Cell Separation Devices, Systems, and Methods

Abstract

Disclosed herein are cell separation devices, methods and systems, as well as compositions and reagents for use in cell separation methods.

We claim: 
     
         1 . A cell separation system, comprising:
 (a) a cartridge comprising
 (i) a processing container comprising at least one input port, a first exit port, and a second exit port; 
 (ii) a second container comprising an input port; 
 (iii) a third container comprising an input port and a first exit port; 
 (iv) a first conduit connecting the first exit port of the processing container and the input port of the second container, wherein the first conduit comprises a first reversible closing device, wherein the second container is transiently fluidically connected to the processing container such that fluid flow only from the processing container to the second container may occur when the first reversible closing device is opened; 
 (v) a second conduit connecting the second exit port of the processing container and the input port of the third container, wherein the second conduit comprises a second reversible closing device, wherein the third container is transiently fluidically connected to the processing container such that fluid flow only from the processing container to the third container may occur when the second reversible closing device is opened; 
   (b) a transfer container comprising at least one port;   (c) an at least third conduit connecting
 (i) the first exit port of the third container to the at least one port of the transfer container, and 
 (ii) the at least one port of the transfer container to the at least one input port of the processing container 
 wherein the at least third conduit comprises at least a third reversible closing device, such that (A) the third container is transiently fluidically connected to the transfer container, and (B) the transfer container is transiently fluidically connected to the processing container; wherein the at least third conduit is configured such that only one of the following may be true
 (I) fluid flow only from the third container to the transfer container may occur when the at least third reversible closing device is opened; or 
 (II) fluid flow only from the transfer container to the processing container may occur when the at least third reversible closing device is opened; and 
 
   (d) a control module configured to control activity in at least the cartridge, and the first and second conduits.   
     
     
         2 . The cell separation system  claim 1 , wherein the transfer container is internal to the cartridge. 
     
     
         3 . The cell separation system of  claim 1 , wherein the at least third conduit comprises a single conduit. 
     
     
         4 . The cell separation system of  claim 3 , wherein the at least third conduit comprises T or Y connector disposed between the third container and transfer container and between the transfer container and the processing container. 
     
     
         5 . The cell separation system of  claim 1 , wherein the at least one port of the transfer container comprises a first input port and an exit port. 
     
     
         6 . The cell separation system of  claim 5 , wherein the at least third conduit comprises:
 (i) a third conduit connecting the exit port of the third container to the input port of the transfer container, wherein the third conduit comprises a third reversible closing device, such that the third container is transiently fluidically connected to the transfer container such that fluid flow only from the third container to the transfer container may occur when the third reversible closing device is opened; and   (ii) a fourth conduit connecting the exit port of the transfer container to the at least one input port of the processing container, wherein the fourth conduit comprises a fourth reversible closing device, such that the transfer container is transiently fluidically connected to the processing container, such that fluid flow only from the transfer container to the processing container may occur when the fourth reversible closing device is opened.   
     
     
         7 . The cell separation system of  claim 1 , wherein the at least one input port of the processing container comprises a first input port and a second input port, wherein the at least third conduit, or the fourth conduit (when present), connects the exit port of the transfer container to the first input port of the processing container. 
     
     
         8 . The cell separation system of  claim 7 , further comprising a first medium input conduit connecting the second input port of the processing container to at least one medium reservoir, wherein the first medium input conduit comprises at least a fifth reversible closing device, wherein the at least one medium reservoir is transiently fluidically connected to the processing container such that fluid flow only from the at least one medium reservoir to the processing container may occur when the at least fifth reversible closing device is opened. 
     
     
         9 . The cell separation system of  claim 5 , wherein the at least one port of the transfer container further comprises a second input port. 
     
     
         10 . The cell separation system of  claim 9 , further comprising a second medium input conduit connecting the second input port of the transfer container to at least one medium reservoir, wherein the second medium input conduit comprises at least a sixth reversible closing device, wherein the at least one medium reservoir is transiently fluidically connected to the processing container such that fluid flow only from the at least one medium reservoir to the transfer container may occur when the at least sixth reversible closing device is opened. 
     
     
         11 . The cell separation system of  claim 1  wherein the cell separation system further comprises a mixer. 
     
     
         12 . The cell separation system of  claim 11 , wherein the mixer comprises a static mixer. 
     
     
         13 . The cell separation system of  claim 11 , wherein the mixer comprises an impeller disposed on an internal surface of a roof of the cartridge. 
     
     
         14 . The cell separation system of  claim 11 , wherein the mixer comprises an impeller spaced away from an internal surface of a roof of the cartridge. 
     
     
         15 . The cell separation system of  claim 11 , wherein the mixer comprises a peristaltic pump comprising a pump conduit having a first end and a second end, wherein the first end of the pump conduit is positioned in the processing chamber, and wherein the second end of the pump conduit is positioned outside of the processing chamber and is connected to the at least one input port of the processing chamber. 
     
     
         16 . The cell separation system of  claim 11 , wherein the mixer comprises a mixing module comprising a bottom portion and a top portion, wherein the cartridge is configured to be positioned in the bottom portion, and wherein the top portion is configured to be removably coupled to the bottom portion. 
     
     
         17 . The cell separation system of  claim 16 , wherein the mixing module includes a rotatable component coupled to the bottom portion, and wherein the rotatable component is configured to rotate the cartridge on its vertical axis by 180 degrees or by 360 degrees. 
     
     
         18 . The cell separation system of  claim 16 , wherein the mixing module is configured to increase a temperature of the cartridge when the cartridge is positioned in the bottom portion of the mixing module. 
     
     
         19 . The cell separation system of  claim 8 , wherein the first medium input conduit and/or the second medium input conduit further comprise a filter. 
     
     
         20 . The cell separation system of  claim 1 , wherein the second container comprises an exit port coupled to a first waste conduit. 
     
     
         21 . The cell separation system of  claim 1 , wherein the processing container further comprises a sterile vent coupled to a second waste conduit. 
     
     
         22 . A cell separation method, comprising either:
 (a1) processing a host liquid having a volume of at least 10 mL (or, alternatively, at least 25 mL, at least 50 mL, at least 75 mL, at least 100 mL, at least 200 mL) in a functionally closed system, wherein the host liquid comprises (i) target cells, and (ii) buoyant reagents, wherein the processing comprises contacting the target cells and buoyant reagents for a time and under conditions suitable to promote attachment of the cells to one or more of the buoyant reagents to generate attached target cells;   (b1) applying a vectorial force, such as centrifugation, to the host liquid within the functionally closed system to cause the attached target cells to stratify within the host liquid; and   (c2) sequestering the attached target cells to an area within the functionally closed system;   or comprising:   (a1) providing a host liquid, wherein the host liquid comprises attached target cells, wherein each attached target cell comprises
 (i) a binding agent bound to at least one cellular epitope on a target cell, 
 (ii) a first linker bound to the agent, wherein the first linker comprises a first oligonucleotide having a first complementary region; 
 (iii) a buoyant label comprising a second linker bound to the buoyant label, wherein the second linker comprises a second oligonucleotide having a second complementary region, wherein the second complementary region is perfectly complementary to the first complementary region, wherein the second complementary region is hybridized to the first complementary region to form a hybrid, and wherein the hybrid of the first and second complementary regions has a calculated Tm of at least 40° C.; 
   (b1) applying a vectorial force, such as centrifugation, to the host liquid to cause the attached target cells to stratify within the host liquid;   (c1) sequestering the attached target cells; and   (d1) subjecting the attached target cells to a temperature of 37° C. or less after step (c) for a time sufficient to dehybridize the first complementary region and the second complementary region to release the buoyant labels from the target cells.   
     
     
         23 . A product selected from the group consisting of:
 (I) a cell suspension, comprising
 (a1) a liquid medium having a volume of at least 1 mL (or, alternatively, at least 2 ml, 5 ml, 10 ml, 15 ml, 30 ml, 25 mL, at least 50 mL, at least 75 mL, at least 100 mL, at least 200 mL); and 
 (b1) desired cells suspended in the liquid medium, wherein the desired cells are selected from the group consisting of hematopoietic stem and progenitor cells, mesenchymal stem and progenitor cells, endothelial progenitor cells found in normal blood, placental/cord blood, bone marrow, white blood cells, granulocytes, mononuclear cells, lymphocytes, monocytes, T-cells, B-cells, NK cells, the stromal vascular fraction cells resident in adipose tissue, cultured cells, genetically modified cells, and sub-populations of such desired cells, wherein the desired cells are present in a liquid medium and wherein the desired cells make up at least 80% (or alternatively, at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or more) of cells in the cell suspension; 
 wherein the desired cells viability is greater than 90%, or greater than 95%, or greater than 97%, or greater than 99%; and wherein the cell suspension is present within a functionally closed cell separation system, or is directly obtained from the functionally closed cell separation system without further processing; 
   (II) a kit comprising
 (a2) a primary binding agent comprising an agent capable of binding to at least one cellular epitope on a target cell; 
 (b2) a first linker bound to the agent, wherein the first linker comprises a first oligonucleotide having a first complementary region; 
 (c2) a buoyant label; 
 (d2) a second linker bound to the buoyant label, wherein the second linker comprises a second oligonucleotide having a second complementary region, wherein the second complementary region is perfectly complementarity to the first complementary region, and wherein a hybrid of the first and second complementary regions has a calculated Tm of at least 40° C.; and 
   (III) a composition comprising:
 (a3) a primary binding agent comprising an agent capable of binding to at least one cellular epitope on a target cell; 
 (b3) a first linker bound to the agent, wherein the first linker comprises a first oligonucleotide having a first complementary region; 
 (c3) a buoyant label; 
 (d3) a second linker bound to the buoyant label, wherein the second linker comprises a second oligonucleotide having a second complementary region perfectly complementary to the first complementary region, wherein a hybrid of the first and second linkers' complementary regions has a calculated Tm of at least 40° C. 
 wherein the first linker and the second linker are hybridized to each other.