IP Library › Granted Patent US 12,590,296
Granted Patent B2
US 12,590,296 · App. 17/776,053 · Granted Mar 31, 2026

Method of culturing immortalized human hepatic progenitors or cells

Inventors: Georges Baffet (Le Rheu, FR); Marie Cuvellier (Rennes, FR); Frédéric Ezan (Rennes, FR); Sophie Rose (Rennes, FR)
Assignees: INSERM (INSTITUT NATIONAL DE LA SANTÉ ET DE LA RECHERCHE MÉDICALE); UNIVERSITÉ DE RENNES 1; ÉCOLE DES HAUTES ÉTUDES EN SANTÉ PUBLIQUE (EHESP)
C12N5/0671G01N33/5067C12N2501/33C12N2501/39C12N2513/00C12N2533/30C12N2533/54
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Quick Facts
Patent No.
US 12,590,296
App. No.
17/776,053
Granted
Mar 31, 2026
Kind
B2
Abstract

A method of obtaining 3D cell structures including differentiated human hepatic cells. The method includes: a first step of culturing stem cell-derived or immortalized human hepatic progenitors in a non-adherent culture vessel, preferably a low or ultra-low attachment culture vessel; a second step of transferring the stem cell-derived or immortalized human hepatic progenitors to a culture medium including methacrylated gelatin (GelMa), thereby embedding the stem cell-derived or immortalized human hepatic progenitors in a GelMa matrix; and a third step of covering the GelMa matrix with culture medium and culturing the stem cell-derived or immortalized human hepatic progenitors embedded in the GelMa matrix, thereby obtaining 3D cell structures including differentiated human hepatic cells. Also, methods for engineering an artificial liver model or an artificial liver organ, and for assessing in vitro the metabolism, toxicity and/or therapeutic effects of a compound.

Claims (23)

1 . A method of obtaining 3D cell structures comprising differentiated human hepatic cells, said method comprising:

a) culturing stem cell-derived or immortalized human hepatic progenitors in a non-adherent culture vessel for a duration ranging from 12 h to 24 h;

b) transferring the stem cell-derived or immortalized human hepatic progenitors to a culture medium comprising methacrylated gelatin (GelMa), thereby embedding the stem cell-derived or immortalized human hepatic progenitors in a GelMa matrix; and

c) covering the GelMa matrix with culture medium and culturing the stem cell-derived or immortalized human hepatic progenitors embedded in the GelMa matrix;

wherein the stem cell-derived or immortalized human hepatic progenitors differentiate into human hepatic cells and cluster into 3D cell structures after being embedded in the GelMa matrix, thereby obtaining 3D cell structures comprising differentiated human hepatic cells.

2 . The method according to claim 1 , wherein at step a) the non-adherent culture vessel is a low or ultra-low attachment culture vessel.

3 . The method according to claim 1 , wherein at step b) the stem cell-derived or immortalized human hepatic progenitors are transferred to a culture medium comprising methacrylated gelatin (GelMa) at a final concentration ranging from about 1% (w/v) to about 20% (w/v).

4 . The method according to claim 1 , wherein at step c) the stem cell-derived or immortalized human hepatic progenitors embedded in the GelMa matrix are cultured for at least seven days.

5 . The method according to claim 1 , wherein the culture medium comprises insulin and a glucocorticoid.

6 . The method according to claim 1 , further comprising:

d) incubating the 3D cell structures comprising differentiated human hepatic cells embedded in the GelMa matrix with a collagenase or an enzyme mixture with collagenolytic activity, thereby isolating the 3D cell structures comprising differentiated human hepatic cells from the GelMa matrix.

7 . The method according to claim 1 , wherein the differentiated human hepatic cells are human hepatocytes or hepatocyte-like cells.

8 . The method according to claim 1 , wherein the stem cell-derived or immortalized human hepatic progenitors are hepatocellular carcinoma-derived and/or transformed human hepatic progenitors.

9 . The method according to claim 1 , wherein the stem cell-derived or immortalized human hepatic progenitors are undifferentiated HepaRG cells.

10 . A method for engineering an artificial liver model or an artificial liver organ comprising differentiated human hepatic cells, the method comprising culturing stem cell-derived or immortalized human hepatic progenitors according to the method of claim 1 with at least another type of cells, wherein the stem cell-derived or immortalized human hepatic progenitors embedded in the GelMa matrix are contacted with the at least another type of cells.

11 . The method according to claim 10 , wherein said at least another type of cells is embedded in a matrix.

12 . The method according to claim 10 , wherein said at least another type of cells is another type of hepatic cells.

13 . The method according to claim 10 , wherein the stem cell-derived or immortalized human hepatic progenitors are cultured with stellate cells and/or endothelial cells.

14 . An in vitro method for assessing the metabolism, toxicity and/or therapeutic effects of a compound, the method comprising:

a) obtaining 3D cell structures comprising differentiated human hepatic cells according to the method of claim 1 ;

b) contacting the 3D cell structures comprising differentiated human hepatic cells with a compound; and

c) assessing the metabolism, toxicity and/or therapeutic effects of the compound on the 3D cell structures comprising differentiated human hepatic cells.

15 . The in vitro method according to claim 14 wherein the differentiated human hepatic cells are human hepatocytes or hepatocyte-like cells.

Assignments (2)
MERGER Recorded Oct 19, 2023
From: UNIVERSITE DE RENNES I
To: UNIVERSITE DE RENNES
Reel/Frame 065490/0667 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jun 8, 2022
From: BAFFET, GEORGES; CUVELLIER, MARIE; EZAN, FRÉDÉRIC; ROSE, SOPHIE
To: INSERM (INSTITUT NATIONAL DE LA SANTÉ ET DE LA RECHERCHE MÉDICALE); UNIVERSITÉ DE RENNES 1; ÉCOLE DES HAUTES ÉTUDES EN SANTÉ PUBLIQUE (EHESP)
Reel/Frame 060134/0883 →
Priority Claims (1)
EP 19306473 · Nov 15, 2019 · regional
Continuity (1)
Related Publication 20220396773A1 · Dec 15, 2022
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