# US 2020/0399592 A1 · Suspension culture of human embryonic stem cells

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- Number: US 2020/0399592 A1
- Document ID: US20200399592A1
- Status: Published application
- Kind: A1
- Application number: 16/862,559
- Filed: 2020-04-29
- Published: 2020-12-24
- Earliest claimed: 2005-06-22
- Assignees: Asterias Biotherapeutics, Inc.
- Applicants: Asterias Biotherapeutics, Inc.
- Inventors: Ramkumar Mandalam; Yan Li; Isabelle Nadeau-Demers
- Web page: https://patentgrep.com/patent/US20200399592A1

## CPC

- C12N 5/0606: {Pluripotent embryonic cells, e.g. embryonic stem cells [ES] (embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696)}
- C12N 2501/115: Basic fibroblast growth factor (bFGF, FGF-2)
- C12N 2501/125: Stem cell factor [SCF], c-kit ligand [KL]
- C12N 2501/15: Transforming growth factor beta (TGF-β)
- C12N 2501/26: Flt-3 ligand (CD135L, flk-2 ligand)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

This disclosure provides an improved system for culturing human embryonic stem cells. The cells are cultured in suspension so as to maximize the production capacity of the culture environment. The new culture system of this invention allows for bulk proliferation of hES cells in a more cost-effective manner, which facilitates commercial production of important products for use in human therapy.

## Claims

### Claim at position 1

-18. (canceled)

### Claim 19

A system or kit for culturing human pluripotent stem (hPS) cells in suspension, comprising a nutrient medium that contains at least 40 ng/mL fibroblast growth factor (FGF).

### Claim 20 (depends on 19)

The system or kit of claim 19, further comprising one or more extracellular matrix components or microparticles for adding to the medium.

### Claim 21 (depends on 20)

The system or kit of claim 20, wherein the one or more extracellular matrix components are selected from human laminin and human fibronectin.

### Claim 22 (depends on 20)

The system or kit of claim 20, wherein the microparticles are coated with one or more soluble or suspended extracellular matrix components.

### Claim 23 (depends on 19)

The system or kit of claim 19, wherein the medium further comprises one or more of transforming growth factor beta (TGFβ), stem cell factor (SCF), and Flt3 ligand (Flt3L).

### Claim 24 (depends on 19)

The system or kit of claim 19, wherein FGF is basic FGF (bFGF).

### Claim 25 (depends on 24)

The system or kit of claim 24, wherein the concentration of bFGF is about 80 ng/mL.

### Claim 26 (depends on 19)

The system or kit of claim 19, further comprising instructions for use.

### Claim 27 (depends on 19)

Use of the system or kit of claim 19 for culturing cells wherein culturing cells comprises:
a) suspending cells in the nutrient medium;
b) maintaining the cells in suspension while culturing;
c) changing the medium periodically;
d) optionally splitting the culture from time to time so as to reduce cell density; and finally
e) harvesting cells from the culture.