# US 9,040,297 B2 · Methods of expanding embryonic stem cells in a suspension culture

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- Number: US 9,040,297 B2
- Document ID: US9040297B2
- Status: Granted
- Kind: B2
- Application number: 12/309,817
- Filed: 2007-08-02
- Published: 2009-12-17
- Granted: 2015-05-26
- Earliest claimed: 2006-08-02
- Assignees: Technion Research & Development Foundation Limited
- Applicants: Michal Amit; Joseph Itskovitz-Eldor
- Inventors: Michal Amit; Joseph Itskovitz-Eldor
- Web page: https://patentgrep.com/patent/US9040297B2

## 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 5/0031: {Serum-free culture media}
- C12N 5/0037: {Serum-free medium, which may still contain naturally-sourced components}
- C12N 5/0043: {Medium free of human- or animal-derived components}
- C12N 2501/115: Basic fibroblast growth factor (bFGF, FGF-2)
- C12N 2501/15: Transforming growth factor beta (TGF-β)
- C12N 2501/2306: Interleukin-6 (IL-6)
- C12N 2501/235: Leukemia inhibitory factor [LIF]
- C12N 2533/12: Glass
- C12N 2533/90: Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

A method of expanding and maintaining human embryonic stem cells (ESCs) in an undifferentiated state by culturing the ESCs in a suspension culture under culturing conditions devoid of substrate adherence is provided. Also provided are a method of deriving ESC lines in the suspension culture and methods of generating lineage-specific cells from ESCs which were expanded in the suspension culture of the present invention.

## Claims

### Claim 1

A method of expanding and maintaining human pluripotent stem cells in a pluripotent undifferentiated state, the method comprising culturing the human pluripotent stem cells in a suspension culture under culturing conditions which allow expansion of the human pluripotent stem cells in the pluripotent undifferentiated state for at least 5 passages without adherence of said cells to a substrate or to feeder cells, wherein said substrate is selected from the group consisting of an extracellular matrix, a glass microcarrier and a bead, wherein said conditions comprise culturing the cells in a serum-free culture medium, and wherein said medium comprises a TGFβ isoform, thereby expanding and maintaining the human pluripotent stem cells in the pluripotent undifferentiated state.

### Claim 2

A method of deriving an embryonic stem cell line, the method comprising:
(a) obtaining an embryonic stem cell from a pre-implantation stage blastocyst, post-implantation stage blastocyst and/or a genital tissue of a fetus; and
(b) culturing said embryonic stem cell in a suspension culture, under culturing conditions which allow expansion of said embryonic stem cells in a pluripotent undifferentiated state for at least 5 passages without adherence of said cells to a substrate or to feeder cells, wherein said substrate is selected from the group consisting of an extracellular matrix, a glass microcarrier and a bead, wherein said conditions comprise culturing the cells in a serum-free culture medium, wherein said medium comprises a TGFβ isoform,
thereby deriving the embryonic stem cell line.

### Claim 3 (depends on 1)

A method of generating lineage-specific cells from human pluripotent stem cells, the method comprising:
(a) culturing the human pluripotent stem cells according to the method of claim 1 to thereby obtain expanded, pluripotent undifferentiated human pluripotent stem cells; and
(b) subjecting said expanded, undifferentiated human pluripotent stem cells to culturing conditions suitable for differentiating and/or expanding lineage specific cells;
thereby generating the lineage-specific cells from the human pluripotent stem cells.

### Claim 4 (depends on 1)

A method of generating embryoid bodies from human embryonic stem cells, the method comprising:
(a) culturing the human embryonic stem cells according to the method of claim 1 to thereby obtain expanded, pluripotent undifferentiated human embryonic stem cells; and
(b) subjecting said expanded, undifferentiated human embryonic stem cells to culturing conditions suitable for differentiating said human embryonic stem cells to embryoid bodies;
thereby generating the embryoid bodies from the human embryonic stem cells.

### Claim 5 (depends on 1)

A method of generating lineage-specific cells from embryonic stem cells, the method comprising:
(a) culturing the human embryonic stem cells according to the method of claim 1 to thereby obtain expanded, pluripotent undifferentiated human embryonic stem cells;
(b) subjecting said expanded, undifferentiated human embryonic stem cells to culturing conditions suitable for differentiating said expanded, undifferentiated human embryonic stem cells to embryoid bodies; and
(c) subjecting cells of said embryoid bodies to culturing conditions suitable for differentiating and/or expanding lineage specific cells;
thereby generating the lineage-specific cells from the human embryonic stem cells.

### Claim 6

A culture medium comprising serum replacement, a soluble interleukin-6 receptor (sIL6R), a soluble interleukin-6 (IL6) and basic fibroblast growth factor (bFGF), wherein the culture medium is suitable for expanding and maintaining human pluripotent stem cells in a pluripotent undifferentiated state for at least 5 passages.

### Claim 7

A culture medium comprising at least 2000 units per milliliter (u/ml) leukemia inhibitor factor (LIF), wherein the culture medium is suitable for expanding and maintaining human pluripotent stem cells in a pluripotent undifferentiated state for at least 5 passages.

### Claim 8 (depends on 6)

A cell culture comprising cells and the culture medium of claim 6.

### Claim 9

A cell culture comprising human embryonic stem cells and a culture medium which comprises at least 2000 u/ml of leukemia inhibitor factor (LIF), wherein said culture medium is suitable for expanding and maintaining said human embryonic stem cells in a pluripotent undifferentiated state for at least 5 passages.

### Claim 10 (depends on 1)

The method of claim 1, wherein said expansion comprises obtaining at least 9×1015 cells from a single embryonic stem cell following 3 months.

### Claim 11 (depends on 1)

The method of claim 1, wherein a medium of said suspension culture is serum-free, serum replacement-free, xeno-free, feeder-free and protein carrier-free.

### Claim 12 (depends on 1)

The method of claim 1, wherein said TGFβ isoform is a TGFβ isoform 1 (TGFβ1).

### Claim 13 (depends on 1)

The method of claim 1, wherein said TGFβ isoform is a TGFβ isoform 3 (TGFβ3).

### Claim 14 (depends on 12)

The method of claim 12, wherein said TGFβ1 is present at a concentration of at least 0.06 ng/ml.

### Claim 15 (depends on 13)

The method of claim 13, wherein said TGFβ3 is present at a concentration of at least 0.5 ng/ml.

### Claim 16 (depends on 6)

The culture medium of claim 6, wherein said bFGF is present at a concentration of at least 2 ng/ml.

### Claim 17 (depends on 5)

The method of claim 5, further comprising isolating lineage specific cells following step (b).

### Claim 18 (depends on 1)

The method of claim 1, wherein the pluripotent stem cells cultured in said suspension culture exhibit normal chromosomal karyotype following at least 2 passages.

### Claim 19 (depends on 1)

The method of claim 1, wherein the pluripotent stem cells cultured in said suspension culture exhibit a doubling time of at least 20 hours.

### Claim 20

A method of expanding and maintaining human pluripotent stem cells in a pluripotent undifferentiated state for at least 5 passages the method comprising culturing the human pluripotent stem cells in a suspension culture under culturing conditions which allow expansion of the human pluripotent stem cells in the pluripotent undifferentiated state without adherence of said cells to a substrate or to feeder cells, wherein said substrate is selected from the group consisting of an extracellular matrix, a glass microcarrier and a bead, wherein said conditions comprise culturing the cells in a culture medium, which comprises a soluble interleukin-6 receptor (sIL6R), wherein said sIL6R is present at a concentration of 15-30 ng/ml, thereby expanding and maintaining the human pluripotent stem cells in the pluripotent undifferentiated state for at least 5 passages.

### Claim 21 (depends on 20)

The method of claim 20, wherein said medium further comprises soluble interleukin-6 (IL6).

### Claim 22

A method of expanding and maintaining human pluripotent stem cells in a pluripotent undifferentiated state for at least 5 passages, the method comprising culturing the human pluripotent stem cells in a suspension culture under culturing conditions which allow expansion of the human pluripotent stem cells in the pluripotent undifferentiated state without adherence of said cells to a substrate or to feeder cells, wherein said substrate is selected from the group consisting of an extracellular matrix, a glass microcarrier and a bead, wherein said conditions comprise culturing the cells in a culture medium which comprises leukemia inhibitor factor (LIF), wherein said LIF is present at a concentration of at least 2000 units per milliliter (u/ml), thereby expanding and maintaining the human pluripotent stem cells in the undifferentiated state for at least 5 passages.

### Claim 23 (depends on 1)

The method of claim 1, wherein said culturing conditions are non-dynamic culturing conditions.

### Claim 24 (depends on 8)

The cell culture of claim 8, wherein said cells are embryonic stem cells and whereas said culture medium is capable of maintaining said embryonic stem cells in an undifferentiated state in a suspension culture.

### Claim 25 (depends on 22)

The method of claim 22, wherein said LIF is present at a concentration of at least 3000 units per milliliter (u/ml).

### Claim 26 (depends on 1)

The method of claim 1, wherein said pluripotent stem cells are embryonic stem cells.

### Claim 27

A method of expanding and maintaining human pluripotent stem cells in a pluripotent undifferentiated state for at least 5 passages, the method comprising culturing the human pluripotent stem cells in a suspension culture without adherence of said cells to a substrate or to feeder cells under conditions comprising a serum-free culture medium selected from the group consisting of: a culture medium comprising a soluble interleukin-6 receptor (sIL6R) at a concentration of at least 10 nanogram per milliliter (ng/ml) and soluble interleukin-6 (IL6), a culture medium comprising at least 2000 units per milliliter (u/ml) leukemia inhibitor factor (LIF), and a culture medium which comprises a TGFβ isoform, thereby expanding and maintaining the human pluripotent stem cells in the pluripotent undifferentiated state for at least 5 passages.

### Claim 28 (depends on 27)

The method of claim 27, wherein said TGFβ isoform is a TGFβ isoform 1 (TGFβ1).

### Claim 29 (depends on 27)

The method of claim 27, wherein said TGFβ isoform is a TGFβ isoform 3 (TGFβ3).

### Claim 30 (depends on 27)

The method of claim 27, wherein said pluripotent stem cells are embryonic stem cells.

### Claim 31 (depends on 6)

The culture medium of claim 6, wherein said sIL6R is present at a concentration of 15-30 nanogram per milliliter (ng/ml).

### Claim 32

A culture medium comprising serum replacement, a soluble interleukin-6 receptor (sIL6R) at a concentration of 15-30 nanogram per milliliter (ng/ml), a soluble interleukin-6 (IL6) and basic fibroblast growth factor (bFGF) at a concentration of at least 2 ng/ml, wherein said culture medium is suitable for expanding and maintaining said human embryonic stem cells in a pluripotent undifferentiated state for at least 5 passages.

### Claim 33 (depends on 32)

The culture medium of claim 32, wherein said soluble IL6 is provided at a concentration of 25 ng/ml.

### Claim 34

A culture medium comprising at least 2000 units per milliliter (u/ml) leukemia inhibitor factor (LIF) and basic fibroblast growth factor (bFGF) at a concentration of at least 2 ng/ml, wherein said culture medium is suitable for expanding and maintaining said human embryonic stem cells in a pluripotent undifferentiated state for at least 5 passages.

### Claim 35 (depends on 34)

The culture medium of claim 34, wherein said bFGF is at a concentration of 4 ng/ml bFGF.

### Claim 36 (depends on 12)

The method of claim 12, wherein said medium further comprises basic fibroblast growth factor (bFGF).

### Claim 37 (depends on 36)

The method of claim 36, wherein said bFGF is present at a concentration of at least 2 ng/ml.

### Claim 38 (depends on 36)

The method of claim 36, wherein said TGFβ1 is present at a concentration of at least 0.06 ng/ml and wherein said bFGF is present at a concentration of 2 ng/ml.

### Claim 39 (depends on 13)

The method of claim 13, wherein said medium further comprises basic fibroblast growth factor (bFGF).

### Claim 40 (depends on 39)

The method of claim 39, wherein said bFGF is present at a concentration of at least 2 ng/ml.

### Claim 41 (depends on 39)

The method of claim 39, wherein said TGFβ3 is present at a concentration of at least 0.5 ng/ml and wherein said bFGF is present at a concentration of at least 2 ng/ml.

### Claim 42 (depends on 1)

The method of claim 1, wherein said pluripotent stem cells are capable of forming teratomas containing representative tissues of all three embryonic germ layers.

### Claim 43 (depends on 1)

The method of claim 1, wherein said pluripotent stem cells express OCT4.

### Claim 44 (depends on 1)

The method of claim 1, wherein said TGFβ isoform is present in said medium throughout said at least 5 passages.

### Claim 45

A method of expanding and maintaining human pluripotent stem cells in an undifferentiated state, the method comprising culturing the human pluripotent stem cells in a suspension culture under culturing conditions devoid of substrate adherence, and which allow expansion of the human pluripotent stem cells in the undifferentiated state for at least 5 passages, thereby expanding and maintaining the human pluripotent stem cells in the undifferentiated state.

### Claim 46 (depends on 45)

The method of claim 45, wherein said culturing conditions comprise a serum-free culture medium selected from the group consisting of: a culture medium comprising a soluble interleukin-6 receptor (sIL6R) at a concentration of at least 10 nanogram per milliliter (ng/ml) and soluble interleukin-6 (IL6), a culture medium comprising at least 1000 units per milliliter (u/ml) leukemia inhibitor factor (LIF), a culture medium comprising an IL6RIL6 chimera and a culture medium which comprises a TGFβ isoform.

### Claim 47 (depends on 45)

The method of claim 45, wherein said substrate comprises components of extracellular matrix, a glass microcarrier or beads.

### Claim 48 (depends on 45)

The method of claim 45, wherein said culturing is performed in a culture vessel having an internal surface designed such that the human pluripotent stem cells cultured therein are unable to adhere or attach to said surface.