[go: up one dir, main page]

WO1988006467A1 - Dispositif d'assistance hepatique utilisant des lignes de cellules transformees - Google Patents

Dispositif d'assistance hepatique utilisant des lignes de cellules transformees Download PDF

Info

Publication number
WO1988006467A1
WO1988006467A1 PCT/US1987/000405 US8700405W WO8806467A1 WO 1988006467 A1 WO1988006467 A1 WO 1988006467A1 US 8700405 W US8700405 W US 8700405W WO 8806467 A1 WO8806467 A1 WO 8806467A1
Authority
WO
WIPO (PCT)
Prior art keywords
hepatocytes
transformed
blood
membrane
face
Prior art date
Application number
PCT/US1987/000405
Other languages
English (en)
Inventor
Joseph M. Viles
Paul V. Hart
Original Assignee
Viles Joseph M
Hart Paul V
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to US06/803,564 priority Critical patent/US4675002A/en
Priority claimed from US06/803,564 external-priority patent/US4675002A/en
Application filed by Viles Joseph M, Hart Paul V filed Critical Viles Joseph M
Priority to JP62501627A priority patent/JP2739758B2/ja
Publication of WO1988006467A1 publication Critical patent/WO1988006467A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/067—Hepatocytes
    • C12N5/0671—Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
    • A61M1/3687—Chemical treatment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
    • A61M1/3687—Chemical treatment
    • A61M1/3689—Chemical treatment by biological cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention is broadly con ⁇ cerned with improved extracorporeal liver assist devices and methods designed to support a human or animal patient suffering from hepatic failure or insufficiency. More particularly, it is concerned with such an assist device designed for perfusion of the patient's blood, and wherein use is made of a semipermeable membrane supplemented with trans- formed hepatocytes subsequently reverted to the somatic phenotype thereof.
  • liver assist device Any clinical use of a Wolf et al. liver assist device would require a continuing supply of primary hepa ⁇ tocytes.
  • hepa ⁇ tocytes should preferably be taken from the same species as patient, and should moreover be histo- compatible with the specific patient being treated, it will become apparent that the requirement for a continuing supply of primary hepatocytes presents a daunting if not insurmountable obstacle to re ⁇ peated use of such liver assist device.
  • liver assist device and method which provides needed liver function for patients suffering from hepatic failure while overcoming the practical problems associated with proposals making * use of primary hepatocytes cul- tured onto semipermeable membranes.
  • the present invention largely overcomes the problems noted above, and provides a greatly improved liver assist device having production and operational characteristics making it extremely useful for ongoing, clinical treatment of patients suffering from hepatic failure or insufficiency.
  • the present invention is based upon the principle of utilization of initially transformed hepatocytes cultured onto a perfusion membrane, and then reverted to the soma ⁇ tic phenotype thereof.
  • Use of transformed hepato ⁇ cytes in this fashion takes advantage of certain known properties of such transformed cells, i.e., their ability to be serially subcultured in vitro virtually forever, and the characteristic high proliferation rate and loss of contact inhibition common to such cells.
  • use is made of virally transformed cells which are temperature sensitive.
  • Papovavirus species such as Simian Virus 40 (SV40) are particu ⁇ larly preferred for hepatocyte transformation.
  • the temperature sensitivity of the transformed hepato ⁇ cytes also permit reversion to the somatic state by the simple expedient of elevating the hepatocytes a few degrees (e.g., from 37 to 41° C).
  • Initially transformed hepatocytes are cultured onto one face of a semipermeable membrane.
  • a plurality of interconnected, tube and shell multiple hollow capillary membrane cartridges are employed, and the transformed hepa ⁇ tocytes are cultured on the exterior surfaces (i.e., the shell side) of the capillary membranes.
  • Such culturing involves an initial inoculation of the cartridge(s) followed by circulation of an appropriate growth medium through the shell side thereof until a confluent monolayer of transformed hepatocytes is developed.
  • the temperature of the circulating growth medium is elevated (and the entire tube and shell membrane device may be placed bodily within a temperature regulated warming water bath) in order to thermally shock the transformed hepatocytes and cause rever ⁇ sion thereof to the somatic phenotype. Thereafter, cell division (proliferation) ceases, and the cultured cartridges are ready for use as an extra ⁇ corporeal liver assist device.
  • the patient's blood is withdrawn and passes into con ⁇ tact with the face of the semipermeable membrane remote from the hepatocyte layer.
  • this involves passing the patient' s blood through the lumen of the plural hollow fiber capillaries.
  • molecular species dissolved in the patient's blood such as bilirubin
  • the overall liver assist device includes, in addi ⁇ tion to the cultured membrane cartridges or their equivalent, means for passing the patient's blood in serial fashion from the patient into and through the perfusion cartridges, and also a second fluid loop for passage of bathing fluid into and through the shell sides of the cartridges in order to remove wastes therefrom.
  • Figure 1 is a schematic representation of an overall liver assist device in accordance with the invention, shown operatively connected to a patient during a liver assist treatment;
  • Fig. 2 is a partially schematic side view illustrating the construction of a preferred multi ⁇ ple-cartridge liver assist perfusion assembly
  • Fig. 3 is a plan view of the chamber depicted in Fig. 2. Description of the Preferred Embodiments
  • Altered growth pattern i.e. , loss of contact inhibition, - j r
  • Altered morphology Altered morphology
  • SV40 transforming virus Q in accordance with the present invention
  • SV40 induces the following particular properties -in transformed cells (Molecular Biology of Tumor ' Viruses, Part 2: DNA Tumor Viruses, John Tooze, Editor, Cold Spring Harbor Monographs, 10B (1980), 5 Library of Congress Accession No. QR 372 06 M64 1980 Part 2): Growth high or indefinite saturation density different, usually reduced, serum requirement growth in agar or methocel suspension--anchor- c age independence tumor formation upon injection into suscepti ⁇ ble animals not susceptible to contact inhibition of move ent growth in a less-oriented manner growth on monolayers of normal cells
  • Transformed cells show many, if not all, of these properties, which are not shared by untrans- formed parental cells.
  • transformed hepa tocyte is defined as primary hepatocytes which have been treated wi th a trans- 35 formant and which exhibi t at leas t the following properties : (1) Increased capacity to persist in serial subcultures ;
  • the papova- virus group and particularly SV40 , is preferred as a transforming agent.
  • Human cells are semipermis- sive of SV40 infection, that is, they can be in ⁇ fected and they support viral replication, but they do not all complete the lytic cycle. The survivors of the lytic cycle are transformed and exhibit the desired growth characteristics; however, they do continue to release SV40 virions after months of culture.
  • SV40 has a much more restricted oncogenic potential than other papovaviruses ; Considering the history of the exposure to SV40, it would seem probable that it is not oncogenic in humans, and indeed to date the only reported case of SV40 associated human disease is a single case of malig ⁇ nant melanoma.
  • a wide variety of attenuated SV40 mutants are available, particularly group A mutants which are defective in early gene (A) function; they induce SV40 T antigen synthesis and stimulate the replication of host cell DNA, but fail to. produce any viral DNA or capsid antigens under certain experimental conditions. These mutants in the (A) gene region produce a heat labile T anti ⁇ gen, necessary to maintain transformation, which ceases to function at elevated temperatures.
  • Transformation by these temperature sensitive SV40 mutants is therefore reversible by simply increas ⁇ ing the temperature of the culture.
  • the transformed cells cease to produce SV40 viral DNA or SV40 capsids, lose their transformed characteristics, and revert to the original somatic phenotype. Transformation with temperature sensitive SV40 thus permits switching from the transformed phenotype back to the somatic phenotype by elevating the temperature a few degrees; this then allows the utilization of the proliferation rate of the transformed state to grow rapidly large area confluent monolayers on a semipermeable membrane substrate, whereupon virus production can be stopped and reversion effected to the somatic phenotype for metabolic perfusions.
  • Transformation of hepatocytes using viral agents such as SV40 is accomplished using known techniques. Broadly speaking, primary hepatocyte cultures are enzymatically dispersed and cultured, followed by Infection with the viral transformant. Thereafter, the transformed hepatocytes are serial ⁇ ly subcultured to select the stably transformed cells from abortive or transiently transformed cells. The stable cells are then used to establish a line for clinical use. Specific details of a preferred transformation procedure are set forth in the following Example.
  • the cells can be cultured onto semipermeable membranes to form a liver assist device.
  • a liver assist device for example, the specific details of such a colonization procedure are within the skill of the art, and an exemplary method is set forth in the Example.
  • the most preferred membrane structure for use in connection with the present invention com ⁇ prises a plurality (usually three) of upright tube and shell cartridges containing an outermost tubu ⁇ lar shell along with a plurality of vertically oriented synthetic resin capillaries within each shell.
  • Appropriate sealing structure is provided for maintaining the shell side of the cartridges (i.e., exteriorly of the capillaries) physically separate from the lumen or Interior of the capil ⁇ laries.
  • appropriate input and output ports are provided for continuous flow of respec ⁇ tive fluids through the tube and shell portions of the cartridges.
  • Model DC30 One particularly preferred apparatus of the type described is produced by the Amicon Cor ⁇ poration, Scientific Systems Division, of Danvers , Massachusetts as Model “DC30.” This device is described in a technical bulletin published by Amicon Corporation entitled “High-Yield Hollow Fiber Concentration/Dialysis System for Rapid Processing of Macromolecular Solutions DC30” ; this bulletin is hereby expressly incorporated by refer ⁇ ence into the present application.
  • the Model DC30 device comprises three upright tube and shell cartridges of the type described, together with appropriate and fluid control devices.
  • the Amicon devices can be provided with various types of internal capillaries, in terms of their exclusion limit, which effectively limits the size of mole ⁇ cules permitted to diffuse through the capillary walls.
  • the hollow fiber capillaries should have an exclusion limit of between 30,000 and 50,000 daltons, with the lower limit being preferred.
  • an overall liver assist device 10 in accordance with the invention is schematically depicted in Fig. 1.
  • the device 10 Includes a membrane asssembly broadly referred to by the numeral 12, providing a blood input 14, a blood output 16, a bathing solution inlet 18 and a bathing solution outlet 20.
  • the device 12 further Includes at least one semiperme ⁇ able membrane capable of supporting hepatocytes 0 prepared in accordance with the methods of the present invention.
  • the assembly 12 is provided with respective flow paths for the blood and bathing solution, which paths are separated by the described membrane.
  • ⁇ ⁇ - The device 10 further includes a blood input line 22 which is operatively coupled to assembly input 14, and includes a conventional pump 24.
  • a blood output line 28 is also provided as illustrated. Input line 2'2 and output line 28 are
  • 25 arterial-venous connections to the patient could be fashioned, depending upon the discretion of the treating physician.
  • the device 10 further includes a bathing fluid reservoir 34 with a solution input line 36
  • a conventional pump 38 is interposed within line 36 for purposes of circulating fluid through assembly 12.
  • a solution output line 40 is operatively coupled between output 20 and a waste reservoir 42
  • Figs. 2-4 illustrate a modified membrane assembly broadly denoted with the numeral 44. It is to be understood in this respect that the assembly 44 could be used in the overall system 10, and in that event would function as the schematically depicted assembly 12. In any event, the assembly 44 com ⁇ prises three banks 46, 47 and 48 each containing five upright tube and shell hollow capillary cart ⁇ ridges 50. Each of these cartridges is identical and includes a large number of hollow fiber capil ⁇ laries therewithin; the preferred cartridges are Amicon ® units having a 30,000 dalton exclusion limit.
  • the assembly 44 further includes a tri- furcated input header 52 having a common blood input line 54 and branch lines 56, 57 and 58 re ⁇ spectively operatively coupled to and feeding the upper ends of the cartridges 50.in each bank 46, 47 and 48.
  • a pressure gauge 60 is operatively con ⁇ nected to the end of branch line 58 remote from input 54, in order to facilitate monitoring of the pressure conditions within the assembly.
  • a similar trifurcated blood output header 62 is located adjacent the bottoms of the respective cartridges 50, and is operatively connected to each bank thereof in the same manner as input header 58.
  • the header 62 includes a common blood output line 64 as illustrated, and a pressure gauge 66 coupled to the branch thereof associated with bank 48.
  • the input and output headers 52 and 62 are operatively con ⁇ nected to short input and output pipes 68, 70 to the lumen or interior of the hollow fiber capil ⁇ laries contained within each cartridge 50.
  • blood from a patient passes through input 54 and the branches 56, 57 and 58 for passage downwardly through the lumen of the capillaries contained within the respective cartridges 50, whereupon such blood passes outwardly through the pipes 70 for return to the patient through header 62 and output line 64.
  • the overall assembly 44 also includes a bathing solution input line 72 which is likewise of trifurcated construction, and is in turn operative ⁇ ly connected to each of the cartridges 50 of the separate banks 56, 57 and 58. It will be noted in this connection that the line 72 is connected to the cartridges 50 adjacent the lower ends thereof. Output for the bathing solution is provided by means of an output line 74 adjacent the upper ends of the cartridges 50 and operatively connected to each of the latter.
  • the lines 72, 74 are coupled to the shell sides of the respective cart ⁇ ridges 50, i.e., so that bathing solution passes through the cartridges exteriorly of the tubular capillaries.
  • the line 54 would be operatively connected to the artery of a patient, whereas the line 64 would be connected to an appropriate vein.
  • the pump 24 associated with the overall apparatus would then be activated, in order to withdraw blood from the patient and pass the same through the assembly, with blood flow being from top to bottom.
  • bath ⁇ ing solution from reservoir 34 would be passed through the shell sides of the respective cart ⁇ ridges in countercurrent relationship to the blood flow, i.e., from bottom to top.
  • pump 38 would be activated.
  • Example sets forth the presently preferred details of the invention in terms of preparation and use of the liver assist device.
  • a liver assist device which includes a plurality of hollow fiber tubes defining semipermeable membranes having confluent monolayers of hepatocytes on the exteni- ous surfaces thereof.
  • host cell hepatocytes are prepared from donor liver tissue derived -from biopsy or autopsy specimens. Advan ageously, the liver tissue is human, and is histocompatible with the ultimate patient to minimize any possible immune response problems during subsequent perfu ⁇ sion procedures..
  • Tissue blocks of 1 mm. cubed, derived by mincing larger samples are immersed in 20 ml. of Ca -free buffer (Buffer 1, Table I) containing 0.5 mM EGTA and incubated at 41° C. with agitation for 10 minutes. The EGTA-buffer solution is then decanted and the tissue rinsed several times with wash buffer (Buffer III, Table 1) . 20 ml.
  • Buffer III wash buffer
  • the cell suspension is filtered through sterile nylon stocking and divided equally into two 50 ml. ster ⁇ ile centrifuge tubes. The cells are centrifuged for two minutes at 50g, the dissociation medium decanted and a 10 ml. aliquot of wash buffer (Buf ⁇ fer III, Table 1) with 10% FCS added to the centri ⁇ fuge tube. The pelleted cells are gently resus-- pended and centrifuged again for two minutes at 50g. The supernatant is decanted and the cells resuspended again in 10 ml. wash buffer (Buffer III, Table 1). A 0.1 ml. aliquot is withdrawn and added to 0.8 ml.
  • wash buffer Buffer III, Table 1
  • wash buffer Buffer III, Table 1
  • trypan blue dye 0.1 ml. 0.4% trypan blue dye.
  • a sample is placed on a hemocytometer and the number of viable (gold) cells and the number of nonviable cells (blue) determined. Cell yield and percentage viability are calculated. The remainder is centri ⁇ fuged for two minutes at 50g. the supernatant decanted and sufficient Liebowitz L--15 medium with
  • the cultured monolayers of primary he- patocytes are then superinfected with tsA28 SV40 virus (10 -10 virus particles per cell) suspended in Liebowitz L-15 medium with 10% FCS added. After 90 minutes incubation at 37° C. for virus attach ⁇ ment in the humidified air environment described previously, the unattached viruses are poured off and fresh aliquots (3 ml.) of the Liebowitz L-15 medium supplemented with 10% FCS are added to each dish. The cells are then kept in culture with fresh aliquots (3 ml.) of the described medium added every 24 hours until the foci of transformed cells begin to appear, which usually will take about 1 week.
  • the clones Upon identification of plaques of trans ⁇ formed cells, the clones are dispersed with trypsin and are serially subcultured using conventional techniques to select out the stably transformed cells (i.e., those which possess the capacity to persist in prolonged serial subculture) from the abortive, or transiently transformed cells.
  • the stably transformed cells are then used to establish a cell line of high growth and division rates with reduced requirements (1%) for FCS concentration in the growth medium. If desired, continued shedding of SV40 virions may be suppressed by culturing the stably transformed cells in the L-15 medium supple ⁇ mented with anti-SV40 serum derived from animals immunized to SV40.
  • the stably transformed cell lines are essentially immortal and can be maintained inde ⁇ finitely in cell culture.
  • the transformed cell line is used to inoculate hollow fiber tube bun ⁇ dles; the method of Wolf et al., "Bilirubin Con ⁇ junction by an Artificial Liver Composed of Cul ⁇ tured Cells and Synthetic Capillaries", Trans. Amer. Soc. Artif. Int. Organs, (1975, pages 16-27) may be employed for this purpose.
  • the Wolf et al. paper is expressly incorporated herein by refer ⁇ ence.
  • the technique involves a circumfusion procedure wherein the selected tube and shell semipermeable membrane cartridge(s) such as those described above are sterilized, and the primary hepatocytes inoculated (e.g., 10 -10 cells) into the shell side of each of the cart ⁇ ridge ⁇ ) i.e., for growth on the exterior surfaces of the semipermeable tubes). Thereupon, an appro ⁇ priate growth medium for the transformed hepato ⁇ cytes is circulated through the shell sides of the seeded cartridge(s) for .growth-promoting contact with the hepatocyte.
  • the primary hepatocytes inoculated e.g. 10 -10 cells
  • an appro ⁇ priate growth medium for the transformed hepato ⁇ cytes is circulated through the shell sides of the seeded cartridge(s) for .growth-promoting contact with the hepatocyte.
  • the Amicon ® hollow fiber cartridges described above are em ⁇ ployed, with the Inner hollow fiber tubes having an exclusion limit of 30,000 daltons in order to prevent exchange of released SV40 virions into the inner capillary lumen and prevent exchange of blood proteins and hepatocyte-secreted proteins across the capillary membrane barrier.
  • Rigorous separa ⁇ tion of virions and dissolved proteins by careful selection of exclusion limits of the capillary membrane reduces the possibility of virus transmis ⁇ sion to the patient and any patient immune respons ⁇ es to the host hepatocytes.
  • the transformed hepatocytes colo ⁇ nize the outside of the hollow fiber capillaries.
  • each cartridge is inoculated with from 10 4-107 hepatocytes
  • the circulating growth medium employed is Leibowitz L-15 medium supplemented with 1% fetal calf serum maintained at a temperature of 37° C.
  • the incubation temperature is raised to 41° C. by elevation of the temperature of the circulating growth medium and immersion of the tube and shell device into a warming water bath maintained at 41° C. This causes the transformed cells to revert to the somatic phenotype, cease virus replication, cease cellular DNA replication, and resume contact inhi ⁇ bition characteristics.
  • the amount of time needed to achieve a confluent monolayer basically depends upon the size of the inoculum.
  • the maximum density to which TsA28 SV40 transformed human cells will 2 proliferate is about 120,000 cells/cm , and this density can normally be achieved in about 6 days with an inoculum of 4,000 cells/cm .
  • the number of cells in the inoculum is determined by the total surface area of the capillary fibers and the time required to reach maximum density is determined by the number of cells in the inoculum.
  • an inoculum of a total of 5 x 107 cells assuming a 40% plating efficiency, will yield a usable device after 6 days of incubation. Doubling the size of the inoculum will reduce incubation time by about 1 day.
  • a four-fold in ⁇ crease in the size of the inoculum will reduce incubation time by about another day.
  • the maximum density noted above represents a condition of an overgrown monolayer, the confluent monolayer being a minimal acceptable configuration; an overgrown monolayer, i.e., two or more monolayers thick would be an ideal condition, particularly since nutrient media, i.e., Leibowitz L-15 and the patient's blood, would bath opposite side of the cell layer.
  • This stable, confluent monolayer of somatic pheno ⁇ type hepatocytes grown on the hollow fiber capil ⁇ lary substrate can then be used to supplement liver function by perfusion of the patient's blood through the capillary fiber lumen.
  • the cartridges can be removed from the roller rack and connected to a large reservoir of Leibowitz L-15 medium with 1% FCS and a pump to circulate the medium through the extracapillary spaces.
  • the pumping system will then continuously recirculate the medium for an additional five days at about 500 ml/minute, during which time colonization of the outside of the entire surface area of the hollow fiber capillaries will occur and a confluent monolayer of trans ⁇ formed hepatocytes will cover all of those surfaces.
  • this medium should be 2 circulated at a rate of 0.06 ml/min/cm (cap ⁇ illary surface area), i.e. about 500 ml/min for each HP10 cartridge.
  • an identical solution should be counter currently circulated through the capillary lumen at the same flow rate.
  • the cartridges are then placed In a 41° C. water bath and the growth medium is circulated through the extracapillary spaces of the cartridge at 41° C.
  • the Leibowitz L-15 with 1% FCS and 5 mg % (5 mg/100 ml) bilirubin is pumped at 0.06 ml/ in/cm through the capil ⁇ lary lumen.
  • This provides the newly reverted hepatocytes a substrate to conjugate; the shell side bathing medium could then be ana ⁇ lyzed for the presence of bilirubin conjugates to determine efficacy of the metabolic assist function.
  • the capillary lumen are then flushed with buffered saline to ready the cartridges for use; these are advantageously intercon ⁇ nected to form a plural cartridge device of the type described.
  • heparinization is established by Injection of an initial dose of 300 units/Kg of body weight of heparin. During perfusion, additional heparin titration may be used If necessary to keep the activated clotting time greater than 400 seconds.
  • prota ine sulfate is injected to reverse the heparinization.
  • regional heparinization may be used. In this procedure, heparin is added to the afferent loop of the liver assist device tubing for anticoagulative purposes, with the addition of protamine sulfate to the efferent loop to effect reversal.
  • the actual perfusion protocol is estab ⁇ lished using techniques similar to convention ⁇ al bypass perfusion.
  • Silicon rubber tubular shunts are operatively placed between the radial artery and vein to establish intra- luminal blood flow rates of up to 350 cc/min.
  • Arterial-venous fistuals, fashioned surgically by side-to-side anastamosis of the radial artery and forearm vein may also be used, after adquate maturing of the surgical anas ⁇ tomosis.
  • blood flows of from about 100 to 350 cc./min. are adequate through the extracorporeal liver assist device of the invention.
  • Such blood flow is directed through the lumen of the membrane tubes, (i.e. , through the tube side of the cart ⁇ ridge ⁇ )) for contact with the faces of the membrane tubes remote from the hepatocyte layers.
  • a supplementary liver function perfusion should involve blood flow through the device for a period of from about 4 to 10 hours, three to five times weekly.
  • a bathing solution is concurrently passed through the shell sides of the cart ⁇ ridge ⁇ ) for contact with the hepatocytes.
  • This bathing solution is preferably Leibowitz L-15 medium supplemented with 1% fetal calf serum, and maintained at a temperature of about 41° C.
  • molecules such as bilirubin dissolved in the patient's blood will diffuse through the capillary membranes to the hepatocyte layers simultaneously with nutrients from the blood.
  • the bilirubin and other molecular species will then be available for takeup and metabolism (conjugation in the case of bilirubin) and consequent excretion of wastes by the hepatocytes into the shell sides of the cartridge(s) .
  • the cultured hepatocytes will assume the complex functions ordinarily provided by the normal liver in vivo.
  • additional uses could include alleviation of the clinical symptoms associated with a wide variety of inherited metabolic diseases such as familial hyperchloesterolemia, gener ⁇ alized gangliosidoses and other storage dis ⁇ eases akin to Tay-Sach' s syndrome, and I cell disease by providing additional unimpaired metabolic capacity to offset metabolic capaci ⁇ ty lost to genetic lesions.
  • metabolic diseases such as familial hyperchloesterolemia, gener ⁇ alized gangliosidoses and other storage dis ⁇ eases akin to Tay-Sach' s syndrome, and I cell disease by providing additional unimpaired metabolic capacity to offset metabolic capaci ⁇ ty lost to genetic lesions.
  • Salt concen ra ions are given in milligrams per 1000 ml. of final solution, pH was adjusted to pH 7.4 with ION NaOH. All solutions were sterilized by filtration through a millipore membrane filter (0.22u) .

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Vascular Medicine (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Cell Biology (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Anesthesiology (AREA)
  • Veterinary Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Public Health (AREA)
  • Cardiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Hematology (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Dispositif (10) et méthode d'assistance hépatique extracorporels améliorés qui emploient une membrane (12) de perfusion du sang cultivée avec des hépatocytes initialement transformées jusqu'à ce que soit développée une monocouche confluente, après quoi les hépatocytes sont retournées au phénotype somatique à des fins de perfusion. L'utilisation d'hépatocytes transformées permet la sous-culture en série pour maintenir une alimentation clinique de cellules au patient, alors que les caractéristiques de prolifération in vitro et la perte d'inhibition de contact des hépatocytes transformées, assurent une division cellulaire rapide et la formation d'une couche sur les membranes de perfusion. Les hépatocytes transformées viralement, sensibles à la température, sont préférées, de sorte que la réversion des cellules peut être accomplie par changement de température. Les hépatocytes transformées peuvent être cultivées sur des surfaces extérieures de cartouches (50) de membranes capillaires multiples et ultérieurement retournées en élevant leur température.
PCT/US1987/000405 1985-12-02 1987-02-24 Dispositif d'assistance hepatique utilisant des lignes de cellules transformees WO1988006467A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US06/803,564 US4675002A (en) 1985-12-02 1985-12-02 Liver assist device employing transformed cell lines
JP62501627A JP2739758B2 (ja) 1987-02-24 1987-02-24 形質転換細胞株を用いる肝臓補助装置

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
US06/803,564 US4675002A (en) 1985-12-02 1985-12-02 Liver assist device employing transformed cell lines

Publications (1)

Publication Number Publication Date
WO1988006467A1 true WO1988006467A1 (fr) 1988-09-07

Family

ID=25186858

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1987/000405 WO1988006467A1 (fr) 1985-12-02 1987-02-24 Dispositif d'assistance hepatique utilisant des lignes de cellules transformees

Country Status (1)

Country Link
WO (1) WO1988006467A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1063289A1 (fr) * 1998-03-03 2000-12-27 JMS Co., Ltd. Clones céllulaires du foie pour foie artificiel et dispositif d'assistance hépatique extracorporel
EP0952769A4 (fr) * 1996-04-23 2006-04-05 Smith & Nephew Bioreacteur a gradient de diffusion et dispositif hepatique extracorporel
EP0854912A4 (fr) * 1995-10-10 2006-04-12 Rhode Island Hospital Separation et culture d'hepatocytes du porc
CN109550093A (zh) * 2017-09-23 2019-04-02 孙奕 细胞膜人工肝脏

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA871264A (en) * 1971-05-18 F. Swenson Harvey Dialyzer
DE2832870A1 (de) * 1978-07-27 1980-02-07 Franz Hans Eduard Prof Dr Med Geraetekonzept zur behandlung eines patienten bei akuter oder chronischer niereninsuffizienz
US4229290A (en) * 1978-03-14 1980-10-21 Raj Ghen M G Compact low surface area dialyzer method and apparatus
US4514295A (en) * 1981-08-05 1985-04-30 Fresenius Ag Dialysis apparatus
US4559034A (en) * 1981-04-16 1985-12-17 Kawasumi Laboratories, Inc. Line for use in body fluid treatment
US4596550A (en) * 1982-09-24 1986-06-24 Baxter Travenol Laboratories, Inc. Method and apparatus for ultrafiltration measurement in a two pump dialysis system

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA871264A (en) * 1971-05-18 F. Swenson Harvey Dialyzer
US4229290A (en) * 1978-03-14 1980-10-21 Raj Ghen M G Compact low surface area dialyzer method and apparatus
DE2832870A1 (de) * 1978-07-27 1980-02-07 Franz Hans Eduard Prof Dr Med Geraetekonzept zur behandlung eines patienten bei akuter oder chronischer niereninsuffizienz
US4559034A (en) * 1981-04-16 1985-12-17 Kawasumi Laboratories, Inc. Line for use in body fluid treatment
US4514295A (en) * 1981-08-05 1985-04-30 Fresenius Ag Dialysis apparatus
US4596550A (en) * 1982-09-24 1986-06-24 Baxter Travenol Laboratories, Inc. Method and apparatus for ultrafiltration measurement in a two pump dialysis system

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0854912A4 (fr) * 1995-10-10 2006-04-12 Rhode Island Hospital Separation et culture d'hepatocytes du porc
EP0952769A4 (fr) * 1996-04-23 2006-04-05 Smith & Nephew Bioreacteur a gradient de diffusion et dispositif hepatique extracorporel
EP1063289A1 (fr) * 1998-03-03 2000-12-27 JMS Co., Ltd. Clones céllulaires du foie pour foie artificiel et dispositif d'assistance hépatique extracorporel
CN109550093A (zh) * 2017-09-23 2019-04-02 孙奕 细胞膜人工肝脏

Similar Documents

Publication Publication Date Title
US4853324A (en) Liver assist device employing transformed cell lines
US5605835A (en) Bioreactor device with application as a bioartificial liver
EP0817833B1 (fr) Dispositif filtrant
US6472200B1 (en) Device and method for performing a biological modification of a fluid
US5270192A (en) Biological artificial liver
JP4023828B2 (ja) 人工腎臓および腎臓疾患治療へのその利用
WO1992007615A1 (fr) Foie bio-artificiel
US6858146B1 (en) Artificial liver apparatus and method
ES2227607T3 (es) Soporte solido para uso en cultivo celular, especialmente para el cultivo de celulas hepaticas, reactor biologico que contiene dicho soporte solido y su uso en un sistema de higado bioartificial.
Cieslinski et al. Tissue engineering of a bioartificial kidney
Flendrig et al. Semipermeable hollow fiber membranes in hepatocyte bioreactors: a prerequisite for a successful bioartificial liver?
JP2002514128A (ja) 体液の生物学的修飾を実施する装置およびその方法
US20030129736A1 (en) Device and method for performing a biological modification of a fluid
US4675002A (en) Liver assist device employing transformed cell lines
US20110190679A1 (en) Extracorporeal cell-based therapeutic device and delivery system
WO1989001967A1 (fr) Purification du sang a l'aide de cellules renales cultivees
US20090081296A1 (en) Extracorporeal cell-based therapeutic device and delivery system
Nagaki et al. Development and characterization of a hybrid bioartificial liver using primary hepatocytes entrapped in a basement membrane matrix
JP2003501078A (ja) 細胞から組織を操作するためのバイオリアクターデザインとプロセス
Rozga et al. Artificial liver evolution and future perspectives
EP0579804B1 (fr) Foie biologique artificiel
Stefanovich et al. Extracorporeal plasma perfusion of cultured hepatocytes: effect of intermittent perfusion on hepatocyte function and morphology
WO1988006467A1 (fr) Dispositif d'assistance hepatique utilisant des lignes de cellules transformees
CA1287314C (fr) Machine pour aider la fonction hepatique dans laquelle on utilise des ligneescellulaires transformees
JP2739758B2 (ja) 形質転換細胞株を用いる肝臓補助装置

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU BR DK JP NO SU

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE FR GB IT LU NL SE