Apparatus and method for culturing and preserving tissue constructs

Abstract

A disclosure is made of various apparatus and methods for culturing and preserving cells and tissue in ways that minimize contamination potential, direct cells to reside in desired areas, allow uniform cell distribution during seeding, provide optimal growth conditions by controlling the amount of medium residing in proximity of cells, allow desired compounds and molecules to reside in proximity of the cells, allow co-culture, provide for efficient scale up, allow a desired shape of tissue to be created while retaining a closed system, and allow cryopreservation and reconstitution of cell and tissue while retaining a closed system. The apparatus and methods can be combined to prevent the need to remove the tissue from the enclosure at any point during the sterilization, seeding, culturing, cryopreservation, shipping, or restoration process. Also disclosed is an apparatus and method of pipette interface with a container in a manner that blocks contaminants from entering the container.

1 . A pipette access port for receiving a pipette having a pipette tip, said pipette being retainingly carried by a pipettor, comprising: 
 a) an elastomeric seal including structure defining a thin walled access opening presenting an access opening perimeter and access opening cross-section and having an access opening depth, said access opening resiliently increasing in cross-section when said pipette is inserted into said access opening thereby sealingly engaging the tip of said pipette;    b) structure presenting a void volume in communication with said access opening, said void volume presenting a void volume depth greater than said access opening depth, and said void volume presenting a void volume cross-section greater than said access opening cross-section, said pipette tip residing in said structure defining said void volume when said pipette is inserted into said access port.    
   
   
       2 . The device according to  claim 1  wherein the thickness of said access opening depth measured immediately adjacent to said access opening perimeter when said pipette is clear of said opening is no greater than about 30% of said access opening cross-section.  
   
   
       3 . The device according to  claim 1  including a pipette stop including structure defining a pipette stop opening for receiving said pipette, said pipette stop opening having a cross-section that exceeds said access opening cross-section, said pipette stop opening oriented generally adjacent to said access opening whereby said pipette sealingly engages the perimeter of said access opening when said pipette is inserted into said access opening through said pipette stop, said pipette stop being engageable with said pipette to limit the distance of the path of travel of said pipette into said access opening cross-section, said stop presenting a stop axis oriented transverse to the said access opening cross-section.  
   
   
       4 . A method of accessing a container septum presenting a container septum surface with a needle while inhibiting contamination of said needle comprised of: 
 a) providing an access needle protection septum comprised of a face and collapsible side walls;    b) providing a needle having a needle tip for fluid transport and locating said needle tip within said access needle protection septum;    c) urging said face of said access needle protection septum against the container septum surface with enough force to collapse said side walls of said access needle protection septum, thereby allowing said needle tip to penetrate both said face of said access needle protection septum and said container septum;    d) delivering to or withdrawing fluid from said container septum;    e) withdrawing said needle from said container septum, and withdrawing said face of said access needle protection septum from said container septum surface such that said side walls of said access needle protection septum extend to their original position and said needle tip is withdrawn within said movable access needle protection septum.    
   
   
       5 . A method of altering the volume of fluid surrounding cells received within a container by performing the steps of: 
 a) providing a container having structure presenting at least one access port and having at least one flexible wall, said container including a cell attachment matrix;    b) introducing an inoculum carrying cells into said container;    c) depositing cells from said inoculum upon said cell attachment matrix;    d) introducing a cell culture medium into said container;    e) altering the distance between said flexible wall of said container and said cell attachment matrix for the purpose of providing benefits including allowing the optimal amount of inoculum to reside above said cell attachment matrix during gravitational seeding, accommodating the optimal medium volume residing in proximity of said cell attachment matrix, altering the cross-sectional area of said container so that the velocity of fluid in proximity of said cell attachment matrix can be changed to a desired rate when said container is perfused at any given flow-rate, relieving pressure increases that may result as medium loses its gas carrying capacity during a temperature increase when batch feeding is utilized, removing gas that comes to reside in said container without having to displace it with medium and potentially dilute important cell produced conditioning agents, changing the volume of space surrounding the cell attachment in order to accommodate the addition of the optimal amount of cyropreservative, changing the volume of space surrounding the tissue in order to accommodate the addition of the optimal amount of reconstitution medium, or allowing microscopic viewing of the tissue.    
   
   
       6 . The method of  claim 5  wherein said flexible wall is comprised of gas permeable material.  
   
   
       7 . A method of inoculating and culturing cells on a cell attachment matrix comprising the steps: 
 a) providing a liquid impermeable housing including structure defining at least one access port, said housing comprised of a flexible material;    b) providing a cell attachment matrix and a frame, and attaching one edge of said cell attachment matrix to said frame and placing said cell attachment matrix into said housing, and securing said frame to said housing;    c) introducing an inoculum into said housing and positioning said housing such that said inoculum resides above said cell attachment matrix;    d) seeding said cell attachment matrix with said inoculum;    e) orienting said housing such that said cell attachment matrix is suspended in the vertical position by said frame such that liquid can surround said cell attachment matrix.    
   
   
       8 . A method of inoculating and culturing cells on a cell attachment matrix comprising the steps of: 
 a) providing a liquid impermeable housing comprised of a flexible material, said housing including structure defining at least one access port;    b) providing a cell attachment matrix and a frame, and attaching one edge of said cell attachment matrix to said frame and placing said cell attachment matrix and frame into said housing and securing said frame to said housing;    c) seeding said cell attachment matrix from said inoculum by centrifuging said liquid impermeable housing at a predetermined speed such that said cells are moved onto and into said cell attachment matrix by centrifugal force;    d) orienting said housing such that said cell attachment matrix is suspended in the vertical position by said frame such that liquid can surround said cell attachment matrix.    
   
   
       9 . A method of inoculating two areas of a cell attachment matrix comprising the steps: 
 a) providing a liquid impermeable housing having at least one access port, said housing comprised of a flexible material;    c) providing a cell attachment matrix and a clamp, attaching one edge of said cell attachment matrix to said clamp and placing said cell attachment matrix and clamp into said housing, and securing said clamp to said housing;    d) introducing a first inoculum into said housing and positioning said housing such that said first inoculum resides above a first area of said cell attachment matrix;    e) seeding said first area of said cell attachment matrix with said first inoculum;    f) introducing a second inoculum into said housing and positioning said housing such that said second inoculum resides above a second area of said cell attachment matrix;    g) seeding said second area of said cell attachment matrix with said second inoculum.    
   
   
       10 . The method according to  claim 7  wherein cells are cultured upon said cell attachment matrix and a predetermined amount of cell culture medium is maintained in proximity of said first area and said second area of said cell attachment matrix by orienting said housing such that said cell attachment matrix is suspended in the vertical position and said housing is constrained by an outer housing in a manner that allows a predetermined amount of said cell culture medium to reside in proximity of said first area and a predetermined amount of said cell culture medium to reside in proximity of said second area of said cell attachment matrix.