Patent Application: US-201515519101-A

Abstract:
a method of making a multi - layer patterned cell assembly is provided . a cell suspension liquid solution containing cells is loaded into a liquid - carrier chamber . the cells in the cell suspension liquid solution are let to settle down to the bottom of the chamber . once the cells in the cell suspension liquid solution have gravitationally settled down to the bottom of the chamber , a hydrodynamic drag force is applied by using a vibration generator with a frequency and acceleration to the cells at the bottom of the chamber . the frequency and acceleration are designed to drag the settled cells into a three - dimensional pattern to form a multi - layer three - dimensional patterned cell assembly . the formed multi - layer three - dimensional patterned cell assembly can be transferred from the liquid - carrier chamber to an incubator to form a tissue culture . the bioengineered construct can be implanted for tissue engineering or other medical applications .

Description:
as shown in fig1 , a vibration generator ( u56001 , 3b scientific , tucker , ga . ), driven by an audio amplifier ( lepai lp - 2020a +, parts express , ohio ) and a function generator ( hp 8116a , hewlett - packard gmbh , germany ), was used to generate vertical vibration . the vertical vibrational acceleration was monitored by an accelerometer ( mma7341l , freescale semiconductor , tex .). the vibration generator was fixed on a metric tilt platform ( edmund optics , n . j . ), which was used to adjust the level of the chamber using a bubble level ( spirit level , hoefer , mass .) as a reference . the metric tilt platform was fixed to a vibration damper ( mcmaster - carr , ga .) to prevent external perturbation . liquid - carrier chambers can be multiple well plate , petri dish , transwell plate or any other commercialized standard cell culture containers or customized containers . the chamber was mounted on the top of the vibration generator using an adapter fitting . the thickness of liquid layer should be less than half wavelength of applied faraday waves . the density of liquid should be smaller than buoyant density of samples to allow samples sink down to the substrate of the liquid carrier chamber . faraday waves can be generated at the liquid surface by vertical vibration of a liquid layer using vibration generator . formation of faraday waves will generate a patterned velocity field , which will further generate a hydrodynamic shear stress on the substrate of the liquid carrier chamber . to design the tissue structure you want to generate , a physical model is used to calculate the corresponding geometry of the liquid carrier chamber , vibrational frequency and acceleration for this tissue structure . the following exemplary procedure could be used : ( 1 ) prepare cells . harvest cells from native tissue , or maintained cell lines . ( 2 ) prepare cell suspension solution . re - suspend one or more types of cells in cell culture medium with designed cell concentration and ratio . the cell concentration is correlated to the thickness of the generated tissue . ( 3 ) load cell suspension solution into the liquid - carrier chamber . wait till all the cells gravitationally settle down to the substrate of the liquid - carrier chamber . normally , it &# 39 ; s take one minutes or more which is determined by liquid thickness , cell size and related density between cells and liquid and fluid viscosity . ( 4 ) applying hydrodynamic drag force . turn on the vibration generator and apply calculated frequency and acceleration . faraday waves are generated on the liquid surface as a result of hydrodynamic instability created by vertical vibration of the liquid layer . faraday waves generate a patterned velocity field inside the liquid layer . this velocity field creates patterned hydrodynamic drag force on the cells on the substrate of the liquid - carrier chamber . cells are dragged to the designed region and form multilayer structure . cells with large size , buoyant density will be assembled under the antinodes of faraday waves inside liquid while cells with small size and buoyant density will be assembled under the nodal region of faraday waves inside liquid . movement of cells can be predicted by the physical model as described herein . the number of layers are correlated to the number of the cells in the chamber . assembly of cells can be completed by applying faraday waves on the liquid surface for 5 to 10 seconds . ( 5 ) tissue culture of the assembled cell encapsulating construct . transfer assembled cells to incubator for tissue culture . culture assembled cells for one or more weeks with specific tissue culture medium based on the tissue type for formation of tissue functions . ( 6 ) these functionalized tissues can be used for tissue - based drug screening or other tissue engineering applications . alternative building blocks can be cell spheroid , cell aggregates , or cell on the microcarriers and the following exemplary procedure could be used : ( 1 ) prepare cell spheroids . prepare cell spheroids from harvest cells from native tissue , or maintained cell lines using commercially - available hanging drop techniques , low affinity plates or other techniques [ 1 ]. ( 2 ) prepare cell spheroid solution . re - suspend one or more types of cell spheroids in cell culture medium with desired number and ratio . the number of cell spheroids determines the thickness of the generated tissue . ( 3 ) load cell spheroid solution into the liquid - carrier chamber . wait till all the cells gravitationally settle down to the substrate of the liquid - carrier chamber . ( 4 ) apply hydrodynamic drag force . turn on the vibration generator and apply calculated frequency and acceleration . faraday waves are generated on the liquid surface as a result of hydrodynamic instability created by vertical vibration of the liquid layer . faraday waves generate a patterned velocity field inside the liquid layer . this velocity field creates hydrodynamic drag force on the cell spheroids on the substrate of the liquid - carrier chamber . cell spheroids will be dragged to the designed region with multilayer structure based on their size and buoyant density . cell spheroids with large size , buoyant density will be assembled under the antinodes of faraday waves inside liquid while cell spheroids with small size and buoyant density will be assembled under the nodal region of faraday waves inside liquid . movement of cell spheroids can be predicted by the physical model developed by our team . the number of layers are determined by the number of the cell spheroids in the chamber . assembly of cells can be completed by applying faraday waves on the liquid surface for 5 to 10 seconds [ 6 ]. an alternative approach are developed to stabilize assembled cells and the following exemplary procedure could be used : ( 1 ) prepare hydrogel prepolymer solution . the hydrogel prepolymer solution can be derived from thermal crosslinking ( e . g ., collagen ), chemical crosslinking ( e . g ., fibrin ) or photo crosslinking ( e . g ., methacrylated gelatin hydrogel ) hydrogels . ( 2 ) prepare cell suspension solution . resuspend one or more types of cells in hydrogel prepolymer solution with desired cell concentration and ratio . the cell concentration determines the thickness of the generated tissue . ( 3 ) load cell suspension solution into the liquid - carrier chamber . wait till all the cells gravitationally settle down to the substrate of the liquid - carrier chamber . ( 4 ) apply hydrodynamic drag force . turn on the vibration generator and apply calculated frequency and acceleration . faraday waves are generated on the liquid surface as a result of hydrodynamic instability created by vertical vibration of the liquid layer . faraday waves generate a patterned velocity field inside the liquid layer . this velocity field creates hydrodynamic drag force on the cells on the substrate of the liquid - carrier chamber . cells will be dragged to the designed region with multilayer structure based on their size and buoyant density . cells with large size , buoyant density will be assembled under the antinodes of faraday waves inside liquid while cells with small size and buoyant density will be assembled under the nodal region of faraday waves inside liquid . the number of layers are determined by the number of the cells in the chamber . assembly of cells can be completed by applying faraday waves on the liquid surface for 5 to 10 seconds . ( 5 ) tissue culture . transfer assembled cells in hydrogel for tissue culture . culture assembled cells for one or more weeks with specific tissue culture medium based on the tissue type for formation of tissue functions . ( 6 ) these functionalized tissues can be used for tissue - based drug screening or other tissue engineering applications . a physical model to describe particle assembly inside the liquid has been developed . the particles on the substrate of the liquid - carrier chamber experience gravity , buoyant , hydrodynamic drag forces and normal force . the forces in vertical direction don &# 39 ; t change particles distribution in x - y plane . the drag force in the x - y planes of liquid is proportional to their velocity fields in the liquid . the physical model of this velocity field created by faraday waves has been developed previously [ 2 - 4 ] and can be expressed as below , where ω is the faraday wave frequency , k is the faraday wavenumber , h is the faraday wave amplitude , h is the thickness of liquid , l is the length of liquid chamber , z is the distance from the point where wave is completely decayed , ζ sh is the standing wave with sub - harmonic frequency ( ω ), ζ h is the standing wave with harmonic frequency ( 2ω ), ζ h , is the standing wave with harmonic frequency ( 2ω ) with phase π / 2 and δ is the stokes characteristic length which is given by f drag = 6πvρ liq r ( u s − u ) where r is the radius of the particle , ρ par is the density of the particle , ρ liq is the density of liquid and u s is the settling velocity which is given by to make the final expression for force potential independent of time before casting it in the form of minimization , the velocity field is time averaged . the time averaged drag force directed along x - y plane can be computed as follows : only a part of sub - harmonic component and the last term of harmonic component will sustain after time - averaging the velocity field . the final expression for the force potential is obtained from the relation , f drag =−∇ u . since , we are interested in the force potential at the bottom of liquid and also the decay length is λ / 2 [ 5 ], z is given by particles in the fluid will fill the bottom of the liquid - carrier chamber from the region with the lowest force potential to the region with the highest force potential . we developed this equation above , which can be used to predict assembly and pattern formation inside liquid . using this equation , we can design assembly of cells , cell spheroids or any other biological entities on the nodal regions or antinode region of faraday waves by tuning parameters such as particle size , particle density , liquid density and excitation frequency . two cell types or biological entities can be assembled into complementary patterns by varying their buoyant densities and sizes . assembly of materials with complementary patterns can benefit many application such as studying cell - to - cell interactions , cell - biomaterials interaction and cell - biochemical interaction . asghar , w . et al . in cancer targeted drug delivery ( eds you han bae , randall j . mrsny , & amp ; kinam park ) ch . 24 , 635 - 665 ( springer n . y ., 2013 ). 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