Patent Application: US-201514965702-A

Abstract:
devices for magnetic 3d culture are described including magnetic lids / bases for single petri plates and adjustable height cap for same . similar devices for multi - magnet culture plates wherein multiwell plates have all adjacent magnets orientated in the opposite polarity , and methods of making same .

Description:
generally speaking the invention relates to a magnetic cell culture plate comprising at least one well having a matching cap fitting over ( or under ) said well , at least one of said well or said cap being having a magnet affixed to said well or cap . alternatively , the invention can comprise just the cap with attached magnet , herein called a magnetic cap or holder or drive , which is sized to fit a culture vessel . generally , the magnet is affixed to said cap with adhesives , fasteners or is snap fit or friction fit with a receptacle designed to accept a magnet , such as a snap fit receptacle . it is preferred however , that adhesives are used to accommodate a range of usage temperatures . the magnetic cap can be used either above or below the vessel , and if used above can lie either over the normal vessel cover or if sterilized and without apertures can replace the cover . preferably , the magnetic cap is used under the plate , and the plate and its cover can be separate devices . in preferred embodiments , the magnetic cap is adjustable so as to adjust the height of said magnet from the vessel . in other embodiments , the position of the magnet on the cap is also adjustable . in one embodiment , the magnetic cap has an adjustable collar that fits outside or inside the cap and which is fitted with grooves or ridges that match ridges or grooves on the magnetic cap , so that unscrewing the collar has the effect of increasing the height of the magnetic cap . instead of using a collar , the magnetic cap can also be fitted with screws or other protrusions that can be lengthened , thus having the same effect . this would allow even a square cap to be easily adjustable . the magnetic cap can also be sized and shaped to fit a multiwell plate . thus , the invention can comprise a magnetic cell culture plate , comprising a plurality of wells in an array ( these wells could be connected by microfluidic channels ), a cap covering said plurality of wells , and a plurality of magnets in an array affixed to said cap , such that each well , or each well in a subset of the wells , has a magnet over said well when said cap is in place over or under said plurality of wells . as above , the invention can comprise just the magnetic cap and magnets , which can be used with commercially available plate ware , and the cap height can be adjustable . in some embodiments , the magnetic cap is merely a plate cover with holes drilled or injection molded therein over each well , and a separate plate having magnetic pins is provided to fit to the cap , the magnets protruding through the hole to varying degrees . in this embodiment , a number of separate plates with magnetic of different lengths or strengths can be provided , again providing the user with an easily customizable magnetic 3d culture hardware . as above , this plate cap can be used above or below a cell culture vessel . in yet another embodiment , the magnetic cap or magnetic drive has a lip ( or rim or vertical edge ) to prevent it from being easily dislodged from the plate or plate cover , and has depressions thereon to receive one or more magnets . preferably , the magnets are snap fit into these depressions , allowing magnets to be switched out for different magnets strengths or shapes . if different magnet shapes are used , the invention can also provide an adaptor for each shape , thus allowing the magnets to be snap fit into the adaptor , and then snap - fit into the drive . in other embodiments , the magnets are more or less permanently affixed , by e . g ., an adhesive . in another embodiment , there is a method of assembling such a multiwell cap or multiwell culture vessel with cap , wherein the magnets are affixed to the cap such that adjacent magnets are in opposite orientation . referring now to the invention in more detail , in fig1 a - e and fig2 a - c there is shown a magnetic holder that fits on the top or the bottom of a single well cell culture dish . in detail fig1 a shows a magnetic holder 1 with a magnet 2 affixed to it . the prototype magnetic holder or drive 1 was made of acrylic , but any polymer , resin , ceramic , glass or metal can be used . inexpensive polymers such as acrylate or polycarbonate are preferred as durable , inexpensive , sterilizable , and transparent , which allows visualization of contents . in this instance , the magnetic holder 1 has a hole 9 through which one can visualize the contents of the plate if an annular magnet 2 is used . the magnetic holder 1 can sit over a petri plate 3 with lid or cover 4 , or can sit under it , depending on the application and / or magnet orientation . a circular or disc shaped magnetic drive 1 is shown in fig1 , but it could be rectangular , triangular , hexagonal , or any other shape defined by a single or combination of magnets . the preferred shapes are compatible with existing cell culture plates and flasks . we have shown the magnet centered on the cap , but it does not have to be . the magnetic holder 1 can be used alone , or can be laid over the existing vessel caps 4 as shown herein . an optional aperture 9 can be included in the holder 1 to facilitate imaging the cultures if needed . the size of this cap will vary depending on the size and shape of the tissue culture vessel and the size and strength of the magnetic field resulting from the magnet being held . as an example , the size of the cap can vary from 5 mm to 100 mm in outer diameter . if this device is to be used to culture large amounts of cells ( such as cultures starting with more than 50m cells per vessel ), this may require larger magnets , therefore holders could vary in size and shape . examples of applications requiring large - scale culture would be the production of recombinant proteins , production of extra - cellular matrix proteins , activation of t - cell by antigen presenting proteins . the height of this cap is a function of magnet strength . stronger magnets may require taller holders . the magnet 2 can be cylindrical or disc shaped , ring or washer shaped , square , triangular and the like , but is not limited to these shapes . here we show a washer shaped magnet 2 with a small hole therein to allow visualization of the culture below . the size of the magnet will vary according to the application . example of size is 32 mm in outer diameter and 6 mm in height . this size could very form 2 mm to 100 mm , but the sizes are not limited . a combination of magnets magnetically held together can also be used . for example , magnetic strength can be increased by stacking magnets , such as disk magnets . fig1 b shows the magnet holder 1 assembled with magnet 2 in the center , and in this instance mounted under petri dish 3 , which is covered with the petri cover 4 . fig1 c shows three such petri plates 3 side by side , but with magnetic holder 1 on top of the petri cover 4 . the minimum distance - l - between the center of magnet holders is defined by the holder size and magnetic strength of magnets , and should be such that the fields do not unduly interfere . the magnet 2 can be held in place on the holder 1 with an adhesive or more preferably , the magnetic holder 1 has a fitting into which the magnet 2 can be snap fit . it can also be held in place with any convention fasteners such as screws and the like . screws or snap fits may be preferred where the magnets 2 are expected to be exchangeable and thus allow easy variation of shape , size and strength . the magnetic holder 1 also keeps the magnet from random displacement relative to the tissue culture dish due to the lip or edge 6 , which fits over the culture plate . in summary , the magnet holder will keep the magnet in place ( either on top or at the bottom ) relative to the tissue culture flask or petri plate . the distance between magnets - l - should be defined relative to the size and shape of the tissue culture flask and / or the strength of the magnet . stronger magnets will require larger distances to keep neighboring magnets from disturbing their respective levitated cultures and / or interacting with each other in a manner that can cause them to be displaced from the top or bottom of the tissue culture dish . this is also important for safety reasons , to prevent fingers from being trapped between very strong magnets , and avoid cross contamination between samples if magnets are displaced and culture media spilled , and / or sample lost . the use of the magnetic drive 1 is not limited to top of the culture plates , but it could also be placed at the bottom of well . when at the bottom , the magnet holder provides increased stability and easier access to cells . fig1 d shows a 6 well magnetic holder 1 with six magnets 2 designed to fit over ( or under ) a six well plate 7 and cover 5 . we have shown the magnets 2 here placed underneath the cover , but we have also made a prototype device where the magnets fit into depressions of the surface of the magnetic holder , and where each depression also has a viewing hole . the magnetic holder 1 can have a lip or edge to hold the device in place over a plate as shown here , or can be a flat surface lacking any protruding lip on the underside , as in our first prototype . however , this is less stable and a lip is preferred . fig1 e shows a 24 - well plate model wherein both the magnetic drive and a special lid are made to fit either over existing plates or a plate and lid combination will both be made . the magnetic drive 121 will be similar to that already described with magnets 122 placed in an array on the drive or holder 121 , which has an array of recesses 129 to receive the magnets 122 . epoxy fixative will then be used to glue 24 ring magnets 122 into the recesses 129 on the drive 121 , thus completing levitator assembly and forming the 24 - well magnetic drive . this will be used with a culture plate 123 and a specially designed lid 124 having 24 wells 126 fitted to receive the magnets and fit inside the 24 wells of the plate 127 . in this way , the magnets can be brought closer to the culture , thus levitating cells , and smaller magnets can be used , thus minimizing interference magnetic fields from neighboring wells . at the same time , the cell cultures are protected from contamination by lid 124 . further , magnet sizes and shapes can easily be switched out by changing holder 121 or the magnets therein . the special lid can be pre - sterilized and thus preserve the sterility of the system . this will allow the magnet drives to be reusable , while the lid will be disposable or designed for single use . the magnetic drive and special lid concept in fig1 e can be adapted with standard or specially designed multiwell plates with 6 , 12 , 24 , 48 , 96 , 384 and / or 1536 wells . in fig2 a there is shown a magnet holder 21 modified with protruding surface 29 for collecting levitating cells . in this embodiment , the magnet is placed inside a cap - shaped protruding surface ( but the surface can be any shape ), such that only this surface contacts media when in use . tissue culture vessel 24 is shown with cells 25 on the bottom . in fig2 b , the holder is moved close enough to the plate 24 so that cells 25 are collected on the protruding surface 29 by attraction to the magnet 22 . once attached to the immersed surface , the cells can be easily removed from solution as in fig2 c . the attached cells can then be counted , separated between levitating and non - levitating cells . the surface attached cells can also be used for analysis , culturing in different flask by displacing the magnet , washing cells off with media or trypsinizing the cells . furthermore , the device can be used to separate , sort , transfer , and evaluate yield of magnetized cells . it is possible to make this protruding surface snap or pressure fit to a protruding edge ( not shown ) on the drive , thus allowing single use , pre - sterilized surface to be prepared and snap fit onto the drive when needed , in the same way that a pipette tip if pressed onto a pipettor . in this embodiment , a simple means for ejecting the cap can also be provided . fig3 and fig4 show multiwell magnet assembly for culturing multiple samples by magnetic levitation . the components of fig3 are a multiwell magnetic drive 31 with an array of magnets 32 protruding therefrom . in more detail , this multiwell magnet holder was made of acrylic or polycarbonate wherein a number of holes were drilled and magnets were inserted therein . in this instance , the magnets were held by either an adhesive or by friction inside a metal holder , heat welding , snap fit , fasteners or any other means can be used to secure the magnets . the multiwell magnetic holder 31 can be used with standard or specially designed multiwell plates 33 with 6 , 12 , 24 , 48 , 96 , 384 and / or 1536 wells 34 . we have shown magnet protruding down into the wells in the multiwell case , and the magnetic holder 31 is used without a plate cover because at some point , the magnets must get close enough to the cells 35 so as to levitate same , but avoid interference effects between well . in other cases the magnets 32 can be sized so as to not protrude into the well , and the device can be combined with a plate magnet 36 , as shown in fig3 b . in such case , the magnetic holder 31 could still be used with a plate cover ( not shown ). once cells reach the meniscus of the media and the smaller magnets 32 have the strength to sustain levitation , this large plate magnet 36 could be removed . also shown in fig3 a is - g -, which is the height of the microwell plate 33 . this height will vary depending on the application and / or magnetic strength of magnets being used . shorter plates of approximately 2 . 5 mm in height , but not limited to it , would be optimum for higher throughput applications , such as when using 96 , 384 , and 1536 plates . the length of multiwell plate is - h - and the depth of the media is - f -. magnets may protrude inside each well to get close enough to the cells to provide the needed magnetic field to levitate cells . with stronger magnets , the magnet can be completely above the wells , and even above the sterile well cover or lid . in some embodiments , these devices could consist of 6 , 12 , 24 , 48 , 96 , 384 , 1536 magnets , or any number in between . the magnetic holder described in fig3 a - b does not have to be used with plates having multiwells , but can be used with fewer wells or even a single well as shown in fig4 a - c wherein the magnetic holder 41 with magnets 42 is used for a large petri plate or culture dish 43 with lid 44 to levitate cells 45 . if needed , the device can also be used with a plate magnet 46 to increase field strength . magnets 42 can also be removed as needed to fit , e . g ., a six well plate . thus , the device is not limited in use to a plate having the same number of wells as there are magnets . the magnet holder 41 with magnets 42 described in fig4 could also be placed under the culture dish . here , cells will be held magnetically at the bottom of the tissue culture dish , allowing them to be printed into different shapes , and depending on the properties of the tissue culture dishes surface ( cell - adhering or non - adhering ). thus , in a typical embodiment , the magnetic holder is sized and shaped so as to fit either above or below the vessel , holding the magnets at a fixed distance d from the culture media , wherein d is & gt ; 0 and is preferably about 0 . 2 - 10 mm . the spacing between magnets will initiate / generate individual levitating cultures , which are held in place and separated by the magnetic field . therefore , this well - less format can be envisioned as an invisible boundary defined by the magnetic field , without internal mechanical barriers , such as found in a multiwell plates . once the individual cultures grow , they may come in contact with each other , which could be a desirable feature for creating larger cultures with improved nutrient flow because of the spacing between individual cultures . this could also be used to generate multiple spheroid bodies within the same culture media . we have obtained proof of principle of such a technique based on label - free monitoring of cells cultured with a prototype 6 well magnetic holder , wherein magnets are held in depressions on the top of a flat piece of plastic , wherein each depression also has a viewing hole drilled therethrough . using the device was as simple as standard 2d techniques , and it was proven to be faster than any other 3d cell culturing product on the market . cells were treated with nanoshuttle ™ ( a proprietary solution from nano3d biosciences ™, inc . containing magnetic nanoparticles that allows cells to levitate in a magnetic field ) and plated into individual wells using media recommended for their specific cell type . the magnetic drive was immediately placed above the culture and magnetic forces gently levitated and guided cells together to quickly induce cell - cell interactions . the cultures were placed in a standard cell culture incubator and , over time , 3d assemblies formed . the location of the culture assembly can be controlled magnetically . however , the morphology of the assemblies and amount of time needed to reach this stage is cell specific . some cells types , such as epithelial , form layered sheets and display squamous morphology while others , such as human umbilical vein endothelial cells , display branching structures . levitated structures can be separated to create multiple samples and viable cells may be removed from the 3d culture for further experimentation . no special additional equipment is required and the magnetic drive is compatible with co - culturing and standard imaging and diagnostic techniques . cells can be maintained for months and toxins can be introduced into culture and examined for any deleterious effects . cultures grown with the magnetic drive provide a model of native tissue , which can be exposed to various drugs and monitored for viability . fig5 a - b shows the drawings of a holder with adjustable magnet positioning ( xyz - axis ), such as height , xyz - axis translation ( lateral positioning ) adjustments of the magnet relative to the tissue culture flask . in detail , the magnetic drive 51 has a magnet 52 , and the magnet can be moved around via positioning screws 57 . this can also be generalized for a multiwell format and the range of motion may be restricted so that each magnet does not move beyond the range of one well . the height of the magnetic drive can also be adjusted , e . g ., by collar 58 with threaded guides for z - axis motion / control . the xy - axis ( horizontal ) adjustment may be desirable for translating the magnetized cells sideways . this can be advantageous or useful when cells accumulate under the view path , therefore obstructing a clear image of the levitated cells . this same xy - axis translating action can also be used to disturb the culture . in fig5 a - b , we show a simple mechanical mechanism for displacing the magnet ( screw action resulting from threads ), however this could be also achieved by electrical ( such as , but not limited to , by using solenoids and electric current , electric magnetic ), and / magnetic actions . an additional mechanism would be a spring - loaded mechanical actions ( similar to a light switch ) in which a single mechanical motion raises the magnet . this example can also be used to reduce or remove the magnetic field so the levitated structures fall to the bottom of the culture dish and then can be magnetically raised back up . one of the applications for removing / reducing the magnetic field would be for higher resolution imaging when it is desirable to have the cells at the bottom of the dish closer to imaging objective or elements , such as in the case of confocal microscopy . in more detail , for multiwell designs , it can work with a lever or other mechanical action instead of a screw . this mechanism is also important for transporting the levitating cultures . when transporting the cultures , if a sudden motion occurs that swings / splashes the media and cells so that the media and cells get too close to the magnet , the magnetic force can pull the cells onto the surface just under the magnet . the cells may then adhere to that surface . by lifting the magnet , it prevents the media and cells from getting close enough to the magnet for them to be attracted to and attached to the surface under the magnet . when the above multiwell plates were manufactured in bulk , it was discovered that it was quite difficult to assemble the plates when all the magnets were in the same orientation due to the repelling fields . the more wells , the smaller the magnets , and the more difficult assembly became . therefore , we began to assemble the plates with alternating magnets , in the orientations as shown in fig6 a and fig6 b . it was not clear at first whether such a plate would function as intended , because there was a distinct possibility that there might be destructive interference , opposing fields cancelling each other out . further , we thought that alternating wells would have cells levitated and repulsed and / or distorted resulting field . however , our tests showed that the fields do not interfere , and may even show less interference . further we found that all cells levitated , regardless of orientation ( probably because the cells freely rotate in the medium ). by contrast , to drive cells to the bottom of the plate ( e . g ., to make sheets or to change the media ), the magnet needed to be under the wells , again regardless of orientation . therefore , henceforth , all multiwell assemblies were made by inserting magnets in opposite polarity . details for 6 , 24 , 96 , 384 , and 1536 well magnetic drives are shown in fig7 - 12 , each of which provides a top view ( a ), and side view ( b ) along the long side of the drive , and a side view on the short side or end of the drive ( c ). the magnetic drives are sized to fit over or under a standard microtitre plate . in several of the embodiments , rims are shown . these can traverse the entire length of each side , but need not and herein a rim is shown only at the center third of each cover . these edges or rims along the outside edge of the drive serve to keep the drive centered over the plate , which can be above or below the magnetic drive . we undertook considerable testing to optimize magnet parameters for the various plates . the results are provided in the table of fig1 . however , a 10 - 25 % variation in strength is certainly possible . the following table provides some useful ranges of strengths . in use , the magnetic drives are used with cell culture plates to levitate cells . first the cells are magnetized , e . g ., using nanoshuttle ™ ( nano3d biosciences , tx ). nanoshuttle ™ is a nanoparticle assembly (˜ 50 nm ) consisting of gold , iron oxide , and poly - l - lysine . the poly - l - lysine will non - specifically bind to cell membranes via electrostatic interactions . nanoshuttle ™- pl will be retained by the cells for roughly 8 days , after which they are released into the extracellular space . if in 3d , nanoshuttle ™- pl will be released into the extracellular matrix , and the 3d culture will retain its magnetic nature . these magnetized cells require magnetic forces ( 30 pn ) only strong enough to aggregate , but not harm cells . further nanoshuttle ™ has been demonstrated to not effect cell proliferation , viability , metabolism , inflammatory or oxidative stress responses , phenotype , and other macro cell functions . cells are magnetized by adding nanoshuttle ™- pl directly to a flask of cells that is 70 - 80 % confluent , and incubating overnight . typically , nanoshuttle ™- pl is added at a concentration of 1 μl / 10 , 000 cells . the next day , treated cells are enzymatically detached with trypsin and resuspended in suitable media . cells can also be magnetized in suspension . briefly , suspension cells are magnetized by mixing them with nanoshuttle ™- pl for ˜ 30 min on a gently rotating orbital shaker . the cells are collected , e . g ., by centrifugation , resuspended in suitable media and ready for use . the cells need to take up enough magnetic nanoparticle so as to be levitated in the magnetic field , but not so much as to disrupt the cells normal metabolism . levels of about 30 - 150 pg / cell , or about 50 pg of magnetic iron oxide are typical . once magnetized , the cells can be levitated for assembly , by adding magnetized cells to a plate , preferably a flat - bottom , ultra - low - attachment plate for maximum levitation efficiency . a magnetic drive as described herein is then placed over or under the cell culture plate . cell assembly typically begins in minutes , and is complete in hours , although different cell types will require different times , and this is typically optimized before an experiment is begun . if desired , the magnetic drive can be left in place even after the cells have assembled into a stable 3d culture . however , typically the drive is removed and the cells studied further . typically , the cells retain their 3d structure once stabilized for about 4 - 8 hrs in the magnetic field . the above descriptions are illustrative only and not intended to unduly limit the invention as defined by the appended claims . the following are incorporated by reference herein in its entirety for all purposes : ansi slas 1 - 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