Temperature-responsive microcarriers for adherent cell culture

Abstract

A method of culturing cells for vaccine production comprises culturing cells in the presence of microcarriers with cell culture media and infecting the cells with a virus. The microcarrier comprises a bead and a coating. The coating comprises a thermo-responsive polymer having a lower critical solution temperature (LCST) of between about 20 °C and about 34 °C. The cells adhere to the coating of the microcarrier at a temperature above the LCST. The microcarrier comprises a polymeric bead, and a hydrophobic polymer. The hydrophobic polymer is a block copolymer which is connected to the bead by a covalent bond or by physical adsorption. The block copolymer comprises at least one hydrophobic block and at least one thermo-responsive block.

Description

TEMPERATURE-RESPONSIVE MICROCARRIERSFOR ADHERENT CELL CULTURECROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to and the benefit of U.S. Provisional Application No. 63 / 696,551 entitled "THERMO-RESPONSIVE POLYMER FOR USE IN BIOLOGICAL APPLICATIONS AND METHOD OF MAKING AND USING THE SAME" (filed on September 19, 2024); and PCT / US25 / 47102 entitled "THERMO- RESPONSIVE POLYMER FOR USE IN BIOLOGICAL APPLICATIONS AND METHOD OF MAKING AND USING THE SAME" (filed on September 19, 2025), which claims the benefit of U.S. Provisional Application No. 63 / 696,551 entitled "THERMO-RESPONSIVE POLYMER FOR USE IN BIOLOGICAL APPLICATIONS AND METHOD OF MAKING AND USING THE SAME” (filed on September 19, 2024). All foregoing applications are incorporated herein by reference in their entireties for all purposes.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted herewith and is hereby incorporated by reference in its entirety. Said .xml copy, created on September 19, 2025, is named TP387495WO1, and is 26,77029,946 bytes in size.BACKGROUND

[0003] Current processes for vaccine production at industrial scale with cells are challenging, as the scale-up of adherent cells involves the detachment and reattachment of cells. Several recent developments have improved vaccine production. First, serum-free cell culture media can eliminate the medium replacement step before viral infection. Second, single-use bioreactors reduce cleaning and sterilization steps compared to stainless steel bioreactors. Third, microcarriers provide an increased surface area and cells can reach higher densities compared to roller bottles.

[0004] There is a significant need for further improvements for scale-up methods of cells on microcarriers used for vaccine production.BRIEF SUMMARY

[0005] A method of culturing cells for vaccine production comprises culturing cells in the presence of microcarriers with cell culture media and infecting the cells with a virus. The microcarrier comprises a bead and a coating. The coating comprises a thermo-responsive polymer having a lower critical solution temperature (LCST) of between about 20 °C and about 34 °C. The cells adhere to the coating of the microcarrier at a temperature above the LCST.

[0006] A method of forming a microcarrier comprises forming a connection between a thermo-responsive polymer and a reactive group on the surface of a polymeric bead. The thermo-responsive polymer has a lower critical solution temperature (LCST) of between about 20 °C and about 34 °C.

[0007] A microcarrier comprises a polymeric bead, and a hydrophobic polymer. The hydrophobic polymer is a block copolymer which is connected to the bead by a covalent bond or by physical adsorption. The block copolymer comprises at least one hydrophobic block and at least one thermo-responsive block. The hydrophobic block comprises a polymer having a Hansen solubility parameter that is within about 1.0 MPa0 5of the Hansen solubility parameter of the polymeric bead. The thermo-responsive block comprises a thermo- responsive polymer having a lower critical solution temperature (LCST) of between about 20 °C and about 34 °C.

[0008] These and other objects and advantages shall be made apparent from the accompanying drawings and the description thereof.BRIEF DESCRIPTION OF THE FIGURES

[0009] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments, and together with the general description given above, and the detailed description of the embodiments given below, serve to explain the principles of the present disclosure.

[0010] FIG. l is a graphic showing how thermo-responsive polymers are able to adhere and detach cells at different temperatures.

[0011] FIG. 2 is a graphic showing “loop structure” and “brush structure” of polymers attached to a polymer bead.

[0012] FIG. 3 is a schematic showing a typical vaccine manufacturing process.

[0013] FIG. 4 is a graph of the quantification of Vero cell attachment and detachment using microcarrier prototypes (non-porous and 150um size) after 30 minutes temperature harvest from Example 2.

[0014] FIG. 5 is a graph of the comparison of Vero cells detachment in different microcarrier prototypes when harvesting using TrypLE enzyme at different incubation times: 4 VS 30 minutes, from Example 3.

[0015] FIG. 6 is a graph of Vero cell adhesion results for all the microcarriers prototypes, from Example 4.

[0016] FIG. 7 is a plot of variable cell density versus time for scale-up of Vero cells on microcarriers in Viral Product! on- Serum Free Medium (VP-SFM).

[0017] FIG. 8 is a plot of TCID50 versus days of post infection for Vesicular stomatitis virus production with Vero cells on microcarriers.

[0018] FIG. 9 is a collection of microscopic images during scale-up of Vero cells on microcarriers.DETAILED DESCRIPTION

[0019] Method of culturing cells

[0020] A method of culturing cells for vaccine production comprises culturing cells in the presence of microcarriers with cell culture media and infecting the cells with a virus. The microcarrier comprises a bead and a coating. The coating comprises a thermo-responsive polymer having a lower critical solution temperature (LCST) of between about 20 °C and about 34 °C. The cells adhere to the coating of the microcarrier at a temperature above the LCST. FIG. 3 shows a typical vaccine manufacturing process.

[0021] The microcarrier comprises a bead. The bead is a support for the coating. In some embodiments, the shape of the bead is spherical, a fiber, a disc, a cube, an ellipsoid, a cylinder, or a torus. In some embodiments, the shape of the bead is spherical. The bead may be porous or non-porous. In some embodiments, porous beads can have a network of interconnected pores or channels within their structure and possess a high surface area due to the presence of pores, allowing for greater cell attachment and growth compared to non-porous beads having a solid, dense structure without internal pores. In some embodiments, a porous bead has a pore volume of 50% to 99% of the total bead volume. A porous bead may be microporous, mesoporous, or macroporous. A microporous bead has an average pore diameter of less than 20 A. A mesoporous bead has an average pore diameter between 20 A and 500 A. In some embodiments, the bead has an average pore diameter of between about 20 A and about 500 A, such as about 150 A to about 500 A, or about 200 A to about 500 A. A macroporous bead has an average pore diameter greater than 500 A. In some embodiments, the bead has an average pore diameter of greater than 500 A, such as about 500 A to about 5000 A, about 500 A to about 3000 A, about 500 A to about 2000 A, about 1000 A to about 5000 A, or about 1000 A to about 3000 A. In some embodiments, pore sizes of beads can be measured by, not limited to, Mercury Intrusion Porosimetry (MIP), and water absorption methods. For example, liquid intrusion porosimetry with water as the intrusion fluid for any macroporous hydrophobic core. Liquid intrusion porosimetry with mercury as the intrusion fluid for any macroporous hydrophilic core. Nitrogen adsorption-desorption porosimetry (e.g., Tristar) with BJH (barrett-joyner-halenda) model analysis for anything that can be dried without shrinking. Inverse size exclusion chromatography with molecules of known size for cores of high (>90%) porosity like agarose. A non-porous bead is one that is not considered porous. In some embodiments, the bead is optically transparent, i.e. it transmits more than 50% of the light.

[0022] In some embodiments, the microcarrier has a diameter of about 10 pm to about 500 pm, such as about 10 pm to about 250 pm, about 10 pm to about 125 pm, about 25 pm to about 500 pm, about 50 pm to about 500 pm, about 50 pm to about 250 pm, about 50 pm to about 125 pm, about 75 pm to about 500 pm, about 75 pm to about 250 pm, about 75 pm to about 125 pm, about 100 pm to about 300 pm, about 125 pm to about 300 pm, about 100 pm to about 200 pm, about 100 pm to about 150 pm, about 125 pm to about 200 pm, about 125 pm to about 150 pm.

[0023] In some embodiments, the bead comprises a polymer. Examples of polymers include polystyrene-co-divinylbenzene, polystyrene, polydivinylbenzene, polyvinyl alcohol, polyacrylamide, polymethacrylate, polyacrylate, polyethylene glycol, polydextran, agarose, alginate, cellulose, dextran, collagen, and combinations thereof, with or without crosslinking. In some embodiments, the bead comprises ceramic; metal, such as stainless steel; glass; or silica.

[0024] The thermo-responsive polymer has a lower critical solution temperature. The critical solution temperature is the temperature at which two partially miscible liquids become completely miscible, forming a single homogeneous phase. In some systems, below the critical solution temperature the mixtures are miscible, when this is the case, the critical temperature is known as the lower critical solution temperature (LCST). Above the LCST the liquids are partially miscible. The cells are able to adhere to the microcarrier above the LCST. When the temperature of the polymer is reduced below the LCST the cells no longer adhere to the microcarrier. In some embodiments, the temperature range for the LCST is between about 20 °C and about 34 °C, such as about 20 °C and about 32 °C, about 22 °C and about 34 °C, about 25 °C and about 34 °C, about 30 °C and about 34 °C, about 20 °C and about 33 °C, and about 20 °C, and about 30 °C.

[0025] Examples of thermo-responsive polymers include: poly(N-isopropyl acrylamide), poly(N-isopropyl methacrylamide), poly(N-n-propyl acrylamide), poly(N,N- diethyl acrylamide), poly(diethylene glycol methyl ether methacrylate), poly(N- vinylcaprolactam), or combinations thereof. In some embodiments, the thermo-responsive polymer comprises poly(N-isopropyl acrylamide). In some embodiments, the thermo- responsive polymer is a co-polymer. In some embodiments, the thermo-responsive polymer is a diblock co-polymer or a triblock co-polymer. In some embodiments, the thermo- responsive polymer comprises poly(N-isopropyl acrylamide) and polystyrene.

[0026] In some embodiments, the thermo-responsive polymer has a structure according to Formula IAFormula IA wherein FG represents a coupling component that is a functional group capable of coupling the thermo-responsive polymer to a microcarrier component. The TG of Formula IA is a terminating group. In some embodiments, the terminating group can be -C(Me)2CN. In some embodiments of the disclosure, the FG is a clickable functional group, a reactive group, a heterobifunctional linker, or a combination thereof. In some embodiments, the clickable functional group can be selected from an azide group, a terminal or di-substituted alkyne group, a tetrazine group, a trans-cyclooctene group, a dibenzocyclooctyne group, or a bicyclo[6.1.0]nonyne group. In some embodiments, the clickable functional group is anazide. In some embodiments, the FG is a heterobifunctional linker that comprises an alkylene or alkylene oxide spacer and a reactive end comprising, for example, an NHS ester, an acid, a sulfo-NHS ester, a biotin group, a maleimide group, a TFP ester, an amine, an STP ester, a haloacetamido group, and the like. Exemplary FG groups that can be used include those contained in various DBCO- containing reagents sold by Vector Laboratories. In some embodiments, the FG is a reactive group, such as a maleimide, a protected amine, an activated ester, or the like.

[0027] In some embodiments, the thermo-responsive polymer can have a structure according to any of the structures provided below. OdT represents attachment to the microcarrier or the microcarrier itself.

[0028] In some embodiments, there is an intermediate layer between the bead and the thermo-responsive polymer. In some embodiments, the intermediate layer comprises at least some reactive groups for attaching the thermo-responsive polymer to the bead. Example reactive handles groups include hydroxyl groups, carboxylic groups, amine groups, halide groups, allyl groups, vinyl groups, maleimide groups, maleic groups, epoxide groups, azide groups, alkyne groups, or thiol groups. In some embodiments, the reactive groups include hydroxyl groups, carboxylic groups, or amine groups. These same reactive groups might be present on the surface of the bead, in which case an intermediate layer might not be needed for thermo-responsive polymer attachment. Not all embodiments require a reactive group for application of the thermo-responsive coating. In some embodiments, the intermediate layer comprises a hydrophilic layer to shield the hydrophobicity of the bead from impacting the performance of the thermo-responsive polymer if the bead is inherently hydrophobic. The hydrophilic layer may or may not contain reactive groups. A hydrophilic layer is a layer that is wetted by water, meaning water spreads over the surface. Many optional reactive groups previously listed make a layer hydrophilic, including but not limited to hydroxyl groups, carboxylic acid groups, amine groups, and some epoxide groups. Other hydrophilic layers may comprise -(CH2CH2O)-, — (CH2CHOHCH2O)— , — (CH2CHOH)— , or - (CH2CH(CH2OH)O)— segments. In some embodiments, the intermediate layer comprises epichlorohydrin-glycidol copolymer or polyacrylic acid-glycidyl ester copolymer. In some embodiments, the intermediate layer is formed by adsorbing or precipitating an amphiphilic molecule or polymer onto the surface of the bead, and then either crosslinking the adsorbed or precipitated molecule or polymer to itself or to the surface of the bead. An amphiphilic molecule or polymer is one that exhibits at least one each hydrophobic and hydrophilic domain. In some embodiments, the intermediate layer is formed by exposing the bead toplasma energy. In some embodiments, the intermediate layer is formed by grafting polymers from or to reactive groups of the bead.

[0029] In some embodiments, the thermo-responsive polymer is attached to the bead in loops. When the polymer is attached to the bead in loops, it is attached to the bead at more than one point. The attachment may be either covalent or through secondary interactions. In some embodiments, the thermo-responsive polymer is attached to the bead in brushes. Beads with a loop structure and a brush structure are shown in FIG. 2. When the polymer is attached to the bead in brushes it is attached to the bead at only one end.

[0030] Microcarriers are useful for culturing cells for the production of vaccines. They provide a high surface area for cells to grow on and can be kept in suspension to allow good exposure of cells to nutrients in cell culture media and dissolved oxygen. They allow for a homogeneous environment with regard to pH and nutrient concentration and facilitate large-scale expansion of adherent cells. The challenge is in harvesting the cells from the microcarriers. Proteolytic enzymes have been used but can negatively impact cell viability, cell density, and virus-like particle yield (VLP) or viral protein yield, as well as cell attachment to new microcarriers.

[0031] Thermo-responsive polymers can address the challenge in harvesting cells from microcarriers, especially without the use of proteolytic enzymes. In some embodiments, the thermo-responsive polymers can be used to form highly-defined and phase-reversible synthetic extracellular matrices (ECMs) that comprise handles for chemical modification (e.g., adding peptides and / or other components to facilitate cell adhesion and / or growth). Cells grown on the thermo-responsive polymers can be easily isolated by manipulating temperature to promote phase changes in the thermo-responsive polymers. In some embodiments, the thermo-responsive polymer exists in liquid form below a LCST point and in solid form above the LCST. By controlling the phase of the thermo-responsive polymer using temperature, cells grown on microcarriers with thermo-responsive polymers can be easily isolated without having to use conventional isolation techniques (e.g., proteolytic enzymes).

[0032] The temperature of the thermo-responsive polymer may be reduced below the LCST so that at least some of the cells detach from the coating of the microcarrier. This is depicted in FIG. 1. The temperature may be reduced by cooling the cell culture media and the microcarrier. The temperature may be reduced by exchanging the cell culture media withcell culture media that is at a lower temperature, thereby cooling the thermo-responsive polymer.

[0033] In some embodiments, the cell culture media is free of trypsin during the culturing the cells and reducing the temperature to detach the cells. In some embodiments, the cell culture media is free of proteolytic enzymes during the culturing of the cells and reducing the temperature to detach the cells. In some embodiments, the cell culture media is free of trypsin during the step of harvesting the cells from the microcarriers. In some embodiments, the cell culture media is free of proteolytic enzymes during the step of harvesting the cells from the microcarriers. Once some or all of the cells have been removed from the microcarriers they may be isolated. Cells may be isolated by, e.g., centrifugation or passing through a strainer or harvestainer.

[0034] During the method of vaccine production, the cells are infected with a virus. In some embodiments, the virus comprises any one or more of varicella zoster virus, vesicular stomatitis virus, Rabies virus, Marek's Disease virus, Hepatitis A virus, Polio virus, and or Adenovirus. In some embodiments, the virus comprises any one or more of Measles virus, Mumps virus, Rubella virus, virus, Influenza A virus, Hepatitis B virus, Rotavirus, Yellow Fever virus, Zika Virus, Coronavirus, Adenovirus, Herpes Simplex Virus, or Japanese Encephalitis Virus. In some embodiments, the virus comprises any one or more of Avian Influenza Virus, Canine Parvovirus, Bovine Viral Diarrhea Virus, Feline Immunodeficiency Virus, Feline Leukemia Virus, Porcine Herpesvirus, Foot-And-Mouth Disease Virus, Bovine Herpesvirus- 1, Newcastle Disease Virus, Bluetongue virus, Equine Encephalomyelitis virus, West Nile virus, Infectious Bovine Rhinotracheitis virus, Peste Des Petits Ruminants Virus, Swine Fever virus, Porcine Reproductive and Respiratory Syndrome virus, or Fowl Pox virus.

[0035] In some embodiments, the cells comprise a cell line selected from any one or more of Vero, MRC-5, MDCK, MDBK, BGM, HEK 293, A549, PK-15, MARC 145, ST, and chicken embryo fibroblasts. In some embodiments, the cell line is Vero. In some embodiments, the cell line is selected from any one or more of Vero, MRC-5, MDCK, MDBK, BGM, HEK 293, or A549; such as Vero, MRC-5, MDCK, MDBK, or BGM. In some embodiments, the cell line comprises Vero.

[0036] Method of forming a microcarrier

[0037] In some embodiments, the bead comprises polyvinyl alcohol which comprises hydroxyl groups which can function as reactive groups. In some embodiments, hydroxyl groups are formed by exposing the bead surface to plasma energy. Example beads include those with polystyrene or polystyrene-co-divinylbenzene beads. In some embodiments, the intermediate layer is formed by adsorbing or precipitating an amphiphilic molecule or polymer onto the surface of the bead, and then either crosslinking the adsorbed or precipitated molecule or polymer to itself or to the surface of the bead. For example, glycidyl groups may be opened on a poly(glycidyl methacrylate) polymer to form hydroxyl groups; a poly(glycidyl) polymer or copolymer may be deposited on the surface and crosslinking it to itself; or polyvinyl alcohol or a polysaccharide may be deposited on the surface and crosslinked.

[0038] Cerium (IV) redox polymerization may be used to polymerize NIPAAM monomer directly to OH modified microcarriers. NIP AM monomer solutions were prepared in DI water followed by addition of nonporous or porous microcarriers. To remove oxygen, the suspension was then sparged with nitrogen for Ih. Catalyst / initiator solution was prepared by dissolving ammonium cerium nitrate in a 1 : 1 ratio by weight of a 5 wt% nitric acid solution in deionized water. The monomer microcarrier solution was sufficiently sparged and the catalyst / initiator solution was then added. The polymerization was then allowed to proceed and was then stopped by exposure to air and fully quenched by the addition of sulfuric acid. The coated microcarriers were then washed several times and dried in vacuo.

[0039] Random copolymers comprise up to 10 mol% 3-(Trimethoxysilyl)propyl methacrylate) (TMSPMA) may be synthesized by controlled free radical polymerization. The TMSPMA-co-NIPAAM random copolymers were attached to hydroxyl modified beads using silanization procedures known to those skilled in the art. For example, 12wt% of polymer was dissolved into 2: 1 ethanol: water mixture and hydroxyl modified beads were dispersed into the polymer solution. Once dispersed, catalytic amounts of acetic acid were added to initiate the reaction. Polymer / bead solutions were then placed on a rotator overnight. Purified reactions solutions were dried in vacuo then characterized by ATR-FTIR to see if NIPAAM was present on the particle surfaces

[0040] Other possible covalent crosslinked methods include attaching monomers to the bead surface then polymerizing monomer modified beads in the presence of NIPAAM monomer. Examples include reacting acid chloride monomers to OH bead (i.e. acryloylchloride), or di vinyl sulfone in presence of DMAP and OH beads, then purifying and polymerizing the monomer modified bead in presence of NIP AM. Another method is to prepare a NIPAAM copolymer with amine reactive groups. This can be done by copolymerizing N-3 aminopropyl methacrylamide or equivalent with NIPAAM, isolating and purifying the polymer and then using an amine-OH crosslinker, such as N,N'- Carbonyldiimidazole (CDI), between the amine functional copolymer and the OH modified bead.

[0041] In some embodiments, the polymer may be connected to the surface of the bead by physical adsorption. In some embodiments, the polymer may be precipitated onto the surface of a bead. Examples include 1) Flash precipitation, which is a rapid solvent exchange methodology, 2) Dry Down coating, and 3) filter coating. Flash coating involves dissolving block copolymers (such as one comprising polystyrene) into a water miscible solvent, such as THF. Nonporous or porous polystyrene microcarriers were then be added to the block copolymer THF solution. The polymer and microcarrier solutions were then placed on a vortex mixer and cold, deionized (DI) water was rapidly added to the polymer / microcarrier solution with excess volume. Rapid solvent exchange into a nonsolvent causes the polystyrene block of the copolymers to precipitate out onto the polystyrene microcarrier surface. The microcarriers were then washed with cold DI water, methanol / water, and methanol, and dried in vacuo overnight.

[0042] Dry down coating involves dissolving a block copolymer into a solvent such as THF. Nonporous or porous polystyrene microcarriers were then added to the block copolymer solution. The solution was allowed to soak for at room temperature, then the solvent was removed via rotary evaporation. Cold, deionized water was added and the solution was then mixed vigorously. The block copolymer coated microcarriers were then washed with cold DI water, methanol / water, and methanol, and dried in vacuo overnight.

[0043] The filter coating method involves dissolving a block copolymer into a solvent such as THF. Nonporous or porous polystyrene microcarriers were then added to the block copolymer solution and allowed to sit at room temperature. The solution was then pipetted over a disposable filter flask and the solvent was removed through vacuum filtration. Cold DI water was added to redisperse the coated microcarriers. The DI water was then removed through vacuum filtration. The washing step was repeated. The washed microcarriers werethen transferred to a centrifuge tube and washed with methanol / water, methanol, and dried in vacuo overnight.

[0044] In some embodiments, the intermediate layer is formed by copolymerizing styrene with NIP AM onto the surface of a nonporous polystyrene bead, also known as a shelling reaction. This is accomplished by dispersing nonporous polystyrene beads into 30v% methanol with 50mM NaCl into a reactor with overhead stirring at 50°C. The reaction was then purged with nitrogen for Ih, followed by addition of the monomers styrene and NIP AM in a 2:3 molar ratio. Once the monomers equilibrated with the dispersed beads for 2h, ammonium persulfate, and sodium metabisulfite with catalyst was added to initiate the shelling reaction and allowed to proceed overnight. Shelled beads were washed 30v% ethanol 5 times then 100v% ethanol 4 times and dried in vacuo overnight.

[0045] In some embodiments, polymers may be attached to beads that comprise a primary amine on the surface. Amine functional beads are first modified dispersed in an aqueous buffer with excess DBCO-spacer-NHS crosslinker. Following removal of excess cross-linker the beads are redispersed in the presence of excess azide terminated PNIPAAM. This results in a copper free click chemistry reaction that modifieds the amine functional bead with an azide- PNIPAM. Attached PNIPAM in this way would result in “brush” like confirmation.

[0046] Microcarrier

[0047] In some embodiments, the microcarrier comprises a polymeric bead and a hydrophobic polymer. The hydrophobic polymer is a block copolymer. The block copolymer comprises at least one hydrophobic block and at least one thermo-responsive block. The hydrophobic block has a Hansen solubility parameter that is within about 1.0 MPa0 5, such as within 0.5 MPa0 5of the solubility parameter of the polymeric bead. The thermo-responsive block comprises a thermo-responsive polymer having a lower critical solution temperature (LCST) of between about 20 °C and about 34 °C.

[0048] A block copolymer is a polymer formed from more than one monomer with regions of repeating monomers, or blocks. In some embodiments, a block copolymer may be a diblock copolymer, or a polymer with two different types of blocks. In some embodiments, a block copolymer may be a triblock copolymer, or a polymer with three different types of blocks. The relative amounts of blocks in the copolymer can have an effect on the propertiesof the block copolymer. In some embodiments, the ratio of hydrophobic block to thermo- responsive block is at least 15% hydrophobic block to 85% thermo-responsive block, such as about 15% to about 85%, about 20% to about 80%, about 23% to about 77%, about 25% to about 75%, about 30% to about 70%, about 35% to about 65%, about 40% to about 60%, about 45% to about 55%, about 50% to about 50%, about 55% to about 45%, about 60% to about 40%, about 65% to about 35%, about 70% to about 30%, about 75% to about 25%, about 80% to about 20%, about 85% to about 15%, about 90% to about 10%, or about 95% to about 5%.

[0049] In some embodiments, the hydrophobic polymer is a diblock copolymer with a hydrophobic block and a thermo-responsive block. In some embodiments, the hydrophobic block comprises polystyrene and the thermo-responsive block comprises polyN- isopropyl acrylamide (PNIPAAm). In some embodiments, the molecular weight of the polystyrene is from about 5kDa to about 25kDa, such as about 7.5kDa. The molecular weight of the PNIPAAm is about 20kDa to about 200kDa, such as about 20kDa to about lOOkDa, about 20kDa to about 50 kDa, about 30kDa to about 50 kDa, or about 46kDa. The number average molecular weight for block copolymers was determined by NMR. In some embodiments, where polystyrene was the first block, 1H NMR is used for end-group analysis to determine the number average molecular weight. In other embodiments, where PNIPAM was first synthesized, or for random copolymers of PNIPAM standard gel permeation chromatography (GPC) / size exclusion chromatography (SEC) is used.

[0050] In example embodiments, molecular weights of linear poly(N- isopropylacrylamide-co-butyl acrylate), linear poly(N-isopropylacrylamide) were measured by standard gel permeation chromatography (GPC) methods. Copolymer compositions were determined from 1HNMR using a 400 MHz Bruker Instrument.

[0051] The first solubility parameter is a Hanson solubility parameter of the polymer bead. The second solubility parameter is a Hanson solubility parameter of the hydrophobic block of the hydrophobic polymer. In some embodiments, the first and second solubility parameters are within about 1.0 MPa0 5, such as within about 0.5 MPa0 5. In some embodiments, hydrophobic block comprises polystyrene having a Hansen solubility parameter ranging from about 15 MPa0 5to about 22 MPa0 5, such as about 15.5 MPa0 5to about 22 MPa0 5, or about 15.5 MPa0 5to about 21.5 MPa0 5, or about 15.6 MPa0 5to about 21.1 MPa0 5. In some embodiments, the hydrophobic block comprises polyvinyl alcoholhaving a Hansen solubility parameter of about 25 MPa0 5to about 27 MPa0 5, such as about 25.5 MPa0 5to about 26.5 MPa0 5, about 25.7 MPa0 5to about 26.3 MPa0 5, or about 25.78 MPa0 5to about 26.3 MPa0 5.

[0052] In some embodiments, the microcarrier comprises a binding agent connected to the surface of the bead. In some embodiments, the binding agent is covalently bound to the surface of the bead. The binding agent is configured to bind to a cell. In some embodiments, the binding agent comprises GFOGER peptide, RGD peptide, Vitronectin peptide, Vitronectin-derived peptide HBP, Laminin Peptide, Laminin SAP / PA molecules, GAG-binding peptides, N-cadherin peptide, Gelling peptide for tumor spheroids, E-cadherin peptides, Soluble FN binder, Laminin Heparin Binding, or Fn-BM.

[0053] Binding agent - Peptide sequences:

[0054] In the present disclosure the singular forms "a", "an" and "the" include the plural reference, and reference to a particular numerical value includes at least that particular value, unless the context clearly indicates otherwise. Thus, for example, a reference to "a material" is a reference to at least one of such materials and equivalents thereof known to those skilled in the art, and so forth.

[0055] The modifier "about" should be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the expression "from about 2 to about 4" also discloses the range "from 2 to 4." When used to modify a single number, the term "about" may refer to plus or minus 10% of the indicated number and includes the indicated number. For example, "about 10%" may indicate a range of 9% to 11%, and "about 1 " means from 0.9 to 1.1.

[0056] When a list is presented, unless stated otherwise, it is to be understood that each individual element of that list and every combination of that list is to be interpreted as a separate embodiment. For example, a list of embodiments presented as "A, B, or C" is to be interpreted as including the embodiments, "A," "B," "C," "A or B," "A or C," "B or C," or "A, B, or C."

[0057] Where present, all ranges are inclusive and combinable. That is, references to values stated in ranges include every value within that range. For example, a range defined as from 400 to 450 ppm includes 400 ppm and 450 ppm as independent embodiments. Ranges of 400 to 450 ppm and 450 to 500 ppm may be combined to be a range of 400 to 500 ppm.

[0058] It is to be appreciated that certain features of the invention which are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. That is, unless obviously incompatible or excluded, each individual embodiment is deemed to be combinable with any other embodiment s) and such a combination is considered to be another embodiment. Conversely, various features of the invention that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any sub-combination. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation. Finally, while an embodiment may be described as part of a series of steps or part of a more general structure, each said step may also be considered an independent embodiment in itself.

[0059] While the present disclosure has illustrated by description several embodiments and while the illustrative embodiments have been described in considerable detail, it is not the intention of the applicant to restrict or in any way limit the scope of the appended claims to such detail. Additional advantages and modifications may readily appear to those skilled in the art. Furthermore, features from separate lists can be combined; and features from the examples can be generalized to the whole disclosure.EXAMPLESExample 1 - workflow

[0060] Vero cells (ATCC CCL-81) were cultured in MEM a with 5% FBS (Gibco™), supplemented with 6 mM L-glutamine, at 37° C and 5% CO2. Cultures were passaged with Trypsin-EDTA (0.05%) and Defined Trypsin Inhibitor (Gibco). Cells were grown in T-flasks or Corning® 125 mL Disposable Spinner Flasks. The control spinner flasks were seeded with Cytodex™ 1 microcarriers (Cytiva) or thermo-responsive microcarriers at 3 g / L and Vero cells at 2.5X105cells / mL and stirred on a magnetic stirrer platform at 30 rpm. Spinner flasks were stirred intermittently after seeding. Intermittent stirring refers to a cycle of stirring for 5 minutes and pausing for 30 minutes, which was repeated 4 times.

[0061] After day 2, 50% of the growth media were exchanged every day and 5 mL samples were taken before each medium exchange. Viable cell densities (VCD) were determined with a NucleoCounter® NC-200™ and NC-250™ (ChemoMetec) and microscopy images were taken with an EVOS® FL Auto Cell Imaging System (Thermo Fisher Scientific). On day 5, cells were harvested or scaled up.

[0062] After 5 days, the cultures were infected with vesicular stomatitis virus (ATCC VR-1415) at a multiplicity of infection (MOI) of 0.01. Supernatant samples were taken every 24 hours. Virus production was determined with a TCIDso assay and titers were calculated following the method by Reed and Muench (a statistical technique used to estimate the median infectious dose (ID50) or the median lethal dose (LD50) of a pathogen or toxin. This method is commonly applied in virology and toxicology to determine the concentration of a substance that causes infection or death in 50% of a test population). Note, for certain budding or lytic viruses, the supernatant can be harvested a number of times to recover the propagated particles, e.g., Rabies Virus, Influenza A virus. For strongly cell-associated viruses, e.g., Varicella Zoster Virus, Hepatitis A the cells need to be detached and the virus recovered by disrupting the cell membrane. This is also the main method of harvesting faster acting lytic viruses, both supernatant and infected cell layers are recovered and disrupted usually by freeze thawing, e.g., Poliovirus, Rotavirus.

[0063] Thermo-responsive microcarrier workflows have several advantages over regular microcarrier workflows, including (a) saving time and labor associated with enzymatic detachment; (b) improving cell viability by removing enzymatic detachment; (c) increase cell attachment in subsequent passages because there is no carryover of trypsin or TrypLE to subsequent cell cultures; and (d) saving cost associated with enzymatic detachment.Example 2 - Quantification of Cell Detachment

[0064] Cell harvest protocols to quantify cell adhesion and detachment of Vero cells in the microcarrier prototypes:

[0065] Cell attachment was quantified by using TrypLE enzyme to ensure complete detachment of the cells from the surface, and afterwards cells were quantified using a hemocytometer. The full protocol will include:

[0066] Protocol to follow for cell harvest:

[0067] Move cells and microcarrier from their original well to an Eppendorf tube.• Rinse well with 200pLDPBS, move to Eppendorf tube.• Short spin• Aspirate supernatant carefully• Add 150 pL ofDPBS• Short spin• Aspirate supernatant carefully• 100 pL TrypLE express or TrypLE Select added• Vortex• Incubate at RT 30 min• Vortex• Added 100 pL of cell media• Vortex• Count cells using: hemocytometer & Nucleo Counter (cells and microcarriers will be taken together because centrifugation risks removing cells from the supernatant as well).

[0068] Cell detachment was quantified by temperature harvest, following the protocol:

[0069] Transfer the content of every well to a new well plate, to avoid adherent cells to the bottom to detach and interfere with the cells detaching from the beads.

[0070] Change half of the media in the wells (250pL) to new fresh media at 24°C and leave plates outside the incubator, on top of a cooling block between 10-15 degrees, placed at room temperature for 30 minutes.

[0071] Take microscopy images of the cells right after media change and 30 min later to follow up on cell detachment. Pipette x8 times, and take new pictures.

[0072] Transfer to a centrifuge tube, centrifuge to resuspend the microcarrier and take the supernatant to a new centrifuge tube. Centrifuge and resuspend the cells in 200uL media. Hemocytometer was used to assess cell detachment.

[0073] Quantification of Vero cell attachment and detachment using microcarrier prototypes (non-porous and 150um size) after 30 minutes temperature harvest is shown in FIG. 4. The quantification of the adherent cells was performed by enzymatic harvest using TrypLE.Example 3 - Comparison to enzymatic detachment

[0074] Vero cells grown in different microcarrier protypes were harvested following protocol in Example 2. After TrypLE addition, the enzyme was incubated for 4 and 30 minutes. Higher detachment results are observed after incubations of 30 minutes, which can be correlated as well with the structural changes of the NIPAAm assisting cell detachment.

[0075] Comparison of Vero cells detachment in different microcarrier prototypes when harvesting using TrypLE enzyme at different incubation times: 4 VS 30 minutes is shown in FIG. 5.Example 4 - Cell adhesion to different beads

[0076] Cell attachment of Vero cells in all the microcarrier prototypes was quantified following the same method as in Example 2. A summary of the Vero cell adhesion results for all the microcarriers prototypes is shown in FIG. 6. It can be observed that larger beads have higher adhesion and also which are the best prototypes providing the highest cell adhesionExample 5 - Cell growth comparison to use with proteolytic enzyme

[0077] Vero cells (ATCC CCL-81) were cultured in MEM Alpha with 10% FBS or VP-SFM (Gibco™), supplemented with 6 mM L-glutamine, in a humidified incubator at 37 C with 5% CO2. Cultures were passaged with Trypsin-EDTA (0.05%) or TrypLE™ (Gibco). Trypsin activity was quenched with Defined Trypsin Inhibitor (Gibco) and cells were washed with medium before determination of viable cell densities with a NucleoCounter® NC-200™ (ChemoMetec).

[0078] Cells were seeded on 3g / L Cytodex 1 microcarriers with a seeding density of 2.5x105 cells / mL in 500mL Spinner Flask. Cells were seeded with continuous or intermittent stirring at 30 rpm. Intermittent stirring includes cycles of 5 minutes of stirring followed by 30 minutes of pausing. 4 cycles were repeated before continuing the culture with continuous stirring. 50% medium was exchanged daily on days 2-4 and days 7-9. On day 5, lOOmL of culture with microcarrier was scaled up to 500mL in two ways. In the first method, cells were washed with DPBS and trypsinized by incubating the microcarriers with trypsin for 30 minutes. Once cells were detached, cells were seeded in 500mL growth medium with new microcarriers. In the second method, cells attached to microcarriers were resuspended in 500mL growth medium with new microcarriers without trypsin to enable direct bead-to-beadtransfer. Both methods included scale up with continuous or intermittent stirring. Cell counts were determined daily using a NucleoCounter® NC-200™ (ChemoMetec).

[0079] For infection on day 10, the growth medium was removed, the microcarriers were washed with DPBS (Gibco), and the medium replaced with Advanced MEM or VP- SFM (Gibco). All media were supplemented with 6 mM L-glutamine. Cultures were infected with vesicular stomatitis virus (ATCC VR-1415) at an MOI of 0.01. Cultures were observed daily for signs of cytopathic effect (CPE). The culture supernatant was used for virus quantification with a TCID50 assay. A summary of the scale-up for the Vero cells on microcarriers is shown in FIG. 7. It can be observed that in the absence of trypsin and intermittent stirring, the variable cell density (VCD) was highest with direct bead-to-bead transfer.Example 6 Cerium (IV) redox polymerization

[0080] Solutions of 1 to 2M NIPAAM monomer were prepared in DI water, followed by the addition of nonporous or porous microcarriers. To remove oxygen, the flask was sealed with a rubber septum and the suspension was then sparged with nitrogen for Ih. Catalyst / initiator solution was prepared by dissolving ammonium cerium nitrate in a 1 : 1 ratio by weight of a 5 wt% nitric acid solution in deionized water. Once the monomer microcarrier solution was sufficiently sparged, it was placed in an ice bath on an orbital shaker. The catalyst / initiator solution was then added via syringe. The polymerization was then allowed to proceed for 3h on an orbital shaker. The polymerization was then stopped by exposure to air and fully quenched by the addition of IM Sulfuric acid. The coated microcarriers were then washed 9 times with IM sulfuric acid followed by washes with DI water until pH was no longer acidic. The microcarriers were then washed with methanol and dried in vacuo overnight at 40°C.Example 7 - Polymerization of azide functional linear Poly(N-isopropylacrylamide-co-butyl acrylate)

[0081] a -terminated azide random linear copolymers with NIP AM and BA were prepared using controlled free radical polymerization methods. Total targeted molecular weights ranged from 10,000 to 25,000g / mol (i.e. degree of polymerization = 150 to 300). BA molo / o was varied in the monomer feed ratio from 0 to 20molo / o in the reactions. For example, 1.04g (9.2 mmol) of NIP AM, 0.103g (0.8 mmol) of BA and 29.9mg (67 pmol) of 2- (dodecylthiocarbonothioylthio)-2- methylpropionic acid 3 -azido- 1 -propanol ester (Azido-CTA; Sigma-Aldrich, St. Louis, MO), were weighed and added to a 25mL round bottomed flask equipped with a stir bar. All monomers and Azido CTA were dissolved in 10 mL of 1,4-dioxane. Once dissolved, 1.37 mg (8.3 pmol) of AIBN was added and the flask was septa sealed and sparged with nitrogen for 45min and subsequently placed in an oil bath at 70 °C for 5h. The polymerization was terminated by exposing the reaction to air and placing in a - 20°C freezer. All copolymers were isolated through removing dioxane by rotary evaporation, redissolving in 2.5 mL THF and precipitating into 40mL cold diethyl ether. The precipitate was then redissolved in 2.5 mL THF and precipitated again into cold diethyl ether and collected by centrifugation. The precipitate was then washed twice with 50mL of cold diethyl ether and dried in vacuo overnight at room temperature. Copolymer molecular weight and compositions were characterized by GPC and 'H NMR, respectively.Example 8 - u-end group removal from azide functional linear PolyfN-isopropylacrylamide- co-butyl acrylate)

[0082] p-end groups (thiocarb onylthio) on synthesized a -terminated azide Poly(NIPAM-co-B A) copolymers 4 were removed using common procedures familiar to those skilled in the art. For example, 400mg (Mn= 14,000 g / mol, 29pmols of thiocarbonylthio) of azide terminated Poly(NIPAM-co-BA), 94mg (570 pmols) of AIBN, and 18.5mg (57 pmols) of BPO were weighed into a round bottomed flask equipped with a stir bar and dissolved into 6mL of dioxane. Once dissolved, the flask was septa sealed and sparged with nitrogen for 45min and subsequently placed in an oil bath at 70 °C overnight. The polymerization was terminated by exposing the reaction to air and placing in a -20°C freezer. The copolymer with removed end-group was isolated by removing dioxane by rotary evaporation, redissolving in 5 mL THF and precipitating into 50 mL cold diethyl ether. Following precipitation, the precipitate was collected by filtration, washed twice with 50mL of cold diethyl ether and dried in vacuo overnight at room temperature. Greater than 90o / o removal of the thiocarb onylthio end-group was confirmed by monitoring the UV absorbance of the thiocarb onylthio group at 310nm during GPC analysis.Example 9 - Polymerization of azide functional linear Poly(N-isopropylacrylamide) andw- end group removal

[0083] Linear homopolymers of only NIP AM were polymerized and the w-end groups were removed identically to the procedures outlined in Example 10 and 11 without the presence of butyl acrylate. Molecular weights varying from 30kDa up to 100 kDa.Example 10 - Conjugation of DBCO modi fied microcarrier to azide PolyfN- isopropylacrylamide)

[0084] DBCO modified microcarrier was conjugated to azide polyfV- isopropyl acrylamide) using copper free click chemistry. For example, 48 mg of azide poly(NIPAM) (Mn = 62.4kDa; 0.8 mol) and microcarrier were weighed into a 2mL microcentrifuge tube. ImL of lx phosphate buffered saline (PBS) was added to dissolve both materials. This vial was then placed at 25 °C for 20h. Reaction of the DBCO group with azide was monitored using UV-vis spectroscopy and recording the absorbance of the DBCO group at 309nm.Example 11 -VSV production with Vero cells on microcarriers

[0085] For infection on day 10, the growth medium was removed, the microcarriers were washed with DPBS (Gibco), and the medium replaced with Advanced MEM or VP- SFM (Gibco). All media were supplemented with 6 mM L-glutamine. Cultures were infected with vesicular stomatitis virus (ATCC VR-1415) at an MOI of 0.01. Cultures were observed daily for signs of cytopathic effect (CPE). The culture supernatant was used for virus quantification with a TCID50 assay. As shown in FIG. 8, the best VSV titer was observed when Trypsin was absent, leading to direct bead-to-bead transfer in VP-SFMExample 12 -Scale up of vero cells on microcarriers

[0086] A collection of microscopy images taken with an EVOS® FL Auto Cell Imaging System during scale-up of vero cells on microcarriers, is as shown in FIG. 9. The parameters for the scale-up, including vero CCL-81 cells, VP-SFM, MEM a + 10% FBS, 6mM L-Glutamine as supplements, 3g / L Cytodex 1 as microcarrier, at a seeding density of 2.5x105 cells / mL. other parameters including, 500mL Spinner Flask, stirring at 30 rpm (continuous or intermittent after seeding), 50% medium exchange days 2-4 and days 7-9, and a scale-up of 1 :5 on day 5

[0087] Referring back to FIG. 3, in some embodiments, cell culture processes used in vaccine manufacturing can include the following steps: A first step can include thawing the cells for scaling up for vaccine production. A second step can include culturing cells in T- flasks, followed by scaling up into roller bottles. After a desired cell density has been reached, the growth medium can be replaced with infection medium. Cells are infected with a virus. After expansion, the virus is harvested. As a first alternative, the second step can include culturing cells in cell factories. Cells can e.g., be scaled up from 2-layer cell factoriesto 10-layer cell factories. After a desired cell density has been reached, the growth medium can be replaced with infection medium. Cells are infected with a virus. After expansion, the virus is harvested. As an alternative, the second step can involve culturing cells on microcarriers. In an example, the microcarrier can be thermo-responsive as described in this disclosure. Cells can, e.g., be scaled up from a spinner flask to large-scale bioreactors, e.g., 2L, 10L, 50L, 200L, 500L, lOOOL, etc. After a desired cell density has been reached, the growth medium can be replaced with infection medium. Cells are infected with a virus. After expansion, the virus is harvested. A third step can include maintaining a working stock of cells using standard cell culture techniques.

[0088] Further, a fourth step can include detaching cells from their growth vessel on the day of seeding, using standard techniques for that particular cell line and the manufacturer’s instructions for the dissociation reagent. For example, in the case of adherent Vero cells (ATCC® CCL-81) and TrypLE cultured in a T-75 flask. A fifth step can include aspirating the cell culture medium and then gently washing the cell monolayer with 5 mL of DPBS. A sixth step can include removing the DPBS and adding 3mL of TrypLE. A seventh step can include incubating for 5 minutes at 37 °C or until the cells have visibly detached. Additionally, the seventh step can include gently tapping the vessel to dislodge the cells.

[0089] Further, an eighth step can include adding an appropriate volume of cell culture medium to resuspend the cells, pipetting up and down to break up any clumps that are present. A ninth step can include performing a count to determine the dilution required, once the cells are resuspended. A tenth step can include seeding cells at 10,000-20,000 cells / cm2 in a T-flask or roller bottle. An eleventh step can include incubating for 3-5 days, until cells have reached -90% confluency, and infecting cells using a standard infection procedure for that virus. Various methods can be used for infection. In some cases, a medium exchange is required, in which case the virus can be mixed into the replacement media. At other times, the volume of virus required is sufficiently small that it can be added directly to the culture. A third variation includes the reduction of culture medium volume, addition of the viral inoculum for a set period of time (e.g., 1 hour), removal of the virus, and addition of fresh medium. Using a virus stock with a known titer, infect cells with a desired Multiplicity of Infection (MOI) value. Sacrificing one or two vessels to get an accurate cell count before infection is recommended to improve the accuracy of the MOI.

[0090] Furthermore, a twelfth step can include observing the cells daily following infection. Once there are visible cytopathic effects, harvest the cells and analyze using the preferred virus quantification method. A thirteenth step can include detaching cells using TrypLE or Accutase per the manufacturer’s protocol, on the day of harvest. In some cases, it is beneficial to save the cell supernatant rather than discarding it, as it may contain floating infected cells. The supernatant can be combined with the dissociated cells to capture the infected cell population. If the virus is cell-associated, the supernatant is discarded, and the cells are lysed. Cells can e.g., be lysed with sonication or freeze-thawing. A final step can include freezing the virus for further processing or quantification. Any of the above steps can have one or more additional steps, one or more steps can be removed, altered, reversed, and can be performed in a different sequence.- 1 -

Claims

CLAIMSWhat is claimed is:

1. A method of culturing cells for vaccine production, comprising: culturing cells in the presence of microcarriers with cell culture media; infecting the cells with a virus, wherein the microcarrier comprises a bead and a coating comprising a thermo- responsive polymer having a lower critical solution temperature (LCST) of between about 20 °C and about 34 °C, and wherein the cells adhere to the coating of the microcarrier at a temperature above the LCST.

2. The method of claim 1, further comprising reducing the temperature of the cells and microcarrier to less than the LCST so that at least some of the cells detach from the coating of the microcarrier.

3. The method of claim 2, wherein the cell culture media is free of trypsin during the culturing of the cells and reducing the temperature to detach the cells.

4. The method of claim 2, wherein the cell culture media is free of proteolytic enzymes during the culturing the cells and reducing the temperature to detach the cells.

5. The method of any one of claims 2-4, further comprising isolating the cells released from the coating of the microcarrier.

6. The method of any one of claims 1-5, wherein the virus comprises any one or more of varicella zoster virus (VZV), vesicular stomatitis virus (VSV), Rabies virus, Marek's Disease virus, Hepatitis A virus, Polio virus, and Adenovirus.

7. The method of claim 1, wherein the cells comprise a cell line selected from any one or more of Vero, MRC-5, MDCK, MDBK, BGM, HEK 293, A549, PK-15, MARC 145, ST, and chicken embryo fibroblasts.

8. The method of any one or more of claims 1-7, wherein the bead of the microcarrier is a polymeric bead.

9. The method of claim 8, wherein the polymeric bead is porous.

10. The method of claim 8, wherein the polymeric bead is non-porous.

11. The method of any one of claims 8-10, wherein the bead has a diameter from about 10 pm to about 500 pm.

12. The method of any one of claims 1-11, wherein the bead comprises any one or more of polystyrene-co-divinylbenzene, polystyrene, polydivinylbenzene, polyvinyl alcohol, polyacrylamide, polymethacrylate, polyacrylate, polyethylene glycol, polydextran, agarose, alginate, cellulose, dextran, collagen, and combinations thereof, with or without crosslinking.

13. The method of any one of claims 1-12, wherein the thermo-responsive polymer comprises poly(N-isopropyl acrylamide), poly(N-isopropyl methacrylamide), poly(N- n-propyl acrylamide), poly(N,N-diethyl acrylamide), poly(diethylene glycol methyl ether methacrylate), poly(N-vinylcaprolactam), or combinations thereof.

14. The method of any one of claims 1-13, wherein the thermo-responsive polymer comprises poly(N-isopropyl acrylamide).

15. The method of any one of claims 1-14, wherein the thermo-responsive polymer is a co-polymer.

16. The method of claim 15, wherein the co-polymer comprises poly(N-isopropyl acrylamide) and polystyrene.

17. The method of claim 15, wherein the thermo-responsive polymer comprises a diblock co-polymer or a triblock co-polymer.

18. The method of any one of claims 12-17, wherein there is an intermediate layer between the bead and the coating.

19. The method of claim 18, wherein the intermediate layer comprises hydroxyl groups, carboxylic groups, or amine groups.

20. The method of claim 13, wherein the thermo-responsive polymer is attached to the bead in loops.

21. The method of claim 13, wherein the thermo-responsive polymer is attached to the bead in brushes.

22. A method of forming a microcarrier, comprising: forming a connection between a thermo-responsive polymer and a reactive group on the surface of a polymeric bead, wherein the thermo-responsive polymer has a lower critical solution temperature (LCST) of between about 20 °C and about 34 °C.

23. The method of claim 22, wherein the connection is a chemical bond.

24. The method of claim 22, wherein the chemical bond is formed by the polymerization of monomers to form the thermo-responsive polymer.

25. The method of claim 22, wherein the reactive group comprises hydroxyl groups, carboxylic groups, or amine groups.

26. The method of claim 22, wherein the chemical bond is formed by grafting of the thermo-responsive polymer to the reactive groups.

27. The method of claim 22, wherein the bead comprises any one or more of polystyrene- co-divinylbenzene, polystyrene, polydivinylbenzene, polyvinyl alcohol, polyacrylamide, polymethacrylate, polyacrylate, polyethylene glycol, polydextran, agarose, alginate, cellulose, dextran, and collagen, with or without crosslinking.

28. The method of claim 22, wherein the thermo-responsive polymer comprises any one or more of poly(N-isopropyl acrylamide), poly(N-isopropyl methacrylamide), poly(N- n-propyl acrylamide), poly(N,N-diethyl acrylamide), poly(diethylene glycol methyl ether methacrylate), and poly(N-vinylcaprolactam).

29. The method of claim 28, wherein the thermo-responsive polymer comprises poly(N- isopropyl acrylamide).

30. The method of any one of claims 22-29, wherein the thermo-responsive polymer comprises a co-polymer.

31. The method of any one of claims 22-29, wherein the polymeric bead is non-porous.

32. The method of claim 22, wherein the connection comprises an intermediate layer.

33. The method of claim 32, wherein the intermediate layer comprises TMSPMA.

34. The method of claim 33, wherein a chemical bond is formed between TMSPMA and a reactive group on the surface of the polymeric bead.

35. The method of claim 34, wherein the chemical bond is formed by the polymerization of monomers to form the thermo-responsive polymer.

36. The method of claim 34, wherein the chemical bond is formed by grafting of the thermo-responsive polymer to the TMSPMA.

37. The method of claim 32, wherein the bead comprises polystyrene-co-divinylbenzene, polystyrene, polydivinylbenzene, polyvinyl alcohol, polyacrylamide, polymethacrylate, polyacrylate, polyethylene glycol, polydextran, agarose, alginate, cellulose, dextran, collagen, or combinations thereof with or without crosslinking.

38. The method of claim 37, wherein the thermo-responsive polymer comprises poly(N- isopropyl acrylamide).

39. The method of claim 22, wherein the connection is by physical adsorption.

40. The method of any one of claims 32-39, wherein the polymeric bead is non-porous.

41. A microcarrier, comprising: a polymeric bead, and a hydrophobic polymer, wherein the hydrophobic polymer is a block copolymer; wherein the block copolymer is connected to the bead, wherein the block copolymer comprises at least one hydrophobic block and at least one thermo-responsive block, wherein the hydrophobic block comprises a polymer having a Hansen solubility parameter that is within about 1.0 MPa0 5of the Hansen solubility parameter of the polymeric bead; and the thermo-responsive block comprises a thermo- responsive polymer having a lower critical solution temperature (LCST) of between about 20 °C and about 34 °C.

42. The microcarrier of claim 41, wherein the block copolymer is connected to the bead by a covalent bond.

43. The microcarrier of claim 41, wherein the block copolymer is connected to the bead by physical adsorption.

44. The microcarrier of any one of claims 41 to 43, wherein a molar ratio of hydrophobic block to thermo-responsive block is at least 15% hydrophobic block to 85% thermo- responsive block.

45. The microcarrier of any one of claims 41 to 44, wherein the hydrophobic block comprises polystyrene and the thermo-responsive block comprises polyN- isopropyl acrylamide (PNIPAAm).

46. The microcarrier of claim 45, wherein a molecular weight of polystyrene is about 7.5kDa, and a molecular weight of PNIPAAm is about 20kDa to about 200 kDa.

47. The microcarrier of any one of claims 41-46, wherein a molar ratio of hydrophobic block to thermo-responsive block is at least 23% hydrophobic block to 77% thermo- responsive block.

48. The microcarrier of any one of claims 41-47, wherein the hydrophobic block comprises polystyrene and the thermo-responsive block comprises polyN- isopropyl acrylamide (PNIPAAm).

49. The microcarrier of any one of claims 41-48, wherein the hydrophobic block comprises polystyrene having a Hansen solubility parameter ranging from about 15 MPa0 5to about 22 MPa0 5.

50. The microcarrier of any one of claims 41-49, wherein the hydrophobic block comprises polyvinyl alcohol having a Hansen solubility parameter of about 25 MPa0 5to about 27 MPa0 5.

51. The microcarrier of any one of claims 41-50, further comprising a binding agent connected to the surface of the bead configured to bind to a cell.

2. The microcarrier of claim 49, wherein the binding agent comprises GFOGER peptide, RGD peptide, Vitronectin peptide, Vitronectin-derived peptide HBP, Laminin Peptide, Laminin SAP / PA molecules, GAG-binding peptides, N-cadherin peptide, Gelling peptide for tumor spheroids, E-cadherin peptides, Soluble FN binder, Laminin Heparin Binding, or Fn-BM.

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