# US 10,104,880 B2 · Cell composition and methods of making the same

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- Number: US 10,104,880 B2
- Document ID: US10104880B2
- Status: Granted
- Kind: B2
- Application number: 12/544,949
- Filed: 2009-08-20
- Published: 2010-02-25
- Granted: 2018-10-23
- Earliest claimed: 2008-08-20
- Assignees: Celularity Inc.
- Applicants: Andy Zeitlin; Gregory Russotti; Shuyang He; Ajai Pal; Hong J. Chen; Thomas Brieva; Ryan Shorr; Brian Murphy
- Inventors: Andy Zeitlin; Gregory Russotti; Shuyang He; Ajai Pal; Hong J. Chen; Thomas Brieva; Ryan Shorr; Brian Murphy
- Web page: https://patentgrep.com/patent/US10104880B2

## CPC

- A01N 1/125: Freeze protecting agents, e.g. cryoprotectants or osmolarity regulators
- A61P 7/00: Drugs for disorders of the blood or the extracellular fluid
- A61P 19/00: Drugs for skeletal disorders
- A61P 19/04: for non-specific disorders of the connective tissue
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- A01N: PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF (preservation of food or foodstuff A23); BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES (preparations for medical, dental or toiletry purposes which kill or prevent the growth or proliferation of unwanted organisms A61K); PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

Provided herein are improved methods for the formulation of compositions comprising placental stem cells, and improved compositions and cell formulations produced thereby.

## Claims

### Claim 1

A method of making a composition comprising homogenous isolated human adherent placental cells that have been passaged in vitro at least two times, comprising:
(a) contacting said cells with a first solution comprising dextran and human serum albumin (HSA) to form a cell-containing solution;
(b) filtering out macro cell clumps from the cell-containing solution;
(c) diluting said cells to no more than about 10±3×106 cells per milliliter with a second solution comprising dextran, dimethylsulfoxide (DMSO), and HSA; and
(d) cryopreserving said cells after step (c), thereby making a composition comprising isolated human adherent placental cells.

### Claim 2 (depends on 1)

The method of claim 1, wherein said dextran is dextran 40.

### Claim 3 (depends on 1)

The method of claim 1, wherein said second solution comprises about 5.5% dextran 40.

### Claim 4 (depends on 1)

The method of claim 1, wherein said second solution comprises about 10% HSA.

### Claim 5 (depends on 1)

The method of claim 1, wherein said second solution comprises about 2.5% DMSO.

### Claim 6 (depends on 1)

The method of claim 1, wherein said second solution comprises about 7.5% to about 9% dextran.

### Claim 7 (depends on 1)

The method of claim 1, wherein said composition comprising cells comprises about 1.0±0.3×106 cells per milliliter to about 5.0±1.5×106 cells per milliliter.

### Claim 8

A method of making a composition comprising cells, comprising:
(a) suspending a plurality of homogenous isolated human adherent placental cells, which have been passaged in vitro at least two times, in a 5.5% dextran 40, 10% HSA solution to form a cell-containing solution;
(b) filtering the cell-containing solution through a 70 μM filter to filter out macro cell clumps from the cell-containing solution;
(c) diluting the cell-containing solution in 5.5% dextran 40, 10% HSA, and 5% DMSO to no more than about 10±3×106 cells/mL;
(d) cryopreserving the cells; and
(e) thawing the cells
to produce said composition comprising cells.

### Claim 9

A method of making a composition comprising cells, comprising:
(a) centrifuging a plurality of homogenous isolated human adherent placental cells, which have been passaged in vitro at least two times, to collect the cells;
(b) resuspending the cells in 5.5% dextran 40;
(c) centrifuging the cells to collect the cells;
(d) resuspending the cells in a 5.5% dextran 40 solution that comprises 10% HSA to form a cell-containing solution;
(e) filtering the cell-containing solution through a 70 μM to 100 μM filter to filter out macro cell clumps from the cell-containing solution;
(f) diluting the cell-containing solution in 5.5% dextran 40, 10% HSA, and 5% DMSO to no more than about 10±3×106 cells/mL;
(g) cryopreserving the cells; and
(h) thawing the cells
to produce said composition comprising cells.

### Claim 10

A method of making a composition comprising homogenous isolated human adherent placental cells, comprising:
(a) providing a plurality of homogenous isolated human adherent placental cells, which have been passaged in vitro at least two times, in a solution comprising 5.5% dextran 40 and 10% HSA to form a solution comprising isolated human adherent placental cells;
(b) filtering said solution comprising homogenous isolated human adherent placental cells to filter out macro cell clumps from the cell-containing solution to produce filtered isolated human adherent placental cells;
(c) diluting said filtered isolated human adherent placental cells with an amount of a solution comprising 5.5% dextran 40, 10% HSA and 5% dimethylsulfoxide (DMSO) sufficient to bring said filtered isolated human adherent placental cells to about 10±3×106 cells per milliliter;
(d) diluting said isolated human adherent placental cells with 10% dextran 40 at a ratio of about 1:1 to about 1:11 isolated human adherent placental cells:dextran 40 to produce said composition;
(e) cryopreserving the cells.

### Claim 11 (depends on 1)

The method of claim 1, wherein said filter is a 70 μM filter.

### Claim 12 (depends on 1)

The method of claim 1, wherein said filter is a 100 μM filter.

### Claim 13 (depends on 1)

The method of claim 1, wherein said isolated human adherent placental cells are CD10+, CD34− and CD105+.

### Claim 14 (depends on 13)

The method of claim 13, wherein said CD10+, CD34− and CD105+ cells are CD200+.

### Claim 15 (depends on 14)

The method of claim 14, wherein said CD10+, CD34−, CD105+ and CD200+ cells are either CD45− or CD90+.

### Claim 16 (depends on 14)

The method of claim 14, wherein said CD10+, CD34−, CD105+ and CD200+ cells are CD45− and CD90+.

### Claim 17 (depends on 1)

The method of claim 1, further comprising concentrating the composition comprising cells to about 5×106 cells per milliliter to 1×108 cells per milliliter.

### Claim 18 (depends on 17)

The method of claim 17, further comprising subcutaneously administering the composition comprising cells to a subject.

### Claim 19 (depends on 8)

The method of claim 8, further comprising diluting the com position comprising cells after step (e) 1:1 to 1:11 with 10% dextran 40.

### Claim 20 (depends on 9)

The method of claim 9, further comprising diluting the composition comprising cells after step (h) 1:1 to 1:11 with 10% dextran 40.

### Claim 21 (depends on 1)

The method of claim 1, wherein said filter is a 70 μM to 100 μM filter.

### Claim 22 (depends on 1)

The method of claim 1, wherein said second solution comprises 5.0% dextran, 10% HSA, and 2.5% DMSO.