Columns for isolation, detection and use of biological cells
This invention relates to devices and methods for purifying, detecting and using biological cells. A variety of cell types including viable tumor, stem, immune and sperm cells can be purified from a complex biological sample using a column, including a pipette tip column. Methods of the invention can aid research, diagnosis and treatment of cancer. Purified viable cells can be detected on the column or eluted from the column and detected. Cells on a column can be used as a stationary phase for liquid chromatography. Cells may be removed, recovered and analyzed.
1. A method for purifying intact cells from a biological sample, comprising:
(a) providing a column comprised of a column body, a top frit and a bottom frit, wherein the column body, top frit and bottom frit define a media chamber, wherein the media chamber contains a loosely packed bed of medium, wherein the loosely packed bed of medium has a bed volume,
(i) wherein the loosely packed bed of medium is comprised of beads, wherein the loosely packed bed of medium is packed in such a way that beads are not compressed, wherein the loosely packed bed of medium has one or more unrestricted flow paths,
(ii) wherein the frits are comprised of pores having a pore size, wherein the pore size is large enough for cells to pass through and wherein the pore size is small enough to retain the loosely packed bed of medium;
(b) providing a biological sample having a sample volume, wherein the sample volume is larger than the bed volume, wherein the biological sample is comprised of intact cells, wherein the intact cells comprise viable cells, and
(c) passing the biological sample through the column, wherein the biological sample is passed through the column in a flowing stream, wherein the flowing stream is passed back and forth through the loosely packed bed of medium multiple times, wherein the cells travel through the unrestricted flow paths in the loosely packed bed of medium, whereby a portion of the intact cells are captured on the loosely packed bed of medium, wherein the biological sample is passed through the column at a rate of at least 0.01 ml/min.
2. The method of claim 1 , wherein the loosely packed bed of medium is a fluidized bed.
3. The method of claim 1 , wherein the method is further comprised of step (d), passing an eluent through the column to elute the intact cells captured in step (c).
4. The method of claim 1 , wherein the beads comprise an affinity group capable of capturing the intact cells in the biological sample.
5. The method of claim 4 , wherein the affinity group comprises an antibody or an aptamer capable of binding a cell surface marker present on the cells.
6. The method of claim 1 , wherein between steps (a) and (b), an antibody is passed through the column, wherein the antibody binds the beads within the loosely packed bed of medium and wherein the antibody is capable of binding a cell surface marker.
7. The method of claim 5 , wherein the cell surface marker is a B-cell surface maker, a T-cell surface marker, a monocyte surface marker or a macrophage surface marker.
8. The method of claim 5 , wherein the cell surface marker is selected from the group consisting of ALCAM, CD2, CD38, CD40LG, CD69, CD83, CD96, CTLA4, DPP4, HLA-DRA, IL12RB1, IL2RA, ITGA1, FRSF4, TNFRSF8, CD70, TNFSF7, CD160, CD28, CD37, CD3D, CD3G, CD247, CD5, CD6, CD7, FAS, KLRB1, KLRD1, NT5E, CD22, CD24, CD37, CD40, CD72, CD74, CD79A, CD79B, CR2, IL1R2, ITGA2, ITGA3, MS4A1, ST6GAL1, CD1C, CHST10, HLA-A, CD80, CD86, FCER2 and CD105.
9. The method claim 1 , wherein following step (c), the captured cells are labelled on the column.
10. The method of claim 1 , wherein the column is a pipette tip.
11. The method of claim 1 , wherein the column is a syringe.
12. The method of claim 1 , wherein the method is automated.
13. The method of claim 1 , wherein the method is performed with a plurality of pipette tip columns operated in parallel.
14. The method of claim 1 , wherein the biological sample is comprised of whole blood, blood, a biological fluid or tissue.
15. The method of claim 1 , wherein the intact cells are pathogenic cells, T cells, B cells, stem cells, sperm cells, mammalian cells, human cells, circulating tumor cells, viruses, bacteria, fungi, parasites or cancer cells.
16. The method of claim 1 , wherein the captured cells are activated B-cells, mature B-cells, cytotoxic T-cells, helper T-cells, activated T-cells, natural killer (NK) cells, monocytes, macrophages, endothelial cells, smooth muscle cells, dendritic cells, mast cells, fibroblasts, epithelial cells or adipocytes.
17. The method of claim 1 , wherein the column is sterile.
18. The method of claim 1 , wherein following step (c), a library of compounds is passed through the column.
19. The method of claim 1 , wherein the biological sample is passed through the column at a rate of at least 0.5 ml/min.
20. The method of claim 1 , wherein the method is performed in 1 hour or less.