Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5082—Supracellular entities, e.g. tissue, organisms
  • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates

Definitions

• the present invention relates generally to high-throughput assay methods that determine the proliferative status of hematopoietic stem and progenitor cells.
• the present invention further relates to kits comprising reagents and instructions for the use thereof to perform the assays of the present invention
• the hematopoietic system is unique in several ways. It is the only system capable of producing at least eight functionally different cell lineages from a single pluripotent stem cell. Assays are available that allow the differential effect of drugs on the various lympho-hematopoietic lineages to be examined. Second, the site of cell production changes during ontological development. This helps in differential sensitivity testing. Third, the site of production in the adult is the bone marrow, which is a significantly different tissue from the functional site of the peripheral circulation. Fourth, compared with other proliferating systems, and almost all other
• ATLANTA 366358vl systems of the body adult hematopoietic stem and progenitor cells are readily accessible.
• Hematopoietic stem and progenator cell lineages can be used to measure parameters that would normally be inaccessible.
• peripheral blood contains mature end cells that can be readily obtained to measure red and white blood cell counts, differential counts and other end stage blood parameters.
• NCI National Cancer Institute
• the peripheral blood also contains circulating populations of stem and progenitor cells that can be isolated and used for hematopoietic status monitoring and hemotoxicity testing.
• the so-called granulocyte-macrophage colony-forming cell (GM-CFC) assay and the enumeration of CD34 + cells (stem and early progenitor cells) currently form the basis of quality control for hematopoietic stem cell transplantation.
• ATLANTA 366358vl In vivo, an insult at the stem or early progenitor cell level requires a certain amount of time for the effect to be detected at the peripheral blood level. The effect may not be observed for weeks, or even months. This does not provide a high level of predictability and is why end stage cell parameters cannot be used to predict the effect of an agent. By the time the effect is observed, adverse reactions by the patient have already occurred.
• colony-forming assays based on stem or progenitor cells, on the other hand, can fulfill the requirements of prediction and sensitivity because they detect the effect of the insult before it is observed in the circulation.
• Colony-forming assays for leukemic cells are also available. In these classic assays, the more primitive the cell to be detected, the longer it takes to detect its progeny in the form of a colony.
• the proliferative potential of the cells being analyzed, and their ability to be stimulated by growth factors in vitro are essential for these assays. This dependency on the amplification compartment inherent in the hematopoietic system is often overlooked and without this component colony-forming assays in general, and especially predictive hemotoxicity testing, could not be performed.
• the proliferative status of primitive stem cells is considered to be quiescent, while the proportion of cells in cell cycle increases with stem cell maturity.
• the stem cell Once the stem cell has become determined with respect to a cell lineage, it enters the amplification compartment for producing the large and constant number of mature cells. With entry into the cell cycle, however, the cell becomes vulnerable to exogenous agents including the cytotoxic drugs typically used in oncology.
• the GM-CFC assay for example, has been used to predict myelosuppression (Prieto, P, Sci. Total Environ. 247, 349-354 (2000)).
• the predictive quality of this assay has been proven by validation studies with alkylating agents (Parchment et al, Toxicol. Pathol.
• ATLANTA 366358vl In the case of cytotoxic drug testing, the target cells have to be in cell cycle. For any drug that relies on cell proliferation, the tissues most affected or damaged by toxicity are those actively engaged in cell proliferation, which includes the bone marrow and the gastrointestinal tract. It therefore follows that hemotoxicity testing could also usefully be extrapolated to, and predictive for, the effects of a potential drug on other proliferating tissues.
• Toxicity in general, and hemotoxicity in particular, can also be correlated with the time of drug administration.
• the therapeutic index of a drug, and hence its toxicity is dependent, in part on the circadian variation in the hematopoietic cell division of rodents (Laerum, O.D, Exp. Hematol. 23, 1145-1147 (1995); Aardal et al, Exp. HemtoL, 11, 792-801 (1993); Aardal, Exp. Hematol. 12, 61-67 (1984); Wood et al., Exp. Hematol, 26, 523-533 (1998)), dogs (Haurie et al, Exp. Hematol.
• Invest. Dermatol. 115, 757-760 (2000)) and the corneal epithelium of the eye exhibit circadian organization.
• S-phase DNA synthesis preferentially occurs in the morning hours rather than in the evening or nighttime hours. This implies cytotoxic agents might be less toxic and exhibit high efficacy if given at a time when the proliferative status of the cells is at a nadir in these tissues.
• CFAs For toxicity testing, large numbers of comparative samples are needed, thereby making the enumeration of manual CFAs for this purpose impractical. CFAs also suffer from a lack of standardized colony enumeration procedures, and the subjectivity and high degree of expertise of the personnel and the time required for accurate enumeration of the colonies. The long culture periods required to visualize the proliferative potential of different cell populations is also a disadvantage. However, the culture period is an inherent property of the cell population and cannot
• the present invention relates generally to kits that provide reagent mixes and instructions for the use thereof, in performing high-throughput assay methods that determine the proliferative status of isolated target cell populations.
• the methods measure the luminescent output derived from the intracellular ATP content of incubated target cells, and correlate the luminescence with the proliferative status of the cells.
• the present invention further relates to kits that provide reagent mixes and instructions for the use thereof in high-throughput assays methods for screening compounds that may modulate the proliferative status of a target cell population.
• the kits of the present invention and methods therein described may be used for determining the proliferative status of any isolated cell line or type.
• the kits and methods of the present invention address the need for rapid assays that will determine the proliferative status of isolated hematopoietic stem and progenitor cells and of subpopulations of differentiated cells thereof
• kits and instructions for the use of the kits in a high-throughput assay method useful for rapidly determining the proliferative status of a population of cells, such as primitive hematopoietic cells, as a function of the ATP content of the cells comprising incubating a target cell population in a cell growth medium having a concentration of fetal bovine serum of between 0% and about 30%, a concentration of methyl cellulose between about 0.4%o and about 0.7%, and in an atmosphere having less than about 7.5% oxygen.
• the cell population is then contacted with a reagent capable of generating luminescence in the presence of ATP.
• the level of luminescence correlates with the amount of ATP in the cell population, wherein the amount of ATP indicates the proliferative status of the target cell population.
• the method of the present invention may further comprises contacting the target cell population with at least one cytokine and may further comprise generating a cell population enriched in hematopoietic stem cells, or a hematopoietic progenitor cell lineage.
• the kits and instructions of the present invention are also suitable for a high- throughput assay method for rapidly identifying a compound capable of modulating the proliferative status of a target cell population.
• a first target cell population is incubated in cell growth medium comprising a concentration of fetal bovine serum between 0% and about 30%, a concentration of methyl cellulose between about 0.4%> and about 0.7%, and in an atmosphere having less than about 7.5% oxygen and, typically, a cytokine.
• the method further comprises providing a first and a second target cell population, and contacting the first and second target cell populations with at least one test compound, contacting the target cell populations with a reagent capable of generating luminescence in the presence of ATP.
• the luminescence generated is detected by the reagent contacting the target cell populations, the level of luminescence indicating the proliferative status of the target cell population.
• the proliferative status of the first target cell populations is compared with the proliferative status of the second target cell population, thereby identifying a test compound capable of modulating the proliferative status of a target cell population.
• kits that comprise a plurality of vessels, each vessel containing one or more of the reagents that, when combined, provide rapid and error-reduced methods for performing the Hematopoietic and/or Hematotoxicity Assays via Luminescence Output (HALO) procedures of the present invention
• HALO Luminescence Output
• Figs. 1A-4B illustrate the correlation between the initial plated cell concentration (0.25, 0.5, 0.75, 1, 1.5 2 x 10 5 /well) and the mean (Figs. 1A, 2A, 3A and 4A respectively) or sum (Figs. IB, 2B, 3B, and 4B respectively) of relative luminescence units (RLU) measured at 4 days (Figs. 1A and IB), 7 days (Figs. 2A and 2B), 10 days (Figs. 3 A and 3B) and 14 days (Figs. 4A and 4B) after culture initiation, as a function of the integration time and/or gain of the plate reader.
• the value 2000 represents an integration time of 2000ms.
• “Max" represents the maximum integration time.
• the values 200, 215, 225 or 250 represent the gains that were used with the respective integration times.
• Fig. 5 shows a direct correlation between clusters counted manually in a conventional quadruplicate assay format and the 96-well plate format of the HALO method
• Fig. 6 shows the correlation on days 7, 10 and 14 between the mean number of cluster counts of erythroid bursts and the mean luminescence (RLU).
• Figs. 7A-7C illustrate histograms showing the number of cell clusters counted manually per well and the relative luminescence units (RLU) per well at day 7 (Fig. 7A), day 10 (Fig. 7B) and day 14 (Fig. 7C) of incubation.
• RLU relative luminescence units
• Figs. 8A-8C illustrate the lack of correlation between cell cluster counts per well and the relative luminescence units (RLU) per well on day 7 (Fig. 8A), day 10 (Fig. 8B) and day 14 (Fig. 8C) of culture incubation.
• RLU relative luminescence units
• Figs. 9A-9C show the correlation between the sum, or mean, of the cell cluster counts with the sum or mean of the relative luminescence units (RLU) measured on day 7 (Fig. 9A), day 10 (Fig. 9B) and day 14 (Fig. 9C) of culture incubation.
• RLU relative luminescence units
• Figs. 10A-10C show the correlation between cell concentration, sum of the replicate cell clusters and mean of the replicate cell clusters on day 7 (Fig. 10A), day 10 (Fig. 10B) and day 14 (Fig. IOC) of culture incubation.
• Figs. 11A-11C show the correlation between a manual 4-well assay and the 96-well assay method of the present invention. The results were plotted as either the sum or mean of the replicates obtained on day 7 (Fig. 11A), day 10 (Fig. 11B) and day 14 (Fig. 11C) of culture.
• Figs 12A and 12B show a comparison between manual cluster counts and luminescence using human bone marrow mononuclear cells.
• Fig. 13 shows a comparison between manual cluster counts and luminescence using mouse bone marrow mononuclear cells.
• Fig. 14 shows stem and multilineage cell assays using frozen non-human primate and rat bone marrow as targets.
• Figs. 15A and 15B show the mean RLU values for each of the DOX concentrations on peripheral blood mononuclear cells measured at day 3 (Fig. 15A) and day 7 (Fig. 15B).
• Fig. 16A and 16B show the effect of DOX on the ADP:ATP ratio of peripheral blood mononuclear cells at day 3 (Fig. 16A) and day 7 (Fig. 16B).
• ATLANTA 366358vl This description is made for the purpose of illustrating the general principles of the invention and should not be taken in the limiting sense.
• kits that comprise vessels, each vessel containing one or more of the necessary reagents mixes and instructions for the use thereof for performing the high-throughput assays of the present invention.
• the kits and instructions of the present invention provide high-throughput assays for detecting and measuring the proliferative status of populations of cells, especially of primitive hematopoietic stem and progenitor cells, and cell lineages derived therefrom.
• the methods of the present invention are especially useful when applied to populations of primitive hematopoietic cells including primary cells isolated from peripheral blood cells and bone marrow cells and hematopoietic stem and progenitor cells.
• the methods of the present invention may be applied to any population of proliferating cells, including cells isolated from tissues and solid tumors.
• the methods of the present invention can also be used to distinguish subpopulations of cells that may differ in the response to cytotoxic inhibitors, or activators such as cytokines The methods may be used to optimize the inhibitors to achieve maximum efficacy against a subpopulation of proliferating cells.
• An optimized dose determined from an isolated small sample of the cell population of a patient, may be administered to the proliferating cells in vivo, wherein the optimized dose may be administered systemically to the human or animal patient having the proliferating subpopulation of cells, thereby reducing the likelihood of potentially harmful side-effects to the recipient patient.
• the high-throughput assay methods of the present invention may also be used to determine the proliferative status of a population of hematopoietic stem or progenitor cells to determine their suitability and acceptability for transplantation into a recipient animal or human patient.
• animal refers to any vertebrate animal other than a human having a population of cells wherein at least one subpopulation of the cells may be proliferating or induced to proliferate.
• animal as used herein also refers to mammals including, but not limited to, bovine, ovine, porcine, equine, canine, feline species, non-human primates including apes and monkies, rodents such as rat and mouse, and lagomorphs such as rabbit and hare.
• tissue refers to a group or collection of similar cells and their intercellular matrix that act together in the performance of a particular function.
• the primary tissues are epithelial, connective (including blood), skeletal, muscular, glandular and nervous.
• cell refers to any cell population of a solid or non-solid tissue including, but not limited to, a peripheral blood cell population, bone marrow cell population, a leukemic cell line population and a primary leukemic cell line population or a blood stem cell population.
• the cells may be hematopoietic cells, including bone marrow, umbilical cord blood, fetal liver cells, yolk sac and differentiating embryonic stem cells or differentiating primordial germ cells or embryonic germ cells.
• the cells may be a primary cell line population including, but not limited to, a leukemic cell line.
• leukemic cell lines include, but are not limited to, an acute lymphocytic leukemia, an acute myeloid leukemia, a chronic lymphocytic leukemia, a chronic myeloid leukemia and a pre-B acute lymphocytic leukemia.
• Such cell lines include, but are not limited to, acute myelogenous leukemia, acute T-cell leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, acute monocytic leukemia and B-cell leukemia.
• target cell population refers to any cell population, especially hematopoietic stem and progenitor cells, or subpopulations thereof, that may be contacted with a test compound, wherein the test compound may modulate the proliferation of the cells in a positive or a negative direction depending upon the compound and the target cell population.
• cell line refers to cells that are harvested from a human or animal adult or fetal tissue, including blood and cultured in vitro, including primary cell lines, finite cell lines, continuous cell lines, and transformed cell lines.
• cell lineage refers to a cell line derived from a stem cell or progenitor cell that is committed to producing a specific functional cell including, but not limited to, mature cells of the a hematopoietic system.
• cell cycle refers to the cycle of stages in the replication of a eukaryotic cell.
• the cycle comprises the four stages Gl, S, G2 and M, wherein the S phase is that portion of the cycle wherein the nucleic acid of the cell is replicated.
• a cell identified as being in the S-phase of the cell cycle is also identified as being a proliferating cell.
• proliferative status refers to whether a population of hematopoietic stem or progenitor cells, or a subpopulation thereof, are dividing and thereby increasing in number, in the quiescent state, or whether the cells are not proliferating, dying or undergoing apoptosis.
• modulating the proliferative status or 'modulating the proliferation refers to the ability of a compound to alter the proliferation rate of a population of hematopoietic stem or progenitor cells
• a compound may be toxic, wherein the proliferation of the cells is slowed or halted, or the proliferation may be enhanced such as, for example, by the addition to the cells of a cytokine or growth factor.
• quiescent refers to cells that are not actively proliferating by means of the mitotic cell cycle.
• Quiescent cells which include cells in which quiescence has been induced as well as those cells which are naturally quiescent, such as certain fully differentiated cells
• Cultured cells can be induced to enter the quiescent state by various methods including chemical treatments, nutrient deprivation, growth inhibition or manipulation of gene expression, and induced to exit therefrom by contacting the cells with cytokines or
• primary cell refers to cells obtained directly from a human or animal adult or fetal tissue, including blood.
• the "primary cells” or “cell lines” may also be derived from a solid tumor or tissue, that may or may not include a hematopoietic cell population, and can be suspended in a support medium.
• the primary cells may comprise a primary cell line.
• hematopoietic stem cells refers to pluripotent stem cells or lymphoid or myeloid (derived from bone marrow) stem cells that, upon exposure to an appropriate cytokine or plurality of cytokines, may either differentiate into a progenitor cell of a lymphoid or myeloid cell lineage or proliferate as a stem cell population without further differentiation having been initiated.
• Hematopoietic stem cells include, but are not limited to, colony-forming cell-blast (CFC-blast), high proliferative potential colony forming cell (HPP-CFC) and colony-forming unit- granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM) cells.
• CFC-blast colony-forming cell-blast
• HPP-CFC high proliferative potential colony forming cell
• CFU-GEMM megakaryocyte
• progenitor and “progenitor cell” as used herein refer to primitive hematopoietic cells that have differentiated to a developmental stage that, when the cells are further exposed to a cytokine or a group of cytokines, will differentiate further to a hematopoietic cell lineage.
• Progenitors and “progenitor cells” as used herein also include “precursor” cells that are derived from some types of progenitor cells and are the immediate precursor cells of some mature differentiated hematopoietic cells.
• progenitor and “progenitor cell” as used herein include, but are not limited to, granulocyte-macrophage colony-forming cell (GM- CFC), megakaryocyte colony-forming cell (Mk-CFC), burst-forming unit erythroid (BFU-E), B cell colony-forming cell (B-CFC) and T cell colony-forming cell (T- CFC).
• GM- CFC granulocyte-macrophage colony-forming cell
• Mk-CFC megakaryocyte colony-forming cell
• BFU-E burst-forming unit erythroid
• B-CFC B cell colony-forming cell
• T- CFC T cell colony-forming cell
• Precursor cells include, but are not limited to, colony-forming unit-erythroid (CFU-E), granulocyte colony forming cell (G-CFC), colony-forming cell-basophil (CFC-Bas), colony-fo ⁇ ning cell-eosinophil (CFC-Eo) and macrophage colony-
• CFU-E colony-forming unit-erythroid
• G-CFC granulocyte colony forming cell
• CFC-Bas colony-forming cell-basophil
• CFC-Eo colony-fo ⁇ ning cell-eosinophil
• cytokine refers to any cytokine or growth factor that can induce the differentiation of a hematopoietic stem cell to a hematopoietic progenitor or precursor cell and/or induce the proliferation thereof.
• Suitable cytokines for use in the present invention include, but are not limited to, erythropoietin, granulocyte-macrophage colony stimulating factor, granulocyte colony stimulating factor, macrophage colony stimulating factor, thrombopoietin, stem cell factor, interleukin-1, interleukin-2, interleukin-3, interleukin-6, interleukin-7, interleukin-15, Flt3L, leukemia inhibitory factor, insulin-like growth factor, and insulin.
• cytokine as used herein further refers to any natural cytokine or growth factor as isolated from an animal or human tissue, and any fragment or derivative thereof that retains biological activity of the original parent cytokine.
• the cytokine or growth factor may further be a recombinant cytokine or a growth factor such as, for example, recombinant insulin.
• cytokine as used herein further includes species- specific cytokines that while belonging to a structurally and functionally related group of cytokines, will have biological activity restricted to one animal species or group of taxonomically related species, or have reduced biological effect in other species.
• cell surface antigen and "cell surface marker” as used herein may be any antigenic structure on the surface of a cell.
• the cell surface antigen may be, but is not limited to, a tumor associated antigen, a growth factor receptor, a viral- encoded surface-expressed antigen, an antigen encoded by an oncogene product, a surface epitope, a membrane protein which mediates a classical or atypical multi-drug resistance, an antigen which mediates a tumorigenic phenotype, an antigen which mediates a metastatic phenotype, an antigen which suppresses a tumorigenic phenotype, an antigen which suppresses a metastatic phenotype, an antigen which is recognized by a specific immunological effector cell such as a T-cell, and an antigen that is recognized by a non-specific immunological effector cell such as a macrophage cell or a natural killer cell.
• cell surface antigens within the scope of the present invention include, but are not limited to, CD3, CD4, CD8, CD34, CD90 (Thy-1) antigen, CD117, CD38, CD56, CD61, CD41, glycophorin A and HLA-DR,
• ATLANTA 366358vl CD133 defining a subset of CD34 + cells, CD19, and HLA-DR.
• Cell surface molecules may also include carbohydrates, proteins, lipoproteins or any other molecules or combinations thereof, that may be detected by selectively binding to a ligand or labeled molecule and by methods such as, but not limited to, flow cytometry.
• cell surface indicator refers to a compound or a plurality of compounds that will bind to a cell surface antigen directly or indirectly, and thereby selectively indicate the presence of the cell surface antigen.
• Suitable “cell surface indicators” include, but are not limited to, cell surface antigen-specific monoclonal or polyclonal antibodies, or derivatives or combinations thereof, and which may be directly or indirectly linked to a signaling moiety.
• the "cell surface indicator” may be a ligand that can bind to the cell surface antigen, wherein the ligand may be a protein, peptide, carbohydrate, lipid or nucleic acid that is directly or indirectly linked to a signaling moiety.
• flow cytometer refers to any device that will irradiate a particle suspended in a fluid medium with light at a first wavelength, and is capable of detecting a light at the same or a different wavelength, wherein the detected light indicates the presence of a cell or an indicator thereon.
• the "flow cytometer” may be coupled to a cell sorter that is capable of isolating the particle or cell from other particles or cells not emitting the second light.
• reagent capable of generating luminescence in the presence of ATP refers to a single reagent or combination of components that, in the presence of ATP, will generate luminescence.
• the amount of luminescence may be reliably related to the amount of ATP present.
• An example of a reagent suitable for use in the present invention is the combination of luciferin and luciferase as described by Crouch et al. (J. Immunol. Meth. 160, 81-88 (2000)) and Bradbury et al. (J. Immunol. Meth. 240, 79-92 (2000) incorporated herein by reference in their entireties.
• toxicity refers to the ability of a compound or a combination of compounds to negatively modulate the proliferation of a population of
• ATLANTA 366358vl hematopoietic stem or progenitor cells ATLANTA 366358vl hematopoietic stem or progenitor cells. It will be understood that the toxicity of a compound or compounds may be effective against one hematopoietic cell lineage and not against another, and may further include the ability of a compound to modulate the differentiation of a hematopoietic stem or progenitor cell.
• the term “differentially distinguishable” as used herein refers to hematopoietic stem and progenitor cells, or any other animal cell, the proliferation status of which may be usefully determined by the assay methods of the present invention and which can be characterized into subpopulations based on, for example, different complements of cell surface markers.
• the terms "a” and “an” as used herein, including the claims, are understood to mean “one” or "more”.
• HALO Hematopoietic and/or Hematotoxicity Assays via Luminescence Output
• IL interleukin
• PBMC peripheral blood mononuclear cells
• PBS phosphate-buffered saline (10 mM phosphate, 138 mM NaCl, 2.7 mM KC1, pH 7.4)
• FBS fetal bovine serum
• BSA bovine serum albumen
• BITSI (B)ovine serum albumin, recombinant human (I)nsulin,iron-saturated (T)ransferrin, (S)erum and (I)MDM
• IMDM Iscove's modified Dulbecco's medium.
• the high-throughput assay methods of the present invention comprise determining the proliferative status of a target cell population by measuring the metabolic activity of samples of proliferating cells as indicated by their ATP content.
• the ATP content can be measured by detecting the luminescence generated by a ATP-dependent reaction requiring, for example, by contacting the cells with an ATP- releasing agent and an ATP luminescence-monitoring agent.
• a suitable system for detecting ATP by the emission of luminescence comprises the combination of luciferin and luciferase, although it is contemplated that any method that will emit a detectable signal, the intensity of which may be correlated to the amount of ATP in a cell culture may be within the scope of the present invention.
• the high-throughput assay (HALO) method of the present invention allows for the detection of actively proliferating target cell populations, especially, but not limited to, hematopoietic stem and progenitor cell lineages that have been induced to undergo proliferation by exposure of the cell population to one or more cytokines. Most hematopoietic cell lineages can be induced to proliferate by contacting the cell population with at least one appropriate cytokine. It is, therefore, contemplated that a cytokine, or combination of cytokines, may be selected to induce the proliferation of a selected cell lineage.
• kits comprising a vessel or vessels that contain reagent mixes required for the HALO method, and instructions for performing this method.
• ATLANTA 366358vl High-throughput assays of hematopoietic stem and progenitor cell proliferation (Hematopoietic and Hematotoxicity Assays via Luminescence Output (HALO))
• the HALO platform of the present invention provides the biotechnology and pharmaceutical industry with a rapid, high-throughput, multifunctional testing system that can be used at all stages of drug development from screening to clinical trials.
• HALO is a proliferation assay with a luminescence readout typically performed in a 96-well plate.
• the present invention provides kits that can allow up to 11 different stem, progenitor and precursor cell populations, from different hematopoietic tissues, from at least five different species, to be detected and quantitatively measured simultaneously.
• Primitive hematopoietic cells can be isolated from suitable animal or human tissues including, for example, peripheral blood, bone marrow, or umbilical cord blood.
• Mononuclear cells for example peripheral blood mononuclear cells (PBMCs) may be further isolated by methods such as density-gradient centrifugation. It is contemplated to be within the scope of the present invention for the primitive cell population to be further subdivided into isolated subpopulations of cells that are characterized by specific cell surface markers. The methods of the present invention may further include the separation of cell subpopulations by methods such as highspeed high-speed cell sorting, typically coupled with flow cytometry.
• the channels of a flow-cytometer and high-speed cell sorter could be set at 530nm, typically used for FITC labeling, 670nm used for APC labeling, and a UV channel, for Hoechst (Ho) 33342 or DAPI staining.
• Fluorescent compensation software such as the System II or Expo 32 (Beckman Coulter) can allow full use of all of these channels.
• Cell subpopulations can be selected based on the presence or absence of cell membrane antigen markers, the intracellular pH, and the cell cycle status. Exemplary methods for selectively distinguishing subpopulations of hematopoietic cells are described, for example, in PCT application Serial No: 20010012620, incorporated herein in by reference in its entirety.
• Multiparameter analysis may be conducted on primary normal and leukemic samples or leukemic cell lines.
• the methods of the present invention may be conducted on primary normal and leukemic samples or leukemic cell lines. The methods of the present invention, however, may
• ATLANTA 366358vl be applied or adapted to any non-leukemic hematopoietic stem or progenitor cell population that might include a subpopulation of proliferating cells.
• An antigen indicator conjugated to APC can be used to selectively detect a normal blood stem cell subpopulation.
• Aliquots of cells may be labeled with panels comprising more than one biomarker.
• An example of one such panel incorporates CD38-FITC, CD34- APC, SNARF and Ho33342.
• Other examples of possible panels can include substituting CD38-FITC with CD117(c-kit)-FTTC, with CD91 (Thy-l)-FITC or with CD133-FITC.
• the procedures of the present invention can provide techniques to analyze combinations of cell markers as described above, or those specific for other lympho-hematopoietic lineages to differentiate the effects of inhibitors on normal different cell subpopulations.
• a similar reasoning can be applied to leukemic cell populations that also show aberrant flow cytometric profiles distinguishable from the normal population.
• a typical example would be chronic myeloid leukemia in chronic phase.
• the leukemic cell population can be defined by a high proportion of CD19 + cells. Therefore, CD19 is a biomarker that can be used to differentiate between leukemic and non-leukemic populations.
• the selected cell subpopulations can then be applied to the high-throughput assays of the present invention described in Examples 1 and 2 below.
• Cell surface indicators may be contacted with the hematopoietic stem or progenitor cells or leukemic cells thereof and the various subpopulations may be selectively separated by techniques such as flow cytometry or by attaching the cell surface indicators directly or indirectly to a separable solid support such as magnetic beads.
• the beads and the attached cells thereon can be isolated by a magnetic field.
• a cell lineage that is induced to proliferate by contacting a first primitive hematopoietic stem or progenitor cell population with a cytokine or combination of cytokines may further be contacted with a test compound that may have a cytotoxic
• ATLANTA 366358vl effect or a cell proliferation enhancing effect may also be determined by comparing the proliferation of the cell lineage in the presence of the test compound, and in its absence from the culture of a second targeted cell population or plurality of second cell populations. It is within the scope of the assay methods of the present invention for a plurality of test compounds to be compared for their cytotoxic effects on one, or a plurality, of proliferating target cell lineages. To these ends, a plurality of hematopoietic stem or progenitor cell populations may, for example, be plated in the wells of a multi-well plate or in individual chambers, thereby allowing rapid testing of multiple samples.
• the high-throughput assays of the present invention may be used to dete ⁇ nine the ability of a test compound to increase the proliferation of a population of hematopoietic stem or progenitor cells.
• proliferation enhancing compounds include, for example, cytokines and growth factors.
• the assay methods of the present invention may also be used with a range of concentrations of the test compound which may be contacted with a plurality of cell populations of the same cell lineage, whereupon the IC50 or the IC90 for the test compound acting against the targeted cell population or a subpopulation thereof may be calculated.
• the high-throughput assay methods of the present invention are also suitable for screening a population of hematopoietic stem or progenitor cells to determine the proliferation status of the cells or subpopulations thereof wherein the proliferative status will indicate the suitability of the stem or progenitor cells for transplantation into a recipient animal or human host.
• the high-throughput assay of the present invention will allow the selection of populations of primitive hematopoietic cell that will likely proliferate and maintain engraftment within the recipient patient.
• ATLANTA 366358v 1 High-throughput assay methods for determining the proliferative status of a target cell population
• target hematopoietic stem and/or progenitor cells may be isolated from animal or human tissues and suspended at cell concentrations ranging from about 1-5 x 10 2 to about 1-2 x 10 5 /ml. Since typical assay volumes are lOO ⁇ l, actual cell concentrations in the assay test vessels may be diluted to 1/10 of the original starting cell concentration.
• the cells are mixed and suspended in methyl cellulose containing 0% to about 30% concentration of fetal bovine serum (FBS), 1% detoxified bovine serum albumin (BSA), iron-saturated human transferrin at a final concentration of 1 x
• FBS fetal bovine serum
• BSA detoxified bovine serum albumin
• the methyl cellulose concentration in the assays of the present invention is between about 0.4% and about 0.7%, with a preferred concentration for most cell populations of about 0.7%.
• One exemplary medium is Iscove's Modified Dulbecco's Medium (IMDM, Life Technologies, Rockville, MD) although other suitable media capable of supporting the growth of hematopoietic cells may also be used.
• IMDM Iscove's Modified Dulbecco's Medium
• Low fetal bovine serum concentrations of between 0% and 10% can also be used.
• Iscove's Modified Dulbecco's Medium obtained from Invitrogen/Gibco (Carlsbad, CA) is prepared in small amounts (100-150ml) using sterilized, 17.3 MOhm water. A 1.75% methyl cellulose stock solution containing alpha-thioglycerol was prepared in IMDM.
• the volumes of all reagents are dependent upon the final volume(s) required for the study. The final volume of reagents is, in turn, dependent on the amount of methyl cellulose that can be dispensed in multiples of standard volumes using a repeater syringe.
• the components may be dispensed into tubes using electronic pipettes as follows: 5% FBS, 20% BIT, growth factors, methyl cellulose and IMDM. The components are thoroughly mixed
• ATLANTA 366358vl on a vortex mixer and cells are added and mixed again.
• the tubes are centrifuged briefly to 500 rpm so that the components are removed from the walls of the tubes.
• lOO ⁇ l of reagent mix is dispensed into replicate wells of a white, multi-well plate.
• the luminescence plate has a clear base so that cell growth can be observed under the inverted microscope.
• Culture plates are incubated at 37° Celsius in a humidified atmosphere containing 5% C0 2 and 5% 0 2 . On the day of analysis, the plates are transferred to a humidified incubator with 5% C0 at 22° Celsius to equilibrate. Reagents for ATP determination. Using a multichannel pipette, 125 ⁇ l of ATP-releasing reagent (ATP releasing reagent) is added to each well, mixed and returned to the 22° Celsius incubator for 15mins. Thereafter, 20 ⁇ l of ATP luminescence-monitoring reagent (ATP luminescence-monitoring reagent) is added and the luminescence read immediately.
• ATP-releasing reagent 125 ⁇ l of ATP-releasing reagent
• 20 ⁇ l of ATP luminescence-monitoring reagent ATP luminescence-monitoring reagent
• Data from the plate reader is used to calculate the mean, standard deviation and percent variation automatically for graphical presentation and/or statistical evaluation respectively.
• a lO ⁇ M ATP standard can be performed on the day of analysis to provide quality control for the reagents and equipment as well as a reference to which all values can be calculated.
• the high-throughput assay method of the present invention further includes contacting a hematopoietic stem or progenitor cell population with at least one cytokine that can induce the proliferation of the stem or progenitor cell population.
• the cytokine, or a combination of cytokines may be selected to induce the differentiation and proliferation of selected subpopulations of, for example, hematopoietic cell lineages.
• Exemplary cytokines include, but are not limited to erythropoietin, granulocyte-macrophage colony stimulating factor, granulocyte colony stimulating factor, macrophage colony stimulating factor, thrombopoietin, stem cell factor, interleukin-1, interleukin-2, interleukin-3, interleukin-6, interleukin-7, interleukin-15, Flt3L, leukemia inhibitory factor. Additional growth factors, alone or in combination, may also be included to boost the proliferative status of a particular culture of cells, including such factors as insulin-like growth factor, insulin and
• ATLANTA 366358vl recombinant insulin examples include cytokines or combinations thereof that may be used in the assay methods of the present invention and the specific targeted stem, progenitor or precursor cell types, and the resulting expanded cell lineages are given in Example 3 and Table 3 below.
• the stock cell culture is aliquoted into sample chambers. While sample chambers may be the wells of a multi-well tissue culture plate, and preferably a or 96-well plate, it is also contemplated to be within the scope of the present invention to conduct the assays of the present invention in any other suitable reaction vessels including, but not limited to, individual tubes, wells of plates and the like. Culture plates with a well surface area of about 35mm 2 and a low ring of about 2mm high are especially useful and allow colonies to be counted that are against the wall of the ring. Preferably the sample chambers are not tissue culture treated.
• Plastic that is sterilized and tissue culture treated exhibits different surface properties than plastic that is not sterilized by radiation and not tissue cultured treated.
• the change in surface properties results in cells preferentially adhering to the plastic and growing more rapidly than colony-forming cells. This is especially true if the cell suspension contains macrophages and other microenvironmental cell components.
• the surfaces of individual wells of a multi-well plate may not be treated homogenously and may result in complete growth inhibition in a significant number of wells. This can be a random event such that although 8-12 replicates may have been plated, up to 5 wells on a single plate might exhibit no growth whatsoever. Unwanted preferential adherence and growth may be avoided by using "non-sterile" and untreated plates. All tissue culture articles made from "virgin" plastic under very high temperatures, when released from the mold, are sterile. Contamination problems are unlikely. Non-treated and non-irradiated plates allow superior growth for the methods of the present application.
• multi-well plates that reduce background light emission or scatter when the plates are being enumerated in the plate reader may also be used. While it is desirable to use replicate reactions, it is to be understood that a single reaction sample may be used for dete ⁇ nining the proliferative
• ATLANTA 366358vl status of cells for each data point can be incubated in a humidified atmosphere having a low oxygen tension for a period preferably extending to about 10 days but also to at least about 14 days.
• a suitable oxygen concentration range is from about 3.5% oxygen to about 7.5% oxygen, most preferably about 5.0% oxygen, and further comprising about 5% C0 2 as described by Bradley et al. (J. Cell Physiol. 97, 517-522 (1968) and Rich & Kubanek (Exp. Hemat. 52, 579-588 (1982) incorporated herein by reference in their entireties.
• the assay can be used to rapidly and quantitatively determine: (a) the proliferative status of a hematopoietic stem or progenitor cell population or of cells of a specific progenitor and differentiation lineage and compare such in parallel assays; (b) if cells from a particular source exhibit a normal or abnormal proliferative capacity; and (c) whether a compound (e.g.
• the assay even of multiple samples, can be completed within 30 min, calculated from the time of adding the ATP releasing agent to the conclusion of the luminescence measurement.
• a high-throughput stem/progenitor cell assay (HT-SPCA) of the present invention does not count colonies or differentiate between colony types. Rather, the
• ATLANTA 366358vl HT-SPCA of the present invention measures the proliferation status of cells within the colonies by determining the amount of ATP being produced by the cells.
• some cells in the cultures will begin to proliferate and fonn aggregates or clusters.
• the proliferative status of the cell population may be limited due to their late stage of differentiation. Thus, a small colony may ensue within a short incubation period, but cell proliferation will rapidly cease.
• the culture conditions include ⁇ -thioglycerol to maintain molecules in a reduced form, and the cultures are incubated under low oxygen tension of between about 3.5% oxygen and about 7.5% oxygen, both conditions reducing oxygen toxicity.
• the cell aggregate or colony can be maintained in a stagnant or non-proliferative state for between about 2 and about 3 weeks.
• Other cells that are developmentally more primitive, for example, stem and progenitor cells, have a greater proliferative capacity and will begin to form colonies after a certain lag period of time. These cells will continue to divide throughout the whole of the incubation period. Eventually, the proliferative capacity of the cells within these colonies will also decrease and finally cease.
• the concentration of fetal bovine serum is between about 0% and 10%. In another embodiment of the method of the present invention, the concentration of methyl cellulose is about 0.7%.
• the concentration of oxygen in the atmosphere is about 5%.
• Another embodiment of the method of the present invention further comprises the step of contacting a target cell population with at least one cytokine and optionally may further comprise the step of generating a cell population enriched in hematopoietic stem cells.
• One embodiment of the method of the present invention comprises the step of generating a target cell population enriched in at least one hematopoietic progenitor cell lineage.
• the primitive hematopoietic cells are hematopoietic stem cells.
• the primitive hematopoietic cells are hematopoietic progenitor cells.
• the population of primitive hematopoietic cells comprises hematopoietic stem cells and hematopoietic progenitor cells.
• the primitive hematopoietic cells are primary hematopoietic cells.
• the target cell population is isolated from animal tissue selected from the group consisting of peripheral blood, bone marrow, umbilical cord blood, yolk sac, fetal liver and spleen.
• the animal tissue is obtained from a human. In one embodiment of the method of the present invention, the animal tissue is selected from bone marrow, yolk sac, fetal liver and spleen.
• the animal is a mammal.
• the mammal is selected from the group consisting of cow, sheep, pig, horse, goat, dog, cat, non- human primates, rodents, rabbit and hare.
• the animal tissue is human tissue further selected from the group consisting of peripheral blood, bone marrow, and umbilical cord.
• the primary hematopoietic stem cells are isolated from peripheral blood.
• Still another embodiment of the method of the present invention further comprises the step of selecting a differentially distinguishable subpopulation of primitive hematopoietic cells from the population of primitive hematopoietic cells, wherein the subpopulation of cells is defined by cell surface markers thereon.
• the step of selecting a differentially distinguishable subpopulation of primitive hematopoietic cells from the population of primitive hematopoietic cells comprises the steps of contacting the population of primitive hematopoietic cells with a cell surface marker indicator capable of selectively binding to a cell surface marker of a differentially distinguishable subpopulation of cells, and selectively isolating the subpopulation of cells binding the at least one indicator.
• the cell surface marker is selected from the group consisting of CD3, CD4, CD8, CD34, CD90 (Thy- 1) antigen, CD117, CD38, CD56, CD61, CD41, glycophorin A, HLA-DR, CD133 defining a subset of CD34 + cells, CD19, and HLA-DR.
• the cell surface marker is CD34 + .
• the subpopulation of differentially distinguishable primitive cells is selectively isolated by magnetic bead separation.
• the subpopulation of differentially distinguishable primitive cells is selectively isolated by flow cytometry and cell sorting.
• the population of primitive hematopoietic cells comprises at least one stem cell lineage selected from the group consisting of colony-forming cell-blast (CFC-blast), high proliferative potential colony forming cell (HPP-CFC) colony-forming unit- granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM).
• the population of primitive hematopoietic cells comprises at least one hematopoietic progenitor cell lineage selected from the group consisting of granulocyte-macrophage colony-forming cell (GM-CFC), megakaryocyte colony-forming cell (Mk-CFC), macrophage colony-forming cell (M-CFC), granulocyte colony forming cell (G- CFC), burst-forming unit erythroid (BFU-E), colony-forming unit-erythroid (CFU-E),
• GM-CFC granulocyte-macrophage colony-forming cell
• Mk-CFC megakaryocyte colony-forming cell
• M-CFC macrophage colony-forming cell
• G- CFC granulocyte colony forming cell
• BFU-E burst-forming unit erythroid
• CFU-E colony-forming unit-erythroid
• CFC-Bas colony-forming cell-basophil
• CFC-Eo colony-forming cell-eosinophil
• B-CFC B cell colony-forming cell
• T-CFC T cell colony-forming cell
• the reagent capable of generating luminescence in the presence of ATP comprises luciferin and luciferase.
• the cytokine is selected from the group consisting of erythropoietin, granulocyte- macrophage colony stimulating factor, granulocyte colony stimulating factor, macrophage colony stimulating factor, thrombopoietin, stem cell factor, interleukin-1, interleukin-2, interleukin-3, interleukin-6, interleukin-7, interleukin-15, Flt3L, leukemia inhibitory factor and combinations thereof.
• the at least one cytokine is stem cell factor, interleukin-6 and Flt3L.
• the at least one cytokine is macrophage colony stimulating factor, interleukin-1, interleukin-3, interleukin-6 and stem cell factor.
• the at least one cytokine is erythropoietin, granulocyte-macrophage colony stimulating factor, granulocyte colony stimulating factor, stem cell factor, interleukin-3, interleukin-6, and stem cell factor and Flt3L.
• the at least one cytokine is selected from the group consisting of erythropoietin, erythropoietin and interleukin-3, erythropoietin and stem cell factor and erythropoietin, stem cell factor and interleukin-3.
• the at least one cytokine is further selected from granulocyte-macrophage colony stimulating factor, granulocyte-macrophage colony stimulating factor and interleukin-3, and granulocyte- macrophage colony stimulating factor, interleukin-3 and stem cell factor.
• the at least one cytokine is further selected from the groups consisting of thrombopoietin, and
• the at least one cytokine is further selected from interleukin-2, and interleukin-7, Flt3L and interleukin-15. In still another embodiment of the method of the present invention, the at least one cytokine is selected from the group consisting of interleukin-7, and interleukin-7 and Flt3L.
• the at least one cytokine is erythropoietin. In another embodiment of the method of the present invention, the at least one cytokine is selected from the group consisting of granulocyte-colony stimulating factor and granulocyte-macrophage colony stimulating factor.
• the at least one cytokine is selected from the group consisting of interleukin-3, and interleukin-3 and stem cell factor.
• the at least one cytokine is selected from the group consisting of granulocyte-macrophage colony stimulating factor, interleukin-3 and interleukin-5.
• the at least one cytokine is selected from the group consisting of macrophage colony stimulating factor, macrophage colony stimulating factor and granulocyte- macrophage colony stimulating factor and interleukin-3, and granulocyte-macrophage colony stimulating factor.
• One embodiment of the method of the present invention further comprises the step of identifying a population of primitive hematopoietic cells having a proliferative status suitable for transplantation into a recipient patient.
• Another aspect of the present invention is a high-throughput assay method for rapidly identifying a population of having a proliferative status suitable for transplantation into a patient, comprising the steps providing a cell population comprising primitive hematopoietic cells, incubating the cell population in cell a
• ATLANTA 366358vl growth medium comprising between 0% and 30% fetal bovine serum and a concentration of methyl cellulose between about 0.4% and about 0.7%, and in an atmosphere having between about 3.5%> and about 7.5% oxygen, contacting the primitive hematopoietic cell population with at least one cytokine selected from the group consisting of erythropoietin, granulocyte-macrophage colony stimulating factor, granulocyte colony stimulating factor, macrophage colony stimulating factor, thrombopoietin, stem cell factor, interleukin-1, interleukin-2, interleukin-3, interleukin-6, interleukin-7, interleukin-15, Flt3L, leukemia inhibitory factor, insulinlike growth factor, and insulin, contacting the cell population with a reagent capable of generating luminescence in the presence of ATP, and detecting luminescence generated by the reagent contacting the at least two cell populations, the level of luminescence indicating the prolife
• the hematopoietic stem cell lineage is selected from the group consisting of colony-forming cell-blast (CFC-blast), high proliferative potential colony forming cell (HPP-CFC) colony- forming unit-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM).
• CFC-blast colony-forming cell-blast
• HPP-CFC high proliferative potential colony forming cell
• CFU-GEMM megakaryocyte
• contacting the target cell population of with a cytokine generates a cell population enriched in at least one hematopoietic progenitor cell lineage.
• the population of primitive hematopoietic cells comprises at least one hematopoietic progenitor cell lineage selected from the group consisting of granulocyte-macrophage colony-forming cell (GM-CFC), megakaryocyte colony- forming cell (Mk-CFC), macrophage colony-forming cell (M-CFC), granulocyte colony forming cell (G-CFC), burst-forming unit erythroid (BFU-E), colony-forming
• GM-CFC granulocyte-macrophage colony-forming cell
• Mk-CFC megakaryocyte colony- forming cell
• M-CFC macrophage colony-forming cell
• G-CFC granulocyte colony forming cell
• burst-forming unit erythroid BFU-E
• CFU-E unit-erythroid
• CFC-Bas colony-forming cell-basophil
• CFC-Eo colony-forming cell-eosinophil
• B-CFC B cell colony-forming cell
• T-CFC T cell colony- forming cell
• Yet another aspect of the present invention is a high-throughput assay method for rapidly identifying a compound capable of modulating the proliferative status of a population of primitive hematopoietic cells, comprising providing target cell population, incubating the cell population in cell a growth medium comprising between 0% and 30% fetal bovine serum and a concentration of methyl cellulose between about 0.4%> and about 0.7%, and in an atmosphere having between about 3.5% and about 7.5% oxygen, contacting the target cell populations with at least one cytokine selected from the group consisting of erythropoietin, granulocyte- macrophage colony stimulating factor, granulocyte colony stimulating factor, macrophage colony stimulating factor, thrombopoietin, stem cell factor, interleukin-1, interleukin-2, interleukin-3, interleukin-6, interleukin-7, interleukin-15, Flt3L, leukemia inhibitory factor, insulin-like growth factor, and insulin, providing a first and a
• the hematopoietic stem cells are selected from the group consisting of colony-forming cell-blast (CFC-blast), high proliferative potential colony forming cell (HPP-CFC)
• contacting the first and second target cell populations of primitive hematopoietic cells with at least one cytokine generates cell populations enriched in at least one hematopoietic progenitor cell lineage.
• the at least one hematopoietic progenitor cell lineage selected from the group consisting of granulocyte-macrophage colony-forming cell (GM-CFC), megakaryocyte colony-forming cell (Mk-CFC), macrophage colony-forming cell (M- CFC), granulocyte colony forming cell (G-CFC), burst-forming unit erythroid (BFU- E), colony-forming unit-erythroid (CFU-E), colony-forming cell-basophil (CFC-Bas), colony-forming cell-eosinophil (CFC-Eo), B cell colony-forming cell (B-CFC) and T cell colony-forming cell (T-CFC).
• One embodiment of the method of the present invention further comprises the steps of contacting a target cell population with at least two concentrations of a test compound, and calculating the IC50 of the test compound.
• Another embodiment of the method of the present invention further comprises the steps of contacting a target cell population with at least two concentrations of a test compound and calculating the IC90 of the test compound.
• kits of the present invention provide the components and instructions for their use in the HALO procedure, thereby providing an easy to use, flexible assay system whereby the effects of both positive and negative regulators of hematopoiesis can be studied using a number of different cell populations, in different species of interest.
• ATLANTA 366358vl In the HALO methods that form the basis of the kits of the present invention, ATP is released from the cells using ATP-releasing reagent and a 15 min incubation. Thereafter, the ATP luminescence-monitoring reagent is added and the plates read immediately. Thirty minutes later, an ADP converting reagent (ADP-CR) is added which converts ATP to ADP and the plates are read every 5 mins for a further 15-20 mins. Three values are then used to calculate the kinetics of the reaction. The first reading (A) is that immediately after addition of ATP luminescence-monitoring reagent. The second reading (B) is just prior to addition of ADP-CR, when the decline in ATP is maximal.
• ADP-CR ADP converting reagent
• the third reading (C) is taken at a time when the conversion of ADP to ATP has reached a plateau.
• the ADP:ATP ratio is then calculated from the simple equation: (C-B)/A.
• This procedure was performed to obtain the results shown in Fig. 16.
• the procedure is preferably performed by the automatic addition of ATP luminescence-monitoring reagent and ADP-CR using a luminometer equipped with two injectors.
• a plating mixture may be necessary that has as many as 14 components, including the cells. Once assembled and mixed, these plating mixtures must then be divided into replicate wells of a multiple-well plate, and the cells allowed to grow.
• the assay staging pre-combines serum and serum replacement components, such that addition of a "serum” cocktail in a single standardized volume, compatible with the same repeating syringe dispensers described above, results in the desired concentrations of each component in each final
• BITSI ATLANTA 366358vl plating mixture being assembled.
• This assay component is designated BITSI.
• BITSI Bovine serum albumin (50mg/ml), recombinant human Insulin (50 ⁇ g/mi) iron-saturated Transferrin (lmg/ml), Serum, and IMDM, although it is contemplated that the amounts and final concentrations of the individual components of BITSI may be varied in accordance with the requirements of a particular cell line being cultured
• the bovine serum albumin, insulin and transferrin may be a single reagent (for example, BIT, Stem Cell Technologies, Vancouver, Canada), that is combined in a 4:1:3 ratio with serum and IMDM respectively.
• IMDM IMDM
• BITSI volume of BITSI required to one that can be dispensed with positive displacement syringe dispensers.
• FBS pre-screened for human cell growth can be used, whereas for mouse and rat cells, FBS pre-screened for murine cell growth is preferred. In each case, a final assay concentration of 5% serum/20% BIT is achieved.
• Combinations of growth factors may be used to stimulate the proliferation of each lineage tested using the HALO procedure. For example, 6 different growth factors are required to stimulate the proliferation of CFC-GEMM, and three different growth factors are required to stimulate the proliferation of BFUe, GM-CFC and Mk- CFC.
• Each of the growth factors required for a particular lineage can be combined, in appropriate proportions, into a lineage-specific growth factor mix, thereby thereby obviating the need to add each of the required growth factors separately.
• a combination of IL-3, IL-6, SCF, GM-CSF, EPO, and G-CSF, added respectively at, for example, doses of 1, 2, 3, 4, 10 and 20ul per ml of plating mixture, can be used to stimulate the proliferation of CFC-GEMM. 40 ⁇ l of this GEMM-specific cocktail would be required for each 1ml of lineage-specific plating mixture being prepared.
• BFU-E for BFU-E, IL-3, SCF, and EPO, respectively at doses of 1, 3 and lOul per ml of plating mixture, could be premixed and 14 ⁇ l of the resulting BFU-E-specific cocktail would be required per ml of plating mixture being prepared.
• 8 ⁇ l of a 1:3:4 premix of IL-3, SCF, and GM-CSF is used to stimulate the proliferation of GM-CFC
• 5 ⁇ l of a 1 :2:2 premix of IL-3, IL-6 and TPO would be
• kits of the present invention Large volumes of pre-mixed growth factor combinations are provided by the kits of the present invention, aliquoted, and frozen ready for use
• kits of the present invention therefore, provide lineage-specific plating mixtures from three premixed reagent mixes, each mix preferably provided in separate vessels (i.e. 2.5X methyl cellulose, a 1.75% methyl cellulose base may contain 2.5X final concentration of ⁇ -thioglycerol, BITSI reagent, growth factor cocktails). Volumes of each mix vary depending upon the final volume of plating mixture required for each lineage under test.
• An additional component to be added to the final mix is a standardized volume of cell suspension containing a predetermined concentration of target cells, and sufficient IMDM to take account of variations in the volume of growth factor cocktails added to different lineages, thereby adjusting each plating mixture to an appropriate final volume.
• the kits may further comprise a luminescence plate that, preferably, is a non-treated, non-sterile multi-well plate.
• the kits of the present invention also comprise instructions for the use of the kit in preparing and performing the HALO procedure.
• the HALO procedure comprises the following stages: (i) assembly of lineage-specific plating mixtures from liquid components, i.e. BITSI, growth factor cocktails, target cells and medium; (ii) addition of methyl cellulose/ -TG mix using positive displacement liquid handling devices; (iii) distributing of aliquots, for example, 100 ⁇ l, of each plating mixture onto multi- well plates using positive-displacement liquid handling devices; (iv) addition of ATP releasing reagent to each of the culture wells; (v) addition of ATP luminescence- monitoring reagent to each of the culture wells; and (vi) addition of ATP-CR to each of the culture wells if apoptosis is to be measured.
• liquid components i.e. BITSI, growth factor cocktails, target cells and medium
• addition of methyl cellulose/ -TG mix using positive displacement liquid handling devices distributing of aliquots, for example, 100 ⁇ l, of each plating mixture onto multi- well plates using positive-displacement
• Stages (i) and (iv) are can be performed using automated liquid handling workstations, and stages (v) and (vi) can be performed using a luminometer equipped with reagent injectors.
• the liquid handling workstation should be confined during this part of the procedure to a sterile environment, either by containing such a workstation within a laminar flow hood, or by using it only within the confines of a separate clean room supplied with positive pressure HEPA-filtered air.
• the liquid handling workstation should, therefore have a footprint small enough to allow enclosure within a laminar air-flow hood.
• any such workstation should be capable of (a) using sterile tips capable of dispensing a wide range of sample volumes, (b) transferring reagents to and from tubes, and to 96 well plates and (c) dispensing ATP releasing reagent using either 8-tip, 12-tip or 96-tip manifolds.
• sterile tips capable of dispensing a wide range of sample volumes
• transferring reagents to and from tubes and to 96 well plates
• dispensing ATP releasing reagent using either 8-tip, 12-tip or 96-tip manifolds.
• a semi-automated embodiment of the HALO procedure of the present invention would be as follows. Firstly, liquid reagent cocktails, i.e. BITSI and lineage-specific growth factor cocktails, cell suspensions, and medium are assembled into an appropriate number of lineage-specific plating mixtures, optionally using an automated liquid handling workstation. Aliquots of a 2.5X methyl cellulose/ ⁇ -TG mix may be added manually to these plating mixtures, using a repeater pipette and a disposable positive-displacement syringe tip.
• liquid reagent cocktails i.e. BITSI and lineage-specific growth factor cocktails, cell suspensions, and medium are assembled into an appropriate number of lineage-specific plating mixtures, optionally using an automated liquid handling workstation. Aliquots of a 2.5X methyl cellulose/ ⁇ -TG mix may be added manually to these plating mixtures, using a repeater pipette and a disposable positive-displacement syringe tip.
• lOO ⁇ l aliquots of each would then be dispensed manually, again using a repeater pipette and disposable syringe tips, onto 96-well plates containing agents under test in appropriate concentration ranges. After incubating assay plates for an appropriate length of time, aliquots of ATP releasing reagent would be added to the wells of each plate under test, using a robotic liquid handling workstation. Plates would be incubated at room temperature for 15 minutes as per manual assays, and
• ATLANTA 366358vl would then be transferred to an injector-equipped luminometer, where of ATP luminescence-monitoring reagent would be injected automatically into each well immediately prior to measurement of ATP-generated bioluminescence. If apoptosis measurements were also to be made, aliquots of ATP-CR would subsequently be injected automatically (from a second injector), and the concomitant conversion of ADP to ATP measured over time. Automating the ATP measurement stage of the assay improves the high-output capacity of the assay system. Similarly, partially automating the "front-end" of the assay significantly increases the throughput of assay plates.
• kits of the present invention are defined for use with human cells, it can also be used to culture non-human primate (both rhesus and cynmologus primates) and canine hematopoietic cells. If the kits are defined for use with mouse cells, they can also be used to culture rat hematopoietic cells.
• ATLANTA 366358vl Transfer sufficient medium to a tube so that it will cover the whole bone. (Some of the medium provided with the kit can be used for this purpose).
• PB peripheral blood
• BM bone marrow
• CB umbilical cord blood
• DGSM density gradient separation medium
• ATLANTA 366358vl cells on top taking care not to mix the DGSM with the cells. 5. Once all the cells have been layered in this way, centrifuge the tube(s) at 400 x g for 25min at room temperature without using the centrifuge brake to slow the rotor. 6. Carefully remove the tubes from the centrifuge without mixing the contents and aspirate the upper diluted plasma layer to approximately 5- 10mm above the interface with the DGSM. The interface contains the mononuclear cells.
• Non-human primate and canine peripheral blood and bone marrow Both of these sources need to be processed in the same manner as human cells to separate the mononuclear cell fraction.
• the same protocol can be followed if non- human primate or canine hematopoietic tissue, but ensure that the correct density- gradient medium is used to separate the mononuclear cells from different species.
• ATLANTA 366358vl Table 1 Recommended Cell Concentrations for Different Species, Cell Types, Cell Preparations and Cell States for the HALO Platform
• Step 2 HALO preparation and cell culture
• a typical HALO kit contains the 3 component mixes required to culture cells:
• Methyl cellulose is a viscous, water soluble, semi-solid medium for immobilizing cells.
• the amount of methyl cellulose provided is for the number of 96-well plates determined by the kit. However, if dispensed carefully, there is sufficient methyl cellulose so that several smaller experiments can be performed using the plate(s) provided.
• a repeater pipette may be used which uses a positive-displacement syringe is used to dispense the methyl cellulose mix.
• Dispensing the master mix Once all the component mixes have been added together, a repeater pipette to dispense the master mix into individual wells. Using normal syringes with needles may result in inaccurate dispensing and greater variation between replicate wells.
• Plate configuration The configuration of the 96-well plate for an experiment is arbitrary. However, the number of replicates and the way in which the reagents required to measure luminescence are added, will determine the plate configuration. For example, if performing 4 or 8 replicates per culture point, we recommend configuring the plate in columns, that is, Al to Dl and El to HI etc. for quadruplicate cultures and Al to HI, A2 to H2 etc. for 8-replicate cultures are possible. If performing 6 or 12 replicates, we recommend configuring the plate in rows, that is, A1-A6 and A7 to A12 for 6 replicates and A1-A12, Bl to B13 for 12 replicates are possible.
• addition of the ATP releasing reagent and ATP luminescence-monitoring reagent can be performed using an 8-channel pipette from left to right across the plate. If performing row replicates, an 8- or 12-channel pipette can be used to dispense the reagents from the top to the bottom of the plate. To calculate the quantities of reagents required:
• ATLANTA 366358vl Total number of wells Number of sample categories x Number of replicates
• Total volume ( ⁇ l) to be prepared (Number of wells x lOO ⁇ l) + 20%.
• ATLANTA 366358vl Before adding the target cells, ensure that a single cell suspension has been prepared.
• Dispense lOO ⁇ l of the reagent master mix into each of the replicate wells This can be performed with a syringe and needle, but a repeater pipette is recommended for ease of use, reproducibility and to reduce variation.
• Step 3 Luminescence measurement with and without manual enumeration
• Luminescence kit components Prior to measuring luminescence, remove the ATP standard and the ATP monitoring reagent are thawed at room temperature or at 22° Celsius - 23° Celsius. The ATP releasing reagent is equilibriated at room temperature or at 22° Celsius - 23° Celsius.
• Luminometer (Set luminometer parameters to: maximum integration time, 1-2 sec; initial gain (if required) at 225; shake duration (if required) to 15s orbital; measurement temperature, 22° Celsius. If the luminometer has an injector, gain and shaking control is not necessary. 8- or 12-channel pipette. Reagent reservoirs for an 8- or 12-channel pipette.
• the performance of an ATP dose response prior to each luminescence measurement has 3 functions: 1. It tests whether the reagents are working properly.
• Adhesive plate covering film To help keep the plate(s) sterile, adhesive, air permeable, sterile covering film is provided so that the part of the plate that is not being used can be covered and kept sterile until required. If using the adhesive film provided, the plate cover should be removed in a laminar air-flow hood and replaced with the film to ensure sterility.
• a PU is a cluster of 8 or more cells that can exist individually or as part of a growing colony. Clusters can grow from the center outward producing an evenly distributed entity having a single central mass of cells. This would be considered a single PU.
• a cluster may consist of an irregular shape and be composed of one or more areas in which a concentration of cells can be seen. These areas usually appear darker than the rest of the cluster. Each of these areas is considered a PU and has to be counted.
• each protuberance from a developing cluster is also considered a PU.
• Each PU is derived from a single cell.
• ATLANTA 366358vl First dilution to l ⁇ M: Remove lOO ⁇ l of the supplied ATP solution (at lO ⁇ M) and transfer it to vial #1. Add 900 ⁇ l of the medium provided in the kit. Mix by vortexing. This ATP concentration is l ⁇ M.
• ATLANTA 366358vl 16 Using new tips, add lOO ⁇ l of ATP luminescence-monitoring reagent to the wells of the first column, mixing as described in Step 13 and discard tips.
• ATP releasing reagent and ATP luminescence-monitoring reagent are performed is the same manner as that for ATP. 1. Place the sample plate(s) in a humidified incubator set at 22° Celsius-23°
• Example 2 Measurement of the ATP content of incubated hematopoietic stem or progenitor cells
• the reagents from the ViaLight HSTM kits were prepared for use. If necessary, the number of cell clusters (aggregates) or colonies that had developed in the wells of the incubated 96-well plates could be counted under an inverted microscope to ensure that a correlation between the sum, or mean, of the ratio of clusters/colonies to the relative luminescence units (RLU) was obtained (see below).
• the ATP luminescence-monitoring reagent was reconstituted as described by the manufacturers by adding 10ml of the supplied buffer to the lyophilized reagent and waiting 15 mins. Alternatively, 1ml of the buffer was used to reconstitute the reagent and the latter was then aliquoted into 1.5ml microtubes and frozen while protected from light. Aliquots were then thawed and diluted to 1ml final volumes using the supplied buffer as needed. The ATP monitoring reagent was protected from light at all times.
• the required quantity of ATP releasing reagent was transferred into the reagent trough and lOO ⁇ l aliquots were transferred, using a multi-tip pipette, to each row or column of wells of the 96-well plated previously incubated as described in Example 1 above. After dispensing the reagent to one row or column, the contents of the wells were mixed at least 4-5 times with the pipette, so that the reagents mixed well with the methyl cellulose master mixes. Addition of the reagents diluted the methyl cellulose and mixing ensured that the cells came into contact with the ATP releasing reagent. This step had to be performed in a similar manner for all wells.
• the plates were typically incubated in the dark for 5min, although the incubation could proceed for up to 30min without loss of sensitivity.
• ATLANTA 366358vl The required amount of ATP luminescence-monitoring reagent was transferred to a new, clean trough and 20 ⁇ l of the reagent pipetted into each of the wells while ensuring that the contents of each well was mixed thoroughly. The plates were immediately transferred to a plate reader and the luminescence measured using an integration time of 1000ms.
• the gain was adjusted such that the luminescence from the ATP standard was approximately 20,000 RLU. By adjusting the gain of the machine to obtain this number of RLU, and then reading the remaining wells of the plate (containing the incubated cells), no overflow values occurred, thereby obviating the need for a second or multiple reading. In all cases, the luminescence was measured in the shortest possible time period possible, because the luminescence decreased rapidly with time.
• ATLANTA 366358vl days (Figs. 4A and 4B) after culture initiation, as a function of the integration time and/or gain of the plate reader.
• the value 2000 represents an integration time of 2000ms.
• “Max” represents the maximum integration time.
• the values 200, 215, 225 or 250 represent the gains that were used with the respective integration times.
• PU proliferative units
• FIG. 5 shows a direct correlation between clusters counted manually in a conventional quadruplicate assay fonnat and the 96-well plate format of the HALO method derived from peripheral blood erythropoietic progenitor cells (burst-forming units-erythroid, BFU-E).
• Fig. 6 shows the correlation on days 7, 10 and 14 between the mean number of cluster counts of erythroid bursts and the mean luminescence (RLU). Although there are very low numbers of BFU-E clusters on day 7, the slope of the curve is steep because of the high cell proliferation occurring at this time.
• Example 3 Hematopoietic stem and progenitor cell lines and their associated cytokine effectors Hematopoietic stem and progenitor cells are induced to differentiate into hematopoietic cell subpopulations by exposure to one or more growth factors/cytokines, as shown in Table 3 below.
• ATLANTA 366358vl Example 4: Proliferation of hematopoietic stem and progenitor cells measured by colony counting and ATP determination
• Figs. 7A-7C When cell proliferation was measured as a function of time in culture, some aggregates or colonies contained cells that were proliferating, while others were not, as shown in Figs. 7A-7C. Wells, therefore, could contain few colonies, but still exhibit high cell proliferation. The results shown in Figs. 7A-7C show that the number of cell clusters counted per well does not correlate with the cell proliferation as detected using the luminescence of the present invention.
• the number of colonies in a well did not generally correlate with the RLU from that well, as shown in Figs. 8A-8C.
• the luminescence measurements were made over the whole area of each and every replicate well, and not from the individual cell aggregates or colonies within these areas.
• the sum of the luminescent values of aggregates or colonies, or the mean of the aggregates or colonies from all replicate wells, can be predicted to correlate with the sum or mean of the luminescence emitted
• ATLANTA 366358vl from the replicate wells, as shown in Figs. 9A-9C.
• Example 5 Use of high-throughput stem/progenitor cell assays to determine the ability of a test compound to modulate the proliferation of hematopoietic stem and progenitor cells
• the HT-SPCA of the present invention is used to test dose responses for a variety of compounds that can interact with hematopoietic stem and progenitor cells.
• the agents either stimulate or inhibit and/or kill hematopoietic cells. Increasing doses of an agent can stimulate cells, but then be inhibitory by being toxic and causing necrosis. Other agents can be toxic at high doses, but induce apoptosis at lower concentrations.
• hematopoietic stem and progenitor cells include, 5-fluorouracil (5-FU), hydroxyurea, cytosine arabinoside (ara-C). busulphan, 3'azido- 3'deoxythymide (AZT), cycloheximide, actinomycin D, etoposide, BCNU, doxorubicin, cisplatin (low hemotoxicity) and carboplatin.
• Growth factors known to inhibit the proliferation of stem and progenitor cells such as interferon- ⁇ (IFN ⁇ ),
• TGF- ⁇ tumor necrosis factor- ⁇
• TGF ⁇ transforming growth factor- ⁇
• Neutraceuticals include, for example, the anti-inflammatory phytochemicals, black and green tea polyphenols, resveritrol, limonene and curcumin.
• mononuclear cells derived from peripheral blood, bone marrow and cord blood are used.
• CD34+ cells derived from these tissues can also be used.
• HT-SPCA can be used to detect and predict hemotoxicity against hematopoietic stem and progenitor cell populations. To validate the inhibition/hemotoxicity of the agents, both manual CFA and the HT-SPCA are performed in parallel. One of the end points is to determine the IC50 and IC90 for the drug.
• Example 7 Measurement of oxidative damage.
• Oxidative damage is indicated in a wide range of pathological conditions such as carcinogenesis, diseases associated with inflammation and ischemia-reperfusion injury as well as in normal metabolic activity. Xenobiotics, environmental toxins and radiation induce damage by generating free radicals and reactive oxygen species (ROS) that can lead to mutation and cancer.
• ROS reactive oxygen species
• the assay system developed for the HALO methods of the present invention provides a procedure to measure oxidative stress and DNA damage based on that developed for flow cytometry. Stem and multilineage progenitor cell assays are staged in exactly the same manner as for HALO. After an incubation period of 5-10 days, 250 ⁇ l of PBS are be added to the wells, the contents mixed and the plates centrifuged at 300 g for 10 min at room temperature. Fluid (300 ⁇ l) will be aspirated from each
• Optilyse Reagent is a combination cell fixative and lysis reagent for erythrocytes. The plates are centrifuged again, the fluid aspirated and lOO ⁇ l of OxyDNA reagent added and incubated for 20 mins at 37° Celsius. Fluorescence is then measured in a plate reader by excitation at 455nm and emission at 530nm (for FITC) or 590nm (for Texas Red).
• Example 8 HALO multilineage testing platform.
• the three stem cell populations to be tested were the colony-forming cell - blast (CFC-blast), the high proliferative potential colony-forming cells (HPP-CFC) and the colony-forming cell - granulocyte, erythroid, macrophage, megakaryocyte (CFC-GEMM).
• the HALO platform of the present invention can be used to detect colony-forming cells of the erythropoietic progenitor (BFU-E) and precursor (CFU-E) cells, granulocyte-macrophage progenitor (GM-CFC) and granulocyte (G-CFC) and macrophage (M-CFC) precursor cells, megakaryocyte progenitor cells (Mk-CFC) and T- and B-lymphocyte progenitor cells (T-CFC and B-CFC).
• Figs. 12A and 12B show a comparison between manual cluster counts and luminescence using bone marrow mononuclear cells (BMMNC) as targets cultured in 5% FBS and 20% serum supplements as a function of time.
• BMMNC bone marrow mononuclear cells
• T- and B-cell colony formation can also be detected. This can be substantially increased if T-lymphocytes are activated with phytohemaglutin (PHA) and B-lymphocytes are stimulated with pokeweed mitogens (PWM). Cluster counts can parallel the luminescence measurements and, therefore, clusters represent the "proliferative units" being measured by the luminescence readout.
• PHA phytohemaglutin
• PWM pokeweed mitogens
• ATLANTA 366358vl Example 9: HALO as a multispecies platform.
• HALO can be used not only to study virtually all lympho-hematopoietic colony-forming cell populations from human target tissue (peripheral blood, bone marrow and cord blood), but also those from different animal species (non-human primate, dog, rat and mouse).
• Fig. 13 shows the response of mouse bone marrow cells with time performed simultaneously with the human study depicted in Figs. 12A and 12B.
• Fig. 14 shows stem and multilineage cell assays using frozen non-human primate and rat bone marrow as targets.
• HALO human bone marrow cells grown under the same lineage- specific conditions used for the HALO technique were immunophenotyped.
• the following cell populations/lineages have been analyzed by HALO: Multipotential stem cells (CFC-GEMM) stimulated with EPO, GM-CSF, G-CSF, IL-3, IL-6 and SCF; Erythropoietic progenitor cells (BFU-E) stimulated with EPO, IL-3 and SCF; Erythroid precursor cells (CFU-E) stimulated with EPO alone; Granulocyte- macrophage progenitor cells (GM-CFC) stimulated with GM-CSF, IL-3 and SCF, and granulocyte (G-CFC) and macrophage (M-CFC) precursor cells stimulated with G- CSF and M-CSF respectively; megakaryocyte progenitor cells (Mk-CFC) stimulated with TPO, IL-3 and IL-6
• CFC-GEMM Multipotential stem cells
• the CFC-blast population is stimulated with either IL-3 and IL-6 or IL-3 and SCF while the HPP-CFC stem cell population requires the same growth factor combination as CFC-GEMM, but with the addition of IL-1 and M-CSF.
• Eosinophil precursor cells are stimulated with IL-3, interleukin-5 (IL-5) and GM-CSF, and Baso- CFC are stimulated with IL-3 alone.
• a particular advantage of the methods of the present invention in the field of immunotherapy is the ability to grow and detect macrophage-derived and lymphocyte-derived dendritic cells.
• ATLANTA 366358vl Multipotential stem cells were stimulated with EPO, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), IL-3 and SCF; BFU-E were stimulated with EPO, IL-3 and SCF; GM-CFC were stimulated with GM-CSF, IL-3 and SCF, while M-CFC were stimulated with macrophage colony-stimulating factor (M-CSF).
• EPO granulocyte-macrophage colony-stimulating factor
• G-CSF granulocyte colony-stimulating factor
• M-CFC macrophage colony-stimulating factor
• Mk-CFC were stimulated with thrombopoietin (TPO), IL-3 and interleukin-6 (IL-6), while T-CFC were stimulated with interleukin-2 (IL-2) and phytohemaglutinin (PHA) and B-CFC were stimulated with interleukin-7 (IL-7) and pokeweed mitogens (PWM).
• TPO thrombopoietin
• IL-6 interleukin-6
• PHA phytohemaglutinin
• B-CFC were stimulated with interleukin-7 (IL-7) and pokeweed mitogens (PWM).
• Example 11 HALO multitasking capability-measurement of cell proliferation and apoptosis in the same assay.
• Fresh human PBMNCs were cultured under BFU-E-stimulating conditions, together with 3 concentrations of DOX, namely lng/ml (low), lOng/ml (medium) and lOOng/ml (high) as per a typical HALO test procedure.
• Replicate plates were used so that the effects of DOX could be examined at 2 time points, namely days 3 and 7.
• APOGLOWTM detection kit (Cambrex BioScience)
• ATP releasing reagent and ATP luminescence-monitoring reagent were added and the luminescence read immediately. A second luminescence reading was taken when the nadir ATP value occurred.
• FIG. 15A and 15B show the mean RLU values for each of the DOX concentrations measured at the 2 time points. Whereas there is little or no effect of the lowest DOX concentration, both the medium and highest concentrations can be seen to decrease the luminescence indicating a reduction in growth potential of the treated cells.
• the effect on the ADP:ATP ratios are shown in Figs. 16A and 16B.
• the increase in ADP:ATP ratio is due to apoptosis of cells with low proliferative capacity.
• BFU-E and immediate progeny are proliferating rapidly and are being induced into apoptosis by the presence of DOX.
• ATLANTA 366358vl Example 12: Measurement of cell proliferation and apoptosis in one procedure
• ADP adenosine diphosphate
• Figs. 15 and 16 The ratio of ADP:ATP provides a determination of apoptosis. More precisely, the ADP:ATP ratio differentiates between necrosis and apoptosis. If cells are stimulated, ATP levels will increase with no change in the ADP levels. Cell proliferation is indicated. Since apoptosis is an energy-requiring process, ATP is essential for many of the early events of apoptosis. When ATP levels decline to a point where basic metabolic function can no longer process, the cells will die.
• ATP is released from the cells using ATP releasing reagent and a 15 min incubation. Thereafter, the ATP luminescence-monitoring reagent is added and the plates read immediately. Thirty minutes later, an ADP converting reagent (ADP-CR) is added which converts ATP to ADP and the plates are read every 5 mins for a further 15-20 mins. Three values are then used to calculate the kinetics of the reaction.
• the first reading (A) is that immediately after addition of ATP luminescence-monitoring reagent.
• the second reading (B) is just prior to addition of ADP-CR, when the decline in ATP is maximal.
• the third reading (C) is taken at a time when the conversion of ADP to ATP has reached a plateau.
• the ADP:ATP ratio is then calculated from the simple equation: (C-B)/A. This procedure was performed to obtain the results shown in Fig. 16. The procedure is preferably performed by the automatic addition of ATP luminescence-monitoring reagent and ADP-CR using a luminometer equipped with two injectors.
• the ATP luminescence-monitoring reagent should not be thawed until needed and should not be re-frozen once thawed. This reagent is light sensitive. Keep in the dark.

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