Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Solid support and method of culturing cells on said solid...
Reexamination Certificate
1998-06-02
2002-04-16
Naff, David M. (Department: 1651)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Solid support and method of culturing cells on said solid...
C435S177000, C435S325000, C435S370000, C435S289100
Reexamination Certificate
active
06372495
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the field of the cultivation of cells, especially of adherent tissue cells such as liver cells. More in particular, the invention relates to the field of biological methods and reactors for the cultivation and/or maintenance of cells, especially liver cells, and to the use of such methods in a bio-artificial liver system (BAL).
BRIEF DESCRIPTION OF THE PRIOR ART
It is generally known that most tissue cells require a solid support on which to grow and divide.
Although it is possible to culture adherent tissue cells in ordinary vessels, such as glass bottles or Petri dishes, during which the cells adhere to the wall of the vessel, usually special reaction vessels or bottles with a high surface area are used so as to provide increased capacity for cell attachment. One way to improve said surface area is to use a solid support for cell adherence. Such solid supports are known in the art; examples include glass beads, microcarriers and cellulose fibers.
A special problem in the cultivation of adherent cells—compared to the cultivation of cells in suspension or in confluent layers—is to provide sufficient nutrients and/or oxygen to the cells and/or provide for sufficient removal of waste products and/or carbon dioxide. This is especially a problem with cells that put stringent demands on both oxygenation as well as the removal of waste products, such as liver cells.
The non-availability of suitable solid supports and methods for the in vitro cultivation of liver cells has over the last 40 years severely hindered the development of the so-called bio-artificial liver (BAL) systems, systems that could be used in patients with liver defects for the support and/or replacement of the natural liver function.
As acute liver failure has a very poor prognosis and is usually fatal to the patient within days or even hours [see for instance Devlin et al., Hepatology Vol. 21, No. 4 (1995), pages 1018-1024 and Lake and Sussman, Hepatology, Vol. 21, No. 3 (1995), pages 879-882, describing the general problems in the art of the treatment of liver failure, both incorporated herein by reference], because livers for transplant are not readily available, a BAL system that could support and/or replace liver function, for instance during the time the patient awaits for a liver to become available for transplant and/or to bridge the period until the liver of the patient sufficiently recovers and/or regenerates by itself and/or as a result of treatment, would be highly desirable.
However, due to the abovementioned lack of suitable methods and/or materials for cultivating and/or maintaining liver cells in vitro, the bio-artificial liver systems from the prior art have so far proved insufficient, because they do not fully replace all the functions carried out by the liver of the patient in vivo, because they have insufficient capacity, and/or because the time during which they are therapeutically effective is too limited for practical use.
The history of bio-artificial liver systems has been described in a number of recent articles, notably Nyberg et al., the American Journal of Surgery, Vol. 166, November 1993, p. 512-521, and Sussman and Kelly, Scientific American, May-June 1995, p. 59-77, incorporated herein by reference.
As described in these articles, the earliest liver support systems were based on hemodialysis, charcoal hemoperfusion, or cross-hemodialysis either between humans or between humans and animals. Also, extra-corporeal liver perfusion has been tried.
All these systems have been found to be insufficient. As stated by Nyberg et al.:
based on the limited success achieved by early liver support techniques, the concept evolved that liver functions essential for survival would be best provided by mammalian liver preparations that allowed sustained or repetitive application. These liver preparations, commonly referred to as hybrid or bio-artificial systems, contain biological components within a synthetic framework. Biological components may include isolated liver enzymes, cellular components, slides of liver or cultured hepatocytes. Hepatocytes may be implanted in the patient or perfused extra-corporally. Hepatocytes systems have shown the greatest promise for bio-artificial liver support. When compared with cellular component and isolated enzyme systems, hepatocyte systems should supply a greater number of liver functions, since they utilize intact, metabolically active liver cells ( . . . ). One major advantage of the hepatocyte bio-artificial liver over traditional hepatocyte transplantation and earlier support techniques, such as cross-circulation and extra-corporal liver perfusion, is that the bio-artificial liver can be constructed from semipermeable materials that provide a barrier between the hepatocytes and the host immune system. As a result bio-artificial liver therapy may be performed without immunosuppression, and hepatocytes from different species (xenocytes) may be used within the bio-artificial liver.
The disadvantages of bio-artificial liver systems include ( . . . ) the problem of maintaining normal hepatocyte viability and function at the high cell density necessary for clinical application. For example, when hepatocytes are grown on a plastic surface with standard cell culture medium, they lose their gap junctions in about 12 to 24 hours; they also flatten and become a granular; tissue specific functions are lost in 3 to 5 days, followed by hepatocyte death within 1 to 2 weeks. As a result, improved techniques of cell culture have become necessary for the application of bio-artificial liver support systems.
A number of different approaches to the cultivation of hepatocytes and related cells for use in or as BAL-systems have been described. However, the prior art hepatocyte systems also suffer from problems with regard to capacity and effective working time, see Sussman and Kelly:
With regard to the provision of sufficient metabolic capacity, it is not clear exactly how much liver necrosis is fatal. Animal experiments suggest that at least 30% of the liver's original function must be preserved in order to survive. The adult human liver contains approximately 1000 gm of hepatocytes, which are the metabolically active cells. Thus we have proposed that effective liver assistance will require the equivalent of 300 to 400 gm of cells. Two sources of hepatocytes are available: freshly isolated cells (primary cultures) and cells grown in continuous culture (cloned or immortalized cells). Cells that have been isolated from a normal human or animal liver retain many of their functions ( . . . ) the technology has severe limitations.
Artificial livers that use freshly isolated cells have so far provided only a fraction of the necessary metabolic capacity. Hepatocytes do not divide after they have been isolated, so a steady supply of new cells is required. Coupled with the labour-intensive nature of cell preparation, this makes it almost impossible to scale up production to meet current needs in a cost-effective manner. Moreover, freshly isolated cells do not appear to last very long during treatment. A liver assist device that lasts for only 6 to 7 hours, as some have been reported to do, clearly falls short of allowing liver regeneration. Finally, production of any such device using animal cells entails a number of problems, especially in areas of sterility and lot-to-lot variability.
Uchino et al., ASIAO Transactions 1988;23;972-977 describe a hybrid bio-artificial liver composed of multiplated hepatocyte monolayers. A total of 80 grams of cultured adult dog hepatocytes was cultured in a reactor comprising a stack of 200 collagen coated borosilicated glass plates. These hepatocytes were viable and functioned well during 4 weeks in perfusion culture. This bio-artificial liver was tested in anhepatic dogs. The longest survival obtained was 65 hours.
However, a serious drawback of this system, besides the complexity of constructing and using a 200 glass plate-reactor, is that the monolayer culture of hepatocytes on sai
Browdy and Neimark , P.L.L.C.
Meller Mike
Naff David M.
Seed Capital Investments-2 (SCI-2) B.V.
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