Monolayer of PBMCs or bone-marrow cells and uses thereof
The invention relates to peripheral blood mononuclear cell (PBMC) monolayers or bone-marrow cell monolayers and methods for its culture and corresponding uses of said monolayers. The present invention also relates, in some aspects, to screening methods comprising the PBMC monolayer or bone-marrow cell monolayer of the invention for determination of response or lack of response of a disease to a therapeutic agent and/or drug screening methods. In some aspects, the invention further relates to methods for diagnosing a disease or predisposition to a disease in a PBMC donor or bone-marrow cell donor comprising the PBMCs/bone-marrow cells cultured according to the method of the invention and/or to methods for determining whether the disease is likely to respond or is responsive to treatment with a therapeutic agent.
1. A method for determining whether a subject suffering from or predisposed to a disease will respond or is responsive to treatment with a therapeutic agent comprising
(a) isolating viable PBMCs from a blood sample obtained from said subject;
(b) incubating said viable PBMCs of step (a) at a density of about 100 cells per mm 2 to about 30000 cells per mm 2 to form a monolayer of viable cells under conditions that maintain naturally occurring cell-cell interactions and membrane integrity during formation of the monolayer of the viable cells;
(c) contacting said viable PBMCs in said monolayer of step (b) with said therapeutic agent; and
(d) assessing the response of PBMCs to the therapeutic agent by microscopic analysis of said monolayer of step (c),
wherein the viable PBMCs comprise lymphocytes and monocytes, and
wherein the viable PBMCs are not subjected to centrifugation and/or spinning during the formation of the monolayer.
2. The method of claim 1 , further comprising (e) altering or initiating treatment of the subject with said therapeutic agent based on the assessment of step (d).
3. The method of claim 1 , wherein the PBMCs are incubated at a density of about 2000 cells per mm 2 to form a PBMC monolayer.
4. The method of claim 1 , wherein PBMCs are isolated from non-nucleated cells, wherein the PBMCs to be incubated contain less than about 100 non-nucleated cells per PBMC.
5. The method of claim 1 , wherein the monolayer is obtained by a method further comprising or wherein the method further comprises a step of adding a viability dye.
6. The method of claim 1 , wherein subsequent to isolation the PBMCs are maintained/processed/analyzed at about 1 g, i.e. 9.81 m/s 2 .
7. The method of claim 1 , wherein subsequent to isolation the PBMCs are not subjected to centrifugation or spinning.
8. The method of claim 1 , wherein the disease is a hematologic malignancy or a malignancy of myeloid and/or lymphoid tissue.
9. The method of claim 1 , wherein the disease is a myeloproliferative disorder, inflammatory disorder, latent virus infection, cellular growth disorder, cellular chemotaxis disorder, metabolic disorder, or autoimmune disorder.
10. The method of claim 9 , wherein the disease is leukemia or lymphoma.
11. The method of claim 1 , wherein microscopic analysis is confocal microscopic analysis.
12. The method of claim 1 , wherein the monolayer is fixed after it is formed so as to maintain natural occurring naturally occurring cell-cell interactions and membrane integrity.
13. The method of claim 12 , wherein a detectable label is added to the fixed PBMCs.
14. The method of claim 13 , wherein the detectable label is an antibody.