Robotic method for coating a multiwell plate by a polyelectrolyte multilayer film

Abstract

The invention concerns a robotic method for coating the bottom surface of at least one well of a multiwell plate by a polyelectrolyte multilayer film, the multiwell plate obtainable according to the method and the use thereof for cell culture.

1 . A multiwell plate obtainable by a method comprising n successive sequences, n being an integer from 1 to 2000, wherein each sequence comprises the steps of:
 a) robotic deposit of a volume V PE   1  of a solution of a first polyelectrolyte PE 1  on the bottom surface of at least one well of a multiwell plate, wherein the first polyelectrolyte PE 1  is either a cationic polymer comprising amino groups, or an anionic polymer, then   b) robotic aspiration of an aspirated volume V aspPE   1  of said solution of PE 1 , wherein the aspirated volume V aspPE   1  is higher than or equal to V PE   1 , then   c) robotic deposit of a volume V PE   2  of a solution of a second polyelectrolyte PE 2  on said bottom surface, wherein the second polyelectrolyte PE 2  is a cationic polymer comprising amino groups when PE 1  is an anionic polymer, or PE 2  is an anionic polymer when PE 1  is a cationic polymer comprising amino groups, then   d) robotic aspiration of an aspirated volume V aspPE   2  of said solution of PE 2 , wherein the aspirated volume V aspPE   2  is higher than or equal to V PE   2 .   
     
     
         2 . The multiwell plate according to  claim 1 , wherein, for each sequence of the method, at steps a), b), c), d), the bottom surface is tilted with an inclination angle α from 5 to 40° relative to the horizontal plane. 
     
     
         3 . The multiwell plate according to  claim 1 , wherein the anionic polymer is selected from the group consisting of poly(acrylic) acid, poly(methacrylic) acid, poly(glutamic) acid, polyuronic acid, glycosaminoglycans, poly(aspartic acid) and Polystyrene sulfonate, any combination of the polyamino-acids (in the D and/or L forms), and mixtures thereof. 
     
     
         4 . The multiwell plate according to  claim 1 , wherein the cationic polymer comprising amino group is selected from the group consisting of poly(lysine), poly(diallydimethylammonium chloride), poly(allylamine), poly(ethylene)imine, chitosan, polyarginine, Poly(ornithine), polyhistidine, poly(mannosamine), polyallylamine hydrochloride, any combination of the polyamino acids (in the D and/or L forms), and mixtures thereof. 
     
     
         5 . The multiwell plate according to  claim 1 , wherein the polyelectrolyte multilayer film is a poly(L-lysine)/hyaluronan sodium film, a polystyrene sulfonate/polyallylamine hydrochloride film, a poly(L-lysine)/poly(L-glutamic acid) film or a chitosan/poly(L-glutamic acid) film. 
     
     
         6 . The multiwell plate according to  claim 1 , wherein the anionic polymer comprises carboxylic groups, and wherein the method comprises, after the n sequences, the following steps:
 e) reacting said amino and carboxylic groups in the presence of a coupling agent, so as to form amide bonds and to cross-link the polyelectrolyte multilayer film, then   f) treating said cross-linked polyelectrolyte multilayer film with a protein containing solution, so as to incorporate said protein on and inside said cross-linked polyelectrolyte multilayer film.   
     
     
         7 . The multiwell plate according to  claim 1 , wherein the method comprises, after the n sequences, a step e′) of robotic deposit of a volume V PE   3  of a solution of a third polyelectrolyte PE 3  on said bottom surface, wherein:
 polyelectrolyte PE 3  is linked to at least a peptide, and 
 the third polyelectrolyte PE 3  is a cationic polymer comprising amino groups when PE 2  is an anionic polymer, or PE 3  is an anionic polymer when PE 2  is a cationic polymer comprising amino groups. 
 
     
     
         8 . A multiwell plate comprising wells, wherein the bottom surface of m wells is coated by a polyelectrolyte multilayer film,
 wherein m is an integer from 1 to the number of wells of the multiwell plate,   the polyelectrolyte multilayer film comprising n layer pairs, n being an integer from 1 to 2000 and each layer pair comprising a layer of a first polyelectrolyte PE 1  and a layer of a second polyelectrolyte PE 2  of opposite charge,   wherein the first polyelectrolyte PE 1  is either a cationic polymer comprising amino groups, or an anionic polymer,   the second polyelectrolyte PE 2  is a cationic polymer comprising amino groups when PE 1  is an anionic polymer, or PE 2  is an anionic polymer when PE 1  is a cationic polymer comprising amino groups,   the polyelectrolyte multilayer film presenting a coefficient of variation CV of its mean thickness less than or equal to 20.3%,   wherein   
       
         
           
             
               CV 
               = 
               
                 
                   SD 
                   hMEAN 
                 
                 × 
                 100 
               
             
           
         
         SD being the standard deviation 
       
       
         
           
             
               SD 
               = 
               
                 
                   
                     
                       ∑ 
                       1 
                       m 
                     
                     ⁢ 
                     
                       
                         ( 
                         
                           hWELLi 
                           - 
                           hMEAN 
                         
                         ) 
                       
                       2 
                     
                   
                   
                     ( 
                     
                       m 
                       - 
                       1 
                     
                     ) 
                   
                 
               
             
           
         
         
           
             
               hMEAN 
               = 
               
                 
                   
                     ∑ 
                     1 
                     m 
                   
                   ⁢ 
                   hWELL 
                 
                 m 
               
             
           
         
         
           
             
               
                 h 
                 WELL 
               
               = 
               
                 
                   ( 
                   
                     hN 
                     + 
                     hW 
                     + 
                     hC 
                     + 
                     hE 
                     + 
                     hS 
                   
                   ) 
                 
                 S 
               
             
           
         
         hN, hW, hC, hE and hS being film thicknesses determined at the positions N, W, C, E, S inside each well as shown in  FIG. 3 . 
       
     
     
         9 . The multiwell plate according to  claim 8 , wherein the anionic polymer is selected from the group consisting of poly(acrylic) acid, poly(methacrylic) acid, poly(glutamic) acid, polyuronic acid, glycosaminoglycans, poly(aspartic acid) and Polystyrene sulfonate, any combination of the polyamino-acids (in the D and/or L forms), and mixtures thereof. 
     
     
         10 . The multiwell plate according to  claim 8 , wherein the cationic polymer comprising amino group is selected from the group consisting of poly(lysine), poly(diallydimethylammonium chloride), poly(allylamine), poly(ethylene)imine, chitosan, polyarginine, Poly(ornithine), polyhistidine, poly(mannosamine), polyallylamine hydrochloride, any combination of the polyamino acids (in the D and/or L forms), and mixtures thereof. 
     
     
         11 . The multiwell plate according to  claim 8 , wherein the polyelectrolyte multilayer film is a poly(L-lysine)/hyaluronan sodium film, a polystyrene sulfonate/polyallylamine hydrochloride film, a poly(L-lysine)/poly(L-glutamic acid) film or a chitosan/poly(L-glutamic acid) film. 
     
     
         12 . The multiwell plate according to  claim 8 , further comprising a layer of a third polyelectrolyte PE 3  deposited on the top of the polyelectrolyte multilayer film, wherein the third polyelectrolyte PE 3  is linked to at least a peptide, and the third polyelectrolyte PE 3  is a cationic polymer comprising amino groups when the second polyelectrolyte PE 2  is an anionic polymer, or the third polyelectrolyte PE 3  is an anionic polymer when the second polyelectrolyte PE 2  is a cationic polymer comprising amino groups. 
     
     
         13 . The multiwell plate according to  claim 8 , wherein the anionic polymer comprises carboxylic groups, and the polyelectrolyte multilayer film is cross-linked via amide bonds or derivatives thereof formed from the carboxylic groups and the amino groups of the polyelectrolyte multilayer film. 
     
     
         14 . The multiwell plate according to  claim 13 , wherein a protein is incorporated on and inside the cross-linked polyelectrolyte multilayer film. 
     
     
         15 . A multiwell plate comprising wells, wherein the bottom surface of m wells is coated by a polyelectrolyte multilayer film,
 wherein m is an integer from 1 to the number of wells of the multiwell plate,   the polyelectrolyte multilayer film comprising n layer pairs, n being an integer from 1 to 2000 and each layer pair comprising a layer of a first polyelectrolyte PE 1  and a layer of a second polyelectrolyte PE 2  of opposite charge,   wherein the first polyelectrolyte PE 1  is either a cationic polymer comprising amino groups, or an anionic polymer,   the second polyelectrolyte PE 2  is a cationic polymer comprising amino groups when PE 1  is an anionic polymer, or PE 2  is an anionic polymer when PE 1  is a cationic polymer comprising amino groups,
 the polyelectrolyte multilayer film presenting a standard deviation SD less than or equal to 3.9%, 
   wherein   
       
         
           
             
               SD 
               = 
               
                 
                   
                     
                       ∑ 
                       1 
                       m 
                     
                     ⁢ 
                     
                       
                         ( 
                         
                           hWELLi 
                           - 
                           hMEAN 
                         
                         ) 
                       
                       2 
                     
                   
                   
                     ( 
                     
                       m 
                       - 
                       1 
                     
                     ) 
                   
                 
               
             
           
         
         
           
             
               hMEAN 
               = 
               
                 
                   
                     ∑ 
                     1 
                     m 
                   
                   ⁢ 
                   hWELL 
                 
                 m 
               
             
           
         
         
           
             
               
                 h 
                 WELL 
               
               = 
               
                 
                   ( 
                   
                     hN 
                     + 
                     hW 
                     + 
                     hC 
                     + 
                     hE 
                     + 
                     hS 
                   
                   ) 
                 
                 S 
               
             
           
         
         hN, hW, hC, hE and hS being film thicknesses determined at the positions N, W, C, E, S inside each well as shown in  FIG. 3 . 
       
     
     
         16 . The multiwell plate according to  claim 15 , wherein the anionic polymer is selected from the group consisting of poly(acrylic) acid, poly(methacrylic) acid, poly(glutamic) acid, polyuronic acid, glycosaminoglycans, poly(aspartic acid) and Polystyrene sulfonate, any combination of the polyamino-acids (in the D and/or L forms), and mixtures thereof. 
     
     
         17 . The multiwell plate according to  claim 15 , wherein the cationic polymer comprising amino group is selected from the group consisting of poly(lysine), poly(diallydimethylammonium chloride), poly(allylamine), poly(ethylene)imine, chitosan, polyarginine, Poly(ornithine), polyhistidine, poly(mannosamine), polyallylamine hydrochloride, any combination of the polyamino acids (in the D and/or L forms), and mixtures thereof. 
     
     
         18 . The multiwell plate according to  claim 15 , further comprising a layer of a third polyelectrolyte PE 3  deposited on the top of the polyelectrolyte multilayer film, wherein the third polyelectrolyte PE 3  is linked to at least a peptide, and the third polyelectrolyte PE 3  is a cationic polymer comprising amino groups when the second polyelectrolyte PE 2  is an anionic polymer, or the third polyelectrolyte PE 3  is an anionic polymer when the second polyelectrolyte PE 2  is a cationic polymer comprising amino groups. 
     
     
         19 . The multiwell plate according to  claim 15 , wherein the anionic polymer comprises carboxylic groups, and the polyelectrolyte multilayer film is cross-linked by amide bonds or derivatives thereof. 
     
     
         20 . The multiwell plate according to  claim 15 , wherein a protein is incorporated on and inside the cross-linked polyelectrolyte multilayer film.