Method for isolation of afterbirth derived cells

• US 8,673,547 B2
• Filed: 03/07/2008
• Issued: 03/18/2014
• Est. Priority Date: 03/08/2007
• Status: Active Grant

• Current Assignee: Hemacell Perfusion, Inc.
• Original Assignee: Hemacell Perfusion, Inc.
• Inventors: Gage, Frederick A.
• Primary Examiner(s): UNDERDAHL, THANE E
• Application Number:
  US12/530,236
• Publication Number:
  US 20100041009A1
• Time in Patent Office: 2,202 Days
• Field of Search: 435/1.2, 435/283.1
• US Class Current: 435/1.2
• CPC Class Codes: C12N 2509/00 Methods for the dissociation of cells, e.g. specific use of enzymes
• CPC Class Codes: C12N 5/0605 Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly

Abstract

A method for extracting cells from afterbirth tissue, including placing the afterbirth into a perfusion circuit prior to exsanguination, extracting the cells from the afterbirth with digestive enzymes and mechanically recovering cells from the digested afterbirth tissue, and isolating the cells from the perfusate and digestion mix. Also disclosed is a cell line derived from afterbirth using the two-step pulsatile perfusion extraction method.

Claims

1. A method for extracting nucleated cells from afterbirth tissue, comprising the steps of:

a) locating the afterbirth tissue in a perfusion circuit without exsanguination of said afterbirth tissue;

b) extracting nucleated cells from said non-exsanguinated afterbirth tissue by pulsatile perfusion carried out using a rhythmic, intermittent flow of a perfusion solution pulsed at between 50 and 60 pulses per minute to produce a perfusate containing cells; and

c) isolating said nucleated cells from the perfusate.

2. The method of claim 1, further comprising the step of administering an anticoagulant to said afterbirth tissue prior to locating said afterbirth tissue in said perfusion circuit.

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

4. The method of claim 1, wherein said perfusion solution is 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, TRIS base, TRIS hydrochloride, TRIS hydrochloride solution, TRIS 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, Dulbecco'"'"'s Modified Eagle'"'"'s Medium, Dulbecco'"'"'s Modified Eagles Medium/Nutrient Mixture F-12 Ham, Minimum Essential Medium/Nutrient Mixture F-12 Ham, 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 10×

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 L-glutamine 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, 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, Belzer Machine perfusion solution, Celsior, Euro-Collins, HTK, Lactated Ringers, Plasmanate, Hespan, Normal Saline, IGL, Vasosol, and Viaspan.

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

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

6. The method of claim 5, wherein said osmolality is in the range from about 310 to about 350 mOsmols.

7. The method of claim 1, wherein said perfusion solution is perfused through said afterbirth tissue at a systolic perfusion pressure from about 50 to about 150 mmHg, and at a temperature from about 4°

C. to about 40°

C.

8. The method of claim 7, wherein said systolic perfusion pressure is in the range from about 90 to about 120 mmHg, and the temperature is in the range from about 15°

C. to about 20°

C.

9. The method of claim 1, further comprising the step of priming a pump of a perfusion machine used in the method with a priming solution prior to said extracting step.

10. The method of claim 9, wherein said priming solution has the same composition as said perfusion solution.

11. The method of claim 1, wherein said nucleated cells are isolated from said perfusate using at least one method selected from the group consisting of centrifugation, immunomagnetic separation, cell sorting and flow cytometry.

12. The method of claim 1, wherein said pulsatile perfusion simulates a pulsed flow of a beating heart.

13. The method of claim 1, further comprising the step of adding at least one digestive enzyme to the perfusion circuit to produce digested afterbirth tissue.

14. The method of claim 13, wherein said at least one digestive enzyme is added to the perfusion circuit upon completion of said extracting step.

15. The method of claim 14, further comprising the step of extracting the nucleated cells from the digested afterbirth tissue through fractionation of the eluate and/or mechanical disruption of the organ.

16. The method of claim 15, wherein the digestive enzyme is capable of disaggregating cell populations from the cytoskeletal framework and connective tissues of the organ or tissues.

17. The method of claim 15, wherein the digestive enzyme is selected from the group consisting of collagenase and trypsin.

18. The method of claim 1, further comprising the step of culturing the nucleated cells isolated from the perfusate to obtain a cell line from said nucleated cells.

19. A method for extracting nucleated cells from afterbirth tissue, comprising the steps of:

a) locating the afterbirth tissue in a perfusion circuit without exsanguination of said afterbirth tissue;

b) extracting nucleated cells from said non-exsanguinated afterbirth tissue by pulsatile perfusion that simulates a pulsed flow of a beating heart at a rate of 50-60 pulses per minute using a perfusion solution to produce a perfusate containing cells; and

c) isolating the nucleated cells from the perfusate.