Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
W o 93t22427 PCT/US93/03843 - :
METHOO OF CULTURING VIABLE CELLS AND METHOD OF REGULATING THE LEVEL OF A
COMPOUND IN A BODY FLUID.
sAcKGRoUND OF THE INVENTIO~
Field of the Invention
This invention relates, in general, to culturing
viable cells i~ the presence of a non-porGus, semi-
permeabl~, biocompatible film formed from a copolymer
of specific ~ensile ~trength, ultimate elongation and
water absorption characteristics which ca~ permeate
molecules of up to 6,000 - 600,000 molecular weight
while being impermeable to ce~ls and par~iculate
matter. ~n the method of the invention the nu~rients
and the medium from wi~hout are permeated through the
film in~o the cells' environment, and any cell ;
products from within are permeated through the film
away from the cells' environment.
,
Description ~f the Backqround
Standard immune ~uppres~ion poses acute risks
for, e.g., a diabetic patient such as nephrotoxicity
~rom the u~e of cyclosporin, and is ex~remely
difficult to justify in the c~qe of, e.g., young
diabetic patients.
Four methods of immunoloyic~l isolation used up
: ~ 25 to the present time are as follows:
~1). Extra~ascular dif fu8ion cham~ers.
~2)~ Intra~as~ular dIf~lsi~n chambers.
(3). Intra~ascular ultrafiltration chambers.
. (4). Microencaps~lation.
~11 these approaches ha~e detrimental featur~s
that are summarized~below.
'
,.
~a ~C:TITI~ 3~ r
W~93/22427 213'~ PCT/U~93/~384~ '
: "
,
I. They produce hos~ fibrotic response to the .
implant and material~s instability, e.g., in alginate
rnicroencap.sulation.
II. There are limitations to the diffusion of
nutrients across semi-permeable membranes of the
prior art with decreasing permeability as protein
- deposition, blood clotting or fibrous ingrowth block
the passage of nutrients through the pores of the
membrane.
III. A lag ~ime is observed in the permeability
and diffusion of gluco~e and insulin acro~s prior art
sëmi-permeable membrane barriers, resulting in a .
delay of a rea~tion by the cells to the host's
gluco~e levels in blood.
Membranes used in prior art implants and
methods, wi~h the possible exception of
microencapsulation with friable gels, have employed
microporous semi~permeable membranes. Such membranes
: have been fabricated from impermea~le polymers with
20~ pores being introduced into the material through :~
~; ~ processing condi:tions and/or leachable additives.
: ~ MILLIPORE~ and~NUCLEOPORE~, or polycarbonate
microporous~membranes~utillzed by the prior art are
made from inherently~impermeable polymers and do nQt
: 25 s~pport long-term~cell viability. Other semi- .-
~: ~ permeable memb~ane~s~demonstrate poor blood
~: compatibil~ty as:well~ as low permeability proficiency
for ~he transport~of g~uco~e and in~ulin across the
membranes. It~hould~be noted that a microporous
! ~membrane :may have acceptable high permeant flux!i.n a
pressure-dri~en~process such as ultrafiltration, but, ~:
: at the same time:,~ have very low:permeability in a ~:
concentrati~on-;driven process such as the in vivo
:~ method of this:in~ention. When the microporous
: 35 membranes are placed in direct contact with a body
fluid ~uch as~blood,~they accumulate a fibrin layer :~
~; ,:-~: ~.. :
SUB~ITUTE 9HE~
W093/22427 21 3 lL O ~ ~', PCT/US~3/03843
--3--
which becomes a major barrier to mass transpor~ation
through the membrane.
In general, polyetherurethane block or segmented
copolymers exhibit good biocompatibility along with
high strength and elastomeric properties. This
unique combination of properties is due in part to
the two-phase morphology of ~he polyurethane
molecule. In a typical polyurethane, aggregated
aromatic or aliphatic urethane or urea segments
constitute a hard glas~y or semicry~talline phase,
whi-e low glass tranRi~ion ~temperature (T~
oi~gomeric se~ments comprise the liquid-like, rubbery
soft phase or segment. The morphology of a
polyurethane depends on many factors, including hard
and so~ segment chemi try, segment polarity
differences, hard segment content, and hard and soft
segmen~ molecular weights.~
In both polyurethaneureas and polyurethanes, the
chemi~try of the~ ~oft Yegment affects the degree of
phase separation in the polymer, which in turn
affects its bulk and surface properties and
u~equent biocompatibility. Polyurethaneureas,
simi~ar~to the ones disclosed in this patent only as
to their hard~cegment co~positions, have been shown
~ to be resiskant to degrada~ion in several
applications ~Paynter, et al., "The Hydrolytic
Stability of Mitrathané; a Polyurethaneurea - An
:
X-ray Photoelectron Spectroscopy Study", J. Biomed.
Mater. Res.;22:687-698 (1988); Szycher, et al.,
3Q` I "Blood Compatible~Polyurethane Elastomers", J.
Biomater. ~ppl. 2:290-313 (1987)).
The application o~ natural and synthetic pol~mer
membranes to the~separation of gaseous and li~uid
mixtures of low molecular weight has been reported in
a number of reviews. Many studies of membrane
; permeability to slmple low molecular weight (MW)
SUBSTI~tJlTE 5HEE~
W0~3/22427 ~ 1 3 ~ 8 PCT/USg3Jo384~
-4-
permeants have been reported in which the composition
of glassy-rubbery or crystalline-rubbery copolymers
are varied. A polyurethane multipolymer membrane
different from the one disclosed herewith has been
shown to be water and salt permeable. In
thermoplastic segmented block copol~ner~ where one
block or segment is glassy or crystalline (hard
seg~ent) and another is rubbery or liquid-like (soft
segment), the perme~tion of molecules occurs
primarily through ~he sof~ se~ment. The rela~ively
impermeable hard segment, provides phy~ical integrity
to the polymer by virtue o~ its strong intermolecular
interaction~ with like se~ments on adjacent
molecules, ~ven under condition~ which may cause
swelling of the soft segment.
Okkema, et al., discloses a series of polyether
polyurethanes based on polyethylene oxide (PEO),
polytetramethylene oxide (PTMO) and mixed PEO/PTMO
soft segme~ts suitable as blood co~tacting surfaces, --
bu~ with a hard segment content of 55 w~, too high
to be useful in the pr~ent in~ention. (Okkema et
al., "Bulk, Surface, and Blood-Contacting Proper~ie~ ~
of Polyurethanes Modi ied with Polyethylene Oxide", ~.;
J. Biomater~ Sci. Polymer. Edn.1~ 43-62 (1989)).
Takahara, et al., disclo~es the preparation of
Segmented Poly (etherurethaneureas) (SP W) with
hydrophilic and hydrophobic polyether components. ~,
(Takahara et al., "8urface Molecular Mobility and
Platelet Reactivity of (SP W S) with Hydrophilic and
30 ! Hydrophobic Soft Segment ~omp~nents", J. Biomater.
Sci. Polymer. Edn. 1(1l:17-29 (198g)). Platelet
adhesion and dynamic contact angle measured after
a~sorption o~ bovine serum albumin revealed that ~he
SP W s with hydrophilic soft segments had a
; 35 non-adhes iYe surface.
~SUB5T3TUTE 9 HEI~
W0~)3/22427 PCT/US93/03843
Chen, et al., examines the relationship between
structure and properties of polyether based
polyurethanes. (Chen et al., "Synthesis,
Characterization and Permeation Properties of
Polyether Based Polyurethanes", J. Appl. Polym. Sci.
16: 2105-2~1~ (1972)). Of particular interest is the
testing of the transport of water and low molecular
weight salt through polymeric membranes made of
elastomers that are block copolymers consisting of
hard and soft ~egments, with the former acting as
physical crosslin~s.
- U.S. Patent 3,804,786 to Sekmakas disrloses
water-dispersible cationic resins, paxticularly
polyurethane resins prepared by reaction of a
resinous polyepoxide wlth a polyisocyana~e to provide
an hydroxy-functional polyurethane with ter~ia~y
amine functionality. These resins are useful for
elec~rode po~ition at the cathode.
U.S. Patent 3,~26,768 to~Suzuki and Osonol ;
discloses a process for preparing polyure~hane
compositions by dispersion of polyurethane-containing
iqocyanates made from polyols and organic isocyanates ~;
in water under specified conditions. ~:
U.S. Patent:3,852,090 to heonard et al., ~-
discloses the utilization of a urethane film for
waterprooflng a breathable textil;e subs~rate. .
U.S. Patent 4,124,572~to Mao relates to
thermoplastic pol~urethanes prepared by a specified
method. The thus produced elastomers are useful for
l automoti~e products, applications such as cattle ear
tags, coatings and coated abrics. `"
U.S. Patent 4,183~836 to Wolfe, Jr. discloses a
water-~based polyurethane dispersion and its
preparation by reacting an aliphatic diisocyanate
with three crltical active hydrogen compounds to form ;.
:~ a pre-polymer containing carboxyl and fre~ isocyana~e
- .
. .
8~JB$TITUTE~ SHEET
WOg3J2~427 PCT/US93/0384~ ~
21 3-i~88
groups, and then dispersing the pre-polymer in an
aqueous medium with a ~ertiary amine and a diamine.
These dispersions are useful in coating applications
such as textile materials.
U.S. Patent 4,190,566 to Noll et al., relates to
non-ionic, water dispersible polyurethanes with
substantially linear molecular structure and lateral
polyalkylene oxide polye~her chains containing
ethylene oxide units of speci~ied content~ :~
.U.S. Patent 4,~02,880 to Fildes et al.,
discloses sustained release deli~ery means comprising
a~~~iologically active agent, i.e., a drug, a linear
hydrophilic block polyoxyalkylene-polyurethane
copolymer, and optionally a buffex. A single
hydrophilic soft segment is used. Only the hard
segment is ~ydrophobic.
U.S. Patent 4,202,g57 to Bunk, et al., discloses
polyurethane polyether-ba~ed elastomers which are ~.
thexmoplastic and recyclable, and have increa~ed high
temperature resis~ance tha~ makes them suitable for
injection molding.
U.S. Paten~ 4,224,432 to Pechhold et al.,
discloses a polyurethane comprising a reaction
product of a~polymerizate~of tetrahydrofuran and an
alkylene oxidé, an~organic polyisoyanate and a chain
extender which~is an~aliphatic polyol or a polyamine.
U.S. Patent 4,367,327 to Holker et al., relates
to a breathable~pol ~ rethane film for coating fabrics ;~
to make them~wat~rprvo. The polyurethane film
1 ~comprises in stoichiometric amounts ~ hard segment
made of a low;molecular weight diisocyanate with a
~ifunc~ional co~pou~d, and a soft segment comprising
~; ~ polye~hylene glycol. The~mechanical properties of
-the film are impro~ed~by crosslinking with a
triisocyanate.
SUBSTIT~U:TE Sl~
2~ O~
W~93/22427 PCT/US93/03X43
U.S. Patent 4,849,458 to Reed et al., discloses
a hydrophilic, segmented polyether polyure~hane-urea
exhibiting increased tensile strength and elongation
when wet with water. The polymers form clear films
that are permeable to water vapor.
Many of the afore~mentioned materials are
~egmented polyurethane elastomers. Sorne of them,
moreover, ha~e found biomedical applications
virtually without being modified. However, despite
their widespread use, many biomaterials were
originally developed for nonmedical uses. In fact,
~ most polyurethane materials were developed to satisfy
high volume, industrial needs. A most notable
example is DuPont's LYCRA SP~NDEX~, a polyurethane
utiliz~d in the fabrication o circulatory support -
device components. This ma~erial was later sold
under the trade name BIVMER0 Se~mented Polyurethane.
~VCOTHANE-51~ resu1ted ~rom the combination of
two commercially available polymers, a silico~e and a
polyurethane, both of which are widely used as fabric
:
coatings. ~VCQTH~NE-51~ is utilized in biomedicial `'`
de~icea such as~an intra-aortic balloon~ The sole
improvements introduced for its biomedical
applications were;the us~e of highly purified s~iarting
materiials, the ~flltration of the product solution and
clean conditions~for the fabrication of
bIood-contacting surfaces. ~not~-er biomedical
polyurethane,~ AVCOTHANE-610~, also called
CARDIOMAT-610~ and ANGIOFLEX~ are presently being
30~ i ~ used i~ blood pumps and trileaflet heart valves.
The thermoplastic material PELLETHANE~ was ~irst
~` ~ applied~to ~he manuacture~o cannulae for blood
vessels, and later of catheters. This material hiad :~
originally been developed as a~ extrusion molding
resin exhibiting superior hydrolytic stability over
~ their polyester-based~counterparts.
.
.
SUE3:8TITUTE SHE~
W093/22427 2 1 3 4 ~ ~ ~ PCT/USg3/03~ ; `
--8--
Although many polyurethanes and ~-
polyurethaneureas are available commercially, some of
which were discussed above, none forms membranes of
permeability/ strength, flexibility, and
S biocompatiblity required for growing cells by
permitting the passage of nutrients, cell products
and cell waste materials while preventing the passage
of immunological or microbiological substances that
migh~ be detrimental to cell growth and the
manufacture of cell products.
A more complete appreciation of the invention.
- and many of the attendant ad~antages thereof will be
readily percei~ed as the same becomes better
understood by reference to the following detailed
description when considered in connection with the
accompanying figures.
Other objects, advantages and features of the
present invèntion will become apparent to those ;~
skilled in the art from the following discussion.
SUMMARY nF TH I NVENTION
This in~ention relates to a method of culturiny
live viable cells, that compri~es:
: culturing live viable cells under conditions
25 ~ effective to~grow the cells:in the presence of a non~
: porous, semi-permeable, biocompatible film formed
:from~ a copolymer comprising about S to 45 wt% of at
: :: least one hard :segment, and about 95 to 55 wt~ o at
lea~t one soft segment comprising at leas~ one
l hydrophilic, hydrophobic or amphipathic oligomer
: sel~cted from the group:~consisting o~ aliphatic
~ pol:yols, aliphatic a~d aromatic polyamines ~nd ~:
: mixtures thereof;~the film havin~ a tensile strength
: ; :greater than about 350 psi and up to about 10,000
psi, an ultimate elonyation greater than about 300~ ;
:` : and up to about 1,500~, and a water absorption such
.
$UB9iTlT~3TE S;HEIE~
213~8
W093/2~4~7 PCT/US~3/03843
that the sum of the volume fraction of absorbed water
and the hydrophilic volume fraction of the soft
se~ment exceeds abou~ 100% and is up to about 2,000
of the dry polymer volume and exceeds about 50% and
is up to about 95~ of the wet polymer volume; the
film being permeable ~o molecules of up tu about
6 ~ ooo to 6ao r ooo molecular wei~ht and substantially
impermeable to cells and particulate matter and
wherein the nutrients and the medium's components
being permeated through the film in~o the cells'
environmen~, and any cell products being permeated
- t~rough the film outside of the cells' environment.
This inven~ion also relates to a method of
regulating the level of a compound in a body fluid of
a subjçct afflicted by an endogenous defect resulting
in abnormal levels of ~he compound of the body fluid,
in the ub~tan~ial absence of a detrimental
immunological reaction, comprising: :
enclosing cells lacking ~he e~dogenous defect of
the patient's cells in a biocompatible device wherein
at leaet one portion thereof compri^~es a nQn porous,
semi-permeable, biocompatible film substantially
: enclosing the cells, the film formed from a copolymer
~ompri~ing about 5 to 45 wt% of at least one hard
segmen~, and about 95 to 55 wt~ of at least one soft
segment comprising at leas~ one hydrophilic,
hydrophobic :or:amphlpathic ollgomer selected from the
: group ~on~isting of aliphatic polyols, aliphatic and
aromatic pol~amines and mixtures thereof; the film
; 30 I havi~a tensile strength greater ~han about 350 psi
and up to about lO,OOO:psi, an ultimate elongation ::
greater than about 300% and up to about 1,500% and a
water absorption such that the sum of the volume
~ractio~ of absorbed~water and the hydrophilic volume `
fraction of the soft segment exceeds about 100~ and
: up to about 2,000~ of the dry polymer volume and
~.
8UBSTgTUlDE 5HIE~
W093/22427 2 1 3 1 ~ ~ ~ PCT/US93/0384 ! .
-10-
exceeds about 50% and up to about 95~ of the wet
pol~mer volume, and the film being permeable to
molecules of up to about 6,000 to 600,000 molecular
weight and subs~antially impermeable to cells and ~:~
particula~e ma~ter;
implanting the device comprising the cells into
a site in the subject's body where the cells are in
contact with the subject's body fluid; and
allowing the cells to survive at the ;:
implantation site where they are in direct :~
interacti~e contact with the compound and act to :~
~ rëgulate its level in the body fluid.
Also provided herein is a kit for correcting a
metabolic defe~t in a subject, comprising:
a.biocompatible implantable device, wherein at
least one portion thereof comprises a non-porous,
semi-permeable, biocompatible, implantable film
formed from a copolymer~comprising about 5 to 45 w~ :
of at lea~t one:hard segment, and about 95 to 55 wt~
0 of:a~ least~one:so~t segment compri~ing at least one
hydrophilic, hydrophobic or amphipathic oligomer
selected from the group consisting of aliphatic ~i
polyols, aliphatic and~aromatic polyamines and
mixtures thereof; the film having a tensile strength
25 ~ grea~er than about 350::~si and up to ahout lO,i~00
psi, ~an ultimate elongation greater than about 300%
: and~up to about:1~500% and a water absorption such ~j`
that the;:sum:of the volume ~raction of absorbed water ~:
and the hydrophi~lic volume fraction of the ~oft
30 ~ ; ~egme~t exceeds about ~00% and up to about 2,000~ of
~he ~ry polymer:volume and exce~d5 about 50% and up
.
~ to about 95~:o~ the wet polymer ~olume and being
.
permeable:to molecules of-up ~to about 6,000 to
~:: 600,000 moleculàr w~ight and substantially
3S impermeable~ to cells and particulate matter;
.
~: syringe; -~
SUBSTlTUfE SHE~ET'
W093/22427 2 i 3 '~ O 8 ~ PCT/US~3/03~43 ~:
needles; and
instructions for uqe of the kit to insert
preselected cells lacking the metabolic de~ect into
the device and implant the device in the body of a
subject afflicted with the metabolic defect so that
the cells may interact with the subject~s body fluids
and alleviate the symptoms associated with the
defect. :.
This in~en~ion provides a me~hod of culturing
cells in the presence of a device comprising at least
one portion o~ a film comprising a biocompatible,
- hydrophilic, segmented bloc~ polyurethane copolymer. :~
In one of the aspec~s of thi,s invention, the ln
i~o method is applied to the remediation o a ::
metabo~ic defect that produces a detrimental or
undesirable compound in a body fluid such as blood;
that when in direct interactive contact with the
cellQ is transformed into a harmles or desirable
compound and ~hen returned to the body fluid. This
is the ca~e of phenylketonuria, where a genetically- :
engineered cell may transform phenylalanine that
builds up in an infant~s blood stream int~ L-tyrosine
or phenylpyruvic acid; harmless products.
In another aspec~, the method utilizes cells
that comprise product-producing cell~. An example of
this type o~ method is that for countering~ high blood
level~ o~ glucose ln diabetic patien~s. Once insulin
producing cel~ls are implanted in accordance with this
inven~ion, and when acti~ated by increa~ed ylucose
30 l levels in blood,~the ~cells produce insulin which is ! ,~'~
pexmeated lnto the blood stream and distributed to
target cells where they aid in the incorporation of
glucose into the cells.
Still part of this in~ention is a kit for ;~
correctins a metabolic defect in a subjec~, that
comprises: .
~'~
$UBSTlTl~TE~ SHE~ ~:
W~93/22~27 PCT/US93/03~4~
~13~g
-12-
a biocompatible device wherein at least one
portion thereof comprises a non-porous, semi-
permeable, biocompatible implantable film formed from
a copolymer comprising about 5 ~o 45 wt~ of at least ::
one hard segment, and about 95 to 55 wt% of at least
one soft segment comprising at least one hydrophilic,
hydxophobic or amphipathic oligomer selected from the
group consi.sting of aliphatic polyols, aliphatic and
aromatic polyamines and mixtures thereof; the film
having a tensile strength greater than about 350 psi
and up to about 10,000 psi, an ultimate elongation
greater than about 300~ and up to about 1,500% and a
water abisorption such that the sum of the volume
fraction of absorbed water and the hydrophilic volume
fraction o~ the soft segment exceeds about lOO~;and ~
up to about 2,000~ of the dry polymer volume and ~-
exceeds about 50~ and up to about 95% of the wet
polymer volume and being permeable to molecules of up
to about 6,000 to 600,000 molecular weight and
substantially impermeable to cells and particulate
matter;
syringe;
needles; and
instructions for Ui8~ of the kit to insert
'25 preselected:cells lacking the metabolic defect into
the device and implant:of the de~ice in the body of a
subject~ afflicted with the metabolic defect so that
: : the cells may interact with the subject's body fluids
and alle~iate the symptoms associated with the
i 3 O ! defect.
In addi:tion to the above components, the kit may
also comprise cel~ culture medium, either in powdered
form, or in liquid:foxm.
With regard to all of the aforementioned
embodiments of the present invention, a further
embodiment relates~to the use of a hydrogel to
~3UB$TlTUlrE SHIEEJ
~ W~93/22427 2 ~ 3 ~1 0 ~ 8 PCT/~Sg3/03843
-13- ..
immobilize cells to insure an even distribution of
cells. The hydrogel of the present invention is
comprised of greater than about 35~ water. It is
preferably that the hydrogel be an alginate.
~:
Brief Description of the Drawinqs
Figure 1 is a graph showing the results of a
glucoqe tolerance test in mice that compares various
treatment groups. ~`
1 0 ''' ~'
DESCRIPTION OF l~E PREF~RRED EMBODIMENTS .,
~ . This invention aro~e from a desir~ of the
inventors to improve on prior art methods to treat a
variety of diseases; such as diabetes, clotting '.
~.
disorders, orga~ failure and brain dlsfunction, among .
others. ~:
Up to the present time, other technologies tha~
have attempted to res~ore the function of faulty :
: biological systems in~the human body ha~e relied on
the administration: of a missing or defective product
such a~ the administration of insulin or other ~:
; hormones by varlous methods, the ex ~i~o treatment o~ ~:
a human body fluid such as blood, and :the ..
impIantation of a device such as a catheter that i.
.,, i
remain~ connected~through the skin with an outside ex ~
,
~ivo system that eff~cts a therapeutic action on the
~ fluids. The~present~in~ention provides a novel and
: : : unobvious me~hod for:the continuous treatment of
human ~diseases~of genetic origin.
30~'1 In~.the more general method of the invention, the .
:~ method comprises: : ..
: a method of culturing live viable cells,
: comprising culturing live viable cells under
conditions effective to g~ow in the presence of a
non-porous, seml-permeable, biocompatible film formed ~-~
from a copolymer~comprising about S to 45 wt% of at
~: ;
SUE~STITUTE S~I~El'~ ``
W093/~2427 ~ 1 3 ~ 0 8 8 PCT/US93/0384
-14-
least one hard segment,and about 95 to 55 wt% of at
least one soft segment comprising at least one
hydrophilic, hydrophobic or amphipathic oligomer
selected from the group consisting of aliphatic
polyols, aliphatic and aromatic polyamines and ~
mixtures thereof; the film having a tensile strength :
greater than about 350 psi and up to about 10,000 :
p5i, an ultimate elongation greater than about 300
and up to about 1,500~, and a water ab~orption such
that the sum of the volume fraction of absorbed water
and the hydrophi~ic volume fractio~ of ~he soft
segment exceeds about 100~ and up to about 2,000~ of
the dry polymer volume and exceeds about 50~ and up
to about 95~ of the wet polymer volume and the film
being permeable to molecules of up to about 6,000 to
600,000 molecular weight and substantially
impermeable to cells and particulate mat~er; wherein
the nutrients and the medium~s components from
without are permeated through the film into th~
~ 20 cells' en~ironment, and any cell products from within
:: : are permeated back through the film outside o~ the :~
:cells' environment.
This method of culturing viable live cells
permits the passage of nutrients and ~ubstrates from ;~
:~25 the medium~outside of the ~ilm or membrane into the ~:: : : :cell area and,:~lce ~versa! any cell products are ;;
: ~ ;tran~ported out~ of the cell en~ironment into the
medium on the:o~her~:side of:the film or membrane.
Thè`characteristics of the pre~ent non-porous~ semi-
: 30 1 permeable biocompatible film and membranes permits
: : the passage o~ mole~ules ~f:varying molecular weights
while, at the same time, the characteri~tics of the
:~film remains substantially impermeable to cells and ~:
other parti~ula~e~matter. Thus, the film may be
tailored, by~varying its composition, to have a
: ~ predefined cut-o f:molecular weight above which no
:
8UBSTITU~E 5HE3
~; ~ . ., .. .. . ~
W093/22~27 ~ 1 3 1 ~ 8 ~ PC~ S93/03~43
-15- :;
molecules can be transported into the cell .
environment. Thus, the film may be custom tailored :
to preempt the passage of immunolog.ical molecules
such as c~mplement and the like which normally are :
produced by a patient upon implantation of foreign
cells.
Any type of cell that is capable o~ "curing" an
endogenous unctional defect of biochemical origin :
may be used in the practice of the present invention. i`
Substrates, proportions thereof, polymers, :
method of preparation and:forms of cu~tom-tailoring
.
th~ characteristics of the film for diferent ~ .
applica~ions are disc~osed in a co-filed, co-pending ::.
U.S. application entitled "Copolymer~ and Non-Porous,
Semi-Permeable Membrane Thereof and Its Use For
Permeating Molecules of Predetermined Molecular ~-
Weight Range" by Robert Ward and Kathleen White, :~
(At~orney Docket No. SOM 20011) the tex~ of the
portions disclo~ing such information being
incorporated herein by reference. ~:
The preferred polymers to be u ed in the method .;:~-
of the pre~ent invention may be synthesized to have a
: specific permeability to a given permeant and/or to
have a specific molecular weight cutoff, by
2~5 implementing an empirical, yet systematic approach. ~;:
: The empirical:nature of the method is mandated by the
: nature of the phenomenon o~ perme3bilîty throu~h ~ `
: dense~membranes:, the properties of specific permeants
or non-perme~nts, inc~uding: theix ~olubility
30~ ! prope~ties, molecular size and conformation. The
inventors:provide herein a systematic approach to the .
: ~ production o membrane polymers in accordance with
the present inven:tion, which may be used to tailor
membrane propertles for speclfLc applications. This
is described briefly ln~the following paragraphs.
~.' '.:
: : -
SL9B~ITUTE St-lEE~
W093/2~427 P~T/US93/0384~
213~088
-16-
The permeation of solutes through dense
polymeric membranes is determined for the most part
by the diffusivity and solubility of the permeants in
the membrane polymer. If the me~brane polymer
absorbs a significant amount of the solvent, then the
permeation of the solutes will be determined by the
diffusivity and ~olubility of the permeants in the
solvent swollen membrane polymer.
The absorption of a solvent, e.g., water, by the
membrane polymer requires that the polymer have some
ainity ~or the solvent. In addition, by
- définition, the ~olv~nt must be capable of dissolving
the solute/permean~. It follows, thus, that the
absorption of the solvent by the membrane may
lncrease the contribution of the solubility factor to
the permeability coefficient by making the
environ~ent withi~ the membrane polymer more like
the pure solvent than it was in the dry ~ate.
In general, in addition to enhancing the
solubility of the permeant in the membrane polymer, a
lbw molecular weight solven~ will of~en act as a
plasticizer for the membrane polymer. Plasticization
involves a degree of dissolution of the polymer by
the plasticizer. Furthermore, as the le~el of
~25 plasticizer/~olvent increases, the glass ~ran~ition
temperature of the~mixture will generally decrea.se.
A~decreased gla~s~transition temperature suggests
that the pla~ticizer may ~acilita~e the relative
movemen~ o macromolecular chains by inserting
30 1 them~elves ~etween adjacent chains to increase the
interm~lecular spacing there between. In addition to
the above, plasticlzer/solvents may reduce the degree
o~ possible polymer~-polymer interactions through
pecific lnteractlons between the polymer and the
plasticizer/solve~t. A reduction in the soft segment
crystallinity upon hydration, which occurs with
8lJB5TlTWTE SHIEET
~1340~
W093/22427 PCT~US93/03843
-17-
certain membrane polymers of the present invention,
is an example o~ the latter mechanism.
In the case of an isotropic polymer membrane,
significant solvent absorption/swelling will produce
a measurable increase in the physical dimensions of
the membrane, e.g., along each of the x, y and z
axes, by an amount approximately equal to the cube ;
root of the ~olume ~raction of the solvent absorbed
therein. This pro~ides direct evidence that the
polymer chains have increased intermolecular distance
in the swollen state since ~he same number of polymer
- m~cule~ are now contained in a larger tot~ volume.
This increased spacing and facilita~ed movement of
polymer chains may increase permeability by
increa~ing the diffusi~ity contribution to the
permeability coeffiient.
Thu~, the absorption of a solvent by a membrane
polymer may enhance the membranes permeability by
lncreasing both ~he diffu~ivity and the solubility of ;~
a particular permeant. One method of tailoring the
membrane of the present in~ention to ob~ain a
. .
speif ic permeability rate and/or molecular weight ~;
cutoff, is to vary the composition and morphology of
the membrane. This;will effect an enhancement of the
amoun~ of solve~t~absorbed, and of the exten~ of
solubility and~diffusivlty that results from greater
sol~ent abs~orption.
Although ;in~some~instances i~ may not always be
possi~le to make ~ exact quantitati~e predictions of
30; 1 ~ ~he permeation characteristics of the resulting
membrane, the inventors ha~e found that certain
qualitative and quant.itative relationships exist
which guide ~he process. Furthermore, the
permeability of candidate membranes may be performed
with the methods described by the inventors herein~
The structure vs. property relationshlps provided
~..
~3WBSTlTl3TE SHE~
w~3/2~427 2 l 3~la~ ~ PCT/~S93/03~' `
-18-
herein may be used to adjust the permeability
properties of the membrane through an iterative
process of synthesis, membrane casting and
permeability measurement, until ~he desired values
for the intend~d use are attained.
In the examples provided below it is assumed
that the permeant is a water-soluble macromolecule
and that the sol~ent i~ water or an aqueous fluid.
Those skilled in the art will know ~hat similar
approaches may be applied that are suited for other
sol~ent/permeant systems by modifying the soft
segment to facilitate the ab~orption of a non aqueous ~:
solvent, for example.
!
~: '
'
.
'
~3UB~;TITUTE SHI~
~VO 93t22427 21~ ~1 n y 8 PCI/US93/03843
-19~
Table ]: Membrane Polymer Structure Versus Property Relationships
:.
Variable Effee~
~ .
Increasing Soft Segment Molecular Olncreases water absorption a~
Weight constant soft segment hydrophilici~y and
constant soh segment conten~ ( f ~)
~IDcreasespelmeability rate (~tf ~)
~Incr~ases molecular weight cutoff (+~)
~Increases (dry) soft segment crystallin~ty
(~f)
~Derreases (dry) tensile modulus unless
sofe segment crystallizes (~
~lncreasesultimate tensile elongation unless
soft segmen~ crystalliz~s ~)
Increasing Sof~ Segmen~ Increases water absorption at constant
Hydrophilicity soft segment molu:ular weight and
2 Q constani soft segmen~ contene (~ + ~)
~Mayincrease soft segment crystallinity
if hydrophilic segments crystalliæ [+ ~)
Increasing~Hard Segment Content ODecreases pe~meabili~ rate (~
~Increases te~sile stre~ +)
~Increases teDsile modulus (~ + f )
~Increases wet strength (~
,
Increased H2rd Segment Domain ~Increases permeability ra~e at consta~t
Size hard sc~nent content ~+) .;.
.
Mi~ing Two or More Soft ~Increases permeability rate wh~ it
Segments decreases soft segment cTystalli~ity (~)
OCanbe usod to inc~ease solubility of
3 5 : : pera~eant in polymer (by adding groups ;:
which have an a~flnity for permzant) and .
: ~ ~ therefore increases permeability (++)
Crosslinlcing At Low Crosslink Densi~ ~1ncreases permeability if used to o~tain: ~ 40 :~ : streDgth by sign~ficantlyrcducing hard
segmen~ contentO (~)
~Candecrease permeabili~ rate and :.
moleelllar weight cutoff at higher crosslink ~.
:. ` densi~
~: 4 5
: ,
.: !~ ` ` i : I : j
~) and (-) refer to:the nanlre of ~he effect and its intensity:
` ` :: 50 }+~+~ = Strong positive efféct.
Weak ~gative effec~, etc.:
.
8lJBSTlTUTE 5HE~
.
W093/22427 PCT/VS93J03~a~ `
21 3-1088
-20-
The hard segment of the copolymer of the -
invention may preferably have a molecular weight of
ab~ut 160 to 10,000, and more preferably about 200 to
2,000. Its components also have preferred molecular
weights as shown in Table 2 below.
Table 2: Preferred Molecular Weights f~r Hard
Segment Component
Hard Seq. Com~nç~_ Most Preferred MW _Preferred MW
Aromatic Diisocyanates 150-270 100-500
Aliph~tic Diiqocyanates 150-270 100-500
C-hain Extenders 60-200 lB-500
.
Although both the hard and soft segments may be
utilized in a broad range of molecular weights,; Table
: 3 below shows typical useful molecular weight ranges
and preferred molecular weight ranges for 80me
exemplary components of the soft qegment.
'
: ~ BUBSTITUTE SHEI~
,i
WO 93t22427 PC~/US93/03~43
-21- :
Table 3: PrefelTed Molecular Weights for Soft Segment CompoIlents
So~t Se~ment Component Most Preferred MW Preferred MW
Polyethylene o~ide 1000-9,000 200-1,000,000
Polytetramethylene o~ide 1000-9000 500-50.000
Polypropylene o~ide-polyethylene :
o~ides 1000-5,000 500~50r000
Polytetramethylene o~ide-polyethylene .
o~;ides l000-2,000 500-5~,000
Amine~apped polypropylene-polyethylene
o~ides 600-6,000 200-l,000,000
Polycarbonates 300-3,000 200-50,000
Z O Amine~apped poly~etramethylene
D o~ides 500-2,000 200-50,000
Hydro~yl-alkyland amine-cap~
silicones 200-5,Q00 100-20,000
~: .
Silicone-polye~ylene o~cides 5~-5,000 200-1,000,000
Polybutadienes 500-3,00~ 200-50,000
Polyisobutylenes 1,000-5,0~ 50()-10,000 .:. i
:`' ''
The content of hard segment of the copolymer is :~
typically ahout 5 to 45 wt~, the remainder of the :~
polymex consi~ting~ of soft se~ment, which may be a :~
combination o hydrophilic, hydrophobic and
amphipathic oligomers. :~
~: In one preferred:embodiment, the copolymer
comprises about 9 to 30 wt% of thP hard se~ment, and
more preferably 10 to 28 wt% thereof. Similarly, a
typical content of the soft segment is about 91 to 70
wt~, and more preferably about 90 to 72 wt~.
However, other proportions of hard and soft segments
are:also ~uita~le for practicing this in~ention. :~
- 45 A polymer made from this compo~ition will have
the properties described in Table 4 below.
$U~5TIT~JTE 5HE~ :
W093/~2427 . PCT~US93/038~
213~1~8~
-22-
Table 4: Characteristics of Film of the Invention
Charactenstics Range
.-- ~
Tensile ~trength > about 350 and up ~o about lO,OOOpsi
Elongation at Break :> about 300 % and up to about 1,5~%
Water Absorption + ~ about 100% and up to about 2000~ dry wt
Hydroph~lic Soft Segment
> about 50% and up tO ab~ut 95% wet wt
or more preferably
Water absorptioll only > about lO0 !~o and up to about 2000% dry Wt
- > about S0~ and up to abou~ 95% wet wt
Thickness about Sto lOOmicroDs ~ .
~when u~supported)
Thickness about I to lOOmicrons
(when supponed or rein~orced)
This invention a}so providec~ a non-porous,
:~ semi-permeable, biocompatible film that compri~es the
block copolymer of the invention. In a preferred
: ~ embodiment,~the film is formed ~rom the copolymer of
this in~ention. In another preferred embodiment the
.
film is coated onto a support. ~n still another
preferred embodiment, the film is an integrated part
of:the su~strate and is made of the same or similar
~:~ polymer.
In particularly:preferred embodiments, the
non-porous;film of the i~vention is provided in the ~:
form of a-flexible~sheet and a~hollow membrane or
; fiber.~ Typically, the flexible sheet may be prepared
: as a long rollable~sheet of about lO to 15 inches
:~ 4~ I ~ width,and lito 6:~feet length. However, other
dimensions may~:also;be selected. Of particular
importance:is:the thickness of the sheet which may be
; about 5 to lOO microns, and more preferably about 19
:to 25 mi~rons when it is to be u~ed without support
or reinforcement.~
'
: ~
: : : :: .
~3UBSTIITI.JTE SHEI~
W093/22427 ~ 1 3~ 8 PCr/USg3/03843
-23-
The flexible sheet is prepared from the block
copolymer of the invention by methods known in the .
art, typically, by casting, and more preferably by :
casting on a web or release liner. As already
indicated, the composition may be coated as a filrn
onto a substrate. Where permanently supported on a
rein(forcing web, e.g., a fabric, the film or membrane ~
may be thinner, e.g., as thin as about 1 micron, ~.
whereas when used unsupported the thickness may only
be as low as about 5 to 10 microns. :~
When membranes are fabricated from the polymer
o the inve~tion by knife-over-roll casting onto a
release paper, web or liner i~ the form of dry films,
they may have an about 1 to 100 micron nominal ~:~
thickneisse3 on a continuous coating line. A ~ ~
20-foot-long continuous web coater may be utilized ~;
: having, e.g., a maximum web width of 15 inches ~:
e~uipped with two forced-air o~ens. In o~le
particular embodiment, the coater may be modi~ied for ;
0 ~ clean operation by fitting the aix ~nlet ducts with
High Efficiency Particulate Air (HEPA) filters. A ~`
nitrogen-purged co~ter box may be used to hold and
.
:: dispense filtered polymer solutions or reactive ~:
: prepolymer liquids. Howe~er, sther set-ups are also
isuitable. :~
All but:trace amounts of a casting solvent,
e.g~, dimethylformamide may be removed by coater's
hot air ovens~:fitted~with HEPA:filters After :~:
:: : membrane casting, me~brane and substrate may be :~
30 ~j furthejr dried to~reduce~esidual solvent content to
less than ~bout 100 ppm, as determined by li.quid
chroma~ography. The thickness:of the fully-dried
cast films may be~measured:by, e.g., using a spring
~ micrometer sensitive to 0.0001 inch t2.5 ~M) or
:: 35 visually by using:a microscope.
: ~ ':'',
- SUBST3TUTE S~E~
W~3/2~427 PCT/US93/03~4^
~ 13 1 ~)~8
-24- :
The membrane of thi~ invention may have any
shape resulting from a process utilizing a liquid ~;
which is subse~uen~ly con~erted to a solid during or
after fabrication, e.g., solutions, dispersions, 100
solids prepolymer liquids, polymer melts, etc.
Con~erted shapes may also be further modified using
methods such as die cutting, heat sealing, solvent or
adhesive bonding or any of a variety o~ other
commonly-us~d fabrication methods. For example, when
in the form of a hollow tube, the membrane is
generally prepared wi~h a diameter of about 0.5 to 10
- mm , and more preferably about ~ to 3 mm, and a
thicknes~ of about 1 to 100 microns, and more ::
preferably about 19 to 25 microns. The hollow
membrane may easily be prepared in long rollable
form, and be cut to a length of about 0.75 to 31 :~;
inches, and more preferably about 0~5 to 6 inches.
~ny type of c~ll that is sapable of l'curing" an
endogenous functional defec~ of biochemi al origin is
suitable for use herein. The present method may be ~:
pra~ticed with a wide range of genetically-engi~eered
or muta~ed cell~types having many different i::~
: therapeutic applications.
One example ~is the application of the present ~
:25 method to the treatment of diabetes. Another example :;
: is that of the~application of the pre~ent method for ~i~
: the treatment;of phenylke~onuria with cells that are :~.
capable~of producing an enzyme that transforms
phenylalanine into ei~thex tyrosine or phenylpyru~ic
30:l acid whi~ch~are i~nocuous to the human body.
: ~ The method of this i~ventio~ will reduce the
:incidence and severity of micro~a~cular complications
associated~with type I diabetes, and other metabolic ::
;diseases. ~ ;
, . .
.
~; :
$UBSTITUTE SI~IEI~
W093/22427 ~ 1 3 ~ ~ ~ i PCT/~93/03~43
.
-2~- ;
The present method utilizes a device that ~:
contains the pertinent cells for correcting a defect
in a subject and provides~
(1) protection to the cultured cells from
macrophages and the immune system;
(2) maintains cell viability for extended
periods;
(3) permits the free pa~sage of nutrients,
secretagogues and cell products;
(4) presents a biocompatible surface to ~he `:
body fluids and tissues;
. (5) simple surgical implantation an~, if :~
neces~ary, cell replenishment;
(6) the impleme~tation of the device utilized in
the in yivo metho~ provided herein is easily fixed in
place and easily removed, when necessary.
The present method overcome~ a major obstacle to
long-~exm survival of cell grats: immunological
rejection of the allografted or xenografted tissue.
The viable exogenous cells may be introduced in
the human body by means of a variety of de~ices. ~
Examples of such devices have been disclosed in a ::
: U.S. application entitled "Biocompatible,
Therapeutic, Implantable Device" by Robert S. Ward, ~
Robert Kuhn and Veronlca Jean Chater, (Serial No. :
07/874,342) the ~ections of the text describi~g the
vaxious devices for implanting live viable exogenous
cells into the human body being incorporated herein
: by reference.
, ~ 30 1 O~e meth~d of the invention relies on the ln
vivo implantatlon of a dev1ce containing viable cells ~`
: : c~p~ble of performing a function that is d~fective in
the human ~u~ject in which it is implanted. for
:exa~ple,: the method~of the present invention may be
practiced on a diabetic person by implanting ins~lin-
~ producing cells in ~a biocompatible, implantable
~,
~ SUBSTlTlJTE SHE~ ~
~;
wo g3/22427 2 1 ~ ~1 0 8 ~ PCT/US9310384~
-26-
device wherein at least a portion thereof comprises a
film or membrane as described herein, the device ; .
surrounding the cells and placing them in isolation
inside the human body. Thus, ~he method may be
conducted by growing the cells in isolation within
the human body, where they are placed in contact with
a human fluid such as blood in direct continuous
interaction therewith. In the case of a diabetic,
the insulin-producing cells are implanted at a site
such as a blood vessel, intestinal ca~ity or in or -~.
around an organ, among other sites, and allowed to .
in~eract with the person's blood. The protein or ;;.
hormone, such as insulin produced by the cells is :~
permeated out of the de~ice through the no~-poroous,
semi-permeable film or membrane and into the blood
stream, where~rom it can reach target cells. Any
glucose precent in blood will thein be capable of :;:
en~e~ing into the human body's cells for utilization -
and metabolism. Accordingly, the undesirable high ;~
:~ 20 blood levels of glucose suffered by diabetics go
~ down. ,,:
:~ In a preferred e~bodiment, the live viable cellsare not only capable~of producing insulin but, in
~addition,: the production:of the hormone is
regulatable in~response to the le~els of glucose in :~
~; the~ blood. However,~other cells may also be ;``:j~
utilized. :~
: The ~use of a~non-porous, self-support.ing, semi-
permeable membrane~such as the one utilized herein
30 : ! ~ maintains an en~ironmental immuno-isolation for the .i
cells while perml~tting the passage of nutrients, ;;~
secretagogues and cell product~. The present method
usea a strong, dense, water-swollen membrane or film, ~`
that is permeable to ~he body fluid components that
must be in contact with:the cells and the device, and
;~ to the products~produced by the cells. ~'~
SUBSTITUTE $HE~
213~1~88
W~93/22427 PCT/U~93/03~43
-27-
In a particular embodiment of the in vitro
method of the inven~ion, the cells comprise product-
secreting cells. The products may be hormone~ such
as thyroid, pancreatic/ and other hormones, or
recombinant proteins produced subsequent to genetic
modification.
In another preferred embodiment, the cells
comprise insulin-producing cells. In a more
preferred embodiment, the production of insulin by
the cells is regulatable by ~hanges in the level of
gluco~e in thé me~ium.
- -- Examples of cells tha~ rnay he used for the ;~
production of insulin in ~itro are unmodiied
mammalian islets of Langerhans, insulin producing
. 15 recombinant prokaryotic or eukaroytic cells and~
glucose~regulated insulin-producing eukaryotic cells
: arising from;homologous recom~ination or mutation,
: In genexal, to practice the method of the ;~
invention, the c~ may be selected from prokaryotic
~20 and eukaryotic cells.: Among these, the cells may be
~ ~ seIected from the group consisting of immoxtalized ~-
; cells, li~e~tissue cells, and primary culture cells. ~:
These:are obtained by methods ~nown in the art that
~ ~ need not be further described herein or commercially
: ~; 25 available sources. See, for example, Gazdar, A.D.,
: Chick, W.L~ Oiej: H.K. et al.," Continuous, Clonal,
In~ulin and~Somatosta~in-Secreting Cell Lines
Esta~lished From~a Transplantable Rat Islet Cell
~ Tumor. Proc.~Natl.~:Acad. Sci. USA, 77:3519-3523
!~ 30 1 ~ (I380); a~d Santerre, R.F., Cook, R.A., Crisel,
: R.M.D:.~ et ~ "Insulin Synthesls In Clonal Cell Line
of Simian Virus:~40~-;TransfQrmed Hamster Pancreatic
Beta Cells," Proc~.~Natl. Acad. Sci U5A 78:`4339-4343
(1981)~ :
.
:: : :~
$UBSTITUl E SHEET ~ -
W093/22427 PCT/US93/~38a~
2 1 3 4 0 8 ~
-28-
In a particularly preferred embodiment of the
method of the invention, the devlce utilized here ::.
substantially encloses the cells.
In a most preferred embodiment of the method of
the invention, the cel~s that are enclosed within the .
device are immobilized in a hydrogel that is
comprised of greater than about 35~ water. The :.
hydrogel maintains an e~en distribution of cells
within the device. This insures the optimal ~;
diffusion of the intracellular products out of the .~.
implanted de~ice. Su~pending the cells within a
- h-ydrogel has al~o been found to provide enhanced cell
viability.
When the method of ~he invention i5 practiced in ;
vi~o, .it further comprises implanting the deYice
comprising the cells into a subject's body; and ;'
wherein the culturing step is conducted in vivo and
in the substantial ab~ence Qf a det~imental ~.
immunological response. ;~
: 20 As already indicated, the filmlor membrane ~
utilized in the present method pre~ents the passage -~
:;: : into ~he cell en~ironment of immunological molecules ~;
that could b~e:elicited~by the oreign cell~s presence ~.
in the subject'~s~body. : .:~
: One method of the in~ention relies on the in
v~vo implantation.of::a device containing live viable
cells capable~of performing a function that is
~ ~ : de~ectivè in~the human subject in which it is .~;;
: : implanted.~ For example, the method of the present
30 1 invention may be~practiced on a diabetic person by !.
implanting insu:lin-producing cells in a
~: biocompatible, implantable device, wherein at least a ..
portion thereof~:compri~se~ a film or membrane as ~::
described herein,~ the device surrounding the cells ~;
and placing them in;isolation inside the human body. .:i.
: Thus, the method may be conducted by growing the .-.
;
SUBSTITUTI~ SHE~
W093/~2427 PCT/US~3/03X43. ~.
-29- :
cells in isolation within the human body, where they
are placed in contact with a human fluid such as
blood in direct continuous interaction therewith. In
the case of a diabetic, the insulin-producing cells ;:
are implanted at a site such a~ a blood ves~el, :~
intestinal cavity, or in or around an organ, among ~:~
other sites, and allowed to interact with the
person~ blood. The protein, or hormone, e~g.,
insulin produced by the cells is permeated out of the
de~ice through the non-porou~, semi-permeable film or
membrane and into ~he blood stream, whexefrom it can
- rëach target cells. Any glucose present in blood ~ :
will then be capable of entering into the human
body's cells for utilization and metabolism.
Accordingly, the undesirable high blood levels of
glucose suffered by diabetics go down.
; In a preferred embodiment, the li~e ~iable cells
are not onIy capable of producing in uIin but, in
: addition, the production~of the hormone i3
~ 20 regulatable in response to the le~els of glucose in
:
: : blood. This is the case of cells such as
s
reco~binant, insulin producing, mammalian cells, and
the like, and in~particular the use of retro~iral
vectors capable of expressing proteins such as
insulin.~ Howe~er, `other cells may also be utilized.
~ccording:t:o: recent estima~es, approxima~ely ;-
400,000 Americans have inæulin-dependent type I
diabetes tha~ is:~;characterized by ~eficient insulin
: production:and/or relea~e. The method of this ~:~
30 ~1 inve~ion is hel:pful in the treatme~t of diabetes by
implantati~n::of~an "artificial pancreas" comprising a
biocompatible device having ~t least one permaable `:;
area permitting the~pas~age of molecules up to a
prede~ermined molecular weight but keeping ou~ cells
and othe~ part~iculate matter, the device con~aining
in~ulin-producing~cells capable of pro~iding
. ,~
':
~3~1B5TlTlJTE~ SgtE~ ::
W093/22427 PCT/US93/03B4~
~3 34a8~ ` ~
-30-
approxlmate normal glycemia through insulln release
in response to changing glucose concentrations in the
area of the device that is in contact with the semi-
permeable film or membrane.
In one particularly preferred embodiment, the
cells implanted are the su~ject~s own defective cells ;~
and they are genetically engineered to overcome the
defect prior to implantation.
The method of the invention may be practiced by
planting the de~ice loaded with the cells in a
multiplicity of sites in ~he subject's body.
Examples of the site~ are in and around an organ, in
and around the omental pouch, intra~aginally, ;~
intradermally, subcutaneously, in~r~cavitarily,
intraperitoneally and intravascularly, amony others. -
When the method is practiced by implanting the
device subcutaneously, the implantation may be
conducted anywhere in the subject's body, depending
~0 on the size and nature of the p~oduct produced by the ,~
cells or the substance present in a body fluid that
needs to be exposed to the cells to affect their
release of the product(s). One example is in the
area adjacent to where Iymph returns to the
circulation. The administration of hormones, such as
growth hormoNe~or other prote}ns products, may be
. . .
practiced in accordance with the method of the
invention by implanting the de~ice subcutaneously,
. . .
e.g., under the~arm. Other area~ of implantation
30 ' could~be in the fat pad~underithe epidermis, in the
intestinal cavity, and the like~ ~;
One preferred embodiment is where the method of ~;
the invention is practiced by implanting a de~ice
such as a catheter ha~lng at least a portion thereof
made of the film of the invention, in a vessel, such
as an artery of a~subject.
~.
S~STI~UT~ s~
W093/22427 2 1 ~ 4 ~ ~ 8 PCT/US93/~384~ ~
Examples of cells that can be utilized, although ~:
nonregulatable, in the case of production of lnsulin
are pancreatic tumour cell lines such as hamster
insulinoma, and rat insulinoma. Cell lines such as
these may be selected and/or mutated repeatedly in
order to transform them into glucose-regulatable
insulinomas.
The presen~ method may counter a metabolic fault
in a subject in multiple ways. One example i5 that
wherein the subject's cells do not produce sufficient
amounts of a ce~tain compound, e.g., a hormone such
as insulin, or thyroxine, or clotting factors as in
treating hemophilia~ A second example is where
undesirable levels o~ m~tabolites accumulate in the
circula~ion due to the patients inability to alter
them. Another example is that where the medium
comprise~ a detrimental or unde~irable component that
: when in direct interactive contact with cells are
tran~formed into harmless or desirable components
that are returned ~o the medium. Thus, e~g., an
enzyme present:in this cell metabolizes a medium
component and renders it harmle~s. ~-
In another a~pect, ~his invention pro~ides a
method of regulating:the level of a compound in a
~25 : body fluid of a~subje~ct aflicted by an endogenous
deEect resulting ln~abnormal levels of the compound
:: in a bvdy fluid, in the substantial ab~ense of a
detrimental immunolo~ical reaction, the method ~.
~: comprising the~step of:
, ~ 30 1 : enclo~ing cells lacking the endogenous defect dæ
: ~ the patient':s cells in a device, ~herein a~ least one
,
po.rtion thereof~compxises a non-porous, semi- ::
: permeable, biocompatible film substantially enclosing :~
the cells, the film formed from a copolymer
comprislng about:5 to 45 wt~ o at lea~t one hard :~
segment,and about~95 ~o SS wt% of at Ieast one soft
, .
~UBSTIT~ SHE~
segment comprising at least one hydrophilic,
hydrophobic or amphipathic oligomer selected from the
group consisting of aliphatic polyols, aliphatic and
aromatic polyamines and mixtures thereof; ~he film
having a tensile strength greater than about 350 psi
and up to about 10,000 psi, an ultimate elongation
greater than about 300% and up to about 1,S00~ and a
water absorption such that the sum of the volume
raction of absorbed water and the hydrophilic ~olume
fraction of ~he soft segment exceeds about 100% and
up to about 2,000~ of the dry polymer volume and
exceeds about 50~ and up to about 95% of ~he wet
polymer volume, and the film being permeable to
molecules of up to about 6,000 to 600,000 molecular
weight and substantially impermeable to cells and
particulate matter î
implanting ~he device comprising the cells into
a site in ~he subjec~'s body where the cells are in
contact with the subiect's body fluid; and
allowing the cells to survive at the
implantation site where they are in direct
interactive contac~ with the compound and act to
regulate its le~el ln the body flui~.
In a most preferred embodiment of the method,
the body fluid~comprises blood and the endogenous
defect is a substantially higher than normal level of
the compound in blood. In another embodiment the
endogenous defect~is a substantially lower than
normal level of a compound in blsod. These are cases
of hormonal imbalances whether an exce~s ~f hormone
:or deficiency exists. An example where the
endogenous defect is a substantially higher than
normal level of a comp~und in blood is that where
glucose's le~els in blood increase due to the lack of
insulin. A case where the endogenous defect is a
substantially lower than normal level of a compound
W093/22427 2 1 3 I ~ ~ ~ PCT/US93/03843
in blood is that where there is a hormonal deficiency
that needs to be replenished, such as the case of
thyroxine, growth hormone, and the like.
The types of cells that are preferred for the
practice of this method are those where the
compensation of the enzyme effect of the compound
le~els being regulated i5 in~ersely regulatable by
the level of the compound present therein.
AB in the case of the in vitro method, examples
of cells suitable for practicing this invention are
immortalized cells, li~e tissue cells, and primary
culture cells. In the case of a method for treating
insulin ~eficiency cells such as unmodified mammalian
islets of Langerhans, or glucose-regulated insulin-
produc~ing eukaryotic cells arising from transgènic
techniques; homologous recombination or muta~ion can
be used. These cells are known in ~he art, may be
produced by cloning of wild type genes from the same
or different species or by, transgenic techniques.
~20 In addition, the cells may be produ~ed by mutation by
means other than cloning. Examples of these are
radiation~, mutagenesis and selection.
In one aspect of~his invention, the method is ~-
applied to a patient that is a diabatic and the cells
25 ~ ~comprise g1ucose-regu1atable, insulin-producing
cells. The c;ells may be the subject's own insulin-
defecti~e~cells, and they may be isolated from the
subject's body and then genetically engineered or
~ o~herwise mutated to produce glurose-regula~able,
I ~ 30 i insulin-producing cells and then implanting thèm in
this same ~ubje~ct.
Typica1ly,~ce11s~used i~ the case of the
dia~etic are mammalian islets o~ Langerhans which
:
~ contain glucose-regulated, insulin-producing cells.
; ~ 35 ~ Having now ~enerally described this invention,
the same will be better unders~ood by reference ~o
:
~: S LJE~S~ITLJTE SHEE~
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213'~8 :::
'.
-34-
specific examples, which are included herein for ;~:
purposes o illustration only, and are not intended ~
to be iimiting of the invention or any embodiment `::
thereof, unless so specified.
~;~
EXAMPLE5 ~
In order to serve as an implantable immunologic ::;
barrier, an appropriate membrane material should meet
a number of criteria. ;:
(1) The material must possess :~
sufficient strength~and be sufficiently ;:
resistant to biodegradation to ~unction ~:
well for a~ least one year in the body.
(2) The material must be nontoxic to
any living cells lodged within it and to
tissue surroundin~ it.
(3) The material must allow the
passage of sufficient nutrients and oxygen :;~
through the mem~ranP to support the
maintenance and/or growth of the cells
: :~ there within. ~
: (4:)~ :The membrane must allow the rapid
transport of~physiologi~c signals in both ~
: directions, e.g.:, ylucose for nourishing ~',
~ islet cells, and~cell products of interest, ;
: ~ e.g., insulin:pro~uced ~y islets~
(5) The membrane must prote~t any
: ~ cells ~odged therewithin rom the cellular
30~ l immune system, an~ optionally, prevent the
pas~age of immunoglobulins su~h as IgG, ~.
: thus ensuring that complemen~ mediated cell :~ .
lysis can not occur.
: 35 STUDIES QN TRANSPORT:OF_MOLECULES_ACROSS PTG ::
MEMBRANES ~;~
: .:,.
:: .
SUB5TITUTE S~IE~ ~:
~ .. . , . ~ .. . . ....... .. . . .
W093/22~27 2 i ~ 1 ~ 8 8 PCT/USg3/03843
-35- ~;
The experiments described below were designed to ;~
test the transport of molecules across a polyurethane
membrane according to the lnvention. The diffusion
of glucose, insulin, and serum components of varying
chemistry across a membrane was examined.
Example l: Transport of Glucose Acxoss th~ Membrane
Two methods were tested to examine the diffusion
of substances across a membrane. In the irst
me~hod, membrane sheets were cast from the copol~ner
as disclosed and claimed in co-pending, co-filed
- patent application sf Robert S. Ward and Kathleen
White (Ser. No. 07/874l336) incorporated herein by
reference. The membrarles were then placed into
two-compar~ment diffusion chamber of 5 ml volume each
to separate the two compartments. Phosphate bu~fered ;~
saline (PBS) was placed on one side and a te~t
solution containing the test compound was placed on
the other side.
A 10 mg/ml solution of glucose in PBS was
utilized for this study of glucose diffusion
experiment.
The diffusion of glucose across the membrane
occurs within a short time. Glucose rapidly crosses
the membrane and;rea`ches e~uilibrium within about 2
hours. If enough glucose can cross the membrane ~.
within ~ period of time of up to 0.5 hr. the membrane `~,
being examined~was conqidered suitable. The ~ested
me~brane permitted the passage of more than 1500 ~g
! ` glu~ose/ml in the first~half hour. This is a ~ast
transport of glucose~through a membrane.
~xam~le 2:~ Transport of Proteins Th~ouqh a Membrane
Membrane tubes instead of planar membranes were
cast as described~ n a co-pending, co-filed
SUi3g~TITilJTE~ SHEI~
W~93/22427 2 ~ 3 ~ O ~ 8 PCT/US93/038~.~
application of Robert S. Ward and Kathleen A. White
(Ser. No. 07/874,336) incorporated herein by
reference. The tube was illed with a l mg/ml
solution of test protein containing a trace amount of
the protein labeled with 125-I. After sealing both
ends of the tube with a hea~ sealing device, it was
placed into 1 ml PBS in a 1.5 ml Eppindorf centrifuge
tube. Aliquots of PBS were removed at various times
and the amoun~ of radioactivity in each sample
determined. To each sample was ~dded an equal volume
of 20% TCA~ the sample was mixed, and the precipit~te
- collected ~s a pellet by centrifugation. The
radioactivity present in the pellet was determined.
Only TC~-precpitable, and hence protein bound, ccunts
were used for calculating the rates of diffusion.
The amount of protein crossing the cross~sectional
area of a known membrane ln a given time can be
calcula~ed based on the original specific activity of
the protein placed in the tube. The permeahility
coefficie~ts, p, are shown in Table 5. As can be
~een, the permeability is not simply a function of
molecular size.
TABLE 5: Permea.bilitY of Membrane Used In In Vivo Srudies: Tubes and Insulets
~: 2 5
PE~MEANT- MOLECULAR WEIGHT (Daltons) _ P gcrnJsqcm~da~/~Jml
Glucose 180 : :~ 96S0 ~
Insulin 6000 2300 .
3 0 Glucagon 3500 400
A~giotensin 1 1400 90 ~
Aproti~n ~ ~ ~ 65no : 25 : -
Alb~im~n : 60000 10 :.
': TgG ~ 15000Q 0
3 5 :~
Ex~m~le 3 _~_in ~ TransPort Across A Mernbrane Tube
For this experiment, the me~rane tubes .:
described and used in Example 2 were filled with ;
0 . 5mg/ml in~u1in in PBS containing a trace amount of
insulin labe1ed with~ 125-I. The tubes were cut and ;;
$UB~3TITUTE SHEEl~ :
W093/22427 2 1 3 ~ ~ ~ 3 PCT/US93/03843
sealed and placed into a 1.5 ml centrifuge tube
containing 1.0 ml PBS. Samples were collec~ed and : -
analyzed as in Example 2. The amount of insulin
crossing the membrane was calculated on the basis of
its original specific activity at a rate of 2-ll x 10- :
10 moles/cm2 within the first 30 minutes, and to reach
e~uilibrium at ~bout 60-120 minutes, as evidenced by
a flattening of the insulin curve as a function of ;
time. Thus, the membrane of the invention was shown
to be suitable for the transpor~ of insulin. The
membrane also showed good strength and handling
~ characteristics.
In summary, the membrane of the in~ention showed
diffusion charact~.ristics that prove it suitable for
uses where molecules of the size of both gluco~e and
insulin mu~t be transported therethrough at a rapid
.
rate. One example is the repl~cement of insulin
levels outside of the membrane in the presence of an
: inc~ease in gluco~e levels. In this case, both
~ 20 glucose and insulin are transported across the
: membrane at reasonable:rates, and both glucose and
: insulin reach e~uilibrium across this mem~rane
between 1 and 2~hours.
~5 Ss~LL_~U~Y~99_ ~ ~
: Studies almed at e~amining the ability of cells
: t~ survi~e and:~unction:in either tubes or Insulets~
composed of~the dense membraned material were
:performed with either establi~hed cell lines or
: 3Q i isol~ted:porcine: islets. Two cell lines ha~e been ~.: obtained from ~TCC and used:~or these studîes. These
: are: RAJI cells :(a lymphoblast-like human ce~l -
established ~rom a Burkitt~lymphoma. These cells
grow in suspension culture and are absolutely ;~
35 ~ dependent upon:the~presence of serum for their --
main~enance:~and growth. These cells are routinely ::
' ':
SUB5TITUTE SHEET
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W0~3/22427 PCr/US93/03~4~ ~
213~38~
-38-
cultured in RPMI 1640 medium supplemented with 10~ -
fetal bovine ser~m (FBS)), and ~OPC-31C cells (These
are an lgG secreting mouse plasmacytoma which grow in
suspension culture. They are routinely cultured in
RPMI 1640 medium supplemented with 10% FBS). Porcine
islets were prepared by a modifi.cation of the method~ ;
of Crowther et al., (Crowther, N.J., Gotfredson,
C.F., Moody, A.J., and Grene I.C. "Porcine Islet ;
Isolation, Cellular Composition and Secretory
Respon~e". Horm. Metabol. Res. ~1: 590 - 595, (1989)
and Ricordi et al.~ (Ricordi, C. Finke, E.IL, and
Lacy, P~E. A Method for The Mass Isolation of I~lets
from the Adult Pig Pancreas". Diabetes 35: 649-653,
(1986)). Following isolation, islets are routinely
cultur d ~n_Yi~e in RPMI containing 10% horse serum
and 11.5 mM gluco e. Cell line~ were cultured at
37C while islets were cultured at room ~emperature.
All cultures were performed under 5% CO2.
In Vitro Culture Studies:
I. Toxicity Studies: ~
.
RAJI or MOPC cells (3 x 104 pex well) were placed
into each well of 6 well tissue culture plates. To
; ~ each well was added~5~ ml of RPMI 1640 medium
25;~ containing ~0~;FBS.~ In~o each well was placed either
nothing (control), 10 inches of a hollow tube
composed of HWU 22B~66 Itest)~ or a rod of USP
negati~e con~rol plastic reference s~andard which
contained an identical surface area. This latter
30 ~l ~ material is a well recognizedlnontoxic material. At
1, 5, 7, and 16 days after seeding the nur~ber of
cells in each welI were determined. A plot of nu~ber"','!~-
of ~ell~ versus days in culture was generated and
used to de~ermine th growth rate of the cells~ No
difference was seen with any of the conditions
demonstrating that HWU 22866 membrane material is not
,~:
$UBSTITIJTE SHE~
W~93t22427 ~ 1 3 4 ~ 8 8 PCT/US93/03~43
. 39
toxic to the cells in culture. Islets do not
proliferate in culture. When islets were cultured
for periods of up to 2 weeks, no decrease in either
islet number or the secretion of insulin was observed
in wells containing the membrane as compared to those
which did not. These studies clearly demonstrate
that the membrane material is not toxic either to
cell lines or to isolated islets.
II. Cell Sur~ival:
Studies were designed to look at the ability of
ceil lines and isle~s to survive and grow within
membranes when their only source of nutrients was the
media outside the membrane device. F'or the~e :~
studie6, both heat sealed tubes and "insulet" devices
were utilized.~ Cells were loaded into the devices as ..
followe. Tubes were filled with media containing ::
: cells, and the ends were then heat sealed after the ::
removal of all air. I~ experiments required shorter
lengths of tubing:, the long tube could be
; compartmentaIized into smaller segments simply by ~:
~ ~: hea~ ~ealing at the~appropriate intervals. With
; : , Insulets, two 26 gauge needles were inserted through
the t~hlck membrane~"O-~ring'1~at approximatley ~.0
angles to each other.: Using a syringe, the cell ::
:: suspension is~p1aced into the insulet. The second
needle allows~the~removal~ of all air from the devi~e.
: ~ Tube~segments~or insulets were then:placed into cell
: ~ culture chamb rs in 6 well plates and:5 ml of culture
30 `I medium was added. Culture medium was replaced
: : week~y. Th~survival and growth of the cells was :~
noted visually~these tubes are optically clear).
Whlle this method does not gi~e quantitative results,
growth of cells lS apparent. Cell number increased
markedly with time. By 6 weeks, th~ tubes were
nearly confluent with cells. Cells continued to grow
8U13~1TUTE $1tE~
W093/~2427 PCT/~S93/0384~
2l3~ass ':
-: .
-40- ,:
for as long as 4 months (the longest we have carried
these out~. At the end of this time period, the :~;
tubes contained a nearly solid mass of cells and were
observed to be ~bulging~ with the cell ma~s. In all ;~
of these studies, the only nutrients required for
growth and maintenance were supplied by diffusion ~:
through the membrane. MOPC cells syn~he~ize and .i~
secrete immunoglobins. At no time were we able to
detect immunoglobin in the out~ide culture media. ~:
This confirms our earlier diffusion studies which :::
~howed that immunoglobins do not cross the membrane :
bàrrier. A buildup in lgG conentration within the
devices could be detected. .:
Islets do not proliferate in culture. :~
Therefore, one does not see an increase in cell .,
number in islets preparation placed inside membrane
devices. Sur~ival of the islets, however, can be
continuously monitored~ These experiments
established that islets can survive for at least 6 :~
mo~ths in _n vitro culture with fresh media being
supplied only outside the tube. The external media
was as~ayed for the presence of insulin and such
as ays found t~hat: in~ulin secretion and diffusion
occu~red throughout this entire period.
: :
Example 4: Cell line ~5udies
Mernbrane ;tubes 10 inches in length were prepared
,,
as described in Example l, autocIaved, and filled
with 7 x 108 Ra~i or MOPC cells. the membranes were ;;
30 1 then heat scaIed at l inch i~tervals. Five one inch ~:
~: : segments were implanted intraperitoneally into ei~her
Swiss-We~s~er or Nude mice. At 2, 4, and 12 weeks,
three mice were;~sacrificed, their implants removed,
:and the viability of the cells within the membrane
3S determined~ Upon:explanation/ the membranes appeared
intact. No alterations in structure could be -~
.",,
~;
8UB9iTlTUTE:SH~
W093/~24~7 2 ~ 3 ~1 0 ~ 8 PCT/~S93/03~43
-41-
observed with either normal or microscopic
examination. By four weeks, the membrane was entlrely
surrounded by a large well vascularized fat pad. No
abnormali~ies in the surrounding tissue was noted.
Similarly, no physiologic changes in the mouse were
observed as a consequence of the implant. The
explanted membranes were opened with a scalpel and
the cells contained within examined for viability.
The cells retrieved from within the membrane were
determined to still be viable by their ability to
exclude trypan blue and their ability to grow under.
in vitro culture condi~ions following retrieYal.
Examination of the cell in the tubes prior to
implantation and sub~equent to their removal xevealed
that they had not only survived but had proliferated
extensively. This occurred in both immunocompromised
(nude) and normal mice demonstrating that the
membrane had successfully protected the cell lines
from the ho~t mouse'~ immune system. In addition, if
the Raji cells has escaped from the membrane device, ~`~
a lymphoma would have been observed in the nude mouse
~tudy. Since this was not found, it can be concluded
that the membrane not only protects the cells within
it from the host immune system but that it also
protects the host fxom cells within.
.
Porcine islets (15,~000; 40,000; or 50,000 each
device) in either RPMI media or RPMI media containing
30 ! Matr~i~el were placed into either membrane tubes or
Insulets as described in Example 1. The de~ices were
then implanted either intraperitoneally or
: .
~subcutaneously into normal BALB/C mice. A~ter 3
months, the~de~ices were removed and tested for the
viability of the islet cells. Quantitation of the
number of islet cells present at the end of the
~,
SUBSTITUTE SHEET
W093t22427 P~T/US93/03843 ~
2 ~ 3 4 0 8 8
-42-
experiment was not possible. We were, however, able
to demonstrate the presence within the device of live
islet cells. In addition, when such a device was
placed into culture in vitro we were able to measure
insulin in the cultu~e media. This could only occur
if the islet cells had survived and continued to
function after the three month period in the animal.
Since the.se studles were performed in normal mice,
these studies demonstrated the xenograted cells were
protected from the host immune sys~em.
,
Exam~le 6: Treq~ t ~iabetic mice
To demonstrate the ability of porcine islets to
function in vi~o within the membrane, diabetic mice
. 15 were implanted with devices prepared as described in
Example 1. Diabetes was induced in BABL/C mice by
the injection of S~reptozotocin (STZ). Animals were
considerred to be diabetic when their fasting blood
glucose levels~were greater than 300 mg/dl for three
conse~utive measuremen~s. Devices containing 50,000 ~;
porcine isl~ts each were implanted either
intrapcritoneally or subcutaneously into diabetic
mice. The~e~an1mal~ were considered to be cured if
~heir fasting blood gluc~e levels returned to 200
2~5 mg/dl or less.~Control diabetic animals received
implanted de~ices with~no is~lets. Blood glucose
levels were monitored;~weekly. Within two weeks
following implanta~ion, animals began to show a
reduct~ion in~blood gluco~e levels. Figure shows
30~ i typical re~ul~s~for animals implanted for either 4 or
7 weeks. The 0 tlme~point repre~ents the fasting
blood glucose~leveIs~.~ As can be seen, diabetic
"controls" had fasting blood glucose levels greater
than 3~0 mg/dl while~those or normal or implanted
m1ce are about 100mg/dl. The presence of porcine
: iBletB within~the~dev1ce has clearly regulated the
~ 8U~BSTITUTE SHE~
W0~3/22427 ~ 1 3 ~ 8 PCT/US93/03~3
~43-
blood glucose levels of the implanted diabetic mice.
A glucose tolerance test more closely demonstrates
the ability of the animal to respond to the
physiologic stress of increased glucose levels.
These are performed by injecting the mice
intrapcritoncally with 3 mg of glucose per gram body
weight at ~ime 0. At 30, 60, 90, 120, and 180
minutes blood glucose levels were determined. Aq can
be seen in Figure 1 the normal mice show a slight
rise in glucose levels and rapidly return to normal
by 120 min post injection. In contrast, diabetic
mfce increase and show no ~endancy to return to pre-
~hallenge level~ during the course of the experiment. `~
The diabe~ic animals implanted with devices
containing porci~e islets show an initial rise
similar to that observed in the diabetic mice. In
. ~
contrast, however, these animals return to more `~
normal levels of glucose during the course of the
experiment. This experiment clearly demons~rates
that porcine islets survive in the membrane device in ~`
normal mice, that they continue to function and can `-
ameliora~e their diabetic state. In a true sen~e,`
this device is functioning as an artificial pancreas.
It has been demonstrated ~ha~ the membrane
~25 material u~ed in the practice of this invention is
not~ to~ic to cells~and that serum dependent cell
lines~can survive and grow within the membrane for
extended~periods~of time so~long as serum is kept in
the medium outside the membrane devices. Two serum
30~ l ~dep~ndent ceill lines, Raji and MOPC-31C have beeh
grown inside the membranes for up to six months. Not
only have they survived but they have prolierated ;~
norm~lly. Devices~containing these cells ha~e also
~ be~en~placed into normal mice. These cells survived
and grew for longer~than 4 months. They obviously ~`
protected the cells from the host immune system. In
; ,
SU~3$TIT~TE 5HEEl~ - :
`;
WO9~/22427 PC~/US93/03~43
213~088
-44-
addition, they prote~ted the host from the cells
within the membrane as no evidence of invasion or
tumor formation was observed. Islets have been
cultured inside devices composed of this membrane
material and have survived in vitro for periods
greater than six months. Throughout this time period
they continued to function normally releasing insulin
both basally and in response to secretagogue
challenyes.
:LO
Example 7: Improved Product Trans~ort and Cell
Viabilt~ Throuqh the Use_oE Hydroqels
It has been found that optimal diffu~ion of the
intracellular produc~s out of the membrane implant is
achie~ed, in part, by maintaining an e~en
distribution of the c211~ within t~e membrane. It
has been known in the art to use hydrophilic natural
polymers to encapsulate mammalian cells. Generally,
mammalian ceIls have been microencapsulated through
crosslinking using, for example, alginate ~nd
polylysine. One drawback to microencapsulation using
certain crosslinked hydrophilic polymers is the
tendency of crosslinked polymers to biodeyrade.
It is also known that alg nate~ and some
vegetable yums are capable o forming hiyh wa~er
content gels sui~able as encapsulating agents. Gels
where the water content is higher than 99.5% have
been produced. The high water content of these gels
provides these gels with high permeabili~y to the
30 1 various permeants important to hybrid artificial
organs. Even at very high water contents, these gels
are very ~iscous and thus capable of immobilizing
cells dispersed within the gel. However, one
drawback to the use of some acrylic hydroyels is
their biodegradability, particularly when the yels
''
$UBSll ITUTE ~HEI~
~31~88
W093/22427 PCT/U~93/03X43
.'.:. .
~ `~
-45- ~:
. .~
are implanted as microcapsules directly into tlssue. ..
................................................................... ............. ~
By placing these high water content hydrogels
within the membranes of the instant invention, it has
5 been found that the hydrogel compositions ~o not
rapidly biodegrade once implanted. Thus a preferred :.
embodiment of the instant invention is the use of a
hydrophilic gel with a water content 2 of about 35
water but preferably ~ 90% water inside of a dense,
semi-permeable polymer rne~brane. The dense membranes
of the instan~ invention provide imm~noisolation and
necessary selective permeability while al~o providing ~-
an absolute barrier to cell~. The water 3wollen gel
fixes the position of the cells within the dense
membrane (e.g. in the shape of a hollow iber) to ..
prevent bunching. ~In addition, the high water :-
content of the~water swollen ge~ pro~ides low
resistance to the permeability of species leaving or
:~ arriving at the con~ained cells.
Further, it has been observed that cells fixed
in a hydrogel exhibit improved viabili~y within the
implantable~device.~
: The dense~membrane may also provide a biostable ~.,
protective layer to the water-~wollen gel, ~h~s
.,
~25~ preventing or reducing:bisdegradation of the yel~ -;
Suitable::dense membranes include but are not
limited to~hydrophili~ or amphipathic polyurethanes '~
: haviny 2 about 20~:water but preferably 2 50% water
: but less e~uilibrium watex content than the water~
3Q I ~ swollen gei.~ Water content is measured in water ~
37C and expres~ed a~ a percentage o~ the wet weight
of the sample after maximum welght gain has occurred. ;.
This valuè is determined:by weighing the wet polymer
: ~ and again after the~polymer has been dried to :~
~ equilibration~at standard laboratory conditions, e.g.
-23~C and 50~ relative humidity.
..
: . .,
.' ~ `.
$UBSTITI,ITE SHlEEr
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-46-
Suitable water-swellable gels are alginates
(e.g. sodium alginate, ammonia alginates, potassium
alginates, propylene glycol alginates, algins), guar
gum, gum tragacanth, locust bean gum, methocel,
xanthan gum, polyethylene oxide, polypropylene oxide,
dextrans, acrylates, methacrylates, polyvinyl ~::
alcohol, polyvinyl pyrolidone and combina~ions of
the above.
Those yum~ or resins capable of ~crosslinking"
may be used cro~slinked or linear. For example,
sodium alginate may be:used "as i5" or conver~ed to
it-s l~soluble calcium form.
The~most preferred hydrogel i9 calcium alginat~
wherein the water content is greater than about 90%.
With regard to all of ~he embodiments of the
present invention, it is preferred tha~ a hydrogel,
that comprise~ greater than ahou~ 35~ water, be used
: to suQpend the cells. The hydrogel serves to
immobilize the cells within the membrane, thus
2 0 insuring an even cell distribution within the
membrane.
The most preferred embodiment of the instant
invention compri~ies the implantation of a den~e ~:
: ~ membrane: in the:form of hollow fibers where the ~-~
2:5 hollow ~iber is~;filled with calcium ~lginate with a
water conten~ greater:~:than about 90%. However, it ~;
i~ understood that the geometry of ~he ~ense membrane ~:
: can ~e ln any ~orm including sheets, larger diameter
tubes, e~c.
30 I ~TIhe inventi~n now being fully de~cribed, it will
be apparent to one of~:ordinary skill in the art that
many changes and:modifications can be made thereto ::
~: wi~hout departing from:th spirit or scope of the . ~:~
invention as ~e~ forth herein.
~, . :
: : ;.;
8UBSTIITUTE SHEET
