# US 9,752,138 B2 · Methods and apparatuses for isolating and preparing stem cells

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- Number: US 9,752,138 B2
- Document ID: US9752138B2
- Status: Granted
- Kind: B2
- Application number: 14/045,828
- Filed: 2013-10-04
- Published: 2014-04-03
- Granted: 2017-09-05
- Earliest claimed: 2009-04-30
- Assignees: AdiStem Ltd.
- Applicants: AdiStem, Ltd.
- Inventors: Bill Paspaliaris; James Andrew Farrant Thornton
- Web page: https://patentgrep.com/patent/US9752138B2

## CPC

- C12N 13/00: Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
- A61K 35/12: Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells (vaccines or medicinal preparations containing antigens or antibodies A61K39/00)
- C12N 5/0667: {Adipose-derived stem cells [ADSC]; Adipose stromal stem cells}
- C12N 2500/36: Lipids
- C12N 2501/135: Platelet-derived growth factor [PDGF]
- C12N 2501/70: Enzymes
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)

## Abstract

The present invention relates to a method of preparing a population of stem cells for autologous implantation to a subject. The cells are activated by irradiating the cells with one or more wavelengths of yellow and red and/or green light. In particular, the cells are irradiated with 575-595 nm (5-20 mW), and 630-635 nm or 660-670 nm (10-100 mW) and/or 510-540 nm (10-60 mW) of monochromatic light for 30-60 mins. Preferably the stem cells are adipose-derived stem cells. Also the invention relates to therapeutic applications of the activated stem cells.

## Claims

### Claim 1

A method of preparing a population of adipose-derived stem cells (ADSC) for autologous implantation to a subject, comprising:
(i) collecting adipose tissue and blood from a subject,
(ii) isolating ADSCs cells from the adipose tissue and platelet rich plasma (PRP) from the blood,
(iii) activating the isolated ADSCs by irradiation with yellow light at a wavelength of 575-595 nm and at 5-20 mW, red light at wavelength of 630-635 nm and at 10-100 mW, and green light at a wavelength of 510-540 nm and at 10-60 mW, for 30-60 minutes,
(iv) mixing the activated ADSCs with the PRP, thereby preparing a population of ADSCs for autologous implantation.

### Claim 2 (depends on 1)

The method of claim 1, wherein the ADSCs are irradiated with yellow light at a wavelength of 595 nm and at 20 mW, red light at wavelength of 635 nm and at 60 mW, and green light at a wavelength of 535 nm and at 60 mW, for 30-60 minutes.

### Claim 3 (depends on 1)

The method of claim 1, wherein the isolated ADSCs in step (ii) are exposed to one or more growth factors.

### Claim 4 (depends on 3)

The method of claim 3 wherein the growth factors are Epidermal Growth Factor (EGF); Platelet-Derived Growth Factor (PDGF); Fibroblast Growth Factor (FGF); Transforming Growth Factor-β (TGF-β); Erythropoietin (EPO); Insulin-like Growth Factor-I (IGF-I); Insulin-like Growth Factor-II; and/or Tumor Necrosis Factor-α (TNF-α).

### Claim 5 (depends on 3)

The method of claim 3 wherein the PRP comprises one or more growth factors.

### Claim 6 (depends on 1)

The method of claim 1, wherein step (i) comprises exposing the adipose tissue to a lipid dissolving agent.

### Claim 7 (depends on 6)

The method of claim 6 wherein said lipid dissolving agent is lecithin.

### Claim 8 (depends on 1)

The method of claim 1, wherein step (i) comprises exposing the adipose tissue to collagenase.

### Claim 9 (depends on 1)

The method of claim 1, wherein the adipose tissue is isolated from the subject by liposuction and/or lipoplasty.

### Claim 10 (depends on 1)

The method of claim 1 comprising:
i) processing the sample of adipose tissue from the subject to obtain a concentrated population of ADSC, said processing comprising: removing free lipid and single cell components of the tissue by rinsing; disaggregating the tissue using mechanical forces, collagenase and lecithin digestion, separating and concentrating the ADSC using centrifugation; and
ii) activating the population of ADSC by irradiating the cells with yellow light at a wavelength of 575-595 nm and at 5-20 mW, red light at wavelength of 630-635 nm and at 10-100 mW, and green light at a wavelength of 510-540 nm and at 10-60 mW, for 30-60 mins, and incubating the ADSCs in the presence of PRP prepared from the patient to be treated at 30° C. to 38° C. for 5-120 minutes.