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WO1992003536A1 - Autotransplantation de cellules de schwann favorisant la reparation du systeme nerveux - Google Patents

Autotransplantation de cellules de schwann favorisant la reparation du systeme nerveux Download PDF

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Publication number
WO1992003536A1
WO1992003536A1 PCT/US1991/005817 US9105817W WO9203536A1 WO 1992003536 A1 WO1992003536 A1 WO 1992003536A1 US 9105817 W US9105817 W US 9105817W WO 9203536 A1 WO9203536 A1 WO 9203536A1
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Prior art keywords
explant
cells
schwann cells
fibroblasts
culture substrate
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PCT/US1991/005817
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English (en)
Inventor
Richard P. Bunge
Patrick M. Wood
Naomi Kleitman
Thomas K. Morrissey
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University Of Miami And Its School Of Medicine
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Publication of WO1992003536A1 publication Critical patent/WO1992003536A1/fr

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    • 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
    • C12N5/0622—Glial cells, e.g. astrocytes, oligodendrocytes; Schwann 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
    • A61K35/30—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

Definitions

  • the present invention relates to methods of promoting nervous system repair comprising transplanting autologous
  • Schwann cells may be used to facilitate neuronal regrowth, diminish neuron loss secondary to axonal injury, decrease denervation atrophy of muscle, provide for more effective remyelination, and to obviate the problem of immune rejection
  • Oligodendrocytes are the CNS counterparts of Schwann cells, but differ from Schwann cells in that oligodendrocytes
  • the yelin sheath improves nerve conduction velocity. Loss of the myelin sheath (demyelination) drastically alters nerve impulse conduction. Restoration of myelin (remyelination) is one of the simplest types of nervous system repair.
  • One type of nervous system repair is believed to involve the migration of cells, such as Schwann cells, into areas of injury (Blakemore, 1983, in “Spinal Cord Reconstruction", Kao et al., eds., Raven Press, NY, pp. 281- 291) .
  • Migration and axonal regeneration may require the presence of appropriate extracellular matrix (ECM) protein.
  • ECM extracellular matrix
  • the usefulness of peripheral nerve in CNS repair may be limited by the absence of appropriate ECM for the migration of Schwann cells into the CNS.
  • de la Torre (1982, Brain Research Bulletin £:545-552) inserted cell-free bovine-derived collagen into transected rat spinal cord and observed evidence of some degree of nerve regeneration through the graft.
  • Blakemore (1984, J. Neurolog. Sci. 6 :265-276) created areas of primary demyelination in spinal cord, and then placed autologous peripheral nerve tissue in the subarachnoid space over the lesions; subsequently, remyelination was observed to be limited to axons in the vicinity of blood vessels.
  • Blakemore (Id.) suggested that Schwann cells migrated from the transplanted tissue into the lesion via the perivascular space, but failed to remyelinate axons distant from blood vessels because of the absence within the CNS of suitable extracellular matrix (ECM) .
  • ECM extracellular matrix
  • central nervous system CNS lesions with a peripheral nerve environment may promote axonal regeneration of central neurons (David and Aguayo, 1981, Science 214:931-933; Davis et al., 1985, J. Neurosci. 5:2662-2671; Kleitman et al., 1988, Exp. Neurol. 102:298- 306).
  • Vidal-Sanz et al. (1987, Jr. Neurosci. 7:2894-2909) observed that peripheral nerve grafts could be used to guide lesioned optic nerve axons from the eye to the tectum to form terminal synapic contacts in the superior colliculus.
  • Natl. Acad. Sci. 82:6330-6334 produced lesions in the septo-hippocampal system of adult rats, bridged that gap with collagen-supported cultures consisting of Schwann cells, extracellular matrix, degenerating neuronal processes and myelin, and observed evidence of regeneration of CNS neurons in vivo.
  • Schwann cells have neurotrophic activities and/or mediating neurite attachment and growth are well established (Bunge and Bunge, 1983, TINS, 6:499-505; Muir et al., 1989, Neurochem. Res., 14:1003-1012; Rende et al., 1990, Soc. Neurosci. Astr. 16:807) .
  • Schwann cells in the distal nerve stump re-express both nerve growth factor (NGF) (Lemke and Chao, 1988, Development 102:499-504; Heumann et al., 1987, J. Cell Biol.
  • NGF nerve growth factor
  • NGFr NGF receptors
  • NGF-NGFr complex is then internalized and transported to the cell body to exert its neurotrophic activity (Johnson et al., 1987, J. Neurosci. 7:923-929).
  • Schwann cells from transected nerves and outgrowing neurites also re-express cell adhesion molecules (CAMs) , including nerve-CAM (N-CAM) and LI (Nieke and Schachner, 1985, Differentiation 30:141-151; Daniloff et al., 1986, J. Cell Biol. 103:929-945; Martini and Schachner, 1988, J. Cell Biol. 106:1735-1746). Both molecules may promote adhesion between Schwann cells and axons (Seilheimer and Schachner, 1988, J. Cell Biol. 107:341-351), facilitate the extension of regenerating neurites on the surface of Schwann cells (Daniloff et al., 1986; J. Cell Biol.
  • This collagen was more three-dimensional than the ammonia-gelled collagen we utilized; collagen preparations vary in organization (Bunge, et al., 1987, In: Progress in Brain Research; Seil, Herbert & Carlson (eds.), Elsevier, 71:61-74) and ability to support central neurite growth (Kleitman et al., 1988, J. Neurosci. 8:653-663) depending upon the gelling procedure employed.
  • BDNF brain derived neurotrophic factor
  • the present invention relates to methods of promoting nervous system repair comprising transplanting autologous Schwann cells into a region of nervous tissue injury.
  • Schwann cells for autologous grafting may be harvested from a patient in need of such treatment and then propagated in culture.
  • the present invention provides for cell culture methods which yield essentially pure populations of Schwann cells in substantial numbers which may preferably be derived from segments of adult peripheral nerve.
  • cells are passaged for a minimal number of times before use.
  • autologous Schwann cells may be applied directly to areas of nerve injury or disease or may, alternatively, be incorporated into a vehicle comprising support matrix material for transplantation.
  • autologous Schwann cells may be comprised in a prosthetic device which may be used to bridge lesions in the peripheral or central nervous systems.
  • the autologous transplantation methods of the invention may be used to promote neuronal regrowth, reduce neuronal loss secondary to axonal injury, decrease denervation atrophy of muscle, and to obviate the problem of immune rejection associated with transplantation of heterologous Schwann cells. Furthermore, the methods of the present invention may be applied to the treatment of central as well as peripheral nervous system lesions.
  • FIGURES Figure 1 Photomicrograph of a substantially pure population of human Schwann cells prepared from adult peripheral nerve.
  • F-80 or F-120 channels as compared to SC-0 and empty channels and **:p ⁇ 0.05 indicates statistical significance between sciatic nerve autografts and F-120 channels.
  • Figure 3 Light micrographs of toluidine blue-stained transverse sections taken 4 mm distally to the proximal suture in (A) CD-80, (B) F-80 channels and (C) sciatic nerve autografts 3 weeks postimplantation.
  • B and C) Myelinated axons (MA) with associated Schwann cells are seen. Note the presence of degenerating figures (DF) in the nerve autografts. In A, B, and C, scale bar 10 ⁇ m.
  • FIG. 4 Light micrographs of toluidine blue-stained transverse sections taken 4 mm distally to the proximal suture in (A) empty, (D) F-CD- ⁇ and (G) F-80 channels 3 weeks postimplantation. Note the increase in cable surface area between the regenerated cables. At higher magnification, light micrographs of transverse sections taken respectively 4 and 8 mm distally to the proximal suture in (B, C) empty, (E,F) F-CO- ⁇ and (H,I) F-80 channels showing nerve microfascicles with blood vessels (BV) and numerous myelinated axons (MA) .
  • BV blood vessels
  • MA myelinated axons
  • Figure 6 Number of blood vessels per mm 2 of cable surface area in transverse sections taken 2, 4, 6 and 8 mm from the proximal suture in empty, F-CO- ⁇ , F-40, F-80, F-120 and CD-80 channels and in sciatic nerve autografts 3 weeks post-implantation. Data represents means + SEM. The Games- Howell test was used to test statistical significance between treatments. **:p ⁇ 0.05 indicates statistical significance between F-CO- ⁇ channels and either F-40, F-80, F-120, CD-80 or empty channels, *:p ⁇ 0.05 indicates statistical signficance between sciatic nerve autografts and either type of channel.
  • a patient's own cells may be employed to foster regeneration of either the peripheral or the central nervous system.
  • small segments of the patient's own peripheral nervous system ay be obtained by biopsy and the cell type known to favorably influence neural regeneration (the cells of Schwann) , may be separated from a variety of other cells that are present in the peripheral nerve trunk.
  • the present invention provides, for the first time, methods of preparing essentially pure populations of Schwann cells from adult nerve; prior to the present invention, substantially purified Schwann cell populations could be prepared from embryonic but not from mature peripheral nerve. After separating the cells of Schwann, a variety of agents may be used to expand their numbers by causing them to proliferate in tissue culture dishes.
  • the cells may be utilized either in suspension or may be comprised in a cell- containing prosthesis for insertion into damaged areas of the central or peripheral nervous system.
  • the construction of this prosthesis may involve the combination of these cultured cells with a variety of organic or inorganic materials to assemble a prosthesis of the correct shape to promote regeneration of specific parts of the nervous system.
  • the prostheses may then be surgically implanted into the nervous system, either to enhance remyelination of demyelinated nerve fibers, to influence neural survival or to enhance the regeneration of nerve fibers.
  • the cells may be treated while in tissue culture by methods which allow the introduction of new genetic material in order to provide them with added growth potential or with the biological activities which they may not otherwise express, thereby enhancing the regenerative or functional activity of nerve cells.
  • Schwann cells may be infected with a retrovirus carrying the genetic code for the production of the enzyme tyrosine hydroxylase, allowing the cells to produce and release L-dopa, a compound which acts to relieve the symptoms of Parkinson's disease. If transplanted into the striatu , these cells may alleviate the movement disorder of this common neurological disease. Because the cells of Schwann appear to be active in influencing the regeneration in both the central and peripheral nervous system neurons, the cellular prostheses of the invention may be useful in the treatment of a variety of central (as well as peripheral) nervous system lesions.
  • the advantages of the present invention include the following.
  • the present invention makes available a quantity of autologous graftable material surpassing the amount of nerve tissue available for grafting in a particular patient.
  • the nerve prostheses of the invention comprise few or no fibroblasts, thereby avoiding the formation of fibroblast-associated scar tissue, which is believed to interfere with nerve regeneration and repair.
  • the detailed description of the invention is divided into the following subsections:
  • Nervous system lesions which may 5 be treated in a patient (including human and non-human mammalian patients) according to the invention include, but are not limited to, the following lesions of either the central or peripheral -lervous systems:
  • traumatic lesions including lesions caused by physical injury or associated with surgery, for example lesions which sever a portion of the nervous system, or compression injuries;
  • ischemic lesions in which a lack of oxygen in a portion of the nervous system results in neuronal injury or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia;
  • malignant lesions in which a portion of the nervous system is destroyed or injured by malignant tissue which is either a nervous system associated malignancy or a malignancy derived from non-nervous system tissue;
  • infectious lesions in which a portion of the nervous system is destroyed or injured as a result of infection, for example, by an abscess or associated with infection by human immunodeficiency virus, Lyme disease, tuberculosis, syphillis, herpes zoster or herpes simplex virus infection;
  • degenerative lesions in which a portion of the nervous system is destroyed or injured as
  • Schwann cells for autotransplantation may be harvested from regions in which removal of nerve tissue is preferably associated with as little clinical consequence as is practical. If the patient who is to receive the autotransplant has damaged tissue as a result of injury or surgery, Schwann cells may be harvested from the damaged tissue.
  • nerve tissue to be used as a source of Schwann cells may be harvested from peripheral nerve, and preferably sensory nerve, which may be obtained by a surgical procedure such as nerve biopsy.
  • peripheral nerve which may be used according to the invention include, but are not limited to, the sural nerve of the ankle, the saphenous nerve, or the brachial or antibrachial nerve of the upper limb, and are preferably sensory nerves.
  • Additional cell types may also be used for autotransplantation according to the invention, including, but not limited to, oligodendrocytes, retinal cells, glial cells (e.g., olfactory glial cells or glial cells from enteric nerve plexices) , or astrocytes. Such cells may be obtained by biopsy or may be recovered from resected tissue.
  • cells harvested from a patient for autotransplantation may be allowed to proliferate in culture so as to expand their numbers or so as to activate the cells prior to transplantation.
  • Cells harvested from a patient may be cultured together with other cell types, or may be cultured so as to select for the growth or survival of one specific population of cells.
  • substantially pure cultures of Schwann cells may be obtained using the following procedure.
  • a portion of peripheral nerve may be cultured as an explant having preferably dimensions of about 1 X 1 mm in Dulbecco's Modification of Eagle's medium (MEM; or a similar basic culture medium) supplemented with 10% fetal bovine serum on plastic tissue culture substratum in a 7% CO atmosphere at 37 C.
  • MEM Dulbecco's Modification of Eagle's medium
  • fibroblasts may be observed to grow out from the explants, which may be periodically transferred to fresh culture containers at a frequency of about once a week for a period of about 3 to 5 weeks.
  • the explants may then be dissociated using, for example, about 1.25 units/ml of dispase and about 0.05 percent collagenase with 15 percent fetal calf serum and 50 mM HEPES buffer (pH 7.4) in Dulbecco's MEM.
  • the dissociated cells should be greater than about 90 percent Schwann cells. Following dissociation, the cells may be used for autotransplantation or, preferably, may be allowed to proliferate in culture.
  • Proliferation may be induced by culturing the purified Schwann cells in the presence of co- cultured axons or glial growth factor (GGF) and forskolin at concentrations of about 20 ⁇ g/ml and 2 ⁇ M, respectively, preferably on a polylysine over plastic substratum.
  • GGF glial growth factor
  • Other agents which may induce Schwann cells to proliferate and which may be used according to the invention include, but are not limited to cholera toxin, laminin, fibronectin, nerve growth factor, platelet derived growth factor, fibroblast growth factors, and transforming growth factor beta; other proliferation inducing agents may become known in the art and are envisioned by the present invention.
  • cultured Schwann cells produced by the method described supra or any method known in the art
  • differentiated Schwann cell functions including, but not limited to, the ability to associate with sensory neurons in culture or the ability to produce myelin. This may be accomplished by establishing substantially pure populations of sensory neurons in culture, seeding the Schwann cells onto these neuronal populations and observing the cell interactions over a period of several weeks. 5.2.2. ADDITIONAL METHODS FOR SELECTING CELLS FOR AUTOTRANSPLANTATION
  • cells which may be used for autotransplantation may be selected from mixed populations of cells using cell selection techniques widely known in the art.
  • cell selection techniques may include, but not be limited to, the selective proliferation of the cell type of interest using specific growth factors or defined culture conditions, the selection of cells of interest by exposing the cells to antibody which selectively binds to the cell type of 0 interest, such that antibody binds to the cell type of interest and such binding is detectable, using, for example, rosetting techniques, magnetic beads, colorimetric techniques, fluorescence techniques including fluorescence activated cell sorting, or affinity chromatography using, for 5 example, a Staphylococcus protein A surface to collect antibody-bound cells, to name but a few of the techniques available.
  • the cell type of interest may be selected by selectively destroying cell types which are not of interest, using, for example, compounds or culture 0 conditions which inhibit the growth or are toxic to undesirable cell types, or by using antibodies to destroy undesirable cell types by, for example, complement mediated cell lysis or antibody dependent cell-mediated cytotoxicity.
  • Schwann 5 cells may be selected using antibodies to the NGF receptor, such as the antibody disclosed in Peng, et al. (1982, Science 215: 1102-1104) .
  • fibroblasts from peripheral nerve could be selectively destroyed, either via antibody directed toward a fibroblast specific antigen such as, for Q example, the thy-1 antigen and may either be separated from Schwann cells by techniques including, but not limited to, fluorescence activated cell sorting or complement mediated lysis, or may be selectively destroyed on the basis of higher proliferative activity relative to Schwann cells by agents 5 selectively toxic to actively dividing cells, including, but not limited to, methotrexate and cytosine arabinoside or fluorodeoxyuridine, or may be destroyed during passaging using active complement in the presence of Thy-1 antigen.
  • agents 5 selectively toxic to actively dividing cells including, but not limited to, methotrexate and cytosine arabinoside or fluorodeoxyuridine, or may be destroyed during passaging using active
  • cells may be autotransplanted into a nervous system lesion directly after harvesting or, alternatively, after culturing.
  • the cells may preferably be comprised in a pharmacologically suitable carrier such as a physiologically compatible vehicle, including, but not limited to, culture medium, including Dulbecco's Modified Eagle's Medium, RPMI 1640, Fisher's, Iscove's, or McCoy's medium to name but a few; or preferably, a solid, semisolid, gelatinous, or viscous support medium including, but not limited to, collagen, collagen- glycosaminoglycan, fibrin, polyvinyl chloride, polyamino acids such as polylysine or polyornithine, hydrogels, agarose, dextran sulfate or silicone, to name but a few.
  • a physiologically compatible vehicle including, but not limited to, culture medium, including Dulbecco's Modified Eagle's Medium, RPMI 1640, Fisher's, Isco
  • the support medium may, in specific embodiments, comprise growth factors or relevant extracellular matrix proteins, such as laminin.
  • gelatinous vehicles include, but are not limited to, collagen, collagen-glycosaminoglycan, hydrogels, agarose, dextran sulfate or silicone.
  • cells may be introduced into a liquid phase of the vehicle which is subsequently treated such that it becomes more solid.
  • cells may be added to unpolymerized vehicle which is then caused to polymerize.
  • cells may be added to type I collagen solubilized in acetic acid in H 2 0.
  • this mixture is brought to neutral pH by addition, for example, of NaOH, and to isotonicity by the addition of salts it should gel in several minutes time at room temperature.
  • cells Prior to gelling, cells can be intermixed into the mixture and thereby may be dispersed throughout the matrix after gelling. If the vehicle permits, cells may simply be mixed with the vehicle.
  • the vehicle in which the vehicle has a solid structure, the vehicle may be molded into a shape which may conform to the shape of the lesion.
  • the vehicle comprising cells may either be applied directly to the lesion or may be partially or completely enclosed in a second material which may allow the vehicle to retain a particular shape while permitting desirable regeneration through the vehicle.
  • the second material may be applied before or after insertion of the vehicle into the patient.
  • a semisolid vehicle comprising autologous cells may be applied to a spinal cord lesion which extends to the surface of the cord, creating a gap.
  • a second substance such as a plastic or hydrogel, may then be placed at the surface of the cord over the gap so as to confine the vehicle and cells within the lesion.
  • a solid tube filled with semi-solid vehicle comprising autologous cells may be used to bridge a gap in a peripheral nerve, an optic nerve, or other parts of the nervous system.
  • autologous cells may be introduced into a nervous system lesion using any method known in the art.
  • the autologous cells comprise Schwann cells or more preferably, substantially pure populations of Schwann cells.
  • Methods of introducing autologous cells include, but are not limited to, surgical techniques which expose the neurological lesion and permit introduction of autologous cells in a suitable vehicle, as well as techniques which can inject autologous cells in suitable vehicle into a neurological lesion without exposing the lesion.
  • a portion of the spinal cord has been destroyed by a crush injury
  • surgical techniques including laminectomy may be used to expose the affected region of the spinal cord.
  • the damaged central region of the cord is often removed by macrophage action, leaving a fluid-filled cyst.
  • This cyst may be visualized by imaging techniques (such as ultrasound) and an injection of cells plus vehicle may then be made directly into the cystic defect.
  • This injection may contain autologous Schwann cells comprised in a semi-solid vehicle such as solubilized collagen (see supra) which should solidify shortly after mixing.
  • the initially semi-solid character of the vehicle permits effective filling of the lesioned area; subsequent solidification may obviate the need to otherwise fix the vehicle in place.
  • a solid vehicle such as a cylindrical sleeve comprising a solid polymer exterior and an interior comprising a gel and autologous cells preferably Schwann cells
  • the affected nerve may then be surgically exposed and the lesioned area removed and replaced with the nerve prosthesis, which may be sutured or otherwise fixed in place.
  • autologous cells in suitable, preferably liquid, vehicle may be injected into the affected area, preferably using radiologic guidance, being careful to limit the injected material to a volume easily accommodated by the tissue so as to avoid increased pressure within the tissue and/or intracranially.
  • the present invention may be utilized to influence the regeneration of the nervous system in a specific human patient.
  • the present invention may be used to treat a patient who has received a severe vertebral injury resulting in the fracture of vertebral bone which has severely crushed that portion of the nervous system within the vertebral canal referred to as the cauda equina. If severe, such an injury may result in motor paralysis, sensory loss, and loss of visceral functioning. Furthermore, the crushing injury may be expected to cause a severe fibrosis of the nerve roots within the cauda equina.
  • the patient may be examined to ascertain, utilizing both neurological and modern imaging techniques, that the neural injury is to the cauda equina.
  • the spinal cord itself is not damaged; rather, damage is limited to the nerve roots of the cauda equina.
  • a biopsy of the patient's peripheral nerve may be taken by removing, for example, a 3-5 inch segment of the sural nerve posterior to the lateral malleolus on the lateral aspect of the ankle. After removal from the body the nerve segment may be placed immediately in sustaining fluids such as a tissue culture medium and cooled. Subsequently, the area of incision may be sutured.
  • the patient may expect to suffer a small area of anesthesia at the lateral aspect of the foot, but there should be no motor deficiency, inasmuch as this is primarily a sensory nerve at the level that the nerve has been removed.
  • the surgery should have no immediate consequences inasmuch as the patient may already be anesthetic below the level of the cauda equina lesion. If the lesion in the cauda equina region involved nerve roots that provide nerve fibers to the sural nerve it may be desirable to use an alternate biopsy site in as much as the sural nerve may have undergone degenerative changes secondary to the injury. In this case, it may be advantageous to use a cutaneous branch of one of the sensory nerves of the arm (see supra) .
  • the segment of nerve may be immediately taken to a tissue culture facility and treated in one of several alternative ways.
  • the nerve is first stripped of its epineural lining with the use of very fine forceps.
  • the nerve may be cut into a multitude of small pieces approximately 0.5mm in diameter which may be placed in tissue culture on plastic surfaces. These may be maintained under standard tissue culture conditions in a medium comprising a minimal essential medium and 10% serum.
  • the fragments of nerve may be treated with digestive enzymes to allow direct dissociation of the cells.
  • the mixed cellular elements obtained may then be propogated in tissue culture.
  • these fragments may be carried in the culture dishes as described (supra) for approximately one week.
  • degenerative changes may occur in the nerve resulting from its amputation from connection to nerve cell bodies.
  • Axons, as well as the surrounding myelin sheaths may degenerate, but the Schwann cells which have been related to myelin sheaths (as well as Schwann cells which would be related to unmyelinated fibers) may, during this period of time, undergo a gradual increase in number.
  • Periodic transplantation of these fragments of nerve to new culture dishes may allow many of the fibroblasts to escape from the explant, leaving the explant enriched in Schwann cells.
  • the outgrowth is progressively enriched in the Schwann cells.
  • These fragments may then be harvested by detaching them from the culture dish and exposing the cells to proteolytic enzymes which have little effect on the living cells but which are effective in dissociating the connective tissue framework within these fragments. From this dissociation several thousand to several hundred thousand Schwann cells may be obtained.
  • This Schwann-cell enriched population may then be expanded from several thousands of cells into populations which may number in the hundreds of thousands to several million.
  • a number of methods may be used to stimulate Schwann cell proliferation, including a) the use of a protein factor called glial growth factor (used in conjunction with forskolin; b) the use of fragments of axolem a obtained by fractionation techniques; c) the use of a variety of growth factors such as transforming growth factor beta, or acidic or basic fibroblast growth factor which are known to be effective on Schwann cells of certain species, especially when combined with agents which raise intracellular cyclic AMP; and d) the use of certain molecules as substratum (for example it has been shown that certain Schwann cells proliferate in the presence of serum, when they are grown on substratum containing the extracellular matrix molecule laminin, but not when they are grown on collagen substratum) .
  • the cells may be optionally treated to introduce new genetic material to enable them to produce desired proteins.
  • a gene encoding a specific cell adhesion molecule which is known to promote nerve fiber growth, or a gene for a specific growth factor molecule (such as the gene for neurotrophin-3 (NT-3)) believed to sustain neuronal health. It is anticipated that under ideal conditions expansion may occur at the rate of 1-2 doublings per week so that it may take several weeks to obtain the required number of cells.
  • the purity of the cells may be monitored by utilizing antibodies which specifically mark Schwann cells (such as antibodies to S100 protein) and by undertaking complement mediated cell lysis of non-Schwann cell contaminants (as described supra) at the time when the cells are transferred from one dish to another as the numbers expand.
  • antibodies which specifically mark Schwann cells such as antibodies to S100 protein
  • complement mediated cell lysis of non-Schwann cell contaminants as described supra
  • the cultured cells may be combined with an encasing matrix, thereby providing a tubular prosthesis which has the correct shape and length to insert into an area of neural injury.
  • Various organic polymers (poly amino acids) or hydrogels are examples of materials known to be compatible with body tissues, and which are also known to provide hospitable housing for live cells, as well as other materials discussed in Section 5.3, supra, may be used.
  • a considerable length of a tubular prosthesis may be needed which would provide an envelope which is porous to allow nutrients to be exchanged between the external aspect of the tubular prosthesis and the enclosed Schwann cells.
  • the envelope may desirably have characteristics which would accept suturing.
  • An estimate of the length of the prosthesis may be undertaken, knowing the number of nerve roots that are involved in the crush injury and the overall dimensions of the lesion which contain these crushed roots. For example, if 12 nerve roots were to be repaired over a distance of two inches, a total of 24 inches of prosthesis several millimeters in diameter would be required, much more material than would be available from the use of an autograft in which the sural nerve would be directly used as an implant into the region of lesion.
  • the patient may be prepared for surgery, and the injury site may be exposed by laminectomy. At the site of nerve root crush the entangled roots and the fibrous scar in which they are embedded may be physically removed.
  • the individual nerve roots may then be identified so that each nerve root as it egresses from the cord above would be connected to the proper nerve root as it courses toward its destination distal to the site of injury below.
  • the availability of extensive prosthetic material according to the invention may allow the prosthesis to be sewn between the divided central and peripheral aspect of the nerve root. In this way the region of injury may be removed from the body and a bridge inserted to replace the region of injury for each of several critical nerve roots. After closing the dura over this repair site, the patient may then be expected to experience a relatively long period of recovery in which nerve fibers may grow across the region of bridging to reach targets which would have previously been denervated.
  • ventral roots may be observed to effectively reach the distal segment and to follow that distal nerve segment to the regions of the muscles which would have been denervated. It may be expected that this process may take several months or longer, inasmuch as the distance of nerve growth required in this case may be between 10 and 15 inches and the rate of nerve growth as it would be observed in standard peripheral nerve injury is at the rate of about one inch per month.
  • this type of Schwann cell containing prosthesis may be used to foster nerve regeneration in which the distances for regeneration may be much shorter. Of particular interest are those patients in which spinal cord injury has occurred in the cervical region. Many of these patients have partial or complete loss of motion within the muscles of their arms.
  • the distance between the site of injury and the portion of the spinal cord which may be intact and able to signal movement within the upper extremity is relatively short; in certain cases only one or two inches.
  • the ability to obtain some regrowth to attain some control over motor neurons distal to a cervical site of injury may be extraordinarily beneficial to a patient who may have little useful movement in certain of the arm muscles.
  • the lesion often results in the loss of central cord tissue which becomes a fluid-filled cystic space. This space would provide a repository for the cellular implant, which in this case may not require an enveloping sleeve in as much as the retained peripheral cord tissue would contain the injected material.
  • Laminectomies were performed at T7 and T8 and a small longitudinal incision was made dorsolaterally in the dura and cyst wall on the left side. Two grafts were introduced through this slit and positioned with their axes oriented mainly in the rostrocaudal direction. No immunosuppression was used. Animals were perfused with fixative (Kuhlengel et al., 1990, J. Comp. Neurol. 293:74-91) at 14, 28, and 90 to 180 days after implantation and processed for light microscopy (semi-thin plastic sections stained with toluidine blue or paraffin sections stained with the Sevier-Munger silver stain) and for electron microscopy.
  • the grafts filled the lesion cavity and closely apposed much of the cavity laterally.
  • the graft reached at least portions of the rostral and caudal ends of the cavity. Macrophage accumulation sometimes prevented the graft from contacting host tissue.
  • axonal ingrowth of the same magnitude as that present at 28 days was observed.
  • Myelinated axons were outnumbered by those only ensheathed by SCs at all time periods. Axons within the implant always appeared in relation to SCs, either in contact with them or surrounded by their basal lamina. As seen by silver staining, the axons followed parallel paths, at least for some distance; the spiralled configuration of the collagen may have constrained many of them to a roughly linear trajectory along the graft. Only at the graft-host interface was profuse axonal branching observed. Astrocytic gliosis at the interface was minimal.
  • SC-collagen grafts are being studied.
  • the appearance of numerous SC- yelinated axons in dorsal regions of lesioned but not implanted cord suggests some ingrowth from dorsal root ganglia (Salvatierra et al., 1990, Soc. Neurosci. Abstr. 16:1282).
  • Silver stained sections suggest that at least some axons traveling through the grafts originate in the spinal cord; the presence of axons in close relation to Schwann-like cells in zones of degenerating corticospinal tract caudal to the lesion suggests that some axons are able to re-enter host tissue and extend for some distance when accompanied by SCs.
  • Our present results utilizing purified SC populations clearly establish the ability of SCs to influence the capacity of regeneration in the spinal cord.
  • Channels containing Schwann cells were compared to nerve autografts, empty channels or channels filled with the laminin-containing hydrogel alone. By three weeks post implantation, regenerating axons had grown into all grafts. Sciatic nerve autografts supported extensive regeneration, containing 4000-5000 myelinated axons at the graft midpoint. The ability of tubes containing Fisher Schwann cells to foster regeneration was dependent on the density of Schwann cells. At the graft midpoint, F-120 channels contained nearly as many myelinated axons as sciatic nerve autografts, and significantly more than F-80, F-40 or control channels.
  • the nerve cable in Schwann cell containing tube consisted of larger, more organotypic fascicles than acellular control channels.
  • heterologous (CD) Schwann cells elicited a strong immune reaction which impeded regeneration; myelinated axons were seen no more than 2mm into the graft channel.
  • CD heterologous
  • the present study shows that cultured adult syngeneic Schwann cells seeded in high density in permselective synthetic guidance channels support extensive peripheral nerve regeneration. Successful regeneration depends upon immune compatibility between donor and host, indicating the importance of developing large populations of autologous adult human Schwann cells for clinical use in the repair of nerve injury.
  • Schwann cell cultures Schwann cells from adult , rat sciatic nerves were isolated according to a modified technique by (Morrissey et al., 1991, J. Neurosci., in press). Schwann cells were harvested from peripheral nerves following a period of "in vitro Wallerian degeneration". Sciatic nerves either from adult male inbred Fisher 344 rats (Taconic, Germantown, NY) weighing 250-300 g or from adult male outbred CD rats (Charles River Laboratories, MA) weighing 400 to 450 g were collected into Dulbecco's Modified Eagles
  • DMEM DMEM
  • Vitrogen ® -coated Collagen Corporation, Palo Alto, CA
  • Petri dishes Fisher Scientific
  • FCS Frco Lab
  • penicillin/streptomycin 1,000 U/ml
  • DMEM-FCS DMEM-FCS
  • HBSS Ca -Mg -free Hanks Balanced Salt Solution
  • trypsin Sigma, St. Louis, MO
  • collagenase Sigma
  • hyaluronidase Sigma
  • the culture medium was replaced with mitogenic medium containing DMEM, FCS, forskolin (2 ⁇ M) (Sigma) and pituitary extract (10 ⁇ g/ml) (Collaborative Research Inc., Bedford, MA) a day later (Porter et al., 1986, J. Neurosci. 3:170-3078).
  • mitogenic medium containing DMEM, FCS, forskolin (2 ⁇ M) (Sigma) and pituitary extract (10 ⁇ g/ml) (Collaborative Research Inc., Bedford, MA) a day later (Porter et al., 1986, J. Neurosci. 3:178).
  • mitogenic medium containing DMEM, FCS, forskolin (2 ⁇ M) (Sigma) and pituitary extract (10 ⁇ g/ml) (Collaborative Research Inc., Bedford, MA) a day later (Porter et al., 1986, J. Neurosci. 3:178).
  • mitogenic medium containing DMEM, FCS,
  • Guidance channel preparation consisted of a 60:40 acrylonitrile vinylcholoride (PAN/PVC) copolymer tubing with a 1.12 mm ID and a 0.126 ⁇ m-thick wall. These tubes were fabricated using a dry-jet wet spinning technique (Cabasso 1980, Encyclopedia of Chemical Technology, Kirk-Othner, 12:492-517; Aebischer et al., 1991, Biomaterials 12:50-56). The tubes featured a smooth inner skin connected to a partially fenestrated outer skin by a trabecular network. The inner skin provided a permselective barrier, with a molecular weight cut-off of 50,000 Da. Prior to seeding, the tubing was cleaned and sterilized as previously described (Aebischer et al., 1988, Brain Res. 454:179-187).
  • PAN/PVC acrylonitrile vinylcholoride
  • Rat syngeneic (inbred) Schwann cells were collected by centrifugation after treatment with 0.05% trypsin and 0.02% EDTA (Sigma) for 5 min at 37°C. The cells were pelleted, washed in DMEM and the number of live cells was evaluated with trypan blue exclusion using a hemocytometer. Pelleted cultured Schwann cells were gently suspended in a 70:30 (v:v) solution of DMEM:laminin-containg hydrogel (Matrigel ) (Collaborative
  • DMEM-FCS:laminin-containing hydrogel at a final density of 80 x 10 cells/ml (CD-80 channels) .
  • the filled tubes were then placed in DMEM at room temperature to allow for gellation of the cell suspension and cut into lOmm-long pieces. These tubes were closed at each end using PAN/PVC copolymer glue.
  • the seeded channels were kept in DMEM for
  • Control channels were filled with a 70:30 ⁇ solution of DMEM:Matrigel in a similar way (F-CO- ⁇ channel) .
  • the closing caps were removed at the time of implantation.
  • Arrangement of the cells in the channels Prior to implantation, tubes collected i) after filling, ii) on the day of surgery and iii) 5 days post-seeding were fixed overnight in 2.5% paraformaldehyde in 0.l M PBS at pH 7.4 and observed under either scanning electron microscopy (SEM) or light microscopy.
  • SEM scanning electron microscopy
  • the channels were post-fixed, dehydrafted in dimethylaminomethyl phenol (DMP) , critical point dried and observed using a Hitachi 2700 microscope.
  • the 217c antibody was generously provided by Dr. J. de Vellis (University of California at Los Angeles, CA) . Contamination with fibroblasts was assessed using a rabbit polyclonal fibronectin antibody, generously supplied by Dr. R. Morris (National Institute for Medical Research, London, England) .
  • fibroblast identification cells were fixed with 95% ethanol: 5% glacial acetic acid (v:v) solution at -20°C for 20 min and incubated overnight at 4°C with rabbit anti-fibronectin antiserum (1:20,000) with 0.2% Triton X-100 in buffer.
  • Tubes collected on the day of implantation were fixed overnight in 3% paraformaldehyde in 0.1 M PBS at pH 7.4.
  • the specimens were frozen on dry ice and 20 ⁇ m-thick longitudinal sections were obtained using a Reichert-Jung 1800 cryocut (Cambridge Instruments) .
  • the sections were mounted on APTES-coated glass slides and reacted with the
  • Sciatic nerve autografting The left sciatic nerve was exposed and cut both 3 and 11 mm distal to the tibio-peroneal bifurcation. The 8 mm-long piece of sciatic nerve was removed and rotated to align the distal end of the nerve segment with the proximal nerve stump and the proximal end of the nerve piece with the distal nerve stump. The nerve autograft was secured in place using 4 to 6 10.0 nylon suture epineurial stitches. Six rats received sciatic nerve autografts for 3 weeks.
  • Arrangement of the cells in the channels Cell viability as observed by trypan blue exclusion was above 95% at the time the channels were seeded. Scattered round cells filled the lumen of the tubes. After a day in culture, a cellular cable free of attachment from the channel's wall was observed at both SEM and light microscopy. Cells were aligned along the longitudinal axis of the tubes, arranging themselves side-by-side and end-to-end. A similar pattern was observed after 5 days in culture. The aligned cells were 217c-positive, indicating that seeded Schwann cells expressed NGF receptors at the time of implantation.
  • ® outbred or inbred Schwann cells suspended in Matrigel were round in shape, and separated from the inner wall of the channels by an acellular gel.
  • the regenerated cables consisted of blood vessels and nerve microgascicles with unmyelinated and myelinated axons and associated Schwann cells surrounded by an epineurial-like sheath. Myelinated axons extended up to section S2 in all the tubes but were never seen at level S4
  • Syng ⁇ neic Schwann cells Cable cross-sectional area: In all the channels, the cable surface area (CSA) decreased up to section S4, then increased toward the distal nerve stump (Figure 2A) . Cables extending in both F-120 and F-80 channels were larger than cables regenerated in empty, F-CD- ⁇ and F-40 channels ( Figure 3A) . There was no significant difference in CSA between cables regenerated in F-40 tubes as compared to empty and F-CO- ⁇ channels ( Figure 2A) .
  • cables contained nerve microfascicles and blood vessels surrounded by a thin epineurial-like layer ( Figures 4A, 4D, and 4G) .
  • the percentage of the CSA covered with an epineurial-like structure was similar in all channels.
  • microfascicles contained both unmyelinated and myelinated axons with associated Schwann cells ( Figures 3B, 4B, 4C, 4H, 41, 5A and 5B) .
  • Myelinated axons The number of myelinated axons decreased along the length of all of the channels ( Figure 2B) . At any section along the tubes, cables regenerated in
  • F-40, F-80, and F-120 channels contained more myelinated axons as compared to empty and F-CD- ⁇ channels, except at S8 where F-40 channels contained less myelinated axons than empty channels ( Figure 2B) .
  • the myelinated axon population increased ( Figure 2B) ;
  • F-120 channels contained significantly more myelinated axons as compared to F-80 channels at both S2 and S4 and the myelinated axon population was signficantly higher in F-80 channels as compared to F-0 channels from S6 to S8.
  • cables extending in F-CO- ⁇ tubes contained less myelinated axons as compared to cables regenerated in empty tubes ( Figure 3B) ; however, the difference was not statistically significant.
  • Blood vessels The number of blood vessels per mm 2 of CSA did not vary significantly between empty, F-40,
  • the present study shows that cultured adult syngeneic Schwann cells harvested after their isolation from degenerated sciatic nerve segments and suspended in a laminin-containing hydrogel are able to form an oriented central cable in synthetic guidance channels. Such Schwann cell cable enhanced peripheral nerve regeneration through permselective guidance channels in a seeding density- dependent fashion.
  • Our findings show that myelinated axon population increased in channels containing syngeneic Schwann cells as compared to channels filled with laminin- containing hydrogel. Although the laminin-containing hydrogel itself impeded regeneration, as previously reported (Valentini et al., 1987, Exp. Neurol.
  • the physical organization of the Schwann cells in the tubes may have also influenced the outcome of regeneration.
  • channels were filled with an organized cellular cable aligned along their main axis.
  • Schwann cells themselves were also oriented in the same direction, lining end-to-end and side-by-side. It is likely that the presence of this pre-existing Schwann cell cable enhanced regenerative processes. It has been demonstrated that the formation of an organized central fibrin cable between the stumps of transected peripheral nerves is critical for successful regeneration (Williams and Varon, 1985, J. Comp. Neurol. 12:851-860; Aebischer et al., 1990, Brain Res. 531:211-218).
  • prefilling silicone elastomer channels with an organized fibrin matrix enhances the rate of regeneration of peripheral nerves (Williams, 1987, Neurochem. Res. 12:851-860).
  • the lined Schwann cells may have served as a scaffold for elongating axons and initial regenerative events were not needed for neurite outgrowth.
  • the Schwann cell seeding density in the tubes influenced the outcome of regeneration.
  • Non-syngeneic adult Schwann cells seeded in per selective guidance channels impeded regeneration.
  • Myelinated axons did not extend farther than 2 mm within channels seeded with non-syngeneic Schwann cells whereas myelinated axons and bridged the nerve gap in channels seeded with syngeneic Schwann cells at a similar density.

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Abstract

L'invention concerne des procédés favorisant la réparation du système nerveux et consistant à transplanter des cellules de Schwann autologues dans une région d'une lésion des tissus du système nerveux. Dans des modes particuliers de réalisation de l'invention, des cellules de Schwann pour une greffe autologue peuvent être prises chez un patient ayant besoin d'un tel traitement puis elles peuvent se propager en culture. La présente invention fournit des procédés de culture de cellules qui produisent essentiellement des populations pures de cellules de Schwann en quantité substantielle et qui peuvent être dérivées de préférence de segments de nerfs périphériques adultes.
PCT/US1991/005817 1990-08-15 1991-08-15 Autotransplantation de cellules de schwann favorisant la reparation du systeme nerveux WO1992003536A1 (fr)

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US5849585A (en) * 1995-05-10 1998-12-15 Genetech, Inc. Isolating and culturing Schwann cells
US5920373A (en) * 1997-09-24 1999-07-06 Heidelberg Engineering Optische Messysteme Gmbh Method and apparatus for determining optical characteristics of a cornea
US6033660A (en) * 1995-05-10 2000-03-07 Genentech, Inc. Method of treating a nervous system injury with cultured schwann cells
WO2000018414A1 (fr) * 1998-09-29 2000-04-06 Diacrin, Inc. Transplantation de cellules neuronales pour le traitement des lesions ischemiques dues a une attaque
WO2002061052A2 (fr) * 2001-01-31 2002-08-08 Interface Biotech A/S Procede perfectionne de culture de cellules de mammifere in vitro pour des procedes d'implantation/de transplantation de cellules autologues
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CN111655336A (zh) * 2017-11-17 2020-09-11 纽约州立大学研究基金会 使用x射线微束辐射治疗受损周围神经的方法
EP3929281A1 (fr) 2020-06-24 2021-12-29 Fachhochschule Technikum Wien Construction cellulaire comprenant des cellules de schwann ou des cellules similaires à des cellules de schwann et une matrice biocompatible

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Cited By (27)

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EP1632238A1 (fr) 1994-04-15 2006-03-08 Neurotech S.A. Cellules encapsulées adaptées pour être implantées dans le liquide de l'humeur vitrée de l'oeil
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
US5795790A (en) * 1994-07-20 1998-08-18 Cytotherapeutics, Inc. Method for controlling proliferation and differentiation of 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
US5833979A (en) * 1994-07-20 1998-11-10 Cytotherapeutics, Inc. Methods and compositions of growth control for cells encapsulated within bioartificial organs
US5840576A (en) * 1994-07-20 1998-11-24 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
EP1983053A2 (fr) 1994-07-20 2008-10-22 Neurotech USA, Inc. Contrôle de la distribution des cellules au sein d'organes bioartificiels
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
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
US5935849A (en) * 1994-07-20 1999-08-10 Cytotherapeutics, Inc. Methods and compositions of growth control for cells encapsulated within bioartificial organs
US6392118B1 (en) 1994-07-20 2002-05-21 Neurotech S.A. Mx-1 conditionally immortalized cells
US6033660A (en) * 1995-05-10 2000-03-07 Genentech, Inc. Method of treating a nervous system injury with cultured schwann cells
US5849585A (en) * 1995-05-10 1998-12-15 Genetech, Inc. Isolating and culturing Schwann cells
US5721139A (en) * 1995-05-10 1998-02-24 Genentech, Inc. Isolating and culturing schwann cells
US6495364B2 (en) * 1995-05-23 2002-12-17 Neurotech, S.A. Mx-1 conditionally immortalized cells
US5920373A (en) * 1997-09-24 1999-07-06 Heidelberg Engineering Optische Messysteme Gmbh Method and apparatus for determining optical characteristics of a cornea
WO2000018414A1 (fr) * 1998-09-29 2000-04-06 Diacrin, Inc. Transplantation de cellules neuronales pour le traitement des lesions ischemiques dues a une attaque
WO2002061052A2 (fr) * 2001-01-31 2002-08-08 Interface Biotech A/S Procede perfectionne de culture de cellules de mammifere in vitro pour des procedes d'implantation/de transplantation de cellules autologues
WO2002061052A3 (fr) * 2001-01-31 2002-12-12 Interface Biotech As Procede perfectionne de culture de cellules de mammifere in vitro pour des procedes d'implantation/de transplantation de cellules autologues
US7147647B2 (en) 2002-04-26 2006-12-12 Medtronic, Inc. Sintered titanium tube for the management of spinal cord injury
CN110343171A (zh) * 2012-02-29 2019-10-18 百深公司 周围神经的IgG刺激髓鞘再生
CN111655336A (zh) * 2017-11-17 2020-09-11 纽约州立大学研究基金会 使用x射线微束辐射治疗受损周围神经的方法
US11511136B2 (en) 2017-11-17 2022-11-29 The Research Foundation For The State University Of New York Method for treating damaged peripheral nerves using x-ray microbeam irradiation
EP3929281A1 (fr) 2020-06-24 2021-12-29 Fachhochschule Technikum Wien Construction cellulaire comprenant des cellules de schwann ou des cellules similaires à des cellules de schwann et une matrice biocompatible
WO2021260137A1 (fr) 2020-06-24 2021-12-30 Fachhochschule Technikum Wien Construction cellulaire comprenant des cellules de schwann ou des cellules du type cellules de schwann et une matrice biocompatible

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