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  • 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
  • C12N5/0619—Neurons
• • 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
• • C—CHEMISTRY; METALLURGY
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  • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
  • C12N2506/08—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from cells of the nervous system

Definitions

• This invention relates to methods for culturing neural tissue, and includes methods for ameliorating/treating impairment to the central nervous system (CNS) due to injury and/or disease by transplantation of cultured neural tissue to the site of injury.
• CNS central nervous system
• Transplantation of neural tissue into the mammalian CNS is a potential therapeutic treatment for neurological and neurodegenerative disorders including epilepsy, stroke, Huntington's disease, head injury, spinal injury, pain, Parkinson's disease, myelin deficiencies, neuromuscular disorders, neurological pain, amyotrophic lateral sclerosis, Alzheimer's disease, and affective disorders of the brain.
• neurological and neurodegenerative disorders including epilepsy, stroke, Huntington's disease, head injury, spinal injury, pain, Parkinson's disease, myelin deficiencies, neuromuscular disorders, neurological pain, amyotrophic lateral sclerosis, Alzheimer's disease, and affective disorders of the brain.
• fetal ventral mesencephalic tissue has been demonstrated to be a viable graft source in Parkinson's disease. [Lindvall et al, 1987; 1990; Bjorklund, 1992].
• fetal striatal tissue has been utilized successfully as graft material in Huntington's disease [Isacson et al, 1986; San
• Neurologically dysfunctional animals have been transplanted with non-fetal, non- neuronal cells/tissue.
• the major advantage of this type of transplantation protocol is that the graft source is not a fetal source and, thereby, circumvents the ethical and logistical problems associated with acquiring fetal tissue [Bjorklund and Stenevi, 1985; Lindvall et al, 1987]. It would be useful to be able to also use neural grafts of both fetal and non-fetal neuronal cells and primary and secondary cell cultures to improve the graft integration (form connections) with the CNS of the recipient (i.e. the host).
• spinal cord injury in mammals is characterized by immediate and severe loss of sensory, motor, and reflex function below the level of injury.
• Transplantation strategies permit recovery of motor function both by specific mechanisms (i.e. permitting the regeneration of particular descending and segmental pathways, and restoring specific synaptic input to denervated targets within the spinal cord) as well as by nonspecific mechanisms (such as providing trophic support for injured neurons or by providing a terrain that permits axonal elongation).
• Animal experiments indicate that transplants of fetal spinal cord tissue mediate recovery of function after spinal cord injury in both neonatal and adult mammals, suggesting that the mechanisms underlying transplant-mediated recovery differ between newborn and adult.
• transplants of fetal CNS or peripheral nervous system (PNS) tissue can influence the response to injury and mediate recovery of function after injury.
• Transplants may replace particular populations of neurons, permit the re-establishment of specific connections between the host and the transplant, or replace or restore levels of neurotransmitters, hormones, or neurotrophic factors lost by the injury [references 1-5].
• the suprasegmental control over the intrinsic segmental circuitry of the spinal cord tissue may serve as a bridge to permit the regrowth of axons from spinal and supraspinal levels across the site of spinal cord injury to reach targets within the cord caudal to the lesion.
• Transplants of fetal spinal cord tissue also provide a population of neurons at the site of injury that may serve as a relay to convey supraspinal control to levels caudal to the lesion site.
• the fetal transplants may provide a source of diffusible trophic support for immature and mature axotomized neurons [6-8], may inhibit the formation of a glial scar at the site of injury [9-10], may inhibit local toxic influences at the site of injury [11], may replace particular neuronal populations lost by injury [12], and may provide a favorable substrate (mechanical, extracellular matrix, guidance cues) to support axonal growth across the injury [13-15].
• Most of the current studies of spinal cord injuries have examined the response of the acutely injured spinal cord.
• Embryonic noradrenergic neurons taken from the locus coeruleus [18,19] and embryonic serotoninergic neurons from the embryonic raphe nuclei [20-21] survive, differentiate, and extend axons within the denervated mature or developing spinal cord.
• embryonic monoaminergic neurons extended axons up to 2cm within the host spinal cord.
• transplanted raphe neurons indicated that the transplanted embryonic neurons are capable of establishing morphologically appropriate synapses with denervated targets within the spinal cord [20-21], such as the intermediolateral cell column, dorsal horn laminae, and ventral horn.
• Immature astrocytes are able to support the growth of dorsal root axons into the spinal cord in the adult, and some of these axons form structurally normal synapses within the cord
• the functional effect of such grafts has not been examined
• Other studies have transplanted astrocytes into the injured spinal cord [37] dorsal column
• the authors suggest that the grafted astrocytes lead to functional improvement compared with lesion-only control [37] They suggest that the effect on the host nervous system is indirect - that is, that the astrocytes may exert a trophic effect on host dorsal column neurons and prevent their atrophy [37]
• Transplants of fetal spinal cord (and other embryonic CNS tissues) survive and mature when placed into the injured spinal cord under a variety of lesion conditions. After spinal cord hemitransection or transection at birth or at maturity, transplants of fetal spinal cord tissue survive, grow, and differentiate [7,12,43,44]. Neuroanatomic connections are established between the host CNS and the fetal tissue (whole tissue or suspension) transplants. Such transplants appear to have a wide range of effects on the host nervous system.
• Transplants may alter tissue oxygen tension levels in the lesion area [11]. Abnormal metabolism in the spinal cord may contribute to some of the secondary pathological changes after spinal cord injury [10]. Transplants may also limit the reactive glial responses with the injured spinal cord [10]. Studies of long-term injury models suggest that fetal spinal cord transplants may even reverse some of the gliosis that had developed [10]. In addition, transplants provide trophic support for immature and mature axotomized neurons [6,7,13]. Such trophic support is able to rescue permanently immature axotomized brain stem-spinal neurons from retrograde cell death [6,13] and prevent the retrograde retraction (dieback) of mature corticospinal axons after injury [45]. Thus, the transplants may improve function by limiting some of the secondary consequences of spinal cord injury. It would be useful to have additional methods of providing the transplants.
• transplants of fetal spinal cord tissue rescue immature axotomized brain stem-spinal neurons from injury-induced retrograde cell death in a target-specific manner [13].
• these rescued neurons are able to regenerate and to extend axons both into the transplant, and through it, to reach normal targets within the host spinal cord caudal to the lesion site [9,13,26,46].
• neurons within the transplants send their axons into the host spinal cord [7,13,47].
• transplants are anatomically in a position to serve both as a bridge and as a relay.
• Axons of host origin form synapses within the transplanted tissue in both neonatal-and adult-lesioned animals [12,13,47,48].
• the transplant After injury in the adult, the transplant is in a position to serve as a relay to convey supraspinal control to the host spinal cord caudal to the injury There is no evidence for the ability of mature neurons to use the transplants as a bridge for injured axons to cross the lesion site
• microcarriers and microcarrier technology emerged from the need for mass production of anchorage dependent animal cells in culture These cells must be propagated in large reactors (up to several hundred liters in volume) in order to produce adequate amounts of human and animal cells for veterinary drugs, such as viral vaccines, interferons and a long list of important recombinant proteins.
• the main advantage of the MC technology over classical culture methods are High surface-to-volume ratio (which can be varied easily by changing the MC concentration) leading to high cell number per unit volume, a possibility to monitor and control environmental conditions (e.g pH, dissolved oxygen, dissolved CO 2 and the concentration of medium components), easy sampling of suspended cell-MC aggregates for microscopic observations, chemical analysis or enumeration - an option not available with most other culture techniques, and ease in scaling MC cultures up to large volumes simply by gradually increasing the reactor volume
• microcarriers currently in use are beaded or cylindrical, made of dextran, gelatin, cellulose, polystyrene or polyacrylamide, and some have large pores which enable cell growth on the inner surface, as well as on the outer periphery of the MCs.
• MC-cultures can be grown either in large reactors, laboratory spinners or in small plastic dishes. A detailed description of the MC-technology was previously described [Reuveny 1990].
• the MC-cell-culture system is used not only for production of large quantities of cells, but also as a different tool for the manipulation and study of cells in culture. Enzymatic dissociation is not required for cell transfer, cell physiology studies on processes such as ion transport, cell interaction, cell differentiation etc. These performances can be examined at any time during cultivation.
• the tridimensional growth pattern which cells achieve on MCs enables their long survival in culture, reaching a high degree of cell differentiation.
• cells grown on MCs are used for reconstruction of embryonal neuronal and muscular implants and can be easily processed for microscopic and biochemical analysis.
• CNS central nervous system
• skeletal myoblasts and cardiomyocyte propagation and differentiation in vitro on MCs were previously described [Shahar, 1990, Shahar et al, 1994].
• cell-MC aggregates in suspension is the only type of culture which allows sampling of cell-aliquots at any time during cultivation, without interfering with the ongoing culture.
• the cylindrical DE-53 (Whatman, UK) pre-swollen microgranular DEAE anion exchange was found most suitable for neuronal growth, when compared to the spherical beads. Being positively charged, these MCs allow a quick adherence of cells to their surface. In addition, their elongated cylindrical form (80-400 x 40- 50 ⁇ m) offers a desirable substrate for regenerating long nerve-fibers.
• dissociated CNS cells grown on MCs are arranged in a tridimensional pattern, close to the m vivo situation They mature to the stage where synapse interconnections and myelination are established [Shahar et al 1983, Shahar and Reuveny 1987]
• the MCs of choice for growing muscle cells were found to be Cultispher-GL (Percell Biolytica AB, Sweden) These macroporous beaded MCs, 140 to 320 ⁇ m in diameter, are made of gelatin and are, therefore, biodegradable.
• a cell culturing method includes the steps of culturing neural tissue in a suspension culture with biodegradable microcarriers thereby allowing formation of a three dimensional cellular-microcarrier aggregate. This aggregate is then transferred and replated in plastic dishes with a suitable culture media including coating the plastic dishes with a matrix gel composed of hyaluronic acid and laminin.
• a method of ameliorating impairment of the central nervous system in a mammal afflicted with neurological or neurodegenerative disorders includes the steps of culturing neural tissue of the type needed to treat the impairment in a suspension culture with biodegradable microcarriers thereby allowing formation of a three dimensional cellular-microcarrier aggregate.
• This aggregate is then transferred and replated in plastic dishes with a suitable culture media including coating the plastic dishes with a matrix gel composed of hyaluronic acid and laminin.
• the cellular-microcarrier aggregates are then harvested and transplanted to the site of impairment of the central nervous system thereby providing amelioration of the impairment.
• the method also can include the step of irradiating with a light source, generating light at a wavelength being within the range of 380-1200nm, at least one of the cultures prior to transplantation.
• the method further provides for irradiating the site of impairment and transplantation with the light source after transplantation.
• Fig.l is an electron micrograph of tissue explants prepared from the spinal cord of rat embryos and cultured on microcarriers;
• Fig. 2a is an electron micrograph of the Cultispher GL microcarrier
• Fig. 2b is an electron micrograph of the DE-53 microcarrier
• Fig.3 a is a photograph showing a rat with complete paralysis of the legs following spinal cord transection
• Fig.3b is a photograph showing a rat with active movement of both legs after embryonal nerve cell implantation in the transected spinal cord followed by low power laser treatment;
• Fig.4a is a photograph showing diffuse sprouting of axons (marked by arrows) at the site of nerve cell implantation in the transected spinal cord of the rat followed by laser therapy;
• Fig.4b is a photograph showing proliferation of fibroblasts and blood capillaries at the site of the transected spinal cord in a non-treated rat;
• Fig.5a is a photograph showing bundles of nerve fibers oriented in different directions around microcarriers (marked by arrows) at the implanted and laser treated site of spinal cord transection of the rat;
• Fig.5b is a photograph showing perikarya at the implanted and laser treated site of spinal cord transection of the rat.
• Fig.5c is a photograph showing a fibrotic scar rich in capillaries at the site of spinal cord transection of a non-treated rat. DETADJED DESCRD7TION OF THE INVENTION
• the present invention provides a method of ameliorating impairment of the central nervous system in a mammal afflicted with neurological or neurodegenerative disorders.
• the patient can be afflicted with neurological or neurodegenerative disorders including epilepsy, stroke, Huntington's diseases, CNS injury, pain, Parkinson's disease, myelin deficiencies, neuromuscular disorders, neurological pain, amyotrophic lateral sclerosis, Alzheimer's disease, and affective disorders of the brain.
• the present invention also provides a method of treating an injury to the central nervous system, which may be a closed or open head injury or a spinal cord trauma.
• ameliorating is meant that the functional deficits that are associated with the impairment of the central nervous systems are, at least, in part reversed.
• the amelioration is effective to achieve improvement including but not limited to improved survival rate or more rapid recovery, or improvement or elimination of symptoms and other indicators as are selected as appropriate measures by those skilled in the art.
• the method includes the steps of culturing neural tissue of the type needed as is known in the art to treat the impairment.
• the culture system is a two stage system allowing first the formation of a three dimensional cell aggregate to mimic cellular interactions in vivo.
• the second stage is a replating of the aggregates in plates in what can be considered a two dimensional system to embed the aggregates in a suitable matrix for transplantation.
• the present invention then allows for the use of the cultured neuronal entities for reconstructing embryonal implants. These implants (which can be light irradiated and exposed during cultivation to different neuronal growth factors) are subsequently implanted.
• the first stage is accomplished by culturing the neural tissue in a suspension culture with biodegradable microcarriers thereby allowing formation of a three dimensional cellular- microcarrier aggregate.
• a cellular-microcarrier aggregate is comprised of tissue growing in culture attached to, or supported by, the microcarriers.
• the tissue may comprise tissue explants, dissociated cells, or a combination of both tissue explants and dissociated cells.
• microcarriers provide an optimal substrate for three-dimensional growth and differentiation of CNS slices from fetal and adult origin.
• the neuronal slices attach to the MCs forming neuronal entities which are cultured in suspension for days to weeks in the first step.
• neural tissue includes dissociated cells, in vitro genetically manipulated cells and tissue explants.
• the type of neural tissue used is as known in the art to treat the impairment.
• the neuronal cells and tissue to be transplanted can be derived from embryonic spinal cord cells or adult forebrain neuronal precursor cells [Goldman, 1995].
• the neural tissue to be transplanted can be glial cells, such as, for example oligodendrocytes and Schwann cells from fetal and adult origins. These implants, composed of cultured central and peripheral myelin forming cells, are intended for transplantation to ameliorate neuronal disorders resulting in demyelinating effects.
• the neural tissue to be transplanted can be fetal ventral mesencephalic tissue.
• the neural tissue to be transplanted is fetal striatal tissue.
• the method of the present invention provides for the use of neuronal cells or appropriate in vitro genetically manipulated cells for ameliorating the impairment to the central nervous system.
• the cell line has been genetically engineered to provide factors necessary for the amelioration of the impairment to the central nervous system.
• the neuronal cells can be of fetal or adult origin, or neuronal or other cells which may have been genetically modified, as set forth, for example, in United States Patent 5,082,670 to Gage et al and incorporated in its entirety by reference.
• the neural tissue to be cultured comprises at least one of primary neuronal cells, or tissue explant cultures derived from embryonal or neonatal spinal cord specimens, or oligodendrocyte cell cultures from adult brain biopsies as appropriate for ameliorating the impairment to the central nervous system.
• the cultured tissue is genetically engineered, to provide at least one factor necessary for the amelioration of the impairment to the central nervous system.
• the nucleic acid encoding these factors is introduced into the cells or tissue, using viral vectors or any other method as in known in the art
• These factors may include neurotrophic agents such as vasoactive intestinal peptide, neuroprotective agents such as superoxide dismutase, extracellular matrix components such as hyaluronic acid and laminin, integrins, cadhedrins, adhesive molecules, growth factors such as any of the FGFs, IGFs, TBF ⁇ s, PDGF, EGF, BMP and the like, growth factor receptors, hormones, hormone receptors, ribozymes and antisense RNAs
• the aggregates are collected at any time during cultivation in suspension and are transferred and plated on a hyaluronic acid and laminin (see US Patent 5,703,205) matrix gel (HA/LAM) as stationary organotypic long-term cultures as described herein
• a composite implant, suitable for transplantation, is formed by this second step of culturing the cellular-microcarrier aggregates in HA/LAM
• the composite implants are then harvested and transplanted to the site of impairment of the central nervous system thereby providing amelioration of the impairment
• microcarriers used in the present invention have the following properties:
• the MCs have functional groups on their surface enabling cell attachment, spreading and growth These groups can be either positively charged (tertiary, quaternary and sometimes primary amines) or negatively charged (tissue culture treated polystyrene or glass) or uncharged gelatin beads An optimal degree of charge should be chosen, in order to ensure cell attachment but not generation of toxic effect on the cells
• the buoyant density of the MCs should be slightly above that of the culture medium (1 03-1 1) to allow the suspension of MCs by slow agitation
• Each MC should be 100-250 ⁇ m in size to allow the adherence and spreading of several hundreds of cells on its surface while in suspension
• the size distribution of the MCs should be as narrow as possible to guarantee culture homogeneity
• the MC should be rigid with a smooth surface to allow cell spreading Its matrix should not be toxic and preferably transparent
• MCs should be autoclavable
• the MCs which answer these criteria and are currently in use are beaded or cylindrical, made of dextran, gelatin, cellulose, polystyrene or polyacrylamide Some of these matrices have large pores which enable cell growth on the inner surface, as well as on the outer periphery of the MCs
• the method also can include the step of irradiating, with a light source generating light at a wavelength being within the range of 380-1200nm, the culture prior to transplantation, as set forth in United States Patent 4,966,144 incorporated in its entirety by reference
• a coherent light source such as a laser is used.
• the method further provides for irradiating the site of impairment and transplantation with the light source after transplantation
• the present invention further provides a method of treating spinal cord injury in a mammal needing such treatment
• the method includes the steps of culturing fetal spinal cord tissue in a suspension culture with biodegradable microcarriers allowing the formation of cellular-microcarrier aggregate This aggregate is then harvested and the cellular-microcarrier aggregate embedded in matrix gel including hyaluronic acid as described herein below
• the cellular-microcarrier aggregate embedded in hyaluronic acid gel is then transplanted at the site of spinal cord injury
• the site is then covered with a thin coagulated fibrin-based membrane
• the site of injury and transplantation is irradiated with a series of irradiations from a light source generating light at a wavelength within the range of 380-1200nm, preferably with a coherent light source, as set forth in United States Patent 4,966,144 incorporated in its entirety by reference
• the light irradiation is done daily for fourteen days with each treatment for thirty minutes
• the MC-culture technique is ideal for the reconstruction of neuronal implants and for biochemical analysis since the MCs provide space for the anchorage of a large quantity of cells which are required for these studies Furthermore, although neuronal cells grown as a monolayer are accessible to morphological and electrophysiological studies, they are usually cultured for no longer than 2-3 weeks and samples cannot be collected without destroying the culture Neuronal or muscular cells grown on MCs in suspension are, on the other hand, long-term cultures (months) and can be sampled at any time during cultivation without interfering with the ongoing culture
• Explants smaller or larger than 300 ⁇ m (which is about the average size for the MCs) are prepared by manual cutting (3 and 7mm Beaver eye blades Becton Dickenson) or using a mechanical cutter (Mcllwan tissue chopper) Approximately a hundred explants are mixed with 3-4 drops of a 1% MC suspension in PBS The mixture is added to a 35mm plastic dish containing 2ml of nutrient medium The explants quickly attach to the MCs forming large floating aggregates which can be maintained in suspension for several weeks When grown in suspension, these explants can be exposed for hours, days or weeks to various factors, hormones, neuro-toxic and neuro-protective agents or drugs, replacing in many aspects the need for m vivo tests
• the present invention includes a second step which follows the culturing of cells or explants on MCs in suspension
• This second step consists of collecting the suspended aggregates and planting them on a bidimensional gel-coated substrate
• a viscous milieu of high molecular weight hyaluronic acid (HA) admixed with murine laminin (LAM) is used to coat the main central area of a 35mm plastic dish
• the coating gel is prepared by mixing a volume of 0.3ml of HA from a 1% solution with 0 6ml of Hank's Buffered Salts Solution (BSS) containing lOOmg of LAM (Sigma)
• BSS Hank's Buffered Salts Solution
• This volume (of 0.9ml HA/LAM mixture) is sufficient for coating eight 35mm plastic dishes (about lOOml/dish), each dish containing about 12mg of LAM
• the HA/LAM coated dishes are left for one hour in a CO 2 incubator and an amount of
• the aggregates firmly attach to the viscous substrate and exhibit, during the days following plating, an intensive neuronal sprouting, together with an active cell-migration.
• the new outgrowth further develops into a network in which the neuronal and glial cells can be easily visualized and followed.
• HA was introduced as a viscous growth permissive milieu [Robinson et al, 1990]. It is a natural occurring high molecular weight polymer (2.5-3.0 x 10 6 dalton) which belongs to the glycosaminoglycan family. Compounds of this family are composed of repeating units of uronic acid (glucuronic acid) and N-acetyl hexosamine (N-acetylglucosamine). In a hydrophilic environment, HA imbibes large amounts of water molecules [Katchalsky 1964; Laurent 1964; Ruohslahti 1988; Preston et al. 1965]. Under these conditions HA forms hydrated gels of a manipulated viscosity dependency.
• HA is a major component of the ECM which is considered an optimal environment for repair regeneration and wound healing. Later in life HA is found in joints, synovial fluids, in the genital tract and in other tissue matrices, such as cartilage and the nervous system [Gahwiler 1984; Yasuhara et al 1994].
• HA is the ligand of many cell surface receptors and cell membrane proteins [Asher and Bigmani 1992; Knudson and Knudson 1993]. Further advantages related to HA in vivo are: a non-antigenic substance, humidity holder, elastic rheological lubricant, antiangiogenic agent, and an antioxidant [Balazs and Denlinger 1988; Toole 1992].
• HA In vitro, HA serves as a growing milieu, traps ions, cells and growth factors and helps cell motility. In addition, it has been reported to modulate neuronal migration and neurite outgrowth [Kapfhammer and Schwab 1992; Thomas et al. 1993]. HA is a biodegradable molecule sensitive to degrading enzymes, such as hyaluronidases and chondroitinases.
• Laminin is an adhesive glycoprotein-ligand composed of three sub-units with a molecular weight of 900,000 dalton.
• LAM possesses the RGD (Arg-gly-asp-ser) sequence recognized by the transmembranal structure of the most common integrin (a ) ⁇ LAM- integrin is known as a major cell-matrix binding structure.
• the present invention provides for the combination of both HA and LAM to form the matrix gel.
• the combination of HA and LAM provides both a flexible, elastic bonding and a tight, rigid bonding of cell-matrix.
• Slices, 300 ⁇ m thick, of the whole spinal cord (SC) with attached dorsal root ganglia (DRG) are prepared from 14 day rat or mouse embryos [Shahar et al. 1991].
• the slices are attached to DE-53 MCs and are grown in suspension for 3-4 days. During this time, cultures can be exposed to growth factors such as: NGF, bFGF, IGF1, EGF, etc.
• the floating SC- MCs aggregates are then collected and plated on HA/LAM coated 35mm plastic dishes where they can be maintained in culture for several weeks. During the first week following plating, an intensive nerve-fiber regeneration takes place from both the SC and the DRG explants.
• the active fiber regeneration is accompanied by a massive outgrowth of non- neuronal dividing cells. Only a few neurons have been observed to migrate from the SC slices, mainly from regions which are in contact with the MCs. The whole SC explant becomes flattened but still maintains its tridimensional structure. Some SC slices, however (usually the smaller ones), often spread and single inter neurons and motor neurons become evident in their structures. These explants usually remain attached to single or a few MCs.
• the DRG are usually disintegrated into single cells or groups of a few cells which become flattened, each surrounded by a few satellite glia cells.
• Brain regions like hippocampus, hypothalamus etc. are usually dissected from mature fetuses or newborns. Therefore, slices which are made from the whole region are too large and should be further sectioned in 300 ⁇ m 3 pieces, which are of the appropriate size to be attached to MCs.
• the growth pattern of brain explants on MCs is basically similar to that described for the SC explants. However, brain-MC cultures, unlike SC explants, usually do not become heavily myelinated from plating.
• the floating cell-MC explants can be exposed to neuro-toxic and neuro-protective agents, either separately or simultaneously.
• the rate and the intensity of neuronal and glial cell-migration from explants following their plating on HA/LAM serves as an indicator for the evaluation of the tropic, toxic or neuro-protective efforts.
• Applicants have used the MC-culture technique for the reconstruction of embryonal rat SC implants for transplantation into SC transected adult paraplegic rats [Rochkind et al 1997], Cells or explants were prepared from SC of rat embryos and cultured on MCs as described herein (see Fig. 1). Two types of MCs were used (Fig. 2): the cylindrical DE-53 and the Cultispher GL. Due to the DE-53 MCs being positively charged, almost all the SC cells or explants promptly attached to them and formed large aggregates which remained floating in the nutrient medium. The Cultispher GL MCs were chosen because of their biodegradability.
• the final in vitro reconstructed implants contained embryonal SC cells or explants attached to MCs and embedded in a viscous solution of high molecular weight hyaluronic acid in phosphate buffered saline (PBS) and laminin (HA/LAM). They were made either from aggregates that were cultured 3-4 days in suspension or from explants 3-4 days after plating on HA/LAM.
• PBS phosphate buffered saline
• H/LAM laminin
• the surgical procedure included exposure of the SC through a dorsal approach and removal of Th7-Th8 laminae. Subsequently the SC was completely transected under a microscope using a scalpel. As a result, complete paralysis was induced to the lower legs. Finally, the muscular and cutaneous planes were sutured and the operated area was irradiated transcutaneously by low power laser (Medi-Robot laser system, 780nm, 250mW for 30min /day) for 14 post-operative days to enhance the neuro-regenerative repair process. Recently, it was shown that low power laser irradiation increased the sprouting of neurons in culture [Wollman et al. 1996] and when applied following brain and SC transplants in animals [Rochkind 1992]
• SC conductivity somato-sensory evoked potentials- SSEPs
• evoked potentials appeared in 9 (69%) of the 13 rats in this group (5 on both sides of the scalp and 4 on one side only)
• only one rat (12 5%) of the 8 that were implanted but not treated with laser showed conductivity in one leg
• the post-operative follow-up after 3 and 6 months showed that in the implanted rats which received laser irradiation, the re-establishment of leg movements was most effective and occurred in more than 90% of the operated animals (Figs 3 a and b)
• FIG 4a Histopathological sections taken three days following implantation and laser treatment showed sprouting of fibers from the implanted neurons (Fig 4a), compared to proliferation of fibroblasts and blood capillaries in the non-treated rats (Fig 4b) Histopathological examination of the implanted and laser treated area 3-6 months after operation, showed many bundles of nerve fibers oriented in different directions around the MCs (Fig 5a) and a few perikarya (Fig 5b), as compared with a fibrotic scar rich in capillaries which was observed in the control rats (Fig 5c)
• Reier PJ Annotation. Neural tissue grafts and repair of the injured spinal cord J Neuropathol Appl Neurobiol 1985; 1 1 : 81-104.

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