Cell Separation Devices, Systems, and Methods
Disclosed herein are cell separation devices, methods and systems, as well as compositions and reagents for use in cell separation methods.
1 . A cell suspension, comprising
(a) a liquid medium having a volume of at least 1 mL; and
(b) 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, 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 the liquid medium and wherein the desired cells make up at least 80% of the suspended cells;
wherein the desired cells viability is greater than 90%;
wherein the number of viable desired cells is at least 1×10 3 ; 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.
2 . The cell suspension of claim 1 , wherein the desired cells are selected from the group consisting of CD3+ cells, CD4+ cells, CD235a, CD14+, CD19+, CD56+, CD34+, CD117 + , KDR + , SIRPA + , ASGR1 + , OCLN + , GLUT2 + , SLC6A1 + , TRA-1-60 − , SSEA4 − , AP − (alkaline phosphatase), SSEA3 − , TDGF1 − , or CD349 − cells.
3 . The cell suspension of claim 1 , wherein the cell suspension is present in a cell suspension removal stream via the transfer container of the functionally closed cell separation system.
4 . The cell suspension of claim 1 , wherein the number of viable desired cells is at least at least 2×10 6 .
5 . The cell suspension of claim 1 , wherein the desired cells comprise a buoyant label attached to the cells.
6 . A composition comprising:
(a) at least one binding agent covalently or non-covalently linked to at least one first linker, the at least one binding agent able to bind to at least one molecular target on the cells in a cell suspension; and
(b) at least one buoyant label covalently or non-covalently linked to at least one second complementary linker, the at least one buoyant label exhibiting a density substantially different from the density of the liquid medium in which the cells to be separated are suspended.
7 . A kit comprising
(i) a primary binding agent comprising an agent capable of binding 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;
(iv) 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.
8 . A composition comprising:
(i) a primary binding agent comprising an agent capable of binding 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;
(iv) 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.
9 . The composition of claim 8 , wherein the composition further comprises a target cell bound to the primary binding agent.
10 . The composition of claim 8 , wherein the calculated Tm of a hybrid of the first and second complementary region is between 40° C. and about 60° C.
11 . The composition of claim 8 , wherein the calculated Tm is greater than or equal to 40° C., as calculated using the nearest-neighbor two-state model:
Tm
(
°
C
.
)
=
Δ
H°
Δ
S°
+
R
ln
[
oligo
]
-
273.15
where ΔH° (enthalpy) and ΔS° (entropy) are the melting parameters calculated from the sequence and the published nearest neighbor thermodynamic parameters under the ionic conditions used, R is the ideal gas constant (1.987 calK −1 mole −1 ), [oligo] is the molar concentration of an oligonucleotide, and the constant of −273.15 converts temperature from Kelvin to degrees of Celsius.
12 . The composition claim 8 , wherein the primary binding agent is selected from the group consisting of antibodies, oligonucleotides, aptamers, molecularly imprinted polymers, carbohydrates, proteins, peptides, enzymes, small molecules, lipids, fatty acids, metal atoms, metal ions or synthetic polymers.
13 . The composition of claim 8 , wherein the primary binding agent comprises an antibody.
14 . The composition of claim 8 , wherein one of the first linker and the second linker further comprises biotin and the other further comprises streptavidin.
15 . The composition of claim 8 wherein the buoyant labels are selected from the group consisting of gas-filled bubbles, hollow polymers, glass beads, microporous based with entrained gas, droplets of an immiscible liquid, gold nanoparticles, and silver nanoparticles.
16 . The composition of claim 8 wherein the buoyant labels comprise gas-filled bubbles.
17 . The composition of claim 16 , wherein the gas-filled bubbles comprise perfluorocarbon gas cores encompassed by lipid, phospholipid, protein or carbohydrate shells.
18 . The composition of claim 16 , wherein the gas-filled bubbles comprise perfluorocarbon gas cores encompassed by phospholipid shells.
19 . The composition of claim 16 , wherein the gas-filled bubbles have a diameter between about 1 um and about 6.5 um.