Separation apparatus and method

A method of enriching a selected cell from a cell mixture containing one or more other cell types with densities different from that of the selected cell type, comprising

• adding the cell mixture to a centrifugation device having
• side walls and a closed bottom,
• a constriction member disposed within the device, said constriction member positioned and constructed to retain fluid in the bottom portion of the device below the constriction member, when the device is inverted,
• a closed top,
• at least a first and a second port in said top, said first port positioned for introduction of fluid material into said device,
• a closed fluid channel communicating between said second port and the bottom of the device below said constriction member, and
• a cell separation medium contained in the bottom portion of the device and extending above said constriction member to a level above an opening formed by said constriction member, such that cells which are captured at an interface between the cell-separation medium and a lower-density medium, after centrifugation, are discharged with the lower-density medium when the device is inverted;
• centrifuging said centrifugation device at a gravitational force sufficient to pellet cells having specific densities greater than the specific density of the density gradient material in said device;
• and
• collecting from said device a cell fraction containing the selected cell.

A method of enriching a natural killer cell from a cell mixture, comprising

• layering the cell mixture onto a cell separation medium in a centrifuge tube, said cell separation medium characterized by a specific density of 1.0605.+-.0.0005 gr/ml isotonicity with the natural killer cell,
• said tube having side walls, a closed bottom, and a constriction member disposed within the tube, wherein said constriction member is positioned and constructed to retain fluid in the bottom portion of the tube below the constriction member when the tube is inverted, said cell separation medium contained in the bottom portion of the tube and extending above said constriction member to a level above an opening formed by said constriction member, such that cells which are captured at an interface between the cell-separation medium and a lower-density medium, after centrifugation, are discharged with the lower-density medium when the tube is inverted,
• centrifuging said centrifuge tube at a gravitational force sufficient to pellet cells having a specific density of greater than said specific density of said separation medium, and
• collecting the natural killer cell from the upper portion of said tube.

A method of enriching a natural suppressor cell from a cell mixture, comprising

• layering the cell mixture onto a cell separation medium in a centrifuge tube, said cell separation medium characterized by a specific density of 1.0605.+-.0.0005 gr/ml isotonicity with the natural suppressor cell,
• said tube having side walls, a closed bottom, and a constriction member disposed within the tube, wherein said constriction member is positioned and constructed to retain fluid in the bottom portion of the tube below the constriction member when the tube is inverted, said cell separation medium contained in the bottom portion of the tube and extending above said constriction member to a level above an opening formed by said constriction member, such that cells which are captured at an interface between the cell-separation medium and a lower-density medium, after centrifugation, are discharged with the lower-density medium when the tube is inverted,
• centrifuging said centrifuge tube at a gravitational force sufficient to pellet cells having a specific density of greater than said specific density of said separation medium, and
• collecting the natural killer cell from the upper portion of said tube.

A method of enriching a dendritic cell from a cell mixture, comprising

• layering the cell mixture into a cell separation medium in a centrifuge tube, said cell separation medium characterized by a specific density selected from the group consisting of 1.0720.+-.0.0005 gr/ml, 1.0610.+-.0.0005 gr/ml and 1.0565.+-.0.0005 gr/ml and isotonicity with the dendritic cell,
• said tube having side walls, a closed bottom, and a constriction member disposed within the tube, wherein said constriction member is positioned and constructed to retain fluid in the bottom portion of the tube below the constriction member when the tube is inverted, said cell separation medium contained in the bottom portion of the tube and extending above said constriction member to a level above an opening formed by said constriction member, such that cells which are captured at an interface between the cell-separation medium and a lower-density medium, after centrifugation, are discharged with the lower density medium when the tube is inverted,
• centrifuging said centrifuge tube at a gravitational force sufficient to pellet cells having a specific density of greater than said specific density of said separation medium, and
• collecting said dendritic cell from the upper portion of said tube.

A method of enriching a cytotoxic T lymphocyte from a cell mixture, comprising

• layering the cell mixture onto a cell separation medium in a centrifuge tube, said cell separation medium characterized by a specific density selected from the group consisting of 1.0720.+-.0.0005 gr/ml, 1.0610.+-.0.0005 gr/ml and 1.0565.+-.0.0005 gr/ml and isotonicity with the T lymphocyte,
• said tube having side walls, a closed bottom, and a constriction member disposed within the tube, wherein said constriction member is positioned and constructed to retain fluid in the bottom portion of the tube below the constriction member when the tube is inverted, said cell separation medium contained in the bottom portion of the tube and extending above said constriction member to a level above an opening formed by said constriction member, such that cells which are captured at an interface between the cell-separation medium and a lower-density medium, after centrifugation, are discharged with the lower-density medium when the tube is inverted,
• centrifuging said centrifuge tube at a gravitational force sufficient to pellet cells having a specific density of greater than said specific density of said separation medium, and
• collecting said T lymphocyte from the upper portion of said tube.

A method of enriching a hematopoietic progenitor CD34.sup.+ cell from cell mixture derived from bone marrow, comprising

• layering the cell mixture onto a cell separation medium in a centrifuge tube, said cell separation medium characterized by a specific density of 1.0685.+-.0.0005 gr/ml and isotonicity with the hematopoietic progenitor CD34.sup.+ cell,
• said tube having side walls, a closed bottom, and a constriction member disclosed within the tube, wherein said constriction member is positioned and constructed to retain fluid in the bottom portion of the tube below the constriction member when the tube is inverted, said cell separation medium contained in the bottom portion of the tube and extending above said constriction member to a level above an opening formed by said constriction member, that cells which are captured at an interface between the cell-separation medium and a lower-density medium, after centrifugation, are discharged with the lower-density medium when the tube is inverted,
• centrifuging said centrifuge tube at a gravitational force sufficient to pellet cells having a specific density of greater than said specific density of said separation medium, and
• collecting the hematopoietic progenitor CD34.sup.+ cell from the upper portion of said tube.

A kit, comprising:

• a) a centrifugation apparatus, which includes:
• (i) a centrifugation device having side walls and a closed bottom;
• (ii) a constriction member disposed within the device, said constriction member positioned and constructed to retain fluid in the bottom portion of the device below the constriction member, when the device is inverted;
• and
• (iii) a cell separation medium contained in the bottom portion of the device and extending above said constriction member to a level above an opening formed by said constriction member, such that cells which are captured at an interface between the cell-separation medium and a lower-density medium, after centrifugation, are discharged with the lower-density medium when the device is inverted;
• and
• b) a cell type-specific binding agent directly or indirectly linked to carrier particles, said particles having a specific density that is at least 0.001 gr/ml greater than the specific density of said cell separation medium.