Multiple stage affinity process for isolation of specific cells from a cell mixture

Claim 2

An affinity method of providing high purity fractions of desired cells by separating the desired cells from a mixture of cells in a first media using plural surfaces having ligands, with a high affinity for the desired cells, immobilized thereon comprising the steps of: (a) contacting one of the surfaces with the first media mixture of cells to permit some of the desired cells to be bound to the one surface, (b) separating the unbound cells and the first media from the bound cells, (c) removing the bound cells from the one surface, and resuspending them in a second media, (d) contacting a different one of the surfaces with the second media to permit some of the removed cells to be bound thereto, (e) separating the unbound cells and the second media from the different surface, and (f) removing the bound cells of step (d) from the different surface to provide the high purity fraction of desired cells.

Claim 3

The method as set forth in claim 1 which includes the additional steps of resuspending the removed cells of step (f) in a third media and repeating steps (d) and (e) with a third one of the surfaces with the removed cells of step (f).

Claim 4

The method as set forth in claim 2 wherein the surfaces each comprise the bottom of a different container.

Claim 5

The method as set forth in claim 1 wherein the surfaces each comprise the bottom of a different container.

Claim 6

The method as set forth in claim 2 wherein the surfaces comprise the surfaces of magnetic particles and the unbound cells and media are separated from the bound cells by separating the magnetic particles therefrom, desorbing the bound cells from the magnetic particles, and resuspending the desorbed cells together with fresh ligand immobilized magnetic particles in a different media.

Claim 7

The method as set forth in claim 1 wherein the surfaces comprise the surfaces of magnetic particles and the unbound cells and media are separated from the bound cells by separating the magnetic particles therefrom, desorbing the bound cells from the magnetic particles, and resuspending the desorbed cells together with fresh ligand immobilized magnetic particles in a different media.

Claim 8

The method as set forth in claim 1 wherein both unbound and bound cells from each contacting step are separately resuspended and contacted with additional affinity coated surfaces in a counter-current manner until the number of unwanted cells in the bound fraction and the number of desired cells in the unbound fraction have reached predetermined low values.

Claim 9

The method as set forth in claim 2 wherein both unbound and bound cells from each contacting step are separately resuspended and contacted with additional affinity coated surfaces in a counter-current manner until the number of unwanted cells in the bound fraction and the number of desired cells in the unbound fraction have reached predetermined low values.

Claim 10

The method as set forth in claim 2 wherein the surfaces comprise the interior wall of roller bottles.

Claim 11

The method as set forth in claim 1 wherein the surfaces comprise the interior wall of roller bottles.

Claim 12

The method as set fort in claim 1 wherein each of the surfaces has a different ligand, each ligand having a different affinity for the desired cells immobilized thereon.

Claim 13

An affinity method of providing high purity fractions of desired cells by separating the desired cells from a mixture of cells in a first media using plural surfaces having ligands, with a high affinity for the desired cells, immobilized thereon comprising. the steps of: flowing media in a first sense, introducing particles to provide the surfaces into the media, the particles having a density relative to that of the media to move in a second sense opposite the first sense, introducing the cell mixture into a mid-portion of the flowing media, and withdrawing the undesired cells and media at the downstream end of media flow, withdrawing the particles and desired cells bound thereto at the upstream end of media flow.

Claim 14

An affinity method of providing high purity fractions of desired cells by separating the desired cells from a mixture of cells in a first media using plural surfaces having ligands, with a high affinity for the desired cells, immobilized thereon comprising. the steps of: flowing media in a first sense, introducing particles to provide the surfaces into the media, the particles having a density relative to that of the media to move in a second sense opposite the first sense, introducing the cell mixture into a mid-portion of the flowing media, and withdrawing the undesired cells and media at the downstream end of media flow, withdrawing the particles and desired cells bound thereto at the upstream end of media flow.

Claim 15

The method of claim 12 wherein the particles are more dense than the media and the flow is upward.

Claim 16

The method of claim 12 wherein the particles are less dense than the media and the flow is downward.

Claim 17

An affinity method of providing high purity fractions of desired cells by separating the desired cells from a mixture of cells in a first media using plural surfaces having ligands, with a high affinity for the desired cells, immobilized thereon comprising the steps of: (a) contacting one of the surfaces with the first media mixture of cells to permit some of the desired cells to be bound to the one surface, (b) contacting a second surface with the unbound cells from the one surface, (c) contacting a third surface with the bound cells from the one surface, (e) contacting a fifth surface with the bound cells from the second surface and the unbound cells from the third surface, (f) contacting a sixth surface with the bound cells from the third surface, (h) contacting a seventh surface with the bound cells from the fifth surface and the unbound cells from the sixth surface, (i) contacting an eighth surface with the bound cells from the seventh surface, (j) and removing the bound cells from the sixth and eighth surfaces to provide desired cells of high purity and yield.

Claim 18

An affinity method of providing high purity fractions of desired cells by separating the desired cells from a mixture of cells in a first media using plural surfaces having ligands, with a high affinity for the desired cells, immobilized thereon comprising the steps of: (a) contacting one of the surfaces with the first media mixture of cells to permit some of the desired cells to be bound to the one surface, (b) contacting a second surface with the unbound cells from the one surface, (c) contacting a third surface with the bound cells from the one surface, (e) contacting a fifth surface with the bound cells from the second surface and the unbound cells from the third surface, (f) contacting a sixth surface with the bound cells from the third surface, (h) contacting a seventh surface with the bound cells from the fifth surface and the unbound cells from the sixth surface, (i) contacting an eighth surface with the bound cells from the seventh surface, (j) and removing the bound cells from the sixth and eighth surfaces to provide desired cells of high purity and yield.