Thermoresponsive cell culture supports

Abstract

The present invention relates to a cell culture support comprising a substrate and a polymeric blend layer bound to the substrate. The polymeric blend layer comprises at least one thermoresponsive polymer and at least one coupling agent. The coupling agent is a non-protein coupling agent that has functional thiol, ester, epoxy, or aldehyde groups. The cell culture support further includes cells supported by the polymeric blend layer, wherein the thermoresponsive polymer provides for temperature induced detachment of the cells and/or cell sheets.

What is claimed is: 
     
         1 . A cell culture support comprising:
 a substrate;   a polymeric blend layer bound to said substrate, wherein the polymeric blend layer comprises at least one thermoresponsive polymer and at least one coupling agent, and wherein the coupling agent is a non-protein coupling agent and has functional thiol, ester, epoxy, or aldehyde groups; and   cells supported by said polymeric blend layer, wherein said thermoresponsive polymer provides for temperature induced detachment of said cells.   
     
     
         2 . The support of  claim 1 , wherein the substrate is selected from the group consisting of polymeric materials, glasses, ceramics, metals, metal oxides, hydrated metal oxides, and combinations thereof. 
     
     
         3 . The support of  claim 1 , wherein the at least one thermoresponsive polymer is selected from the group consisting of poly (N-isopropylacrylamide) (PNIPAAm), poly(N,N-diethylacrylamide) (PDEAAm), poly(N-vinlycaprolactam) (PVCL), poly[2-(dimethylamino) ethyl methacrylate] (PDMAEMA), and poly(ethylene oxide) (PEO), 2-oxazoline (Pox), such as poly(2-ethyl-2-oxazoline), poly(2-cyclopropyl-2-oxazoline), poly(2-n-propyl-2-oxazoline), poly(2-isopropyl-2-oxazoline); or the group consisting of poly(2-oxazine) (POZIs), such as poly(2-ethyl-oxazine), poly(2-n-propyl-oxazine), and combinations thereof. 
     
     
         4 . The support of  claim 1 , wherein the at least one thermoresponsive polymer is poly (N-isopropylacrylamide) represented by the formula: 
       
         
           
           
               
               
           
         
       
     
     
         5 . The support of  claim 1 , wherein the at least one network forming adhesion promoter is an aminosilane. 
     
     
         6 . The support of  claim 1 , wherein the at least one coupling agent is selected from the group consisting of 3-Acetoxypropyltrimethoxysilane (AOPTMS), 3-Mercaptopropyltrimethoxysilane (MPTMS), Triethoxysilylbutyraldehyde (TESBA), 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane (ECHETMS), 3-(2,3-epoxypropoxy)propyltrimethoxysilane (EPPTMS), and combinations thereof. 
     
     
         7 . The support of  claim 1 , wherein the at least one coupling agent is Triethoxysilylbutyraldehyde (TESBA), represented by the formula: 
       
         
           
           
               
               
           
         
       
     
     
         8 . The support of  claim 1 , wherein the at least one coupling agent is 3-Mercaptopropyltrimethoxysilane (MPTMS), represented by the formula: 
       
         
           
           
               
               
           
         
       
     
     
         9 . The support of  claim 1 , wherein the polymer blend is characterized as having a thermoresponsive polymer to coupling agent ratio (TRP:CA), by mass, of from about 99:1 to about 10:90, and wherein the polymer blend layer is cured to produce a film. 
     
     
         10 . The support of  claim 1 , wherein the polymeric blend layer is devoid of adhesive proteins, plasma treatment, and e-beam treatment. 
     
     
         11 . The support of  claim 1 , wherein the support supports said cells such that said temperature induced detachment is achieved without mechanical agitation or enzymatic aided detachment methods. 
     
     
         12 . A method of making a cell culture complex comprising:
 providing a substrate;   blending at least one thermoresponsive polymer and at least one coupling agent having functional thiol, ester, epoxy, or aldehyde groups to provide a polymeric blend;   applying a thin film of said polymeric blend to the substrate to provide a polymeric blend layer on the substrate;   curing the polymeric blend layer on the substrate to provide a cell culture support; and   depositing cells onto said cell culture support, wherein the cells may optionally further comprise medium, to provide a cell culture complex.   
     
     
         13 . The method of  claim 12 , wherein the cultured cell layer comprises cells further characterized as anchor dependent cells. 
     
     
         14 . The method of  claim 12 , wherein the cultured cell layer comprises cells further characterized as adhesive cells. 
     
     
         15 . The method of  claim 12 , wherein the cultured cell layer comprises cells selected from the group consisting of fibroblasts, myoblasts, myotube cells, corneal cells, vascular endothelial cells, smooth muscle cells, cardiomyocytes, dermal cells, epidermal cells, mucosal epithelial cells, mesenchymal stem cells, ES cells, iPS cells, osteoblasts, osteocytes, chondrocytes, fat cells, neurons, hair root cells, dental pulp stem cells, β-cells, hepatocytes, and combinations thereof. 
     
     
         16 . The method of  claim 12 , wherein the cultured cell layer comprises cells and medium. 
     
     
         17 . The method of  claim 12 , wherein the thin film of said polymeric blend is spin-coated onto the substrate. 
     
     
         18 . The method of  claim 12 , wherein the polymeric blend layer is devoid of adhesive proteins, plasma treatment, and e-beam treatment; and wherein the detachment of the cultured cell layer is devoid of proteolytic enzymes or mechanical agitations. 
     
     
         19 . The method of  claim 12 , wherein the step of blending further includes creating a first solution of the at least one coupling agent in ethanol, creating a second solution of the at least one thermoresponsive polymer in ethanol, and combining the first solution with the second solution to provide the polymeric blend. 
     
     
         20 . The method of  claim 12 , wherein the step of blending further includes creating a mixture of the at least one coupling agent and the at least one thermoresponsive polymer and dissolving the mixture in ethanol to provide the polymeric blend.