Note: Descriptions are shown in the official language in which they were submitted.
2 ~ O ~ ~ 5 9
W096/09876 ~CT~S94/10935
Artificial Liver Apparatus and Method
This application is a continuation-in-part of U.S.
Patent Application Serial No. 07/943,777, filed September
11, 1992, now abandoned.
R~r~CUND OF THE INVENTION
1. Field of the Invention
This invention relates to an device and a process for
the extracorporeal purification of blood and plasma.
Specifically, it relates to a purification device which,
while similar in structure to hemodialysis devices used
in the treatment of renal insufficiencies, uses novel
biological means to perform many of the functions of a
normal human liver. The device and method are therefore
intended to assist in the treatment and support of
patients suffering from liver disease or who have
undergone transplantation of liver tissue.
2. HistorY of the Prior Art
To best understand the invention, an overview of
principles of anatomy and physiology relating to human
liver function and disease is useful. The liver is an
organ divided into two principal lobes made up of
functional units called lobules. A lobule consists of
cords of hepatic cells arranged radially around a central
vein. Between these cords are sinusoid spaces lined with
phagocytic cells known as Rupffer cells. Oxygenated
blood is provided to the liver via the hepatic artery
while deoxygenated blood leaves the liver via the hepatic
portal vein. Branches of these vessels deliver blood to
the sinusoids, where oxygen, most nutrients and certain
toxins are extracted into the hepatic cells.
More specifically, as glucose-rich blood passes
through the liver, excess glucose is removed and stored
as the polysaccharide glycogen. When the level of glucose
in the blood drops below normal, glycogen will be broken
down into glucose which is released by the hepatic cells
W096/~876 ~ 5 ~ PCT~S94/10935
into the blood stream. The liver also assists protein
metabolism by extracting and storing excess amino acids
in the bloodstream for use in the construction of many
plasma proteins, such as albumin. Bile, a solution of
salts, bilirubin, cholesterol and fatty acids which
assists in the emulsification of fats and intestinal
absorption of lipids, is also produced by hepatic cells.
It is not, however, normally secreted into the bloodstream
by these cells but is instead transported to, and stored
in, the gallbladder.
Of greater importance to this invention is not the
liver's role in digestion of food but its role in
regulating the concentration of wastes and toxins in the
blood. Hepatic cells contain enzymes which either break
down toxins carried in the blood, transform them into less
harmful substances or, failing either of those processes,
stores them. For example, metabolism of amino acids will
result in the release of free amino acids and nitrogenous
wastes, the latter of which are converted by hepatic cells
to urea. In moderate amounts, this urea is harmless and
is easily excreted by the kidneys and sweat glands. "Old"
red blood cells and certain bacteria can also be destroyed
and, in the case of the former, recycled by the Kupffer
cells.
In short, the liver is vital to maintaining the body's
normal biochemical state. Impairment or loss of its
function can, therefore, be fatal. A concise summary of
known possible derangements of hepatic metabolism can be
found in Podolsky, et al., "Derangements of Hepatic
Metabolism" Ch. 315, Principles of Internal Medicine, 10th
ed., pp. 1773-1779, 1983. The medical art has developed
several approaches to the treatment of, or compensation
for, liver disease, damage and failure. In addition,
humans (as well as many other species) are capable of
regenerating lost or damaged liver tissue.
However, although supportive and pharmaceutical
treatments or transplantation may alleviate or reverse
2 ~ 5 ~
W096/09876 PCT~S94/10935
many symptoms of liver disease, these methods all require
time which an acutely ill patient may not have. Further,
while undergoing treatment, support for any loss of normal
liver function must be provided to maintain or approximate
metabolic homeostasis. A means, therefore, is needed
which can perform the cleansing functions of the liver
when it cannot, thus increasing the time available for
treatment.
Extracorporeal liver perfusion (i.e., pumping blood
through foreign liver tissue) has been a proposed means
for treatment and support for many years, with mixed
success. An example of the use of repeated liver
perfusions for long-term hepatic support can be found in
Abouna, et al., "Long-Term Hepatic Support by Intermittent
15Liver Perfusions", The Lancet, pp. 391-396, (Aug. 22,
1970), which reports maintenance of a patient suffering
from liver failure for 76 days using periodic liver
perfusions. However, despite attempts to use liver tissue
from 5 different species, immunological and other
20biochemical reactions limited the use of the perfusions
and the patient died before a suitable transplant donor
could be found.
Isolated hepatocyte transplantation has also be
performed, again with mixed results (see, e.g., Makaowa,
25L., et al., Can. J. Surg., 24:39-44, 1981, and Demetriou,
et al., Proc. Natl. Acad. Sci. USA, 83: 7475-7479, 1986).
In contrast, extracorporeal methods of purifying blood
and plasma; i.e., by hemodialysis, hemoperfusion or
hemofiltration are well-known and established in the art
30for treatment of renal insufficiencies. The major goal
of these methods is to maintain fluid and electrolyte
balance and rid the body of waste products.
In renal hemodialysis, blood is pumped into a dialyzer
containing an artificial semipermeable membrane suspended
35in a dialysis solution. With a concentration gradient
established across the membrane for a particular
substance, flow from the blood into the dialysis bath will
W096/09876 ~ PCT~S94/10935
occur. This method can be used to successfully lower the
concentration in blood of urea and in plasma of potassium.
Net removal of substances whose concentrations should not
be altered in blood or plasma, such as sodium in the
latter, can be removed by establ;sh;~g a hydrostatic
pressure gradient across the membrane, creating a
convective pathway for movement of solutes across the
membrane. Details concerning the structure of a
conventional hemodialysis device as well as means for
controlling fluid temperature, dialyzate concentration,
and fluid flow therein are set forth in several existing
patents, including, representatively, U.S. Patent No.
5,011,607 to Shinzato, U.S. Patent 4,923,598 to Schal,
U.S. Patent 4,894,164 to Polaschegg, and U.S. Patent
5,091,094 to Veech.
In operation, blood is removed or pumped directly from
the patient into the dialyzer and flows along one side of
the membrane. The dialysis solution is pumped in
counterflow across the membrane; effluent blood is
returned to the patient.
Hemodialysis according to the method outlined above
is most effective for the removal of small molecular
weight species that are water-soluble and not protein-
bound. As a result, it is principally used in therapy for
renal insufficiencies, although it may be used in the
treatment of certain drug overdoses. To use the method
effectively to compensate for loss of liver function,
however, additional strategies for blood detoxification
are required.
To that end, a number of implantable and
extracorporeal bioartificial liver devices have been
proposed in the art and tested in clinical trials (see,
e.g., the review in Nyberg, et al., Am.J.Surg., 166:512-
521, 1993). Although the design and operation of such
devices have varied widely, they share common elements.
For example, the devices typically utilize isolated
hepatocytes to metabolize solutes from blood which pass
5 9
w096/09876 PCT~S94/10935
through as well as one or more permeable membranes. Of
the devices which utilize isolated hepatocytes, the
hepatocytes are typically anchored onto a supporting
substrate to facilitate cellular differentiation and
5 aggregation.
For example, using many of the concepts disclosed in
the parent application of this continuation-in-part
application, a bioartificial liver containing hepatocytes
bound to collagen-coated microcarrier beads (CYTODEX 3
beads, a trademarked product of Pharmacia) in a hollow
fiber containing bioreactor was tested and described as
producing more efficient metabolite transfer than systems
which entrap hepatocytes within gel or gel droplets
(Rozga, et al., Biotech. and Bioengineering, 43:645-653,
1994; see also, Miura, et al., Artif.Org., 10:460-465,
1986 [re use of a calcium alginate gel as a cell support],
and Cai, et al., Artif.Org., 12:383-393, 1988
[microencapsulation of cells in a gel]).
Other approaches to the use of isolated hepatocytes
in an artificial liver have attached the cells to
microcarriers and placed them into a chromatography column
for perfusion (Demetriou, et al., Ann.Surg., 259-271,
1986), onto hollow fibers (Jauregui, et al., J.Cell
Biochem., 45:359-365, 1991; see also, U.S. Patent No.
5,043,260), onto glass plates stacked in a module and
perfused with oxygenated medium (Uchino, et al., ASAIO
Proc., 34:972-977, 1988), onto asialoglycoprotein polymers
(Akaike, et al., Gastroenterol., 28 Supp. 45-52, 1993),
and into beds packed with matrix-forming materials such
as glass beads (Li, et al., In Vitro Cell Dev.Bio.,
29A:249-254, 1993). The efficacy of these approaches has
been limited by relatively short periods of cell viability
(as short as a few hours; Demetriou, et al.), difficulties
in forming cell aggregates, and diminished contact between
the cells and nutrients, metabolites and toxins (as a
result of immobilization of the cells onto a substate
which masks a portion of the cell surface).
W096t09876 ~ PCT~S94/10935
Over time, t~c-hn;ques to improve cell viability and
aggregation have been im~Gved (see, e.g., published
patent application 93-272876 tWO 9316171], which describes
a system similar to that disclosed in Li, et al., In Vitro
Cell Dev.Bio., supra). However, it has been generally
accepted in the art that hepatocyte aggregation and
function sufficient for use in extracorporeal liver
support are dependent at least on attachment of the cells
to a substrate or matrix, if not also immobilization of
the cells (see, e.g., Rotem, et al., Biotech. and
Bioengineering, 43:654-660, 1994 thepatocytes are
anchorage dependent cells]; Miura, et al., Biomatter
Artif.Cells Artif.Org., I8:549-554, 1990 thepatocyte
aggregation is required for proper cell function; to that
end, immobilization of the cells is preferred]; Rozga,
et al., Ann.Surg., 2I7:502-511, 1993 [attachment of cells
to microcarriers enhances cell function and
differentiation]; and, Rozga, et al., Biotech. and
Bioengineering, supra tattachment of cells to
microcarriers or entrapment of cells in a gel preferred]).
In contrast, conventional hemodialysis utilizing a
membrane against a suspension of free (i.e.,
"unattached"), isolated hepatocytes has not been shown to
be clinically effective in providing liver support (see,
e.g., Olumide, et al., Surgery, 82:599-606, 1977). Thus,
the bioartificial liver devices that utilize isolated
hepatocytes which are presently being developed and tested
in the art anchor the cells to a substrate, a process
which entails a relatively delicate manufacturing step
informing the cell/substrate attachment, and risks damage
to the cells.
8UNMARY OF THE INVENTION
The invention consists of an device and method for
using it to purify (i.e., detoxify) a biological fluid
such as blood or plasma, whereby blood or separated plasma
is circulated through a bioreactor having at least one
semi-permeable membrane passing therethrough. The
2 ~ O ~ 1 5 9
Wog6los876 PCT~S94/10935
semipermeable membranes may be in tube, film or hollow
fiber form (preferably the latter), and are surrounded by
a sterile cell culture medium in solution for mainten~nce
of hepatocytes and/or a hepatoma cell line (as explained
further below, the term "hepatocyte" will, unless context
otherwise requires, refer both to isolated hepatic cells
and a combination of those cells with Kupffer bile duct
epithelial and endothelial cells and, in some instances,
fibroblasts). Soluble proteins, glucose and toxins in the
blood or plasma diffuse across the membrane into the
culture medium for metabolism by the hepatocytes.
In one embodiment, the hepatocytes utilized in the
invention are attached to biologically compatible
microcarrier particles. Circulation of the microcarriers
in the cell culture medium is assisted by means such as
an alternating magnetic field to affect magnets embedded
within the microcarriers. As the diffused molecules
(i.e., solutes) come into contact with the bound
hepatocytes they are taken up by the cells and broken
down, transformed or stored according to normal hepatic
cell function with respect to said molecules. Effluent
blood or plasma (after recombination with a previously
separated fraction of red blood cells) is then returned
to the patient.
In the preferred embodiment of the invention,
unattached hepatocytes (i.e., cells which are not attached
to a substrate or otherwise immobilized) are utilized.
Means are provided to facilitate cell aggregation within
the device (i.e., in situ) to provide extracorporeal liver
support to a patient in need of such support.
Additional purification means such as a hemofiltration
device, means for adsorption onto an activated charcoal
column or other resin adsorbents and/or a conventional
dialysis device may also be provided as needed to remove
certain toxic drug or waste products not broken down or
stored by the hepatocytes (such as urea excreted thereby
into the culture medium). Means may also be provided in
W096/09876 PCT~S94/10935
the device to remove any antibodies formed to the
hepatocytes or, for example, to xenogeneic grafts of liver
tissue in transplant patents not captured by the
hepatocytes.
BRIEF n~P~PTPTION OF T~E DRA~ING8
FIGURE 1 is a schematic representation of a preferred
emho~iment of the device of FIGURE 1 for use with
unattached hepatocytes.
FIGURE 2 is a schematic representation of an
alternative embodiment of the device of the invention,
including preferred additional purification means.
FIGURE 3 is a graph illustrating the percentage of rat
hepatocytes viable after up to 21 hours of circulation of
a population of hepatocytes through the device of FIGURE
l. Viability was determined by trypan blue exclusion.
FIGURE 4 is a graph illustrating the percentage of
porcine hepatocytes viable after up to 6 hours of
circulation of a population of hepatocytes through the
device of FIGURE l.
FIGURE 5 is a graph illustrating the percentage of rat
hepatocytes viable after up to 6 hours of circulation of
a population of hepatocytes through the device of FIGURE
l as used for in vivo, extracorporeal purification of
plasma of a male pig.
FIGURE 6 is a graph illustrating the extent to which
the ammonia concentration within fluid circulating through
the blood loop of the device of FIGURE 1 decreased over
time with circulation of a population of rat hepatocytes
through the hepatocyte loop of FIGURE l.
FIGURE 7 is a graph illustrating the extent to which
the urea concentration within fluid circulating through
the blood loop of the device of FIGURE 1 increased over
time with circulation of a population of rat hepatocytes
through the hepatocyte loop of FIGURE l.
DE8CRIPTION OF THE PREFERRED EMBODIMENT8
A. I~ol~tion and Prepar~tion of Cell~.
A principal feature of the invention is its use of
~o ~
wos6lo9876 PCT~Ss4/1os35
living, isolated liver cells to assist in the purification
of substances out of blood or plasma which would, absent
liver disease, impairment or failure, be removed by the
patient's own liver. For most embodiments, the cells will
be hepatocytes of nonhuman, human, or xenogeneic origin.
However, it will be understood by those of skill in the
art that hepatoma cells may also be used as the liver
cells of the invention. Thus, while hepatocytes are the
preferred liver cells due to the lesser risk of
pathogenesis that they pose, the word "hepatocyte" and the
disclosure herein will be understood to encompass hepatoma
cells, such as the human hepatoma cell line Accession No.
CRL 8024, which is available from the American Type
Culture Collection, Rockville, Maryland.
Preparation of hepatocytes for use in the invention
is as follows:
Methods for isolation of human hepatocytes suitable
for use in the method of the invention are known in the
art (see, e.g., Tak~h~h;, et al., Artif.Org., 17:653_659,
1993). The use of human hepatocytes in an artificial
liver system may reduce the risk of immunologic reaction
by the patient to contact with nonhuman hepatocytes.
However, the availability of human hepatocytes is
nPcecc~rily limited. Therefore, of nonhuman hepatocytes,
porcine hepatocytes are preferred for use in the invention
principally because of their availability and physiologic
similarity to human hepatocytes. It is also anticipated
that, for use of the system with liver transplant
patients, porcine hepatocytes will be particularly useful
where porcine tissue can be modified to be a xenogeneic
organ source for human transplantation. It will be
appreciated by those skilled in the art, however, that
other mammalian species may also be suitable sources for
the hepatocytes of this invention.
Using porcine tissue as an example, therefore,
hepatocytes in lobules are retrieved from the liver
immediately following slaughter of the animal. The
W096/~876 ~ PCT~S94/10935
-- 10 --
lobular tissue is screened for disease according to means
known in the art. Hepatocytes are then isolated from the
lobular tissue by enzymatic (collagenase) digestion
according to means known in the art and purified (see,
e.g., t~çhniques described in Berry, et al., J. Cell.
Biol. 43:506-520, 1969; Seglen, Methods Cell Biol. 13:29-
83, 1976; and Example I, infra). Depending on the method
of isolation used, Kupffer cells and other cellular
components of the lobular tissue (such as bile duct
epithelial cells) may also be included with the
hepatocytes. If desired, the hepatocytes may be further
purified by pelleting the cells at low speed (e.g., 50
Xg), in a reagent such as the 60% PERCOLL solution
supplied commercially by Sigma Chemical of St. Louis, MO.
Because the biochemical states which contribute to or
cause liver disease are not completely understood, it is
not yet known the extent to which each of these cell
populations may contribute or be necessary to the function
of the invention. It is expected that the use of purified
populations of hepatocytes will be preferred; however, as
used herein, the term "hepatocyte" should be understood
to encompass hepatic cells or fibroblasts alone or in
combination with Kupffer cells, bile duct epithelial and
endothelial cells. It may also be desirable to utilize
cells which have been genetically altered to produce or
metabolize liver specific products such as albumin or
clotting factors. These cells could be used exclusively
or in a cocktail with native hepatocytes.
Once purified, one or more hepatocytes may be attached
for use in one embodiment of the invention to a
biologically inert carrier, preferably beads of 150-200
~m in diameter. Preferred for use in this regard are
natural polymer materials such as dextran beads, available
under the trade name CYTODEX 3 by Pharmacia LKB
Biotechnology of Uppsala, Sweden. Equivalent materials
such as agarose beads (available from Bio-Rad, Inc. of
Richmond, California) or other substrates such as glass
w096/09876 ~ ~ 0 1 ~ 5 ~ PCT~S94/10935
-- 11 --
may also be used. Therefore, although for convenience the
substrate to be used to support the hepatocytes in the
invention will be referred to as beads, those of skill in
the art will know of, or can readily ascertain, other
~u~o~ substrates suitable for use in this embodiment of
the invention.
The beads will preferably be coated with denatured
collagen or equivalent biological material to permit
attachment of the hepatocytes thereto. Collagen is the
preferred surface because exogenous fibronection is not
required for attachment of hepatocytes to collagen.
Examples of suitable methods can be found in
"Microcarrier Cell Culture: Principles and Methods"
available on request from Pharmacia LKB Biotechnology, in
Gjessing, et al., Exp. Cell. ~es. 129:239-249, 1980, and
in Demetriou, et al., Science, 23:1190-1192, 1986, the
disclosures of which are incorporated herein by this
reference to illustrate knowledge in the art concerning
cell attachment to microcarrier substrates. For example,
using a commercially available spinner vessel, 10 mg. of
dextran beads can be added to each milliliter of cell
culture. Hepatocytes, to a final concentration in the cell
culture medium of 1 x 105 cells/ml., are added to the
beads, mixed gently and incubated at 37C. Periodic
stirring may be necessary as the cell culture medium
volume is increased if attachment is not achieved readily.
However, in the preferred embodiment of the invention,
the hepatocytes will not be attached to a substrate or
otherwise immobilized. Therefore, for use in this
embodiment of the invention, hepatocytes will be isolated
as described above and will be stored as described below.
The attached or unattached hepatocytes may be
preserved by maintaining them at a temperature of less
than -75C in a sterile environment. Preferably, they
will be frozen and stored in a container of liquid
nitrogen and maintained according to substantially the
same method used to preserve isolated spermatocytes for
W096/09876 ~ PCT~S94/10935
- 12 -
artificial insemination. A particularly suitable method
for cryopreservation of hepatocytes is described in Rozga,
et al. Biotech. and Bioengineering, 43:645-653, 1994. An
alternative method for cryopreserving hepatocytes is
described in Dixit, et al., Transplantation, 55:616-662,
1994, wherein cells are encapsulated and frozen in calcium
alginate. By freezing the hepatocytes, it may be possible
to store the cells for periods of several years, although
storage for periods of less than a year would be
preferred. However, most preferably, to the extent
practical, the hepatocytes will be used shortly after
isolation.
For use of the hepatocytes, the cells will be
suspended in a biocompatible culture medium. If frozen,
the cells may be thawed and washed in a suitable buffer,
such as phosphate buffered saline. Preferably, the
culture medium will contain nutrients and other
supplements to maintain the viability of the hepatocytes,
which nutrients and supplements are known to, or may be
readily ascertained by, those of ordinary skill in the
art. An example of a culture medium suitable for use in
hepatocyte maintenance is the medium sold under the trade
name RPMI-1640 by Grand Island Biologicals, Grand Island,
New York. Another suitable and commonly used culture
medium is the WAYMUTH medium supplied commercially by
GIBC0 Laboratories, Grand Island, NY. Preferably, the
medium will be supplemented with salts or nutrients known
to those skilled in the art depending on the needs of the
particular cells used in the system (i.e., which may vary
depending on whether cells other than hepatocytes are
included, the length of use of the hepatocytes, the
specific substances to be metabolized, etc.). Such
additives may include one or more amino acids, nutrients
and stabilizers such as alanine, serine, asparagine,
albumin, aminoleulinic acid, oleic acid, dexamethasone,
thyroxine, tocopherol, glucagon, insulin and gentamicin,
all of which are commercially available. A suitable
W096/09876 ~ ~ O ~ PCT~S94/10935
- 13 -
supplemented culture medium is described in detail in
Example I, infra. Alternatively, particularly if the
hepatocytes are to be used for purification purposes
within about 6 hours of isolation or thawing, the culture
medium may be any blood compatible fluid, such as saline.
B. Bio~rtificial Liver Devices 8uit~ble for Use in the
Nethods of the Invention
l. Construction and Operation of a Device Preferred
for Use With Unattached Hepatocytes
Referring now to the drawings, FIGURE 1 depicts a
preferred embodiment of the device of the invention for
use primarily with unattached hepatocytes, while FIGURE
2 depicts suitable modifications of the device of FIGURE
l. The devices of the invention are generally are
composed of a purification bioreactor having at least one
semipermeable membrane therethrough, and at least two
pumps or other means for circulation of the hepatocytes
and blood or plasma through the device. Generally, each
device encompassed by the invention will include a first
conduit (i.e., biological fluid loop 20) and a second
conduit (i.e., hepatocyte loop 5), which conduits are
separated by at least one semi-permeable membrane. All
of the components of the device (including connectors,
clamps, luer-locks, tubing, and containers) will
preferably be sterilizable.
In addition, means for sensing and controlling flow
temperature and pressure of the dialyzate and blood, as
well as determining the relative concentrations of solutes
therein, are known in the art and, where noted below, may
be suitable for use in the device of this invention. For
purposes of illustration, reference may be made for these
details to the references identified in the background
section, supra.
Fluids in biological fluid loop 20 will enter it
substantially at body temperature and should be returned
to the patient at substantially the same temperature.
Maintenance of this fluid temperature will be achieved by
q ~
W096/09876 ~ ~ ~ q ~ ~ ~ PCT~S94110935
- 14 -
external temperature control means for blood and plasma
known to those skilled in the art; e.g., means used to
maintain the temperature of fluids during plasmapheresis,
blood bank storage or transfusion. Means for temperature
control of fluid during renal hemodialysis and/or
hemofiltration are also described in U.S. Patent 4,894,164
(disclosing a ter~nique for cooling blood exposed to
heated dialyzate during renal hemodialysis). A proposal
for maintaining the blood temperature at 34C could also
be utilized with the process of this invention (see, e.g.,
Maggiore, et al . Proc. EDTA 18:597-602, 1981). An oxygen
source (i.e., oxygenator 9) will also be provided to
supply oxygen to the hepatocytes.
One particularly suitable means for temperature
control places most of the components of the device within
an insulated incubator sufficient to maintain the
temperature of the fluids circulating within the device
at about body temperature (e.g., 37C for humans).
Suitable incubator materials will be known to, or can be
readily ascertained by those of skill in the art and
include foam or glass in a durable container, such as
stainless steel.
It should be noted that operation of the entire
system, including control of the valve means and the
initiation and cessation of flow into and out of mixing
vessel 1 and stations within the hepatocyte loop 5, as
well as flow into, through and out of the biological fluid
loop 20, will preferably be substantially continuous
within a purification cycle. Control of the system will
preferably be by control means (not shown) such as a
microprocessor with manual override capabilities according
to means well-known in the art and used in the control of
devices for renal hemodialysis and hemofiltration.
Alternatively or preferably in addition to these
electronic control means, manual control means (not shown)
will be provided throughout the system to allow a skilled
operator to open and close the flow valves into and out
w096/09876 ~ S ~ PCT~S94/1093S
of each station within biological fluid loop 20, control
the pump means 8, 14 and 15, and control flow into and out
of hepatocyte loop 5.
In addition, although pump means are depicted in
FIGURE l at particular locations, it will be appreciated
by those skilled in the art of mer-h~n;cal engineering that
one or more of such pump means may be included in the
loops at sites other than the ones shown.
Referring to FIGURE l, a preferred embodiment of the
device includes a mixing vessel l into which unattached
and uncontaminated (i.e., "new") hepatocytes will be
introduced and circulated to facilitate their aggregation
prior to introduction of the cells into the purification
bioreactor 7 via hepatocyte loop S. The mixing vessel l
will include a hepatocyte introduction port 2 (for
introduction of new hepatocytes into mixing vessel l),
sampling port 3 (for the removal of contaminated (i.e.,
"old"), hepatocytes and culture medium from mixing vessel
l), hepatocyte circulation outlet port 4, hepatocyte
circulation inlet port 5 and a waste outlet 6.
Conveniently, these ports may be formed of tubing attached
to and through a wall of mixing vessel l.
For example, where mixing vessel l is formed of glass,
ports 2-6 may be formed of glass tubes fused into and
through the wall of vessel l at suitable locations, such
as those shown in FIGURE l. The ports may also be
attached to mixing vessel l with a luer-lock or equivalent
fitting attached to the port via flexible tubing that can
be closed using a hose clamp or similar clamping means.
Other designs and materials for ports 2-6 will be apparent
to those of ordinary skill in the art and will not,
therefore, be described in detail here.
The hepatocyte loop itself (exclusive of stations
identified infra) will be a conduit formed of arterial
flexible tubing or biologically compatible materials
similar or identical to those used to form mixing vessel
l and purification bioreactor 7. Hepatocyte loop 5 will
W 0 96/09876 ~ 5 ~ PC~rrUS94/10935
- 16 -
be sealably attached to mixing vessel 1 at outlet port 4
and inlet port 5. Conveniently, the seal attachment may
be made by an O-ring and O-ring joint clamp, the latter
of which will allow adjustments to be made to increase or
decrease the tension on the O-ring seal.
The mixing vessel 1 and purification bioreactor 7 must
be of a biologically compatible, non-cytotoxic material
and will be preferably be of a polymer compound such as
acrylonitrile butadiene styrene ("ABS") resin plastic,
polypropylene, polysulfone, glass or equivalent materials
composed to meet USP XXI Class VI toxicity testing
stAn~rds .
The size of mixing vessel 1 may vary, but will
generally be sufficient to retain a number of hepatocytes
equivalent to at least 1% of the approximate weight of the
liver in the mammalian species to be treated; i.e., a
"theoretical minimum" number of cells. For example, in
humans, the theoretical minimum number of cells which
would be expected to be effective in extracorporeal
purification of blood or plasma is about 1% of the liver's
hepatocytes or about 2 billion cells. The mixing vessel
will, therefore, be sufficient in size to contain at least
a theoretical minimum number of hepatocytes suspended in
a sterile, biologically compatible fluid such that the
2S hepatocytes may be circulated within mixing vessel l;
i.e., the cells will not be packed to the point of
immobilization within the mixing vessel.
The size of the purification bioreactor 7 will depend
on the volume of blood or plasma to be circulated through
the system. It is expected that this volume will vary
from l/2 pint to 4 pints, with the former being the volume
expected to be circulated in a pediatric application and
the latter being the upper limit expected to be available
for adult applications with use of plasma expanders and
plasma blood products.
Purification bioreactor 7 is in fluid communication
with a source of biological fluid (e.g., blood or plasma)
w096/09876 ~ 5 ~ PCT~S94/10935
- 17 -
to be treated by external connections which form a
circulation loop for the biological fluid (e.g., blood or
plasma) to be treated. The purification bioreactor will
preferably be a hollow fiber bioreactor. In a hollow
fiber bioreactor, the circulation of biological fluid
through purification bioreactor 7 will occur through a
multiplicity of capillaries (representatively depicted as
7' in FIGURE 1) formed of semipermeable membranes which
will allow the exchange therethrough of soluble proteins
(which may include antibodies) and metabolic waste
products of sizes which will vary according to the
application.
To this end, the membrane pore size is expected to
vary upward to a pore large enough to permit passage of
proteins therethrough, with the larger pore size intended
for removal of proteins such as antibodies. It should be
noted that where larger pore sizes are used, certain
desirable substances (e.g., plasma proteins) may diffuse
into the culture medium. Means for return of these
substances are provided below; because of this phenomenon,
however, it is expected that pore sizes of about 0.2 ~m
in diameter will be most practical and will result in
significant fluid convection, which is the primary force
responsible for solute transport across the membranes that
form the hollow fibers.
A commercially available and suitable product for use
as purification bioreactor 7 is the ZYMAX hollow fiber
bioreactor (ZYMAX is a registered trademark of Microgon,
Inc. of Laguna Hills, California). The ZYMAX product
comprises a collection of 3600 hollow fibers formed of a
semipermeable membrane material having a pore size of 0.2
~m formed of mixed esters of cellulose which allows free
passage of soluble proteins and metabolic wastes
therethrough. The fibers are contained within a ported,
biologically compatible and non-cytotoxic polysulfone
housing; fittings, including clamps, gaskets, hoses and
plugs necessary to construct the bioreactor from the
W096/09876 ~ 5 ~ PCT~S94/10935
- 18 -
manufacturer. Other suitable hollow fiber bioreactors are
the Z22M-060-OlX bioreactor from Microgon (which has only
670 fibers, but uses fibers of longer length [about 550
mm potted end] than many conventional bioreactors), and
tOTHER E~AMPLE8????????]
The total number of hollow fibers (shown as 7' in
FIGURE 1) required is ~epen~ent on the volume of culture
medium contained within purification bioreactor 7, which
is in turn dependent on the volume of biological fluid to
be purified within the inventive system (which will
determine the number of hepatocytes to be suspended within
the culture medium). For example, given a known volume
of blood or plasma and the volume and permeability of the
hollow fibers (approximately 200 milliliters in the lumens
and 0.2 ~m respectively for a single ZYMAX bioreactor),
the total number of fibers and volume of culture medium
can be estimated. As a general principle, the total
surface area of the hollow fibers should be maximized with
respect to the volume of culture medium to ensure
contacting of solutes from the blood and plasma with
hepatocytes circulating through purification bioreactor
7 in hepatocyte loop 5.
Within purification bioreactor 7, hepatocyte loop 5
comprises the extracapillary space within the bioreactor
housing, while biological fluid loop 20 comprises the
intracapillary space; i.e., the lumens of the hollow
fibers 7' contained within bioreactor 7. The movement of
hepatocytes through the hepatocyte loop is driven by pump
14 (e.g., a peristaltic pump), while the movement of blood
or plasma through the biological fluid loop is driven by
pump 8 (e.g., a blood monitor pump).
As indicated in the background discussion preceding
this disclosure, it is highly desirable that the
hepatocytes in an artificial liver system aggregate
together. For example, it has been previously shown that
unaggregated, single hepatocytes in culture die relatively
rapidly compared to cells in aggregates. For that reason,
W096l09876 ~ PCT~S94/10935
-- 19 --
prior art artificial liver systems provide an
extracellular solid substrate to allow the hepatocytes
to aggregate within a bioreactor and/or culture the cells
in a manner which encourages the formation of aggregate
spheroids before use of the cells in an artificial liver
system.
The process by which hepatocytes aggregate requires
energy. Thus, although not absolutely necessary to their
survival, the viability of hepatocytes for use in an
10 artificial liver system has been shown to be enhanced by
the presence of oxygen, particularly in the early stages
of aggregation (see, e.g., Rotem, et al., Biotech. &
Bioengineering, 43:654-660, 1994 [oxygen partial pressure
of about 0.064 mmHg required for half-maximal attachment
15 of a single layer of hepatocytes to a collagen-based
substrate, while about 0.13 mmHg was shown to be required
for the cells to spread across the substrate]). Where
hepatocytes in an artificial liver system or culture are
present in multiple layers, it has been suggested that
20 oxygenation of the cells beneath the surface of the top
layer may require shortening the diffusion distance
between hepatocytes and the oxygen source or increasing
the concentration of oxygen present in the early stages
of aggregation (Rotem, et al., supra at 659).
In the preferred embodiment of the invention, the
hepatocytes are neither immobilized onto a solid substrate
nor precultured into aggregate spheroids. Instead, the
cells are introduced directly into a circulation
suspension of blood compatible fluid (preferably saline
30 or a hepatocyte culture medium as described supra) and
encouraged to aggregate in situ. This utilization of the
hepatocytes of the invention eliminates the work involved
in preculturing the cells, eliminates the work involved
in attaching the cells to a solid substrate, ensures that
35 the entire surface of each cell is available for
interaction with other cells, oxygen, nutrients and
metabolites in the culture medium, minimizes the potential
W096/09876 ~ 5 ~ PCT~S94/10935
- 20 -
damage to each cell inherent in the handling involved in
preculturing the or immobilizing the cells, and shortens
the time between isolation and use of cells in the method
of the invention.
To ensure adequate oxygenation of unattached
hepatocytes in the circulation suspension, mixing vessel
1 is filled with a blood compatible fluid which is driven
by pump 15 from a feed reservoir 13 or other fluid source
through line 18. The fluid is oxygenated by oxygenator
9, which may be any conventional fluid oxygenator known
in the art. For oxygenation, recycle pump 14 is activated
such that the fluid in line 18 passes into line 19 through
a valved feed line 12 at outlet port 4 and through
oxygenator 9. Leaving oxygenator 9, the culture medium
passes pH probe 10 and dissolved oxygen probe 11.
The pH and oxygen probes will be set at desired set
points and the gas composition of the culture medium
adjusted accordingly. The culture medium will be
maintained at a pH of about 7-8, preferably about 7-7.5,
and most preferably about 7.35. The oxygen composition
of the culture medium will be maintained between about 0
and 20%, preferably not lower than about 5%. Most
preferably, the oxygen composition will be about that of
saturated air at the time that the hepatocytes are
introduced into the culture medium. The oxygen
composition is adjusted by increasing or decreasing the
concentration of oxygen introduced into the culture medium
in oxygenator 9, while the pH of the culture medium may
be controlled by increasing or decreasing the
concentration of carbon dioxide introduced into the
culture medium at oxygenator 9, as appropriate. The
balance of the gaseous phase will be nitrogen. Gas is
introduced into the culture medium in the oxygenator in
a direction of flow opposite the direction of flow of the
circulation fluid. AS the culture medium enters mixing
vessel 1 through outlet port 4, gas leaves the system
through waste outlet port 6 into degassing vessel 16.
w096/09876 ~ 5 ~ PCT~S94/10935
Flow through the biological fluid loop will be as
follows. Blood or plasma for purification will be either
removed from the patient for introduction into the system
or, preferably, flow will be generated directly from the
patient. In the preferred embodiment, vascular access
will be provided according to medically accepted
t~chn; ques such as those used for acute or chronic renal
dialysis (e.g. catheterization, direct anastomosis of
native vessels or artificial grafts).
In the most preferred embodiment, the biological fluid for
purification will be plasma. After removal of blood from
the patient, the plasma fraction is separated from the red
blood cells by plasmapheresis, using a method and device
(not shown) well known in the art. Plasmapheresis may be
performed independently and out of the biological fluid
loop, but the means therefor will preferably be included
in the loop so that direct vascular access to the patient
is with the plasmapheresis device.
The plasma fraction (or unseparated blood where
plasmapheresis is not performed) passes through biological
fluid loop 20 to hollow fibers 7' via a connection conduit
(not shown) to each fiber or, for the preferred embodiment
using a bioreactive housing such as the ZYMAX product, to
each such housing wherein said conduits will be provided
and attached thereto according to the housing
manufacturer's specifications. The structure of the
conduits may vary widely according to the number and
structure of the hollow fibers 7' used, but will be formed
of a biologically compatible, non-cytotoxic material
similar to that disclosed above for use to form mixing
vessel l or an arterial flexible tubing known in the art.
Valve means 2l and 22 can be provided at the input and
output ports (not shown) of the biological fluid loop 20.
Preferably, valve means 21 will be pressure regulated (by,
for example, pressure regulator 24 shown in FIGURE 2) so
effluent blood returned to the patient is at an adequate
pressure. Suitable valve means are well known in the art
W096/09876 ~ PCT~S94/10935
and are not, therefore, described further herein.
Flow of blood or plasma through biological fluid loop
20 can be generated by the pressure gradient between the
fluid pressure of the blood or plasma as it leaves the
patient, but will preferably be assisted by a low pressure
mechanical pump means 8 prec~;ng the input portal to
biological fluid loop 20, which will preferably be fitted
with a pressure regulator (not shown) to prevent any back
surge. A suggested placement of flow sensor 25 and
control means 30 are schematically depicted in FIGURE 2.
As suitable means for flow control are well-known in the
art, no further description thereof will be provided here.
It may also be necessary to provide means for release of
air (not shown) from purification bioreactor 7; again,
such means are well-known in the art and not described
further here.
Noncellular components of blood or plasma introduced
into bioreactor 7 via biological fluid loop 20 will
exchange across the membranes comprising hollow fibers 7'
with the culture medium in the hepatocyte loop. Once the
exchange is complete, hepatocytes are introduced into
mixing vessel l through hepatocyte introduction port 2,
then additional culture medium added as needed to ensure
that the mixing vessel l remains completely filled with
fluid. Alternatively, the hepatocytes may be introduced
into mixing vessel l as the culture medium is being
exchanged with the plasma.
In this respect, those of skill in the art will
appreciate that the use of unattached hepatocytes requires
a delicate balance to be achieved between the flow rate
in the hepatocyte loop sufficient to facilitate contact
between, and aggregation of, the hepatocytes, while not
causing the cells to shear or otherwise degrade. At the
same time, the need to shorten the time necessary to
detoxify a therapeutically beneficial quantity of blood
or plasma must be balanced against the need to optimize
the rate at which solute transport across the membranes
W096t09876 ~ PCT~S94110935
which comprise hollow fibers 7' into the hepatocyte loop
(i.e., into the extracapillary space of bioreactor 7) will
occur.
To this end, the direction of flow in the biological
- 5 fluid loop 20 will be substantially opposite the direction
of flow in the hepatocyte loop 5. Through activation of
recycle pump 14 in a direction opposite that used to
oxygenate the culture medium, the cell-containing
circulation fluid in the hepatocyte loop will flow from
mixing vessel l through bioreactor 7, past probes lO and
11 and through oxygenator 9 at a rate of about 20 to 80
milliliters of fluid/minute, preferably at a rate of about
65 milliliters of fluid/minute. At the same time, through
activation of blood monitor pump 8, blood or plasma in the
biological fluid loop will be maintained at a rate of
about 20 to 250 milliliters fluid/minute, with a rate of
150-200 milliliters fluid/minute being preferred.
It should be noted that the flow rate in the
biological fluid loop which may be effectively and simply
controlled through the use of a fluid reservoir. By
ret~;ning blood or plasma in this reservoir, a store of
fluid is available for processing according to the method
of this invention at a substantially higher flow rate and
volume than would be possible if flow was directly and
entirely from the patient. Using the reservoir,
therefore, fluid would be collected from the patient,
directed to the reservoir, then removed through valve
means and pumped through the biological fluid loop as
otherwise described herein.
Returning to the passage of flow through biological
fluid loop 20, as blood or plasma passes through hollow
fibers 7', soluble proteins and metabolic waste products
will pass therethrough into the culture medium by virtue
of the osmotic pressure gradient between the fluid in the
lumens of the hollow fibers and the culture medium in
bioreactor 7, as well as the so-called transmembrane
pressure.
Wog6tos876 2 ~ 5 ~ PCT~S94/10935
- 24 -
It will be appreciated that, unlike the process of
renal dialysis, no counterflow across hollow fibers 7' is
normally provided or desired in this device. Although it
can be expected that there may be some back diffusion of
5solutes which reach a concentration equilibrium vis-a-vis
the fluid in loop 20 and the culture medium, such
diffusion is preferably avoided by repl~n;chment of the
culture medium, compensated by additional purification
means (e.g., those shown as 40 and 50 in FIGURE 2), as
10well as minimized by the concentration gradient across
hollow fibers 7'.
Counterflow may be important, however, where the pore
size of the membrane is required to be large; e.g. when
diffusion of larger molecular weight species out of blood
15is sought. With those species, however, may for example
diffuse plasma proteins which should be returned to the
patient. In such an application, a multiplicity of
channels may be used to capture plasma proteins and return
them to the biological fluid loop utilizing a counterflow
20(see, e.g., the counterflow pump means described in U.S.
patent No. 5,011,607). For example, if the filtrate to
be removed were known to be a certain diameter, smaller
plasma proteins (e.g., albumin) could be captured by a
second and smaller semipermeable membrane bounded
25oppositely by a channel (not shown) leading back to
biological fluid loop 20. The remaining filtrate, being
too large to diffuse across the second membrane, would
remain in solution in the culture medium for metabolism
by the hepatocytes.
30Alternatively, in absence of means to return desirable
solutes to the patient, such solutes, in particular plasma
proteins, may be independently returned to the patient via
known medical practices. This is not, however, a
preferred approach because of the risk of infection from
35foreign blood products.
As a general principle, proteins and waste products
(hereafter "solutes") in solution in the culture medium
W096/09876 ~ PCT~S94/10935
- 25 -
susceptible to being metabolized or stored by the
hepatocytes will be absorbed by those hepatocytes which
they contact. However, it is expected that the degree of
absorption achieved will be limited in at least two
respects.
First, certain solutes will remain in solution because
they do not contact a hepatocyte, or do not contact a
viable hepatocyte; i.e., one capable of absorption of
filtrate. It is this latter possibility that gives rise
to the second limitation; i.e., hepatocytes may die,
become damaged or otherwise be rendered incapable of
performing their expected functions in the system.
As it would not be possible to determine or predict
when function may be lost to a given hepatocyte, it may
be necessary to replace all or a percentage of the
hepatocytes used in the invention through sampling port
3. However, as used in the device and according to the
method described above, the hepatocytes circulating in the
device should remain viable for at least about 6 hours,
the period of time which would be expected to be expended
in a single purification treatment of an adult human.
Further, because solutes not absorbed by hepatocytes
(due to the factors discussed above or, as would be true
of certain toxins, because the solutes are not
susceptible to being metabolized by the liver) will remain
in the culture medium, additional removal means may be
required and periodic replacement of the culture medium
will be necessary.
To that end, hepatocyte loop 5 may include a
replenishment station (not shown) at sampling port 3
wherein flow through the loop will be temporarily
interrupted for removal from and replacement of
hepatocytes and, if necessary, culture medium in the
system. The replenishment station could comprise a
variety of structures, including a sterile and ported tank
or conduits having valve means (not shown) from which
"used" cells and medium would flow and into which "new"
W096/09876 ~ 5 Q PCT~S94/10935
- 26 -
cells and medium would be introduced.
Preferably, this removal and reintroduction would
occur at predetermined intervals throughout a purification
cycle to ensure optimal performance of the process.
Because it would be somewhat impractical to recycle all
of the hepatocytes and culture medium during a single
purification cycle, removal and replacement of a
statistical percentage thereof is preferred. This
percentage, as well as the intervals at which it will be
removed and replaced, will vary according to the volume
of medium, cells and biological fluid in the system and,
once these values are known, can be determined according
to accepted statistical calculations known in the art.
Alternatively, or in conjunction with the above method,
the entire volume of cells and medium could be replaced
after each cycle.
On completion of a purification cycle, blood is
returned to the patient or plasma returned first to the
plasmapheresis device for recombination with the patient's
red blood cells, then returned to the patient via conduits
identical to those described above for introduction of
blood or plasma into biological fluid loop 20. It will
be appreciated that an immunoassay for IgM and/or known
liver function tests may be performed to assist in
verification of proper and effective performance of the
inventive process prior to or after return of the blood
to the patient. Depending on the course of treatment
indicated and length of cycle required, the purification
cycle could be repeated up to two or three times in a
single day.
With respect to the additional removal means, once
flow in biological fluid loop 20 has passed through
bioreactor 7, the fluid therein may pass through one or
more additional purification means stations before being
returned to the patient or, alternatively, before being
p~c~ again through bioreactor 7. Alternatively, and
depending on the substance to be additionally removed,
W096/09876 ~ S ~ PCT~S94110935
these means could be included within hepatocyte loop 5.
For example, it will be appreciated that for certain
metabolic waste products excreted into the culture medium
by the hepatocytes; e.g., urea, removal means will be best
situated in the hepatocyte loop 5.
As the biochemical states which contribute to or cause
liver failure or damage may vary according to clinical
cause (e.g., hepatitis, alcohol or toxic contamination,
drug overdose) and are not completely known or underætood,
it is not clear what extracorporeal liver functions will
be required for a particular patient. Nor is it clear
which of those functions will be completely or competently
performed by the hepatocytes, and which may have to be
completed or performed by additional means. However,
examples of such means are well known in the medical arts,
are representively depicted in FIGURE 2 as additional
purification means 40 and 50, and may include:
- Adsorbent means for removal of solutes such as urea
onto an activated charcoal column or, coated charcoal
(such as the filter sold under the trade name "GAMBRO")
other resin adsorbents (a commercially available adsorber
is available as the "DT" machine [detoxifier] from Ash
Medical, Inc. West Lafayette, Indiana).
- Conventional dialysis means for removal of small
molecular weight species that are water soluble and not
strongly bound to protein(s).
- Immunoreactive procedures to remove natural IgM
antibodies present in human serum that can react with
porcine xenoantigens. Examples of immunoabsorbent
materials include dialysis membranes sold by VWR
Scientific under the trademarks SPECTRA/POR 6, SPECTRA/POR
7 and SPECTRA/POR l. Lectins attached to SEPHAROSE 6mb
beads can also be used to isolate IgM antibodies (these
beads are commercially available from Pharmacia LkB).
Isolation and purification means may also be used manually
or by automation according to techn;ques well known in the
art; an example of such means is the product sold by
~ r n
W096/09876 ~ C ~ U ~ PCT~S94/10935
- 28 -
Pierce, Rockford, Illinois, under its trademark IMMUNOPURE
~IgM Purification Kit).
- Means for hemofiltration (i.e., means where blood
is passed through an extracorporeal circuit through a
hemofilter by which an ultrafiltrate of uremic toxins is
withdrawn and an equal quantity of uremic-toxin-free blood
is added in proportion the volume of blood via an exchange
element).
The presence or absence of such additional
purification means in either loop will increase or
decrease the time needed to complete a given purification
cycle by the time needed to perform each additional
purification step. It will be appreciated, however, that
these steps may be performed through use of the means
described independent of the inventive system.
It should be understood that sound medical practice
dictates that bioreactor 7 may, and preferably will, be
disposed of and replaced or sterilized following each
purification cycle and will not be used with more than one
patient. It is expected that, depending on the course of
treatment indicated, each purification cycle would last
several hours if performed at a constant rate. In
addition, it will be appreciated by those of ordinary
skill in the art that more than one bioreactor may be
utilized in parallel by providing additional conduit,
fitting and pump means such as those described supra with
respect to the single bioreactor system depicted in
FIGURES 1 and 2.
C. Construction and Operation of a Device For Use With
AttachQd Hepatocytes in the Method of the Invention.
It will be appreciated by those of skill in the art
that the device described supra may be used in detoxifying
blood or plasma with attached hepatocytes. However, where
attached hepatocytes are used, it will not be necessary
to insure that the flow rate of fluid in the hepatocyte
loop is sufficient to facilitate cell aggregation.
Instead, the rate of flow will be such that contact
w096/09876 ~ PCT~S94/10935
- 29 -
between the attached hepatocytes is minimized to avoid
damage thereto. In addition, the attached hepatocytes may
be introduced directly into bioreactor 7 via a
repl~nichment station and/or sampling port in the
hepatocyte loop. To this end, the device may be
simplified as schematically depicted in FIGURE 2.
The use of attached hepatocytes in an artificial liver
system presents the drawbacks discussed supra, such as
decreased surface area for contact between the hepatocytes
and solutes to be metabolized. To compensate for the loss
of cellular surface area, means are provided in this
embodiment of the device to circulate the hepatocytes
within the bioreactor.
This circulation must, however, be performed gently
to avoid contacting the inner surface of bioreactor 7,
thus damaging or destroying the hepatocytes. Because of
this concern, mechanical mixing of the culture medium or
shaking of bioreactor 7 are possible but not preferred
methods of achieving circulation of the hepatocytes within
the bioreactor. Instead, a preferred method of
circulation is the application of an alternating magnetic
field to the bioreactor.
According to this method, a low frequency AC power
supply 31 (e.g., 60 cycles/second) and at least one pair
of opposing coils 32 electrically connected thereto are
used to generate an alternating, low frequency magnetic
field. Coils 32 will preferably be situated opposite one
another and externally to bioreactor 7; preferably, they
will be housed in a nonconductive housing and attached to
opposite external walls of the reactor by any suitable
attachment means. A very moderate circulating flow within
the culture medium will be generated by response of
magnets placed within a percentage of the dextran beads
or other microcarriers 23 to which the hepatocytes are
attached (shown in FIGURE 2 as beads 23A having an "X"
therethrough representative of the magnetic particles
within selected beads; preferably, a magnetic particle
W096/09876 ~ 5 q PCT~S94/10935
- 30 -
will be placed in at least l out of approximately lO
beads). Beads having magnetic particles therein are
commercially available from Dynal, Inc. of Great Neck, New
York and are sold under the trade name DYNABEADS M-450.
Another useful method of achieving circulation while
maximizing contact of solutes with the attached
hepatocytes is achieved using a boundary layer pump. Most
useful with film or sheet (rather than hollow fiber)
membranes, a boundary layer creates a stream of culture
medium which moves along the face of the membrane and
carries attached hepatocytes therewith. Alternatively,
and particularly if the use of hollow fiber membranes is
desired, hepatocytes and culture medium could be
introduced into an axial conduit (not shown) through the
inner diameter of fibers of a sufficient diameter to
accommodate said conduit and passed therethrough using a
pump to generate centripetal force to spiral or circulate
the hepatocytes and medium in the conduit.
Boundary layer pump means, and the use thereof to move
solid articles, are described in U.S. Patent No. 4,335,994
to Gurth. Such pump means (not shown) are available from
Discflo Corporation in Santee, California and are sold
under the trademark DISCFL0 PUMP. Instructions for and
assistance with the attachment and use of the DISCFL0 PUMP
are available from Discflo Corporation.
Except for these modifications, the operation and
structure of this alternative embodiment of the device of
the invention is as disclosed with respect to the
preferred embodiment described su pra .
Examples regarding the use of the inventive devices
and methods are provided below. The examples are intended
to illustrate rather than limit the scope of the
invention. Standard abbreviations (e.g., ~Iml~l for
milliliters, "min" for minute, etc.) are used.
W0 96/09876 ~ PCl`tUS94/10935
-- 31 --
D~A~lPLE I
l~V Vl~ UATION OF T~lE ARTIFICIAL LIVE:R
DBVICE OF FIG~RE 1 ~8ING IlNATTP~l2n HEPA.G~ .~D
Hepatocytes were isolated from Sprague-Hawley rats
and/or farmer's pigs after slaughter using the techn;que
described in Li, et al., lCITB?], 1990. Briefly, the
t~chnique was perfomed using collagenase (0.5~ w/v, type
1) perfusion. The isolated cell population was determined
to have over 80~ viability as assessed by trypan blue
exclusion. Following isolation, the hepatocytes were
suspended in WAYMUTH 752/L culture medium supplemented
with 11.2 mg/l alanine, 12.8 mg/l serine, 24 mg/l
asparagine, 0.168 mg/ml aminolevulinic acid, 0.393 mg/l
dexamethasone, 0.03 mg/l glucagon, 20 units/; insulin, and
84 mg/l gentamicin.
The mixing vessel, bioreactor and biological fluid
loop were filled with culture medium and equilibrated to
a 20% oxygen concentration in a total medium volume of
about 350 ml. Ammonia in a concentration of about 20
ug/ml was added to the biological fluid loop as a model
waste product for metabolism in the artificial liver
device. Approximately 200 million viable rat hepatocytes
were injected into the mixing vessel, while an equivalent
number of porcine hepatocytes were injected into the
mixing vessel of a second device. Circulation of fluid
through the hepatocyte and biological fluid loops was
begun and maintained at a flow rate of about 65 ml per min
in both loops.
The number of rat hepatocytes still viable in the
system after 21 hours is graphically depicted in FIGURE
3, while the number of porcine hepatocytes still viable
after 6 hours (the total test period applied to the
porcine cell containing device) is depicted in FIGURE 4.
Viability was determined by a conventional technique;
i.e., trypan blue exclusion staining.
FIGURES 5 and 6 respectively depict the decrease in
ammonia concentration obtained in the culture medium in
W096/~876 ~ 5 ~ PCT~S94/10935
- 32 -
both devices over a period of 6 hours. As shown in the
FIGURES, a substantial decrease in ammonia concentration
was obtained. It is predictable based on these data and
the data reported in Example II, infra, that the ammonia
concentration in blood or plasma can be reduced in a human
by a factor of about 10%.
EXAMPLE II
IN VIVO EVALUA~ION OF THE ARTIFICIAL LIVER
DEVICE OF FIG~RE 1 UgING UNAT~R~n HEPA~
The device of FIGURE 1 was prepared for use with rat
hepatocytes as described in Example I, except that culture
medium was not introduced directly into the biological
fluid loop. A venous connection between the biological
fluid loop of the device and the femoral veins of a young
male pig (weight=about 26 kg) was established. Blood
flowing from the femoral veins of the pig was perfused
through the bioreactor for about 6 hours at a flow rate
of about 150 ml/min, with continuous return of blood to
the pig.
The vital signs and blood chemistry of the pig were
observed throughout the 6 hour perfusion period.
Hepatocytes were collected from the mixing vessel through
the sampling port periodically and the viability of the
cells evaluated by trypan blue exclusion staining.
FIGURE 5 depicts the percetnage of hepatocytes still
viable during the perfusion period at the time points
indicated in the FIGURE.
FIGURE 6 depicts the decrease in ammonia concentration
measured in blood sampled from the biological fluid loop
of the device at the time points indicated in the FIGURE.
FIGURE 7 depicts the increase in urea concentration
measured in the same blood samples at the same time
points.
3~ As shown in the FIGURES, the observed efficacy of the
system and viability of the hepatocytes circulated through
the system as used in vivo was comparable to the in vitro
W096t09876 ~ 5 ~ PCT~S94/10935
efficacy and viability measurements reported in Example
II. The pig did not suffer any detrimental health effects
attributable to the use of the artificial liver device.
Although preferred embodiments of the inventive device
and methods are disclosed herein, it will be appreciated
by those skilled in the art that modifications may be made
to the embodiments disclosed without departing from the
spirit or scope of the invention, which is defined by the
claims appended hereto.
