[0001] This application claims priority on provisional application Ser. No. 60/028,152 filed on Oct. 18, 1996, the entire contents of which are hereby incorporated by reference.
[0002] The present invention relates to a serum-free medium which can support the growth and proliferation of normal human hematopoietic CD34
[0003] The growth of these cells is becoming more important in view of recent developments in clinical regimens for combatting diseases such as cancer, myeloproliferative diseases and autoimmune diseases. However, many media are not suitable for culturing normal bone marrow cells, especially CD34
[0004] Since not all cells will proliferate in a single (universal) serum-free medium, care must be taken in the development of each medium. Such a medium is invaluable in the expansion of specific hematopoietic lineages for bone marrow transplantation. Such a medium will allow the potential to store small amounts of bone marrow or subsets of the bone marrow cell population (such as by freezing) and at a later time expand the cells by thawing the cells and growing them in vitro for transplantation purposes. The inventor has developed such a medium that can support CD34
[0005] It is one object of the invention to provide a serum-free medium comprising a basal medium, an effective amount of essential fatty acid, an effective amount of cholesterol, transferrin in an amount of 120 to 500 μg/ml or an effective amount of an iron salt and an effective amount of insulin growth factor, wherein said medium supports the proliferation and differentiation of normal CD34
[0006] It is another object of the invention to provide a serum-free medium comprising a serum-free culture medium which supports the proliferation and differentiation of CD34
[0007] It is another object of the invention to provide a method for growing normal CD34
[0008] Various growth factors and/or cytokines for driving proliferation and differentiation of the cells can optionally be added to the medium used to culture the cells. By means of adding various cytokines, the composition of the cell population can be altered with respect to the types of cells present in the population.
[0009] The term “serum-free” is used herein to mean that all whole serum is excluded from the medium. Certain purified serum components, such as human serum albumin, can be added to the medium.
[0010] The basal medium is preferably Iscove's modified Dulbecco's medium (IMDM). Other such basal media might be used, such as McCoy's 5a or a blend of Dulbecco's modified Eagle's Medium and Ham's-F12 media at a 1:1 ratio. The requirements of the basal medium are that it provide i) inorganic salts so as to maintain cell osmolality and mineral requirements (e.g., potassium, calcium, phosphate, etc.), ii) essential amino acids required for cell growth, that is, amino acids not made by endogenous cellular metabolism, iii) a carbon source which can be utilized for cellular energy metabolism, typically glucose, and iv) various vitamins and co-factors, such as riboflavin, nicotinamide, folic acid, choline, biotin, and the like, as my be required to sustain cell growth. Glutamine is one of the amino acids which may be added to the medium of the present invention in an effective amount. The glutamine concentration is usually between 100 and 500 μg/ml, preferably between 125 and 375 μg/ml and most preferably between 150 and 300 μg/ml. Because of its instability, glutamine is sometimes added just before use of the media.
[0011] The basal medium also typically contains a buffer to maintain the pH of the medium against the acidifying effects of cellular metabolism, usually bicarbonate or HEPES. The pH of the basal medium is usually between 6.8 and 7.2. The composition of IMDM is shown in Table I, below:
TABLE I Iscove's Modified Dulbecco's Medium Component mg/L L-Alanine 25.0 L-Arginine HCl 84.0 L-Asparagine · H 28.40 L-Aspartic Acid 30.0 L-Cystine · 2HCl 91.24 L-Glutamic Acid 75.0 L-Glutamine 584.0 Glycine 30.0 L-Histidine HCl · H 42.0 L-Isoleucine 104.8 L-Leucine 104.8 L-Lycine HCl 146.2 L-Methionine 30.0 L-Phenylalanine 66.0 L-Proline 40.0 L-Serine 42.0 L-Threonine 95.2 L-Tryptophan 16.0 L-Tyrosine, 2Na · 2H 103.79 L-Valine 93.6 Biotin 0.013 D-Ca Pantothenate 4.00 Choline Chloride 4.00 Folic Acid 4.00 i-Inositol 7.00 Nicotinamide 4.00 Pyridoxal HCl 4.00 Riboflavin 0.40 Thiamine HCl 4.00 Vitamin B 0.013 Antibiotics omitted 2-a-Thioglycerol (7.5E-5M) omitted CaCl 215.86 KCl 330.0 KNO 0.076 MgSO 97.67 NaCl 4505. NaH 108.69 Na 0.0173 Glucose 4500. Phenol Red · Na 15.34 Sodium Pyruvate 110.0 NaHCO 3024. HEPES 25 mM 5958. CO 5% medium contains 5% CO
[0012] Albumin is preferably supplied in the form of human serum albumin (HSA) in an effective amount for the growth of cells. HSA provides a source of protein in the media. Moreover, protein acts as a substrate for proteases which might otherwise digest cell membrane proteins. Albumin is thought to act as a carrier for trace elements and essential fatty acids. HSA is greatly advantageous over protein derived from animals such as bovine serum albumin (BSA) due to the reduced immunogenic potential of HSA. The HSA may be derived from pooled human plasma fractions, or may be recombinantly produced in such hosts as bacteria and yeast, or in vegetable cells such as potato and tomato. Preferably, the HSA used in the present formulations is free of pyrogens and viruses, and is approved regulatory agencies for infusion into human patients. The HSA may be deionized using resin beads prior to use. The concentration of human serum albumin is 1-8 mg/ml, preferably 3-5 mg/ml, most preferably 4 mg/ml.
[0013] The albumin mentioned above could be substituted by a soluble carrier/essential fatty acid complex and a soluble carrier cholesterol complex which can effectively deliver the fatty acid and cholesterol to the cells. An example of such a complex is a cyclodextrin/linoleic acid, cholesterol and oleic acid complex. This is advantageous as it would allow for the replacement of the poorly characterized albumin with a well defined molecule. The use of cyclodextrin removes the need for the addition of human/animal serum albumin, thereby eliminating any trace undesired materials which the albumin would introduce into the media. The use of cyclodextrin simplifies the addition of specific lipophilic nutrients to a serum-free culture.
[0014] Three cyclodextrins which are employable are α-, β-, and γ-cyclodextrins. Among them, β-cyclodextrin appears to be the best. In this invention dealing with the expansion of CD34
[0015] The lipophilic substances which can be complexed with cyclodextrin include unsaturated fatty acids such as linoleic acid, cholesterol and oleic acid. The linoleic acid, cholesterol and oleic acid are present in effective amounts and can be present in equal proportions such that the total amount is 0.001 to 100 μg/ml, preferably 0.1 to 10 μg/ml. The preparation of such complexes is known in the art and is described, for example, in U.S. Pat. No. 4,533,637 of Yamane et al, the entire contents of which is hereby incorporated by reference.
[0016] A source of iron in an effective amount and in a form that can be utilized by the cells can be added to the media. The iron can be supplied by transferrin in an effective amount. The transferrin may be derived from animal sera or recombinantly synthesized. It is understood that when transferrin is derived from an animal source, it is purified to remove other animal proteins, and thus is usually at least 99% pure. The transferrin concentration is usually between 80 and 500 μg/ml, preferably between 120 and 500 μg/ml, more preferably between 130 and 500 μg/ml, even more preferably between 275 and 400 μg/ml and most preferably 300 μg/ml. Alternatively, an iron salt, preferably a water soluble iron salt, such as iron chloride (e.g. FeCl
[0017] Insulin may also be added to the media of the present invention in an effective amount. The insulin concentration is between 0.25 and 2.5 U/ml, more preferably 0.4-2.1 U/ml, most preferably 0.48 U/ml. In the conversion of Units to mass, 27 U=1 mg. Therefore, incorporating the conversion, the insulin concentration is approximately between 9.26 μg/ml and 92.6 μg/ml, more preferably 14.8 μg/ml-77.8 μg/ml, most preferably 17.7 μg/ml. It is again understood that human insulin is more preferable than animal insulin. Highly purified recombinant insulin is most preferred. An insulin like growth factor such as insulin like growth factor 1 and insulin like growth factor 2 may be used in place of insulin in an amount which provides substantially the same result as a corresponding amount of insulin. Thus, the term “insulin growth factor” includes both insulin and insulin like growth factors.
[0018] The addition of other lipids to the above essential reagents could enhance the proliferative potential of precursor cells. These components, however, are preferably not added unless they are necessary for a particular experiment or to grow a particular type of cell. Optionally, triglycerides and/or phospholipids may be included as additional sources of lipid. A preferable source of lipid contains a mixture of neutral triglycerides of predominantly unsaturated fatty acids such as linoleic, oleic, palmitic, linolenic, and stearic acid. Such a preparation may also contain phosphatidylethanolamine and phosphatidylcholine. Another source of lipid is a human plasma fraction precipitated by ethanol and preferably rendered virus-free by pasteurization.
[0019] Other ingredients which can optionally be added to the media are cited in the following references: Smith et al, WO 95/06112, Yamane et al, U.S. Pat. No. 4,533,637, Ponting et al, U.S. Pat. No. 5,405,772. The entire contents of all of these references are incorporated by reference.
[0020] When the media is to be used to grow cells for introduction into a human patient, the media preferably does not contain ingredients such as bovine serum albumin, mammalian serum, and/or any natural proteins of human or mammalian origin (as explained above). It is preferable that recombinant or synthetic proteins, if they are available and of high quality, are used. Most preferably, the amino acid sequences of the recombinant or synthetic proteins are identical to or highly homologous with those of humans. Thus, the most preferable serum-free media formulations herein contain no animal-derived proteins and do not have even a non-detectable presence of animal protein.
[0021] In the most ideal system, optional components which are not necessary are preferably not added to the medium. Such optional components are described in the prior art cited above and may be selected from the group consisting of meat extract, peptone, phosphatidylcholine, ethanolamine, anti-oxidants, deoxyribonucleosides, ribonucleosides, soy bean lecithin, corticosteroids, and EX-CYTE, myoinositol, monothioglycerol, and bovine or other animal serum albumin.
[0022] The medium of the present invention is of course aqueous and is made using distilled water. The medium is formulated from freely soluble materials. Thus, the order of the addition of the ingredients is not particularly important to the invention. Typically, the basal medium is made first and the remaining components required for growth of bone marrow cells in the absence of serum are then added to the basal medium.
[0023] The most ideal system, as described in this invention, is one wherein the serum-free media is made fresh on the day that it is to be added to the culture. However, when storage previous to use is necessary, it may be desirable to add certain compounds. Reducing agents such as α-monothioglycerol and β-mercaptoethanol, which are thought to diminish free-radical formation, may be added to the serum-free media formulations. This will enhance stability of the serum-free media during storage for lengths of time of up to 20 days or longer. Additionally, in these less than preferred circumstances, antibiotics may also be added to the media as a precaution against bacterial contamination.
[0024] All of the ingredients in the medium, including the ingredients in the basal medium, are present in amounts sufficient to support the proliferation and differentiation of CD34
[0025] The medium is formulated and sterilized in a manner conventional in the art. Typically, stock solutions of these components are made filter sterilized. A finished medium is usually tested for various undesired contaminants, such as mycoplasma or virus contamination, prior to use.
[0026] In the art of tissue culture it has for some time been desired that a serum-free medium be found that supports the proliferation and differentiation of CD34
[0027] Recent studies have shown that an early progenitor/stem (CD34
[0028] The medium of the present invention, a formulation suitable for use in human therapeutic protocols, has two types of utility in transplant therapies as described above. First, the media can be used in the expansion of the CD34
[0029] In either of the above cases, the presence of appropriate growth factors, such as interleukins (IL), colony stimulating factors (CSF), and the like, will influence the rate of proliferation and the distribution of cell types in the population. Cytokines used for the expansion and differentiation of early progenitor cells are stem cell factor, interleukin-1 and interleukin-6. Cytokines used to stimulate proliferation and differentiation of mid-progenitor cells are interleukin-3 and granulocyte-macrophage colony stimulating factor. Cytokines which promote the differentiation of specific blood cell types are granulocyte colony stimulating factor, macrophage colony stimulating factor and erythropoietin. For transplantation purposes, the GM-colony forming cells are among the most important. The myeloid population is absolutely necessary for the transplant patient to survive. The role which each of these cytokines play in hematopoiesis is under intense investigation in the art and it is expected that eventually it will be possible to faithfully recapitulate hematopoiesis in vitro.
[0030] The second utility is in “ex-vivo purging” protocols. In a therapy of this type, “normal” (non-tumorigenic) CD34
[0031] The medium is specially adapted to selectively promote the growth of CD34
[0032] When supplemented with SCF, IL-3, IL-6 and G-CSF at 50 ng/ml each, the media of the present invention can maintain a distinct population of cells with the immature CD34
[0033] The invention is illustrated by the Examples below, which are not intended to be limiting of the scope of the invention.
[0034] In order to develop a medium that can be used for human clinical CD34
[0035] In the experiments in this Example, the Serum Free Media 1 and Serum Free Media 2 had the same compositions of ingredients except as described below.
Serum Free Serum Free Media 1 Media 2 Insulin 0.48 U/ml 0.14 U/ml Transferrin 300 μg/ml 100 μg/ml
[0036] The data in Table II illustrates that in three separate experiments using CD34TABLE II Proliferation of CD34 normal bone marrow cultured for 14 days in Serum Free Medium 1, Serum Free Medium 2 and serum-containing medium each supplemented with the cytokines; SCF, IL-3, IL-6 and G-CSF at 50 ng/ml each. The data is from three separate experiments using CD34 different donors. The spent medium and cytokines were replaced with fresh medium and cytokines every 7 days. Initial Seeding Day 14 Density × Density × Medium 10 10 Expt. Serum Free 2 95 1 Medium 1 Serum Free 2 60 Medium 2 IMDM + 20% 2 78 FBS Expt. Serum Free 2 140 2 Medium 1 Serum Free 2 36 Medium 2 IMDM + 20% 2 14 FBS Expt. Serum Free 2 100 3 Medium 1 Serum Free 2 62 Medium 2 IMDM + 20% 2 52 FBS
[0037] In another experiment we compared the Serum Free Medium 1 with Serum Free Medium 2, serum-containing medium and three commercially available serum-free media developed especially for hematopoietic cells. The CD34TABLE III Proliferation of CD34 normal human bone marrow and cultured for 14 days in various media plus the cytokines SCF, IL-3, IL-6 and G- CSF at 50 ng/ml each. The spent medium and cytokines were replaced with fresh medium and cytokines every 7 days. Initial Day 14 Seeding Level × Density × Medium 10 10 Serum Free 1 100 Medium 1 Serum Free 1 62 Medium 2 IMDM + 20% FBS 1 52 AIM V 1 36 X-VIVO 10 1 60 StemPro 34 1 30
[0038] Table IV illustrates that mobilized CD34TABLE IV Proliferation of mobilized CD34 cells after 14 days of culture in the noted medium containing the cytokines SCF, IL-3, IL- 6 and G-CSF at 50 ng/ml each. The data is from three separate experiments using CD34 cells. The spent medium and cytokines were replaced with fresh medium and cytokines every 7 days. Initial Seeding Day 14 Density × Density × Medium 10 10 Expt. 1 Serum Free 2 132 Medium 1 Serum Free 2 80 Medium 2 IMDM + 20% FBS 2 132 Expt. 2 Serum Free 2 90 Medium 1 StemPro 34 2 62 IMDM + 20% FBS 2 100 Expt. 3 Serum Free 2 180 Medium 1 StemPro 34 2 32 IMDM + 20% FBS 2 170
[0039] In another experiment we compared the Serum Free Medium 1 and IMDM plus 20% FBS both containing the added cytokines for the ability to support the growth of CD34
[0040] In another experiment using cord blood CD34TABLE V Proliferation of cord blood CD34 after 14 days of culture in the noted medium containing the cytokines; SCF, IL-3, IL-6 and G-CSF at 50 ng/ml each. The spent medium and cytokines were replaced with fresh medium and cytokines every 7 days. Day 14 Initial Seeding Density × Medium Density × 10 10 Expt. 1 Serum-Free 2 200 Medium 1 IMDM + 20% 2 129 FBS Expt. 2 Serum-Free 1 186 Medium 1 Serum-Free 1 153 Medium 2 IMDM + 20% 1 134 FBS Stem Pro 1 88 AIM V 1 49 X-VIVO 10 1 123 Expt. 1 Serum-Free 2 200 Medium 1 IMDM + 20% 2 129 FBS Expt. 2 Serum-Free 1 186 Medium 1 Serum-Free 1 153 Medium 2 IMDM + 20% 1 134 FBS Stem Pro 1 88 AIM V 1 49 X-VIVO 10 1 123
[0041] The medium of the present invention has several advantages. First, the medium of the present invention is useful in the growth of CD34
[0042] The invention being thus described, various modifications of the materials and methods set forth will be obvious to one of skill in the art. Such modifications are within the scope of the invention as defined by the claims below.