Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Animal or plant cell
Reexamination Certificate
2001-02-09
2002-04-16
Marx, Irene (Department: 1651)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Animal or plant cell
C435S385000, C435S384000, C435S404000, C435S405000, C435S407000
Reexamination Certificate
active
06372210
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a serum-free medium which can support the growth and proliferation of normal human hematopoietic CD34
+
cells purified from sources such as normal human bone marrow, the peripheral blood of patients treated with cytokines (termed mobilized CD34
+
cells) or umbilical cord blood.
The growth of these cells is becoming more important in view of recent developments in clinical regimens for combatting diseases such as cancer, myeloproliferative diseases and autoimmune diseases. However, many media are not suitable for culturing normal bone marrow cells, especially CD34
+
cells because of their high proliferative capability. Therefore, a need exists for developing a serum-free medium which can support the proliferation and differentiation of CD34
+
cells.
SUMMARY OF THE INVENTION
Since not all cells will proliferate in a single (universal) serum-free medium, care must be taken in the development of each medium. Such a medium is invaluable in the expansion of specific hematopoietic lineages for bone marrow transplantation. Such a medium will allow the potential to store small amounts of bone marrow or subsets of the bone marrow cell population (such as by freezing) and at a later time expand the cells by thawing the cells and growing them in vitro for transplantation purposes. The inventor has developed such a medium that can support CD34
+
cellular proliferation and in the presence of the appropriate cytokine(s), expand specific cell types/lineages. An advantage to this medium is that it contains components derived from U.S. Pharmaceutical grade components that will permit it to be used in clinical regimens.
It is one object of the invention to provide a serum-free medium comprising a basal medium, an effective amount of essential fatty acid, an effective amount of cholesterol, transferrin in an amount of 120 to 500 &mgr;g/ml or an effective amount of an iron salt and an effective amount of insulin growth factor, wherein said medium supports the proliferation and differentiation of normal CD34
+
cells or comprising a basal medium, an effective amount of fatty acid, an effective amount of cholesterol, an effective amount of transferrin or an effective amount of an iron salt and insulin in an amount of 0.25 to 2.5 U/ml or an effective amount of insulin like growth factor, wherein said medium supports the proliferation and differentiation of normal CD34
+
cells.
It is another object of the invention to provide a serum-free medium comprising a serum-free culture medium which supports the proliferation and differentiation of CD34
+
cells which comprises an effective amount of human serum albumin, transferrin in an amount of 130 to 500 &mgr;g/ml and insulin in an amount of 0.25 to 2.5 U/ml, wherein said human serum albumin, transferrin, and insulin are each present in an amount effective for supporting the proliferation and differentiation of CD34
+
cells.
It is another object of the invention to provide a method for growing normal CD34
+
cells which comprises cultivating said cells in one of the above defined media or in a serum-free medium comprising: human serum albumin; transferrin; and insulin, wherein each of said human serum albumin, transferrin and insulin is dissolved in a serum-free basal medium.
Various growth factors and/or cytokines for driving proliferation and differentiation of the cells can optionally be added to the medium used to culture the cells. By means of adding various cytokines, the composition of the cell population can be altered with respect to the types of cells present in the population.
DETAILED DESCRIPTION OF THE INVENTION
The term “serum-free” is used herein to mean that all whole serum is excluded from the medium. Certain purified serum components, such as human serum albumin, can be added to the medium.
Basal Medium
The basal medium is preferably Iscove's modified Dulbecco's medium (IMDM). Other such basal media might be used, such as McCoy's 5a or a blend of Dulbecco's modified Eagle's Medium and Ham's-F12 media at a 1:1 ratio. The requirements of the basal medium are that it provide i) inorganic salts so as to maintain cell osmolality and mineral requirements (e.g., potassium, calcium, phosphate, etc.), ii) essential amino acids required for cell growth, that is, amino acids not made by endogenous cellular metabolism, iii) a carbon source which can be utilized for cellular energy metabolism, typically glucose, and iv) various vitamins and co-factors, such as riboflavin, nicotinamide, folic acid, choline, biotin, and the like, as my be required to sustain cell growth. Glutamine is one of the amino acids which may be added to the medium of the present invention in an effective amount. The glutamine concentration is usually between 100 and 500 &mgr;g/ml, preferably between 125 and 375 &mgr;g/ml and most preferably between 150 and 300 &mgr;g/ml. Because of its instability, glutamine is sometimes added just before use of the media.
The basal medium also typically contains a buffer to maintain the pH of the medium against the acidifying effects of cellular metabolism, usually bicarbonate or HEPES. The pH of the basal medium is usually between 6.8 and 7.2. The composition of IMDM is shown in Table I, below:
TABLE I
Iscove's Modified Dulbecco's Medium
Component
mg/L
L-Alanine
25.0
L-Arginine HCl
84.0
L-Asparagine · H
2
O
28.40
L-Aspartic Acid
30.0
L-Cystine · 2HCl
91.24
L-Glutamic Acid
75.0
L-Glutamine
584.0
Glycine
30.0
L-Histidine HCl · H
2
O
42.0
L-Isoleucine
104.8
L-Leucine
104.8
L-Lycine HCl
146.2
L-Methionine
30.0
L-Phenylalanine
66.0
L-Proline
40.0
L-Serine
42.0
L-Threonine
95.2
L-Tryptophan
16.0
L-Tyrosine, 2Na · 2H
2
O
103.79
L-Valine
93.6
Biotin
0.013
D-Ca Pantothenate
4.00
Choline Chloride
4.00
Folic Acid
4.00
i-Inositol
7.00
Nicotinamide
4.00
Pyridoxal HCl
4.00
Riboflavin
0.40
Thiamine HCl
4.00
Vitamin B
12
0.013
Antibiotics
omitted
2-a-Thioglycerol (7.5E-5M)
omitted
CaCl
2
· 2H
2
O
215.86
KCl
330.0
KNO
3
0.076
MgSO
4
(anhyd)
97.67
NaCl
4505.
NaH
2
PO
4
108.69
Na
2
SeO
3
· 5H
2
O
0.0173
Glucose
4500.
Phenol Red · Na
15.34
Sodium Pyruvate
110.0
NaHCO
3
3024.
HEPES 25 mM
5958.
CO
2
(The air in the jar over the
5%
medium contains 5% CO
2
and air)
Albumin
Albumin is preferably supplied in the form of human serum albumin (HSA) in an effective amount for the growth of cells. HSA provides a source of protein in the media. Moreover, protein acts as a substrate for proteases which might otherwise digest cell membrane proteins. Albumin is thought to act as a carrier for trace elements and essential fatty acids. HSA is greatly advantageous over protein derived from animals such as bovine serum albumin (BSA) due to the reduced immunogenic potential of HSA. The HSA may be derived from pooled human plasma fractions, or may be recombinantly produced in such hosts as bacteria and yeast, or in vegetable cells such as potato and tomato. Preferably, the HSA used in the present formulations is free of pyrogens and viruses, and is approved regulatory agencies for infusion into human patients. The HSA may be deionized using resin beads prior to use. The concentration of human serum albumin is 1-8 mg/ml, preferably 3-5 mg/ml, most preferably 4 mg/ml.
Soluble Carrier/Fatty Acid Complex
The albumin mentioned above could be substituted by a soluble carrier/essential fatty acid complex and a soluble carrier cholesterol complex which can effectively deliver the fatty acid and cholesterol to the cells. An example of such a complex is a cyclodextrin/linoleic acid, cholesterol and oleic acid complex. This is advantageous as it would allow for the replacement of the poorly characterized albumin with a well defined molecule. The use of cyclodextrin removes the need for the addition of human/animal serum albumin, thereby eliminating any trace undesired materials which the albumin would introduce into the media. The use of cyclodextrin simplifies the addition of specific lipophilic nutrients to a serum-free culture.
Th
Afremova Vera
Marx Irene
Quality Biological, Inc.
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