Patent Application: US-201314016495-A

Abstract:
the invention relates to a novel multiwell array comprising a plurality of wells , the majority of which have a unique cross - sectional shape whereby each well within the array can be identified and so tracked whilst the contents of the array are investigated . the invention also concerns a computer and a programmable data storage device for use with the array and an imaging apparatus including any one or more of the afore features or aspects .

Description:
pdms ( sylgard 184 ) was obtained from dow corning , platinic acid catalyst , divinylpdms ( dms - v01 ). and dimethyldiethoxysilane ( dmdes ) was purchased from fluorochem . polymethylhydrosiloxane ( m w 1 , 700 - 3 , 200 ), potassium hydroxide , paraformaldehyde ( pfa ), araldite 506 , hexamethyldisilazane ( hmds ), triethoxysilane ( teos ), propidium iodide ( pi ), hoechst 33342 , rhodamine phalloidin and aminopropryltriethoxysilane ( aptes ) were obtained from sigma - aldrich ( uk ) black liquid ink whiteboard ( easyflo , pentel ) was purchased from wh smiths . ethanol and methanol were purchased from fisher scientific ( uk ). epoxy resin 301 - 2 ( exotech , usa ) was obtained from jp kummer ( uk ). cell culture medias d - mem and rpmi , foetal bovine serum ( fbs , heat shocked for 30 minutes at 56 ° c . before being filtered using a 1 . 2 μm filter ) and penicillin / streptomycin supplied by invitrogen ( uk ). pdms was mixed in a ratio of 10 : 1 prepolymer to curing agent and cast on a coverslip either 150 - 170 μm thick or to a glass plus coverslip thickness of 200 μm . the polymer was degassed for 30 mins by placing in a vacuum before being baked at 110 ° c . for 1 hr and allowed to cool . laser machining was performed in two separate ways . firstly , glass or pdms cover slips were coated in whiteboard ink . for circular wells , wells of 15 nm or 20 μm , were ablated into glass or pdms individually using a 157 nm f 2 eximer laser ). for encoded microwells , first a mask was made for the microwell design in a metal sheet using 795 nm ti : sa femtosecond laser . the wells were then manufactured by ablating the coated glass or pdms under a 192 nm laser containing the mask . after ablation the samples were cleaned up by sonicating for 20 mins in methanol , ethanol , 50 % ethanol / distilled water and distilled water . glass cover slips were further cleaned by wet etching in 7m koh for 1 hour followed by washes in distilled water . a positive mould of the glass cover slips with the wells was made using pdms . cover slips were placed under vacuum and the standard pdms polymer mix was added . the vacuum was removed and sample degassed and baked as above . the cover slips were removed from the pdms leaving a positive mould for the resin . epoxy resin was made by mixing the prepolymers 11 : 8 . 7 : 0 . 3 araldite 506 : epoxy hardener : epoxy accelerator or 100 : 35 part a : part b ( 301 - 2 ). the polymer was then poured onto the pdms mould allowing 35 μl of resin for each cover slip ( this allows for a thickness approximately 160 μm , similar to a no . 0 glass cover slip ). the resin was degassed in a vacuum for 30 minutes before being baked at 60 ° c . overnight for araldite or 80 ° c . for three hours using 301 - 2 and allowed to cool ( see fig1 ). the hydrophobic pdms surface was modified to make the surface more hydrophilic . first the pdms was placed under uv / ozone for 90 minutes to create hydroxyl groups on the surface . the surface was immediately immersed in distilled water to maintain the groups . the surface was then immersed in 5 mm aptes , 5 mm teos solution or water for 2 hours at room temperature . these surfaces were subsequently cleaned by sonicating in ethanol and water ( twice ) for five minutes and backed in a drying oven overnight . the surfaces were subsequently stored in methanol ( aptes ) or water ( pdms , teos or hydroxyl - modified ). surfaces were sterilised by immersing the cover slips in ethanol for ten minutes followed by three five minute washes in sterile phosphate buffered saline ( pbs ). adherent cells were plated onto the cover slips resting in 12 well plates at 100 , 000 - 200 , 000 cells per well and allowed to adhere overnight in complete media ( d - mem , 10 % fbs and 100 u / ml penicillin with 100 μg / ml streptomycin ). before imaging , the cover slips were washed thrice in sterile pbs and placed in imaging media ( rpmi medium lacking phenol red , containing 10 % fbs and 10 μm na - hepes buffer ph 7 . 4 ). non - adherent cells were plated directly onto the coverslip after washing . 1 - 2 ml of cells were removed from the culture flask and centrifuged at 800 × g for 1 min in a microcentrifuge , the sample was resuspended in clear serum free media and washed a further two times . finally the cells were resuspended in imaging medium at 500 , 000 per ml . cover slips were ideally held in place using a custom - made cover slip holder ( see fig1 ). cells were imaged live and fixed under the confocal microscope . for live cells , cells were imaged in imaging media at 37 ° c . during uptake of alexa488 or alexa633 labelled transferrin or after exposure to a pro - apoptotic cpp with 1 μg / ml propidium iodide ( pi ) added to the sample . for fixed cells , cells were fixed in 2 % paraformaldehyde ( pfa ) in pbs for 15 minutes , washed in pbs and permeabilised in 0 . 02 % triton for 10 mins . the cells are washed once and incubated with 0 . 2 μg / ml of hoescht33342 and 1 μg / ml rhodamine - phalloidin for ten minutes . cells are washed then mounted on a coverslip . live cells were fixed in 1 % glutaraldehyde for 30 minutes washed and post - fixed in 1 % osmium tetroxide for a further 30 minutes . cells were washed and dehydrated in 10 min steps of 50 %, 70 %, 80 %, 90 % and three neat ethanol washes . the cells were dried either in co 2 using a critical point drier or using hexamethyldisilazane ( hmds ), whereby cells were washed twice in hmds and allowed to air - dry in a fume hood . the cover slips were splutter - coated in gold and imaged using an sem . pdms was made using different ratios of short chained divinylpdms with pdms prepolymer . these mixes were added with a curing agent at a ratio of 1 : 1 with the short chained pdms and 1 : 10 with the long chained pdms . platinic acid was added to the mix at 1 ul of 20 mg / ml ( in thf ) to 100 mg short chained divinylpdms . the different mixes were degassed in a vacuum for 30 minutes and baked at 110 ° c . for 1 hour and allowed to cool as before . pieces of pdms were super - glued onto a mounting block for the microtome and attempts were made to section the material at different thicknesses . different ratios of the epoxy 301 - 2 were used ranging from 100 : 10 to 100 : 70 ( part a : part b , part a being diglycidyl ether of bisphenol a and part b being polyoxypropylenediamine ) in steps of 10 and cured as before . this produced a range of epoxies of different hardness which were then sectioned on an ultramicrotome using a glass knife at two thicknesses 1 μm and 100 nm . microwells were moulded of 301 - 2 as above at a ratio of 100 : 20 and were backfilled with and hardened as before . the microwell containing epoxies were again sectioned on an ultramicrotome using a glass knife . different microwell designs were developed to enable correlative microscopy . after sectioning it would be very difficult to determine a well position if an ordered array of round wells were used ( see fig2 a ). two ways were developed to overcome this problem , firstly , wells were designed to be different distances away from each other ( see fig3 ). when a section is taken , as long as there are at least three wells in a 2d arrangement , it is possible to calculate an individual well position ( see fig2 b ). the second method used bumps on the edge of the well to give the well its own code . each well is now individual so its position can be easily calculated . there are three parts to this well type : i . the well itself , a place for the cell to rest ii . the code around the edge , this is a simple , for example , binary code where each well is numbered ( e . g . 1001101 for say well 77 or 0011111 for say well 31 ) iii . the final part is a marker such as a triangle denoting the order in which the code is read , this is important due to the possibility of a section being in any orientation after it has been sectioned and handled ( see fig5 ) a similar system was devised where the shape of the well was an octagon with various edges missing or present , this also used a binary code ( see fig4 ). whilst we used round extensions to denote the code , any shape can be used . differences could also have been made to the code , if there are two different shapes at any one position a tertiary code could be used ( e . g . 010221 would encode for well 106 , [ 3 0 + 2 × 3 1 + 2 × 3 2 + 3 4 ]) if there are three shapes a quaternary system can be used and so on . it may also be possible to encode for an 8 × 8 array using an 8 - bit binary form of hexadecimal , in this case the array would be divided into two sections , the number and the letter e . g . 1a being 00011010 ( 0001 is binary for 1 , 1010 is binary for a ) and 7d being 01111101 ( 0111 = 7 , 1101 = d ). the binary system was chosen as this is the simplest form . another important feature which could be used independently of the above or incorporated therein is a funnel shaped extension . this is where the relative size of the extrusion changes with depth , e . g . smaller at the bottom of the well and wider at the top of the well . although more difficult to manufacture , this would provide a way of estimating the depth of the slice within the well . pdms is a hydrophobic material and doesn &# 39 ; t promote cell binding it is however , non - toxic and has been used in a variety of microfluidic - cell applications . by modifying the surface we were able to reduce cytophobicity of the surface . the pdms surface has been shown , by various research groups , to be hydroxylated in a variety of ways using oxygen plasma etching , uv ozone or by chemical reaction . in most cases , however , the hydroxylated surface has shown to be short lived and requires further modification to keep the surface viable . we achieved this by using aptes ( aminopropyltriethoxysilane , see fig9 ), when the surface was modified using aptes there was improved cell - surface binding than with unmodified surface or dmdes modified surface . live movies show kg1a cells residing in the grids after exposure to an apoptotic peptide . cells uptake propidium iodide and it is possible to see blebbing occurring on the surface of the cells . this blebbing is not present during exposure to the control peptide ( see fig6 ). cells have also been imaged using scanning electron microscopy . kg1a cells were again imaged under scanning electron microscopy in both round well arrays and coded arrays ( see fig7 ). images show similar cell morphology between cells in the array and cells resting on the surface indicating the well don &# 39 ; t have an effect on cell uptake . correlative light - sem images were also taken of kg1a cells resting in round wells . localisation is consistent between the light images and sem images to indicate the cells have not moved during the fixation and preparation process ( see fig8 ). first attempts at slicing the epoxy resin 301 - 2 at the recommended ratio of 100 : 35 part a ( diglycidyl ether of bisphenol a ): part b ( polyoxypopylenediamine ) was not possible due to the strength of the material . different ratios of the two parts of the epoxy were attempted to determine the most suitable material for cutting , aiming for the material to have similar properties to araldite ™. most ratios still proved too strong for sectioning leaving two ratios 100 : 20 and 100 : 60 most effective . the final material is still rigid and strong but soft enough to allow sectioning with the glass knife at both 1 μm and 100 nm thicknesses . whilst both 100 : 20 and 100 : 60 showed suitability it was decided to continue using 100 : 20 for sectioning the wells . a copy of the coded wells was made from the ablated glass using a pdms intermediate , and backfilled using araldite ™. sectioning of the araldite ™: 301 - 2 epoxy block was much easier with sections of both araldite ™ and 301 - 2 possible . sectioning through the wells also produced some success , however , the wells were at an angle to the blockface making it difficult to read the code produced ( see fig1 ). it can therefore be seen that we have developed a highly useful way of correlating data obtained on cellular images using a variety of imaging techniques . our methodology involves the manufacture of unique multiwall arrays wherein the individual shape of each well within the array is different from any other well within the array and this difference is used as a way of naming and so identifying each well within the array . 1 . fernandez - suarez , m . and a . y . ting , fluorescent probes for super - resolution imaging in living cells . nat rev mol cell biol , 2008 . 9 ( 12 ): p . 929 - 43 . 2 . huang , b ., et al ., three - dimensional super - resolution imaging by stochastic optical reconstruction microscopy . science , 2008 . 319 ( 5864 ): p . 810 - 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well arrays for 3 d shape control and high resolution analysis of single cells . lab on a chip , 2007 . 7 ( 8 ): p . 1074 - 1077 . 10 . mcdonald , j . c ., et al ., fabrication of microfluidic systems in poly ( dimethylsiloxane ). electrophoresis , 2000 . 21 ( 1 ): p . 27 - 40 . 11 . verkade , p ., moving em : the rapid transfer system as a new tool for correlative light and electron microscopy and high throughput for high - pressure freezing . j microsc , 2008 . 230 ( pt 2 ): p . 317 - 28 .