Clamping insert for cell culture

Abstract

A clamping insert for cell culture having a lower support with a hollow member engagable in a well of a microplate, the hollow member being open at both ends and including a support surface at one end, and first tightening elements. The clamping insert further having an inner holder sized to be fitted inside the hollow member, and second tightening elements arranged to cooperate with the first tightening elements of the hollow member to tighten reversibly between the inner holder and the support surface of the lower support a porous substrate. The inner holder further including an open channel keeping free the porous substrate. Retaining elements intended to cooperate with the well of the microplate are provided to allow the clamping insert to be suspended in the well, and sealing elements are provided to hermetically seal the contact area between the clamping insert and the porous substrate.

What is claimed is: 
     
         1 . A clamping insert for cell culture comprising:
 a lower support comprising:   i. a hollow member sized to be engaged in a well of a microplate, said hollow member having an upper end with a first opening, a lower end with a second opening, an interior wall extending between the first and second openings and defining a open channel therebetween, and a support surface at lower end, said support surface extending inwardly from the interior wall and including a central opening so as to defining an open passage between an uppermost surface of the hollow member and a lowermost surface of the hollow member, and   ii. a first tightening element, and   iii. a retaining element comprising flanges extending, at the upper end, outwardly from an outside wall of the hollow member oppositely to the lower end comprising said support surface, wherein said retaining element is adapted to cooperate with an upper surface of the well of the microplate to allow the hollow member a) to be suspended in the well of the microplate, via a lower surface of the retaining element resting on the upper surface of the well of the microplate, and b) to divide the well of the microplate into an apical compartment and a basolateral compartment in communication via a porous substrate;   an inner holder sized to be fitted inside the open channel of the hollow member, and comprising a lowermost surface and a second tightening element that cooperates with said first tightening element of the hollow member to clamp reversibly said porous substrate between the lowermost surface of the inner holder and the support surface of the lower support, said inner holder comprising an open channel extending from an uppermost surface of the inner holder to the lowermost surface of the inner holder and arranged to provide access to the porous substrate;   wherein the inner holder is situated entirely within the open channel of the hollow member; and   a seal, that when placed in contact with said porous substrate, hermetically seals a contact area between said porous substrate and one of a group consisting of the inner holder and the lower support.   
     
     
         2 . The clamping insert of  claim 1 , wherein an inner wall of the inner holder also comprises a guiding groove oriented longitudinally, allowing passage of a pipette or of a TEER (Trans Epithelial Electrical Resistance) electrode. 
     
     
         3 . The clamping insert of  claim 1 , wherein said seal is interposed between the inner holder and the porous substrate and/or interposed between the porous substrate and the lower support. 
     
     
         4 . The clamping insert of  claim 1 , wherein the inner holder and/or the lower support is/are made of PEEK, polycarbonate, polystyrene or other plastics compatible with cell culture. 
     
     
         5 . An assembly of a clamping insert for cell culture comprising:
 a lower support comprising:   i. a hollow member sized to be engaged in a well of a microplate, said hollow member having an upper end with a first opening, a lower end with a second opening, an interior wall extending between the first and second openings and defining a open channel therebetween, and a support surface at lower end, said support surface extending inwardly from the interior wall and including a central opening so as to defining an open passage between an uppermost surface of the hollow member and a lowermost surface of the hollow member, and   ii. a first tightening element, and   iii. a retaining element comprising flanges extending, at the upper end, outwardly from an outside wall of the hollow member oppositely to the lower end comprising said support surface, wherein said retaining element is adapted to cooperate with an upper surface of the well of the microplate to allow the hollow member a) to be suspended in the well of the microplate, via a lower surface of the retaining element resting on the upper surface of the well of the microplate, and b) to divide the well of the microplate into an apical compartment and a basolateral compartment in communication via a porous substrate;   an inner holder sized to be fitted inside the open channel of the hollow member, and comprising a lowermost surface and a second tightening element that cooperates with said first tightening element of the hollow member to clamp reversibly said porous substrate between the lowermost surface of the inner holder and the support surface of the lower support, said inner holder comprising an open channel extending from an uppermost surface of the inner holder to the lowermost surface of the inner holder and arranged to provide access to the porous substrate;   wherein the inner holder is situated entirely within the open channel of the hollow member;   a seal, that when placed in contact with said porous substrate, hermetically seals a contact area between said porous substrate and one of a group consisting of the inner holder and the lower support; and   a porous substrate clamped between the lower support and the inner holder, so that the porous substrate can be removed from the insert only by untightening and disassembling the inner holder and the lower support.   
     
     
         6 . The assembly of  claim 5 , wherein the porous substrate is made of Si3N4. 
     
     
         7 . The assembly of  claim 5 , wherein the porous substrate is micro machined. 
     
     
         8 . The assembly of  claim 5 , wherein the porous substrate comprises a silicon nitride membrane supported on a silicon frame. 
     
     
         9 . The assembly of  claim 5 , integrating pads and electrical circuits for electrical measurements, wherein the substrate is provided with integrated electrodes and in that the insert is equipped with contact pads designed to be in contact with the electrodes. 
     
     
         10 . A method for clamping a porous substrate with an insert as claimed in  claim 1 , comprising the following steps:
 unfitting the inner holder and the lower support,   positioning said porous substrate on the support surface of the lower support,   positioning the inner holder so as to clamp the porous substrate between the inner holder and the lower support, and   tightening the inner holder with the lower support utilising a dedicated tool comprising some driving structures intended to cooperate with corresponding structures arranged in the inner holder.