# US 12,180,507 B2 · Engineered platforms to stabilize both hepatocytes and endothelial cells in vitro

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- Number: US 12,180,507 B2
- Document ID: US12180507B2
- Status: Granted
- Kind: B2
- Application number: 15/749,733
- Filed: 2016-08-05
- Published: 2018-08-09
- Granted: 2024-12-31
- Earliest claimed: 2015-08-05
- Assignees: Colorado State University Research Foundation
- Applicants: Colorado State University Research Foundation
- Inventors: Salman R. Khetani; Mitchell J. Durham; Brenton R. Ware
- Web page: https://patentgrep.com/patent/US12180507B2

## CPC

- C12N 5/067: {Hepatocytes}
- A61K 35/407: Liver; Hepatocytes
- A61K 35/44: Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
- G01N 33/5014: {for testing toxicity}
- C12N 2502/1323: Adult fibroblasts
- C12N 2502/28: Vascular endothelial cells
- C12N 2503/02: Drug screening
- C12N 2533/52: Fibronectin; Laminin
- C12N 2533/54: Collagen; Gelatin
- C12N 2533/90: Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
- C12N 2535/10: Patterned coating
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The disclosure relates to in vitro cultures of human hepatocytes, and in particular co-cultures systems including human hepatocytes, non-parenchymal cells, and human endothelial cells, and the use of the co-cultures in developing and screening drugs.

## Claims

### Claim 1

A composition comprising a population of human hepatocytes, a population of murine embryonic fibroblasts, and a population of human endothelial cells selected from human primary liver sinusoidal endothelial cells (LSECs) or human umbilical vein endothelial cells (HUVECs) in co-culture in vitro,
wherein the hepatocytes are disposed in a micropattern on a culture substrate,
wherein the micropattern comprises a predetermined two-dimensional pattern of multiple microdots, the micropattern defined by a microdot diameter and an edge-to-edge spacing between each of any two neighboring microdots, and
wherein the hepatocytes occupy the microdots, the murine embryonic fibroblasts occupy the inter-microdot space, and the population of human endothelial cells is positioned on top of a layer of material comprising at least one extracellular matrix protein that is disposed on the population of human hepatocytes and the population of murine embryonic fibroblasts.

### Claim 2 (depends on 1)

The composition of claim 1, further comprising fibronectin.

### Claim 3 (depends on 1)

The composition of claim 1, wherein the human hepatocytes are primary human hepatocytes.

### Claim 4 (depends on 1)

The composition of claim 1, wherein the human hepatocytes are derived from pluripotent stems cells.

### Claim 5 (depends on 4)

The composition of claim 4, wherein the human hepatocytes are derived from induced pluripotent human stem cells.

### Claim 6

A method of culturing a population of human hepatocytes in vitro, comprising co-culturing the population of human hepatocytes with a population of murine embryonic fibroblasts, and a population of human endothelial cells selected from human primary liver sinusoidal endothelial cells (LSECs) or human umbilical vein endothelial cells (HUVECs),
wherein the hepatocytes are disposed in a micropattern on a culture substrate,
wherein the micropattern comprises a predetermined two-dimensional pattern of multiple microdots, the micropattern defined by a microdot diameter and an edge-to-edge spacing between each of any two neighboring microdots,
wherein the hepatocytes occupy the microdots, the murine embryonic fibroblasts occupy the inter-microdot space, and the population of human endothelial cells is cultured on top of a layer of material comprising at least one extracellular matrix protein that is disposed on the population of human hepatocytes and the population of murine embryonic fibroblasts.

### Claim 7 (depends on 6)

The method of claim 6, further comprising co-culturing the population of human hepatocytes with the population of murine embryonic fibroblasts and the population of human endothelial cells with fibronectin.

### Claim 8 (depends on 6)

The method according to claim 6, wherein the human hepatocytes are primary human hepatocytes.

### Claim 9 (depends on 6)

The method according to claim 6, wherein the human hepatocytes are derived from pluripotent stems cells.

### Claim 10 (depends on 6)

The method according to claim 6, wherein the human hepatocytes are derived from induced pluripotent human stem cells.

### Claim 11 (depends on 2)

The composition of claim 2, wherein the fibronectin is disposed on the culture substrate.

### Claim 12 (depends on 7)

The method of claim 7, wherein the fibronectin is disposed on the culture substrate.

### Claim 13 (depends on 1)

The method according to claim 1, wherein the murine embryonic fibroblasts are 3T3-J2 murine embryonic fibroblasts.