Ultrasonic cavitation derived stromal or mesenchymal vascular extracts and cells derived therefrom obtained from adipose tissue and use thereof

• US 8,440,440 B2
• Filed: 12/12/2011
• Issued: 05/14/2013
• Est. Priority Date: 12/27/2010
• Status: Expired due to Fees

• Current Assignee: Stroma Cell Therapeutics LLC
• Original Assignee: Intellicell Biosciences, Inc.
• Inventors: Victor, Steven
• Primary Examiner(s): Landau, Sharmila G.
• Assistant Examiner(s): PYLA, PAUL D
• Application Number:
  US13/323,030
• Publication Number:
  US 20120164113A1
• Time in Patent Office: 519 Days
• Field of Search: None
• US Class Current: 435/173.9
• CPC Class Codes: A61K 35/35 Fat tissue; Adipocytes; Stromal cells; Connective tissues adipose-derived stem cells A61K35/28; collagen A61K38/39
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 19/00 Drugs for skeletal disorders
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 25/06 Antimigraine agents
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: C12N 13/00 Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
• CPC Class Codes: C12N 5/0667 Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
• CPC Class Codes: G01N 2333/70503 Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
• CPC Class Codes: G01N 2333/70596 Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025

Abstract

Methods of treating using adipose tissue using ultrasonic cavitation to dissociate the fat cells and blood vessels contained within adipose tissue and thereby obtain mesenchymal or stromal vascular fractions for use in human subjects are provided. These methods preferably do not include the use of any exogenous dissociating enzymes such as collagenase and result in increased numbers of the cells which constitute the mesenchymal or stromal vascular fractions (about 10-fold greater) than methods which use collagenase to isolate these cells.

Claims

1. A method for recovering stromal vascular fraction cells from blood vessels contained in or proximate to human adipose tissue comprising treating about 45-60 cc of adipose tissue comprising fat cells, blood vessels, and a stromal vascular fraction in a 60 cc tube with ultrasonic cavitation for about 10 minutes using a 14 mm ultrasonic rod,

wherein ultrasonic cavitation is effected by slowly increasing cycle and amplitude once the ultrasonic rod is submerged into the tube containing the adipose tissue,

wherein after 5 minutes, the ultrasonic process is stopped and the ultrasonic rod is adjusted up in the tube and continued for the 5 minutes, and

wherein the fat cells and blood vessels are exploded,

thereby releasing substantial numbers of intact stromal vascular fraction cells while substantially maintaining the viability of the cells constituting the stromal vascular fraction.

2. The method of claim 1, wherein said method does not include the addition of an enzyme that breaks down collagen or a endopeptidase.

3. The method of claim 1, wherein the adipose tissue is obtained from the stromal compartment of the body of a living or non-living donor, mesenchymal compartment of the body of a living or non-living donor, a liposuction derived aspirate, solid fat obtained from a human cadaver, or solid fat obtained from a living donor.

4. The method of claim 1, wherein the adipose tissue is comprised in phosphate buffered saline, normal saline, or another biologically acceptable liquid.

5. The method of claim 1, wherein a high intensity ultrasonic processor or high intensity cavitation device is used.

6. The method of claim 1, wherein after ultrasonic cavitation the resultant composition is allowed to settle or is centrifuged resulting in the separation of fat and stromal vascular fraction cells into a supernatant and a pellet, respectively, optionally, the stromal vascular fraction cells may be isolated from the pellet.

7. The method of claim 1, wherein after ultrasonic cavitation the stromal vascular fraction cells are assayed for the presence of adipose-derived stem cells including CD34 and/or Thy-1 or CD90 expressing stem cells.

8. The method of claim 7, wherein the stromal vascular fraction cells are assayed by flow cytometry.

9. The method of claim 7, wherein after ultrasonic cavitation the stromal vascular fraction cells are fractionated using fluorescence activated cell sorting (FACS) based on cell surface antigens which are specific to adipose-derived stem cells.

10. The method of claim 9, wherein the resultant isolated adipose-derived stem cells are expanded in culture.

11. The method of claim 9, wherein the resultant isolated adipose-derived stem cells are infused into a patient.

12. The method of claim 1, wherein the stromal vascular fraction comprises endothelial cells, endothelial progenitor cells, mesenchymal stem cells, macrophages, dendritic cells, Kupffer cells, platelets, granulocytes, B cells, T cells, NK cells, lymphocytes, megakaryocytes, neutrophil granulocytes, osteoclasts, or neutrophils.

13. The method of claim 1, wherein the stromal vascular fraction and cells derived therefrom are used in a cosmetic surgery application, to promote wound healing, are used in a tissue filler or in association with breast augmentation or reconstruction, tissue engineering, or burn treatment.

14. The method of claim 1, wherein after ultrasonication the stromal vascular fraction cells are poured into a specimen tube for filtering.

15. The method of claim 1, wherein the stromal vascular fraction cells are divided into 2 parts to which optionally are added equal amounts of 0.9% sodium chloride and these parts are centrifuged resulting in a layered sample with liquid on the bottom with a pellet and fat on top.

16. The method of claim 15, wherein centrifugation is effected for about 3 minutes at 500 RPM.