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
1999-12-21
2001-04-17
Tate, Christopher R. (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...
C435S001100, C435S001200, C435S002000, C435S325000, C435S370000, C435S373000, C435S401000, C435S289100, C435S297100, C435S297200, C435S298100, C435S299100, C435S375000
Reexamination Certificate
active
06218182
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods and apparatuses for culturing three-dimensional mammalian tissue, especially liver tissue. More particularly, the present invention relates to bioreactors capable of growing and sustaining three dimensional tissue cultures comprising multifunctional cells that operate via a diffusional or osmotic pressure gradient on either side of the cultured tissue.
The present invention is suited to the culturing of three-dimensional mammalian tissues for purposes of transplantation or implantation in vivo, in vitro toxicology testing, continuously producing biological cell products such as proteins, delivery of genes to tissue for ex vivo gene therapy, and as the primary component of an extracorporeal organ assist device. In particular, the present invention is suited to the culturing of three-dimensional liver tissue, and the use of liver tissue in a bioreactor as an extracorporeal liver assist device.
BACKGROUND OF THE INVENTION
The past decade has shown great advances in the area of growing tissues and organs in vitro (Langer et al., 1993, “Tissue Engineering,”
Science
260:920-926). One such system for culturing three-dimensional tissues is described in U.S. Pat. No. 5,266,480 to Naughton et al. The culture system of Naughton et al. involves seeding stromal cells from a tissue of interest onto a porous substrate. As the stromal cells grow in this environment, they produce an extracellular matrix and deposit growth factors that contribute to the development of a three-dimensional tissue. This static cell culture milieu provides the necessary microenvironment for cell-cell and cell-matrix communication as well-as an adequate diffusional environment for delivery of nutrients and removal of waste products. When the stromal tissue has grown and has developed into a three-dimensional tissue, it is ready for the seeding of the parenchymal cells of interest. The resulting system provides an “in vivo” environment for the full differentiation of the tissue. This system has been used to culture bone marrow tissue (Naughton et al., 1987, “Hematopoiesis on Nylon Mesh Templates. I. Long Term Culture of Rat Bone Marrow-Cells,”
J. Med.
18:219-250; Naughton et al., 1989, “Modulation of Long-Term Bone Marrow Culture by Stromal Support Cells,”
Ann. NY Acad. Sci.
554:125-140); skin tissue (Landeen et al ., 1992, “Characterization of Human Dermal Replacement,”
Wounds
4:167-175; Naughton et al., 1989, “A Physiological Skin Model for In Vitro Toxicity Studies,” 183-189,
Alternative Methods in Toxicology. In Vitro Toxicology: New Directions
Vol. 7, (A.M. Goldberg, ed.) Mary Ann Liebert Publishers, New York; Slivka et al., 1993, “Characterization, Barrier Function, and Drug Metabolism of an In Vitro Skin Model,”
J. Invest. Dermatol.
100:40-46; U.S. Pat. No. 5,266,480); and liver tissue (Naughton et al., 1991, “Long Term Liver Cell Cultures as Potential Substrates for Toxicity Assessment,” 193-202,
In Vitro Toxicology: Mechanisms and New Technology
(A.M. Goldberg, ed.) Mary Ann Liebert Publishers, New York).
While many methods and bioreactors have been developed to grow tissue masses for the purposes described above, these bioreactors do not adequately simulate in vitro the mechanisms by which nutrients and gases are delivered to tissue cells in vivo. Cells in living tissue are “polarized” with respect to diffusion gradients. Differential delivery of oxygen and nutrients, as occurs in vivo by means of the capillary system, controls the relative functions of tissue cells and perhaps their maturation as well. Thus, prior art bioreactors that do not simulate these in vivo delivery mechanisms cannot be used to culture a wide variety of three-dimensional tissues.
The tissue culturing system and bioreactors of the present invention improve on the prior art methods of culturing three-dimensional tissues by using diffusion gradients to deliver nutrients to, while simultaneously removing metabolic waste products from, the three-dimensional tissue culture. Such a diffusion-driven delivery mechanism enhances delivery of nutrients and removal of waste products and simulates in vitro the diffusional mechanisms whereby nutrients are delivered to mammalian cells in vitro, thereby optimizing the growth and differentiation of cell cultures grown in vitro. Thus, a wide variety of three-dimensional tissues having multifunctional cells can be cultured and sustained using the present invention.
Currently available bioreactor techniques for growing tissue masses in general include hollow fiber techniques, static maintenance reactor systems, fluidized bed reactors, and flat-bed, single-pass perfusion systems.
The most commonly used bioreactors involve hollow fibers. Hollow fiber reactors generally use numerous hollow fiber membranes of appropriate composition and porosity for the cells being cultured. They are often referred to as artificial capillary systems. (See, for example, U.S. Pat. No. 4,200,689 to Knazek et al.) Culture medium flows through the middle of the hollow fibers, and the cells are located on the outside of the fibers and in the spaces between the fibers. Nutrients flow through the hollow fibers to the cells. This type of bioreactor is not capable of growing thick tissue, as the cells only grow in the small interstitial spaces between the hollow fibers.
Hollow fibers are also disclosed in U.S. Pat. No. 5,081,035 to Halberstadt et al. In this system, cells are also grown in the interstitial spaces of an array of capillary tubes. Convective forces are used to maintain a constant nutrient gradient to all of the cells growing in the interstitial areas of the bioreactor. This method is also limited to growing cells in the small areas between the fibers, and cannot be used for a thick tissue.
Another hollow fiber device is described in U.S. Pat. No. 3,997,396 to Delente. In this system, cells are attached to the interstitial spaces of a hollow fiber bundle. An oxygen carrier is passed through the center of the fibers while the cells are incubated in a nutrient medium. As with the previously described hollow fiber devices, this method does not provide a thick cultured tissue and does not utilize osmotic pressure differentials to deliver nutrients to the cells from the nutrient medium.
Yet another hollow fiber device is described in WO 90/13639 to Tolbert et al. However, this system does not utilize osmotic pressure differentials to deliver nutrients to the cultured tissue mass.
A single-pass perfusion bioreactor system is described in Halberstadt et al., 1994, “The In Vitro Growth of a Three-Dimensional Human Dermal Replacement Using a Single-Pass Perfusion System,”
Biotechnology and Bioengineering
43:740-746. In this system a tissue is cultured on a mesh contained in a teflon bag. Media containing nutrients is pumped through the bag using a peristaltic pump. Nutrients in the media diffuse into the tissue and waste products secreted into the media are carried away. While this system has been used to culture relatively thick skin tissue, the method does not drive the delivery of nutrients by way of a diffusion or osmotic pressure gradient.
Generally, the prior art has not found a way to culture a wide variety of three-dimensional tissues. As discussed above, the prior art bioreactors do not simulate in vivo nutrient delivery mechanism, and therefore these bioreactors cannot be used to culture a wide variety of tissues.
A system to provide hepatic assist to patients awaiting transplants or with limited functioning livers has also long been sought. Unlike the kidney, the function of which is basically only to filter, or unlike the heart, the function of which is purely mechanical, the liver has functions that are complex and involve the removal, chemical conversion, and addition to the blood of a multitude of chemicals, or combinations of these functions. Past methods of providing an artificial liver have failed to provide a device that is as effective as a human liver, and which can be used by patients with a wide range of liver malfunctions. The
Halberstadt Craig R.
Naughton Brian A.
Sibanda Benson
Advanced Tissue Sciences
Pennie & Edmonds LLP
Tate Christopher R.
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