Claims for Patent: 5,397,706
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Summary for Patent: 5,397,706
Title: | Serum-free basal and culture medium for hematopoietic and leukemia cells |
Abstract: | A basal serum-free medium and a hematopoietic cell growth and differentiation-promoting serum-free medium based thereon are provided for the maintenance, cultivation, growth and differentiation of erythroid progenitor cells, other hematopoietic progenitor cells, and leukemia cells in which the effects of various growth factor compounds can be quantitatively evaluated. Both media are wholly serum-free and contain no intrinsic growth factor compounds. The hematopoietic growth and differentiation medium consists essentially of the basal serum-free medium to which has been added at least one primarily but not exclusively growth-promoting agent selected from heme or hemin, interleukin-3 and recombinant human stem cell factor (and optimally all of them), and at least one primarily but not exclusively cell differentiation -promoting agent selected from erythropoietin (Epo), insulin-like growth factor (IGF) and a retinoid (and optimally including all of them). |
Inventor(s): | Correa; Paulo N. (Concord, Ontario, CA), Alexrad; Arthur A. (Willowdale, Ontario, CA) |
Assignee: | |
Application Number: | 08/103,419 |
Patent Claims: | 1. A basal serum-free medium for maintaining cells selected from the group consisting of hematopoietic stem cells, hematopoietic progenitor cells and leukemia cells, the medium
consisting of:
a minimum essential medium; from 1 .mu.g/mL to 100 mg/mL of each of adenine deoxyriboside, thymine deoxyriboside, guanine deoxyriboside and cytosine deoxyriboside; from 1 .mu.g/mL to 100 mg/mL of each of adenine riboside, uridine riboside, guanine riboside and cytosine riboside; from 0.1 mM to 20 mM final molarity of L-glutamine; from 1 mg/mL to 100 mg/mL of an albumin which is deionized, fatty-acid free and globulin-free, and which is selected from the group consisting of bovine serum albumin, human serum albumin and recombinant albumin; from 1 .mu.g/mL to 1 mg/mL of a human or bovine transferrin; from 0.1 .mu.g/mL to 100 .mu.g/mL of a phosphatidyl choline; from 0.1 .mu.g/mL to 100 .mu.g/mL of a C.sub.16 -C.sub.24 unsaturated fatty acid; from 0.1 .mu.g/mL to 100 .mu.g/mL of a cholesterol; from 1 .mu.M to 1 mM final molarity of at least one bioacceptable antioxidant; and from 0.1 .mu.g/mL to 250 .mu.g/mL of at least one antibiotic substance wherein (i) the medium is essentially free of burst promoting activity, and (ii) the cells do not proliferate in the medium. 2. The basal serum-free medium of claim 1 wherein the phosphatidyl choline is synthetic L-.alpha.-phosphatidyl choline dipalmitoyl. 3. The basal serum-free medium of claim 2 wherein the unsaturated fatty acid is linoleic acid or oleic acid. 4. The basal serum-free medium of claim 3 wherein the cholesterol is porcine liver cholesterol. 5. The basal serum-free medium of claim 4 wherein the antioxidant is selected from the group consisting of .beta.-mercaptoethanol, .alpha.-thioglycerol and combinations thereof. 6. The basal serum-free medium of claim 5 wherein the antibiotic substance is selected from the group consisting of sodium penicillin G, streptomycin sulphate and combinations thereof. 7. The basal serum-free medium of claim 6 further including from 0.1 mg/mL to 10 mg/mL of a bio-acceptable semi-solid or viscous matrix material selected from the group consisting of methylcellulose, agar and agarose. 8. A serum-free culture medium for the growth and differentiation of cells selected from the group consisting of hematopoietic stem cells, hematopoietic progenitor cells, and leukemia cells, whereby said medium is free from indigenous cell growth factors and consists of: (a) a basal serum-free medium as defined in claim 1; (b) at least one growth-promoting agent selected from the group consisting of heme or hemin in an amount of from 50 .mu.M to 1 .mu.mM, interleukin-3 in an amount from 0.1 ng/mL to 1 .mu.g/mL, and recombinant human stem cell factor in an amount from 0.5 ng/mL to 1 .mu.g/mL; and (c) at least one cell differentiation-promoting agent selected from the group consisting of erythropoietin in an amount of from 0.1 ng/mL to 1 .mu.g/mL, insulin-like growth factor in an amount from 1 pg/mL to 1 .mu.g/mL, and a retinoid in an amount from 0.1 nM to 0.1 mM. 9. The culture medium of claim 8 wherein the cell differentiation promoting agent is erythropoietin. 10. The culture medium of claim 9 wherein the growth promoting agent is interleukin-3. 11. The culture medium of claim 10 wherein the growth promoting agent is recombinant human stem cell factor. 12. The culture medium of claim 11 which includes insulin-like growth factor 1. 13. The culture medium 12 which includes hemin as the growth promoting agent. 14. The culture medium of claim 13 which includes a retinoid selected from the group consisting of retinoic acid and lower alkyl esters of retinoic acid. 15. The culture medium of claim 14 wherein the retinoid is retinyl acetate. 16. The culture medium of claim 14 wherein the retinoid is all-trans retinoic acid. 17. The culture medium of claim 16 wherein the basal serum-free medium consists of: a minimum essential medium; from 1 .mu.g/mL to 100 mg/mL of each of adenine deoxyriboside, thymine deoxyriboside, guanine deoxyriboside and cytosine deoxyriboside; from 1 .mu.g/mL to 100 mg/mL of each of adenine riboside, uridine riboside, guanine riboside and cytosine riboside; from 0.1 mM to 20 mM final molarity of L-glutamine; from 1 mg/mL to 1 mg/mL of an albumin which is deionized, fatty-acid free and globulin-free, and which is selected from the group consisting of bovine serum albumin., human serum albumin and recombinant albumin; from 1 .mu.g/mL to 1 mg/mL of a human or bovine transferrin; from 0.1 .mu.g/mL to 100 .mu.g/mL of synthetic L-.alpha.-phosphatidyl choline dipalmitoyl; from 0.1 .mu.g/mL to 100 .mu.g/mL of a C.sub.6 -C.sub.24 unsaturated fatty acid selected from the group consisting of linoleic acid, oleic acid and combinations thereof from 0.1 .mu.g/mL to 100 .mu.g/mL of a cholesterol; from 1 .mu.M to 1 mM final molarity of at least one bioacceptable antioxidant selected from the group consisting of .beta.-mercaptoethanol, .alpha.-thioglycerol and combinations thereof; and from 0.1 .mu.g/mL to 250 .mu.g/mL of at least one antibiotic substance selected from the group consisting of sodium penicillin G, streptomycin sulphate and combinations thereof. 18. The culture medium of claim 16 wherein the basal serum-free medium consists of: a minimum essential medium; from 1 .mu.g/mL to 100 mg/mL of each of adenine deoxyriboside, thymine deoxyriboside, guanine deoxyriboside and cytosine deoxyriboside; from 1 .mu.g/mL to 100 mg/mL of each of adenine riboside, uridine riboside, guanine riboside and cytosine riboside; from 0.1 mM to 20 mM final molarity of L-glutamine from 1 mg/mL to 100 mg/mL of an albumin which is deionized, fatty-acid free and globulin-free, and which is selected from the group consisting of bovine serum albumin, human serum albumin and recombinant albumin; from 1 .mu.g/mL to 1 mg/mL of a human or bovine transferrin; from 0.1 .mu.g/mL to 100 .mu.g/mL of synthetic L-.alpha.-phosphatidyl choline dipalmitoyl; from 0.1 .rho.g/mL to 100 .mu.g/mL of a C.sub.16 -C.sub.24 unsaturated fatty acid selected from the group consisting of linoleic acid, oleic acid and combinations thereof; from 0.1 .mu.g/mL to 100 .mu.g/mL of a cholesterol; from 1 .mu.M to 1 mM final molarity of at least one bioacceptable antioxidant selected from the group consisting of .beta.-mercaptoethanol, .alpha.-thioglycerol and combinations thereof; from 0.1 .mu.g/mL to 250 .mu.g/mL of at least one antibiotic substance selected from the group consisting of sodium penicillin G, streptomycin sulphate and combinations thereof; and from 0.1 mg/mL to 10 mg/mL of a bio-acceptable semi-solid or viscous matrix material selected from the group consisting of methylcellulose, agar and agarose. 19. The basal serum-free medium of claim 1 further including from 100 nM to 1 mM of d-.alpha.-tocopherol or an ester thereof. |
Details for Patent 5,397,706
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