# US 12,005,084 B2 · Composition derived from mammalian umbilical cord and Whartons jelly for use in therapeutic and regenerative applications

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- Number: US 12,005,084 B2
- Document ID: US12005084B2
- Status: Granted
- Kind: B2
- Application number: 16/731,911
- Filed: 2019-12-31
- Published: 2020-04-30
- Granted: 2024-06-11
- Earliest claimed: 2016-07-26
- Assignees: Equi-Stem Biotechnologies LLC
- Applicants: Equi-Stem Biotechnologies LLC
- Inventors: Todd Flower
- Web page: https://patentgrep.com/patent/US12005084B2

## CPC

- A61K 35/51: Umbilical cord; Umbilical cord blood; Umbilical stem cells
- A61K 9/0014: {Skin, i.e. galenical aspects of topical compositions (non-active ingredients are additionally classified in A61K47/00; A61K9/0009, A61K9/0021, A61K9/7015, A61K9/7023 take precedence; cosmetic preparations A61K8/00, A61Q; preparations for wound dressings or bandages A61L26/00)}
- A61K 35/28: Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
- A61K 35/44: Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
- C12N 5/0605: {Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly}
- 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)
- 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 preparation of a solution used for therapeutic and regenerative applications having a concentration of at least about 0.05 microgram of total protein extract is disclosed. The method includes collecting an umbilical cord, incubating the umbilical cord in a solution containing antibiotics for sterilization, washing the umbilical cord in a buffered salt solution, homogenizing the umbilical cord in the buffered salt solution so as to produce an aqueous solution, applying an ultrasonic processer to the aqueous solution to sonicate and disrupt umbilical cord tissue, placing the aqueous solution into a centrifuge to separate the aqueous solution into a soluble component and a non-soluble component, filtrating and discarding the non-soluble component of the aqueous solution from the centrifuge through a 0.2 micron filter, and measuring protein concentration of the soluble component to insure at least about 0.05 microgram of total protein extract.

## Claims

### Claim 1

A method of preparation of a solution used for therapeutic and regenerative applications having a concentration of at least 0.05 micrograms of total protein extract, the method comprising the steps of:
a) collecting an umbilical cord;
b) incubating the umbilical cord in a solution containing antibiotics for sterilization at a temperature not exceeding 40 degrees Centigrade;
c) washing the umbilical cord in a first buffered salt solution;
d) homogenizing the umbilical cord in a second buffered salt solution so as to produce an aqueous solution comprising umbilical cord tissue proteins, cytokines, growth factors, and RNA, wherein said proteins comprise Brain Derived Neurotropic Factor (BDNF), Glial Derived Neurotrophic Factor (GDNF), Stromal Cell Derived Factor 1 (SDF-1), Angiopoietin-2, Chemokine Ligand 16 (CXCL-16), Neutrophil-Activating Protein-2 (Nap-2), Glucocorticoid-induced Tumor Necrosis Factor Receptor (GITR), and Fibroblast Growth Factor 20 (FGF-20);
e) applying an ultrasonic processer to the aqueous solution to sonicate and disrupt umbilical cord tissue, wherein the ultrasonic processor is applied at a power output of 300 W at a frequency of 20 kHz, wherein sonication occurs in 3 second cycles of 2 seconds on and 1 second off for a total duration of 5 minutes;
f) placing the aqueous solution into a centrifuge at about 5,500-7,500 rpm to separate the aqueous solution into a soluble component and a non-soluble component;
g) filtrating the non-soluble component of the aqueous solution from the centrifuge through a 0.2 micron filter and discarding the non-soluble component that was filtered; and
h) measuring protein concentration of the soluble component to ensure at least 0.05 microgram of total protein extract.

### Claim 2 (depends on 1)

The method of claim 1, wherein the solution that is used for therapeutic and regenerative applications has a therapeutically effective dose ranging from 0.001 μg/ml to 5 μg/ml.

### Claim 3 (depends on 2)

The method of claim 2, wherein the step of collecting the umbilical cord further comprises collecting an umbilical cord from a mammal at the time of birth of an offspring of the mammal.

### Claim 4 (depends on 3)

The method of claim 3, wherein the step of homogenizing the umbilical cord further comprises breaking up the umbilical cord in the second buffered salt solution using a blender for 5 minutes at room temperature so as to produce an aqueous solution, including minced tissue less than 1 mm in size.

### Claim 5 (depends on 4)

The method of claim 4, wherein the step of placing the aqueous solution into a centrifuge further comprises placing the aqueous solution into a centrifuge at about 6,000 rpm.

### Claim 6 (depends on 5)

The method of claim 5, wherein the soluble component is combined with a pharmaceutically acceptable carrier, resulting in a mixture.

### Claim 7

A method of preparation of a solution for topical application having a concentration of at least 2 micrograms of total protein extract, the method comprising the steps of:
a) collecting a mammalian umbilical cord;
b) incubating the umbilical cord in a solution containing antibiotics for sterilization at a temperature not exceeding 40 degrees Centigrade;
c) washing the umbilical cord in a first buffered salt solution;
d) homogenizing the umbilical cord for 5 minutes in a second buffered salt solution so as to produce an aqueous solution comprising umbilical cord tissue proteins, cytokines, growth factors, and RNA, wherein said proteins comprise Brain Derived Neurotropic Factor (BDNF), Glial Derived Neurotrophic Factor (GDNF), Stromal Cell Derived Factor 1 (SDF-1), Angiopoietin-2, Chemokine Ligand 16 (CXCL-16), Neutrophil-Activating Protein-2 (Nap-2), Glucocorticoid-induced Tumor Necrosis Factor Receptor (GITR), and Fibroblast Growth Factor 20 (FGF-20);
e) applying an ultrasonic processer to the aqueous solution to sonicate and disrupt the umbilical cord tissue, wherein the ultrasonic processor is applied at a power output of 300 W at a frequency of 20 kHz, wherein sonication occurs in 3 second cycles of 2 seconds on and 1 second off for a total duration of 5 minutes;
f) placing the aqueous solution into a centrifuge at 5,500-7,500 rpm to separate the aqueous solution into a soluble component and a non-soluble component;
g) filtrating the non-soluble component of the aqueous solution from the centrifuge through a 0.2 micron filter and discarding the non-soluble component that was filtered; and
h) measuring protein concentration of the soluble component to ensure at least 2 micrograms of total protein extract.

### Claim 8 (depends on 7)

The method of claim 7, wherein the soluble component of the aqueous solution has a concentration of at least 2 micrograms of total protein extract, resulting in a therapeutically effective dose.

### Claim 9 (depends on 8)

The method of claim 8, wherein the step of collecting the umbilical cord further comprises collecting an umbilical cord from a mammal at the time of birth of an offspring of the mammal.

### Claim 10 (depends on 9)

The method of claim 9, wherein the step of homogenizing the umbilical cord further comprises breaking up the umbilical cord in the second buffered salt solution using a blender for 5 minutes at room temperature so as to produce an aqueous solution including minced tissue less than 1 mm in size.

### Claim 11 (depends on 10)

The method of claim 10, wherein the step of measuring the protein concentration of the soluble component further comprises measuring additional characteristics of the soluble component.

### Claim 12 (depends on 11)

The method of claim 11, further comprising mixing the soluble component with liposomes, resulting in a mix.

### Claim 13 (depends on 12)

The method of claim 12, further comprising mixing the mix with cream as a vehicle for application to a mammal, so as to produce a mixed cream.