JP2739758B2 - Liver assist device using transformed cell line - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Background of the Invention 1.Field of invention   The present invention relates to a human suffering from a disease of liver failure or liver dysfunction.
Or improved extracorporeal liver replacement to assist animal patients
The present invention relates to an auxiliary device and a method of using the same. More specifically, the shape
Transformed hepatocytes (which then revert to their somatic phenotype)
For blood perfusion of patients using a semi-permeable membrane supplemented with
The present invention relates to the auxiliary device devised in the above. 2.Description of conventional technology   Medical science advances in artificial organs in recent years
Brought important research. One example is artificial
Kidney dialysis machines are now relatively commonplace
And is used daily by patients with renal failure. this
In some devices, blood is drawn from the patient and then dialysed.
Together with the dialysis room; generally removed by the patient's kidneys
Impurities in the patient's blood spread through the semipermeable membrane of the dialysis room.
And is removed by the dialysate.   Also, in the past, the body to assist patients with liver failure
External liver assist devices have been proposed. For example, Wolf et al., “Culture
Vibration by artificial liver composed of cells and synthetic capillaries
Lilvin conjugation ",Trans.Amer.Soc.Artif.Int.Organs(197
5), pp. 16-27. In the method of Wolf et al.
-Fiber semipermeable membrane shell-tube cartridge is human
Used as engineered liver. This device is a
From a number of elongated semi-permeable membrane capillaries in a well
Made up of Wolf et al. Reported that hepatocytes in normal liver
A solid tissue mass similar in morphology to the ivo mechanism
Cultured on the outer surface of the capillary membrane to form
Secondary hepatocytes were utilized. Growing on hollow fiber capillaries
These primary hepatocytes were isolated from normal liver of surviving animals.
Complex like bilirubin uptake conjugation and secretion
Had the ability to perform its functions.   Numerous other cell types, such as Chinese Hamster
-Ovarian cells, monkey kidney cells, chicken and mouse
Embryonic fibroblasts, Chang hepatoma cells, W138 cells and Will
Hamster embryo fibroblasts transformed with
The growth on low fiber capillaries was successful. further,
Organs shown by cells grown on hollow fiber capillaries
The function is Chic et al. (Science 187: 847-848 (1975);Tran
s.Amer.Soc.Artif.Int.Organs 21: 8-15 (1975))
Has been proven. This study was performed on hollow fiber capillaries.
Maintenance of pancreatic β-cells in culture and during in vitro culture
The continuous production of insulin by these pancreatic β cells has been established.
It was proof.   Specific literature on the previous study, as well as other statements in the background art
The offering includes: Knazek, R.A. (1974).Fed.Proc.Fed.Am.Soc.Exp.Biol.
33: 1978-81. Knazek, R.A .; Gullino, P.M .; Kohler, P.O; and Dedrick, R.
I. (1972).Science 178: 65-67. Knazek, R.A .; Kohler, P.O .; and Gullino, P.M. (1974).
Exp.Cell Res.84: 251-254. Knisely, M.H .; Reneau, D.D .; and Bruley, D.F. (1969).
Angiology 20: 1-56. Kruse, P.F .; and Miedema, E. (1965).J. Cell. Biol.27:
273. Nettesheim, P .; and Makinodan, T. (1967). In “Method
s in Developmental Biology. "P.471. Crowell-Collie
r, New York. Rose, G.G. (1967).J. Cell. Biol.32: 108. Russ, M.B. (1976) .M.S.Thesis, University of Delawar
e, Newark. Schratter, P. (1974). “Synthetic capillaries for cell culture
tube".Am.lab., October: 33-38. Schratter, P. (1976). “Cell culture with synthetic capillaries”.
Methods in Cell Biol.XIV: 95 Thomlinson, R.H. and Gray, L.M. (1955).Br.J.Cancer
16: 841. Tromwell, O.A. (1958).Exp.Cell.Res.16: 118-147. White, A.; Handler, P.; And Smith, E.L. (1973) .In “Pr
inciples of Biochemistry ", 5th ed.p.905.McGraw-Hil
l, New York. Wolf, C.F.W. (1978).Intern.J.of Art.Organs.1: 45-
51.   Some types of liver assist devices developed by Wolf et al.
Although it has been shown that it can be used, the clinical use of this device
Many critical issues that prevent widespread use
Is left. As described earlier, Wolf et al.
Vesicles were used. These cells do not normally divide, and therefore are
It cannot be transmitted in subculture. As a result, Wolf et al.
The continuous use of primary hepatocytes for clinical use of liver assist devices
Will be required. Such hepatocytes are preferably affected
Should be collected from the same species as
Should be histocompatible with the particular patient
Considering the need for a continuous supply of primary hepatocytes,
Overcoming the repeated use of organ assist devices
Clearly, this can cause very difficult obstacles.
Would.   Therefore, it is advisable to use primary hepatocytes cultured on semipermeable membranes.
It is necessary for patients with hepatic failure while overcoming the practical problems
An improved liver assist device that provides the required liver function and
There is clearly a need in the art for methods and methods. Summary of the Invention   The present invention solves the above-mentioned problems and continues the treatment of patients with liver failure.
Manufacturing and operationally extremely useful for clinical procedures
To provide a highly improved liver assist device with the characteristics of
Offer.   Briefly, the present invention relates to an initially transformed liver cell.
Vesicles (cultured on perfused membranes and then reverted to the somatic phenotype)
Is used). This way
Use of transformed hepatocytes in such transformed cells
Some known properties of (ie, virtually forever
their ability to be continuously subcultured in ro, as well as
Very high growth rates and contact inhibition common to species cells
Disappearance). In a particularly preferred embodiment, the
Temperature-sensitive cells transformed by
You. Papobails such as cyamine virus 40 (SV40)
Is particularly suitable for the transformation of hepatocytes. Transformed
The temperature sensitivity of hepatocytes that have been isolated may
Somatic by simple treatment to increase from 37 ° C to 41 ° C)
To return to the state.   The first transformed hepatocytes are on one side of the semipermeable membrane
Cultivated. In a preferred embodiment, a plurality of interconnected
Shell-tube multiple hollow capillary membrane cartridge
The transformed hepatocytes are located on the outer surface of the capillary membrane.
(Ie, on the shell side). This type of culture is
Initial inoculation of cartridges, followed by circulation of appropriate growth media
Ring (shell side until confluent monolayer of transformed hepatocytes appears)
Circulated through). At this point, the transformed liver
Heat Shock Cells to Return to Somatic Phenotype
Raise the temperature of the circulating growth medium to wake it up
(In a warm water bath with temperature control of the entire shell-tube membrane state)
Can be put in). Then, cell division (proliferation)
No, the cultured cartridge is an extracorporeal liver assist device
Ready to use at any time.   In blood perfusion, the patient's blood is removed and the hepatocyte layer is removed.
Circulates the blood while in contact with another semipermeable membrane surface
Ring. In a preferred embodiment of the invention, a large number of patient blood
Through the lumen of a hollow fiber capillary tube. This message
During the time, molecular species dissolved in the patient's blood (eg,
Rubin) diffuses through semipermeable membrane capillaries and then hepatocytes
It is taken up and metabolized by the layers. Metabolism from hepatocytes
Waste liquid is circulated countercurrently on the shell side of the cartridge.
Are sequentially removed by   When performing the above method, the liver assist device must be
In addition to the membrane cartridges or their equivalent,
Blood from the patient to the perfusion cartridge
Means to further remove the waste
A second fluid circuit leading to the shell side of the cartridge is also included. BRIEF DESCRIPTION OF THE FIGURES   FIG. 1 shows a operatively accessible patient during a liver assist procedure.
Schematic of the overall liver assist device according to the invention, continued
Is;   FIG. 2 shows a preferred multi-cartridge liver assisted perfusion device.
A partial schematic side view showing the configuration of   FIG. 3 is a plan view of the chamber shown in FIG. Description of the preferred embodiment   The concept of cell transformation is generally well known and usually
Functionally (ie, altered by the transformant)
Later by the nature of the cell). Tumor virus
In the case of cell-induced transformation, the transformed cells
Shows the following properties (Enders "Simian virus 40 to human
And hamster kidney cell responses indicate virus
Transformation of cells by "Cell Transformation, p.113-1
52): 1. Changes in growth pattern, ie, loss of contact inhibition; 2. Morphological changes; 3. increase in growth rate; 4. Increased ability to continue serial subculturing; 5. Metabolic changes; 6. Chromosomal abnormalities; 7. reduced ability to support the growth of infectious virus; 8. Prevent re-infection with transformants and other viruses
Increased resistance; 9. emergence of new cellular antigen components; 10. Tumorigenic ability.   Furthermore, the optimal transforming virus according to the present invention, SV
40, induces the following properties in transformed cells (Mole
cular Biology of Tumor Viruses, Part 2: DNA Tumor Vi
ruses,John Tooze, Editor, Cold Spring Harbor Monogra
phs, 10B (1980), Library of Congress Accession No. Q
R 372 06 M64 1980 Part2): Multiplication   High or endless saturation of cell density^a   Different (generally reduced) serum requirements^a   Growth in agar or Methocel suspension ...
Scaffold independent   Tumor formation when injected into susceptible animals   Less susceptible to contact hindrance of movement   Undirected growth^a   Growth of normal cells on a monolayer^a Surface   Increased cohesiveness of plant lectins^a   Changes in composition of glycoproteins and glycolipids   Loss of tight junction   Emergence of fetal antigen   Virus-specific transplantation antigen   Different dyeing properties   Increased nutrient transport speed   Increased secretion of proteases or activators^a Intracellular   Destruction of cytoskeleton   Changes in the amount of cyclic nucleotides Virus evidence   Detectable virus-specific antigen protein   Detection of viral DNA sequences   Presence of viral mRNA   Virus can be extracted in some cases. Transformed cells are those that are not shared by untransformed parent cells.
Shows many, if not all, of the properties.   Some of the above properties marked with a isolate transformed cells
It was the basis for doing so.   In the present invention, the term "transformed hepatocytes" refers to
Primary liver treated with convertible material and exhibiting at least the following properties:
Defined as a cell: (1) increased ability to continue continuous subculture; (2) increased in vitro growth rate; (3) Loss of contact inhibition.   A variety of transformants to achieve transformation
Can be used, but according to the invention
It is preferred to use a conversion material. Papova virus
The group, especially SV40, is advantageous as a transformant.
Human cells are semi-permissive for SV40 infection, i.e.
They infect and support viral replication, but all cells
Does not complete the cell lysis cycle. Cell lysis
The viable cells of the cycle are transformed to achieve the desired growth characteristics.
Show. However, after several months of cultivation, SV40
Continue releasing ruth particles. SV40 is another Papova virus
Has limited tumorigenicity. Course of exposure to SV40
Given that it probably does not cause human tumors
Apparently not, in fact, the only SV40 reported to date
The only relevant human disease is malignant melanoma. a diversity of
Attenuated SV40 mutants are used, especially in the early genes
(A) a group A mutant deficient in function is used,
Induce the synthesis of the SV40 T antigen and replicate host cell DNA.
Stimulates, but under experimental conditions viral DNA or cap
Does not produce sid antigen. (A) This in the gene region
These mutants have the thermolabile T required to maintain transformation.
Produces antigens (which fail at elevated temperatures). Therefore,
Transformation with these temperature-sensitive SV40 mutants is performed at the culture temperature.
It can be reversed simply by raising. When the temperature is raised, the trait
Converted cells produce SV40 viral DNA or SV40 capsid.
Cease production, lose their transforming properties, and
Revert to somatic phenotype. Thus, the temperature sensitive SV40
Transformation is achieved by raising the temperature by several degrees.
Enables switching from a conversion phenotype to a somatic phenotype,
And a large area confluent cell monolayer on the semipermeable membrane support
Use of the growth rate in the transformed state to grow crabs
to enable. Once a confluent cell monolayer is formed,
Lus production is stopped and somatic phenotype due to metabolic perfusion
Is returned.   Transformation of hepatocytes using viral material such as SV40
Is achieved by known techniques. Roughly speaking, the primary liver
Cell cultures are dispersed and enzymatically cultured and then
Infect Rus transformants. And then transformed
Hepatocytes were continuously subcultured to obtain stable transformed cells.
From sterile or transiently transformed cells. That
Thereafter, a cell line for clinical use is established using the stable cells.
Suitable transformation methods are described in detail in the Examples below.
Will be.   After a stable transformed cell line has been established,
Vesicles are cultured on a semi-permeable membrane to make a liver assist device
You. Again, the details of the colony formation method are described in the art.
It is within the scope of the operation, and the specific method is described in
I will explain in detail.   The optimal membrane structure used in connection with the present invention is
A large number of synthetic resin capillaries vertically arranged in the
Multiple (usually three) uprights, including the outermost tubular shell
Tube-shell cartridge. car
Capillary on the shell side of the cartridge (ie, outside the capillary)
To maintain a physical separation from the lumen or interior of the vessel
A suitable seal structure is used for the second step. In addition, cart
Each passing through the ridge tube and shell
Suitable inlets and outlets for continuous flow of fluid
Be killed.   A particularly preferred device of the above type is Massachusetts
Amicon Corporation, Scientific Systems D, Denver
Provided by ivision as model "DC30". This device
From Amicon Corporation for “High Speed Processing of Macromolecule Solutions”
High yield hollow fiber concentration / dialysis system DC30 "
Described in a technical report issued to
The report is incorporated herein by reference. Model DC
30 devices, with appropriate fluid controls,
Includes three upright tube-shell cartridges.
This device from Amicon is capable of diffusing through the capillary wall.
Aspects of those exclusion limits that effectively limit child size
It is possible to install various types of internal capillaries
Wear. For the purposes of the present invention, hollow fiber capillaries
Should have an exclusion limit of 0,000-50,000 daltons,
Lower limits are preferred.   Referring now to the drawings, FIG.
A physical liver assist device 10 is schematically depicted. This equipment
The device 10 has a blood inlet 14, a blood outlet 16,
Includes a membrane assembly indicated by numeral 12 having a liquid outlet 20
No. Apparatus 12 further comprises a liver prepared by the method of the present invention.
And at least one semi-permeable membrane capable of supporting cells. Also,
Assembly 12 consists of blood and blood separated by the aforementioned membrane.
Each channel is provided for a bath and a bath.   The device 10 has a blood inlet line connected to the assembly inlet 14.
In 22 (including conventional pump 24). Blood spill rye
28 is also provided as shown. Inflow lines 22 and
And the outflow line 28 is a suitable arteriovenous implant implanted in the patient's forearm
Operablely connected to fistula 30, 32 (this
Line 22 is connected to the patient's radial artery while
In 28 is connected to the forearm vein). Of course, to patients
Other arteriovenous connections can be used at the doctor's discretion
Would.   Apparatus 10 further includes a bath reservoir 34 into which a bath inlet is provided.
Line 36 is connected and connected to inlet 18. Aseptic solution
Midway on line 36 to circulate through
A conventional pump 38 is disposed. The bath outflow line 40 is shown
To operate the outlet 20 and waste storage 42 so that
Connect in a state.   Referring next to FIGS. 2-3, there is shown at 44.
An improved membrane assembly is illustrated. Ase
The embry 44 is used in the overall system 10 and its
Function as the schematically illustrated assembly 12.
And should understand. In any case, assemble
Lee 44 includes three banks 46, 47 and 48, each bank
Is a five upright tube-shell hollow capillary cartridge
Including 50. Each of these cartridges is the same
It contains many hollow fiber capillaries in it.
The preferred cartridge has an exclusion limit of 30,000 daltons
Amicon having Unit. Assembly 44
Blood inflow line 54 and branch lines 56, 57 and
And 58 (5 cartridges in banks 46, 47 and 48 respectively)
0 is connected to the upper end and supplies blood to the upper end)
And a three-way inlet header 52 having Pressure gauge
60 facilitates monitoring of pressure conditions within the assembly
To the end of the branch line 58 away from the inflow line 54
Continued. A similar trifurcated blood inflow header 62
Inflow headers located close to the bottom of each cartridge 50
-52 connected to the respective banks. H
The hopper 62 includes a common blood outflow line 64 as shown.
Pressure gauge 66 contacts its branch line associated with bank 48.
Continued. Inflow and outflow headers 52 and 62 are
Inside hollow fiber capillaries contained in cartridge 50
Short inflow and outflow pipes 6 connected to the cavity or interior
Connected to 8,70. Therefore, blood from the patient is
Route 54 and branch lines 56, 57 and 58
Passes down the lumen of the capillary contained in the cartridge 50
At this point, the blood passes through header 62 and outflow line 64
Pass through pipe 70 to return to the patient.   The overall assembly 44 also branched into three as well
A bath liquid inlet line 72 having a
Operate on cartridges 50 of each of inks 56, 57 and 58
It is connected in a possible state. In this connection, line 72
Can be connected close to the lower end of the cartridge 50.
You will understand. Outflow of solution is at the top of cartridge 50
Brought by the closely connected outflow line 74
You. Due to the construction of the inflow and outflow lines 72, 74, the bath
Pass through the assembly in countercurrent relation to the liquid flow.
You will understand. In addition, lines 72 and 74 are
Each cartridge should pass through the cartridge outside the tube.
Connected to the shell side of the cartridge 50.   In use of the assembly 44, the inflow line 54 is connected to the patient
Outflow line 64 to the appropriate vein.
Will be continued. Then built into the whole device
Activate pump 24 to draw blood from the patient and
Blood is passed through the assembly from top to bottom. at the same time,
The solution from the reservoir 34 is flushed with the blood flow in countercurrent
(From bottom to top) pass through the shell side of each cartridge
Let For this purpose, the pump 38 is activated.   The following examples relate to the preparation and use of a liver assist device.
3 illustrates preferred details of the invention. Example   This example has a confluent monolayer of hepatocytes on the outer surface.
Liver containing multiple hollow fiber tubes with defined semipermeable membranes
A gut assist device is made.   In the first step, the host hepatocytes are biopsied or
Obtained from donor liver tissue from autopsy specimens
You. Advantageously, the liver tissue is of human origin and subsequent
Minimizes possible immune response problems during the flow procedure
To be histocompatible with the end patient.   Primary hepatocyte cultures were obtained from Seglen,Methods in Cell Biolo
gy,Using a modification of collagenase digestion described in Vol.XIII
Enzymatic dispersion of solid liver tissue
Can be Finely chop a larger sample 1mm^Threeof
The sized tissue mass is treated with Ca containing 0.5 mM EGTA.²⁺Incongruence buffer
Solution (Buffer I, Table I) in 20 ml and stir at 41 ° C for 10 minutes
Incubate while heating. Then, EGTA-buffer was added.
Decant and tissues are washed with wash buffer (buffer III, Table I).
Wash twice. 10mM Ca²⁺Together with 0.05% collagenase (Sigma
Standard buffer (Buffer II, Table I) containing 20m
l and incubate the tissue at 41 ° C for 60 minutes.
To Thereafter, the dissociated cells are removed using a pipette.
Remove every minute and place in an ice-cooled 250 ml flask.
After each 10 minute interval, leave an additional 20 ml of dissociation medium.
In addition to the existing non-dissociated tissue, incubate while stirring.
Continue the session. After collecting each aliquot of dissociated cells,
Add 2 ml of FCS (fetal calf serum) to the cell suspension.   After dissociation of the tissue, the cell suspension is transferred to a sterile nylon stocking
And place in two 50 ml sterile centrifuge tubes.
Divide by amount. The cells are centrifuged at 50g for 2 minutes and dissociation medium
Decant and wash this centrifuge tube with 10% FCS
Add 10 ml of buffer (Buffer III, Table I). Precipitated cells
Is gently resuspended and centrifuged again at 50 g for 2 minutes. Supernatant
And the cells are washed with wash buffer (buffer III, Table I) 10
Resuspend in ml. Remove 0.1 ml aliquot and wash
0.8 ml of buffer (buffer III, Table I) and 0.4% trypanb
Add to 0.1 ml of roux dye. The sample is sent to a hemocytometer.
The number of viable (gold) cells and the number of non-viable (blue) cells.
Measure. Determine cell yield and percent viability. Remaining
The cells are centrifuged at 50 g for 2 minutes, the supernatant is decanted, and 10% FCS
Containing Leibowitz L-15 medium in a sufficient amount,
5 × 10⁶Dilute to viable cells. In this medium
Gently suspend the cells and aliquot the plastic
Transfer to bird dish.   3 ml of suspension (about 4 × 10⁶Live cells)
Tissue culture dishes (6 mm) are humidified (ie, saturated) empty
Incubate at 37 ° C. in air environment. This incube
During the incubation period, some of the inoculated cells
After attachment and incubation, the non-adherent cells
Discard by letting it out. 10% in each culture dish
3 ml fresh aliquot of Leibowitz L-15 medium with FCS
And add the culture to the same conditions as for the initial incubation.
Incubate for another 24 hours under this condition
During adherence, adherent cells flatten to form a monolayer
I do.   Cultured monolayer primary hepatocytes were from Leibo with 10% FCS.
tsA28 SV40 virus (cells) suspended in witz L-15 medium
Per 10⁶−10⁷Virus particles). Will
90 minutes at 37 ° C in the above humidified air environment for adhesion
After the incubation, release the non-adherent virus, and
3m fresh aliquot of Leibowitz L-15 medium with FCS
Add 1 to each culture dish. After that, cells are
While adding 3 ml aliquots every 24 hours
Until the colonies of the cells begin to appear (usually
Continue the culture.   When identifying plaques of transformed cells, clones
Trypsin-dispersed, then stable transformed cells
(That is, the ability to continue long-term continuous subculture
From sterile or transiently transformed cells
Subculture continuously using conventional techniques to select
You. Then, use stable transformed cells to grow and
Of high FCS rate and FCS concentration in growth medium
A cell line with reduced (1%) is established. SV if desired
The continued shedding of 40 virions was SV40
L-supplemented with anti-SV40 serum from animals immunized against
By culturing stable transformed cells in 15 media
Can be suppressed.   A stable transformed cell line is essentially immortal
It can be maintained indefinitely by culture. Liver assist device
When using this transformed cell line,
Inoculate the fiber tube bundle. For this, Wolf et al.
“For an artificial liver composed of cultured cells and synthetic capillaries
Bilirubin conjugation ",Trans.Amer.Soc.Artif.Int.Organ
s,(1975, pp. 16-27). W
This article by olf et al. is incorporated herein by reference.   Basically, this technique is a reflux method, in which
Pre-determined tube-shell semipermeable membrane cartridges are sterile
And transfer the primary hepatocytes to the shell side of each cartridge.
(Ie, growth on the outer surface of the semipermeable tube)
Inoculate (for example, 10^Four−10⁷cell). in this case,
A suitable growth medium for transformed hepatocytes is
Of inoculated cartridges for growth-promoting contact
Circulated through the lure side. In fact, the released SV40
Prevents transfer of ills particles into capillary lumen and reduces blood tamper
Capillaries for proteins secreted by dendrites and hepatocytes
30,000 daltons to prevent passage through the membrane barrier
Above with horror fiber tube with removal limit
Amicon Hollow fiber cartridges are used
You. Carefully select the exclusion limit of the capillary membrane to remove virus particles
Strict separation of offspring and dissolved proteins
Virus transmission in the elderly and patient immunity to host hepatocytes
The likelihood of a reaction is reduced.   Transformed hepatocytes during inoculation and reflux culture
Form colonies outside hollow fiber capillaries
You. Advantageously, 10 for each cartridge^Four−10⁷Hepatocytes
Inoculate and maintain the circulating growth medium used at a temperature of 37 ° C.
In Leibowitz L-15 medium supplemented with 1% fetal bovine serum
is there. When a confluent cell monolayer is formed (usually
8 days after circulating the growth medium), raise the temperature of the circulating growth medium.
Tube-shell in a warm water bath maintained at 41 ° C.
By immersing the device, the incubation temperature
Increase to 41 ° C. This restores transformed cells to the somatic phenotype.
Stop virus replication and cellular DNA replication
And resume the nature of the contact inhibition. Confluent single layer
The amount of time it takes to form a seed depends essentially on the amount of inoculum
Is done. Growth of human cells transformed with TsA28 SV40
Maximum confluency of about 120,000 cells / cm^TwoAnd this density
Is usually 4,000 cells / cm^TwoAchieved in about 6 days with inoculum
it can. The number of cells in the inoculum is the full table of capillary fibers
Determined by area, and the time to reach maximum confluence is
Determined by the number of cells in the inoculum. 9,00 total surface area
0cm^TwoFor Amicon capillary fiber cartridges,
5 × 10 in total⁷The inoculum of cells is assumed to have a plating efficiency of 40%.
Equipment that can be used after 6 days of incubation.
Will result in Doubling the volume of inoculum
The incubation time will be reduced by about one day.
Increasing the volume of inoculum by a factor of 4 during incubation
The time will be reduced by another day. Above confluence
Indicates overgrown monolayer, confluent monolayer is acceptable
This is the smallest possible single-layer profile. In particular, the nutrient medium (Sanawa
That is, Leibowitz L-15 and the patient's blood)
When passing the contralateral side, the overgrown monolayer (ie,
A thickness of two or more layers) would be an ideal condition. Hollow
Of somatic phenotypes grown on fiber capillary supports
A stable confluent monolayer of hepatocytes is then
Liver machine by perfusing the patient's blood through the burr lumen
Used to assist in performance.   The most preferred specific inoculation protocol is described below.
RU: 1. Sterile Amicon with 30,000 dalton exclusion limit
The cartridge is fully loaded in the space outside the capillary of the cartridge
Until 1
Fill the Leibowitz L-15 medium. 2. Place the cartridge in the extra-capillary space
5 × 10⁷Add inoculum of transformed hepatocytes. 3. To evenly distribute the suspended cells over the outer surface of the capillary
Stir gently to allow the cells to settle on the outer surface.
Let 4. After 4 hours, place the cartridge on the roller rack
And rotate the cartridge very slowly at 1 rpm to prevent adhesion
Redistribute the cells onto the outer capillary surface. Rotation speed is 20 hours
Should be reduced to 1/5 rpm. 5. After the initial 24-hour application period, lower the cartridge.
Remove from the rack and remove 1% FCS from Leibowitz L-
15 Large medium storage tank and medium through extra-capillary space
Connect to pump for circulation. 6. After that, 5 × 10⁷form
Add an additional inoculum of transgenic hepatocytes and cover the entire outer surface of the capillary.
Stir gently to evenly distribute the suspended cells
You. After a settling / adhering period of 4 hours, use the pump system to store
The medium from the storage tank is slowly circulated through the extra-capillary space.
Let 7. Using a pump system, culture medium at about 500 ml / min for another 5 days
Is continuously circulated while the hollow fiber capillary
Colonies are formed on the outer surface and confluence of transformed hepatocytes
A monolayer will cover all of these surfaces. Bird
That is, this medium is 0.06 ml / min / cm^Two(Capillary surface area) speed
Degree, i.e. about 500 ml / min for each HP10 cartridge
Should be cycled in degrees. At the same time, the same solution at the same flow rate
It should circulate countercurrently through the capillary lumen. 8. The cartridge is then placed in a 41 ° C water bath and the medium is
Circulate through the outer capillary space of the cartridge at 41 ° C.
This causes the hepatocytes to revert to their somatic phenotype. Sa
24 hours later, 1% FCS and 5mg% (5mg / 100ml)
0.06 ml / min / cm of Leibowitz L-15 containing rubin^TwoWith hair
Pump through the tubule lumen. This is a new return
Hepatocytes are provided with a substrate to be conjugated. Then on the shell side
The bath medium was used to determine the efficacy of metabolic support
Will be analyzed for the presence of the bin conjugate. Hitota
Once the function and functionality have been determined, flush the capillary lumen with buffered saline.
To make the cartridge ready for use.
You. Advantageously, these cartridges are interconnected.
Thus, a multiple cartridge device of the type described above is produced.   In perfusion, the patient's blood is first coagulated in an auxiliary device.
Measures are taken to prevent settling. With liver failure coagulation disorder
For patients who do not have an initial dose of 300 units / kg (body weight) of heparin
Heparin is administered by injecting the initial dose. Perfusion
In order to maintain activated clot formation time of 400 seconds or more,
Additional heparin titration can be used if desired.
Immediately before stopping perfusion, injection of protamine sulfate and
Antagonize phosphorus administration. Coagulation disorders (secondary disorders of liver failure)
If you have a recent abdominal surgery
Heparin administration is used. In this way, heparin
Added to the import circuit of liver assist devices for anticoagulation,
Protamine sulfate is added to the export circuit to antagonize it.
available.   The actual perfusion protocol is similar to conventional bypass perfusion
Is achieved using the techniques described above. Silicone rubber tubular flow divider
The radial artery to establish a luminal blood flow of 350cc / min
And placed between the veins. In addition, the radial artery and forearm vein
Created surgically by side-to-side anastomosis
Arteriovenous fistula also develops after full maturation of the surgical anastomosis
Can be used.   In the case of human perfusion, it passes through the extracorporeal liver assist device of the present invention
Preferably, the blood flow is about 100-350 cc / min. This
Blood flow like that of the membrane tube
Through the lumen of the membrane tube to make contact with the surface of the
Introduced (ie through the tube side of the cartridge)
Is done. Generally, supplemental hepatic perfusion is performed 3-5 times a week,
Allow blood to pass through this device for approximately 4-10 hours
Should.   At the same time blood flows into the device, the bath
Circulate countercurrently through the shell side of the cartridge for contact
Ring. The bath is preferably supplemented with 1% fetal bovine serum.
Leibowitz L-15 medium fed and maintained at a temperature of about 41 ° C
It is.   During perfusion, the amount of bilirubin dissolved in the patient's blood
Such molecules pass through the capillary membrane with nutrients from the blood.
Will spread to the hepatocyte layer. Then bilirubin
And other molecular species are taken up by hepatocytes and metabolized (bilirubin
Is conjugated), and the waste products are then lysed by hepatocytes.
It will be discharged to the shell side of the cartridge. Like this
Cultured hepatocytes are usually the complex that normal liver plays in vivo.
Will perform miscellaneous functions.   In addition to general liver function assistance in cases of chronic liver failure,
To make up for metabolic capacity lost by genetic disorders
Give the family an unimpaired metabolic capacity
Hypercholesterolemia, systemic gangliosidosis
Tay-Sach's Syndrom
e) and a wide range of other storage diseases similar to I-cell disease
Alternative uses to reduce clinical symptoms associated with genetic metabolic disorders
The device of the present invention is also used.

   ────────────────────────────────────────────────── ─── Continuation of front page    (72) Inventor Hart, Paul Buoy               66048, Lee, Kansas, United States               Wunworth, North Esplanade               603                (56) Reference US Pat. No. 4,675,002 (US, A)

Claims (1)

(57) [Claims] A first surface having a monolayer of hepatocytes and a second surface opposite thereto, and a solution for removing waste products while contacting the blood with the second surface while contacting the hepatocytes; For use as a liver assist device including means for passing through,
A method for producing a semipermeable membrane, comprising preparing a population of primary hepatocytes, transforming the hepatocytes to proliferate the primary hepatocytes in vitro and reduce contact inhibition thereof, Culturing the transformed hepatocytes on the first surface of the membrane until a monolayer is formed on the first surface of the membrane, and returning the cultured transformed cells to their somatic phenotype. How to make a film. 2. 2. The hepatocyte is transformed by a virus.
The described method. 3. The method according to claim 1, wherein the culturing step comprises the steps of sowing the transformed hepatocytes on the first surface and passing a culture medium for promoting the growth of the transformed hepatocytes into contact with the hepatocytes. 4. A first surface having a monolayer of hepatocytes and a second surface opposite to the first surface, and a solution for removing waste products at the same time as passing the blood in contact with the second surface; A semipermeable membrane for use as a liver assisting device including means for passing, provided that a monolayer of hepatocytes prepares a population of primary hepatocytes, proliferates primary hepatocytes in vitro and reduces contact inhibition thereof Transforming the hepatocytes to form a monolayer of transformed hepatocytes on the first surface, and culturing the transformed hepatocytes on the surface until a monolayer of transformed hepatocytes is formed on the first surface; Characterized by the step of reverting to a somatic phenotype. 5. A liver assist device for extracorporeal blood treatment for assisting an organism suffering from liver failure or liver dysfunction, the device comprising a semi-permeable membrane having a first surface and a second surface opposite to the first surface, Means for passing a monolayer of hepatocytes on one side and blood drawn from said organism while contacting the second side while simultaneously passing a solution for removing waste products in contact with the hepatocytes; A liver assisting device comprising using, as a hepatocyte layer, primary hepatocytes that were first transformed to grow hepatocytes in vitro and reduce their contact inhibition, and then returned to a somatic phenotype.