Patent Application: US-76539910-A

Abstract:
the invention provides a nutrient medium composition and associated methods for lengthening the useful life of a culture of muscle cells . disclosed is a method of culturing mammalian muscle cells , including preparing one or more carriers coated with a covalently bonded monolayer of trimethoxy - silylpropyl - diethylenetriamine ; verifying deta monolayer formation by one or more associated optical parameters ; suspending isolated fetal rat skeletal muscle cells in serum - free medium according to medium composition 1 ; plating the suspended cells onto the prepared carriers at a predetermined density ; leaving the carriers undisturbed for cells to adhere to the deta monolayer ; covering the carriers with a mixture of medium 1 and medium 2 ; and incubating . a cell nutrient medium composition includes neurobasal , an antibiotic - antimycotic composition , cholesterol , human tnf - alpha , pdgf bb , vasoactive intestinal peptides , insulin - like growth factor 1 , nap , r - apolipoprotein e2 , purified mouse laminin , beta amyloid , human tenascin - c protein , rr - sonic hedgehog shh n - terminal , and rr - agrin c terminal .

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 , or 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 , however , 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 6661 f52 , 22 × 22 mm no . 1 ) were cleaned using an o 2 plasma cleaner ( harrick pdc - 32g ) for 20 minutes 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 qq 100 ° c ., rinsed with toluene , reheated to approximately 100 ° c ., and then oven dried [ 28 ]. 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 al kα excitation were obtained [ 28 ]. the skeletal muscle was dissected from the thighs of the hind limbs of fetal rats ( 17 - 18 days old ). the tissue was collected in a sterile 15 ml centrifuge tube containing 1 ml of 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 minutes in a 37 □ c water bath at 50 rpm . after 30 minutes the trypsin solution was removed and 4 ml of 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 minutes 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 minutes . the non - attached cells were removed and then centrifuged on a 4 % bsa cushion [ 28 ]. 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 one hour . the final medium was prepared by mixing medium one ( table 1 ) and medium two ( table 2 ) in a 1 : 1 v / v ratio . fig1 indicates the flowchart of the culture protocol . tables 1 and 2 indicated the growth factor and hormone supplement compositions of medium one and medium two . the cells were maintained in a 5 % co 2 incubator ( relative humidity 85 %). the full medium was replaced after four days with nbactiv4 medium according to the protocol in fig1 [ 34 ]. thereafter three - fourth of the medium was changed every three days with nbactiv4 . nbactiv4 ™ ( available from brainbits llc ) comprises all of the ingredients in neurobasal ™, b27 ™, and glutamax ™. nbactiv4 ™ also comprises creatine , estrogen , and cholesterol . coverslips were prepared for immunocytochemical analysis as previously described . briefly , 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 ) for 10 minutes , and blocked for 2 h with 10 % goat serum and 1 % bsa . cells were incubated overnight with primary antibodies against embryonic myosin heavy chain ( f1 . 652 ) ( dilution & gt ; 1 : 5 ), neonatal myosin heavy chain ( n3 . 36 ) ( 1 : 5 ) ( developmental studies hybridoma bank ), ryanodine receptor ( ab9078 , millipore ) ( 1 : 500 ) and dihydropyridine binding complex ( α1 - subunit ) ( mab 4270 , millipore ) ( 1 : 500 ) diluted in the blocking solution . cells were washed with pbs and incubated with the appropriate secondary antibodies for two hours in pbs . after two hours the coverslips were rinsed with pbs and mounted on glass slides and evaluated using confocal microscopy [ 25 , 28 , 31 ]. achrs were labeled as described previously by incubating cultures with 5 × 10 - 8 m of α - bungarotoxin , alexa fluor ® 488 conjugate ( b - 13422 ; invitrogen ) for 1 . 5 h at 37 ° c . [ 12 , 31 ]. following incubation in α - bungarotoxin , the cultures were fixed as above for subsequent staining with embryonic myosin heavy chain ( f1 . 652 ) antibodies . whole - cell patch clamp recordings were performed in a recording chamber located on the stage of a zeiss axioscope 2fs plus upright microscope as described previously [ 25 , 33 ]. the chamber was continuously perfused ( 2 ml / min ) with the extracellular solution ( leibovitz medium , 35 ° c .). patch pipettes were prepared from borosilicate glass ( bf150 - 86 - 10 ; sutter , novato , calif .) with a sutter p97 pipette puller and filled with intracellular solution ( k - gluconate 140 mm , egta 1 mm , mgcl 2 2 mm , na 2 atp 2 mm , phosphocreatine 5 mm , phosphocreatine kinase 2 . 4 mm , hepes 10 mm ; ph = 7 . 2 ). the resistance of the electrodes was 6 - 8 mω . voltage clamp and current clamp experiments were performed with a multiclamp 700a amplifier ( axon laboratories , union city , calif .). signals were filtered at 2 khz and digitized at 20 khz with an axon digidata 1322a interface . data recording and analysis were done with pclamp 8 software ( axon laboratories ). membrane potentials were corrected by subtraction of a 15 mv tip potential , which was calculated using axon &# 39 ; s pclamp 8 program . sodium and potassium currents were measured in voltage clamp mode using voltage steps from a − 85 mv holding potential . action potentials were evoked with 1 second depolarizing current injections from a − 85 mv holding potential [ 25 , 28 ]. static contact angle and xps analysis was 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 were similar to previously published results [ 24 , 25 , 28 , 29 , 31 ]. based on the ratio of the n ( 401 and 399 ev ) and the si 2p3 / 2 peaks , xps measurements indicated that a reaction - site limited monolayer of deta was formed on the coverslips [ 35 ]. development of the serum free medium formulation and culture timeline for long - term survival and maturation of myotubes the serum free medium composition was developed empirically . the final medium is derived from two different medium compositions described in tables 1 and 2 . table 1 constitutes the same medium composition used previously for a motoneuron - muscle co - culture and adult spinal cord neurons culture [ 26 , 27 , 30 , 31 ]. table 2 is composed of twelve additional factors that had been shown to promote skeletal muscle maturation and neuromuscular junction formation separately . the final medium was prepared by mixing these two media in a 1 : 1 v / v ratio . after first 4 days of culture the whole medium was replaced with nbactiv4 medium [ 34 ]. thereafter , every three days three - fourth medium was changed with nbactiv4 . the culture technique has been illustrated in the flowchart ( fig1 ). using this new medium formulation and timeline , myotubes were successfully cultured for more than 50 days . fig2 indicates 50 days old myotubes in culture . as the myotubes aged and grew they began to form the characteristic anisotropic ( a band ) and isotropic ( i band ) banding pattern observed with in vivo muscle fibers [ 22 , 23 ]. this banding pattern is caused by differential light diffraction due to the organization of myofibril proteins forming sarcomeres within the myotubes [ 22 , 23 ]. the arrowheads in the images ( fig2 a - d ) indicate myotubes where sarcomeric organization has occurred and is visualized by the appearance of a and i bands . the myotubes formed were evaluated for the expression of fetal mhc to establish a baseline as comparison to our previous results [ 28 ]. in fig3 , the myotubes phenotypes formed at approximately day 50 in vitro are shown . the myotubes ranged from having clustered nuclei ( fig3 a - d ) to having diffuse nuclear organization ( fig3 e - h ). the arrowheads in the images indicate the characteristic striations . in order to determine if the myotubes were maturing in a physiologically relevant way as they aged in vitro , the expression of neonatal mhc protein was evaluated . after approximately 50 days in vitro 25 % of the myotubes expressed neonatal mhc ( fig4 a - m ). additionally , the myotubes were stained for clustering of acetylcholine receptors ( achr ) using alpha bungarotoxin ( fig5 b , f ). this clustering of the achr receptors , induced by the motoneuron protein agrin in vivo , are locations on the myotube where neuromuscular junction formation occurs . the presence of ryanodine ( ryr ) receptor and dihydropyridine ( dhpr ) receptor clusters , as well as their colocalization in vivo , represents the development of excitation - contraction coupling apparatus in skeletal muscle myotubes [ 19 , 21 - 23 ]. the clustering of both ryr and dhpr receptors was observed on the myotubes after 30 days in culture ( fig5 a - d ). the clustering and colocalization of the ryr + dhpr clusters was observed with different myotube morphologies ( fig5 e - l ). this functional adaptation illustrated that the medium formulation facilitated not only the structural maturation but also the functional maturation of myotubes in this in vitro system . the clustering of the ryr + dhpr receptors was also observed in the 70 day old myotubes , indicating that the older myotubes maintained their functional integrity ( fig6 a - f ). the myotubes contracted spontaneously in the culture and the contractions began generally by day four and continued throughout the life of the culture . most of the myotubes expressed functional voltage gated sodium , potassium and calcium ion channels as reported previously [ 28 ]. the voltage clamp electrophysiology of the myotubes indicated the inward and outward currents that demonstrate functional sodium and potassium channels ( fig7 a ). the current clamp study indicated the single action potential fired by the myotubes ( fig7 b ). herein we have documented the development of a system for long - term in vitro functional , skeletal muscle culture . this system was developed in response to a need for more physiologically relevant skeletal muscle myotubes for functional in vitro systems . for our specific research , they were needed for a realistic model of the stretch reflex arc development and to be integrated with bio - mems cantilevers for screening applications . the results indicate we achieved three significant structural modifications within the myotubes , causing both the developmental profile and functionality of the fibers to better mimic in vivo physiology . it is believed that this skeletal muscle maturation resulted from modifications to the cell culture technique , a new medium formulation and the use of nbactiv4 as the maintenance medium . the presently described serum - free medium supplemented with growth factors was developed to support the survival , proliferation and fusion of fetal rat myoblasts into contractile myotubes . the rationale for selecting the growth factors was based on the distribution of their cognate receptors in the developing myotubes in rat fetus [ 1 - 11 ]. 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 used for motoneuron - muscle co - culture and adult spinal cord neuron culture [ 26 , 27 , 30 , 31 ]. table 2 lists the twelve additional factors we have identified in muscle development and neuromuscular junction formation . the use of nbactiv4 for the maintenance of the cells provided unexpected results in that it significantly improved the survival of the skeletal muscle derived myotubes despite the original development of nbactiv4 for the long - term maintenance and synaptic connectivity of fetal hippocampal neurons in vitro [ 34 ]. we observed a ratio of 25 % neonatal to 75 % embryonic mhc expression of the myotubes , which contrasts with the previous study in which mhc expression was strictly embryonic . we believe that the myotubes matured in this culture system because the long - term survival provided adequate time for the myotubes to respond to the additional growth factors , which activated the necessary signaling pathways to achieve mhc class switching [ 20 ]. this suggests that a different growth factor profile could be utilized to activate alternative signaling pathways and drive myotube differentiation down other pathways . for example , the effects of adding steroid hormones like testosterone to the system could be critically examined . the colocalization of ryr and dhpr clusters in the myotubes indicated the formation of excitation - contraction coupling apparatus and was another indicator of functional maturation in the fibers . excitation - contraction coupling is the signaling process in muscle by which membrane depolarization causes a rapid elevation of the cytosolic ca 2 + generating contractile force [ 36 ]. the close proximity of the dhpr and ryr complexes occurs at specialized junctions established between the transverse tubule and sarcoplasmic reticulum ( sr ) membranes in skeletal muscle myotubes [ 37 ]. at these junctions , t - tubule depolarization is coupled to ca 2 + release from the sr resulting in muscle contraction [ 38 - 40 ]. this structural adaptation represents a significant functional change due to the fact that excitation - contraction coupling is required for successful extrafusal muscle fiber development as well as neuromuscular junction formation [ 19 , 21 - 23 ]. this improved model provides the potential to study excitation - contraction coupling in a defined system as well as myotonic and myasthenic diseases . the development of sarcomeric structures , the excitation - contraction coupling apparatus and mhc class switching in the skeletal muscle myotubes is a result of the improvements to the model system documented in this research . this improved system along with the new findings support the goal of creating physiologically relevant tissue engineered muscle constructs and puts within reach the goal of functional skeletal muscle grafts . furthermore , we believe this serum - free culture system will be a powerful tool in developing advanced strategies for regenerative medicine in muscular dystrophies , stretch reflex arc development and integrating skeletal muscle with bio - hybrid prosthetic devices . 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 . 1 . arnold h h , winter b . muscle differentiation : more complexity to the network of myogenic regulators . curr opin genet dev . 1998 october ; 8 ( 5 ): 539 - 44 . 2 . olson e . activation of muscle - specific transcription by myogenic helix - loop - helix proteins . symp soc exp biol . 1992 ; 46 : 331 - 41 . 3 . olson e n . interplay between proliferation and differentiation within the myogenic lineage . dev biol . 1992 december ; 154 ( 2 ): 261 - 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