Patent Application: US-201414299802-A

Abstract:
the invention provides a method of co - culturing mammalian muscle cells and mammalian motoneurons . the method comprises preparing one or more carriers coated with a covalently bonded monolayer of trimethoxysilylpropyl diethylenetriamine ; suspending isolated fetal mammalian skeletal muscle cells in serum - free medium according to medium composition 1 ; suspending isolated fetal mammalian spinal motoneurons in serum - free medium according to medium composition 1 ; plating the suspended muscle cells onto the one or more carriers at a predetermined density and allowing the muscle cells to attach ; plating the suspended motoneurons at a predetermined density onto the one or more carriers and allowing the motoneurons to attach ; covering the one or more carriers with a mixture of medium composition 1 and medium composition 2 ; and incubating the carriers covered in the media mixture .

Description:
the present invention will now be described more fully hereinafter with reference to the accompanying drawings , in which preferred embodiments of the invention are shown . unless otherwise defined , all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains . although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention , suitable methods and materials are described below . any publications , patent applications , patents , and other references mentioned herein are incorporated by reference in their entirety . in case of conflict , the present specification , including any definitions , will control . in addition , the materials , methods and examples given are illustrative in nature only and not intended to be limiting . accordingly , this invention may be embodied in many different forms and should not be construed as limited to the illustrated embodiments set forth herein . rather , these illustrated embodiments are provided so that this disclosure will be thorough and complete , and will fully convey the scope of the invention to those skilled in the art . other features and advantages of the invention will be apparent from the following detailed description , and from the claims . glass coverslips ( thomas scientific 6661f52 , 22 × 22 mm no . 1 ) were cleaned using an o2 plasma cleaner ( harrick pdc - 32g ) for 20 min at 100 mtorr . the deta ( united chemical technologies inc . t2910kg ) films were formed by the reaction of the cleaned glass surface with a 0 . 1 % ( v / v ) mixture of the organosilane in freshly distilled toluene ( fisher t2904 ). the deta coated coverslips were then heated to approximately 100 ° c ., rinsed with toluene , reheated to approximately 100 ° c ., and then oven dried ( das et al ., 2006 ). surfaces were characterized by contact angle measurements using an optical contact angle goniometer ( ksv instruments , cam 200 ) and by x - ray photoelectron spectroscopy ( xps ) ( kratos axis 165 ). xps survey scans , as well as high - resolution n1s and c1s scans utilizing monochromatic ai ka excitation were obtained ( das et al ., 2006 ). skeletal muscle was dissected from the thighs of the hind limbs of fetal rat ( 17e18 days old ). briefly , rats were anaesthetized and killed by inhalation of an excess of co 2 . this procedure was in agreement with the animal research council of university of central florida , which adheres to iacuc policies . the tissue was collected in a sterile 15 ml centrifuge tube containing 1 ml phosphate - buffered saline ( calcium - and magnesium - free ) ( gibco 14200075 ). the tissue was enzymatically dissociated using 2 ml of 0 . 05 % of trypsin - edta ( gibco 25300054 ) solution for 30 min in a 37 ° c . water bath at 50 rpm . after 30 min the trypsin solution was removed and 4 ml hibernate e / 10 % fetal bovine serum ( gibco 16000044 ) was added to terminate the trypsin reaction . the tissue was then mechanically triturated with the supernatant being transferred to a 15 ml centrifuge tube . the same process was repeated two times by adding 2 ml of l15 / 10 % fbs each time . the 6 ml cell suspension obtained after mechanical trituration was suspended on a 2 ml , 4 % bsa ( sigma a3059 ) ( prepared in l15 medium ) cushion and centrifuged at 300 g for 10 min at 4 ° c . the pellet obtained was washed 5 times with l15 medium then resuspended in 10 ml of l15 and plated in 100 mm uncoated dishes for 30 min . the non - attached cells were removed and then centrifuged on a 4 % bsa cushion ( das et al ., 2006 ). the pellet was resuspended in serum - free medium according to the protocol illustrated in fig1 and plated on the coverslips at a density of 700 - 1000 cells / mm 2 . the serum - free medium containing different growth factors and hormones was added to the culture dish after 1 h . the final medium was prepared by mixing medium 1 ( table 1 ) and medium 2 ( table 2 ) in a 1 : 1 v / v ratio . fig1 indicates a flowchart of the culture protocol . tables 1 and 2 list the growth factor and hormone supplement compositions of medium one and medium two . the cells were maintained in a 5 % co2 incubator ( relative humidity 85 %). the entire medium was replaced after four days with nbactiv4 medium according to the protocol in fig1 ( brewer et al ., 2008 ). as described in ( brewer et al ., 2008 ), nbactiv4 ™ ( available from brainbits llc ) comprises all of the ingredients in neurobasal ™ ( table 3 ), b27 ™ ( table 4 ), and glutamax ™ ( table 5 ). nbactiv4 ™ may also comprise creatine , estrogen , and cholesterol . thereafter three - fourths of the medium was changed every three days with nbactiv4 ™. rat spinal motoneurons were purified from ventral cords of embryonic day 14 ( e14 ) embryos . briefly , rats were anaesthetized and killed by inhalation of an excess of co 2 . this procedure was in agreement with the animal research council of university of central florida , which adheres to iacuc policies . ventral spinal cord cells from the embryo were collected in cold hibernate e / glutamax / antibiotic - antimycotic / b27 . the cells were dissociated with 0 . 05 % trypsin - edta ( invitrogen ) treatment for 15 min . the dissociated cells were layered over a 4 ml step gradient optiprep diluted 0 . 505 : 0 . 495 ( v / v ) with hibernate e / glutamax / antibiotic - antimycotic / b27 and then made to 15 %, 20 %, 25 % and 35 % ( v / v ) in hibernate e / glutamax / antibiotic - anti - mycotic / b27 followed by centrifugation for 15 min , using 200 g at 4 ° c . after centrifugation , four bands of cells were obtained . the motoneurons with large somas constituted the uppermost band . these cells present in the uppermost band were collected in fresh hibernate e / glutamax / antibiotic - anti - mycotic / b27 and centrifuged for 5 min at 200 g and 4 ° c . the pelleted motoneurons were re - suspended in plating medium then plated on top of muscle cells at a density of 100 cells / mm 2 . motoneuron plating was performed 30 min after plating of the muscle cells . coverslips were rinsed with pbs , fixed in 20 ° c . methanol for 5 - 7 min , washed in pbs , incubated in pbs supplemented with 1 % bsa and 0 . 05 % saponin ( permeabilization solution ), and blocked for 30 min in a permeabilization solution + 10 % goat serum ( blocking solution ). cells were incubated overnight with primary antibody against neonatal mhc ( n3 . 36 , igg , developmental studies hybridoma bank ) diluted ( 1 : 5 ) in the blocking solution . cells were washed with pbs and incubated with alexafluor secondary antibody ( invitrogen ) ( diluted in pbs ) for 2 h . the secondary antibody solution was removed and the cells were rinsed using pbs . the coverslips were dried and mounted on glass slides using vectashield + dapi mounting medium ( vector laboratories h - 1200 ) and viewed on a confocal microscope ( ultraview lci , perkinelmer ). co - cultures were processed for immunocytochemistry as described above . next , cells were incubated overnight at 4 ° c . with rabbit anti - neurofilament m polyclonal antibody , 150 kd , ( chemicon , ab1981 , diluted 1 : 2000 ) and neonatal mhc ( n3 . 36 , igg , developmental studies hybridoma bank diluted 1 : 5 ). after overnight incubation , the coverslips were rinsed three times with pbs and then incubated with the alexafluor secondary antibodies ( invitrogen ) for 2 h . after rinsing three times in pbs , the coverslips were mounted with vectashield + dapi mounting medium onto glass slides . the coverslips were visualized and images collected using a confocal microscope ( ultraview lci , perkinelmer ). controls without primary antibody were negative . achrs were labeled as described previously by incubating cultures with 5 × 10 − 8 m of α - bungarotoxin , alexa fluor ® 488 conjugate ( molecular probes , b - 13422 ) for 1 . 5 h at 37 ° c . before observation ( das et al ., 2007 ( neuroscience )). labeled cultures were fixed with glacial acetic acid and ethanol , washed with pbs , dried , mounted and examined by confocal microscopy . the coverslips which were used for double staining with achr + synaptophysin for locating the nmjs were processed further . after 1 . 5 h of α - bungarotoxin labeling of the achr receptors , the coverslips were fixed , blocked , permeabilized and incubated overnight with synaptophysin antibody ( mab368 , diluted 1 : 1000 ; millipore / chemicon ), the pre - synaptic marker present in motoneuron axonal terminals . statistics were calculated using the following procedure . one coverslip was randomly selected from each experiment ( typically , there are six coverslips per experiment ). 25 non - overlapping fields of view were used to characterize each coverslip . at the magnification used , 25 fields covers over 40 % of the surface area of the coverslip . static contact angle and xps analysis were used for the validation of the surface modifications and for monitoring the quality of the surfaces . stable contact angles ( 40 . 64 °± 2 . 9 / mean ± sd ) throughout the study indicated high reproducibility and quality of the deta surfaces and these characteristics were similar to previously published results ( das et al ., 2004 ; das et al ., 2007 ( nat . protocols ); das et al ., 2007 ( neuroscience ); das et al ., 2006 ; das et al ., 2003 ). based on the ratio of the n is ( 401 and 399 ev ) and the si 2p 3 / 2 peaks , xps measurements indicated that a reaction - site limited monolayer of deta was formed on the coverslips ( stenger et al ., 1992 ). the formation of the maximal number of neuromuscular junctions was observed using the temporal growth factor application technique described in fig1 . upon plating of the motoneurons and skeletal muscle , the cells were treated with medium containing factors that promoted both growth and survival as well as enhancement of nmj formation ( table 1 , table 2 ). after 3 days in culture , the entire medium was removed and switched to a minimal formulation , nbactiv4 , which facilitated both long - term survival and further development of the nmjs ( fig1 ). further , three - fourths of the nbactiv4 medium per well was removed and replaced with an equal volume of fresh nbactiv4 medium . when compared to the continuous application of growth factors , the timed application resulted in cultures that lasted for up to 7 weeks as opposed to 10 - 12 days . phase contrast microscopy was used to visualize motoneuron axons appearing to interact with skeletal muscle myotubes between days 12 - 15 ( fig2 , a - d ). some of the axonal processes appear to branch and terminate on the myotubes . furthermore , many of the myotubes exhibited characteristic striation patterns observed after sarcomere formation when the fibers reached approximately 25 - 30 days in culture ( fig3 , a - d ). quantification of the appearance of striations after this time indicated that the co - cultures contained about twice the number of myotubes showing striations . the characteristic protein expression patterns of the motoneurons and myotubes in co - culture were evaluated at day 25 . immunocytochemistry was used to visualize the neurofilament protein expression in the motoneurons and neonatal myosin heavy chain ( mhc ) expression for the myotubes ( fig4 , a - b ). motoneuron processes were clearly indicated interacting with the skeletal muscle myotubes . a band / i band formation was more visible in the myotubes after staining with the neonatal myosin heavy chain antibody . the immunocytochemical analysis supported the morphological analysis , which had indicated the presence of striations in double the number of myotubes as observed with the muscle only controls . in order to determine neuromuscular junction formation using this novel medium formulation , the clustering of achrs using alpha - bungarotoxin and their colocalization with synaptophysin vesicles was analyzed immunocytochemically . the colocalization of these two synaptic markers indicated the proximity of pre - synaptic and post - synaptic structures and was a positive indication of nmj formation . this technique was used to identify the colocalization of synaptophysin vesicles with the achr clusters ( fig5 , a - d ). the axon + myotube interactions that did not result in the colocalization of pre - synaptic and post - synaptic structures were also identified ( fig6 , a - b ). the observation of the negative result defines the difference between colocalization and non - colocalization and emphasizes the positive result observed in this system . fig7 illustrates nmj formation between a myotube in culture that did not stain for neonatal myosin heavy chain and a motoneuron . this work documents the substantial improvement of an in vitro model system for nmj formation . specifically , we observed enhanced survivability of the culture resulting in our ability to conduct long - term studies on the motoneuron - skeletal muscle cocultures . this increased survivability resulted in maturation of the skeletal muscle myotubes and a significant improvement in the number of nmjs formed in culture . previously , we developed the first defined culture model to coculture embryonic motoneuron and fetal skeletal muscle , however this model was not suitable for long - term tissue engineering studies and the myotubes in the culture only expressed an early muscle marker , i . e . fetal myosin heavy chain and none of the myotubes exhibited characteristic striations . in this study , significant improvement over our previous motoneuron - skeletal muscle co - culture model system was documented . this new culture model supported long - term co - culture of both motoneuron and muscle , resulted in a more adult - like morphology of the muscle and a higher density of neuromuscular junctions ( nmj ). our findings were supported by morphological and immunocytochemical data . we developed this serum - free medium , supplemented with growth factors that supported the survival , proliferation and fusion of fetal rat myoblasts into contractile myotubes , in a semi - empirical fashion . the rationale for selecting the growth factors was based on the distribution of their cognate receptors in the developing myotubes in rat fetus ( arnold et al ., 1998 ; brand - saberi et al ., 2005 ; olson et al ., 1992 ). tables 1 and 2 reference the literature where these individual growth factors , hormones and neurotransmitters were observed to support muscle and neuromuscular junction development . the composition in table 1 is the formulation used for a previously published medium utilized for motoneuron - muscle co - culture and adult spinal cord neuron culture ( das et al ., 2007 ( neuroscience ); das et al ., 2008 ( exp . neurology ); das et al ., 2005 ; das et al ., 2007 ( biomaterials )). table 2 lists the twelve additional factors identified in muscle development and neuromuscular junction formation that enabled the increased survivability of the system . further addition of the factors in table 2 promoted formation of characteristic striation in the muscle in the culture . the use of nbactiv4 for the maintenance of the cells significantly improved the survival of the skeletal muscle derived myotubes despite the fact that the original purpose of the development of nbactiv4 was for the long - term maintenance and synaptic connectivity of fetal hippocampal neurons in vitro ( brewer et al ., 2008 ). in our previous co - culture model , we did not observe the expression of neonatal mhc proteins in the myotubes . interestingly , when this same medium and protocol was used to culture pure skeletal muscle we observed certain striking differences . the pure muscle culture survived longer , exhibited characteristic striations , but only a very small percentage of myotubes expressed n3 . 36 ( das et al ., 2009 ( biomaterials )). to the best of our understanding , the n3 . 36 expression in skeletal muscle in culture is influenced by the motoneurons either physically or by certain trophic factors secreted in the presence of this modified medium and nbactiv4 . this observation needs further studies in order to dissect the molecular pathways regulating n3 . 36 expression in pure skeletal muscle culture and in skeletal muscle - motoneuron co - culture . also , the potential regulation of mhc class switching independent of neuronal innervation / denervation represents an interesting topic for further study . this system would have applications in developing therapies for muscle - nerve diseases such as als , spinal muscular atrophy , spinal cord injury and myasthenia gravis . the development of robust nmj formation , long - term survival of motoneuron skeletal muscle co - cultures and selective mhc class switching is documented in this research . this improved system supports the goal of creating a physiologically relevant tissue engineered motoneuron skeletal muscle construct and puts within reach the goal of developing functional bio - hybrid devices to analyze nmj activity . this defined model can also be used to map the developmental pathways regulating nmj formation and mhc class switching . furthermore , we believe this serum free culture system will be a powerful tool in developing advanced strategies for regenerative medicine in als , stretch reflex arc development and integrating motoneuron + skeletal muscle with bio - hybrid prosthetic devices . due to the use of a serum - free defined culture system this also has applications for new high - throughput screening systems for use in drug discovery research and toxicology investigations . accordingly , in the drawings and specification there have been disclosed typical preferred embodiments of the invention and although specific terms may have been employed , the terms are used in a descriptive sense only and not for purposes of limitation . the invention has been described in considerable detail with specific reference to these illustrated embodiments . it will be apparent , however , that various modifications and changes can be made within the spirit and scope of the invention as described in the foregoing specification and as defined in the appended claims . akaaboune m , et al . 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