Methods for isolating target cells from blood
Disclosed herein are methods for isolating target cells from blood, involving mixing in an open container an undiluted blood sample having a volume of 10 ml or less, and binding agents, wherein each binding agent comprises (A) a primary binding agent comprising an agent capable of binding to at least one cellular epitope on target cells in the undiluted blood sample, (B) a first linker bound to the primary binding agent, to generate binding agent-attached target cells in the undiluted blood sample; contacting the binding agent-attached target cells in the undiluted blood sample with a plurality of buoyant reagents that include a second linker capable of binding to the first linker to generate an undiluted buoyant reagent-attached target cell mixture; diluting the undiluted buoyant reagent-attached target cell mixture by at least 20% to produce a diluted buoyant reagent-attached target cell mixture; applying a vectorial force, such as centrifugal force, to the diluted buoyant reagent-attached target cell mixture to generate a stratified diluted buoyant reagent-attached target cell mixture; removing the buoyant reagent-attached target cells from the stratified diluted buoyant reagent-attached target cell mixture; and isolating the target cells from the buoyant reagent-attached target cells.
1. A method for isolating target cells from whole blood, comprising:
(a) mixing in an open container
(i) an undiluted whole blood sample having a volume of between 1 ml and 7.5 ml, and
(ii) binding agents, wherein each binding agent comprises (A) a primary binding agent comprising an antibody capable of binding to at least one cellular epitope on target cells in the undiluted whole blood sample, and (B) a first linker bound to the primary binding agent, wherein the first linker comprises a biotin linker,
wherein the mixing occurs for a time and under conditions suitable to promote binding of the primary binding agents to the target cells to generate binding agent-attached target cells in the undiluted whole blood sample;
(b) contacting the binding agent-attached target cells in the undiluted whole blood sample contained in the open container with a plurality of buoyant reagents, wherein each buoyant reagent comprises a second linker bound to the buoyant reagent, wherein the second linker comprises streptavidin, wherein the contacting occurs for a time and under conditions suitable to promote binding of the second linker to the first linker to generate an undiluted buoyant reagent-attached target cell mixture;
(c) diluting the undiluted buoyant reagent-attached target cell mixture contained in the open container by at least 20% to produce a diluted buoyant reagent-attached target cell mixture;
(d) applying a vectorial force to the diluted buoyant reagent-attached target cell mixture to generate a stratified diluted buoyant reagent-attached target cell mixture;
(e) removing the buoyant reagent-attached target cells from the stratified diluted buoyant reagent-attached target cell mixture; and
isolating the target cells from the buoyant reagent-attached target cells; wherein the target cells are not platelets or red blood cells.
2. The method of claim 1 , wherein the buoyant reagents comprise gas-filled bubbles.
3. The method of claim 2 , wherein the gas-filled bubbles comprise perfluorocarbon gas cores encompassed by lipid or phospholipid shells.
4. The method of claim 2 , wherein the gas-filled bubbles have a mean size volume of greater than 6 μm 3 and less than 10 μm 3 .
5. The method of claim 2 , wherein the gas-filled bubbles have a mean size diameter of between 1.5 μm and about 3 μm.
6. The method of claim 2 , wherein the gas-filled bubbles are present in the contacting step at a concentration of at least 4×10 8 per ml.
7. The method of claim 1 , wherein the streptavidin is present on the buoyant reagent at a density of greater than 20,000 molecules per um 2 .
8. The method of claim 1 , wherein the streptavidin is present on the buoyant reagent at a density of greater than 25,000 molecules per um 2 .
9. The method of claim 1 , wherein the streptavidin is present on the buoyant reagent at a density of greater than 26,000 molecules per um 2 .
10. The method of claim 1 , wherein the undiluted whole blood sample has a volume of 1 ml to 5 ml.
11. The method of claim 1 , wherein the undiluted whole blood sample has a volume of 1 ml to 3 ml.
12. The method of claim 1 , wherein the diluting comprises diluting the undiluted buoyant reagent-attached target cell mixture by between 20% and 500% to produce the diluted buoyant reagent-attached target cell mixture.
13. The method of claim 1 , wherein the removing step comprises removing the buoyant reagent-attached target cells from the stratified diluted buoyant reagent-attached target cell mixture using a pipette or syringe.
14. The method of claim 1 , wherein the isolating step comprises isolating the target cells from the buoyant reagent-attached target cells by sonicating or applying positive pressure to degas the microbubbles.
15. The method of claim 1 , wherein the target cells comprise cells of surface immunophenotypes comprising one or more of CD45+, CD3+, CD4+, CD8+, CD25+, CD14+, CD16+, CD19+, CD56+, CD34+, CD117+, CD235a, CD349 − , T cell receptor (TCR) alpha, gamma, beta & delta.
16. The method of claim 1 , wherein the target cells comprise CD3 + cells.
17. The method of claim 1 , wherein the target cells are isolated with a purity of at least 85%.
18. The method of claim 1 , wherein the target cells are isolated with a purity of at least 90%.
19. The method of claim 1 , wherein the target cells are isolated with a viability of at least 80%.
20. The method of claim 1 , wherein the target cells are isolated with a viability of at least 90%.
21. The method of claim 1 , wherein at least 50% of the target cells in the whole blood sample are isolated.
22. The method of claim 1 , wherein at least 70% of the target cells in the whole blood sample are isolated.