Read Cat. No. 10639, StemPRO-34® SFM Complete Med.Insert No., 3838 text version

STEMPRO-34 SFM Complete Medium

Cat. No. 10639 includes: STEMPRO-34 SFM Cat. No.: 10640 500 mL STEMPRO-34 Nutrient Supplement Cat. No.: 10641 13 mL CAUTION:

Human origin materials are non-reactive (donor level) for anti-HIV 1 & 2, anti-HCV, and HBsAg. Handle in accordance with established biosafety practices.

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of, and ability to control the processes of hematopoiesis, ex vivo, has been the requirement to supplement culture systems with one, or more, animal sera. Sera can vary from lot to lot in its' ability to support cell specific growth in culture. Serum-free culture systems eliminate this major variable in the study of hematopoiesis ex vivo. In recent years, several laboratories have developed serum-free 30,31,32 culture media for both murine and human hematopoietic cells 33 and Sandstrom, have recently reviewed the subject. Product Description STEMPRO-34 SFM is a serum free medium developed to support the + growth of human hematopoietic progenitor cells (e.g. CD34 ). The + formulation was optimized using freshly isolated CD34 bone marrow cells from normal donors. STEMPRO-34 SFM is supplied without L-Glutamine. A 4OX nutrient supplement is supplied as a frozen liquid (-5 to -2O°C). These two components are combined at the time of use. L-Glutamine (GIBCO Cat. No. 25030) must be added at the time of use to a final concentration of 2 mM. NOTE: STEMPRO-34 SFM is manufactured without cytokines and hematopoietic growth factors, giving the investigator the freedom to use any factor or combination of factors required in ones' studies. Optimal cell growth requires the addition of hematopoietic growth factors. Precautions Aseptic addition of STEMPRO-34 Nutrient Supplement to STEMPRO 34 SFM is required prior to use. DO NOT USE THIS PRODUCT IF: 1. Product packaging appears compromised. 2. STEMPRO-34 Nutrient Supplement is received thawed. 3. STEMPRO-34 SFM appears cloudy or a visible precipitate is observed. Storage All products should be stored in the dark. Store medium at 2 to 8°C. Store nutrient supplement -5 to -20°C. NOTE: The nutrient supplement may be thawed, aliquoted and refrozen once. Do not subject to repeated freeze/thaw cycles. When aliquoting care must be taken to reduce the potential for dehydration. We recommend storing in a well sealed container with minimal head space. Most frost-free freezers have heat/cool cycles that will impact the product. We recommend storage in a Non-FrostFree freezer at -20°C. Instructions for Use When ready to use thaw the frozen STEMPRO-34 Nutrient Supplement in a 37°C water bath, mix well by gently vortexing and aseptically transfer the complete contents of the bottle to the STEMPRO-34 SFM. Swirl to obtain a homogenous complete medium. Aseptically add L-Glutamine (Cat. No. 25030) to a final concentration of 2 mM, by adding 5 mL 200 mM L-Glutamine to 500 mL medium. STEMPRO-34 SFM Complete Medium should be stored at 2 to 8°C, in the dark. The complete medium has a shelf life of 30 days when stored as recommended. The thawed nutrient supplement has a shelf life of 14 days at 2 to 8°C, in the dark. STEMPRO-34 SFM Complete Medium requires the addition of cytokines or growth factors for extended support of progenitor cells (see Performance Testing). Our research program has shown that normal bone marrow cells can be held at 2 to 8°C, in the complete medium, without added growth factors, for 5-7 days without a significant loss of cell viability as determined by trypan blue dye exclusion. Both cell phenotype

Intended Use STEMPRO-34 SFM Complete Medium is used to support the growth of human hematopoietic progenitor cells. It is intended for laboratory research use only. Background In 1961, Till and McCulloch demonstrated that, in the mouse, there was a cell of bone marrow origin which formed macroscopic nodules of regenerating hematopoietic cells within the spleens of lethally 1 irradiated mice (i.e. CFUs/ Pluripotent Stem Cell). Trentin and his colleagues subsequently demonstrated that both spleen colony development and bone marrow regeneration were influenced by 2 microenvironmental factors. In the mid 1960's, in order to better understand the mechanisms of normal and aberrant hematopoiesis, investigators began trying to grow bone marrow cells, ex vivo, using both suspension and semi-solid tissue culture systems. The early 3 studies of Bradley and Metcalf, as well as those of Pluznik and 4 Sachs, demonstrated that serum alone was not sufficient to support the growth of myeloid progenitors in culture. Cell growth required the presence of undefined factor(s), found in conditioned media of selected cell types. Michael Dexter and his colleagues developed the long-term bone marrow culture (LTBMC) which does not require the use of conditioned media, or specific cytokines, and appears to 5 mimic, in vitro, the hematopoietic inductive microenvironment. Long term bone marrow cultures have brought us full circle, opening avenues of basic research not possible in the past. Similar long term culture procedures have been established using human bone 6,7 8 marrow as well as the marrow from other animal species. Today it is possible to expand hematopoietic cells in culture by adding combinations of recombinant cytokines or hematopoietic growth factors directly to the culture system. Several distinct factors, with growth promoting activity toward primitive hematopoietic cells have been identified, cloned and are now routinely manufactured as 9,10 11,12,13 recombinant molecules. These include Erythropoietin, IL-3, 14,15 16 17,18,19,20,21,22 23 GM-CSF, G-CSF, SCF, and Il-11, to cite only a few. Combined with our increased ability to expand hematopoietic cells, ex vivo, it is now possible to identify hematopoietic progenitor cells on the basis of several criteria other than cell morphology, including: 24,25 immunophenotype (CD antigen expression) and CFU potential in 26 semi-solid culture systems. In order to better understand the mechanism(s) of hematopoiesis, a great deal of research has focused on the use of recombinant cytokines, combined with the measurement of CD antigen expression and CFU potential, to monitor both total cell and lineage specific expansion in liquid culture 27,28,29 systems. However, a major obstacle to our full understanding

and colony forming ability are unaffected when cells are held under these described conditions.

Performance Testing STEMPRO-34 SFM and STEMPRO-34 Nutrient Supplement are tested at the time of manufacture for pH, osmolality, endotoxin and sterility. In addition, each lot of medium and 40X frozen nutrient supplement is performance tested in an applications specific assay of cell growth + under defined conditions, utilizing CD34 cells isolated from freshly drawn human bone marrow from normal donors. Under our defined experimental conditions, test samples of STEMPRO-34 SFM Complete Medium, when supplemented with Interleukin 3 (IL-3), GM-CSF, and Stem Cell Factor, must be found acceptable for their ability to support cell proliferation when compared to the level of growth obtained using a control medium of IMDM and 20% FBS. References:

1. Till, J.E., McCulloch E.A., A Direct Measurement of the Radiation Sensitivity of Normal Mouse Bone Marrow Cells. Radiat. Res., 14: 213222 (1961). Trentin, J.J., Influence of Hematopoietic Organ Stroma (Hematopoietic Inductive Microenvironment) on Stem Cell Differentiation. In: Regulation of Hematopoiesis, Ed. Gordon, A.S., Volume 1, Appleton-CenturyCrofts, New York, pp. 161-168 (1970). Bradley, T.R. and Metcalf, D. The Growth of Mouse Bone Marrow Cells in vitro., Aust.J. Expl. Biol. Med. Sci., 44: 287-300 (1966). Pluznik, D.H. and Sachs, L. The Induction of Normal Mast Cells by a Substance from Conditioned Medium., Expl. Cell Res., 43: 553-563 (1966). Dexter, T.M.,, Conditions Controlling the Proliferation of Haemopoietic Stem Cells in vitro., J. Cell Physil. 91: 335-344 (1977). Greenberg, H.M.,, Human Granulocytes Generated in Continuous Bone Marrow Culture are Physiologically Normal, Blood 58: 724-732 (1981). Eaves, C.J., Methodology of Long-Term Culture of Human Hemopoietic Cells., J. Tiss. Cult. Method., 13: 55-62 (1991). Eastment, C.E. and Ruscetti, F.W., "Evaluation of Hematopoiesis in Long-Term Bone Marrow Culture: Comparison of Species Differences", In: Long-Term Bone Marrow Cultures, Droc Foundation Series, 18: 97118, Allan R. Liss Inc., New York, USA (1984). Lin, F.K., Cloning and Expression of the Human Erythropoietin Gene., Proc. Natl. Acad. Sci. USA, 82(22): 7580-7584. (1985). Stone, W.J.,, Treatment of the Anemia of Predialysis Patients with Recombinant Human Erythropoietin: a Randomized, Placebo-Controlled Trial., Am.J. Med. Sci., 296(3): 171-179 (1988). Fung, M.C.,, Molecular Cloning of cDNA for Murine Interleukin-3., Nature 307 (5948): 233-237 (1984). Yokota, T.,, Isolation and Characterization of a Mouse cDNA Clone that Expresses Mast-Cell Growth Factor Activity in Monkey Cells., Proc. Natl. Aca. Sci. USA., 81: 1070-1074 (1984). Ganser, A.,, Effects of Recombinant Human Interleukin-3 in Patients with Normal Hematopoiesis and in Patients with Bone Marrow Failure. Blood, 76(4): 666-676 (1990). Wong, G.G.,, Human GM-CSF: Molecular Cloning of the Complementary DNA and Purification of the Natural and Recombinant Proteins., Science, 228(4701): 810-815 (1985). Sieff, C.A.,, Human Recombinant Granulocyte-Macrophage Colony-Stimulating Factor: A Multilineage Hematopoietin., Science, 230(4730): 1171-1173 (1985). Souza, L.M.,, Recombinant Human Granulocyte ColonyStimulating Factor: Effect on Normal and Leukemic Myeloid Cells. Science, 232(4746): 61-65 (1986). Copeland, N.G.,, Mast Cell Growth Factor Maps Near the Streel Locus on Mouse Chromosome 10 and is Deleted in a number of Steel Alleles., Cell, 63: 175-183 (1990). Flanagan, J.G.,, The Kit Ligand: A Cell Surface Molecule Altered in Steel Mutant Fibroblasts., Cell, 63: 185-194 (1990). Zsebo, K.M.,, Identification, Purification and Biological Characterization of Hematopoietic Stem Cell Factor from Buffalo Rat Liver-Conditioned Medium., Cell, 63: 195-201 (1990).


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20. Martin, F.H.,, Primary Structure and Functional Expression of Rat and Human Stem Cell Factor DNAs., Cell, 63: 203-211 (1990). 21. Zsebo, K.M.,, Stem Cell Factor is Encoded at the Sl Locus of the Mouse and Is the Ligand for the c-kit Tyrosine Kinase Receptor., Cell, 63: 213-224 (1990). 22. Huang, E.,, The Hematopoietic Growth Factor KL Is Encoded by the SL Locus and Is the Ligand of the c-kit Receptor, the Gene Product of the W Locus., Cell, 63: 225-233 (1990). 23. Paul, W., Molecular Cloning of a cDNA Encoding Interleukin 11, a Stromal Cell-Derived Lymphopoietic and Hematopoietic Cytokine., Proc. Natl. Acad. Sci. USA, 87(19): 7512-7516 (1990). 24. Sieff, C.,, Change in Cell Surface Antigen Expression During Hemopoietic Differentiation, Blood, 60(3): 703-713 (1982). 25. Terstappen LWMM,, Differentiation and Maturation of Growth Factor Expanded Human Hematopoietic Progenitors Assessed by Multidimensional Flow Cytometry., Leukemia, 6(10): 1001-1010 (1992). 26. Keating, A. and Toor P., In Vitro Clonal Culture of Human Hematopoietic Progenitor Cells. In: Methods in Molecular Biology V; Animal Cell Culture Ed:Pollard, J.W. and Walker, J.M. Humana Press Clifton, New Jersey, pp. 339-346 (1990). 27. Srour, E.F., Brandt, J.E., Briddell, R.A., Grigsby, S., Leemhuis, T and Hoffman, R., Long-Term Generation and Expansion of Human Primitive Hematopoietic Progenitor Cells In Vitro., Blood, 81(3): 661-669 (1993). 28. Mayani, H., Dragowska, W. and Lansdorp, P.M., Cytokine-Induced Selective Expansion and Maturation of Erythroid Versus Myeloid Progenitors From Purified Cord Blood Precursor Cells., Blood 81(12): 3552-3558 (1993). 29. Shapiro, F. Yao, T., Raptis, G., Reich, L., Norton, L. and Moore MAS, + Optimization of Conditions for Ex Vivo Expansion of CD34 Cells From Patients With Stage IV Breast Cancer., Blood 84(15): 3567-3574 (1994). 30. Monette, F.C. and Signounas, G., Growth of Murine Multipotent Stem Cells in A Simple "Serum-free" Culture System: Role of Interleukin-3, Erythropoietin and Hemin., Exp.Hematol., 16: 250-255 (1988). 31. Sonoda, Y. and Ogawa, M., Serum-Free Culture of Human Hemopoietic Progenitors in Attenuated Culture Media., Am. J. Hematol., 28: 227-231 (1988). 32. Lansdorp, P.M. and Dragowska, W., Maintenance of Hematopoiesis in Serum-Free Bone Marrow Cultures Involves Sequential Recruitment of Quiescent Progenitors., Exp. Hematol., 21: 1321-1327 (1993). 33. Sandstrom, C.E.,, Review: Serum-Free Media for Cultures of Primitive and Mature Hematopoietic Cells., Biotech. and Bioengnr. 43: 706-733 (1994).

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For research use only. CAUTION: Not intended for human or animal diagnostic or therapeutic uses.

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June 2001

Form No. 3838



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Cat. No. 10639, StemPRO-34® SFM Complete Med.Insert No., 3838

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