Pulsatile perfusion extraction method for non-embryonic pluripotent stem cells

• US 20070190649A1
• Filed: 01/18/2007
• Published: 08/16/2007
• Est. Priority Date: 01/18/2006
• Status: Active Grant

• Current Assignee: Hemacell Perfusion
• Original Assignee: Hemacell Perfusion
• Inventors: Gage, Fred
• Granted Patent: US 9,944,900 B2
• Time in Patent Office: Days
• US Class Current: 435/325
• CPC Class Codes: A01N 1/021 Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
• CPC Class Codes: A01N 1/0236 Mechanical aspects
• CPC Class Codes: A01N 1/0247 for perfusion, i.e. for circulating fluid through organs, blood vessels or other living parts
• CPC Class Codes: A01N 1/0289 Pressure processes, i.e. using a designated change in pressure over time
• CPC Class Codes: A61L 27/3683 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
• CPC Class Codes: A61L 27/3834 Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
• CPC Class Codes: A61L 27/507 for artificial blood vessels apparatus for applying cells on a blood vessel prosthesis A61F2/062
• CPC Class Codes: C12N 5/0605 Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
• CPC Class Codes: C12N 5/0647 Haematopoietic stem cells; Uncommitted or multipotent progenitors

Abstract

A method for extracting stem cells from a non-embryonic stem cell source, including providing a non-embryonic stem cell source including stem cells; perfusing the non-embryonic stem cell source with a pulsatile flow of a perfusion solution to produce a perfusate including stem cells and a perfused non-embryonic stem cell source; and isolating the stem cells from the perfusate to produce isolated stem cells, is provided. Also provided is a non-embryonic stem cell line derived from a non-embryonic stem cell obtained using the pulsatile perfusion extraction method.

Claims

1. A method for extracting stem cells from a non-embryonic stem cell source, comprising:

providing a non-embryonic stem cell source comprising stem cells;

perfusing the non-embryonic stem cell source with a pulsatile flow of a perfusion solution to produce a perfusate comprising stem cells and a perfused non-embryonic stem cell source; and

isolating the stem cells from the perfusate to produce isolated stem cells.

2. The method of claim 1, wherein perfusing comprises

first placing the non-embryonic stem cell source into a perfusion circuit of a pulsatile perfusion machine;

and

operating the pulsatile perfusion machine to perfuse the non-embryonic stem cell source with a pulsatile flow of perfusion solution under conditions sufficient to produce a perfusate comprising stem cells.

3. The method of claim 1, wherein the non-embryonic stem cell source comprises one or more members selected from the group consisting of human placenta and human umbilical cord.

4. The method of claim 2, further comprising

prior to first placing, priming the pulsatile perfusion machine with a priming solution.

5. The method of claim 4, wherein the priming solution comprises the perfusion solution.

6. The method of claim 1, further comprising

prior to perfusing, administering an anticoagulant to the non-embryonic stem cell source.

7. The method of claim 6, wherein the anticoagulant comprises one or more members selected from the group consisting of heparin and warfarin sodium.

8. The method of claim 1, wherein perfusing is carried out under conditions that simulate conditions of the non-embryonic stem cell source in vivo.

9. The method of claim 1, wherein perfusing comprises perfusing the non-embryonic stem cell source with a pulsatile flow of perfusion solution at a systolic perfusion pressure of from about 50 to about 150 mmHg and at a temperature of from about 4°

C. to about 40°

C.

10. The method of claim 9, wherein perfusing comprises perfusing the non-embryonic stem cell source with a pulsatile flow of perfusion solution at a systolic perfusion pressure of from about 90 to about 120 mmHg and at a temperature of from about 15°

C. to about 20°

C.

11. The method of claim 1, wherein the perfusion solution comprises cell culture media.

12. The method of claim 1, wherein the perfusion solution comprises one or more members selected from the group consisting of BES, BIS-TRIS, BIS-TRIS propane, EPPS, Gly-Gly, HEPES, HEPES sodium salts, MES hydrate, MES sodium salts, MOPS, MOPS sodium salts, PIPES, TAPS, TAPS sodium salts, TAPSO TES, Tricine, Trizma®

base, Trizma®

Hydrochloride, Trizma®

hydrochloride buffer solution, Trizma®

Preset crystals, Alsever'"'"'s Solution, Ames Medium, Basal Medium Eagle, Click'"'"'s Medium, Dulbecco'"'"'s Modified Eagle'"'"'s Medium—

high glucose, Dulbecco'"'"'s Modified Eagle'"'"'s Medium-low glucose, Dulbecco'"'"'s Modified Eagle'"'"'s Medium/Nutrient Mixture F-12 Ham, Dulbecco'"'"'s Phosphate Buffered Saline, Earle'"'"'s Balanced Salts, Gey'"'"'s Balanced Salt Solution, Glasgow Minimum Essential Medium, Grace'"'"'s Insect Medium, Hanks'"'"' Balanced Salt Solution, IPL-41 Insect Medium, Iscove'"'"'s Modified Dulbecco'"'"'s Medium, Iscove Modified Dulbecco'"'"'s Medium, Krebs-Henseleit Buffer Modified, Krebs-Ringer bicarbonate buffer, L-15 Medium (Leibovitz), McCoy'"'"'s 5A Medium, MCDB 105 Medium, MCDB 110 Medium, MCDB 131 Medium, MCDB 153 Medium, MCDB 201 Medium, Medium 199, Mega Cell™

Dulbecco'"'"'s Modified Eagle'"'"'s Medium, Mega Cell™

Dulbecco'"'"'s Modified Eagles Medium/Nutrient Mixture F-12 Ham, Mega Cell™

Minimum Essential Medium Eagle, Mega Cell™

Minimum Essential Medium/Nutrient Mixture F-12 Ham, Mega Cell™

RPMI-1640 Medium, Minimum Essential Medium Eagle, NCTC 109 Medium, Nutrient Mixture F-10 Ham, Nutrient Mixture F-12 Ham, RPMI 1640, RPMI 1640 Medium with L-glutamine and sodium bicarbonate, RPMI 1640 HEPES Modification with 25 mM HEPES without L-glutamine, RPMI 1640 medium Modified with 20 mM HEPES and L-glutamine and sodium bicarbonate, RPMI 1640 Medium with sodium bicarbonate without L-glutamine, RPMI 1640 Medium Dutch Modification with sodium bicarbonate and 20 mM HEPES without L-glutamine, RPMI 1640 medium 10X without glutamine, folic acid and sodium bicarbonate, RPMI 1640 medium modified with sodium bicarbonate without methione, cystine and L-glutamine, RPMI 1640 medium modified with sodium bicarbonate without Lglutamine and phenol red, RPMI 1640 medium HEPES modification, with L-glutamine 25 mM HEPES without sodium bicarbonate, RPMI 1640 medium with L-glutamine without glucose and sodium bicarbonate, RPMI 1640 medium modified with L-glutamine without phenol red and sodium bicarbonate, RPMI 1640 medium powder, AutoMod™

cell cultured tested, Schneider'"'"'s insect medium, Shields and Sang M3 insect medium, Shields and Sang M3 insect medium, TC-100 insect medium, TNM-FH insect medium, Tyrode'"'"'s salts, Waymouth MB 752/1 medium, Williams'"'"' medium E, Hanks, Eagles, Albumin, Beizer Machine perfusion solution, Celsior, Euro-Collins, HTK, Lactated Ringers, Plasmanate, Hespan, Normal Saline, IGL, Vasosol, and Viaspan.

13. The method of claim 1, wherein the perfusion solution comprises one or more members selected from the group consisting of a colloidal agent, an anti-edema agent, an antioxidant, an anti-inflammatory agent, a vasodilator, an antimicrobial agent, and a combination of any two or more thereof.

14. The method of claim 1, wherein the perfusion solution comprises a colloidal agent, an anti-edema agent, an antioxidant, an anti-inflammatory agent, and a vasodilator.

15. The method of claim 1, wherein the perfusion solution has a pH temperature, corrected to 37°

C., within a range of 7.35 to 7.45, and an osmolality in the range of from about 300 to 400 mOsmols.

16. The method of claim 15, wherein the osmolality is in the range of from about 310 to about 350 mOsmols.

17. The method of claim 1, wherein isolating comprises subjecting the perfusate comprising stem cells to one or more of density gradient centrifugation, magnet cell separation, affinity cell separation, flow cytometry, and cell sorting, to produce isolated stem cells.

18. The method of claim 1, further comprising cryopreserving the isolated stem cells.

19. A method for producing a non-embryonic stem cell line, comprising:

culturing the isolated stem cells of claim 1 in stem cell culture media to produce the non-embryonic stem cell line.

20. A method for producing a vascular tissue graft, comprising

after perfusing, collecting the perfused non-embryonic stem cell source of claim 1;

and

isolating arteries and/or veins from the perfused non-embryonic stem cell source to produce the vascular tissue graft.