Title:  Mechanically elongated neuronal cells

United States Patent:  6,264,944

Assignee:  Trustees of the University of Pennsylvania (Philadelphia, PA)

Filed:  August 2, 2000

Mechanically elongated neuronal cells and methods of mechanically producing elongated cells are provided. Also provided are methods for transplanting elongated neuronal cells into an animal for treatment of spinal cord injuries and other nerve injuries.

DETAILED DESCRIPTION OF THE INVENTION

The primary functional constituents of the spinal cord are myelinated axons and neurons. Signals travel from brain to body and back via these axons which synapse to spinal neurons communicating with the targeted body region. Paralysis develops when the bi-directional signaling is interrupted due to axon damage which severs communication below the site of injury. A key to recovery from such injury would be axonal transplantation. However, axons, which grow out of neurons and then are guided to adjacent neuronal cells by chemical attractants, have not been able to be grown over the distances required for SCI, distances of centimeters rather than millimeters.

A mechanical device has now been developed to elongate neuronal cells so that two populations of neurons can be connected by stretching axonal cells over distances not possible with other prior art methods. Although previous studies have shown that axons exhibit short-term tolerance to strain or stretching, the ability of axonal cells to tolerate long-term stretch and then to elongate successfully and remain viable has not previously been shown.

To elongate cells, an enclosed cell culture system was developed which comprises a plexiglass box with a removable lid and glass bottom and a gas exchange port. On the inside base, a long rectangular absorbable membrane (substrate; Lactosorb, BioMet, Inc., Warsaw Ind.) for neuron attachment was fixed in place. The biologically absorbable material was chosen as it is more compatible for transplantation into tissue. Another shorter membrane was placed on top leaving an exposed region of the underlying membrane near one end (see FIG. 1). This overlying membrane was fixed to a movable bar that was driven by two steel rods. Movement of the overlying membrane across the underlying membrane was performed by activation of a motor-table assembly (Servo Systems, Inc., Montville, N.J.) and microstepper motors (Pacific Scientific, Rockford, Ill.). Control of the movements was computer driven using a linear table (Aerotech, Irvine, Calif.), an encoder (Remco Encoders, Inc., Goleta, Calif.) and an indexer/driver (Panther, Intelligent Motor Systems, Marlborough, Conn.; QuickStep II Driver Software).

Any cells can be elongated with this device. By "elongated cells" it is meant cells that have been modified so that they have an increased length as compared to cells that have not been stretched with the method of the present invention. In a preferred embodiment of the present invention, the device is used to produce compositions comprising elongated neurons. In addition to the elongated cells, the compositions may further comprise culture media and selected growth factors. Neurons to be elongated can be derived from various animal sources, including humans, and isolated via filtration. Alternatively, neuronal cell lines such as the N-tera2 cell line can be elongated. Using this device, it has been shown that the axons or neurons can be elongated to greater than 0.2 cm after one day of stretching and greater than 1 cm after 5 days of stretching.

The ability of this device to elongate neurons was demonstrated using the N-tera2 cell line and primary rat neurons. Cells (approximately 10 million) were plated over the outside border of the overlying and underlying membrane. The cells remained in culture for three to seven days to allow time for adherence of neurons to the membrane and for the growth of nerve fibers (axons and dendrites), forming a network between the neurons. Accordingly, a single neuron network was established that covered both the overlying and underlying membranes. The driver of the device was then activated and the stepper motors moved the top (overlying) membrane across the underlying membrane at speeds of 3.5 to 7 .mu.m every 5 minutes or 1 to 2 mm/day (see FIG. 1). The movement of the membrane split the neuron culture into two populations, bridged by bundles of axons (see FIG. 2). The axons readily adapted to the stretch even to distances of over one centimeter (see FIGS. 3 and 4).

Using the step rate of 3.5 .mu.m per 5 minutes to progressively move further apart the two halves that had been formed with stretching, it was found that few of no neuronal somata were present in the expanding center region. However, bridging this expanding center region were numerous large bundles of axoms, 3 to 40 .mu.m in diameter. These bundles originated from fascicular tracts of axons that had crossed the dividing line between the underlying and overlying membranes prior to separation. While these tracts had random directional orientations prior to stretching with the method of the instant invention, the axon bundles crossing the expanding gap gradually assumed straight orientations arranged in parallel (see FIG. 4). These bridging axons appeared to readily adapt to stretch event though they had increased their original length or 100 to 200 .mu.m to become longer than 7 mm over 7 days of stretch-growth. These bridging axons grew in girth as well as in length. In particular, the hillocks of the axon bundles at the edges of the neuronal populations became wider during elongation (see FIG. 3). In addition to general thickening, there was a joining together of neighboring axon bundles during stretch-growth. Thus, there were progressively fewer but much broader fasicular tracts of axons bridging the two populations of neurons. With the typical diameter of the axons at less than 1 .mu.m, the larger bundles were estimated to contain more than 1000 axons. Despite the relatively rapid stretch-growth of these axons, when the flasks containing the cells were agitated, lateral movement of the axon bundles was observed, indicating that there remained some slack in the axon bundles and that the center portion of the axon bundles was not attached to the membranes. The regions of the bundles nearest the ends of the gaps, however, did appear to adhere to the underlying membrane.

These results are the first demonstration of substantial progressive growth of large tracts of synapsed CNS axons in response to a continuous mechanical tension. Further, these data show for the first time that mechanically elongating axon bundles consolidate into larger tracts. Moreover, the elongated axon/neuron cultures remained sufficiently viable for use as transplant material. Although these studies were terminated at 7 days of stretch-growth, these was no indication that further elongation could not be achieved with longer times of stretch-growth.

Doubling of the elongation speed from 3.5 .mu.m/5 minutes to 7 .mu.m/5 minutes led to an almost total obliteration of the axon bundles, with only a few remaining that spanned the gap at 3 days of stretch. Therefore, there is a limit to the tolerance of long-term stretch in terms of the rate of stretch, which is lower than the tolerance previously reported for short-term elongation of single axons towed from their growth cones.

Compositions comprising elongated neuronal cells of the present invention are useful as a source of transplant material for patients with SCI as well as other nerve lesions. Methods for transplantation of the cells produced by the method of the instant invention are well known to those of skill in the art of cell transplantation.

In one embodiment, mechanically elongated neurons are implanted at both ends of a lesion proximate to viable cells so that the implanted cells can replace nerve function and reconnect nerves of the individual to remedy or otherwise ameliorate the injury. The neurons are implanted in a location that allows processes which develop therefrom to substitute for the processes of the damaged nerve, thereby repairing the damaged nerve network. Thus, as used herein, the term "at or near a site of said nerve damage" is meant to refer to the location where nerve cells are implanted in order to replace destroyed, damaged or dysfunctional nerve cells and/or restore function resulting from destroyed, damaged or dysfunctional nerve cells. The location is defined as being a site where such implanted cells can develop as replacement cells for destroyed, damaged or dysfunctional nerve cells and make the necessary linkages to restore function lost due to destroyed, damaged or dysfunctional nerve cells.

A transplant strategy is to match the length of the stretched axon cultures with the length of the spinal cord lesion. Transplant would proceed by placing the membrane with the cultured cells into the lesion so that neurons at both ends of the axon bundles are in proximity to viable tissue at the end margins of the spinal cord lesion. In addition to spinal cord repair, the transplant material can be used as a bridge for other types of neural injuries, including optic nerve damage and peripheral nerve damage. Transplant of elongated axons for peripheral nerve damage repair may be most optimal due to the more permissive neural growth environment in the peripheral nervous system compared with the CNS.

The capacity of grafted elongated neurons to promote axonal regeneration and functional recovery in vivo can be demonstrated using an animal model of spinal cord injury. For example, adult rats can be surgically anesthetized and prepared for aseptic surgery. For these studies, rats are first trained in the "staircase test" to assess forepaw function prior to receiving a C3-C4 laminectomy and cervical cord hemisection, which causes loss of function in one upper limb. Immediately following the hemisection, into one group of animals is transplanted a membrane with the elongated axons stretched to the length of the lesion. The membrane with the elongated axons stretched to the length of the lesion is then transplanted so that the neuron populations at each end of the membrane are inserted into viable tissue at each end of the lesion. The second group of animals is left untreated as controls.

Beginning at one week post-transplant, the dorsal spinal cord is examined electrophysiologically to determine whether communication between proximal and distal regions of the lesion had been re-established.

Skilled forelimb function is also assessed using a staircase apparatus consisting of a plastic box with built-in left and right staircases with five steps each. The staircases are separated from each other in such a manner that it is impossible for an animal to reach the right staircase with any limb other than the right forelimb and vice versa. The five steps on each staircase are loaded with small food pellets, and the animals are allowed to acquire as many food pellets as possible using each forelimb independently in a 15 minute period. The number of pellets consumed by the rat are counted at the end of each test period and recorded as "number of pellets taken". Each animal is assessed in the staircase test preoperatively and at 1-, 4- and 8-weeks post-implant. At 8-weeks post-implant, animals are sacrificed and sections of the spinal cord are processed for serotonin (5-HT) immunohistochemistry to identify descending serotonergic fibers.

Similar transplant procedures can be performed in humans. For example, N-Tera2 cells are currently being evaluated for transplantation into the brain in human stroke patients. Prior to transplant, it is preferable to diagnose location and presence of any damage to the spinal cord and the volume of the damage by MRI and CT. Neuronal cells for implant are elongated as discussed above. It is preferred that a volume of cells equal to that of the damaged regions of the spinal cord be elongated. The surgeon then locates the appropriate level(s) of the spine and accesses the spinal canal to remove the damaged regions and other debris which might block nerve regeneration, using known techniques. Next the surgeon places the membrane of elongated neuronal cells into this region so that the elongated axons bridge the length of the lesion and the neuron populations at each end of the membrane are inserted into viable tissue at each end of the lesion. Next the layers surrounding the spinal cord are closed, as are the more superficial layers. In circumstances of the acute application of this technique following trauma, methylprednisolone is administered at the beginning of the surgery in the usual spinal injury dose and is continued for as long as the surgeon considers necessary, which may vary from 1 week to several months. In circumstances in which the cells are histocompatible with the recipient, or other situations under the physician's determination, anti-rejection therapy may not be needed.

Claim 1 of 3 Claims

What is claimed is:

1. A composition comprising integrated elongated neuronal cells resulting from ex vivo machine-driven, physical stretching of already synapsed neurons maintained in culture.

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