# US 10,246,680 B2 · Methods and compositions for feeder-free pluripotent stem cell media containing human serum

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- Number: US 10,246,680 B2
- Document ID: US10246680B2
- Status: Granted
- Kind: B2
- Application number: 15/050,069
- Filed: 2016-02-22
- Published: 2016-08-04
- Granted: 2019-04-02
- Earliest claimed: 2007-10-19
- Assignees: ViaCyte, Inc.
- Applicants: ViaCyte, Inc.
- Inventors: Allan J Robins; Thomas C Schulz
- Web page: https://patentgrep.com/patent/US10246680B2

## 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 5/068: {Stem cells; Progenitors}
- C12N 2500/84: from mammals
- C12N 2500/98: Xeno-free medium and culture conditions
- C12N 2501/15: Transforming growth factor beta (TGF-β)
- C12N 2506/02: from embryonic cells
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention provides compositions and methods for the culture and maintenance of pluripotent stem cells. More particularly, the present invention provides for compositions and methods for culturing, maintaining, growing and stabilizing primate pluripotent stem cells in a feeder-free defined media further comprising human serum, or a soluble attachment component of the human serum, for promoting cell attachment.

## Claims

### Claim 1

A composition comprising human pluripotent stem cells and a medium comprising an effective amount of a soluble human serum attachment component, wherein the human pluripotent stem cells are in contact with the medium in a culture vessel that is not pre-coated with an extracellular matrix and wherein the effective amount of the soluble human serum attachment component promotes growth of the human pluripotent stem cells.

### Claim 2 (depends on 1)

The composition of claim 1, wherein the attachment component is a fraction of human serum having a molecular weight greater than 100 kDa, greater than 150 kDa, greater than 200 kDa, greater than 250 kDa, or greater than 300 kDa.

### Claim 3 (depends on 1)

The composition of claim 1, wherein the effective amount of the soluble human serum attachment component is about 0.5% to about 10% of the medium.

### Claim 4 (depends on 1)

The composition of claim 1, wherein the human pluripotent stem cells are human embryonic stem cells.

### Claim 5 (depends on 1)

The composition of claim 1, wherein the composition further comprises definitive endoderm cells and the medium further comprises Activin A.

### Claim 6 (depends on 1)

The composition of claim 1, wherein the medium further comprises a TGFβ superfamily member.

### Claim 7 (depends on 1)

The composition of claim 1, wherein the medium is feeder-free.

### Claim 8 (depends on 1)

The composition of claim 1, wherein the medium is not conditioned.

### Claim 9 (depends on 1)

The composition of claim 1, wherein the human pluripotent stem cells proliferate in an undifferentiated state.

### Claim 10 (depends on 1)

The composition of claim 1, wherein the human pluripotent stern cells are differentiable in vitro.

### Claim 11 (depends on 1)

The composition of claim 1, wherein the human pluripotent stem cells are dissociated into a single cell suspension.

### Claim 12 (depends on 1)

The composition of claim 1, wherein the culture vessel is a bioreactor.

### Claim 13

A composition comprising human pluripotent stem cells and an effective amount of a soluble human serum attachment component to promote growth of the human pluripotent stem cells in a medium, wherein the cells, attachment component, and medium are in an uncoated culture vessel.

### Claim 14 (depends on 1)

A method of culturing human pluripotent stem cells comprising culturing the composition of claim 1.

### Claim 15 (depends on 14)

The method of claim 14, wherein the attachment component is a fraction of human serum having a molecular weight greater than 100 kDa, greater than 150 kDa, greater than 200 kDa, greater than 250 kDa, or greater than 300 kDa.

### Claim 16 (depends on 14)

The method of claim 14, wherein the human pluripotent stem cells are human embryonic stem cells.

### Claim 17 (depends on 14)

The method of claim 14, wherein the composition is feeder-free.

### Claim 18 (depends on 14)

The method of claim 14, wherein the medium is not conditioned.

### Claim 19 (depends on 14)

The method of claim 14, wherein the medium comprises glucose.

### Claim 20 (depends on 1)

The composition of claim 1, wherein the effective amount of the soluble human serum attachment component promotes growth of the human pluripotent stem cells by reducing deterioration in morphological quality of the human pluripotent stem cells.