Systems and methods for isolating and using clinically safe adipose derived regenerative cells

• US 8,883,499 B2
• Filed: 04/20/2012
• Issued: 11/11/2014
• Est. Priority Date: 12/07/2001
• Status: Expired due to Fees

• Current Assignee: Lorem Vascular Pte. Ltd.
• Original Assignee: Cytori Therapeutics Incorporated
• Inventors: Hedrick, Marc H., Fraser, John K., Schulzki, Michael J., Byrnes, Bobby, Carlson, Grace, Schreiber, Ronda Elizabeth, Wulur, Isabella
• Primary Examiner(s): Lankford, Jr., Blaine
• Application Number:
  US13/452,349
• Publication Number:
  US 20120264200A1
• Time in Patent Office: 935 Days
• Field of Search: None
• US Class Current: 435/366
• CPC Class Codes: A61B 17/00 Surgical instruments, devices or methods, e.g. tourniquets A61B18/00 takes precedence; contraceptive devices, pessaries, or applicators therefor A61F6/00; eye surgery A61F9/007; ear surgery A61F11/00
• CPC Class Codes: A61K 35/28 Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
• CPC Class Codes: C12M 29/04 Filters; Permeable or porous membranes or plates, e.g. dialysis
• CPC Class Codes: C12M 45/00 Means for pre-treatment of biological substances
• CPC Class Codes: C12M 47/04 Cell isolation or sorting purging biological preparations of unwanted cells C12N5/0081, determining the presence or kind of microorganism C12Q1/04
• CPC Class Codes: C12N 5/0667 Adipose-derived stem cells [ADSC]; Adipose stromal stem cells

Abstract

Systems and methods are described that are used to separate cells from a wide variety of tissues. In particular, automated systems and methods are described that separate regenerative cells, e.g., stem and/or progenitor cells, from adipose tissue. The systems and methods described herein provide rapid and reliable methods of separating and concentrating regenerative cells suitable for re-infusion into a subject.

Claims

1. An automated cell processing unit for isolating a concentrated cell population comprising CD31 positive cells from adipose tissue, wherein said cell processing unit is configured to maintain a closed system, comprising:

a tissue collection chamber that is configured to receive a first portion of unprocessed adipose tissue from said subject while maintaining a closed system, wherein said tissue collection chamber comprises an aspiration port;

a first filter that is disposed within said tissue collection chamber, wherein said first filter is configured to substantially retain a first component of said unprocessed adipose tissue and substantially pass a second component of said unprocessed adipose tissue, such that said first filter substantially separates said first component from said second component, and wherein said first component comprises a cell population that comprises adipose-derived stem cells and said second component comprises lipid, blood, mature adipocytes and washing solution;

a processing chamber that is joined to said tissue collection chamber by a conduit such that a closed system is maintained, wherein said processing chamber comprises a first filter, a centrifuge, or a spinning membrane filter, or any combination thereof;

a programmable processing device capable of communication with said tissue collection chamber and said processing chamber, wherein said processing device operates a vacuum or a pump configured to use pressure to control ingress and egress of tissues through said automated cell processing unit; and

an outlet wherein said outlet is configured to allow the aseptic removal of a concentrated population comprising CD31 positive cells obtained from said processing chamber.

2. The automated cell processing unit of claim 1, wherein said automated cell processing unit is configured to mechanically or enzymatically disaggregate said adipose tissue.

3. The automated cell processing unit of claim 1, wherein the programmable processing device comprises software to allow for collection of a set of run data selected from the group consisting of:

a lot number of a reagent, a temperature measurement, a volume measurement, a tissue volume measurement a dose of enzyme introduced into the device;

an operator identity;

a patient identity, and an incubation time.

4. The automated cell processing unit of claim 1, wherein said automated cell processing unit comprises a disposable component and a hardware component.

5. The automated cell processing unit of claim 4, wherein the disposable component comprises the collection chamber.

6. The automated cell processing unit of claim 4, wherein the hardware component comprises a centrifuge.

7. The automated cell processing unit of claim 1, further comprising:

a probe that can connect to at least one of said tissue collection chamber, said processing chamber, said cell concentrator, or a conduit that connects the tissue collection chamber and the cell processing chamber while maintaining a closed system.

8. The automated cell processing unit of claim 7, wherein said probe comprises a sensor.

9. The automated cell processing unit of claim 8, wherein said sensor is selected from the group consisting of an optical sensor, an ultrasonic sensor, and a pressure sensor.

10. The automated cell processing unit of claim 7, wherein said sensor is configured to detect the presence of red blood cells.

11. A method of processing adipose tissue to produce a concentrated population of cells comprising CD31 positive cells, comprising

introducing adipose tissue that comprises a population of cells comprising CD31 positive cells into the automated cell processing unit of claim 1;

and

processing said adipose tissue to obtain a concentrated population of cells comprising adipose-derived CD31-positive cells.

12. The method of claim 11, further comprising a disaggregation step, wherein said adipose tissue is mechanically or enzymatically disaggregated.

13. The method of claim 12, wherein said disaggregation step comprises an enzymatic digestion.

14. The method of claim 13, wherein said enzymatic digestion comprises collagenase treatment.

15. The method of claim 13, wherein said enzymatic digestion comprises treatment with a neutral protease.

16. The method of claim 13, wherein said enzymatic digestion is inhibited by human serum.

17. The method of claim 11, wherein said automated cell processing unit comprises a probe that comprises a sensor.

18. The method of claim 17, wherein said sensor is selected from the group consisting of an optical sensor, an ultrasonic sensor, and a pressure sensor.

19. The method of claim 17, wherein said sensor is configured to detect the presence of red blood cells in said sample.

20. The method of claim 11, further comprising:

receiving an input from a user that identifies whether said concentrated population of cells comprising adipose-derived CD31-positive cells will be administered intravascularly, non-systemically, or both; and

determining the presence of at least one factor selected from the group consisting of adipocytes, free lipids, a proteolytic substance, a platelet aggregating substance, and an endotoxin in said concentrated population of cells comprising CD31-positive cells.

21. The method of claim 20, wherein the presence of an endotoxin, a proteolytic enzyme, a platelet aggregating substance, or free lipids is determined when the input received from said user identifies that said concentrated population of cells comprising CD31-positive cells will be administered intravascularly.

22. The method of claim 21, wherein said determining step is automatically performed.

23. The method of claim 21, wherein said determining step is not automatically performed.

24. The automated cell processing unit of claim 5, wherein said collection chamber comprises a flexible bag.

25. The automated cell processing unit of claim 1, further comprising a support for said collection chamber, said support comprising an internal frame or an external frame.

26. The automated cell processing unit of claim 1, wherein the collection chamber is configured to receive between 5 ml and 2 liters of adipose tissue.

27. The automated cell processing unit of claim 1, further comprising an agitation mechanism that agitates the collection chamber.