WO1999058042A2 - Specially devised neuronal implants for reconstruction of damaged central nervous system - Google Patents

Abstract

Specially devised neuronal implants for reconstruction of damaged central nervous system are prepared by culturing embryonal or neonatal tissue explants in a suspension culture with biodegradable microcarriers allowing formation of cellular-microcarrier aggregates. The cellular-microcarrier aggregates are transferred to plastic dishes coated with a matrix gel composed of hyaluronic acid and laminin, and the cellular-microcarrier aggregates are cultured in a suitable culture medium, forming a composite implant suitable for transplantation.

Description

SPECIALLY DEVISED NEURONAL IMPLANTS FOR RECONSTRUCTION OF DAMAGED CENTRAL NERVOUS SYSTEM

GOVERNMENT SUPPORT

Research in this application was supported in part by grant from the National Institutes of Health (NLNDS) RO1NS33106 and RO1NS29813. The U.S. Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION

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.

DESCRIPTION OF RELATED ART

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. For example, fetal ventral mesencephalic tissue has been demonstrated to be a viable graft source in Parkinson's disease. [Lindvall et al, 1987; 1990; Bjorklund, 1992]. Likewise, fetal striatal tissue has been utilized successfully as graft material in Huntington's disease [Isacson et al, 1986; Sanberg et al, 1994].

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).

For example, 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. In each of the studies cited herein that examine the effects of transplants on recovery of function, despite transplant-mediated recovery of function after spinal cord injury, permanent deficits in reflex and locomotor function persist. This suggests that additional approaches are needed to ameliorate the deficits/impairments due to the spinal cord injury and to restore paralyzed function to significant activity levels.

There are several possible mechanisms by which 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]. After spinal cord injury either at birth or at maturity, 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. At a cellular level, 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. However, few studies [9,16,17] show that chronically injured spinal cord has the capacity for repair and recovery of function. These studies suggest that chronically injured neurons maintain the capacity of axonal regeneration, if provided an appropriate environment such as PNS or fetal CNS tissue. However, the methods as discussed herein of providing such tissue are not always successful.

One classic approach to the study of spinal cord injury has been to interrupt the pathways within the spinal cord surgically, either unilaterally (hemisection) or completely (transection). The clear advantage of transection models is that the lesion is complete, and a contribution of intact host pathways does not confound the behavioral analysis. One major disadvantage of transection models is the requirement for extensive and intensive postsurgical animal care to prevent morbidity and mortality. An additional disadvantage is the difficulty in maintaining an adequate interface between the ends of the transected spinal cord and transplant placed within the lesion site. TRANSPLANTATION IN THE INJURED SPINAL CORD: 1. Intraspinal replacement of Particular Neurons or Neurotransmitters :

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. Following transplantation of cell suspensions, or cell aggregates or whole-tissue transplants, embryonic monoaminergic neurons extended axons up to 2cm within the host spinal cord. Studies of 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.

Transplants of noradrenergic-containing locus coeruleus neurons also project to appropriate areas of gray matter (ventral and intermediate gray) deprived of its normal noradrenergic input [18,19,22], but the extent to which specific synapses with host target neurons are established is uncertain.

Other approaches to restore particular populations of neurons have sought both to replace motor neurons and to restore the circuitry between the motor neurons in the spinal cord and their peripheral targets Host motor neurons depleted either surgically, by the creation of a cavity within the spinal cord or by neonatal axotomy, can be replaced by embryonic spinal cord transplants or by embryonic cell suspensions enriched for motor neurons [23,24] In these experiments, investigators have sought to restore some of the segmental circuitry by providing a bridge of peripheral nerve tissue to guide regrowing axons to targets in the periphery In both approaches, embryonic motor neurons and dorsal root ganglion neurons survive and undergo phenotypic maturation Retrograde neuroanatomic- tracing techniques indicate that transplanted neurons are able to extend axons into the peripheral nerve segment In experiments in which a peripheral nerve bridge was placed between host spinal cord and muscle [24], electrophysiologic stimulation of the peripheral nerve graft elicited end plate potentials These potentials were cholinergic, suggesting that host motor neurons were able to reinnervate skeletal muscle targets The extent to which transplanted neurons also establish functional reinnervation of target structures has not been determined

2 Peripheral Nerve Grafts to Repair Spinal Cord Injury

Another approach to anatomic and functional repair after spinal cord injury has been the use of transplantation strategies to bridge the lesion site Studies, using segments of peripheral nerve to bridge the site of spinal cord transection or to provide an alternative route for medullary neurons to reach spinal cord levels, were important in establishing that specific mature central neurons were capable of regrowth following spinal cord injury [25-28] After spinal cord transection, a bridge of peripheral nerve placed between the cut ends of the spinal cord supports the growth of dorsal root ganglion neurons, axons of propriospinal neurons, and axons of suprasegmental origin into the peripheral nerve bridge [28] Although these population of axons are able to elongate considerable distances (10-35mm) within the peripheral nerve environment [25-28], when they re-enter the CNS environment, they terminate within a few millimeters of the host-graft interface which does not allow for recovery of function Other studies have indicated that chronically injured neurons are also capable of growth through peripheral nerve grafts [16], and this growth can be increased by the administration of exogenous nerve growth factor [29]

3 Grafts of Central and Peripheral Glia for Repair of the Injured Spinal Cord

Based largely on the studies that indicate that elements contained within peripheral nerve grafts are able to support the growth of central axons, some investigations have explored the contribution of cultured peripheral non-neuronal cells to stimulate axon growth in the injured spinal cord [30-33] These studies have demonstrated axons associated with the Schwann cell components within the lesion site, but the origin of the axons within the grafts have not been determined Other studies examined the potential for combinations of cultured Schwann cells and dorsal root ganglion neurons to support the growth of corticospinal axons within a spinal cord lesion site in newborn rats [34] However, corticospinal axons did not enter the Schwann cell-dorsal root ganglion grafts Mixed populations of dissociated dorsal root ganglion neuronal and non-neuronal cells also have been used in the adult spinal cord following contusion injury [31] However, these mixed transplants did not lead to recovery of functions

The ability of immature astrocytes to support axonal growth after spinal cord injury has also been examined [35,36] 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, however, 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]

Since some of the loss of function after spinal cord injury may represent loss of conduction in axons that remain intact [38], replacement of oligodendrocytes or Schwann cells at the site of injury might be expected to restore conduction and lead to improved function Studies in myelin-deficient mutants indicate that transplanted oligodendrocytes or Schwann cells are capable of migrating long distances within the CNS and remyelinating axons [30,39,40] In contrast with the studies that suggest a beneficial role of oligodendrocytes in repair of demyelination of injured CNS axons, other studies have suggested that components of oligodendrocytes are inhibitory to the growth of axons [41,42], and that interfering with this inhibition leads to some modest growth of corticospinal axons (41) 4 Transplantation of Fetal Spinal Cord Tissue Into the Site of Spinal Cord Another strategy to repair the injured spinal cord has been to place transplants of fetal spinal cord tissue directly into the spinal lesion site. 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.

After spinal cord lesions at birth, 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]. In the presence of transplant, 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]. In addition, neurons within the transplants send their axons into the host spinal cord [7,13,47]. Thus, after injury at birth, transplants are anatomically in a position to serve both as a bridge and as a relay.

After spinal cord injury in the adult, neurons within the transplant send their axons into the host spinal cord, and host neurons (corticospinal, raphe-spinal, coeruleospinal, intraspinal, or dorsal root) project axons into the transplant [7,9,12-15,45]. Axons of host origin form synapses within the transplanted tissue in both neonatal-and adult-lesioned animals [12,13,47,48]. 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

Adult brain organotypic cultures on microcarriers A method of culturing adult neurons has been described (Goldman et al, 1997) MICROCARRIERS

The development of microcarriers (MCs) 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 classical methods for propagating anchorage dependent cells in static conditions on bi-dimensional glass or plastic surfaces, offered restricted surface to volume ratio and created a bottleneck in the production of large amounts of cells and cell products In an attempt to provide a system that offers large accessible surfaces for cell growth in relatively small culture volumes, van Wezel in 1978 developed a concept of microcarrier technology In such a system, cells are propagated on the surface of small solid particles which are suspended in the growth medium by slow agitation Once attached to the MCs the cells grow up to confluence on the MCs' surface This method has brought together the necessity of large surfaces on which cells can grow and the homogeneous conditions for cell propagation created by the continuous agitation of the MCs

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 CO₂ 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

MC technology today is well established and a variety of commercial MCs are available Some 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.

This technique is used for propagation of a variety of cells from different origins (mammalian, fish, bird or invertebrate), different tissues (neuronal, muscular, pituitary, etc.) and different cell types (primary cells, diploid cell strains, cell lines, as well as recombinant cell lines). 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. Finally, cells grown on MCs are used for reconstruction of embryonal neuronal and muscular implants and can be easily processed for microscopic and biochemical analysis. PRIMARY CULTURES OF DISSOCIATED NERVE AND MUSCLE CELLS ON MICROCARRIERS

Dissociated fetal central nervous system (CNS) cells, skeletal myoblasts and cardiomyocyte propagation and differentiation in vitro on MCs were previously described [Shahar, 1990, Shahar et al, 1994]. The rational behind introducing the MC-technology for growing dissociated neuronal and muscular cells as primary cultures was to achieve a tridimensional cell-growth, simulating the in vivo situation and enabling more advanced stages of cell-maturation in vitro, allowing prolonged life span in culture. Also, 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.

Among the variety of MCs, 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- 50μm) offers a desirable substrate for regenerating long nerve-fibers. Unlike the bidimensional growth in monolayers, where the neurons reach only a limited stage of maturation, 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 320μm in diameter, are made of gelatin and are, therefore, biodegradable. In spite of the fact that these MCs are not positively charged, muscular cells quickly adhere to the gelatin substrate and form aggregates which are cultured in suspension In the aggregates, as a monolayer cultures, the myoblasts or cardiomyocytes adhered to the MCs and differentiate into myotubes and cardial fibers respectively These become striated and contract spontaneously within each aggregate [Shahar et al 1985, 1994] Another advantage of this culture is that, while muscle cells grown in monolayers usually detach from the dish during the second week in culture due to the active contractions, the contracting cell-MC aggregates can persist in suspension for months

When both neuronal cells and skeletal myoblasts prepared from the same fetus (autologous nerve-muscle co-cultures) are cultured on MCs in suspension, an intensive combined growth and differentiation of both cell types takes place resulting in active contractions of aggregates due to the formation of neuro-muscular junctions [Shahar et al 1987, 1992]

SUMMARY OF THE INVENTION

According to the present invention, a cell culturing method is provided. The 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.

Further according to the present invention, a method of ameliorating impairment of the central nervous system in a mammal afflicted with neurological or neurodegenerative disorders is provided. The method 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.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be understood and appreciated from the following detailed description, taken in conjunction with the drawings in which:

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; and

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.

By 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.

In this two step-culturing technique commercially available microcarriers (MCs; see herein below) 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.

The term 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. For example, for spinal cord injury, the neuronal cells and tissue to be transplanted can be derived from embryonic spinal cord cells or adult forebrain neuronal precursor cells [Goldman, 1995].

Where the impairment to the central nervous system is a myelin deficiency, 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.

Where the impairment to the central nervous system is Parkinson's Disease, the neural tissue to be transplanted can be fetal ventral mesencephalic tissue. For Huntington's Disease 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. In an embodiment 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.

In a preferred embodiment of the present invention 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.

According to another preferred embodiment 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, TBFβs, PDGF, EGF, BMP and the like, growth factor receptors, hormones, hormone receptors, ribozymes and antisense RNAs

For the second step, 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

Preferably, the microcarriers used in the present invention (MCs) 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 100-250μm 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 In addition the MC should be rigid with a smooth surface to allow cell spreading Its matrix should not be toxic and preferably transparent Finally, in order to ensure sterility, 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 In a preferred embodiment, 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 Finally 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 In an embodiment 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

COMBINED SUSPENSION/PLATING ORGANOTYPIC CULTURE-METHOD FOR GROWING CNS EXPLANTS ON MICROCARRIERS

The conventional organotypic culture methods currently in use for growing spinal cord and brain explants from a fetus or newborn require the use of substrates such as' collagen [Bornstein and Murray 1958, Romijn et al. 1988, Notepek et al 1993)] plasma clots [Hild 1957, Gahwiler 1984] or Hyaluronic acid (HA) gel [Levy et al 1996] Adult avian and mammalian forebrain organotypic cultures are successfully grown on a laminin substrate [Goldman 1995]

The main reason for growing tissue explants rather than dispersed cells on MCs is to obtain a larger quantity of neuronal tissue mass for implantation and for biochemical analysis In addition, attachment of CNS explant to MCs is probably the only way to provide an adequate substrate for these explants, which enables them to survive in suspension for long periods Applicants' experience shows that the most suitable MCs for growing CNS explants were found to be the cylindrical DE-53

Explants, smaller or larger than 300μm (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

In order to establish the state of the cells composing the suspended explants and enable their visualization, 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) 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 CO₂ incubator and an amount of 590μl/dish of nutrient medium is added sufficient to cover the coated area with a thin fluid layer The suspended aggregates are collected using a plastic pipette or small spatulas and placed (about 6-10 aggregates) in the center of the coated area.

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. SUBSTRATE FOR GROWING ORGANOTYPIC NEURONAL CULTURES

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 10⁶ 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. These gels are serving in vivo as a space filling substance [Longaker et al, 1989], During the early developmental stages of a fetus, 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].

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 (LAM) 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 )ι 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. Unexpectedly, the combination of HA and LAM provides both a flexible, elastic bonding and a tight, rigid bonding of cell-matrix. EMBRYONAL CNS ORGANOTYPIC CULTURES ON MICROCARRIERS

A. SPINAL CORD

Slices, 300μm 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.

In the following weeks there is further development of the neuronal fibers into a well- established network. The fibers of the DRG neurons, in particular, become thicker and are usually arranged in bundles. Toward the third week in culture, there is an enlargement in size of the perikarya, concomitantly with synaptogenesis and the onset of central and peripheral myelination.

In some flattened SC slices single motor neurons can be visualized.

B. BRAIN

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 300μm³ 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.

During their growth in suspension, the floating cell-MC explants, both from SC or brain regions, 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.

EMBRYONAL SPINAL CORD CULTURES GROWN ON MICROCARRIERS IMPLANTED TO CURE ADULT PARAPLEGIC RATS

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. However, since they are not positively charged, cell or explant attachment efficacy to these MCs is low. Therefore, implants had to be monitored by a scanning electron microscope (SEM) in order to evaluate the amount of MCs bearing cells prior to their implantation. 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. The implants were placed in the transected SC area in direct contact with the two stumps. The implanted area was then covered with a thin coagulated fibrin-based membrane to ensure attachment of implants to the desired site and to prevent bleeding and outflow of cerebrospinal fluid.

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]

Three months after implantation, SC conductivity (somato-sensory evoked potentials- SSEPs) was studied by stimulating the sciatic nerve and measuring latency and amplitude potentials (positive P wave peak) on the surface of the scalp In the group of implanted and post-operative laser treated rats, 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) In comparison, only one rat (12 5%) of the 8 that were implanted but not treated with laser showed conductivity in one leg In the control group, only 3 rats survived and did not show electrical conductivity Analysis of the electrophysiological findings using the Fisher Exact Test was statistically significant (P=00237) In addition, 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)

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)

The above discussion provides a factual basis for the use of a two step culture system in combination with microcarriers and a matrix gel composed of hyaluronic acid and laminin to prepare three-dimensional tissue-microcarrier aggregates for transplantation into the central nervous system A lOmin film of the procedure and outcome of a preferred embodiment of the present invention is available from the Applicants on request

Throughout this application, various publications, including United States patents, are referenced by either author and year, or by number [#], and patents by number Full citations for the publications are listed below. The disclosures of these publications and patents in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

The invention has been described in an illustrative manner, and it is to be understood that the terminology which has been used is intended to be in the nature of words of description rather than of limitation.

It is appreciated that various features of the invention which are, for clarity, described in the contexts of separate embodiments may also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment may also be provided separately or in any suitable subcombination.

It will be appreciated by persons skilled in the art that many modifications and variations of the present invention are possible in light of the above teachings. It is, therefore, to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described.

REFERENCES

1 Bjorklund A Neural transplantation - an experimental tool which clinical possibilities TINS 1991 , 14 3 19-322

2 BergmanBS, Reier PJ Neural tissue transplants rescue axotomized rubrosptnal cells from retrograde death J Comp Neural 1986, 244 86095

3 Brundin P, Strecker RE Preparation and intracerebral grafting ϊ dissociated fetal brain tissue in rats In Conn PM, ed Lesions and transplantation San Diego Acagemic Press, 1991 305-326

4 Dunnett SB, Bjorklund A Neural transplantation, a practical approach London IRL Press, 1992

^* 5 Moorman SJ, Whalen LR Fetal implants in the lesioned spinal cord of the rat In Conn PM, ed Lesions and transplantation San Diego Academic Press, 1991 300-304

6 Breg anBS, Kunkel-Bagden E Effect of target and non-target transplants on neuronal survival and axonal elongation after injury to the developing spinal cord Prog Brain Res 1988, 78 205-212

7 Bregman BS, Reirer PJ Neural tissue transplants rescue axotomized rubrospinal cells from retrograde death J Comp Neurol 1986, 244 86-95

8 Eagleson KL, Cunningham TJ, Haun F Rescue of both rapidly and slow degenerating neurons in the dorsal lateral geniculate nucleus of adult rats by cortically deπved neuron survival factor Exp Neurol 1992, 1 16 156-162

9 Houle JD, Reier PJ Transplantation of fetal spinal cord into the chronically injured adult rat spinal cord J Comp Neurol 1988, 269 535-547

10 Reier PJ, Houle JD The glial scar its bearing on axonal elongation and transplantation approaches to CNS repair In Waxman SD, ed Physiologic basis for functional recovery in neurological disease. New York Raven Press, 1988 87-138

1 1 Stokes BT, Reier PJ Oxygen transport intraspinal fetal grafts - graft host relations Exp Neurol 1991 , 1 1 1 312-323

12 Jakeman LB, Reier PJ, Bregman BS, Wade EB, Kastner PL, Dailey M Differentiation of substantia gelatmosa-like regions in intraspinal and intracerebral transplants of embryonic spinal cord tissue in the rat Exp Neurol 1989, 103 17-33 3 Bregman BS, Bernstem-Goral II, Kunkel-Bagden E CNS transplants promote anatomical plasticity and recovery of function after spinal cord injury J Restor Neurol Neurosci 1991 , 2 327-338 4 Tessler A, Himes BT, Houle J, Reier PJ Regeneration of adult dorsal root axons into transplants of embryonic spinal cord J Comp Neurol 1988, 270 537-548 5 Reier PJ, Bregman BS, Wujek JR Intraspinal transplantation of embryonic spinal cord tissue in neonatal and adults rats / Comp Neurol 1986, 247 275-296 6 Houle JD Demonstration of the potential for chronically injured neurons to regenerated axons into intraspinal peripheral nerve grafts Exp Neurol 1991 , 1 13 1-9 7 Houle JD, Reier PJ Regrowth of calcitonin gene-related peptιde(CGRP) immunoreactive axons from the chronically injured ray spinal cord into fetal spinal cord tissue transplants Neurosci Lett 1989, 103 253-258

.T⁾. 18 Bjoi klund A, Nornes H, Gage FH Cell suspension grafts of noradrenergic locus coeruleus neurons in rat hippocampus and spinal cord reinnervation and transmitter turnover Neuroscience 1986, 18 685-698

19 Commissiong JW Fetal locus coeruleus transplanted into the transected spinal cord of the adult rat Brain Res 1983 , 271 174- 179

20 Foster GA, Rooerts MHT, Wilkinson LS Structural and functional analysis of raphe neurone implants into denervated rat spinal cord Brain Re Bull 19S9, 22 13 1- 137

21 Pπvat A, Mansour H, Rajaofetra N, Geffard M Intraspinal transplants of serotonergic neurons in the adult rat Brain Res Bull 1989, 22 123- 129

22 Nornes H, Bjorklund A, Steveni U Reinnervation of the denervated adults spinal cord of rats by intraspinal transplants of embryonic brain stem neurons Cell Tissue Res 1983, 230 15-35

23 Clowry G, Sieradzan K, Vrbova G Transplants of embryonic motoneurones to adult spinal cord survival and innervation abilities TINS \ 991, 1 14 355-357

24 Horvat JC, Baillet-Derbin C, Ye JH, Rhrich F, Affane F Co-transplantation of embryonicneural tissue and autologous peripheral nerve segments to severe spinal cord injury of the adult rat Guided axiogenesis from transplanted neurons Restor Neurol Neurosci 1991, 2 289-298 5 Aguayo AJ, David S, Bray G Influences of the glial environment on the elongation of axons after injury transplantation studies m adult rodents J Exp Biol 1981 , 95 23 1-240 6 Aguayo AJ, David S, Richardson P, Bray GM Axonal elongation in peripheral and central nervous system transplants Adv Cell Neurobiol 1979, 3 215-234 7 David S, Aguayo AJ Axonal regeneration after crush injury of rat central nervous system fibers innervating peripheral nerve grafts J Neurocytol 1985, 14, 1 -12 8 Richardson PM. McGuiness UM, Aguayo AJ Peripheral nerve autografts to the rat spinal cord studies with axonal tracing methods Brain Res 1982; 237 147- 162 9 Houle JD, Johnson JE Nerve growth factor(NGF)-treated nitrocellulose enhances and directs the regeneration of adult rat dorsal root axons through intraspinal neural tissue transplants Neurosci Lett 1989, 103 17-23 Blakemore WT, Crang aj, Evans RJ, Patterson RC Rat Schwann cell remyelination of demyelinated cat CNS axons evidence that injection of cell suspension of CNS tissue results in Schwann cell remyelination. Neurosci Lett 1987, 77 15-19 Hoovler DW, Wrathall JR Implantation of neuronal suspensions into contusive injury sites in the adult rat spinal cord Ada Neuropathol 1991 , 81 303-3 1 1 Martin D, Sdioenen J, Delree P, Leprince P, Rogister B, Moonen G Grafts of synergic cultured adult dorsal root ganglion-deπved Schwann cells to the injured spinal cord of adult rats-preliminary morphological studies Neurosci Lett 1991 , 124 44-48 Wrathall JR, Kapoor V, Kao CC Observation of cultured peripheral nonneuronnal cells implanted into the transected spinal cord Ada Neuropathol 1984, 64 203-212 uhlengel R, Bunge MD, Bunge RP, Burton I I Implantation of cultured sensory neurons and Schwann cells into lesioned neonatal rat spinal cord II Implant ch.uacteπsticd and examination of corticospinal tract growth J Comp Neurol I W J 74- 1 35. Kliot M, Smith GM, Siegal JD, Silver J. Astrocyte-polymer implants promote regeneration of dorsal root fibers into the adult mammalian spinal cord. Exp Neurol 1990; 109: 57-69

36. Smith GM, Miller RH, Silver J. Changing role of forebrain astrocytes during development, regenerative failure and induced regeneration upon transplantation. J Comp Neurol 1 86, 25 1 :23-43.

37. Berbstein JJ, Goldberg WJ. Grafted fetal astrocytes migration can prevent host neuronal atrophy: comparison of astrocytes from cultures and whole piece donors. Restor Neurol Neurosci 1991, 2: 261-270.

38. Blight AR. Axonal physiology of chronic spinal cord injury in the cat: intracellular recording in vitro. Neuroscience 1983; 10: 1471- 1486.

39. Gout O, Gansmuller A, Baumann N, Gumpel M. Remyelination by transplanted oligodendrocytes of a demyeiinated lesion in the spinal cord of the adult shiverer mouse. Neurosci Lett 1988; 87: 195-199.

40. Rosenbiuth J, Hasegawa M, Shirasaki N, Rosen CL, Liu Z. Myelin formation following transplantation of normal fetal glia into myelindeficient rat spinal cord. J Neurocytol 1990; 19:718-730.

41. Savio T, Schwab ME. Lesioned corticospinal tract axons regenerate in myelin free rat spinal cord. Proc Nat Acad Sci USA 1990; 87: 4130-4133.

42. Schwab ME, Caroni P. Oligodendrocytes and CNS myelin are nonpermissive substrate for neurite growth and fibroblast spreading in vitro. J Neurosci 1991; 1 1 : 2381-2393.

43. Bregman BS. Spinal cord transplants permit the growth of serotonergic axons across the site of neonatal spinal cord transection. Dev Brain Res 1987; 34:265-279.

44. Reier PJ. Annotation. Neural tissue grafts and repair of the injured spinal cord J Neuropathol Appl Neurobiol 1985; 1 1 : 81-104.

45. Bregman BS, Kunkel-Bagden E, McAtee M, O'Neill A. Extension of the critical period for developmental plasticity of the corticospinal pathway. J Co p Neurol 1989; 282: 355-370.

46. Bernstein-Goral H, Bregman BS. Spinal cord transplants support the regeneration of axotomized neurons after spinal cord lesions at birth: a quantative double labeling study. Exp Neurol 1993, 123: 1 18-132.

47. Jakeman LB, Reier PJ. Axonal projections between fetal spinal cord transplants and the adult rat spinal cord: a neuroanatomical tracing study of local interactions. J Comp Neurol 1991; 307: 3 1 1-334.

48. Itoh Y, Tessler A. Ultrastaictural organization of regenerated adult dorsal root axons within transplants of fetal spinal spinal cord. J Comp Neurol 1990; 292: 396-41 1. Goldman S A , et al, "Neural precursors and neuronal production in the adult mammalian forebrain " Frontiers of Neurology A symposium in honor of Fred Plum Annals of the New York Academy of Sciences, Vol 835, December 19, 1997

Bjorklund and Stenevi, "Intracerebral neural grafting a historical perspective" in Bjorklund, A and U Stenevi, eds Neural grafting in the mammalian CNS, Amsterdam Elsevier, 3-11 (1985)

Bjorklund, "Dopaminergic transplants in experimental Parkinsonism Cellular mechanisms of graft-induced functional recovery" Current Biology, 2 683-689 (1992)

Goldman, 1995 "Neuronal Precursor Cells and Neurogenesis in the Adult Forebrain" The Neuroscientist 1(6) 338-350

Isacson et al, "Graft-induced behavioral recovery in an animal model of Huntington's disease" Proc Natl Acad Sci , 83 2728-2732 (1986)

Lindvall et al, "Transplantation in Parkinson's disease two cases of adrenal medullary grafts to the putamen" Ann Neurol , 22 457-468 (1987)

Lindvall et al, "Grafts of fetal dopamine neurons survive and improve motor function in Parkinson's disease" Science, 247 574-577 (1990)

Sanberg et al, "Cell transplantation for Huntington's disease" R G Landes Co , Boca Raton, FL, pp 19-21 (1994) van Wezel A L and C A M van der Velden de Groot, "Large scale cultivation of animal cells in microcarrier cultures" Process Biochem 13 6-8 (1978)

Rochkind, S et al, " A combined procedure utilizing cultured embryonal cell implantation and low power laser treatment to cure paraplegic rats following complete spinal cord transection Proceedings 11th International Congress of Neurological Surgery, Amsterdam, The Netherlands, pp 1903- 1908, July 6-11 (1997)

Claims

CLAIMSWhat is claimed is:

1. A cell culturing method comprising: a culturing embryonal or neonatal tissue explants in a suspension culture with biodegradable microcarriers allowing formation of cellular-microcarrier aggregates; b transferring the cellular-microcarrier aggregates of step a to plastic dishes coated with a matrix gel composed of hyaluronic acid and laminin, said dishes containing a suitable culture medium, and culturing said cellular-microcarrier aggregates.

2. A method according to claim 1 wherein said tissue explant is comprised of neural tissue.

3. A method according to claim 1 or 2 wherein the cultured cellular-microcarrier aggregates form a composite implant which is suitable for transplantation.

4. A composite implant prepared by the steps of a culturing tissue explants in a suspension culture with biodegradable microcarriers allowing formation of cellular-microcarrier aggregate; and b transferring the cellular-microcarrier aggregate of step a to plastic dishes coated with a matrix gel composed of hyaluronic acid and laminin, said dishes containing a suitable culture medium, and culturing said cellular-microcarrier aggregates.

5. A composite implant according to claim 4 wherein said tissue explant is comprised of embryonal, neonatal or adult tissue.

6. A composite implant according to claim 5 wherein said tissue explant is comprised of neural tissue.

7. A composite implant according to any one of claims 4, 5 or 6, which is suitable for transplantation.

8. A method of ameliorating impairment of the central nervous system in a mammal afflicted with neurological or neurodegenerative disorders, the method comprising: a culturing neural tissue of the type needed to treat the impairment in a suspension culture with biodegradable microcarriers allowing formation of cellular- microcarrier aggregates; b transferring the cellular-microcarrier aggregate of step a to plastic dishes coated with a matrix gel composed of hyaluronic acid and laminin, said dishes containing a suitable culture medium, and culturing said cellular-microcarrier aggregate to form a composite implant; and c harvesting said composite implant and transplanting to a site of impairment of the central nervous system.

9. A method according to claim 8 and also comprising the subsequent step of irradiating, with a light source generating light at a wavelength between the range of 380nm-1200nm, at least one of the composite implant prior to transplantation, or the site of impairment following transplantation to the site.

10. A method according to claim 9 wherein the light source comprises a coherent light source.

11. A method according to claim 9 wherein both the composite implant prior to transplantation, and the site of impairment post-transplantation are irradiated with the light source.

12. A method according to claim 8 wherein the impairment of the central nervous system comprises a spinal cord injury and the neural tissue to be cultured comprises embryonic spinal cord cells.

13. A method according to claim 8 wherein the impairment of the central nervous system comprises a spinal cord injury and the neural tissue to be cultured comprises adult forebrain neuronal precursor cells.

14 A method according to claim 8 wherein the impairment of the central nervous system comprises a myelin deficiency and the neural tissue to be cultured comprises glial cells

15 A method according to claim 8 wherein the impairment of the central nervous system comprises Parkinson's disease and the neural tissue to be cultured comprises fetal ventral mesencephalic tissue

16 A method according to claim 8 wherein the impairment of the central nervous system comprises Huntington's disease and the neural tissue to be cultured comprises fetal striatal tissue

17 A method according to claim 8 wherein the tissue to be cultured comprises at least one of primary neuronal cells, 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

18 A method according to claim 17 wherein the cultured tissue has been genetically engineered to provide at least one factor necessary for the amelioration of the impairment to the central nervous system

19 A method according to claim 18 said at least one factor comprises at least one of neurotrophic agents, neuroprotective agents, extracellular matrix components, integrins, cadhedrins, adhesive molecules, growth factors, growth factor receptors, hormones, hormone receptors, ribozymes and antisense RNAs

20 A method of treating spinal cord injury in a mammal, the method comprising a culturing fetal spinal cord tissue in a suspension culture with biodegradable microcarriers allowing formation of cellular-microcarrier aggregate, b transferring the cellular-microcarrier aggregate of step a to plastic dishes coated with a matrix gel composed of hyaluronic acid and laminin, said dishes containing a suitable culture medium, and culturing said cellular-microcarrier aggregate to form a composite implant; and c harvesting said composite implant and transplanting the composite implant to a site of spinal cord injury.

21. A method according to claim 20 which also comprises the step, performed between steps b and c, of embedding the cellular-microcarrier aggregate in a matrix gel including hyaluronic acid, and wherein the composite implant, embedded in hyaluronic acid gel, is transplanted to a site of spinal cord injury.

22. A method according to claim 20 and also comprising the subsequent step of covering said site of spinal cord injury and transplantation with a membrane.

23. A method according to claim 22 wherein said membrane comprises a thin coagulated fibrin-based membrane.

24. A method according to any one of claims 20, 21, 22 or 23 and also comprising the subsequent step of providing at least one irradiation, with a light source generating light at a wavelength of between the range of 380nm-1200nm, to said site of spinal cord injury and transplantation.

25. A method according to claim 24 wherein the light source comprises a coherent light source

26. A method according to claim 24 wherein said at least one irradiation comprises a series of irradiations.

27. A method according to claim 26 wherein the step of providing light irradiation is done substantially daily for substantially fourteen days.

28. A method according to claim 24 wherein the step of providing light irradiation is of substantially thirty minutes duration.

29. A composite implant prepared by the steps of a culturing fetal or neonatal spinal cord tissue in a suspension culture with biodegradable microcarriers allowing formation of cellular-microcarrier aggregate; and b transferring the cellular-microcarrier aggregate of step a to plastic dishes coated with a matrix gel composed of hyaluronic acid and laminin, said dishes containing a suitable culture medium, and culturing said cellular-microcarrier aggregate to form a composite implant; and optionally c embedding said composite implant in a matrix gel including hyaluronic acid, for the treatment of spinal cord injury in a mammal.

30. A composite implant according to claim 29 wherein the treatment comprises transplanting the composite implant, optionally embedded in hyaluronic acid gel, to a site of spinal cord injury.

31. A composite implant according to claim 30 wherein the treatment comprises the subsequent step of covering said site of spinal cord injury and transplantation with a thin coagulated fibrin-based membrane.

32. A composite implant according to claim 30 or 31 wherein the treatment comprises the subsequent step of providing at least one irradiation, with a light source generating light at a wavelength of between the range of 380nm-1200nm, to said site of spinal cord injury and transplantation.

33. A composite implant prepared by the steps of a culturing neural tissue of the type needed to treat an impairment of the central nervous system in a suspension culture with biodegradable microcarriers allowing formation of cellular-microcarrier aggregates; b transferring the cellular-microcarrier aggregate of step a to plastic dishes coated with a matrix gel composed of hyaluronic acid and laminin, said dishes containing a suitable culture medium, and culturing said cellular-microcarrier aggregate to form a composite implant, for the treatment of an impairment of the central nervous system in a mammal afflicted with neurological or neurodegenerative disorders

34 A composite implant according to claim 33 wherein the treatment comprises harvesting the composite implant and transplanting said composite implant to a site of impairment of the central nervous system

35 A composite implant according to claim 34 wherein the treatment comprises the subsequent step of covering said site of impairment of the central nervous system and transplantation with a thin coagulated fibrin-based membrane

36 A composite implant according to claim 34 or 35 wherein the treatment comprises the subsequent step of providing at least one irradiation, with a light source generating light at a wavelength of between the range of 380nm-1200nm, to said site of impairment of the central nervous system and transplantation

37 A composite implant according to claim 33 wherein said impairment of the central nervous system comprises a spinal cord injury and the neural tissue to be cultured comprises embryonic spinal cord cells

38 A composite implant according to claim 33 wherein said impairment of the central nervous system comprises a spinal cord injury and the neural tissue to be cultured comprises adult forebrain neuronal precursor cells

39 A composite implant according to claim 33 wherein said impairment of the central nervous system comprises a myelin deficiency and the neural tissue to be cultured comprises glial cells

40. A composite implant according to claim 33 wherein said impairment of the central nervous system comprises Parkinson's disease and the neural tissue to be cultured comprises fetal ventral mesencephalic tissue.

41. A composite implant according to claim 33 wherein said impairment of the central nervous system comprises Huntington's disease and the neural tissue to be cultured comprises fetal striatal tissue.

42. A composite implant according to claim 33 wherein the neural tissue to be cultured comprises at least one of primary neuronal cells, 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.

43. A composite implant according to claim 42 wherein the cultured tissue has been genetically engineered to provide at least one factor necessary for the amelioration of the impairment to the central nervous system.

44. A composite implant according to claim 4 wherein said at least one factor comprises at least one of neurotrophic agents, neuroprotective agents, extracellular matrix components, integrins, cadhedrins, adhesive molecules, growth factors, growth factor receptors, hormones, hormone receptors, ribozymes and antisense RNAs.