Site News  |   Monitor Keywords  |   Monitor Archive  |   Organizer  |   Account Info  |

Hematopoietic cell culture nutrient supplement

Hematopoietic cell culture nutrient supplement description/claims

The present invention relates to a replacement for the serum supplementation normally required for ex vivo expansion of CD34+ hematopoietic cells and cells of myeloid lineage.

Blood cells in the mammal can be divided into three main categories or families: red cells, white cells of myeloid lineage, and white cells of the lymphocytic lineage. Red blood cells carry oxygen from the lungs to the tissues and cells of the body and transport CO2 from the tissues and cells back to the lungs for elimination. White cells of myeloid lineage include: neutrophils, basophils, eosinophils, megakaryocytes, monocytes/macrophages, and dendritic cells. These cells play a role in the identification and elimination of foreign organisms (e.g. bacteria) or damaged tissue, cells and substances from the body. White cells of lymphocytic lineage are divided into two main subgroups: T lymphocytes (helper cells, killer cells, suppressor cells), which are involved in cell mediated responses to viruses, tumor cells and foreign tissue grafts; and B lymphocytes, which are involved in the production of antibodies which circulate in the blood and react in a chemically specific fashion to foreign materials (e.g. bacteria, foreign proteins).

Embryologically, blood cells are formed in the third week of development from cells of the splanchnic mesoderm (Langman, J., Medical Embryology, 3rd ed., pp. 1-7, Williams and Wilkins Co., Baltimore, Md., 1975). In the yolk sac, hematopoiesis (i.e., blood cell formation) initially occurs in clusters or islands (“blood islands”) of splanchic mesoderm cells. The blood islands then migrate to the liver during fetal development. At full gestation and immediately after delivery, these cells migrate to the bone marrow in the shafts of long bones, ribs, and hips. Throughout the remainder of life, the bone marrow serves as the normal site of blood cell formation, releasing mature cells into the circulation (Wintrobe, M. M., Clinical Hematology, pp. 1-7, Lea and Febiger, Philadelphia, Pa., 1967).

Hematopoiesis is an ordered process by which different blood cells are produced at a rate of 400 billion per day (Koller, M. R. and Palsson, B. O., Ex Vivo 42:909-930 (1993)). Since the early 1960s, researchers have acquired experimental evidence for the hypothesis that each of the various blood cell types are derived from a common “pluripotent” stem cell in the mouse (Till, J. E. and McCulloch, E. A., Radiation Res. 14:213-222 (1961); Becker, A. J. et al., Nature 195:452-454 (1963); Siminovitch, L. et al., J. Cell. Comp. Physiol. 62:327-336 (1963)). Experimental evidence also supports the existence of pluripotent stem cells in humans (Nowell, P. C. and Hungerford, D. A., J. Natl. Cancer Inst. 25:85-109 (1960); Tough, I. M. et al., Lancet 1:411-417 (1961); Barr, R. D. and Watt, I., Acta Haemat. 60:29-35 (1978)).

Hematopoietic cell differentiation occurs in stages (Pimentel, E., Ed., Handbook of Growth Factors Vol. III: Hematopoietic Growth Factors and Cytokines, pp. 1-2, CRC Press, Boca Raton, Fla., 1994). The first stage is represented by the hematopoletic stem cell. Development then diverges along the lymphoid and myeloid lineages as lymphoid progenitor cells and myeloid/erythroid progenitor cells are formed. Development further diverges within each of these lineages. Lymphoid progenitor cells form pre-B and pre-T precursor cells, which subsequently develop into B lymphocytes and T lymphocytes, respectively. Myeloid/erythroid progenitor cells form (1) erythroid burst-forming unit (BFU-E) cells, which eventually develop into erythrocytes; (2) megakaryocyte colony-forming unit (CFU-MEG) cells, which eventually develop into megakaryocytes and platelets; (3) granulocyte/macrophage colony-forming units (CFU-GM), which eventually develop into monocytes, macrophages, and neutrophils; (4) eosinophils; and (5) basophils.

A major focus in the field of experimental hematology continues to be the identification of the most primitive, pluripotent stem cell. One approach has been to identify cell surface markers (such as CD antigens) on the surface of progenitor cells and to correlate these markers with stages of development or differentiation by the cells' ability to form colonies of differentiated cells in methylcellulose culture systems. CD antigen expression has been shown to be modulated during cellular differentiation (Sieff, C. et al., Blood 60:703 (1982)). Hematopoietic stem cells are CD34+ cells. That is, they express the CD34 surface marker. The most primitive known human progenitor cell, which has been characterized as CD34+/CD33−/CD38−, represents only 1 to 2% of all bone marrow cells (Civin, C. I. et al., J. Immunol. 133:157 (1984)).

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 studies of Bradley and Metcalf (Bradley, T. R. and Metcalf, D., Biol. Med. Sci. 44:287-300 (1966)), as well as those of Pluznik and Sachs (Pluznik, D. H. and Sachs, L., Expl. Cell Res. 43:553-563 (1966)), demonstrated that serum alone was not sufficient to support the growth of myeloid progenitors in culture. Cell growth required the presence of factor(s), secreted by other cells (i.e feeder cells) and found in the conditioned media from cultures of these cells. It is now clear that the growth of hematopoietic tissue ex vivo requires the presence of several cytokines or hematopoietic growth factors.

Several distinct factors have been identified, cloned and are now routinely manufactured as recombinant molecules for both research and/or clinical use. These include erythropoietin (Lin, F. K. et al., Proc. Natl. Acad. Sci. U.S.A. 82:7580-7584 (1985); Stone, W. J. et al., Am. J. Med. Sci. 296:171-179 (1988)), interleukin-3 (IL-3) (Fung, M. C. et al., Nature 307(5948):233-237 (1984); Yokota, T. et al., Proc. Natl. Acad. Sci. U.S.A. 81:1070-1074 (1984); Ganaser, A. et al., Blood 76:666-676 (1990)), granulocyte macrophage-colony stimulating factor (GM-CSF) (Wong, G. G. et al., Science 228:810-815 (1985); Sieff, C. A. et al., Science 230:1171-1173 (1985)); granulocyte-colony stimulating factor (G-CSF) (Souza, L. M. et al., Science 232:61-65 (1986)); stem cell factor (SCF) (Copeland, N. G. et al., Cell. 63:175-183 (1990); Flanagan, J. G. et al., Cell 63:185-194 (1990); Zsebo, K. M. et al., Cell 63:195-201 (1990); Martin, F. H. et al., Cell 63:203-211 (1990); Szebo, K. M. et al., Cell 63:213-224 (1990); Huang, E. et al., Cell 63:225-233 (1990)), and interleukin-11 (II-11) (Paul, W. et al., Proc. Natl. Acad. Sci. U.S.A 87:7512-7516 (1990)), to cite only a few.

In 1977, Dexter and his colleagues developed a long-term bone marrow culture (LTBMC) protocol (Dexter, T. M. et al., J. Cell Physiol. 91:335-344 (1977)). Known as “Dexter” culture, this type of cell culture system does not require the use of conditioned media and appears to establish and mimic, in vitro, the hematopoietic environment. Long term cultures have been established using human bone marrow (Grenberger, H. M. et al., Blood 58:724-732 (1981); Eaves, C. J. et al., J. Tiss. Cult. Method. 13:55-62 (1991)), as well as the marrow from other animal species (Eastment, C. E. and Ruscetti, F. W., Evolution of Hematopojesis in Long-Term Bone Marrow Culture: Comparison of Species Differences, in: Long-Term Bone Marrow Cultures, Droc Foundation Series 18, pp. 97-118, Allan R. Liss, Inc., New York, N.Y., 1984).

In long-term culture systems, growth and differentiation of stem cells and early progenitor cells appears to require either direct cell to cell contact or very close proximity between the developing hematopoietic cells and stromal cells (Dexter, T. M. et al., J. Cell. Physiol. 91:335-344 (1977)).

Each of the above cell culture systems (e.g., liquid static culture, semi-solid culture and long term bone marrow culture) appears to have their own unique, critical requirements which must be met before one can culture hematopoietic cells of human or other mammalian species. To date, however, a common requirement, and major disadvantage, of cell culture systems has been the requirement for undefined components contained in animal sera (e.g., fetal bovine serum or horse serum) or in “conditioned cell culture media” for optimal growth.

The use of serum in the culture of hematopoietic cells is disadvantageous for several reasons. Serum is a major source of undefined differentiation factors and thus tends to promote hematopoietic cell differentiation, rather than expansion. The efficiency of serum varies between lots of serum. Some lots of serum have been found to be toxic to cells. Moreover, serum may be contaminated with infectious agents such as mycoplasma, bacteriophage, and viruses. Finally, because serum is an undefined and variable component of a medium to which serum is added, the use of serum prevents the true definition and elucidation of the nutritional and hormonal requirements of the cultured cells.

In view of the many problems associated with the use of serum in the growth of CD34+ hematopoietic cells, laboratories performing work with CD34+ hematopoietic cells must resort to pre-screening serum prior to purchase. However, the pre-screening process is time-consuming and subject to interpretation. Even after a satisfactory lot is identified, storage of large quantities of pre-screened lots of serum at −20° C. and below is problematic.

As a result, researchers have attempted to replace animal sera or conditioned media with serum-free culture media of varying degrees of chemical definition. These attempts have met with varying degrees of success, depending upon the identity of the cell type one is trying to expand. The development of serum-free media has recently been reviewed (Sandstrom, E. E. et al., Biotech. & Bioengin. 43:706-733 (1994); Collins, P. C. et al., Curr. Opin. Biotech. 7:223-230 (1996); McAdams, T. A. et al., TIBTECH 14:341-349 (1996)).

International Patent Application WO 96/39487 discloses a serum-free medium for culturing human mesenchymal precursor cells. U.S. Pat. No. 5,405,772 discloses a serum-free or serum-depleted medium for culturing hematopoietic cells and bone marrow stromal cells. U.S. Pat. No. 4,972,762 discloses a serum-free medium, containing penicillamine and N-acetylcysteine, for growing hybridomas and lymphoid cells.

Research with CD34+ hematopoietic cells, cultivation of CD34+ hematopoietic cells in culture, expansion of CD34+ hematopoietic cells, control of differentiation of CD34+ hematopoietic cells, and explanation of CD34+ hematopoietic cells, is hindered by the necessity for serum. Further, a major problem associated with previously available serum-free media is short shelf-life. Oxidation of the vitamins and lipid ingredients of serum-free media occurs during storage. CD34+ hematopoietic cell growth and expansion cannot be sustained in a serum-free medium which contains oxidated ingredients. Thus, there remains a need for a serum-free medium supplement and a serum-free medium which supports the growth and expansion of CD34+ hematopoietic cells and which can be stored for long periods of time.

The present invention provides a serum-free, eukaryotic cell culture medium supplement comprising or obtained by combining one or more ingredients selected from the group consisting of one or more antioxidants, one or more albumins or albumin substitutes, one or more lipid agents, one or more insulins or insulin substitutes, one or more transferrins or transferrin substitutes, one or more trace elements, and one or more glucocorticoids, wherein a basal cell culture medium supplemented with the supplement is capable of supporting the expansion of CD34+ hematopoietic cells and cells of myeloid lineage, in serum-free culture.

The present invention also provides a serum-free, eukaryotic cell culture medium supplement comprising or obtained by combining one or more antioxidants and one or more ingredients selected from the group consisting of one or more albumins or albumin substitutes, one or more lipid agents, one or more insulins or insulin substitutes, one or more transferrins or transferrin substitutes, one or more trace elements, and one or more glucocorticoids, wherein a basal cell culture medium supplemented with the supplement is capable of supporting the expansion of CD34+ hematopoietic cells and cells of myeloid lineage, in serum-free culture.

The present invention also specifically provides a serum-free, eukaryotic cell culture medium supplement comprising or obtained by combining one or more ingredients selected from the group consisting of N-acetyl-L cysteine, human serum albumin, Human Ex-Cyte®, ethanolamine HCl, human zinc insulin, human iron saturated transferrin, Se4+, hydrocortisone, D,L-tocopherol acetate, and 2-mercaptoethanol, wherein the ingredients are present in an amount which, when the supplement is added to a basal cell culture medium, supports the expansion of CD34+ hematopoietic cells and cells and cells of myeloid lineage, in serum-free culture.

The present invention also provides a method of making a serum-free, eukaryotic cell culture medium supplement, the method comprising admixing the ingredients of the supplement of the invention in any order. The invention specifically comprises admixing water, N-acetyl-L-cysteine, human serum albumin, Human Ex-Cyte®, ethanolamine HCl, zinc insulin, human iron saturated transferrin, a Se4+ salt, hydrocortisone, D,L-tocopherol acetate, and 2-mercaptoethanol.

• Provisional Patent
• Utility Patent

• What Is a Patent?
• What Is a Trademark or Servicemark?
• What Is a Copyright?
• Patent Laws