Tissue dissociating system and method

• US 5,786,207 A
• Filed: 05/28/1997
• Issued: 07/28/1998
• Est. Priority Date: 05/28/1997
• Status: Expired due to Term

• Current Assignee: University Of Pittsburgh (University of Pittsburgh of The Commonwealth System of Higher Education)
• Original Assignee: University Of Pittsburgh (University of Pittsburgh of The Commonwealth System of Higher Education)
• Inventors: Katz, Adam J., Llull, Ramon
• Primary Examiner(s): Redding, David A.
• Application Number:
  US08/864,444
• Time in Patent Office: 426 Days
• Field of Search: 435/262, 435/267, 435/271, 435/283.1, 435/297.1, 435/298.2, 435/308.1, 494/36
• US Class Current: 435/267
• CPC Class Codes: C12M 45/02 by mechanical forces; Stirring; Trituration; Comminuting crushing, pulverizing, disintegrating in general B02C
• CPC Class Codes: C12M 45/05 by centrifugation centrifuges in general B04B
• 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

Abstract

A tissue dissociation method and system for washing and dissociating tissue and producing a cellular suspension for use in autologous, cell-based therapies or research wherein a sterile environment is maintained. More particularly, a system for dissociating tissue comprising a housing unit, a first compartment having a plurality of pores providing for fluid communication between the interior of the first compartment and the interior of the housing unit, a first inlet port that provides communication between the exterior environment and the interior of the first compartment and thereby provides a means for introducing solutions into the interior of the first compartment, a second compartment, a second inlet port for introducing a tissue sample into the system, a means for agitating or rotating the second compartment in the housing unit, a means for controlling the exit of effluent from the housing unit, a first receptacle, and a centrifuge-ready second receptacle for collecting a resulting cell suspension. The method comprises introducing a tissue sample into the system and subjecting it to a washing phase and a dissociating phase wherein the tissue is agitated or rotated. The tissue may also be subjected to a sedentary phase and a phase in which a solution is added to the resulting cell suspension to cease tissue dissociation. At the conclusion of the tissue dissociation process, the resulting cell suspension is collected in a centrifuge-ready receptacle.

Claims

1. A system for dissociating tissue comprising:

a. a housing unit having a means of accessing the interior of said housing unit and having a downwardly inclined floor wherein an aperture exists at the nadir of said inclined floor;

b. a first compartment having a plurality of fluid communicating means between the interior of said first compartment and the interior of said housing unit;

c. a first inlet port that provides communication between the exterior environment and said interior of said first compartment and thereby provides a means for introducing solutions into said interior of said first compartment;

d. a second compartment having pores providing for fluid communication between the interior of said second compartment and said interior of said housing unit and a means for reversibly securing said second compartment in said housing unit while permitting said second compartment to be rotated or agitated;

e. a second inlet port for introducing a tissue sample into said second compartment;

f. a third inlet port wherein said third inlet port has a filter and provides a means for filtered air to enter said housing unit;

g. a means for agitating or rotating said second compartment in said housing unit;

h. a means for controlling the exit of effluent through said aperture;

i. a first receptacle sealably engaging said housing unit; and

j. a second receptacle for collecting a cell suspension wherein said second receptacle is centrifuge-ready and sealably engages said housing unit.

2. A system for dissociating tissue according to claim 1 further comprising a heat source.

3. A system for dissociating tissue according to claim 1 further comprising a vacuum source.

4. A system for dissociating tissue according to claim 1 wherein said first compartment has a reversible means for occluding said plurality of fluid communicating means between said interior of said first compartment and said interior of said housing unit.

5. A system for dissociating tissue according to claim 1 wherein said plurality of fluid communicating means between said interior of said first compartment and said interior of said housing unit are located on the surface of the portion of said first compartment facing said second compartment housed within said housing unit.

6. A system for dissociating tissue according to claim 1 wherein said second compartment is substantially cylindrical in shape.

7. A system for dissociating tissue according to claim 1 wherein said second compartment is substantially cylindrical in shape and said pores of said second compartment are between 50 and 2000 microns.

8. A system for dissociating tissue according to claim 1 wherein said second compartment has extensions radiating out from the exterior surface of said second compartment to facilitate the interaction of materials in said housing unit.

9. A system for dissociating tissue according to claim 1 wherein said means for controlling the exit of effluent through said aperture in the floor of said housing unit is one of a group consisting of:

a. a valve having an open position and closed position;

b. a three-way valve having an open position, an open position with vacuum and a closed position;

c. a valve having an open position, an open position with vacuum, an open position with filter, an open position with filter and vacuum, and a closed position;

d. a panel, slidably and sealingly engaged with said housing unit and having an open position and closed position;

e. a panel, slidably and sealingly engaged with said housing unit and having an open position, an open position with filter, and closed position;

f. a disc slidably and sealingly engaged with said housing unit and having an open position and a closed position;

or

g. a disc slidably and sealingly engaged with said housing unit and having an open position, an open position with filter, and a closed position.

10. A system for dissociating adipose tissue comprising:

a. a housing unit having a means of accessing the interior of said housing unit and having an inclined floor wherein an aperture exists at the nadir of said inclined floor;

b. a first compartment having a plurality of fluid communicating means between the interior of said first compartment and said interior of said housing unit;

c. a first inlet port that provides communication between the exterior environment and said interior of said first compartment and thereby provides a means for introducing solutions into said interior of said first compartment;

d. a second compartment having pores providing for fluid communication between the interior of said second compartment and said interior of said housing unit, and a means for reversibly securing said second compartment in said housing unit while permitting said second compartment to be rotated or agitated;

e. a second inlet port for introducing a tissue sample into said second compartment;

f. a third inlet port wherein said third inlet port has a filter and provides a means for filtered air to enter said housing unit;

g. a means for agitating or rotating said second compartment in said housing unit;

h. a means for controlling the exit of effluent through said aperture in said floor of said housing unit;

i. a heat source;

j. a vacuum source;

k. a first receptacle capable of sealably engaging with said housing unit; and

l. a second receptacle for collecting a resulting cell suspension wherein said second receptacle is centrifuge-ready and sealably engages said housing unit.

11. A method for dissociating tissue comprising:

a. providing a system for dissociating tissue having a housing unit having a first compartment and second compartment located therein wherein said first compartment is superiorly positioned relative to said second compartment and said first compartment has a plurality of fluid communicating means between the interior of said first compartment and the interior of said housing unit, said second compartment having pores providing for fluid communication between the interior of said second compartment and said interior of the housing unit, a first inlet port that provides communication between the exterior environment and said interior of said first compartment and provides a means for introducing solutions into said interior of said first compartment, a second inlet port for introducing tissue into said second compartment, a third inlet port wherein said port has a filter and provides a means for filtered air to enter said housing unit, said second compartment having a means for engaging a rotating or agitating mechanism, a means for rotating or agitating said second compartment, a heat source, said housing unit having a downwardly inclined floor wherein an aperture exists at the nadir of said inclined floor, a means for controlling the exit of effluent through said aperture, a first receptacle sealably engaging said housing unit, and a second receptacle wherein said second receptacle is centrifuge-ready and sealably engages said housing unit;

b. inserting tissue to be dissociated into said second compartment via said second inlet port;

c. inserting a washing solution into said first compartment via said first inlet port;

d. permitting said washing solution to descend through said plurality of fluid communicating means between said interior of said first compartment and said interior of said housing unit, through said pores providing for fluid communication between said interior of said second compartment and said interior of said housing unit, and into said second compartment where it interacts with the tissue inserted therein;

e. placing said means for controlling the exit of effluent through said aperture in an open position such that said effluent exits the housing unit and is collected in said first receptacle;

f. placing said means for controlling the exit of effluent through said aperture in a closed position and disengaging said first receptacle and sealably engaging said second receptacle;

g. inserting a proteolytic enzyme solution into said housing unit;

h. heating said proteolytic enzyme solution using said heat source while agitating or rotating said second compartment within said housing unit using said means for agitating or rotating said second compartment and thereby facilitating tissue dissociation;

i. placing said means for controlling the exit of effluent through said aperture in an open position such that effluent exits said housing unit and is collected in said second receptacle;

j. collecting the cell suspension resulting from tissue dissociation in said second receptacle; and

k. sealing said second receptacle closed.

12. A method for dissociating tissue according to claim 11 wherein said washing solution is introduced into the system under pressure such that the pressure of said washing solution when it interacts with said second compartment dislodges materials occluding said pores of said second compartment.

13. A method for dissociating tissue according to claim 11 further comprising a sedentary step wherein components of said tissue are permitted to separate based on their density properties and placing said means for controlling the exit of effluent through said aperture in an open position such that effluent exits said housing unit and is collected in said first receptacle, wherein said sedentary step is performed before permitting said washing solution to descend through said plurality of fluid communicating means between said interior of said first compartment and said interior of said housing unit.

14. A method for dissociating tissue according to claim 11 further comprising:

a. subjecting said tissue to a sedentary step wherein components of said tissue are permitted to separate based on density properties; and

b. placing said means for controlling the exit of effluent through said aperture in an open position such that effluent exits the housing unit and is collected in said first receptacle, wherein said sedentary step is performed before permitting said washing solution to descend through said plurality of fluid communicating means between said interior of said first compartment and said interior of said housing unit; and

c. inserting a solution that inhibits proteolytic enzyme activity into said housing unit while agitating or rotating said second compartment within said housing unit, wherein said insertion of said solution is performed before placing said means for controlling the exit of effluent through said aperture in an open position such that effluent exits said housing unit and is collected in said second receptacle.

15. A method for dissociating tissue according to claim 11 further comprising agitating or rotating said second compartment while said washing solution is inserted into said system for dissociating tissue and while said proteolytic enzyme solution is inserted into said system.

16. A method for dissociating tissue according to claim 11 wherein said washing solution and said proteolytic enzyme solution are introduced into said system under pressure such that the pressure of said washing solution and said proteolytic enzyme solution dislodges material occluding said pores of said second compartment when said washing solution and said proteolytic enzyme solution interact with said second compartment.

17. A method for dissociating tissue according to claim 11 further comprising inserting a solution that inhibits proteolytic enzyme solution activity into said housing unit while agitating or rotating said second compartment within said housing unit before placing said means for controlling the exit of effluent through said aperture in an open position such that effluent exits said housing unit and is collected in said second receptacle.

18. A method for dissociating tissue according to claim 11 further comprising a step of permitting said washing solution to descend through said plurality of fluid communicating means between said interior of said first compartment and said interior of said housing unit, through said pores providing for fluid communication between said interior of said second compartment and said interior of said housing unit, and into said second compartment where it interacts with the tissue inserted therein while said means for controlling the exit of effluent through said aperture in an open position such that said effluent exits the housing unit and is collected in said first receptacle, wherein said step is performed immediately before placing said means for controlling the exit of effluent through said aperture in a closed position and disengaging said first receptacle and sealably engaging said second receptacle.

19. A method for dissociating tissue according to claim 11 wherein said tissue to be dissociated and introduced into said second compartment is adipose tissue.

20. A method for dissociating tissue according to claim 11 wherein said tissue to be dissociated and introduced into said second compartment is adipose tissue harvested by a liposuction procedure using tumescent solution and further comprising the steps of:

a. subjecting said tissue to a sedentary step wherein components of said tissue are permitted to separate based on density properties; and

b. placing said means for controlling the exit of effluent through said aperture in an open position such that effluent exits said housing unit and is collected in said first receptacle, wherein said sedentary step is performed before permitting said washing solution to descend through said plurality of fluid communicating means between said interior of said first compartment and said interior of said housing unit; and

c. inserting a solution that inhibits proteolytic enzyme solution activity into said housing unit while agitating or rotating said second compartment within said housing unit, wherein said inserting of said solution is performed before placing said means for controlling the exit of effluent through said aperture in an open position such that effluent exits said housing unit and is collected in said second receptacle.