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:
isolating bone-marrow cells from a bone-marrow sample obtained from the subject;
generating, utilizing gravitational forces without centrifugation and on a culture device not treated or coated to promote cell adhesion, a monolayer of bone-marrow cells from the bone-marrow cells by incubating the bone-marrow cells in a solution exposed to the gravitational forces without centrifugation at a density of about 100 cells per mm 2 to about 30000 cells per mm 2 to maintain naturally occurring cell-cell interactions and membrane integrity;
contacting the monolayer of bone-marrow cells that is not exposed to centrifugation with a therapeutic agent; and
assessing a response of the monolayer of bone-marrow cells to the therapeutic agent by microscopic analysis of the monolayer of bone-marrow cells.
2 . The method of claim 1 , further comprising altering or initiating treatment of the subject with the therapeutic agent based on assessing the response of the monolayer of bone-marrow cells to the therapeutic agent.
3 . The method of claim 1 , wherein incubating the bone-marrow cells comprises incubating at a density of about 2000 cells per mm 2 .
4 . The method of claim 1 , wherein:
isolating the bone-marrow cells comprises isolating the bone-marrow cells from non-nucleated cells; and
incubating the bone-marrow cells comprises incubating the bone-marrow cells that include less than about 100 non-nucleated cells per bone-marrow cells.
5 . The method of claim 1 , wherein generating the monolayer of bone-marrow cells further comprises adding a viability dye to the bone-marrow cells isolated from the bone-marrow sample prior to fixation, wherein the viability dye verifies that the bone-marrow cells are living.
6 . The method of claim 1 , wherein:
isolating the bone-marrow cells comprises isolating the bone-marrow cells that includes healthy bone-marrow cells and cancerous bone-marrow cells; and
generating the monolayer of bone-marrow cells comprises generating the monolayer that contains physiologically-relevant cell-cell interactions of the healthy bone-marrow cells and the cancerous bone-marrow cells.
7 . The method of claim 1 , wherein the method for determining whether the subject suffering from or predisposed to the disease will respond or is responsive to treatment with the therapeutic agent comprises not exposing the monolayer of bone-marrow cells to centrifugation after formation of the monolayer of bone-marrow cells.
8 . The method of claim 1 , wherein assessing the response of the monolayer of bone-marrow cells to the therapeutic agent by microscopic analysis comprises:
generating an image of the monolayer of bone-marrow cells in contact with the therapeutic agent; and
performing an image-based analysis by comparing the image of the monolayer of bone-marrow cells with a reference image of bone-marrow cells to determine to alter or to initiate treatment of the subject with the therapeutic agent.
9 . The method of claim 1 , wherein the disease is at least one of a hematologic malignancy or a malignancy of myeloid and/or lymphoid tissue.
10 . The method of claim 9 , wherein the disease is a myeloproliferative disorder, inflammatory disorder, latent virus infection, cellular growth disorder, cellular chemotaxis disorder, metabolic disorder, or autoimmune disorder.
11 . The method of claim 10 , wherein the disease is leukemia or lymphoma.
12 . The method of claim 1 , wherein microscopic analysis is confocal microscopic analysis.
13 . The method of claim 1 , wherein contacting the monolayer of bone-marrow cells with the therapeutic agent comprises:
fixing the monolayer of bone-marrow cells after it is formed to maintain the naturally occurring cell-cell interactions and the membrane integrity.
14 . The method of claim 13 , further comprising:
determining the naturally-occurring cell-cell interactions in the monolayer of the bone-marrow cells based on cell markers; and
adding a detectable label to the monolayer of bone-marrow cells that is fixed.
15 . The method of claim 13 , further comprising:
adding a detectable antibody-based label to the monolayer of bone-marrow cells that is fixed,
wherein subsequent to staining the monolayer of bone-marrow cells, the detectable antibody-based label minimizes washing requirements of the monolayer of bone-marrow cells within a threshold amount.
16 . The method of claim 1 , wherein isolating the bone-marrow cells from the bone-marrow sample comprises isolating the bone-marrow cells with diagnostic cell markers that include CD117 and CD34 positive cells.
17 . The method of claim 1 , wherein isolating the bone-marrow cells from the bone-marrow sample comprises isolating the bone-marrow cells with diagnostic cell markers that include CD34 and pSTAT5 positive cells.
18 . The method of claim 1 , wherein isolating the bone-marrow cells from the bone-marrow sample comprises isolating cell subpopulations that include neutrophilic metamyelocytes, neutrophilic myelocytes, segmented neutrophils, normoblasts and lymphocytes.
19 . The method of claim 1 , wherein isolating the bone-marrow cells from the bone-marrow sample comprises isolating viable bone-marrow cells.
20 . The method of claim 1 , wherein generating the monolayer of bone-marrow cells comprises forming the monolayer of bone-marrow cells with viable bone-marrow cells.