Cellular microcompartment and preparation processes

Abstract

The invention relates to a cellular microcompartment comprising successively, organized around a lumen, at least one layer of pluripotent cells, an extracellular matrix layer and an outer hydrogel layer. The invention also relates to processes for preparing such cellular microcompartments.

We claim: 
     
         1 . A cellular microcompartment comprising successively, organized around a lumen:
 at least one layer of human pluripotent cells, wherein the human pluripotent cells are polarized and the inner/apical side of the pluripotent cell layer is adjacent to the lumen;   an extracellular matrix layer; an outer hydrogel layer, wherein said outer layer thickness is between 5 to 40 μm.   
     
     
         2 . The cellular microcompartment according to  claim 1 , wherein said microcompartment is closed. 
     
     
         3 . The cellular microcompartment according to  claim 1 , wherein the cell density is between one and several thousand cells per microcompartment. 
     
     
         4 . The cellular microcompartment according to  claim 1 , wherein the outer layer comprises alginate. 
     
     
         5 . The cellular microcompartment according to  claim 1 , wherein the outer layer comprises alginate more than 80% of α-L-guluronate and less than 20% of β-D-mannuronate. 
     
     
         6 . The cellular microcompartment according to  claim 1 , wherein hydrogel layer is alginate with an average molecular mass of 100 to 400 KDa. 
     
     
         7 . The cellular microcompartment according to  claim 1 , wherein said microcompartment has a spherical or elongated shape. 
     
     
         8 . The cellular microcompartment according to  claim 1 , wherein said microcompartment has a diameter or a smallest dimension comprised between 10 μm and 1 mm, preferentially between 50 μm and 500 μm, more preferentially less than 500 μm, even more preferentially less than 400 μm. 
     
     
         9 . The cellular microcompartment according to  claim 1 , wherein the cell density is comprised between one and several thousand cells, preferentially 50 to 1000 cells per microcompartment. 
     
     
         10 . The cellular microcompartment according to  claim 1 , wherein the thickness of the extracellular matrix layer represents 5 to 80% of the radius of the microcompartment. 
     
     
         11 . The cellular microcompartment according to  claim 1 , wherein the extracellular matrix layer is attached to the inner side of the hydrogel shell. 
     
     
         12 . A method for preparing a cellular microcompartment of  claim 1 , the method comprising:
 (a) incubating human pluripotent stem cells in a culture medium containing a RHO/ROCK pathway inhibitor;   (b) mixing the pluripotent stem cells from step (a) with an extracellular matrix;   (c) encapsulating the mixture from step (b) in a hydrogel layer;   (d) culturing the capsules obtained in step (c) in a culture medium containing a RHO/ROCK pathway inhibitor;   (e) rinsing the capsules from step (d) to remove the RHO/ROCK pathway inhibitor;   (f) culturing the capsules from step (e) for 3 to 20 days or for 5 to 10 days, and optionally recovering the cellular microcompartments.   
     
     
         13 . The method of  claim 12  further comprising
 dissociating the pluripotent stem cells from step (a) before step (b) by contacting the pluripotent stem cells with an enzyme-free reagent. 
 
     
     
         14 . A method for preparing a cellular microcompartment of  claim 1 , comprising the steps consisting of:
 (a) mixing human differentiated cells with an extracellular matrix and cell reprogramming agents;   (b) encapsulating the mixture from step (a) in a hydrogel layer;   (c) culturing the capsules from step (b) for 10 to 40 days, and optionally recovering the cellular microcompartments obtained.   
     
     
         15 . The method of  claim 14 , wherein each capsule from step (b) contains between 1 and 500 differentiated cells. 
     
     
         16 . The method of  claim 12 , wherein the method further comprises freezing the cellular microcompartments obtained in step (f). 
     
     
         17 . The method of  claim 14 , wherein the method further comprises freezing the cellular microcompartments obtained in step (c).