U.S. patent application number 12/192832 was filed with the patent office on 2009-01-29 for hematopoietic cell culture nutrient supplement.
This patent application is currently assigned to INVITROGEN CORPORATION. Invention is credited to William C. Biddle, Barbara M. Dadey, John P. Daley, Michelle G. Wysocki.
Application Number | 20090028838 12/192832 |
Document ID | / |
Family ID | 21758558 |
Filed Date | 2009-01-29 |
United States Patent
Application |
20090028838 |
Kind Code |
A1 |
Daley; John P. ; et
al. |
January 29, 2009 |
HEMATOPOIETIC CELL CULTURE NUTRIENT SUPPLEMENT
Abstract
The present invention provides a serum-free supplement which
supports the growth of hematopoietic cells in culture. Also
provided are a medium comprising a basal medium supplemented with
the serum-free supplement of the present invention. The present
invention also provides methods for culturing and for
differentiating hematopoietic cells.
Inventors: |
Daley; John P.; (Buffalo,
NY) ; Dadey; Barbara M.; (East Aurora, NY) ;
Biddle; William C.; (Buffalo, NY) ; Wysocki; Michelle
G.; (Cheektowaga, NY) |
Correspondence
Address: |
INVITROGEN CORPORATION;C/O INTELLEVATE
P.O. BOX 52050
MINNEAPOLIS
MN
55402
US
|
Assignee: |
INVITROGEN CORPORATION
Carlsbad
CA
|
Family ID: |
21758558 |
Appl. No.: |
12/192832 |
Filed: |
August 15, 2008 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10716619 |
Nov 20, 2003 |
|
|
|
12192832 |
|
|
|
|
08792299 |
Jan 31, 1997 |
6733746 |
|
|
10716619 |
|
|
|
|
60013149 |
Mar 12, 1996 |
|
|
|
Current U.S.
Class: |
424/93.21 ;
424/93.7; 435/350; 435/351; 435/352; 435/353; 435/354; 435/366;
435/377; 435/384; 435/385; 435/388; 435/407 |
Current CPC
Class: |
C12N 2500/32 20130101;
C12N 2500/38 20130101; C12N 2500/90 20130101; C12N 2501/125
20130101; C12N 2500/25 20130101; C12N 2500/36 20130101; C12N 5/0647
20130101; C12N 2501/23 20130101; A61P 43/00 20180101; C12N 2501/39
20130101 |
Class at
Publication: |
424/93.21 ;
435/407; 435/366; 435/353; 435/354; 435/352; 435/350; 435/351;
435/388; 435/384; 435/385; 424/93.7; 435/377 |
International
Class: |
A61K 35/12 20060101
A61K035/12; C12N 5/06 20060101 C12N005/06; A61P 43/00 20060101
A61P043/00 |
Claims
1. A serum-free, eukaryotic cell culture medium supplement
comprising 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 said supplement is capable of supporting the expansion of
CD34.sup.+ hematopoietic cells.
2. A serum-free, eukaryotic cell culture medium supplement
comprising 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 said supplement is capable of supporting the expansion of
CD34.sup.+ hematopoietic cells.
3. The serum-free, eukaryotic cell culture medium supplement
according to claim 1, wherein said antioxidants are selected from
the group consisting of N-acetyl-L-cysteine, 2-mercaptoethanol, and
D,L-tocopherol acetate, or derivatives or mixtures thereof.
4. The serum-free, eukaryotic cell culture medium supplement
according to claim 1, wherein said albumin is human serum
albumin.
5. The serum-free, eukaryotic cell culture medium supplement
according to claim 1, wherein said lipid agents are selected from
the group consisting of Human Ex-Cite.RTM. and ethanolamine or
derivatives and mixtures thereof.
6. The serum-free, eukaryotic cell culture medium supplement
according to claim 1, wherein said insulin is human zinc
insulin.
7. The serum-free, eukaryotic cell culture medium supplement
according to claim 1, wherein said transferrin is human
iron-saturated transferrin.
8. The serum-free, eukaryotic cell culture medium supplement
according to claim 1, wherein said trace element is Se.sup.4+.
9. The serum-free, eukaryotic cell culture medium supplement
according to claim 1, wherein said glucocorticoid is
hydrocortisone.
10. The serum-free, eukaryotic cell culture medium supplement
according to claim 1, wherein said supplement is concentrated.
11. The serum-free, eukaryotic cell culture medium supplement
according to claim 10, wherein said supplement is concentrated
about 2 to about 100 fold.
12. The serum-free, eukaryotic cell culture medium supplement
according to claim 11, wherein said supplement is about a 40.times.
formulation.
13. A serum-free, eukaryotic cell culture medium supplement
obtained by combining one or more ingredients of claim 1.
14. A serum-free, eukaryotic cell culture medium supplement
according to claim 13, wherein said ingredients comprise one or
more ingredients selected from the group consisting of N-acetyl-L
cysteine, human serum albumin, Human Ex-Cyte.RTM., ethanolamine,
human zinc insulin, human iron saturated transferrin, Se.sup.4+,
hydrocortisone, D,L-tocopherol acetate, and 2-mercaptoethanol.
15. A method of making a serum-free eukaryotic cell culture medium
supplement, said method comprising admixing water with one or more
ingredients according to claim 1.
16. The method according to claim 15, wherein said ingredients
comprise water, N-acetyl-L cysteine, human serum albumin, Human
Ex-Cyte.RTM., ethanolamine HCl, human zinc insulin, human iron
saturated transferrin, a Se.sup.4+ salt, hydrocortisone,
D,L-tocopherol acetate, and 2-mercaptoethanol.
17. A kit comprising a carrier means, said carrier means being
compartmentalized to receive in close confinement therein one or
more container means, wherein a first container means contains the
supplement of claim 1, and wherein optionally a second container
means contains a basal medium.
18. A serum-free eukaryotic cell culture medium comprising a basal
cell culture medium supplemented with the serum-free culture
supplement according to claim 1, wherein said supplemented culture
medium is capable of supporting the expansion of CD34.sup.+
hematopoietic cells.
19. The serum-free eukaryotic cell culture medium according to
claim 18, wherein said basal medium is selected from the group
consisting of Iscove's Modified Dulbecco's Medium, RPMI-1640,
.alpha.-MEM.
20. A serum-free eukaryotic cell culture medium obtained by
combining a basal cell culture medium with the serum-free
supplement according to claim 1, wherein said medium is capable of
supporting the expansion of CD34.sup.+ hematopoietic cells.
21. The serum-free eukaryotic cell culture medium according to
claim 20, wherein said medium is a 1.times. medium.
22. The serum-free eukaryotic cell culture medium according to
claim 20, wherein said medium is a concentrated medium
formulation.
23. The serum-free eukaryotic cell culture medium according to
claim 22, wherein said medium is from about 2.times. to about
100.times. concentrated
24. The serum-free eukaryotic cell culture medium according to
claim 23, wherein said concentrated medium formulation is about a
10.times. formulation.
25. The serum-free eukaryotic cell culture medium according to
claim 23, wherein said concentrated medium formulation is greater
than 10.times. concentrated.
26. A method of making a serum-free eukaryotic cell culture medium,
said method comprising admixing a basal cell culture medium with
the serum-free supplement according to claim 1, wherein said medium
is capable of supporting the expansion of CD34.sup.+ hematopoietic
cells.
27. A serum-free eukaryotic cell culture medium comprising 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, one or more glucocorticoids, one or more inorganic salts,
one or more energy sources, one or more buffering agents, one or
more pyruvate salts, one or more pH indicators, one or more amino
acids, and one or more vitamins, wherein the medium is capable of
supporting the expansion of CD34.sup.+ hematopoietic cells.
28. The serum-free, eukaryotic cell culture medium according to
claim 27, wherein said antioxidants are selecting from the group
consisting of N-acetyl-L-cysteine, 2-mercaptoethanol, and
D,L-tocopherol acetate, or derivatives or mixtures thereof.
29. The serum-free, eukaryotic cell culture medium according to
claim 27, wherein said albumin is human serum albumin.
30. The serum-free, eukaryotic cell culture medium according to
claim 27, wherein said lipid agents are Human Ex-Cyte.RTM. and
ethanolamine.
31. The serum-free, eukaryotic cell culture medium according to
claim 27, wherein said insulin is human zinc insulin.
32. The serum-free, eukaryotic cell culture medium according to
claim 27, wherein said transferrin is human iron-saturated
transferrin.
33. The serum-free, eukaryotic cell culture medium according to
claim 27, wherein said glucocorticoid is hydrocortisone.
34. The serum-free, eukaryotic cell culture medium according to
claim 27, wherein said inorganic salt ingredient comprises one or
more inorganic salts selected from the group consisting of one or
more calcium salts, one or more potassium salts, one or more
magnesium salts, one or more sodium salts, one or more carbonate
salts, and one or more phosphate salts.
35. The serum-free, eukaryotic cell culture medium according to
claim 27, wherein said energy source is D-glucose.
36. The serum-free, eukaryotic cell culture medium according to
claim 27, wherein said buffering agent is HEPES.
37. The serum-free, eukaryotic cell culture medium according to
claim 27, wherein said pyruvate salt is sodium pyruvate.
38. The serum-free, eukaryotic cell culture medium according to
claim 27, wherein said pH indicator is phenol red.
39. The serum-free, eukaryotic cell culture medium according to
claim 27, wherein said amino acid ingredient comprises one or more
amino acids selected from the group consisting of glycine,
L-alanine, L-asparagine, L-cysteine, L-aspartic acid, L-glutamic
acid, L-phenylalanine, L-histidine, L-isoleucine, L-lysine,
L-leucine, L-glutamine, L-arginine HCL, L-methionine, L-proline,
L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine,
and L-valine, and salts and derivatives thereof.
40. The serum-free, eukaryotic cell culture medium according to
claim 27, wherein said vitamin ingredient comprises one or more
vitamins selected from the group consisting of biotin, D-calcium
pantothenate, choline chloride, folic acid, i-inositol,
niacinamide, pyridoxal HCl, riboflavin, thiamine HCl, and vitamin
B.sub.12 and derivatives thereof.
41. The serum-free, eukaryotic cell culture medium according to
claim 27, wherein said ingredients comprise N-acetyl-L-cysteine,
2-mercaptoethanol, human serum albumin, D,L-tocopherol acetate,
Human Ex-Cyte.RTM., ethanolamine, human zinc insulin,
iron-saturated transferrin, Se.sup.4+, hydrocortisone, Ca.sup.2+,
K.sup.+, Mg.sup.2+, Na.sup.+, CO.sub.3.sup.2-, PO.sub.4.sup.3-,
D-glucose, HEPES, sodium pyruvate, phenol red, glycine, L-alanine,
L-asparagine, L-cysteine, L-aspartic acid, L-glutamic acid,
L-phenylalanine, L-histidine, L-isoleucine, L-lysine, L-leucine,
L-glutamine, L-arginine HCL, L-methionine, L-proline,
L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine,
and L-valine, biotin, D-calcium pantothenate, choline chloride,
folic acid, i-inositol, niacinamide, pyridoxal HCl, riboflavin,
thiamine HCl, and vitamin B.sub.12.
42. The serum-free, eukaryotic cell culture medium according to
claim 41, wherein said medium is obtained by combining water and
the ingredients of claim 46.
43. A method of making a serum-free, eukaryotic cell culture medium
said method comprising admixing the ingredients according to claim
41.
44. A composition comprising CD34.sup.+ hematopoictic stem cells in
the serum-free medium of claim 27.
45. The composition according to claim 44, wherein said CD34.sup.+
hematopoietic stem cells are obtained from an animal selected from
the group consisting of human, monkey, ape, mouse, rat, hamster,
rabbit, guinea pig, cow, swine, dog, horse, cat, goat, and
sheep.
46. A method of expanding CD34.sup.+ hematopoietic cells, said
method comprising (a) contacting said cells with the medium
according to claim 27; and (b) culturing said cells under
conditions suitable to facilitate the expansion of said cells.
47. The method according to claim 46, wherein said culturing
further comprises adding a hematopoietic cell growth factor to said
medium.
48. The method according to claim 47, wherein said growth factor is
one or more growth factors selected from the group consisting of
erythropoietin, granulocyte-colony stimulating factor, stem cell
factor, interleukin-3, and granulocyte/macrophage-colony
stimulating factor.
49. A method of providing CD34.sup.+ hematopoietic cells, in
serum-free culture, to a mammal, said method comprising (a)
contacting said CD34.sup.+ hematopoietic cells with the medium
according to claim 27; (b) cultivating said CD34.sup.+
hematopoietic cells under conditions suitable to facilitate the
expansion of said cells in serum-free culture; and (c) introducing
said expanded cells into a mammal.
50. The method according to claim 49, wherein said culturing
further comprises adding a hematopoietic cell growth factor to said
medium.
51. A method of causing CD34.sup.+ hematopoietic cells to
differentiate into a particular type of cell in serum-free culture,
said method comprising (a) contacting said CD34.sup.+ hematopoietic
cells with the medium according to claim 27; (b) cultivating said
cells under conditions suitable to facilitate the expansion of said
cells in serum-free culture; and (c) adding one or more
differentiation factors or changing culturing conditions to induce
differentiation of cells to form a different type of hematopoietic
cell.
52. A method of providing differentiated hematopoietic cells, in
serum-free culture, to a mammal, said method comprising (a)
contacting said CD34.sup.+ hematopoietic cells with the medium
according to claim 27; (b) cultivating said cells under conditions
suitable to facilitate the expansion of said cells in serum-free
culture; (c) adding one or more differentiation factors or changing
culturing conditions to induce differentiation of cells to form a
different type of hematopoietic cell; and (d) introducing said
differentiated cells into a mammal.
53. A method of expanding a recombinant CD34.sup.+ hematopoietic
cells, said method comprising obtaining a recombinant CD34.sup.+
hematopoietic cell containing a nucleic acid molecule which encodes
a protein of interest; and (b) culturing said cell in the medium
according to claim 27 to form a population of recombinant
cells.
54. A method of providing a recombinant CD34.sup.+ hematopoietic
cell to a mammal, said method comprising (a) obtaining a
recombinant CD34.sup.+ hematopoietic cell containing a nucleic acid
molecule which encodes a protein of interest; (b) culturing said
cell in the medium according to claim 27 to form a population of
recombinant CD34.sup.+ hematopoietic stem cells; and (c)
introducing said recombinant cells into a mammal.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a replacement for the serum
supplementation normally required for ex vivo expansion of
CD34.sup.+ hematopoietic cells and cells of myeloid lineage.
BACKGROUND OF THE INVENTION
[0002] 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 CO.sub.2 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).
[0003] 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).
[0004] 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)).
[0005] 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.
[0006] 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.sup.+ cells. That is, they
express the CD34 surface marker. The most primitive known human
progenitor cell, which has been characterized as
CD34.sup.+/CD33.sup.-/CD38.sup.-, represents only 1 to 2% of all
bone marrow cells (Civin, C. I. et al., J. Immunol. 133:157
(1984)).
[0007] 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.
[0008] 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.
[0009] 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).
[0010] 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)).
[0011] 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.
[0012] 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.
[0013] In view of the many problems associated with the use of
serum in the growth of CD34.sup.+ hematopoietic cells, laboratories
performing work with CD34.sup.+ 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.degree. C. and below is
problematic.
[0014] 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)).
[0015] 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.
[0016] Research with CD34.sup.+ hematopoietic cells, cultivation of
CD34.sup.+ hematopoietic cells in culture, expansion of CD34.sup.+
hematopoietic cells, control of differentiation of CD34.sup.+
hematopoietic cells, and explanation of CD34.sup.+ 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.sup.+ 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.sup.+ hematopoietic
cells and which can be stored for long periods of time.
SUMMARY OF THE INVENTION
[0017] 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.sup.+ hematopoietic cells and
cells of myeloid lineage, in serum-free culture.
[0018] 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.sup.+ hematopoietic cells and cells of myeloid lineage, in
serum-free culture.
[0019] 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.RTM., ethanolamine HCl, human zinc insulin, human iron
saturated transferrin, Se.sup.4+, 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.sup.+ hematopoietic
cells and cells and cells of myeloid lineage, in serum-free
culture.
[0020] 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.RTM., ethanolamine HCl, zinc insulin, human iron saturated
transferrin, a Se.sup.4+ salt, hydrocortisone, D,L-tocopherol
acetate, and 2-mercaptoethanol.
[0021] The present invention also provides a method of making a
serum-free, eukaryotic cell culture medium supplement, the method
comprising admixing water, N-acetyl-L cysteine, human serum
albumin, Human Ex-Cyte.RTM., ethanolamine HCl, human zinc insulin,
human iron saturated transferrin, a Se.sup.4+ salt, hydrocortisone,
D,L-tocopherol acetate, and 2-mercaptoethanol, wherein each
ingredient is present in an amount which, when added to a basal
medium, supports the expansion of CD34.sup.+ hematopoietic cells
and cells of myeloid lineage, in serum-free culture.
[0022] The present invention also provides a kit comprising a
carrier means, the carrier means being compartmentalized to receive
in close confinement therein one or more container means, wherein a
first container means contains the supplement of the present
invention, and wherein optionally a second container means contains
a basal medium.
[0023] The present invention also provides a serum-free eukaryotic
cell culture medium comprising a basal cell culture medium
supplemented with the serum-free culture supplement of the
invention, wherein the supplemented culture medium is capable of
supporting the expansion of CD34.sup.+ hematopoietic cells and
cells of myeloid lineage, in serum-free culture.
[0024] The present invention also provides a serum-free eukaryotic
cell culture medium obtained by combining a basal cell culture
medium with the serum-free supplement of the invention, wherein the
medium is capable of supporting the expansion of CD34.sup.+
hematopoietic cells and cells of myeloid lineage, in serum-free
culture.
[0025] The present invention also provides a method of making a
serum-free eukaryotic cell culture medium, the method comprising
admixing a basal cell culture medium with the serum-free supplement
of the invention, wherein the medium is capable of supporting the
expansion of CD34.sup.+ hematopoietic cells and cells of myeloid
lineage, in serum-free culture.
[0026] The present invention also provides a serum-free eukaryotic
cell culture medium comprising 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, one or more
glucocorticoids, one or more inorganic salts, one or more energy
sources, one or more buffering agents, one or more pyruvate salts,
one or more pH indicators, one or more amino acids, and one or more
vitamins, wherein the medium is capable of supporting the expansion
of CD34.sup.+ hematopoietic cells and cells of myeloid lineage in
serum-free culture.
[0027] The present invention specifically provides a serum-free,
eukaryotic cell culture medium comprising the ingredients
N-acetyl-L-cysteine, 2-mercaptoethanol, human serum albumin,
D,L-tocopherol acetate, Human Ex-Cyte.RTM., ethanolamine, human
zinc insulin, iron-saturated transferrin, Se.sup.4+,
hydrocortisone, Ca.sup.2+, K.sup.+, Mg.sup.2+, Na.sup.+,
CO.sub.3.sup.2-, PO.sub.4.sup.3-, D-glucose, HEPES buffer, sodium
pyruvate, phenol red, glycine, L-alanine, L-asparagine, L-cysteine,
L-aspartic acid, L-glutamic acid, L-phenylalanine, L-histidine,
L-isoleucine, L-lysine, L-leucine, L-glutamine, L-arginine HCL,
L-methionine, L-proline, L-hydroxyproline, L-serine, L-threonine,
L-tryptophan, L-tyrosine, and L-valine, biotin, D-calcium
pantothenate, choline chloride, folic acid, i-inositol,
niacinamide, pyridoxal HCl, riboflavin, thiamine HCl, and vitamin
B.sub.12. The present invention also provides a method of making
the medium of the present invention, the method comprising admixing
the ingredients of the medium.
[0028] The present invention also provides a serum-free, eukaryotic
cell culture medium obtained by combining the ingredients water,
N-acetyl-L-cysteine, 2-mercaptoethanol, human serum albumin,
D,L-tocopherol acetate, Human Ex-Cyte.RTM., ethanolamine, human
zinc insulin, iron-saturated transferrin, a Se.sup.4+ salt,
hydrocortisone, a calcium salt, a potassium salt, a magnesium salt,
a sodium salt, a carbonate salt, a phosphate salt, D-glucose,
HEPES, a pyruvate salt, phenol red, glycine, L-alanine,
L-asparagine, L-cysteine, L-aspartic acid, L-glutamic acid,
L-phenylalanine, L-histidine, L-isoleucine, L-lysine, L-leucine,
L-glutamine, L-arginine HCL, L-methionine, L-proline,
L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine,
and L-valine, biotin, D-calcium pantothenate, choline chloride,
folic acid, i-inositol, niacinamide, pyridoxal HCl, riboflavin,
thiamine HCl, and vitamin B.sub.12, wherein the ingredients are
present in an amount which supports the expansion of CD34.sup.+
hematopojetic cells and cells of myeloid lineage.
[0029] The present invention provides a method of making a
serum-free, eukaryotic cell culture medium said method comprising
admixing the ingredients water, N-acetyl-L-cysteine,
2-mercaptoethanol, human serum albumin, D,L-tocopherol acetate,
Human Ex-Cyte.RTM., ethanolamine, human zinc insulin,
iron-saturated transferrin, a Se.sup.4+ salt, hydrocortisone, a
calcium salt, CaCl.sub.2, one or more potassium salts, a magnesium
salt, a sodium salt, a carbonate salt, a phosphate salt, D-glucose,
HEPES, a pyruvate salt, phenol red, glycine, L-alanine,
L-asparagine, L-cysteine, L-aspartic acid, L-glutamic acid,
L-phenylalanine, L-histidine, L-isoleucine, L-lysine, L-leucine,
L-glutamine, L-arginine HCL, L-methionine, L-proline,
L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine,
and L-valine, biotin, D-calcium pantothenate, choline chloride,
folic acid, i-inositol, niacinamide, pyridoxal HCl, riboflavin,
thiamine HCl, and vitamin B.sub.12, wherein the ingredients are
present in an amount which supports the expansion of CD34.sup.+
hematopoietic cells and cells of myeloid lineage.
[0030] The present invention also provides a composition comprising
CD34.sup.+ hematopoietic cells and the serum-free medium of the
present invention, wherein the serum-free medium is capable of
supporting the expansion of CD34.sup.+ hematopoietic cells and
cells of myeloid lineage in serum-free culture.
[0031] The present invention also provides a method of expanding
CD34.sup.+ hematopoietic cells and cells of myeloid lineage, the
method comprising contacting cells with the medium of the present
invention and culturing the cells under serum-free conditions
suitable to facilitate the expansion of the cells.
[0032] The present invention also provides a method of providing
CD34.sup.+ hematopoietic cells to a mammal, the method comprising
contacting CD34.sup.+ hematopoietic cells with the medium of the
present invention, cultivating cells under conditions suitable to
facilitate the expansion of the cells in serum-free culture, and
introducing the expanded cells into a mammal.
[0033] The present invention also provides a method of causing
CD34.sup.+ hematopoietic cells to differentiate into a particular
type of cell in serum-free culture, the method comprising
contacting CD34.sup.+ hematopoietic cells with the medium of the
present invention, cultivating the cells under conditions suitable
to facilitate the expansion of cells in serum-free culture, and
adding one or more differentiation factors or changing culturing
conditions to induce differentiation of cells to form a different
type of hematopoietic cell.
[0034] The present invention also provides a method of providing
differentiated hematopoictic cells to a mammal, the method
comprising contacting CD34.sup.+ hematopoietic cells with the
medium of the present invention, cultivating the cells under
conditions suitable to facilitate the expansion of the cells in
serum-free culture, adding one or more differentiation factors or
changing culturing conditions to induce differentiation of cells to
form a different type of hematopoietic cell, and introducing the
differentiated cells into a mammal.
[0035] The present invention also provides a method of expanding
recombinant CD34.sup.+ hematopoietic cells, the method comprising
obtaining a recombinant CD34.sup.+ hematopoietic cell containing a
nucleic acid molecule which encodes a protein of interest, and
culturing the cell in the medium of the present invention to form a
population of recombinant cells.
[0036] The present invention also provides a method of providing a
recombinant CD34.sup.+ hematopoietic cell to a mammal, the method
comprising obtaining a recombinant CD34.sup.+ hematopoietic cell
containing a nucleic acid molecule which encodes a protein of
interest, culturing the cell in the medium of the present invention
to form a population of recombinant CD34.sup.+ hematopoietic stem
cells, and introducing the recombinant cells into a mammal.
[0037] The invention also relates to a method for regeneration of a
eukaryotic cell culture medium (preferably a serum-free medium)
comprising mixing a sufficient amount of an antioxidant (preferably
N-acetyl-L-cysteine or a derivative thereof) to generate the
medium. After regeneration, the medium may be used to support
growth of any eukaryotic cell, particularly CD34.sup.+
hematopoietic cells.
[0038] The invention also relates to increasing or enhancing
shelf-life of a eukaryotic cell culture medium (preferably a
serum-free medium) comprising mixing the medium with a sufficient
amount of an antioxidant (preferably N-acetyl-L-cysteine or a
derivative thereof) to increase or enhance the shelf-life of the
medium.
BRIEF DESCRIPTION OF THE FIGURES
[0039] FIG. 1 shows the effect of the serum-free StemPro-34.TM.
medium on the kinetics of growth factor driven expansion of an
enriched population of CD34.sup.+ hematopoietic cells, as compared
to the effect of serum.
[0040] FIG. 2 shows the effect of the serum-free StemPro-34.TM.
medium on the preferential expansion of CD34.sup.+ cells from an
enriched population of CD34.sup.+ cells, as compared to the effect
of serum.
[0041] FIG. 3 shows the effect of the serum-free StemPro-34.TM.
medium on expansion index of bone marrow cells, as compared to the
effect of serum.
[0042] FIG. 4 shows the effect of growth factor addition on both
total cell expansion and CD34.sup.+ cell expansion under serum-free
and serum-supplemented culture conditions.
[0043] FIG. 5 shows the effect of recombinant growth factors on the
development of erythroid blast-forming unit (BFU-E) cells in
serum-free and serum supplemented media.
[0044] FIG. 6 shows the results of experiments in which the
concentration of transferrin was optimized.
[0045] FIG. 7 shows experimental results which depict the
optimization and equivalence of Human Ex-Cyte.RTM. to lipids and
fatty acids.
[0046] FIG. 8 shows the effect of 2-mercaptoethanol on the
expansion of human bone marrow cells in serum-free culture.
[0047] FIG. 9 shows the effect of human serum albumin concentration
on expansion of CD34.sup.+ cells.
[0048] FIG. 10 shows the effect of ammonium ion concentration on
the proliferation of CD34.sup.+-selected cells.
[0049] FIG. 11 shows the correlation of the generation of ammonia
as a function of cell expansion.
[0050] FIG. 12 reflects the requirement for N-acetyl-L-cysteine for
optimal long-term performance of the serum-free medium of the
present invention.
[0051] FIG. 13 shows the effect of N-acetyl-L-cysteine
concentration on the performance of the serum-free supplement of
the present invention.
[0052] FIG. 14 shows the effect of basal medium formulation on the
expansion of human bone marrow cells using the serum-free
supplement of the present invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0053] The present invention provides a chemically defined cell
culture supplement and medium which has been developed specifically
for stem cells and progenitor cells of the hematopoietic system.
This formulation is unique in its ability to support the expansion
of CD34.sup.+ cells which are, at the present time, defined as the
earliest hematopoietic stem cell identifiable in bone marrow,
peripheral blood or neonatal cord blood. The supplement and the
medium of the present invention are particularly suited for
supporting the expansion of CD34.sup.+ cells and cells of myeloid
lineage, including BFU-E cells, erythrocytes, CFU-MEG cells,
megakaryocytes, CFU-GM cells, monocytes, macrophages, neutrophils
eosinophils, and basophils. In earlier stages of development, cells
of myeloid lineage express the CD34.sup.+ marker protein. In later
stages of development, cells of myeloid lineage do not express
detectable levels of the CD34.sup.+ marker protein. Whether a cell
of myeloid lineage expresses the CD34.sup.+ marker protein can be
determined by one of ordinary skill in the art using well-known
techniques, such as fluorescence activated cell sorting.
[0054] The supplement and the medium of the present invention can
also be used to culture or expand cells of mesenchymal origin. Such
cells include but are not limited to, chondrocytes, osteoclasts,
osteoblasts, and epithelial cells of skin and vascular tissue. The
supplement and the medium of the present invention can also be used
to culture both primary and immortalized cells of most or all
embryonic origin (e.g., ectodermal derivatives, mesodermal
derivatives, and endodermal derivatives). These cells include cells
of the central and peripheral nervous system (neurons, glial cells,
and astrocytes), epithelial cells (sensory epithelial cells,
epidermal cells (skin, mammary, hair, nails, pituitary gland,
sebaceous gland)), connective tissue cells (cartilage, bone,
striated and smooth muscle, hematopoietic cells, lymphoid cells,
kidney, gonadal cells, adrenal cells) and parenchymal cells of the
liver, pancreas, thyroid, thymus, as well as epithelial linings of
the urinary bladder, urethra, tympanic cavity, and eustachian tube.
These cells can be of human or other mammalian or eukaryotic
origin.
[0055] Using the serum-free supplement or medium of the present
invention, the growth, expansion, and differentiation of
hematopoietic cells can be regulated by the addition of defined
growth factors or other cytokines. Such influence over
hematopoietic cells in culture is not possible when cells are
cultured in serum-supplemented media. Undefined components in serum
obscure the cellular responses to defined factors. Using the
supplement or the medium of the present invention, CD34.sup.+
hematopoietic cells (including hematopoietic stem cells), and cells
of myeloid lineage can be expanded and differentiated in suspension
culture and in the absence of stromal cells, collagen, or support
matrices.
[0056] Stromal cells are bone marrow cells which form a supporting
network for hematopoietic cells in vivo and in vitro. Stromal cells
serve as sites of attachment for hematopoietic cells. Further,
stromal cells secrete cytokines and other growth factors which
hematopoietic cells require for expansion and growth. In culture,
stromal cells form an adherent layer and include endothelial cells,
macrophages, fibroblasts, and adipocytes. Using the supplement or
the medium of the present invention, it is not necessary to
co-culture hematopoietic cells with stromal cells in order to
expand the hematopoietic cell population.
[0057] The serum-free supplement and the medium of the present
invention differ from previously available serum-free media in that
the supplement and medium of the present invention provide for the
growth and expansion of both the putative pluripotent stem cell
population (i.e., CD34.sup.+ cells from bone marrow, peripheral
blood and neonatal cord blood) and differentiated progeny.
[0058] The serum-free supplement and the medium of the present
invention can be used to culture hematopoietic cells derived from a
number of animals, including human, monkey, ape, mouse, rat,
hamster, rabbit, guinea pig, cow, swine, dog, horse, cat, goat, and
sheep. Preferably, human hematopoietic cells are cultured.
[0059] The supplement and the medium of the present invention allow
hematopoietic stem cells and their progeny to respond fully to
human recombinant hematopoietic growth factors or other cytokines.
Until now, the cellular response to defined growth factors has been
hindered by undefined components in serum and the lack of
hematopoietic cell-specific optimization of the media chemistry.
Unlike other serum-free media, the supplement and the medium of the
present invention include an antioxidant or combination of
antioxidants which protect hematopoietic cells from the detrimental
(and often lethal) effects of multiple free radical species and
other toxic moieties known to be generated in the cell culture
environment. Thus, by addition of antioxidants according to the
invention, shelf-life of a medium can be increased or enhanced.
Moreover, to the extent that such detrimental products are formed
during the storage of a medium, addition of antioxidants according
to the invention provide for regeneration of the medium, thereby
enhancing or increasing the capacity or the ability of the medium
to expand cells. The addition of antioxidants to achieve such
beneficial results can be applied to any cell culture media,
particularly serum-free media. Examples of the media which can be
used include media for expansion both primary and immortalized
cells of most or all embryonic origin (see infra). Such media
include, but are not limited to DMEM, Ham's F-10, Ham's F-12, MCDB
series, AIM-5, Keratinocyte SFM, chemically defined keratinocyte
media, AmnioMax, human chondrocyte media, human dermal fibroblast
media, CHO II, CHO III (GibcoBRL).
[0060] In the description that follows, a number of terms
conventionally used in the field of cell culture media and for the
growth of eukaryotic cells are utilized extensively. In order to
provide a clear and consistent understanding of the specification
and claims, and the scope to be given such terms, the following
definitions are provided.
[0061] The term "regeneration" refers to increasing the ability or
capacity of a medium to expand eukaryotic cells. Typically,
eukaryotic cell culture media, particularly serum-free media will,
upon storage, lose or have reduced ability or capacity to support
growth or expansion of cells. In determining the capacity or
ability of a medium to grow or expand cells has been increased,
comparisons can be made between a medium supplemented with an
antioxidant and a control lacking the antioxidant. According to the
invention, the capacity or ability of a medium to facilitate the
growth or expansion of cells is enhanced or increased if the medium
containing the antioxidant facilitates a greater level of growth or
expansion than a medium which does not contain the antioxidant.
[0062] The term "shelf-life" refers to the storage over time of
medium. Typically, media are stored at 4.degree. to 25.degree. C.,
although other storage temperatures may be used. In determining if
a shelf-life has been enhanced or increased, comparisons can be
made to evaluate the effect of storage on capacity or ability of a
particular medium to support cell growth or expansion. According to
the invention, the shelf-life is said to be enhanced or increased
if the stored medium containing the antioxidant outperforms a
control medium, which does not contain the antioxidant and which is
stored for the same period of time as the medium which contains the
antioxidant. Performance is determined by comparing the level of
cell growth or expansion to the level in medium containing the
antioxidant and the control medium.
[0063] The term "albumin substitute" refers to any compound which
may be used in place of human serum albumin (e.g., bovine serum
albumin (BSA) or AlbuMAX.RTM. I) in the supplement of the invention
to give substantially similar results as albumin. Albumin
substitutes may be any protein or polypeptide source. Examples of
such protein or polypeptide samples include but are not limited to
bovine pituitary extract, plant hydrolysate (e.g., rice
hydrolysate), fetal calf albumin (fetuin), egg albumin, human serum
albumin (HSA), or another animal-derived albumins, chick extract,
bovine embryo extract, AlbuMAX.RTM. I, and AlbuMAX.RTM. II.
[0064] Preferably, the albumin is of human origin. Most preferably,
the albumin is human serum albumin. In the supplement and the
medium of the present invention, the concentration of albumin or
albumin substitute which facilitates cell growth, expansion, and
differentiation in culture can be determined using only routine
experimentation.
[0065] The term "transferrin substitute" refers to any compound
which may replace transferrin in the supplement of the invention to
give substantially similar results as transferrin. Examples of
transferrin substitutes include but are not limited to any iron
chelate compound. Iron chelate compounds which may be used include
but are not limited to iron chelates of ethylenediaminetetraacetic
acid (EDTA), ethylene glycol-bis(.beta.-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA), deferoxamine mesylate,
dimercaptopropanol, diethylenetriamine-pentaacetic acid (DPTA), and
trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic adic (CDTA), as
well as a ferric citrate chelate and a ferrous sulfate chelate.
[0066] Preferably, the transferrin is iron saturated transferrin.
Most preferably, the transferrin is iron saturated human
transferrin. In the supplement and the medium of the present
invention, the concentration of the transferrin or transferrin
substitute which facilitates cell growth, expansion, and
differentiation in culture can be determined using only routine
experimentation.
[0067] The term "insulin substitute" refers to any zinc containing
compound which may be used in place of insulin in the supplement of
the invention to give substantially similar results as insulin.
Examples of insulin substitutes include but are not limited to zinc
chloride, zinc nitrate, zinc bromide, and zinc sulfate.
[0068] A number of insulins are known to those of ordinary skill in
the art. See Gilman, A. G. et al., Eds., The Pharmacological Basis
of Therapeutics, Pergamon Press, New York, 1990, pp. 1463-1495.
Preferably, insulin, rather than an insulin substitute, is used in
the supplement and the medium of the present invention. More
preferably, the insulin is zinc insulin. Most preferably, the
insulin is human zinc insulin. In the supplement and the medium of
the present invention, the concentration of the insulin substitute
which facilitates cell growth, expansion, and differentiation in
culture can be determined using only routine experimentation.
[0069] The term "expand" refers to the growth and division, and not
the differentiation, of hematopoietic cells in culture. The term
"differentiation" refers to the development of a cell of a
particular type into a cell of another type. The development of a
pluripotent hematopoietic stem cell into a myeloid precursor is an
example of differentiation. Likewise, the development of precursor
cell into another type of cell is an example of
differentiation.
[0070] The term "antioxidant" refers to molecules which inhibit
reactions that are promoted by oxygen or peroxides. Antioxidants
which may be used in the supplement or the medium of the present
invention include but are not limited to N-acetyl-L-cysteine or
derivatives thereof (see International Patent Application WO
95/00136), 2-mercaptoethanol or derivatives thereof, D,L-tocopherol
acetate or derivatives thereof, ascorbic acid or derivatives
thereof, thiol compounds, such as dithiothreitol and glutathione,
or derivatives of thiol compounds, catalase or derivatives thereof,
cysteine or derivatives thereof, thiolactate or derivatives
thereof, penicillamine or derivatives thereof,
mercaptoethanesulfonic acid or derivatives thereof, and
mercaptopropionic acid, or derivatives thereof. Preferably, the
antioxidants used in the supplement and the medium of the present
invention are N-acetyl-L-cysteine, 2-mercaptoethanol, and
D,L-tocopherol acetate or mixtures or derivatives thereof.
[0071] The term "glucocorticoid" refers to steroid compounds which
are in the glucocorticoid, as opposed to the mineralocorticoid,
class of corticosteroids. The term includes but is not limited to
glucocorticoids such as hydrocortisone, cortisol, dexamethasone,
and derivatives of these compounds. See Gilman, A. G. et al., Eds.,
The Pharmacological Basis of Therapeutics, Pergamon Press, New
York, 1990, pp. 1440-1462. Preferably, the glucocorticoid of the
present invention is hydrocortisone.
[0072] The term "trace element" refers to a moiety which is present
in a cell culture medium in only trace amounts. In the present
invention, this term encompasses Se.sup.4+, Ag.sup.+, Al.sup.3+,
Ba.sup.2+, Cd.sup.2+, Co.sup.2+, Cr.sup.3+, Ge.sup.4+, Se.sup.4+,
Br.sup.-, I.sup.-, Mn.sup.2+, F.sup.-, Si.sup.4+, V.sup.5+,
Mo.sup.6+, Ni.sup.2+, Rb.sup.+, Sn.sup.2+ and Zr.sup.4+ and salts
thereof. Preferably, the trace element is used in the supplement
and the medium of the present invention. Any salt of a given trace
element can be used to make the supplement or the medium of the
present invention. For example, the sodium salt of selenium oxide
(sodium selenite, Na.sub.2SeO.sub.3) is preferably used to provide
selenium. Suitable concentrations of trace element-containing
compounds can be determined by one of ordinary skill in the art.
For example, in the medium of the invention, the concentration of
SeO.sub.3.sup.2- can be about 0.007 to about 0.07 mg/L. In a
preferred embodiment of the medium of the invention, the
concentration of SeO.sub.3.sup.2- is about 0.01 mg/L.
[0073] The term "lipid agent" refers to an agent which provides a
source of lipids or contributes to lipid formation. Suitable lipid
agents which can be used in the supplement and the medium of the
present invention include, but are not limited to, Human
Ex-Cyte.RTM. (Bayer), ethanolamine (or a salt thereof), sitosterol
(a plant steroid), rice hydrolysate (a mixture of proteins and
lipids), LTI Defined Lipid Mixture, a mixture of arachidonic acid,
cholesterol, DL-.alpha.-tocopherol-acetate, ethyl alcohol, linoleic
acid, linolenic acid, myristic acid, oleic acid, palmitric acid,
palmitic acid, Pluronic.RTM. F-68, stearic acid, and Tween.RTM. 80.
Preferably, Human Ex-Cyte.RTM. and ethanolamine are used in the
supplement and the medium of the invention. Suitable concentrations
of a lipid agent ingredient can be determined by one of ordinary
skill in the art.
[0074] The term "ingredient" refers to any compound, whether of
chemical or biological origin, that can be used in cell culture
media to maintain or promote the growth or proliferation of cells.
The terms "component," "nutrient" and "ingredient" can be used
interchangeably and are all meant to refer to such compounds.
Typical ingredients that are used in cell culture media include
amino acids, salts, metals, sugars, lipids, nucleic acids,
hormones, vitamins, fatty acids, proteins and the like. Other
ingredients that promote or maintain growth of cells ex vivo can be
selected by those of skill in the art, in accordance with the
particular need.
[0075] By "cell culture" is meant cells or tissues that are
maintained, cultured or grown in an artificial, in vitro
environment.
[0076] By "culture vessel" it is meant glass containers, plastic
containers, or other containers of various sizes that can provide
an aseptic environment for growing cells. For example, flasks,
single or multiwell plates, single or multiwell dishes, or
multiwell microplates can be used. Further, a bioreactor can be
used to culture cells.
[0077] The terms "cell culture medium," "culture medium" and
"medium formulation" refer to a nutritive solution for culturing or
growing cells.
[0078] The terms "cultivating" and "culturing" are synonymous.
[0079] The term "container means" includes culture vessels, jars,
bottles, vials, straws, ampules, and cryotubes.
[0080] The term "feeding" or "fluid-changing" refers to replacing
the medium in which cells are cultured.
[0081] The term "combining" refers to the mixing or admixing of
ingredients in a cell culture medium formulation.
[0082] The term "contacting" refers to the mixing, adding, seeding,
or stirring of one or more cells with one or more compounds,
solutions, media, etc.
[0083] A "serum-free" medium is a medium that contains no serum
(e.g., fetal bovine serum (FBS), horse serum, goat serum,
etc.).
[0084] By "compatible ingredients" is meant those media nutrients
which can be maintained in solution and form a "stable"
combination. A solution containing "compatible ingredients" is said
to be "stable" when the ingredients do not degrade or decompose
substantially into toxic compounds, or do not degrade or decompose
substantially into compounds that cannot be utilized or catabolized
by the cells in culture. Ingredients are also considered "stable"
if degradation can not be detected or when degradation occurs at a
slower rate when compared to decomposition of the same ingredient
in a 1.times. cell culture media formulation. Glutamine, for
example, in 1.times. media formulations, is known to degrade into
pyrolidone carboxylic acid and ammonia. Glutamine in combination
with divalent cations are considered "compatible ingredients" since
little or no decomposition can be detected over time. See U.S. Pat.
No. 5,474,931.
[0085] A cell culture medium is composed of a number of ingredients
and these ingredients vary from medium to medium. Each ingredient
used in a cell culture medium has unique physical and chemical
characteristics. Compatibility and stability of ingredients are
determined by the "solubility" of the ingredients in solution. The
terms "solubility" and "soluble" refer to the ability of an
ingredient to form a solution with other ingredients. Ingredients
are thus compatible if they can be maintained in solution without
forming a measurable or detectable precipitate. Thus, the term
"compatible ingredients" as used herein refers to the combination
of particular culture media ingredients which, when mixed in
solution either as concentrated or 1.times. formulations, are
"stable" and "soluble."
[0086] A "1.times. formulation" is meant to refer to any aqueous
solution that contains some or all ingredients found in a cell
culture medium. The "1.times. formulation" can refer to, for
example, the cell culture medium of any subgroup of ingredients for
that medium. The concentration of an ingredient in a 1.times.
solution is about the same as the concentration of that ingredient
found in the cell culture formulation used for maintaining or
growing cells. Briefly, a culture medium used to grow cells is, by
definition, a 1.times. formulation. When a number of ingredients
are present (as in a subgroup of compatible ingredients), each
ingredient in a 1.times. formulation has a concentration about
equal to the concentration of those ingredients in a cell culture
medium. For example, RPMI 1640 culture medium contains, among other
ingredients, 0.2 g/L L-arginine, 0.05 g/L L-asparagine, and 0.02
g/L L-aspartic acid. A "1.times. formulation" of these amino acids,
which are compatible ingredients according to the present
invention, contains about the same concentrations of these
ingredients in solution. Thus, when referring to a "1.times.
formulation," it is intended that each ingredient in solution has
the same or about the same concentration as that found in the cell
culture medium being described. The concentrations of medium
ingredients in a 1.times. formulation are well known to those of
ordinary skill in the art. See Methods For Preparation of Media,
Supplements and Substrate For Serum-Free Animal Cell Culture, Allen
R. Liss, N.Y. (1984).
[0087] A 10.times. formulation refers to a solution wherein each
ingredient in that solution is about 10 times more concentrated
than the same ingredient in the cell culture media. RPMI 1640
media, for example, contains, among other things, 0.3 g/L
L-glutamine. A "10.times. formulation" may contain a number of
additional ingredients at a concentration about 10 times that found
in the 1.times. culture media. As will be apparent, "25.times.
formulation," "50.times. formulation," and "100.times. formulation"
designate solutions that contain ingredients at about 25, 50 or 100
fold concentrations, respectively, as compared to a 1.times. cell
culture media.
[0088] The term "inorganic salt" refers to a salt of Ca.sup.2+,
K.sup.+, Mg.sup.2+, Na.sup.+, CO.sub.3.sup.2-, PO.sub.4.sup.3-. Any
salt of each of these moieties can be used to make the medium of
the present invention. For example, the following salts can be
used: CaCl.sub.2, KCL, KNO.sub.3, MgSO.sub.4, NaCl, NaHCO.sub.3,
and NaH.sub.2PO.sub.4water.
[0089] Examples of concentrations of compounds containing
Ca.sup.2+, K.sup.+, Mg.sup.2+, Na.sup.+, CO.sub.3.sup.2-, and
PO.sub.4.sup.3- are as follows. In the 1.times. medium of the
present invention, the concentration of Ca.sup.2+ is about 0.3 to
about 140 mg/L, the concentration of K.sup.+ is about 0.5 to about
250 mg/L, the concentration of Mg.sup.2+ is about 10 to about 100
mg/L, the concentration of Na.sup.+ is about 1200 to about 3700
mg/L, the concentration of CO.sub.3.sup.2- is about 70 to about
2900 mg/L, and the concentration of PO.sub.4.sup.3- is about 7 to
about 500 mg/L. In a preferred embodiment, the concentration of
Ca.sup.2+ is about 45 mg/L, the concentration of K.sup.+ is about
170 mg/L, the concentration of Mg.sup.2+ is about 20 mg/L, the
concentration of Na.sup.+ is about 1900 m/L, the concentration of
CO.sub.3.sup.2- is about 2100 mg/L, and the concentration of
PO.sub.4.sup.3- is about 90 mg/L.
[0090] The term "energy source" refers to a carbohydrate source.
Suitable energy sources which can be used in the supplement and the
medium of the present invention include D-fructose, D-mannose, and
D-galactose. Preferably, the energy source used is D-glucose.
[0091] The term "buffering agent" refers to an agent which acts to
stabilize the hydrogen ion concentration and therefore the pH of a
solution by neutralizing, within limits, both acids and bases.
Suitable buffering agents which can be used in the supplement and
the medium of the present invention include
N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES),
.beta.-glycerol-phosphate, and bicarbonate buffer. Preferably, in
the supplement and the medium of the present invention, HEPES is
used.
[0092] The term "amino acid" refers to amino acids or their
derivatives (e.g., amino acid analogs), as well as their D- and
L-forms. Examples of such amino acids include glycine, L-alanine,
L-asparagine, L-cysteine, L-aspartic acid, L-glutamic acid,
L-phenylalanine, L-histidine, L-isoleucine, L-lysine, L-leucine,
L-glutamine, L-arginine, L-methionine, L-proline, L-hydroxyproline,
L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine.
[0093] "CD34.sup.+ hematopoietic cells" or "CD34.sup.+ cells" are
hematopoietic cells which express the CD34.sup.+ surface marker
protein. Such cells include but are not limited to hematopoietic
stem cells, myeloid progenitor or precursor cells, erythroid
progenitor or precursor cells, and lymphoid progenitor or precursor
cells.
[0094] A preparation of cells enriched for CD34.sup.+ cells can be
obtained from bone marrow, peripheral blood and neonatal cord blood
using methods that are well known by those of ordinary skill in the
art. Various systems are available to those of ordinary skill in
art. For example, the MicroCELLector System.RTM. (Applied Immune
Sciences), the MiniMacs System (Miltenvi Biotec), the StemSep.TM.
system (StemCell Technologies) can be used can be used to isolate
CD34.sup.+ cells. To prepare a preparation of cells enriched for
CD34.sup.+ cells on a larger scale, systems marketed by Baxter
Healthcare and CellPro are available to those of ordinary skill in
the art.
[0095] The terms "hematopoietic stem cell" and "pluripotent
hematopoietic stem cell" refer to a cell which can give rise to any
type of hematopoietic progenitor or precursor cell, including
myeloid progenitor or precursor cells, erythroid progenitor or
precursor cells, and lymphoid progenitor or precursor cells.
Hematopoietic stem cells display a CD34.sup.+/CD33.sup.-/CD38.sup.-
phenotype or a CD34.sup.+/HLD-DR.sup.-/CD38.sup.- phenotype (Daley,
J. P. et al., Focus 18:62-67 (1996); Pimentel, E., Ed., Handbook of
Growth Factors Vol. III: Hematopoietic Growth Factors and
Cytokines, pp. 1-2, CRC Press, Boca Raton, Fla., 1994).
[0096] A "population" of cells refers to any number of cells
greater than one.
[0097] The terms "recombinant CD34.sup.+ hematopoietic cell" refers
to a CD34.sup.+ hematopoietic cell into which a nucleic acid
molecule has been introduced and has become stably maintained. The
nucleic acid molecule can contain a drug resistance gene which aids
in the selection of recombinant CD34.sup.+ hematopoietic cells.
After introduction of the nucleic acid molecule and clonal drug
selection, ES clones are analyzed by either PCR or Southern
blotting methods to verify correct gene targeting.
[0098] The term "nucleic acid construct" refers to a nucleic acid
molecule which contains a sequence which encodes a protein of
interest. Preferably, the nucleic acid construct is an expression
vector which contains the nucleic acid encoding the protein of
interest operably linked to an expression control sequence (i.e., a
promoter and/or an enhancer, regulatory sequences to which gene
regulatory proteins bind and exert control over gene
transcription). Expression vectors which may be used are well known
to those of ordinary skill in the art.
[0099] The term "basal medium" refers to any medium which is
capable of supporting growth of CD34.sup.+ hematopoictic cells, or
other cells, when supplemented either with serum or with the
serum-free supplement of the present invention. The basal medium
supplies standard inorganic salts, such as zinc, iron, magnesium,
calcium and potassium, as well as trace elements, vitamins, an
energy source, a buffer system, and essential amino acids. Basal
media which can be used in the present invention include but are
not limited to Iscove's Modififed Dulbecco's Medium, RPMI 1640,
Minimal Essential Medium-.alpha. (MEM-.alpha.), and other media
disclosed infra. In a preferred embodiment, the basal medium is
Iscove's Modififed Dulbecco's Medium.
[0100] The terms "serum-free culture conditions" and "serum-free
conditions" refer to cell culture conditions that exclude serum of
any type.
[0101] The present invention provides a serum-free, eukaryotic cell
culture medium supplement comprising 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.sup.+ hematopoictic cells in
serum-free culture.
[0102] The present invention also specifically provides a
serum-free, eukaryotic cell culture medium supplement comprising
one or more ingredients selected from the group consisting of
N-acetyl-L cysteine, human serum albumin, Human Ex-Cite.RTM.,
ethanolamine HCl, human zinc insulin, human iron saturated
transferrin, Se.sup.4+, hydrocortisone, D,L-tocopherol acetate, and
2-mercaptoethanol.
[0103] The concentration ranges within which ingredients are
believed to support the growth of CD34.sup.+ hematopoietic cells
are listed in Tables 1-3. These ingredients can be combined to form
the cell culture medium supplement of the present invention. As
will be readily apparent to one of ordinary skill in the art, the
concentration of a given ingredient can be increased or decreased
beyond the range disclosed and the effect of the increased or
decreased concentration can be determined using only routine
experimentation.
[0104] The supplement of the present invention is the
StemPro-34.TM. Nutrient Supplement. The quantitative description of
this formulation, including final concentrations of the
ingredients, is shown in Table 1. The supplement of the present
invention can be concentrated (infra). Preferably, the supplement
of the present invention is about a 40.times. formulation (See
Table 2). In a preferred embodiment, of the present invention, the
concentrated nutrient supplement is admixed with a 1.times. basal
medium (e.g., Iscove's Modified Dulbecco's Medium).
[0105] To make a 40.times. formulation of the Stempro-34.TM.
Nutrient supplement, Human Ex-Cyte.RTM. (Bayer Corp., Kankakee,
Ill.) is added in dropwise fashion to 25% human albumin. To this
mixture is added ethanolamine HCl, followed by sodium selenite,
hydrocortisone, and D,L-tocopherol acetate. Insulin is then
solubilized in 0.05 M HCl. The pH of the insulin solution is then
adjusted to 9.0 and is added to the mixture. Next, human
transferrin is added, followed by 2-mercaptoethanol.
[0106] N-acetyl-L-cysteine is solubilized in double-distilled
water. The pH of the N-acetyl-L-cysteine at this point is
approximately 2.0. To prevent protein denaturation, the pH of the
N-acetyl-L-cysteine is adjusted to 7.0 with 5N sodium hydroxide and
is added to the mixture. The entire mixture is then filtered
through a 0.2 micron low protein binding filter.
[0107] The medium of the present invention is StemPro.TM.-34
serum-free medium (SFM). The quantitative description of this
formulation, including final concentrations of the ingredients
after addition to a basal medium to form a 1.times. formulation, is
shown in Table 3.
[0108] Preferably, the supplement of the present invention is
stored at about 4.degree. C. and most preferably at about
-20.degree. C., although the supplement may be stored at lower
temperatures (e.g., about -80.degree. C.). Preferably, the medium
of the present invention is stored at about 4.degree. C. When the
medium of the present invention includes the ingredient
N-acetyl-L-cysteine, the medium was stable after having been stored
for greater than eighteen months at 4.degree. C. Thus, even after
greater than eighteen months of storage, the medium of the present
invention supports the expansion of CD34.sup.+ hematopoietic
cells.
[0109] As will be apparent to one of ordinary skill in the art, the
ingredients may react in solution. Thus, the present invention
encompasses the formulations disclosed in any of Tables 1-3 as well
as any reaction mixture which forms after the ingredients in any
one of Tables 1-3 are combined.
[0110] The concentrations of the supplement ingredients in Table 3
are obtained when 24.8 mL of the 40.times. supplement formulation
(Table 2) are diluted in one liter of basal medium. Alternatively,
the concentrations of supplement ingredients shown in Table 3 can
be obtained by making the medium of the present invention as a
complete formulation (rather by than adding the supplement to a
basal medium).
[0111] The supplement or the medium of the present invention can be
in liquid form or can be maintained in dry form. The type of liquid
carrier and the method used to dissolve the ingredients into
solution vary and can be determined by one of ordinary skill in the
art with no more than routine experimentation. In general, the
liquid carrier is water.
[0112] The supplement or the medium of the present invention can be
made as a concentrated formulation (greater than 1.times. to
1000.times.) or as a 1.times. formulation. Preferably, the
solutions comprising ingredients are more concentrated than the
concentration of the same ingredients in a 1.times. media
formulation. For example, the ingredients can be 10 fold more
concentrated (10.times. formulation), 25 fold more concentrated
(25.times. formulation), 50 fold more concentrated (50.times.
concentration), or 100 fold more concentrated (100.times.
formulation). In particular, the supplement or the medium of the
present invention can be made by dividing the ingredients into
compatible, concentrated subgroups. See U.S. Pat. No.
5,474,931.
[0113] If the ingredients of the supplement or the medium are
prepared as separate concentrated solutions, an appropriate
(sufficient) amount of each concentrate is combined with a diluent
to produce a less concentrated formulation or a 1.times.
formulation. Typically, the diluent for the subgroups used is water
but other solutions including aqueous buffers, aqueous saline
solution, or other aqueous solutions may be used according to the
invention.
TABLE-US-00001 TABLE 1 StemPro .TM.-34 Nutrient Supplement
Formulation Concentration Range A Preferred in 1X Medium (mg/L)
Embodiment (mg/L) Ingredient (About) (About) Human Serum Albumin
1000-15,000 5000 Human Ex-Cyte .RTM. 1-15 5.0 Ethanolamine 1-25 10
Sodium Selenite 0.0001-0.01 0.005 Hydrocortisone 0.003-0.07 0.04
D,L-Tocopherol 0.005-0.05 0.02 Iron Saturated Human 10-500 100
Transferrin Human Zinc Insulin 1-25 10 N-acetyl-L-cysteine 16-660
160 2-Mercaptoethanol 2-8 4
TABLE-US-00002 TABLE 2 StemPro .TM.-34 Nutrient Supplement
Formulation (40X) Concentration (mg/L) Ingredient (About) Human
Serum Albumin 200,000 Human Ex-Cyte .RTM. 200
Ethanolamine.cndot.HCl 400 Sodium Selenite 0.2 Hydrocortisone 2
D,L-Tocopherol 0.8 Iron Saturated Human 4 Transferrin Human Zinc
Insulin 400 N-acetyl-L-cysteine 7 2-Mercaptoethanol 180
TABLE-US-00003 TABLE 3 StemPro .TM.-34 SFM Medium Formulation
Concentration Range A Preferred in 1X Medium Embodiment (mg/L)
(mg/L) Ingredient (About) (About) Inorganic Salts CaCl.sub.2 1-500
165 KCL 1-500 330 KNO.sub.3 0.008-0.8 0.08 MgSO.sub.4 10-500 100
NaCl 3000-9000 4500 NaHCO.sub.3 100-4000 3000
NaH.sub.2PO.sub.4.cndot.water 10-750 125 Amino Acids L-Alanine
5-250 25 L-Asparagine (free base) 5-150 25 L-Arginine HCl 10-250 84
L-Aspartic Acid 5-125 30 L-Cystine 2.cndot.HCl 1-200 90 L-Glutamic
Acid 5-500 75 Glycine 5-200 30 L-Histidine.cndot.HCl.cndot.water
5-200 42 L-Isoleucine 5-500 105 L-Leucine 25-500 105 L-Methionine
5-500 30 L-Phenylalanine 5-500 70 L-Proline 5-500 40 L-Serine 5-500
40 L-Threonine 5-500 100 L-Lysine.cndot.HCl 25-500 150 L-Tryptophan
2-100 15 L-Tyrosine (disodium salt) 25-500 100 L-Valine 5-500 95
Vitamins Biotin 0.01-1.0 0.01 D-Ca Pantothentate 0.05-10.0 4
Choline Chloride 1-150 4 Folic Acid 0.1-10.0 4.00 i-Inositol 1-75 7
Niacinamide 0.1-10.0 4 Pyridoxal.cndot.HCl 0.1-10.0 4 Riboflavin
0.01-2.0 0.4 Thiamine.cndot.HCl 0.1-10.0 4 Vitamin B.sub.12
0.001-5.0 0.001 Other Components Human Serum Albumin 1000-15,000
5000 Na.sub.2SeO.sub.3 0.001-0.01 0.02 D-Glucose 2000-9000 4500
Phenol Red 0.5-30 15 HEPES 1000-7000 6000 Sodium Pyruvate 10-300
110 Human Ex-Cyte .RTM. 1-15 5 Ethanolamine 1-25 10 Hydrocortisone
0.003-0.07 0.04 D,L-Tocopherol 0.005-0.05 0.02 Iron Saturated Human
10-500 100 Transferrin Human Zinc Insulin 1-25 10
N-acetyl-L-cysteine 16-660 160 2-Mercaptoethanol 2-8 4
[0114] The supplement or the medium or concentrated formulation of
the present invention (both aqueous and dry forms) are typically
sterilized to prevent unwanted contamination. Sterilization may be
accomplished, for example, by ultraviolet light, filtration, or
heat.
[0115] All of the ingredients of the supplement of the present
invention which are of human origin (e.g., human serum albumin,
transferrin, and Ex-Cyte.RTM.) are heat treated, prior to use, by
heating at 60.degree. C. for 10 hours.
[0116] The osmolarity of the 40.times. supplement of the present
invention can be in the range of about 200 to about 400 mOsm, and
is preferably from about 250 to about 350 mOsm. The pH of the
40.times. supplement can be in the range from about 6 to about 8,
is preferably from about 6.4 to about 7.4. The osmolarity of the
medium of the present invention can be in the range from about 200
to about 400, and is preferably from about 270 to about 310 mOsm.
The pH of the medium of the present invention can be in the range
from about 6 to about 8, and is preferably from about 6.8 to about
7.3.
[0117] Those of ordinary skill in the art are familiar with methods
for culturing hematopoietic cells. For example, guidelines for
hematopoietic cell culture are outlined in Freshney, R. I. et al.,
Eds., Culture of Hematopoietic Cells, Wiley-Liss, New York, 1994,
pp. 81-98.
[0118] The present invention also provides a kit comprising a
carrier means such as a box or carton being compartmentalized to
receive in close confinement therein one or more container means
such as vials, tubes, ampules, jars, and the like, wherein a first
container means contains the supplement of the present invention.
Optionally, a second container means contains a basal medium.
Preferably, the container containing the supplement of the present
invention can be stored from about -135 to about 4.degree. C.,
preferably from about -5 to about -80.degree. C., most preferably
from about -5 to about -20.degree. C., and still more preferably at
about -20.degree. C. A container containing the medium of the
invention is preferably stored at about 2 to about 8.degree. C.,
and most preferably at about 4.degree. C.
[0119] The present invention also provides a composition comprising
CD34.sup.+ hematopoietic cells in a serum-free medium, wherein the
serum-free medium, which is supplemented with the serum-free
supplement of the invention, is capable of supporting the growth of
the CD34.sup.+ hematopoietic cells in serum-free culture. Aliquots
of this composition can be frozen at about -80.degree. C. and
below. Aliquots of this composition can be stored indefinitely at
less than or equal to about -135.degree. C. After an aliquot of the
composition has been thawed and opened, using sterile cell culture
technique, the CD34.sup.+ hematopoietic cells can be cultivated in
serum-free culture. Animals from which CD34.sup.+ or equivalent
hematopoietic cells can be obtained include human, monkey, ape,
mouse, rat, hamster, rabbit, guinea pig, cow, swine, dog, horse,
cat, goat, and sheep. Equivalent hematopoietic cells are cells from
non-human species which are regarded by those of ordinary skill in
the art as hematopoietic stem cells, based on the expression of
surface marker proteins.
[0120] It is also routine to freeze hematopoietic cells for use at
a later date. Freezing media generally consist of 5-10% DMSO,
10-90% FBS and 55-85% eukaryotic cell culture media. The supplement
of the present invention can be used as a serum substitute for
cryopreservation and reconstitution purposes. The conditions for
cryopreservation of such cells with the supplement of the invention
include 0.5-95% supplement, 1-10% of a cryoprotectant (e.g.,
dimethylsulfoxide (DMSO)), and 1-90% of a basal medium. CD34.sup.+
hematopoietic cells can be frozen under such conditions at about
-80.degree. C. and below. CD34.sup.+ hematopoietic cells can remain
frozen indefinitely at temperatures less than or equal to about
-135.degree. C.
[0121] The medium of the present invention can be supplemented with
growth factors or other cytokines in order to facilitate the
expansion and/or the differentiation of CD34.sup.+ hematopoietic
cells. CD34.sup.+ hematopoietic cells can be incubated with
specific factors in order to facilitate expansion or to induce
differentiation of the CD34.sup.+ hematopoietic cells into a
particular type of cell. Such factors are well know to those of
ordinary skill in the art. Such factors include, but are not
limited to, interleukins, cytokines, colony stimulating factors,
growth factors, and interferons. Examples of such factors include
but are not limited to stem cell factor, an interleukin (IL) such
as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,
IL-11, IL-12, CSF, G-CSF, thrombopoietin (TPO), GM-CSF,
erythropoietin (EPO), Flt3, Flt2, PIXY 321, and leukemia inhibitory
factor (LIF). Such factors can be used to expand CD34.sup.+ stem
cells or as differentiation factors to cause stem cells to
differentiate into a different cell type.
[0122] The present invention also provides a method of providing
CD34.sup.+ hematopoietic cells to a mammal, the method comprising
contacting CD34.sup.+ hematopoietic cells with the medium of the
present invention, cultivating cells under conditions suitable to
facilitate the expansion of the cells in serum-free culture, and
introducing the expanded cells into a mammal.
[0123] The serum-free supplement of the present invention can also
be used to prepare a hematopoietic cell type of interest for
explantation into a mammal. In this embodiment, cells which have
been caused to differentiate (supra) are introduced into a mammal.
For example, CD34.sup.+ hematopoietic cells which have been caused
to differentiate into a hematopoietic stem, precursor, or
progenitor cell can be introduced into the bone marrow or the
bloodstream of the mammal. The CD34.sup.+ or differentiated cells
can be introduced into, for example, the bone marrow or bloodstream
of the mammal by well-known techniques.
[0124] Having now fully described the present invention, the same
will be more clearly understood by reference to certain specific
examples which are included herewith for purposes of illustration
only, and are not intended to be limiting of the invention.
EXAMPLE 1
Experimental Methods
[0125] In the examples that follow, unless otherwise indicated, the
methods of obtaining bone marrow cells, selecting CD34.sup.+ cells,
assaying cell proliferation, and flow cytometry assays were
performed as described in this Example.
Bone Marrow Cells
[0126] Bone marrow was obtained from a population of screened
normal donors (courtesy of Roswell Park Cancer Institute). Marrow
was aspirated from the posterior iliac crest and placed into
sterile heparinized tubes. In the laboratory, approximately 25 mL
of heparinized bone marrow was diluted with an equal volume of
Hank's Balanced Salt Solution (without calcium or magnesium) (Life
Technologies, Gaithersburg, Md.) and carefully layered over
Nycoprep.TM. 1.077 (Life Technologies) in 50 mL tubes. Samples were
then centrifuged at 800.times.g for 30 minutes at room temperature.
After centrifugation, the band of bone marrow mononuclear cells
(BMMC) at the interface was removed by pipetting. Cells were washed
once with Hank's Balanced Salt Solution (without calcium or
magnesium) and counted using a hemocytometer (using trypan blue dye
exclusion to measure cell viability).
CD34.sup.+ Cell Selection
[0127] For some experiments, CD34.sup.+ cells were separated using
the MicroCELLector.RTM. System according to the manufacturer's
instructions. Briefly, the non-adherent fraction of BMMC was
collected after incubation on Soybean Agglutinin
MicroCELLector.RTM. flasks. Cells were washed with Iscove's
Modified Dulbecco's Medium (IMDM).
[0128] For some experiments, CD34.sup.+ cells were separated using
the MiniMACS system according to the manufacturer's instructions.
Briefly, mononucleated cells were suspended at a concentration of
10.sup.8 cells in 300 .mu.l of buffer (PBS/0.5% human serum
albumin/ACD solution 1:10). Blocking reagent and antibody reagent
from the CD34 Isolation Kit were added simultaneously to the
suspension. The suspension was mixed well and allowed to incubated
at 6 to 12.degree. C. for fifteen minutes. After incubation, the
cells were washed in buffer by centrifugation at 1200 RPM for ten
minutes. The supernatant was removed and the cell pellet was
resuspended in 400 .mu.l of buffer. The colloidal syspenesion of
submicroscopic magnetic beads was added to the cells. Cells were
mixed and incubated at 6 to 12.degree. C. for fifteen minutes.
Cells were again washed by centrifugation, the supernatant was
removed, and the cells were resuspended in 500 .mu.l of buffer.
Separation columns were prepared by rinsing with 2 mL of buffer and
placed in a magnetic field. Cells were applied to the column and
eluted by gravity. CD34.sup.+ cells were retained on the column.
The column was rinsed with 2 mL of buffer. CD34.sup.+ cells were
eluted by removing the column from the magnetic field and flushing
the column with 2 mL of buffer.
CD34.sup.+ Cell Culture
[0129] For a given experiment, the CD34.sup.+ cells that were used
were cells that had been isolated by only one of the above methods.
Unless indicated otherwise, CD34.sup.+ cells were incubated at
37.degree. C. in 5% CO.sub.2/95% air atmosphere. Cells were counted
using a hemocytometer. Cell viability was assessed by the ability
of the cells to exclude trypan blue dye.
Cell Proliferation Assay
[0130] CD34.sup.+ cells were cultured for 6 to 8 days in either
StemPro-34.TM. SFM (Life Technologies) supplemented with
recombinant growth factors or IMDM supplemented with 20% fetal
bovine serum and recombinant growth factors. Unless otherwise
indicated, recombinant growth factors were added at the beginning
of the setup of a cell culture experiment.
[0131] Cells were seeded at an initial density of 2.times.10.sup.4
cells/mL (1.times.10.sup.4 cells/cm.sup.2) in 24-well culture
plates without additional feeding. At the end of the incubation
period, cells were harvested and cell expansion was determined by
hemocytometer counting. Aliquots of harvested cells were removed
for further analysis in colony forming unit assays and phenotypic
analysis by fluorescence activated cell sorting.
Flow Cytometry
[0132] Freshly selected or cultured cell populations were stained
with fluorochrome-labeled monoclonal antibodies as follows.
Aliquots of cells were incubated on ice for 15 minutes with mouse
IgG to block nonspecific binding sites. The cells were then added
to 12.times.75 mm polystyrene tubes containing either the FITC- or
PE-conjugated monoclonal antibodies. These tubes were mixed and
incubated on ice for 30 to 45 minutes. The stained cells were then
washed one time with Dulbecco's Phosphate Buffered Saline (DPBS)
and fixed with 2% formalin/DPBS. Analysis was carried out using a
FACSort apparatus (Becton Dickinson (BD)). The standard panel of
fluorochrome-conjugated antibodies was anti-CD34/HPCA-2 (BD),
anti-CD 14/MoP9 (BD), anti-CD38/T16 (Immunotech, Inc.),
anti-CD13/366 (Coulter Corp.), and anti-CD33/906 (Coulter).
EXAMPLE 2
Proliferation of CD34.sup.+-Selected Cells Under Serum-Free Culture
Conditions
[0133] In order to study the ability of CD34.sup.+ cells to expand
and differentiate under various culture conditions, CD34.sup.+
cells were selected from normal donor bone marrow. Initially, 1.3%
of the total bone marrow cells were CD34.sup.+ (Table 4), in
agreement with reported values (Cinin, C. I. et al., J. Immunol.
133:157 (1984)). The selection process enriched CD34.sup.+ cells to
64% from 1% in freshly aspirated normal bone marrow cells.
[0134] The absence of serum allows the study of the effect of
early-acting or late-acting hematopoietic growth factors on cell
expansion. Various combinations of human recombinant cytokines were
examined for their ability to support proliferation (Table 5).
Using StemPro-34.TM. SFM, it was possible to stimulate variable
degrees of cell proliferation, in the absence of the confounding
effects of serum, by altering the combinations of growth factors
employed.
[0135] In preliminary studies, stem cell factor alone did not
affect expansion under serum-free conditions (data not shown). The
addition of combinations of early-acting (e.g., SCF, IL-1, IL-3)
and mid-acting (e.g., IL-6) pleiotropic cytokines had marginal
growth-promoting effects under serum-free conditions (Table 5). The
stimulation of cell expansion required a combination of early-(SCF,
IL-3), mid-(IL-6) and late-(GM-CSF, EPO) acting differentiation
factors that play a role in hematopoiesis.
TABLE-US-00004 TABLE 4 CD34.sup.+ Population Characteristics of
Bone Marrow Aspirates From Normal Donors Total selected % Purity of
Donors Total BMMC CD34.sup.+ % Total Selected Evaluated
(.times.10.sup.6) (.times.10.sup.5) BMMC CD34.sup.+ cells
MicroCELLector .RTM. System 22 101 .+-. 12 12 .+-. 2 1.3 .+-. 0.1
64 .+-. 2 MiniMACS System 24 61 .+-. 5 18.3 .+-. 2.3 2.9 .+-. 0.3
44 .+-. 4 Data are the mean .+-. S.E.M. % purity of selected
CD34.sup.+ cells was determined by FACS analysis.
[0136] The expansion index in StemPro-34.TM. SFM, with various
cytokines, was compared to that of cells cultured in IMDM
supplemented with 20% FBS, SCF, IL-3, and GM-CSF (Table 5). This
growth factor cocktail (SCF, IL-3, and GM-CSF) is commonly used
when culturing hematopoietic cells in serum-supplemented medium
(Haycock, D. N. et al., Blood 80:1405 (1992)).
TABLE-US-00005 TABLE 5 Cytokine-Driven Proliferation of Selected
CD34.sup.+ Cells in StemPro-34 .TM. SFM Medium SCF IL-1 IL-3 IL-6
GM-SCF EPO Expansion (100 ng/mL) (10 ng/mL) (50 ng/mL) (10 ng/mL)
(25 ng/mL) (6 ng/mL) Index.sup.1 % FBS.sup.2 + - + - + - 24 100 + +
- - - - 5 19 + - - + - - 5 19 + - + - - - 7 27 + - - - + - 12 51 +
- - - - + 19 76 + + - - - + 22 90 + - + - - + 29 120 + - - - + + 30
122 + + + + + - 42 171 Data are a representative study. .sup.1The
expansion index indicates the level of amplification of the cell
population as determined by the equation: Cells at day 7 / 2
.times. 10.sup.4. .sup.2% FBS = experimental group cells at day 7 /
cells in FBS control group at day 7 .times. 100% (FBS control is
cells cultured in IMDM + 20% FBS + SCF + IL-3 + GM-CSF).
EXAMPLE 3
Kinetics of CD34.sup.+ Cell Expansion
[0137] The kinetics of cell expansion in StemPro-34.TM. SFM were
compared to the kinetics of cell expansion in serum-containing
medium. CD34.sup.+ bone marrow cells were seeded at an initial
density of 2.times.10.sup.4 cells/well in 24 well plates. Final
concentrations of human recombinant factors SCF (100 ng/mL), IL-3
(50 ng/mL) and GM-CSF (25 ng/mL) were added to either
StemPro.TM.-34 SFM or IMDM/20% FBS at setup. On the days indicated,
wells were harvested and the cells counted using a hemocytometer
and trypan blue dye exclusion. Results are depicted in FIG. 1.
[0138] In both StemPro-34.TM. SFM and the serum-supplemented
cultures, an initial lag phase (about 3 days) was observed to
precede cell expansion. This initial lag phase may reflect
experimental observations that the majority of hematopoietic stem
cells (i.e., CD34.sup.+/CD33.sup.-/CD38.sup.-) are in a quiescent
G.sub.0 state and require stimulatory signals for expansion and
subsequent differentiation (Traycoff, C. M. et al., Exp. Hematol.
22:1264 (1994)). After 10 days, there were about three times as
many cells in StemPro-34.TM. SFM, compared to the
serum-supplemented culture.
[0139] Cell-doubling times were calculated from the regression
lines of the exponential growth phase of the curves in FIG. 1. In
StemPro-34.TM. SFM, cells displayed a doubling time of 43 hours. In
the serum-supplemented medium, doubling time was 72 hours. These
doubling times reflect the total population kinetics. The cell
population is heterogeneous during exponential growth as cells are
not only growing but are also responding to differentiation
signals.
[0140] One of ordinary skill in the art will readily understand
that the supplement or the medium of the present invention, in
combination with suitable growth factors, facilitates the expansion
of hematopoietic progenitor cells (i.e., CD34.sup.+ cells) to
levels approximately 3.5-fold above those obtained when the same
combination of growth factors is added to medium supplemented with
serum. Thus, one can measure the true proliferative response to
combination of recombinant cytokines in the absence of the apparent
and undefined components present in serum.
EXAMPLE 4
Expansion of CD34.sup.+ Cells
[0141] Changes in the CD34.sup.+ cell population during cell
expansion were examined. CD34.sup.+ bone marrow cells were seeded
at 2.times.10.sup.4 cells per mL in 24 well flat bottomed plates
and culture for 6 to 8 days in either StemPro.TM.-34 SFM for
IMDM+20% FBS. All cultures were supplemented with final
concentrations of the human recombinant growth factors SCF (100
ng/mL), IL-3 (50 ng/mL) and GM-CSF (25 ng/mL) at the time of setup.
Total cell counts were determined by hemocytometer and trypan blue
dye exclusion. CD34.sup.+ cells were enumerated by FACS.
[0142] As shown in FIG. 2, in StemPro-34.TM. SFM, the CD34.sup.+
population of cells increased 3-fold and the total population of
cells increase about 22-fold. In contrast, in serum-supplemented
medium, the CD34.sup.+ population of cells actually declined by 3%,
while the total cell population increased about 13-fold. Thus, the
supplement and the medium of the present invention support the
expansion of CD34.sup.+ cells in culture. These results are the
first, to date, which demonstrate an expansion of CD34.sup.+ cells
in culture.
[0143] The CD34.sup.+ cell population in StemPro-34.TM. SFM was
analyzed for several cell surface markers (Table 6). The cocktail
of cytokines used stimulated the expansion of myeloid cells, as
demonstrated by the increase in CD13 antigen, a protein expressed
by early myeloid precursor cells.
TABLE-US-00006 TABLE 6 CD Antigen Expression On Hematopoietic
Progenitor Cells Day 6-8.sup.1 IMDM + 20% Antigen Day 0.sup.1
StemPro-34 .TM. SFM FBS CD34 64.4 .+-. 4.8 10.8 .+-. 2.6 14.9 .+-.
4.1 CD14 0.5 .+-. 0.2 1.9 .+-. 0.5 8.4 .+-. 1.7 CD33 12.5 .+-. 4.5
16.9 .+-. 7.6 27.2 .+-. 11.5 CD38 67.8 .+-. 4.1 34.8 .+-. 2.4 58.4
.+-. 4.8 CD13 40.0 .+-. 7.1 77.3 .+-. 3.6 73.7 .+-. 4.8 .sup.1Data
are percent positive cells. Data are expressed as mean .+-. S.E.M.
for N = 10.
[0144] The morphological characteristics of CD34.sup.+ cells
present after 6 days of growth in StemPro-34.TM. SFM reflect the
myeloid nature of the cells. The cytoplasm of Wright-Giemsa stained
cells displayed an intensely basophilic character, a large
nuclear-to-cytoplasmic ratio, a delicate chromatin pattern, and
clearly visible nucleoli (Daley, J. P. et al., Focus 18:62-67
(1996)).
EXAMPLE 5
Effect of Growth Factor Addition on Expansion
[0145] The advantageous effect of the supplement and the medium of
the present invention is further depicted in FIG. 3. CD34.sup.+
selected cells from normal bone marrow were plated at a density of
2.times.10.sup.4 cells per mL in StemPro.TM.-34. Human recombinant
growth factors were added to final concentrations of 100 ng/mL SCF
50 ng/mL IL-3, and 25 ng/mL GM-CSF the day (d) indicated and the
cells were incubated for 7 days. The expansion index was calculated
as (day 7 cell density/initial seeding density). The expansion
index was about 4-fold in StemPro-34.TM., as compared to IMDM
supplemented with 20% FBS.
EXAMPLE 6
Effect of Growth Factor Addition on Expansion
[0146] The results in FIG. 3 indicate that StemPro-34.TM. affords
much greater control over cell expansion. Similar results are shown
in FIG. 4. CD34.sup.+ selected cells from freshly harvested normal
bone marrow were cultured at a seeding density of 2.times.10.sup.4
cells per mL in StemPro.TM.-34 or IMDM+20% FBS. As indicated, human
recombinant growth factors were added to final concentrations of
100 ng/mL SCF 50 ng/mL IL-3, 25 ng/mL GM-CSF, and 12 units/mL EPO
at either the time of culture setup or three days after setup.
After 7 days of culturing, the cells were harvested, counted, and
aliquots of cells were stained for FACS analysis. In FIG. 4,
CD34.sup.- cells are differentiated cells that are supported by the
supplement and the medium of the invention. Such cells are red
blood cell precursors (e.g., BFU-E cells) and myeloid precursors
(e.g., CFU-GM and CFU-G cells).
EXAMPLE 7
Effect of Growth Factors on BFU-E Cell Development
[0147] Erythropoietin (EPO) and interleukin 6 (IL-6) stimulate the
differentiation of CD34.sup.+ hematopoietic cells into BFU-E cells.
Differentiation of CD34.sup.+ cells was evaluated using
StemPro-34.TM. SFM. CD34.sup.+ selected cells from normal bone
marrow were plated at a density of 2.times.10.sup.4 cells/mL (in
triplicate) in the indicated medium using a combination of human
recombinant cytokines (SCF (100 ng/mL), IL-3 (50 ng/mL), GM-CSF (25
ng/mL) IL-6 (20 ng/mL) (final concentrations)) and the
concentrations of EPO shown in the inset in FIG. 5.
[0148] After seven days of culturing, aliquots of the expanded cell
population were then plated in a methylcellulose CFU assay.
Briefly, cells to be assayed for colony-forming unit function were
diluted in IMDM prior to plating. Freshly selected CD34.sup.+ cells
were plated at a seeding density of 1.times.10.sup.3 cells/well.
After culturing for 6-8 days, cells were plated at a seeding
density of 2.times.10.sup.4 cells/well. Colonies were enumerated on
day 14 of culture for BFU-E and CFU-GM colonies using standard
methylcellulose systems (StemPro Complete Methylcellulose Medium,
Life Technologies). Data shown in FIG. 5 are for BFU-E cells.
[0149] Serum contains undefined components which promote BFU-E
colony development (Migliaccio, G. et al., Expl. Hematol. 18:1049
(1990)). Thus, the effects of EPO and IL-6 would be difficult, if
not impossible, to interpret using serum-supplemented medium.
Accordingly, it will be evident to one of ordinary skill in the art
that the supplement and the medium of the present invention
facilitate the controlled study of hematopoietic cell
differentiation.
EXAMPLE 8
Optimization of Various Supplement Ingredients
[0150] Several ingredients of the supplement and the medium of the
formulation were shown to have functional activity over a range of
different concentrations. These components included transferrin,
human serum albumin, Human Ex-Cyte.RTM., 2-mercaptoethanol and
N-acetyl-L-cysteine.
A. Transferrin
[0151] Normal human bone marrow cells were cultured in
StemPro-34.TM. SMF medium, to which transferrin was added to the
final concentrations indicated in FIG. 6. After six days in
culture, cells were harvested and counted. A concentration of 100
.mu.g/mL human transferrin proved optimal. As indicated, however,
other concentrations of human transferrin support cell growth.
B. Human Ex-Cyte.RTM.
[0152] CD34.sup.+ cells were cultured in Stem-Pro-34.TM. SFM to
which various lipid/fatty acid mixtures were added to the final
concentrations indicated in FIG. 7. After 6-7 days in culture,
cells were harvested and counted.
[0153] The combination of lipids and fatty acids, as supplied by
Human Ex-Cyte.RTM., worked optimally when Human Ex-Cyte.RTM. was
present in the range of 5 mg/L (5 .mu.g/mL) as shown in FIG. 7.
Human Ex-Cyte.RTM. worked as well as a combination of LDL's and
lipids, supplied as "Intralipids" by Kabi Pharmaceuticals.
C. 2-Mercaptoethanol
[0154] CD34.sup.+ cells were cultured in StemPro-34.TM. SFM, with
or without 0.05 mM 2-mercaptoethanol (final concentration). After
6-7 days in culture, cells were harvested and counted. The results
are shown in FIG. 8. The antioxidant 2-mercaptoethanol used at a
final concentration of 4.34 mg/L enhanced performance approximately
2 fold.
D. Human Serum Albumin Concentration
[0155] CD34.sup.+ cells were cultured in StemPro-34.TM. SFM, to
which the final concentrations of human serum albumin and Human
Ex-Cyte indicated in FIG. 9 were added. After 6-7 days in culture,
cells were harvested and counted. In a preferred embodiment of the
present invention, human serum albumin, is employed at a final
concentration of 5 mg/L when Human Ex-Cyte.RTM. is used at a
concentration of 5 mg/L.
EXAMPLE 9
Identification of Requirement for N-acetyl-L-cysteine for Long-Term
Performance
[0156] Ammonia is a moiety which is toxic to hematopoietic cells.
In culture, ammonia takes the form of the ammonium ion
(NH.sub.4.sup.+). As shown in FIG. 10, ammonium ion breaks down
spontaneously in liquid formulations. The effect of ammonium ion
concentration on cell expansion was evaluated. CD34.sup.+ cells
were seeded at an initial density of 2.times.10.sup.4 cells/well,
in duplicate, in 24 well flat bottomed plates. Media used in these
experiments were freshly made and ammonium was titrated into the
medium at the indicated concentrations. All wells were then
supplemented with human recombinant SCF to 100 ng/mL, IL-3 to 50
ng/mL, and GM-CSF to 25 ng/mL (final concentrations). After 6 days
in culture, cells were harvested and the ammonium concentration was
determined using a calibrated ammonium probe.
[0157] Further, as shown in FIG. 11, the cells themselves generate
even more ammonium over time. CD34.sup.+ cells were seeded at an
initial density of 2.times.10.sup.4 cells/well, in duplicate, in 24
well flat bottomed plates. At the indicated time intervals,
aliquots of medium were analyzed for ammonium using a calibrated
ammonium probe. Aliquots of medium were also taken for cell
counting by hemocytometer.
[0158] Further, oxidative species are generated by interaction of
light with light-sensitive vitamins. Moreover, oxidation of
reactive lipids and fatty acids leads to a time-dependent decline
in the ability of serum-free media to support the growth and
expansion of hematopoietic cells in culture. Thus, the shelf-life
of any medium is severely limited. An advantage provided by the
present invention is the long shelf-life of the supplement and the
medium of the present invention. The present inventors have
discovered that addition of an antioxidant (preferably,
N-acetyl-L-cysteine or its derivatives) to the supplement or the
medium of the present invention results in a dramatically
lengthened shelf life.
[0159] Medium formulated without N-acetyl-L-cysteine shows a marked
decline in performance at about weeks 12-16 after its manufacture.
As can be seen from FIG. 12, the addition of N-acetyl-L-cysteine to
an "aged," inactivated medium improves the performance of the
medium. CD34.sup.+ selected cells from normal bone marrow were
plated at a density of 2.times.10.sup.4 cells/mL in StemPro-34.TM.
SFM formulated with or without N-acetyl-L-cysteine at the times
indicated in FIG. 12. All cultures were supplemented with the
recombinant human growth factors SCF (100 ng/mL), IL-3 (50 ng/mL),
and GM-CSF (25 ng/mL) (final concentrations).
[0160] In a preferred embodiment of the present invention, a final
concentration of addition of 1.0 mM (164 mg/L) N-acetyl-L-cysteine
is used. However, as the results in FIG. 13 indicate, that
N-acetyl-L-cysteine increase the performance of the medium over a
range of concentrations. Inclusion of N-acetyl-L-cysteine in the
supplement or the medium of the present invention, at the time the
supplement or the medium is formulated, increases the shelf-life of
the supplement and the medium.
EXAMPLE 10
[0161] The supplement of the present invention can be used with any
of a number of different basal media to culture hematopoietic
cells. CD34.sup.+ selected cells from normal bone marrow were
plated at a density of 2.times.10.sup.4 cells/mL in RPMI 1640,
MEM-.alpha., IMDM, Medium 199, NCTC 109, or Ham's F10 media. Each
of these media can be purchased from Life Technologies, Inc. All
cultures were supplemented with the recombinant human growth
factors SCF (100 ng/mL), IL-3 (50 ng/mL), and GM-CSF (25 ng/mL)
(final concentrations). As shown in FIG. 14, a number of media
formulations can be used with the supplement of the present
invention to support the growth and expansion of CD34.sup.+
cells.
[0162] All publications, patent applications, and patents are
herein incorporated by reference to the same extent as if each
individual publication, patent application, or patent was
specifically and individually indicated to be incorporated by
reference.
[0163] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims.
* * * * *