Hydrogel Particle Encapsulation and Suspension Media Removal

Abstract

A hydrogel particle encapsulation and suspension media removal method includes combining hydrogel materials, which include a hydrogel polymer, a linker, a cargo suspension, and a suspension media. The hydrogel materials are mixed to form a hydrogel-suspension media mixture. The mixed hydrogel materials are polymerized to form hydrogel particles having encapsulated cargo. The suspension media is substantially removed from the hydrogel-suspension media mixture by: adding a density adjusting material, forming a three-phase solution that includes the density adjusting material settled between the hydrogel particles and the suspension media, centrifuging the three-phase solution, agitating the three-phase solution, centrifuging the three-phase solution, and retrieving the hydrogel particles lacking the suspension media.

1 . A hydrogel particle encapsulation and suspension media removal method, comprising:
 combining hydrogel materials, the hydrogel materials comprising a hydrogel polymer, a linker, a cargo suspension, and a suspension media;   mixing the hydrogel materials to form a hydrogel-suspension media mixture;   polymerizing the hydrogel materials to form hydrogel particles having encapsulated cargo; and   substantially removing the suspension media from the hydrogel-suspension media mixture, comprising:
 adding a density adjusting material; 
 forming a three-phase solution, wherein the three-phase solution comprises the density adjusting material settled between the hydrogel particles and the suspension media; 
 centrifuging the three-phase solution; 
 agitating the three-phase solution; and 
 retrieving the hydrogel particles lacking the suspension media. 
   
     
     
         2 . The method of  claim 1 , wherein mixing the hydrogel materials comprises: 1) accelerating a container holding the hydrogel materials in a first direction, 2) decelerating the container to a stop, 3) accelerating the container in a second direction opposite the first direction back to an initial position, and 4) repeating steps 1-3 a plurality of times. 
     
     
         3 . The method of  claim 1 , wherein forming the three-phase solution comprises centrifuging the hydrogel-suspension media mixture after adding the density adjusting material to provide a forced separation between the suspension media, the density adjusting material, and the hydrogel particles. 
     
     
         4 . The method of  claim 1 , comprising adding a cargo-compatible solution following the step of adding a density adjusting material, wherein the cargo-compatible solution comprises a cargo-compatible surfactant. 
     
     
         5 . The method of  claim 1 , following the step of substantially removing the suspension media, washing the hydrogel particles on a filter and resuspending the hydrogel particles in a storage solution. 
     
     
         6 . The method of  claim 5 , comprising resuspending the hydrogel particles in a mixture of suspension media and storage solution, and repeating the steps of washing the hydrogel particles and resuspending the hydrogel particles in a mixture of suspension media and storage solution having progressively higher ratios of storage solution to gradually resuspend the hydrogel particles in the storage solution in a stepwise manner. 
     
     
         7 . The method of  claim 1 , comprising repeating the steps of agitating the three-phase solution and centrifuging the three-phase solution a plurality of times. 
     
     
         8 . The method of  claim 7 , wherein combining hydrogel materials comprises adding the hydrogel materials to a substantially transparent container such that the steps of agitating the three-phase solution and centrifuging the three-phase solution a plurality of times are repeated until hydrogel/suspension media clumps are substantially broken apart based on a visual inspection through the substantially transparent container. 
     
     
         9 . The method of  claim 7 , comprising adding additional density adjusting material following the steps of agitating the three-phase solution and centrifuging the three-phase solution a plurality of times, and centrifuging the three-phase solution a final time. 
     
     
         10 . The method of  claim 1 , wherein agitating the three-phase solution comprises mechanically agitating the three-phase solution. 
     
     
         11 . The method of  claim 1 , wherein a density of the density adjusting material is from 1.2 g/mL to 1.4 g/mL. 
     
     
         12 . The method of  claim 1 , wherein the density adjusting material comprises one or more surfactants that are added to improve the removal of suspension media. 
     
     
         13 . The method of  claim 1 , wherein the density adjusting material comprises any one or a combination of optiprep or iodixanol. 
     
     
         14 . The method of  claim 1 , wherein centrifuging the three-phase solution comprises centrifuging at 7000 RPM for 3 minutes. 
     
     
         15 . The method of  claim 1 , wherein the three-phase solution comprises an upper phase, a middle phase, and a lower phase, the upper phase comprising the hydrogel particles suspended within a cargo-compatible solution, the middle phase comprising the density adjusting material, and the lower phase comprising the suspension media, and the step of retrieving the hydrogel particles lacking the suspension media comprises collecting the upper phase. 
     
     
         16 . A method for generating and purifying hydrogel particles comprising encapsulated cargo, the method comprising:
 combining hydrogel materials in a container, the hydrogel materials comprising a hydrogel polymer, a linker, a cargo suspension, and a suspension media;   mixing the hydrogel materials to form a hydrogel-suspension media mixture, wherein mixing comprises shaking the container until the hydrogel materials are substantially mixed;   polymerizing the hydrogel materials to form hydrogel particles having encapsulated cargo; and   substantially removing the suspension media from the hydrogel-suspension media mixture, comprising:
 adding a density adjusting material; 
 adding cargo-compatible solution wherein the cargo-compatible solution includes a cargo-compatible surfactant; 
 centrifuging the hydrogel-suspension media mixture with the density adjusting material and cargo-compatible solution to form a three-phase solution comprising: 1) an upper phase comprising the hydrogel particles suspended within the cargo-compatible solution, 2) a middle phase comprising the density adjusting material, and 3) a lower phase comprising the suspension media; 
 agitating the three-phase solution via mechanical agitation; 
 centrifuging the three-phase solution; and 
 retrieving the hydrogel particles lacking the suspension media by collecting the upper phase. 
   
     
     
         17 . The method of  claim 16 , comprising repeating the steps of adding density adjusting material, agitating the three-phase solution, and centrifuging the three-phase solution. 
     
     
         18 . The method of  claim 16 , wherein a density of the density adjusting material is about 1.3 g/mL. 
     
     
         19 . The method of  claim 16 , wherein the upper phase once collected comprises a yield of between 0.1 gram and 1 gram of hydrogel particles. 
     
     
         20 . The method of  claim 16 , wherein the cargo suspension comprises viable cells and the upper phase once collected comprises cell viability of about 93%. 
     
     
         21 . A hydrogel particle encapsulation and suspension media removal method, comprising:
 combining hydrogel materials in a vial via an automated liquid handling system, the hydrogel materials comprising a hydrogel polymer, a linker, a cargo suspension, and a suspension media;   transferring the vial to a shaking device via a robotic arm;   mixing the hydrogel materials via the shaking device to form a hydrogel-suspension media mixture;   transferring the vial from the shaking device to a UV light source via a robotic arm;   polymerizing the hydrogel materials via the UV light source to form hydrogel particles having encapsulated cargo; and   substantially removing the suspension media from the hydrogel-suspension media mixture via the robotic arm and the automated liquid handling system, comprising:
 adding a density adjusting material to the vial; 
 adding a cargo-compatible solution to the vial, wherein the cargo-compatible solution comprises a cargo-compatible surfactant; 
 centrifuging the vial; 
 forming a three-phase solution, wherein the three-phase solution comprises the density adjusting material settled between the suspension media and cargo-compatible solution containing the hydrogel particles; 
 agitating the three-phase solution via an automated mechanical mixing device; centrifuging the vial; and 
 retrieving the hydrogel particles lacking the suspension media. 
   
     
     
         22 . The method of  claim 21 , wherein mixing the hydrogel materials comprises: 1) accelerating the vial in a first direction, 2) decelerating the vial to a stop, 3) accelerating the vial in a second direction opposite the first direction back to an initial position, and 4) repeating steps 1-3 a plurality of times. 
     
     
         23 . The method of  claim 22 , wherein the steps of mixing the hydrogel materials are repeated six times. 
     
     
         24 . The method of  claim 21 , comprising preparing the cargo suspension prior to mixing the hydrogel materials, comprising the steps of:
 growing mammalian cells in a bioreactor until a desired cell number is reached;   harvesting the mammalian cells from the bioreactor via an automated centrifuge system; and   adjusting a cell concentration of the mammalian cells to a desired cell concentration via the automated liquid handling system.   
     
     
         25 . The method of  claim 21 , comprising washing the hydrogel mixture via the liquid handling system. 
     
     
         26 . The method of  claim 21 , comprising examining hydrogel particles from the hydrogel mixture via an automated microscope to ensure correct encapsulation and substantially no clumping of particles.