are plant-derived proteins with the
surprizing ability to selectively bind to subtly different
animal cell types (e.g., T and B cells can be distinquished by
the lectin wheat germ agglutinin). Lectins have found many uses
in biochemistry because of this cell-binding ability, and
consequently can be aquired commercially. They also are in
general rather durable proteins that require no unusual handling
techniques. Antibodies are produced by organisms to bind to
alien substances, and are reknowned for the great selectivity of
this binding.
The major barrier to our use of lectins and antibodies for
affinity phase partitioning was the chemical difficulty of
attaching a certain number of PEG's to a protein without
destroying the activity of the protein. What is required here
is mild, selective chemistry. We explored two routes, the first
being preparation of a cyanuric chloride-activated PEG for
reaction with the protein, and the second being reductive
amination of the protein with PEG aldehyde and sodium
cyanuborohydride. Both methods are effective, athough the
cyanuric-chloride method is preferable because it is somewhat
faster.
Once we had developed our chemical methods it became
necessary to determine how many PEG's were required to cleanly
partition a protein to the top PEG phase of a typical phase
system. This question was answered by preparing several samples
of bovine serum albumin (MW 67,000; our model protein for
development work) having differing numbers of
	 PEG-5000's
attached.	 Partitioning of these materials showed that five
PEG's were sufficient for clean partitioning.
The next step was to determine if PEG substitution had an
effect on protein activity. Previous work by Abuchowski with
enzymes indicated that there would be little loss of activity
(Appendix I). Similarly, we found that lentil lectin with 40%
substitution retained at least 80% of its ability to agglutinate
human red blood cells.
We are now at the point of testing wheat germ agglutin
d
3
O
- 6 -
attached to PEG and antihuman-RBC antibodies attached to PEG.
Initial results are quite promising. This work will be
continued under our new contract.
Countercurrent Chromatography (Appendix V)
Dr. Y. Ito of NIH has designed several versions of a device
called a countercurrent chromatograph which provides
countercurrent movement through a tube of two immiscible
liquids. We have borrGwed two of these instruments to determine
the suitability of the devices for performing cell separations
with the polymer phase systems. To perform this evaluation we
have passed model particles (sheep, human, and dog red blood
(ells: through the machine and used peak shape and computer
analysis to calculate the number of transfers (theoretical
plates) achieved in the Ito device. To summarize, we have found
ghat the device provides the equivalent of about twenty
transfers. The two basic problems we encountered and were unable
to solve were: (1) enhanced gravitational fields throw cells
against the tubing walls causing them to stick; and (2) dramatic
band spreading occurs during removal of cells from the column.
It is our opinion that the traditional countercurrent
distribution apparatus (Albertsson type) is a more effective
apparatus for cell separations with polymer phase systems. On
the other hand the Ito device is quite effective for molecular
separations with nonpolymer phase systems.
Zero-G Apparatus
Cis an initial space experiment we plan to examine
nongraviational mechanisms for phase separation. The two
simplest mechanisms, and the first we will examine, are: (1) Use
of the liquid's interfacial energies to coalesce the droplets of
each phase and thus form the required separated phases. And (2)
use of containers coated with the polymers themselves to cause
preferential wetting of the walls with one phase, thus providing
a separation.
Two things were needed. First we had to develop chemistry
to attach PEG to glass, and second we needed a container which
would provide mixing of the cells in micro-gravity.
	
The first
goal	 was achieved by reacting glass first 	 with
trimethoxvaminopropylsilane and next with PEG (Appendix I).
This procedure effectively coats glass with PEG.
Two designs for apparatus have been considered. The first
design is intended to fit in a locker and to have several
chambers, Figure 1. The set of cells would be shaken manually,
inserted in the holder, and the camera activated. Photographs
would be taken at predetermined times by using the automatic
timing capability of the available Nikon cameras. Mixing would
be provided by steel balls, which would be Wemoved from the
C4
i
- 7 -	 1
light path by attraction to a magnetic plate at the end of the
chamber. This apparatus is very similar to one used on STS-8 by
Milan Bier; our plan is to use this apparatus, and to modify it
for our needs.
The second apparatus is designed to be hand carried by an
astronaut, and consists simply of two tubes with a magnetic
plate at the bottom to remove the mixing ball. Such a simple
tube with Velcro on the back could be shaken, attached to a
wall, and photographed. The information provided would be of
great assistance in designing a more sophisticated experiment
for the first apparatus.
In order to facilitate ready visualization of the mixing
and separation of phases, polymer-dye conjugates will be used.
Commercial blue dextran is available. A colleague from West
Germany, Werner Muller, has donated samples of red and green
PEG. If further variation in color is needed, it can be
obtained by synthesis of the PEG-dye conjugates in our
laboratory.
Contract-Supported Publications
Harris, J.M. and Chenault, A.A. "Countercurrent Partitioning of
Mammalian Cells in the Toroidal Coil Chromatograph," Second
International Conference on Partitioning, Sheffield, England,
1981. 
Harris, J.M., Hundley, N.H., Shannon, T.G., and Struck, E.C.
"Substituted Polyethylene Glycols as Soluble, Recoverable
Phase-Transfer Catalysts," J. Oro. Chera., 47, 4789 (1982).
Harris, J.M., Hundley, N.H., Shannon, T.G., and Struck, E.C.
"Substituted Polyethylene Glycols as Soluble, Recoverable
Phase-Transfer Catalysts," Polymer Preprints, 23, 193 (1982).
Harris, J.M. and Hipps, J.M. "Synthesis of New Derivatives of
Polyethylene Glycol," Abstracts Southeastern Regional ACS
Meeting, 1982.
Harris, J.M. and Case, M.G. "Octadecyl Ethers of Polyethylene
Glycols," ibid.
Harris, J.M., Hundley, N.H., Shannon, T.G., and Struck, E.C.
"Polyethylene Glycols as Phase-Transfer Catalysts. Synthesis of
Derivatives," in "Crown Ethers and Phase Transfer Catalysis in
Polymer Science," Carreher, C. E. and Mathias, L., Eds. Plenum,
1983.
Harris, J.M., Chenault, A.A., Snyder, R.S., and Case, M.G. "Cell
Separations on the Countercurrent Chromatograph," J. Liquid
Chrom., in press.
Harris, J.M., Hovanes, B.A., and Case, M.G. "Synthetic Methods
for Affinity Ligand Attachment," Third International Conference
on Partitioning, Vancouver, Canada (1983).
-7a-
cvncntnn ENT D
4
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FIGURE 3. Schematic diagram of apparatus for nongravitational phase
separation.
FLUORESCENT LIGHT
DIFFUSING SCREEN
CONTROL BOX
Q
5=5 CUVETTE RACK^
I	 I
EXPERIMENT A
=EXPERIMENT B
EXPERIMJIENT C
t
f	 I
t
-e -
Harris, J.M. and Case, M.G. "High G Cell Separations on the
Countercurrent Chromatograph," Ibid.
Harris, J.M., Case, M.G., Hovanes, B.A., Van Alstine, J.M., and
Brooks, D.E. "Purification of Biomaterials by Phase-Partitioning
with PEG-Alkyl Ethers, Industrial and Engineering Chemis.trv,
Product Research and Development, In Press.
Harris, J.M., Yalpani, M., Van Alstine, J.M., Struck, E.C.,
Case, M.G., Paley, M.S. , and Brooks, D.E. "Synthesis of
Polyethylene Gl ycol Derivatives," J. Poly. Sci., Poly ,. Chem,
Ed., In press.
Harris, J.M. and Case, M.G. "Polyethylene Glycol-Ethers as
Recoverable Phase Transfer Agents in Permanganate Oxidations, J.
Org . Chem., In Press.


New polymers for low-gravity purification of cells by phase partitioning

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