Nanoparticulate polycosanol formulations & novel polycosanol combinations

Abstract

The invention provides a culture device comprising a plurality of culture units, wherein each unit comprises a culture chamber, an inlet port for liquid supply of the culture and an outlet port for discharging liquid from the unit, wherein the inlet port is in fluid communication with the culture chamber and the culture chamber is in fluid communication with the outlet port for allowing a liquid flow through the culture chamber. The culture device is particularly suitable for testing immune cells and immunofunction in vitro. Aspects of the invention include a culture device and associated methods for cultivating immune cells and an in vitro method of analysing the effect of a test compound on immune cells.

1 . A culture device having a top side, a bottom side, and at least one lateral side, comprising a plurality of culture units, wherein each unit comprises
 (i) a culture chamber,   (ii) an inlet port for reversibly connecting the unit with an external liquid supply and   (iii) an outlet port for discharging liquid from the unit,   wherein the inlet port is in fluid communication with the culture chamber and the culture chamber is in fluid communication with the outlet port for allowing a liquid flow through the culture chamber,   wherein the inlet port is accessible for connecting an external liquid supply from the top side or lateral side of the culture device, and the outlet port is accessible for connecting a discharge conduct from the top side or lateral side of the device.   
   
   
       2 . The culture device of  claim 1 ,
 wherein the mainstream of the liquid flow traverses the culture chamber within the plane of the culture device.   
   
   
       3 . The culture device according to  claim 1 , wherein the fluid communication is established by a channel between the culture chamber and the inlet port and a channel between the culture chamber and the outlet port. 
   
   
       4 . The culture device according to  claim 1 , wherein the culture chamber or chambers:
 (a) are sealed on at least one side with a gas-permeable foil;   (b) are gas-tightly closed except for openings involved in fluid communication of the culture chamber with the ports;   (c) have a culture volume from 25 to 1000 μl;   (d) are translucent for allowing microscopic inspection of cells present in the culture chambers;   (e) comprises a preformed matrix;   (f) comprises living eukaryotic cell material embedded in a matrix;   (g) comprises living eukaryotic cell material seeded on a matrix; or   (h) any combination thereof.   
   
   
       5 . (canceled) 
   
   
       6 . The culture device according to  claim 5 , further comprising a conduct between the inlet port and the external liquid supply, the conduct comprising a gas permeable conduct that allows equilibrating liquid media to a predefined oxygen and carbon dioxide content. 
   
   
       7 . (canceled) 
   
   
       8 . The culture device according to  claim 4 , wherein the culture chamber has a culture volume from 50 to 250 μl or from, 50 to 100 μl. 
   
   
       9 . The culture device according to  claim 1 , the device comprising 2 to 200 culture units. 
   
   
       10 . The culture device of  claim 1 , wherein test substances and/or stimulatory agents may be added to living cell material in each individual unit separately. 
   
   
       11 . (canceled) 
   
   
       12 . (canceled) 
   
   
       13 . (canceled) 
   
   
       14 . (canceled) 
   
   
       15 . The culture device according to  claim 4 , wherein the matrix is selected from a hydrogel, foam or non-woven fibres. 
   
   
       16 . (canceled) 
   
   
       17 . The culture device according to  claim 4 , wherein the living eukaryotic cell material comprises leucocytes or co-cultures of leucocytes with other cells of interest. 
   
   
       18 . The culture device according to  claim 17 , wherein the leucocytes are whole peripheral blood mononuclear cells, defined subpopulations of peripheral blood mononuclear cells, in vitro differentiated peripheral blood mononuclear cell subpopulations and any co-cultures of these. 
   
   
       19 . The culture device according to  claim 17 , wherein the other cells of interest are selected from the group consisting of endothelial cells, stem cells, follicular dendritic cells, stromal cells and others. 
   
   
       20 . The culture device according to  claim 1 , any wherein:
 (a) the culture unit comprises an additional inlet port for introducing a suspension comprising eukaryotic cells and/or an aqueous matrix-forming composition into the culture chamber;   (b) the culture units further comprise hollow fibre membranes traversing the culture cavity, whereby the hollow fibre membranes are in fluid communication with the culture chamber and the ports for allowing a liquid flow through the culture chamber; or   (c) any combination thereof.   
   
   
       21 . (canceled) 
   
   
       22 . The culture device according to  claim 20 , wherein the hollow fibre membranes are mounted in a separate cassette comprising the culture chamber. 
   
   
       23 . The culture device according to  claim 1 , that provides a microenvironment. 
   
   
       24 . A method of cultivating immune cells, comprising the steps of
 (a) introducing a suspension of immune cells in an aqueous matrix-forming composition into a culture chamber,   (b) solidifying the suspension, thereby forming a matrix having the immune cells embedded therein, and   (c) incubating the immune cells at predefined culture conditions for a predefined period of time, whereby the matrix is continuously perfused with liquid culture media and supplements.   
   
   
       25 . A method of cultivating immune cells, comprising the steps of:
 (a) introducing a suspension of immune cells in an aqueous matrix-forming composition into the culture chamber of at least one culture unit of the culture device according to  claim 1 ,   (b) solidifying the suspension, thereby forming a matrix having the immune cells embedded therein, and   (c) incubating the immune cells at predefined culture conditions for a predefined period of time, whereby the matrix is continuously perfused with liquid culture media and supplements.   
   
   
       26 . A method of cultivating immune cells, comprising incubating immune cells embedded in a matrix at predefined culture conditions for a predefined period of time, whereby the matrix is continuously perfused with liquid culture media and supplements. 
   
   
       27 . A method of cultivating immune cells, comprising incubating immune cells embedded in a matrix in the culture chamber of at least one culture unit of the culture device according to  claim 1 , at predefined culture conditions for a predefined period of time, whereby the matrix is continuously perfused with liquid culture media and supplements. 
   
   
       28 . The method according to  claim 25 , wherein the individual culture units provide comparable conditions. 
   
   
       29 . The method according to  claim 24 , wherein the matrix is selected from a hydrogel, foam, sponge or non-woven fibres. 
   
   
       30 . (canceled) 
   
   
       31 . The method according to  claim 24 , wherein the immune cells are leucocytes or co-cultures of leucocytes with other cells of interest. 
   
   
       32 . The method according to  claim 31 , wherein:
 (a) the leucocytes can be whole peripheral blood mononuclear cells, defined subpopulations of peripheral blood mononuclear cells, in vitro differentiated peripheral blood mononuclear cell subpopulations and any co-cultures of these;   (b) the other cells of interest are selected from the group consisting of endothelial cells, stem cells, follicular dendritic cells, stromal cells and others; or   (c) any combination thereof.   
   
   
       33 . (canceled) 
   
   
       34 . The method according to  claim 24 , wherein:
 (a) the immune cells are incubated at predefined culture conditions for a period of time of up to 8 weeks;   (b) the culture conditions are defined by culture media, supplements, matrices, technically supported micro-environment and gas supply;   (c) cultured cells are provided with gas through a gas permeable membrane that seals at least one side of the culture chamber;   (d) the matrix, culture media composition, cell density and cell mixture allow formation of micro-organoid structures;   (e) the matrix, culture media composition, cell density and cell mixture allow formation of a microenvironment; or   (f) any combination thereof   
   
   
       35 . (canceled) 
   
   
       36 . (canceled) 
   
   
       37 . (canceled) 
   
   
       38 . (canceled) 
   
   
       39 . A method of analysing the effect of a test compound on immune cells, which comprises the steps of
 (a) incubating immune cells embedded in a matrix in the culture chamber of at least one culture unit of the culture device according to  claim 1 , at predefined culture conditions for a predefined period of time, whereby the matrix is continuously perfused with liquid culture media and supplements,   (b) adding the test compound to at least one culture unit through the inlet port,   (c) incubating immune cells as described in step a) in the presence of the at least one test compound   (d) removing at least one sample of the culture media discharged from the outlet port of the at least one culture unit to which the test compound was added, and   (e) analysing the sample for an effect of the test compound on the immune cells.   
   
   
       40 . The method according to  claim 39 , wherein the matrix is selected from a hydrogel, foam, sponge or non-woven fibres. 
   
   
       41 . (canceled) 
   
   
       42 . (canceled) 
   
   
       43 . The method according to  claim 39 , wherein:
 (a) the sample is removed while maintaining incubation of the cells;   (b) in step e) the presence or concentration of soluble factors selected from cytokines, chemokines and antibodies are analysed in the sample;   (c) the test compound, concentration of test compound, time of exposing the cells to the test compound are different between individual culture units;   (d) further comprising analysis of the immune cells by a method selected from the group of immune fluorescence, light microscopy, flow cytometry, fluorescence activated cell sorting, histology, molecular biology techniques and enzyme linked immuno-spot methods after the culture process; or   (e) any combination thereof.   
   
   
       44 . (canceled) 
   
   
       45 . The method according to  claims 25 , which further comprises online monitoring and/or offline monitoring of cell culture conditions in culture media discharged at the outlet port. 
   
   
       46 . The method according to  claim 45 , wherein the cell culture conditions comprise oxygen content, pH and metabolites. 
   
   
       47 . (canceled)