# US 2010/0166718 A1 · Large scale pancreatic islet purification

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- Number: US 2010/0166718 A1
- Document ID: US20100166718A1
- Status: Published application
- Kind: A1
- Application number: 12/645,910
- Filed: 2009-12-23
- Published: 2010-07-01
- Earliest claimed: 2008-12-26
- Assignees: Baylor Research Institute
- Inventors: Shinichi Matsumoto; Masayuki Shimoda
- Web page: https://patentgrep.com/patent/US20100166718A1

## CPC

- A61K 35/39: Pancreas; Islets of Langerhans (Langerhans cells of epidermis A61K35/36)
- A61P 3/10: for hyperglycaemia, e.g. antidiabetics
- C12N 5/0676: {Pancreatic cells}
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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 includes a method of isolating pancreatic islets by density centrifugation wherein the pancreatic islets are loaded in a solution comprising pancreatic islets with the density gradient and the islets are isolated by centrifuging the vessel wherein the pressure on the pancreatic islets is less than 50 Pa and wherein the pancreatic islets are isolated from the gradient, the improvement comprising creating a continuous density gradient in a vessel comprising at least 100 milliliters with a bend tube that reduces convection currents that disrupt the gradient.

## Claims

### Claim 1

A method of isolating pancreatic islets comprising:
creating a continuous density gradient in a vessel comprising at least 100 milliliters with a bend tube that reduces convection currents that disrupt the gradient;
loading a solution comprising pancreatic islets in the density gradient;
centrifuging the vessel wherein the pressure on the pancreatic islets is less than 50 Pa; and
isolating the pancreatic islets from the gradient.

### Claim 2 (depends on 1)

The method of claim 1, wherein the gradient and the solution comprising pancreatic islets are topped with a dilution solution.

### Claim 3 (depends on 1)

The method of claim 1, wherein the pancreatic islets are top-loaded and in which the gradient comprises:
a high-density first layer at the bottom of the vessel,
a second layer comprising the continuous density gradient on the high density first layer,
a third layer on the second layer, the third layer comprising a low-density solution that comprises a pancreatic digest; and
a fourth layer on the third layer comprising a dilution solution.

### Claim 4 (depends on 1)

The method of claim 1, wherein the pancreatic islets are bottom-loaded and in which the gradient comprises:
a high-density first layer comprising a pancreatic digest at the bottom of the vessel; a second layer on the first layer comprising a continuous density gradient; and
a third layer comprising a dilution solution.

### Claim 5 (depends on 1)

The method of claim 1, wherein the gradient comprises a continuous density solution, further defined as comprising a low-density to a high-density by changing the volumetric ratio of low-density to high-density using the bend tube.

### Claim 6 (depends on 1)

The method of claim 1, wherein the gradient comprises a continuous density solution, further defined as comprising a low-density to a high-density comprising Iodixanol to ET-Kyoto solutions produced by changing the volumetric ratio of Iodixanol and ET-Kyoto solution using the bend tube.

### Claim 7 (depends on 1)

The method of claim 1, wherein the gradient comprises a continuous density solution, further defined as comprising a low-density (density: 1.077) to a high-density (density: 1.095-1.125) Iodixanol-ET-Kyoto solutions produced by changing the volumetric ratio of Iodixanol and ET-Kyoto solution using the bend tube.

### Claim 8 (depends on 1)

The method of claim 1, wherein the bottle has a smooth inner surface.

### Claim 9 (depends on 1)

The method of claim 1, wherein the gradient is formed using a bent-tip that reduces convection currents in the gradient when the gradient is created.

### Claim 10 (depends on 1)

The method of claim 1, wherein the vessel is 200, 250, 300, 400, 500, 600, 700, 800, 900 or 1,000 milliliters.

### Claim 11 (depends on 1)

The method of claim 1, further comprising the step of isolating the islet cells from the collection vessel comprising the islet cells.

### Claim 12 (depends on 1)

The method of claim 1, wherein the further comprising the steps of:
isolating the islet cells from the collection vessel comprising the islet cells;
washing the islet cells; and
transplanting the pancreatic islets into a new host.

### Claim 13 (depends on 1)

The method of claim 1, wherein the islets are human islets.

### Claim 14 (depends on 1)

The method of claim 1, wherein the pancreatic islets are cadaveric islets.

### Claim 15

A method of isolating pancreatic islets comprising:
creating a continuous density gradient in a vessel comprising at least 100 milliliters with a bend tube that reduces convection currents that disrupt the gradient, the continuous density solution, further defined as comprising a low-density (density: 1.077) to a high-density (density: 1.095-1.125) prepared using a bend tube;
loading pancreatic islet cells in a dilution solution on the gradient;
centrifuging the vessel wherein the pressure on the pancreatic islets is less than 50 Pa;
separating into two or more collection vessels the gradient from the top to the bottom of the vessel;
selecting the collection vessel with the pancreatic islets; and
isolating the pancreatic islets from the gradient.

### Claim 16 (depends on 15)

The method of claim 15, wherein the gradient and the solution comprising pancreatic islets are topped with a dilution solution.

### Claim 17 (depends on 15)

The method of claim 15, wherein the pancreatic islets are top-loaded and in which the gradient comprises:
a high-density first layer at the bottom of the vessel,
a second layer comprising the continuous density gradient on the high density first layer,
a third layer on the second layer, the third layer comprising a low-density solution that comprises a pancreatic digest; and
a fourth layer on the third layer comprising a dilution solution.

### Claim 18 (depends on 15)

The method of claim 15, wherein the pancreatic islets are bottom-loaded and in which the gradient comprises:
a high-density first layer comprising a pancreatic digest at the bottom of the vessel; a second layer on the first layer comprising a continuous density gradient; and
a third layer comprising a dilution solution.

### Claim 19 (depends on 15)

The method of claim 15, wherein the gradient comprises a continuous density solution, further defined as comprising a low-density (density: 1.077) to a high-density (density: 1.095-1.125) Iodixanol-ET-Kyoto solutions produced by changing the volumetric ratio of Iodixanol and ET-Kyoto solution.

### Claim 20 (depends on 15)

The method of claim 15, wherein the vessel is 150, 200, 250, 300, 400, 500, 600, 700, 800, 900 or 1,000 milliliters.

### Claim 21 (depends on 15)

The method of claim 15, wherein the further comprising transplanting the pancreatic islets into a new host.

### Claim 22 (depends on 15)

The method of claim 15, wherein the islets are human islets.

### Claim 23 (depends on 15)

The method of claim 15, wherein the pancreatic islets are cadaveric islets.

### Claim 24

A method of isolating pancreatic islets comprising:
creating a density gradient in a vessel comprising at least 100 milliliters;
loading a solution comprising pancreatic islets in a dilution solution below the density gradient;
centrifuging the vessel wherein the pressure on the pancreatic islets is less than 50 Pa;
separating into two or more collection vessels the gradient from the top to the bottom of the vessel;
selecting the collection vessel with the pancreatic islets; and
isolating the pancreatic islets from the gradient.

### Claim 25

A method of isolating pancreatic islets by density centrifugation wherein the pancreatic islets are loaded in a solution comprising pancreatic islets with the density gradient and the islets are isolated by centrifuging the vessel wherein the pressure on the pancreatic islets is less than 50 Pa and wherein the pancreatic islets are isolated from the gradient, the improvement comprising creating a continuous density gradient in a vessel comprising at least 100 milliliters with a bend tube that reduces convection currents that disrupt the gradient.

### Claim 26

One or more pancreatic cells isolated by the method comprising:
creating a continuous density gradient in a vessel comprising at least 100 milliliters with a bend tube that reduces convection currents that disrupt the gradient;
loading a solution comprising pancreatic islets in the density gradient;
centrifuging the vessel wherein the pressure on the pancreatic islets is less than 50 Pa; and
isolating the pancreatic islets from the gradient.

### Claim 27 (depends on 26)

The cells of claim 26, wherein the gradient and the solution comprising pancreatic islets are topped with a dilution solution.

### Claim 28 (depends on 26)

The cells of claim 26, wherein the pancreatic islets are top-loaded and in which the gradient comprises:
a high-density first layer at the bottom of the vessel,
a second layer comprising the continuous density gradient on the high density first layer,
a third layer on the second layer, the third layer comprising a low-density solution that comprises a pancreatic digest; and
a fourth layer on the third layer comprising a dilution solution.

### Claim 29 (depends on 26)

The cells of claim 26, wherein the pancreatic islets are bottom-loaded and in which the gradient comprises:
a high-density first layer comprising a pancreatic digest at the bottom of the vessel; a second layer on the first layer comprising a continuous density gradient; and
a third layer comprising a dilution solution.

### Claim 30 (depends on 26)

The cells of claim 26, wherein the gradient comprises a continuous density solution, further defined as comprising a low-density to a high-density by changing the volumetric ratio of low-density to high-density using the bend tube.

### Claim 31 (depends on 26)

The cells of claim 26, wherein the gradient comprises a continuous density solution, further defined as comprising a low-density to a high-density comprising Iodixanol to ET-Kyoto solutions produced by changing the volumetric ratio of Iodixanol and ET-Kyoto solution using the bend tube.

### Claim 32 (depends on 26)

The cells of claim 26, wherein the gradient comprises a continuous density solution, further defined as comprising a low-density (density: 1.077) to a high-density (density: 1.095-1.125) Iodixanol-ET-Kyoto solutions produced by changing the volumetric ratio of Iodixanol and ET-Kyoto solution using the bend tube.