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WO1993014790A1 - Procede de transplantation de cellules dans le cerveau et utilisations therapeutiques de ce procede - Google Patents

Procede de transplantation de cellules dans le cerveau et utilisations therapeutiques de ce procede Download PDF

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Publication number
WO1993014790A1
WO1993014790A1 PCT/US1993/000494 US9300494W WO9314790A1 WO 1993014790 A1 WO1993014790 A1 WO 1993014790A1 US 9300494 W US9300494 W US 9300494W WO 9314790 A1 WO9314790 A1 WO 9314790A1
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cells
disease
brain
cell
support matrix
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PCT/US1993/000494
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English (en)
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Bruce D. Cherksey
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New York University
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Priority to JP5513305A priority Critical patent/JPH07503467A/ja
Priority to CA002128630A priority patent/CA2128630A1/fr
Priority to EP93904541A priority patent/EP0673259A4/fr
Publication of WO1993014790A1 publication Critical patent/WO1993014790A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3813—Epithelial cells, e.g. keratinocytes, urothelial cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/22—Urine; Urinary tract, e.g. kidney or bladder; Intraglomerular mesangial cells; Renal mesenchymal cells; Adrenal gland
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/33—Fibroblasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6923—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0085—Brain, e.g. brain implants; Spinal cord
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/167—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction with an outer layer or coating comprising drug; with chemically bound drugs or non-active substances on their surface
    • A61K9/1676—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction with an outer layer or coating comprising drug; with chemically bound drugs or non-active substances on their surface having a drug-free core with discrete complete coating layer containing drug
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
    • A61L27/3878—Nerve tissue, brain, spinal cord, nerves, dura mater
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • 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
    • C12N5/0613—Cells from endocrine organs
    • C12N5/0614—Adrenal gland
    • 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
    • C12N5/0618—Cells of the nervous system
    • C12N5/0621—Eye cells, e.g. cornea, iris pigmented 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
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/08—Coculture with; Conditioned medium produced by cells of the nervous system
    • 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
    • C12N2531/00—Microcarriers
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/10—Mineral substrates
    • C12N2533/12—Glass

Definitions

  • the invention in the field of neuroscience and medicine relates to methods for implantation or transplantation of cells into the mammalian brain, useful in treating neurological disorders.
  • Neurons or neuronal-like cells can be grafted into the central nervous system (CNS) , in particular, into the brain, either as solid tissue blocks or as dispersed cells.
  • CNS central nervous system
  • Parkinson's disease results from a selective loss of dopaminergic nigrostriatal neurons, resulting in a loss of input from the substantia nigra to the striatum.
  • Solid grafts of tissues potentially capable of producing dopamine such as adult adrenal medulla and embryonic substantia nigra (SN) , have been used extensively for experimental grafting in rats and primates treated with 6-hydroxydopamine (6-OHDA) to destroy dopaminergic cells (Dunnett, S.B. et al.. Brain Res. 215: 147-161 (1981); ibid. 229:457-470
  • Embryonic tissue provides an excellent source of cells which will differentiate in a foreign environment and become integrated with the host tissue.
  • grafts of embryonic SN into 6- OHDA treated rats have been shown to produce dopamine, to reduce apomorphine- or amphetamine-induced rotation, to alleviate sensory deficits and to make synapses in the host striatum (Dunnett et al. , Morisha et al.. Perlow et al. , supra) .
  • Grafted neurons are also spontaneously active, thus mimicking normal adult SN neurons (Wuerthele, S.M. et al.. In: Catecholamines, Part B f (E. Usdin et al.. eds.), A.R. Liss, Inc., New York, pp. 333-341).
  • fetal neural tissue In contrast to successful grafting of fetal neural tissue, mature CNS neurons have never been found to survive in transplants (Stenevi, U. et al.. Brain Res. 114:1-20 (1976)). The reason fetal CNS neurons survive grafting procedures while adult neurons do not, while uncertain, is probably related to several factors. First, fetal neurons are less affected by low oxygen levels than mature neurons (Jilek, L. , In: Developmental Neurobiology, Himwich, W.A. , ed. , CC. Thomas Publisher, Springfield, IL, 1970, pp. 331-369) , and grafting procedures necessarily involve periods of anoxia until an adequate blood supply to the transplant is established.
  • fetal neurons seem to survive best when they are taken during a rapid growth phase and before connections are established with target tissues (Boer, G.J. et al.. Neuroscience 15:1087-1109, (1985)). Also, fetal tissue may be especially responsive to growth (or "survival") factors which are known to be present in the milieu of the damaged host brain (Nieto-Sampedro, M. et al.. Science 217:860-861 (1982); Proc. Natl. Acad. Sci. USA 81:6250-6254 (1984)) .
  • Adrenal medullary cells are derived from the neural crest and, like sympathetic neurons, grow processes in vivo or in vitro in response to nerve growth factor (NGF) (Unsicker, K. et al.. Proc. Natl. Acad. Sci. USA 75:3498-3502 (1978)).
  • NGF nerve growth factor
  • grafting dissociated cells compared to blocks of tissue is that the cells can be precultured with various substances such as growth factors prior to grafting or they can be co- grafted with other cells or substances which promote specific parameters of differentiation.
  • glial cells may have specific regional effects and produce neuronal growth factors (Barbin, G. et al.. Devel. Neurosci. 7:296-307 (1985); Schurch-Rathgeb, Y. et al.. Nature 273:308-309 (1978); Unsicker, K. et al. Proc. Natl. Acad. Sci. USA 81:2242-2246 (1984); Whitaker-Azmitia, P.M. et al.. Brain Res.
  • the semipermeable membrane is of an acrylic copolymer, polyvinylidene fluoride, polyurethane, polyalginate, cellulose acetal, polysulphone, polyvinyl alcohol, polyacrylonitrile, or their derivatives or mixtures and permits diffusion of solute of up to 50 kD molecular weight.
  • This device was said to be useful in treatment of neurotransmitter-deficient conditions, such as Parkinson's disease, by sustained, local delivery of neurotransmitters, precursors, agonists, fragments, etc. , to a target area, especially the brain.
  • the device may be made retrievable so that the contents may be renewed or supplemented, and the cells are protected against immunological response and viral infection.
  • the inventors have made the unexpected discovery that by first culturing cells in vitro on a support matrix, such as 90 ⁇ m diameter glass beads, such cells including mature CNS neurons or cultured cells, can be successfully transplanted into the mammalian brain.
  • a support matrix such as 90 ⁇ m diameter glass beads, such cells including mature CNS neurons or cultured cells.
  • the beads, to which the cells adhere are stereotaxically injected into the recipient's brain. Cells so injected retain their viability and are thus effectively transplanted. They show prolonged survival and viability in vivo r even when transplanted across species barriers.
  • Parkinson's disease results indicate that, not only do these cells survive for prolonged periods, but they continue to function and have therapeutic efficacy. It is an objective of the present invention to overcome the aforementioned deficiencies in the prior work.
  • the present invention provides a method for grafting a cell in the brain of a mammalian subject comprising allowing the cell to attach to the surface of a support matrix in vitro, pre erably by culturing the cell with the matrix, such th - the cell is not encapsulate by the matrix, and iirr -nting the support matrix with the attached cell int_; the brain.
  • the method includes support matrices made of glass or other silicon oxides, polystyrene, polypropylene, polyethylene, polyacrylamide, polycarbonate, polypentene, acrylonitrile polymer, nylon, amylases, gelatin, collagen, natural or modified polysaccharides, including dextrans and celluloses (e.g.
  • nitrocellulose nitrocellulose
  • hyaluronic acid extracellular matrix
  • extracellular matrix agar
  • magnetite agar
  • Preferred support matrices are beads, porous or nonporous, in particular microbeads having a diameter from about 90 to about 150 ⁇ m.
  • the method of the present invention may employ cells of many different types, preferably either cells of neural or paraneural origin, such as adrenal chromaffin cells. Also useful are cell lines grown in vitro. Cells not of neural or paraneural origin, such as fibroblasts, may also be used following transfection with DNA encoding a neuropeptide or an enzyme or set of enzymes which results in production of neurotransmitter, or a neuronal growth factor.
  • the present invention includes a method for treating a neurological disease in a subject which comprises grafting an effective number of cells capable of treating the disease according to the above methods.
  • Diseases which can be treated according to the present invention include, but are not limited to, Parkinson's disease, Alzheimer's disease, Huntington's disease, epilepsy, familial dysautonomia, and traumatic brain injury.
  • Figure 1 is a photomicrograph of a section of the brain of a rat implanted with adult adrenomedullary cells on a glass microbead.
  • the section was fixed an stained histochemically for tyrosine hydroxylase (TH) using horse radish peroxidase.
  • the darkly stained areas indicate cells containing TH.
  • the pattern of staining shows a needle tract entering the section on the right side.
  • the large circular stained area in the center represents the bulk of the implanted cells.
  • the injected cells form a "gland-like" pattern, with some extending back into the needle tract.
  • Figure 2 is a graph showing effects of transplanting cells attached to glass beads on apomorphine-induced turning behavior in rats. Rats received either control beads, or beads to which adrenal chromaffin cells or retinal pigment epithelial
  • FIG. 3 is a graph showing the survival of cells, either transplanted alone or transplanted after incubation with glass beads, transplanted into rat brains.
  • Figure 4 is a photomicrograph showing a glass bead surrounded by associated viable pigmented adrenal chromaffin cells six months after transplantation into a rat brain (Enlargement: 400x) .
  • the present invention is based on the unexpected discovery by the inventors that adult cells or cultured cells which cannot normally be transplanted into a mammalian brain and survive, can be made to survive by first attaching them to a support matrix.
  • a number of different cell types are useful for the present invention.
  • a cell will be selected based on its ability to provide a missing substance to the recipient brain. Missing substances can be neurotransmitters or other neurally-active molecules, the absence of which results in neurological disease or dysfunction. It is important that the transplanted cell not grow as a tumor once it is inserted into the recipient.
  • neural or paraneural origin a cell which is derived from the embryonic neural crest.
  • a preferred example of a cell of paraneural origin is a adrenal medullary chromaffin cell.
  • the precursor cells to the mammalian adrenal medulla are of neural crest origin and possess the potential to develop along either neuronal or endocrine lines of differentiation (Bohn, M.C. et al. , 1981, supr . Devel. Biol. 89:299-308 (1982); Unsicker, K. , Develop. Biol. 108:259-268 (1985)).
  • Chromaffin cells from the rat, monkey, and human adrenal medulla when removed from adrenal cortical influences and exposed to nerve growth factor (NGF) , change from an endocrine to a neuronal phenotype (Notter, M.F. et al.. Cell Tiss. Res. 244:69-70 (1986); Stromberg, I. et al.. Exp. Brain Res. 60:335-349 (1985); Unsicker, K. et al.. 1978, supra..
  • NGF nerve growth factor
  • Another paraneural cell type is a retinal pigment epithelium cell (Song, M-K et al. , J. Cell. Phvsiol. 148:196-203 (1990)).
  • Neural lines may express a tremendous amount of genetic information which corresponds to the genetic expression seen in CNS neurons. Such cells are described in the following references: Kimhi, Y. et al.. Proc. Natl. Acad. Sci. USA 73:462-466 (1976); In: Excitable Cells in Tissue Culture. Nelson, P.G. et al. , eds.. Plenum Press, New York, 1977, pp.
  • a major advantage of these cultured cells has been the potential to manipulate the environment, as well as the cells themselves, in controlling the phenotype and genotype.
  • human neuroblastoma cells from the IMR 32 cell line can survive and express cholinergic markers in primate brain nine months after transplantation (Gash, D.M. et al. f Science 233:1420- 22 (1986)). These cells are preferably treated to render them morphologically and biochemically differentiated in vitro and must be rendered permanently amitotic before implantation, which further aids in their survival (Gash et al.. supra; Gupta, M. et al.. Dev. Brain Res. 19:21-29 (1985)).
  • cell line cells are modulated in vitro with the appropriate growth or differentiation factor and with an amitotic agent before transplantation in order to promote cell survival and prevent expression of the malignant potential. It will be apparent to one of ordinary skill in the art that examination of the neural cell surface of clonal cells in vitro using conventional methods will permit the selection of appropriate cells for use according to the present invention. During normal development and differentiation, the neuronal cell surface undergoes significant changes which account for the migration, recognition and integration of neurons in the CNS.
  • Somatic cell hybridization is a powerful cell biologic tool used not only to generate cell lines with a variety of genotypes but to analyze the mechanisms regulating the expression of various phenotypes of differentiation. Fusing cells which differ in the expression of specific genes allows for the exploration of the mechanisms controlling gene expression while chromosome alterations occur at rates to generate genetically different cell lines.
  • Hybrid cells can be formed which retain the properties of differentiated cells. Hybrids derived from fusion of sympathetic ganglia and neuroblastoma cells can synthesize dopamine (Greene, L.A. et al.. Proc. Natl. Acad. Sci.
  • This neural crest-derived tissue has been involved in clinical trials (see Background) to treat Parkinson's disease.
  • Adult monkey adrenal medulla can be cultured in vitro for at least about three weeks as single cells (Notter, M.F. et al.. Cell Tiss. Res. 244:69-76 (1986)). These cells respond to NGF by phenotypic alteration from the epithelioid, glandular morphology, to a neuronal morphology in which cells show extensive neuritic arborizations containing microtubular arrays.
  • This neuronal phenotype appears to be critical for long term survival of rat medullary cells in host CNS as well as their integration with host tissue (Stromberg, L. et al.. supra) .
  • Transplanted adrenal medulla tissue can correct functional deficits resulting from nigrostriatal dopamine depletion in rats (see, for example, Freed et al.. 1981, supra) . This, however, is thought to be brought about by diffusion of dopamine from the transplant, a phenomenon that decreases three to six months after transplantation. NGF treatment of the transplanted cells induces fiber outgrowth from the transplant into the host and induces a longer lasting behavioral recovery (at least a year) . Indeed, without NGF treatment, few chromaffin cells transplanted either to rat (Freed et al.. supra) or rhesus monkey caudate nucleus (Morihisia, J.M. et al.. Exp. Neurol. .84 . :643-653 (1984) using prior art techniques survive.
  • retinal pigment epithelial cells secrete dopamine and other factors and may be used for brain implants according to the present invention (Li, L. et al.. Exp. Eye Res. 47:771-785 (1988); Lui, G.M. et al.. Proc. Int'l. Soc. Eve Res. 6:172 (1990); Li, L. et al.. Inv. Ophthal. Vis. Sci. 31(SUPP1) :595 (1990, abstr. 2915-13); Sheedlo, H.J. et al.. ibid.. abstr. 2916-14; Fektorovich, E.G. et al.. ibid. (abstr. 2917-15) ; Song, M-K et al.. supra) .
  • an additional embodiment of the present invention is directed to transplantation of cells attached to a support matrix combined with the treatment, either in vitro prior to transplant, in vivo after transplant, or both, with the appropriate growth/differentiation factor.
  • Human adrenal medullary cells can be maintained in vitro having the neuronal phenotype for at least nine weeks (Notter, M.F. et al. , Schmitt Neurol. Sci. Svmp.. June 30, 1987, abstr.). NGF induces this conversion from the glandular state. Adrenal medullary cells co-cultured with C6 glioma cells exhibit extensive neuritic arborization and intimate contact with glioma cells. These astrocytic cells, treated with antimitotic agents to inhibit mitosis, are known to produce a growth factor similar to NGF which sustains sympathetic neurons in vitro (Barde, Y.A., Nature 274:818 (1978)).
  • glial cells may play an important role in functional recovery of neurons and may be an important source of trophic factors (Doering, L.C. et al.. J. Neurolog. Sci. .63.:183-196 (1984); Gumple, J. et al.. Neurosci. Lett. 2-1.' 307-311 (1984)). Therefore, another embodiment of the present invention involves co-culture of neural or paraneural cells with glial cells, their co-incubation with a support matrix, followed by implantation of the support matrix carrying both cell types. In additional embodiments of the present invention, cells which are not of neural or paraneural origin, but which have been altered to produce a substance of neurological interest, are used.
  • a preferred cell type is a human foreskin fibroblast which is easily obtained and cultured, and survives upon transplantation into the rat brain using the methods of the present invention (see Example III) .
  • the cells are genetically altered, using methods known in the art, to express neuronal growth factors, neurotransmitters, neuropeptides, or enzymes involved in brain metabolism. (See, for example, Gage, F.H. et al.. Neuroscience 23:795-807 (1987); Rosenberg, M.B. et al.. Science 242:1575-1578 (1988); Shimohama, S. et al- . Mol. Brain Res. 15:271-278 (1989); which are hereby incorporated by reference) .
  • Standard reference works setting forth the general principles of recombinant DNA technology and cell biology, which are hereby incorporated by reference, include Watson, J.D., et al. , Molecular Biology of the Gene. Volumes I and II, Benjamin/Cummings Publishing Co., Inc., Menlo Park, CA (1987) ; Darnell, J.E. et al.. Molecular Cell Biology. Scientific American Books, Inc., New York, NY (1986); Lewin, B.M., Genes II. John Wiley & Sons, New York, NY (1985) ; Old, R.W. et al.. Principles of Gene Manipulation: An Introduction to Genetic Engineering. 2nd Ed., University of California Press, Berkeley, CA (1981) ; Maniatis.
  • the recombinant DNA molecules useful for the methods of the present invention can be produced through any of a variety of means, such as, for example, DNA or RNA synthesis, or more preferably, by application of recombinant DNA techniques. Techniques f° r synthesizing such molecules are disclosed by, for example, Wu, R.
  • the gene or genes of interest are cloned from a library of expression vectors which has been prepared by cloning DNA or, more preferably, cDNA (from a cell capable of expressing the gene) into an expression vector.
  • the library is then screened for members capable of expressing the gene product of interest, such as a neurotransmitter-synthesizing enzyme, using antibody binding with an antibody specific for the gene product.
  • DNA, or more preferably cDNA is extracted and purified from a cell which is capable of expressing the gene product of interest.
  • the purified cDNA is fragmentized (by shearing, endonuclease digestion, etc.) to produce a pool of DNA or cDNA fragments.
  • DNA or cDNA fragments from this pool are then cloned into an expression vector in order to produce a library of expression vectors whose members each contain a unique cloned DNA or cDNA fragment.
  • An "expression vector” is a vector which (due to the presence of appropriate transcriptional and/or translational control sequences) is capable of expressing a DNA (or cDNA) molecule which has been cloned into the vector and of thereby producing a polypeptide or protein. Expression of the cloned sequences occurs when the expression vector is introduced into an appropriate host cell.
  • An appropriate mammalian host cell would be any mammalian cell capable of expressing the cloned sequences. Procedures for preparing cDNA and for producing a genomic library are disclosed by Sambrook et al. (supra) .
  • a DNA sequence encoding a product or products the expression of which is desired for the present invention may be recombined with vector DNA in accordance with conventional techniques, including blunt-ended or staggered-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Techniques for such manipulations are disclosed by Sambrook et al. , supra. and are well known in the art.
  • a nucleic acid molecule such as DNA, is said to be "capable of expressing" a polypeptide if it contains nucleotide sequences which contain transcriptional and translational regulatory information and such sequences are “operably linked” to nucleotide sequences which encode the polypeptide.
  • An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gene expression.
  • regulatory regions needed for gene expression may vary from organism to organism, but shall in general include a promoter region which, in prokaryotes, contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal the initiation of protein synthesis.
  • promoter region which, in prokaryotes, contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal the initiation of protein synthesis.
  • Such regions will normally include those 5'-non-coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and the like.
  • the non-coding region 3' to the gene sequence coding for the protein may be obtained by the above-described methods.
  • This region may be retained for its transcriptional termination regulatory sequences, such as termination and polyadenylation.
  • the transcriptional termination signals may be provided. Where the transcriptional termination signals are not satisfactorily functional in the expression host cell, then a 3• region functional in the host cell may be substituted.
  • Two DNA sequences are said to be operably linked if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region sequence to direct the transcription of the coding sequence, or (3) interfere with the ability of the coding sequence to be transcribed by the promoter region sequence.
  • a promoter region would be operably linked to a DNA sequence if the promoter were capable of effecting transcription of that DNA sequence.
  • a promoter is a double-stranded DNA or RNA molecule which is capable of binding RNA polymerase and promoting the transcription of an "operably linked" nucleic acid sequence.
  • a "promoter sequence” is the sequence of the promoter which is found on that strand of the DNA or RNA which is transcribed by the RNA polymerase.
  • a “promoter sequence complement” is a nucleic acid molecule whose sequence is the complement of a "promoter sequence.” Hence, upon extension of a primer DNA or RNA adjacent to a single-stranded "promoter sequence complement” or, of a "promoter sequence,” a double-stranded molecule is created which will contain a functional promoter, if that extension proceeds towards the "promoter sequence” or the “promoter sequence complement.” This functional promoter will direct the transcription of a nucleic acid molecule which is operably linked to that strand of the double-stranded molecule which contains the "promoter sequence” (and not that strand of the molecule which contains the "promoter sequence complement”) .
  • the promoter sequences useful for producing cells for the present invention may be either eukaryotic or viral. Suitable promoters are repressible, or, more preferably, constitutive.
  • Useful eukaryotic promoters include the promoter of the mouse metallothionein I gene (Hamer, D., et al.. J. Mol. Appl. Gen. .1:273-288 (1982)); the TK promoter of Herpes virus (McKnight, S., Cell 31:355-365 (1982)); the SV40 early promoter (Benoist, C. , et al.. Nature (London) 290:304-310 (1981)).
  • a preferred promoter in particular for human fibroblasts, is the collagen promoter (Prockop, D.J. et al.. N. Eng. J. Med. 301:13-23. 77-85 (1979); Eyre, D.R. , Science 207:1315-1322 (1980); Martin, G.R. et al.. Trends Bioch. Sci. 10:285-287 (1985); which references are hereby incorporated by reference) .
  • cells attached to, or mixed with, a support matrix, according to the invention which are frozen and stored in a frozen state using methods well known in the art. Following thawing, the matrix-bound cells are implanted into a recipient brain.
  • the number of cells needed to achieve the purposes of the present invention is variable depending on the size, age, weight of the subject, the o nature of the underlying disease, other concurrent therapies, and the like. This can be determined by one of skill in the art without undue experimentation.
  • an effective number of cells attached to a support matrix are in the range of about 5 1 x 10 3 to about 1 x 10 7 cells, more preferably about 5 x 10 3 to about l x l ⁇ 6 cells.
  • the effective amount of transplanted cells can be determined in terms of mass of cells added to a volume of beads, for example 40 mg of cell mass per ml of o beads.
  • Materials of which the support matrix can be comprised include those materials to which cells adhere following in vitro incubation, and on which cells can grow, and which can be implanted into a 5 mammalian brain without producing a toxic reaction, or an inflammatory or gliosis reaction which would destroy the implanted cells or otherwise interfere with their biological or therapeutic activity.
  • Such materials may be synthetic or natural chemical substances or substances having a biological origin.
  • the matrix materials include, but are not limited to, glass and other silicon oxides, polystyrene, polypropylene, polyethylene, polyvinylidene fluoride, polyurethane, polyalginate, polysulphone, polyvinyl alcohol, acrylonitrile polymers, polyacrylamide, polycarbonate, polypentene, nylon, amylases, gelatin, collagen, natural and modified polysaccharides, including dextrans and celluloses (e.g. nitrocellulose) , agar, and magnetite. Either resorbable or non-resorbable materials may be used. Also intended are extracellular matrix materials, which are well-known in the art (see below) . Extracellular matrix materials may be obtained commercially or prepared by growing cells which secrete such a matrix, removing the secreting cells, and allowing the cells which are to be transplanted to interact with and adhere to the matrix.
  • the support matrix of the present invention is distinguished from that described by Aebischer and his colleagues (see above) in that, according to the present invention, the cells are attached to or coating the surface of the support; they are not encapsulated within a closed compartment, where their survival would be questionable given the exclusion capacity of the disclosed encapsulating supports. Furthermore, the support matrix of the present invention presents no requirement that the material have particular permeability properties, such as the selective permeability of the Aebischer devices which allow low molecular weight substances to cross but exclude larger molecules (>50 kD) .
  • the cells used for transplantation are generally on the "outer surface" of the support.
  • the support may be solid or porous. However, even in a porous support, the cells are in direct contact with the external milieu without an intervening membrane or other barrier. Thus, according to the present invention, the cells are considered to be on the
  • outer surface of the support even though the surface to which they adhere may be in the form of internal folds or convolutions of the porous support material which are not at the exterior of the particle or bead itself.
  • the configuration of the support is preferably spherical, as in a bead, but may be cylindrical, elliptical, a flat sheet or strip, a needle or pin shape, and the like.
  • a preferred form of support matrix is a glass bead.
  • Another preferred bead is a polystyrene bead. Bead sizes may range from about 10 ⁇ m to 1 cm in diameter, preferably from about 90 to about 150 ⁇ m.
  • various microcarrier beads see, for example. Fisher Biotech Source 87-88. Fisher Scientific Co., 1987, pp. 72-75; Sigma Cell Culture Catalog. Sigma Chemical Co., St. Louis, 1991, pp. 162-163; Ventrex Product Catalog.
  • the solid matrix may optionally be coated on its external surface with factors known in the art to promote cell adhesion, growth or survival.
  • factors include cell adhesion molecules, extracellular matrix, such as, for example, fibronectin, laminin, collagen, elastin, gycosaminoglycans, or proteoglycans (see: Albers, B. supra, pp. 802-834) or growth factors, such as, for example, NGF.
  • the growth- or survival-promoting factor or factors may be incorporated into the matrix material, from which they would be slowly released after implantation in vivo.
  • cells growing on, or mixed with, resorbable matrices such as, for example, collagen
  • resorbable matrices such as, for example, collagen
  • cells attached to the matrix of the invention may be implanted into the spinal cord, or placed in, or adjacent to, the optic nerve.
  • the matrix material on which the cells to be implanted grow, or with which the cells are mixed may be an endogenous product of the implanted cells themselves.
  • the matrix material may be extracellular matrix or basement membrane material which is produced and secreted by the very cells to be implanted.
  • Parkinson's Disease can be treated according to the present invention by implanting dopamine-producing cells in the recipient's striatum.
  • Alzheimer's disease involves a deficit in cholinergic cells in the nucleus basalis.
  • a subject having Alzheimer's disease or at risk therefor may be implanted with cells producing acetylcholine.
  • Huntington's disease involves a gross wasting of the head of the caudate nucleus and putamen, usually accompanied by moderate disease of the gyrus.
  • a subject suffering from Huntington's disease can be treated by implanting cells producing the neurotransmitters gamma amino butyric acid (GABA) , acetylcholine, or a mixture thereof.
  • GABA neurotransmitters gamma amino butyric acid
  • the support matrix material to which such cells are attached is preferably implanted into the caudate and putamen.
  • Epilepsy is not truly a single disease but rather is a symptom produced by an underlying abnormality.
  • each epileptic subject will have damage or epileptic foci which are unique for the individual.
  • Such foci can be localized using a combination of diagnostic methods well-known in the art, including electroencephalography, computerized axial tomography and magnetic resonance imaging.
  • a patient suffering from epilepsy can be treated according to the present invention by implanting the support matrix material to which GABA-producing cells are attached into the affected site. Since blockers of glutamate receptors and NMDA receptors in the brain have been used to control experimental epilepsy, cells producing molecules which block excitatory amino acid pathways may be used according to the invention. Thus implantation of cells which have been modified as described herein to produce polyamines, such as spermidine, in larger than normal quantities may be useful for treating epilepsy.
  • the methods of the present invention are intended for use with any mammal which may experience the beneficial effects of the methods of the invention. Foremost among such mammals are humans, although the invention is not intended to be so limited, and is also applicable to veterinary uses.
  • Adrenal chromaffin cells from adult rats were prepared according to known methods (see Example IV, below; see also, Inoue, M. et al.. A. J. Phvsiol. 257 (Cell Physiol.) :C906-912 (1989)). After sacrifice by nembutal anesthesia followed by decapitation adrenal glands were removed. The medulla was freed of capsular and cortical material by careful microdissection. The tissue was cut into 2 or 3 pieces and incubated for 30 minutes with 0.2% collagenase in calcium-free balanced salt solution containing 140 mM NaCl, 5 mM KC1, 5.2 mM MgCl 2 , 5 mM
  • HEPES 10 mM D-glucose, at pH 7.4.
  • the preparation was shaken gently by bubbling through 99.9% 0 2 .
  • the tissue was washed three or four times with the above salt solution and then triturated gently in a pasteur pipet.
  • the dissociated chromaffin cells were transferred to cell culture flasks containing serum free medium (Ventrex PC 1) and were cultured overnight at 37°C in a 5% C0 2 atmosphere. Under these conditions, the cells retain their viability for no more than a few days. Sterilized glass beads (Ventrex, 90 micron) were added to the culture flask. The cells attached to the beads and essentially formed a monolayer on the beads. Within 24 hrs. of preparation in vitro. 1 to 5 ⁇ l aliquots of the cell-carrying bead suspension were injected into the brains of anesthetized adult rats.
  • Adrenal medullae were removed from adult guinea pigs according to methods described in Examples I and IV and subjected to the procedure described in Example I.
  • the cells on glass microbeads were injected into rat brain.
  • EXAMPLE III Cultured human foreskin fibroblasts were attached to glass microbeads as described above and injected into the rat brain. The cells were localized by immunofluorescence using human IgG which selectively bound to the fibroblasts, followed by a fluorescein- conjugated goat anti-human IgG antibody. The implanted human cells survived for a period of at least 21 days in a completely healthy state. No signs of immunological rejection were evident.
  • a lesion was induced in the SN by stereotaxic application of the selective neurotoxic agent, 6-OHDA.
  • Rats were anesthetized using sodium pentobarbital, 45 g/kg, i.p. Animals were placed in a Kopf-type stereotaxic instrument. For lesioning the right side, the following coordinates were used: Rostral Caudal: -4.8mm; Dorsal Ventral: -8.1mm; Lateral Medial: -1. , -2.0mm; Jaw Bar: -3.3mm. After the induction of Nembutal anesthesia, the rat's head was shaved using an Oster clipper with surgical blade. The rat was then placed into the stereotaxic apparatus and its head approximately positioned. A 3/4 inch rostral-caudal incision was made midline in the cranium.
  • the skull was gently scraped using a #10 scalpel blade to remove the pericranial membranes.
  • the bregma was located and the injection needle was placed directly over this landmark.
  • Stereotaxic coordinates were then corrected for the individual animal using the position directly over the bregma as the zero-value coordinates.
  • the rostral- caudal coordinate was subtracted from the zeroed value and the lateral-medial coordinate was subtracted from the zeroed value.
  • the needle was then lowered to touch the skull and the point of contact was marked with a pencil.
  • the needle was then raised out of the way and a hole was drilled through the cranium using a Dremel Flex- shaft drill equipped with a #253 handpiece and a #6 dental bit.
  • the needle was repositioned above the opening in the skull and then lowered into the hole so that it sat just inside the entrance.
  • the stereotaxic coordinate of this position was recorded as the dorsal-ventral zero value with the needle at the dura mater.
  • the dorsal-ventral value was then subtracted from this value to give the desired final coordinate.
  • the needle was then lowered to the corrected dorsal ventral coordinate for the injection.
  • the 6-OHDA hydrobromide in a vehicle of isotonic (0.9%) saline containing 0.2 mg/ml ascorbic acid was used at a concentration of 8 ⁇ g/4 ⁇ l.
  • the agent was prepared immediately prior to injection to minimize oxidative degradation of this drug and was kept on ice until it was used.
  • the drug was injected using a perfusion pump attached to a 23 gauge needle at a rate of 1 ⁇ l/min until a total volume of 4 ⁇ l had been injected. Prior to removal, the needle was allowed to remain in place for an additional 5 minutes to allow for infiltration of the drug into the desired area.
  • the surgical site was closed using Clay-Adams 9mm wound clips, and the rats were allowed to recover. After the surgery, the animal was placed into a warm container and allowed to recover from the anesthesia prior to its return to its normal housing in Berg Institute. By the next day, the rats had regained normal behavior.
  • Rats were scored on the basis of turns per minute. Rats exhibiting less than 7 apomorphine-induced turns per minute were rejected from the study. Rats were tested at weekly intervals until the rotational behavior stabilized, usually at three to four weeks post- injection, and remained stable for an additional two testing periods. In general, rats exhibited 9 to 10 apomorphine-induced turns per minute at this time, and were ready for transplant studies.
  • the free adrenal was transferred to a 60mm culture dish containing Ventrex PC-1 supplemented medium.
  • the adrenal was minced as finely as possible using a scalpel.
  • the medium was carefully decanted and replaced with 10 ml sterile complete PC-1 medium containing 0.1% Trypsin/0.2% collagenase.
  • the adrenal was incubated in this solution for 30 ins. in a 37°C incubator in an atmosphere of 5% carbon dioxide in air.
  • the cell mixture was made homogeneous by repeatedly drawing it up and down through a sterile 10 ml plastic pipette and was then passed through a 100 ⁇ m cell sieve. The clean filtrate was placed in a sterile tube and centrifuged at 200 x g for 5 min. The supernatant was decanted and the cell-containing pellet was resuspended in complete PC-1 medium, which included the "serum-like” factors, (3-4 ⁇ __. if cells were to be cultured on microcarriers) . The mixture was shaken gently and placed in a 37°C incubator.
  • Ventrex glass microcarriers (Ventreglas, 90-120 ⁇ m) were sterilized by placing the beads in 10 ml of sterile distilled water per 1 gram of beads and heating to 121°C for 15 min. The solution was allowed to cool to room temperature and the water was discarded. The microcarriers were then suspended into a small volume of culture medium and allowed to stand for 30 mins. The medium was removed and the beads (approximately 0.21 g) were added to the previously described cell preparation. The resulting mixture was shaken for 2 hours and an additional 4 ml of complete PC-1 was added. The culture flask was then incubated overnight at 37°C in a 5% C0 2 atmosphere to allow the cells to adhere to the microcarriers.
  • Additional cell types which have been testing by the present inventors using this methodology include human retinal pigment epithelial cells and human foreskin fibroblasts. These cell types have been tested only for survival in the recipient animal•s brain, and have given positive results in experiments lasting up to four months, (see Example I) .
  • the cells were injected into caudate/putamen region of the brain using a stereotaxic injection.
  • the Atlas coordinates used for the injection were: Rostral Caudal: -3.14; Dorsal Ventral: -7.4; Lateral Medial: -5.0; Jaw Bar: -3.3. Corrected values were derived as previously described.
  • the stereotaxic coordinates required further correction for the actual opening of the needle rather than its tip. This was accomplished by measurement of the tip-opening distance (typically 1 - 2 mm) and appropriate reduction of the values given above.
  • the tip-opening distance typically 1 - 2 mm
  • the suspension containing microcarrier-adhering cells was injected at a rate of 4 ⁇ l/min until a final volume of 20 ⁇ l had been injected.
  • the total number of beads injected varied somewhat due to sedimentation during the injection, but was calculated to be about 170-200 beads. Current estimates are that approximately 175 beads
  • each holding 2-5 cells need to be injected to obtain an excellent therapeutic response. This number extrapolates to 50,000 to 75,000 beads in the human, occupying a volume of about 0.5 ml.
  • Rats were anesthetized with sodium pentobarbital, 60 mg/mg i.p.. the chest cavity was opened to expose the heart.
  • a 22 gauge butterfly needle was inserted into the bottom of the left ventricle, and the right atrium was cut.
  • the butterfly needle was attached to a peristaltic pump and isotonic saline was pumped through the heart until the solution leaving the cut atrium appeared clear. In general, 200 - 400 ml of saline were required.
  • the perfusion solution was then changed to 1% glutaraldehyde/ 1% paraformaldehyde/ 0.1 M sodium phosphate, pH 7.2. The perfusion was continued with this fixative until the liver achieved a whitish color.
  • the sections were stained as appropriate and mounted on gelatin-coated slides.
  • a filtered solution containing 3 g gelatin, 0.3 g chromium potassium sulfate in 600 ml distilled water was freshly prepared and the slides were individually immersed once in this solution.
  • the slides were allowed to dry in open air overnight.
  • the section was then mounted on the slide and dehydrated by immersing for 5 minutes each in 70% alcohol, 90% alcohol, 95% alcohol, twice in 100% alcohol and twice in xylene. Coverslips were then mounted using Permount solution.
  • a second effect observed in these studies relates to the fact that 6-OHDA lesioned rats do not maintain a normal growth curve.
  • the rats which have been implanted with adrenal chromaffin cells or retinal pigment epithelium cells on beads regain a normal growth curve.
  • Histological analysis of the brains of rats exhibiting no or only limited effects of the transplanted cells indicated that only a very small number of cells had been injected. In such nonresponders, injection coordinates were found to have been inaccurate.
  • the average rotation after cell transplant was reduced from 9 + 0.5 turns/min for non-transplanted controls to 5.1 ⁇ 1.2 turns/min for transplanted rats.
  • Statistical analysis using Student's t test yielded a p-value between 0.05 and 0.001, indicating a high level of statistical significance of this difference.
  • Adrenal chromaffin cells (prepared as described in Example IV) in numbers ranging from 10 3 to 10 7 were injected into the caudate-putamen area of rat brains in volumes of 5-20 ⁇ l. At various times thereafter ranging from 1 to 180 days (see Figure 3) , animals were sacrificed, and their brains were sectioned into 20-50 ⁇ m sections. The sections were examined at 400x and the densities of cells were calculated from the cells/microscopic field. Results were expressed as the % of cells surviving (viable cells as a percent of injected cells) . Each experimental group contained 6 rats.
  • Figure 4 is a photomicrograph of a section of a rat brain six months after transplantation of adrenal chromaffin cells showing deeply pigmented viable cells attached to the bead and in the adjacent area.

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Abstract

Selon un procédé destiné à greffer une cellule dans le cerveau d'un mammifère, on fixe la cellule à une matrice support de façon à ce que la cellule se fixe à la surface de la matrice, et on implante dans le cerveau la matrice support et la cellule fixée. Les matrices support préférées sont en verre ou sont faites de microbilles en plastique, solides ou poreuses, d'un diamètre d'environ 90 à environ 125 νm. Ce procédé utilise des cellules de types différents, de préférence des cellules d'origine neurale ou paraneurale, telles que des cellules chromaffines surrénales. On utilise également des lignées cellulaires cultivées in vitro. Les cellules qui ne sont pas d'origine neurale ou paraneurale, telles que des fibroblastes, peuvent être également utilisées selon la modification génétique pour exprimer un produit neural désiré tel qu'un neurotransmetteur ou un facteur de croissance neuronal. Le procédé est utilisé pour traiter des maladies neurologiques telles que la maladie de Parkinson, la maladie d'Alzheimer, la maladie d'Huntington, l'épilepsie et les lésions traumatiques du cerveau.
PCT/US1993/000494 1992-01-23 1993-01-21 Procede de transplantation de cellules dans le cerveau et utilisations therapeutiques de ce procede WO1993014790A1 (fr)

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US5776747A (en) * 1994-07-20 1998-07-07 Cytotherapeutics, Inc. Method for controlling the distribution of cells within a bioartificial organ using polycthylene oxide-poly (dimethylsiloxane) copolymer
US5795790A (en) * 1994-07-20 1998-08-18 Cytotherapeutics, Inc. Method for controlling proliferation and differentiation of cells encapsulated within bioartificial organs
US5833979A (en) * 1994-07-20 1998-11-10 Cytotherapeutics, Inc. Methods and compositions of growth control for cells encapsulated within bioartificial organs
US5834029A (en) * 1994-07-20 1998-11-10 Cytotherapeutics, Inc. Nerve guidance channel containing bioartificial three-dimensional hydrogel extracellular matrix derivatized with cell adhesive peptide fragment
US5840576A (en) * 1994-07-20 1998-11-24 Cytotherapeutics, Inc. Methods and compositions of growth control for cells encapsulated within bioartificial organs
US5843431A (en) * 1994-07-20 1998-12-01 Cytotherapeutics, Inc. Controlling proliferation of cells before and after encapsulation in a bioartificial organ by gene transformation
US5853717A (en) * 1994-07-20 1998-12-29 Cytotherapeutics, Inc. Methods and compositions of growth control for cells encapsulated within bioartificial organs
US5858747A (en) * 1994-07-20 1999-01-12 Cytotherapeutics, Inc. Control of cell growth in a bioartificial organ with extracellular matrix coated microcarriers
US5935849A (en) * 1994-07-20 1999-08-10 Cytotherapeutics, Inc. Methods and compositions of growth control for cells encapsulated within bioartificial organs
US6156572A (en) * 1994-07-20 2000-12-05 Neurotech S.A. Bioartificial extracellular matrix containing hydrogel matrix derivatized with cell adhesive peptide fragment
US6392118B1 (en) 1994-07-20 2002-05-21 Neurotech S.A. Mx-1 conditionally immortalized cells
US6495364B2 (en) * 1995-05-23 2002-12-17 Neurotech, S.A. Mx-1 conditionally immortalized cells
EP0927043A4 (fr) * 1996-04-08 2004-09-01 Univ New York Medical Ct Procede de transfert d'un gene dans le systeme nerveux central
EP1073420A4 (fr) * 1998-05-14 2004-09-08 Abraham Shahar Implants neuronaux speciaux, pour la reconstruction du systeme nerveux central endommage

Also Published As

Publication number Publication date
EP0673259A1 (fr) 1995-09-27
AU3586993A (en) 1993-09-01
JPH07503467A (ja) 1995-04-13
CA2128630A1 (fr) 1993-08-05
EP0673259A4 (fr) 1995-12-06

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