CA2114311C - Monoclonal wb b-cell line - Google Patents
Monoclonal wb b-cell lineInfo
- Publication number
- CA2114311C CA2114311C CA002114311A CA2114311A CA2114311C CA 2114311 C CA2114311 C CA 2114311C CA 002114311 A CA002114311 A CA 002114311A CA 2114311 A CA2114311 A CA 2114311A CA 2114311 C CA2114311 C CA 2114311C
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- 210000003719 b-lymphocyte Anatomy 0.000 claims abstract description 11
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- 102100026144 Transferrin receptor protein 1 Human genes 0.000 claims abstract description 8
- 101001018097 Homo sapiens L-selectin Proteins 0.000 claims abstract description 7
- 102100033467 L-selectin Human genes 0.000 claims abstract description 7
- 239000012980 RPMI-1640 medium Substances 0.000 claims abstract description 7
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- 101000777636 Homo sapiens ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 1 Proteins 0.000 claims abstract description 6
- 101001057504 Homo sapiens Interferon-stimulated gene 20 kDa protein Proteins 0.000 claims abstract description 6
- 101001055144 Homo sapiens Interleukin-2 receptor subunit alpha Proteins 0.000 claims abstract description 6
- 101000878605 Homo sapiens Low affinity immunoglobulin epsilon Fc receptor Proteins 0.000 claims abstract description 6
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- 230000004913 activation Effects 0.000 claims abstract description 3
- 238000004114 suspension culture Methods 0.000 claims abstract 2
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 claims description 9
- 241000701044 Human gammaherpesvirus 4 Species 0.000 claims description 4
- 102000005613 PAX5 Transcription Factor Human genes 0.000 claims description 4
- 108010045055 PAX5 Transcription Factor Proteins 0.000 claims description 4
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- 108050007852 Tumour necrosis factor Proteins 0.000 claims description 2
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- 210000004369 blood Anatomy 0.000 claims description 2
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- 102000004169 proteins and genes Human genes 0.000 abstract description 9
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- 108091058545 Secretory proteins Proteins 0.000 abstract 1
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- 210000003934 vacuole Anatomy 0.000 abstract 1
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- 239000012581 transferrin Substances 0.000 description 4
- 230000007774 longterm Effects 0.000 description 3
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 210000001744 T-lymphocyte Anatomy 0.000 description 2
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- 102000007238 Transferrin Receptors Human genes 0.000 description 2
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- 230000012202 endocytosis Effects 0.000 description 2
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- 239000012679 serum free medium Substances 0.000 description 2
- 230000002459 sustained effect Effects 0.000 description 2
- 108010027122 ADP-ribosyl Cyclase 1 Proteins 0.000 description 1
- 102000018667 ADP-ribosyl Cyclase 1 Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 108010008286 DNA nucleotidylexotransferase Proteins 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108091006975 Iron transporters Proteins 0.000 description 1
- 102000003729 Neprilysin Human genes 0.000 description 1
- 108090000028 Neprilysin Proteins 0.000 description 1
- 239000004809 Teflon Substances 0.000 description 1
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- 230000001640 apoptogenic effect Effects 0.000 description 1
- 238000003782 apoptosis assay Methods 0.000 description 1
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- 210000000601 blood cell Anatomy 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 210000004524 haematopoietic cell Anatomy 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 230000005522 programmed cell death Effects 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
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- 238000011105 stabilization Methods 0.000 description 1
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- 238000004627 transmission electron microscopy Methods 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0635—B lymphocytes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
- C12N2500/95—Protein-free medium and culture conditions
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Hematology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
A human B-cell line (designated WB) was selectively cloned from peripheral blood of a healthy 48 year-old Chinese male by use of a serum-free defined medium. The in vivo pre-activated B-cells required an 8-week "incubation period" before large clusters of cells with apical cytoplasmic projections directed radially outwards became apparent, and their gradual proliferation was observed. Within a week, sufficient cell clusters predominate to allow a 1:2 split twice weekly. Phase-contrast microscopy showed individual cells as 10u to 20u fusiforms with abundant phase-dense granules. By electron microscopy, each cell revealed numerous inclusion bodies, mitochondria, stratified rough endoplasmic reticulum (RER), carbohydrate inclusions, and a heterochromatic thick-rimmed, irregular large nucleus containing a prominent nucleolus. These EBV-negative WB cells expressed the CD19 and CD20 pan-B antigens, IgG, and activation markers DR, DQ, Leu 8, CD23, CD25, CD38, and CD71.
After 6 months of subculture, the cell line was adapted to culture in a protein-free RPMI
1640 medium. Interestingly, the cells could sustain autonomous growth and replication in semi-suspension culture in the absence of any exogenous protein. Under electron microscopy, these protein-independent cells exhibited ringed nuclei, frequent binucleism, dense elongated mitochondria, and carbohydrate inclusions. In addition, there was abrogation of DR, DQ, Leu 8, CD23, CD25, CD38, and CD71. The conditioned medium (CM) from either the serum-free or the protein-free WB culture contained two novel proteins, namely 21 and 53 kilodaltons (Kd) moeities with respective necrotic and growth bioactivity. Hence, availability of this cell line would allow for easy purification of these important secretory proteins, and for the production of human monoclonal antibodies. Also, further study of the cell line may provide the answer as to how cancer cells could gain autonomy in a milieu of apoptosis.
DESCRIPTION OF FIGURES
Figure 1.
Transmission-electron micrograph of a representative WB cell showing inclusion bodies, stratified rough endoplasmic reticulum (RER), and a heterochromatic thicked-rimmed, irregular large nucleus with a prominent nucleolus. Magnification: 26,000X.
Figure 2.
Transmission-electron micrograph of a ringed nucleus surrounding a central vacuole displayed by an occasional protein-independent WB cell. Magnification: 26,000X.
Figure 3.
Transmission-electron micrograph of a representative binucleism shown by several protein-independent WB cells. Magnification: 26,000X.
Figure 4.
Transmission-electron micrograph of dense elongated mitochondria and inclusion bodies found in the protein-independent WB cell. Magnification: 26,000X.
After 6 months of subculture, the cell line was adapted to culture in a protein-free RPMI
1640 medium. Interestingly, the cells could sustain autonomous growth and replication in semi-suspension culture in the absence of any exogenous protein. Under electron microscopy, these protein-independent cells exhibited ringed nuclei, frequent binucleism, dense elongated mitochondria, and carbohydrate inclusions. In addition, there was abrogation of DR, DQ, Leu 8, CD23, CD25, CD38, and CD71. The conditioned medium (CM) from either the serum-free or the protein-free WB culture contained two novel proteins, namely 21 and 53 kilodaltons (Kd) moeities with respective necrotic and growth bioactivity. Hence, availability of this cell line would allow for easy purification of these important secretory proteins, and for the production of human monoclonal antibodies. Also, further study of the cell line may provide the answer as to how cancer cells could gain autonomy in a milieu of apoptosis.
DESCRIPTION OF FIGURES
Figure 1.
Transmission-electron micrograph of a representative WB cell showing inclusion bodies, stratified rough endoplasmic reticulum (RER), and a heterochromatic thicked-rimmed, irregular large nucleus with a prominent nucleolus. Magnification: 26,000X.
Figure 2.
Transmission-electron micrograph of a ringed nucleus surrounding a central vacuole displayed by an occasional protein-independent WB cell. Magnification: 26,000X.
Figure 3.
Transmission-electron micrograph of a representative binucleism shown by several protein-independent WB cells. Magnification: 26,000X.
Figure 4.
Transmission-electron micrograph of dense elongated mitochondria and inclusion bodies found in the protein-independent WB cell. Magnification: 26,000X.
Description
SPECIFICATION
This invention encompasses the clonal selection of a normal B-cell line, designated WB, upon long-term culture in a serum-free defined medium. This innovative procedure was based upon the upregulation of the transferrin receptor (CD71) by pre-activated blood cells for the endocytosis of transferrin, a source of organic iron. It was achleved by applying selective pressures for survival of pre-activated peripheral blood mononuclear cells (PBMC) through the use of a standard tissue culture medium cont~in;ng 1% insulin, transferrin, and albumin respectively, as protein-source. By means of such a serum-free defined medium, isolated PBMC were subjected to long-term culture, and after an initial 8-week attrition of all other PBMC except pre-activated B-cells which sustained proliferation, these B-cells were confirmed by phenotype. Continuous subcloning of surviving B-cells resulted in establishment of the monoclonal WB cell line. The cell line was negative for the Epstein-Barr virus (EBV) despite the donor's serum being positive for anti-EBV antibody. Under phase-contrast microscopy, individual cells were 10u to 20u fusiforms with cytoplasmic projections and phase-dense granules. By transmission electron microscopy (TEM), each representative cell (Figure 1) displayed numerous inclusion bodies, stratified rough endoplasmic reticulum (RER), and a heterochromatic thick-rimmed, irregular large nucleus with a prominant nucleolus. The WB cell line exhibited immunoglobulin (Ig) G with Kappa (K) light-chain, the CD19 and CD20 pan-B antigens, and activation markers DR, DQ, Leu 8, CD23, CD25, CD38, and CD71. Expression of both Leu 8 and CD38 antigens indicated that the cell line was highly activated with inter-cell and intra-cellular signals operational. The cell line did not express CD10, t~rm;n~l deoxynucleotidyl transferase, and the common myelomonocytic and T-cell lineage antigens. After stabilization in serum-free cultures for 6 months, the cell line was subjected to progressive adaptation into a protein-free RPMI
1640 medium. Surprisingly, the WB cells maintained normal growth and replication amongst apoptotic ones in this stringent environment for more than 6 months, a feat yet achieved by other mammalian cell lines. These protein-independent cells developed ringed nuclei (Figure 2), binucleism (Figure 3), with numerous dense elongated mitochondria and inclusion bodies (Figure 4). Also, their phenotype showed abrogation of DR, DQ, Leu 8, CD23, CD25, CD38. and CD71.
~, D
211~311 Phenotypic characterization of the previously established WEE-l B-cell line cloned by manual selection of B-cell clusters from PBMC cultures grown in teflon (trade-mark) jars demonstrated the acquisition of the transferrin receptor (CD71) with long-term in vitro culture. Since endocytosis of transferrin was functional and adaptation of the WEE-1 cells to serum-free culture attainable, I exploited this capacity of the pre-activated B-cells to effect an efficient iron transporter for their selective expansion from other PBMC.
Separated PBMC were cultured at 106 per ml in 15 ml serum-free medium in duplicate tissue culture (TC) flasks incubated upright to maintain microaerophilic and microadhesive conditions. Initially, the cultures developed T-cells, and mature monocytes which adhered to the bottom of the TC flasks; after 8 weeks many buoyant B-cell clusters became apparent.
Within a week, enough clusters predominate to allow a 1:2 split twice weekly in fresh serum-free medium. After several subcultures, the monoclonal WB cell line exhibited a doubling time of approximately 30 hours, and its viability was consistently more than 95%.
Since transferrin-independent uptake of iron-organic anion chelates by a membrane-based transport system could be functional in cultured cell lines, the WB cell line was gradually weaned into a protein-free RPMI 1640 basal medium by doubling subcultures. At the onset there was enhanced proliferation, followed by cultures harbouring a few cells that underwent programmed cell death (apoptosis). This ph~nnm~nnn declined with continued subcultures, and after three months, apoptosis was min;m~l. By phase-contrast microscopy, these cells appeared more transparent than their parental counterparts; a consequence of protein deprivation in spite of sustained equivalent rates of growth and replication.
The conditioned medium (CM) obtained from either the serum-free or the protein-free WB cell culture contained two novel bioactive proteins one of which resembled the 21 kilodaltons (Kd) WEE-l blood tumour necrosis factor (BTNF or TNF-y) with similar cytotoxic function, and the other a 53 Kd growth factor. This 53 Kd B-cell specific activator protein (BSAP) enabled a number of established haematopoietic cell lines to proliferate in a protein-free medium beyond two months. The WB cell line could provide a unique resource for simple purification of such invaluable cell products, and production of human monoclonal antibodies for therapeutic use.
D
This invention encompasses the clonal selection of a normal B-cell line, designated WB, upon long-term culture in a serum-free defined medium. This innovative procedure was based upon the upregulation of the transferrin receptor (CD71) by pre-activated blood cells for the endocytosis of transferrin, a source of organic iron. It was achleved by applying selective pressures for survival of pre-activated peripheral blood mononuclear cells (PBMC) through the use of a standard tissue culture medium cont~in;ng 1% insulin, transferrin, and albumin respectively, as protein-source. By means of such a serum-free defined medium, isolated PBMC were subjected to long-term culture, and after an initial 8-week attrition of all other PBMC except pre-activated B-cells which sustained proliferation, these B-cells were confirmed by phenotype. Continuous subcloning of surviving B-cells resulted in establishment of the monoclonal WB cell line. The cell line was negative for the Epstein-Barr virus (EBV) despite the donor's serum being positive for anti-EBV antibody. Under phase-contrast microscopy, individual cells were 10u to 20u fusiforms with cytoplasmic projections and phase-dense granules. By transmission electron microscopy (TEM), each representative cell (Figure 1) displayed numerous inclusion bodies, stratified rough endoplasmic reticulum (RER), and a heterochromatic thick-rimmed, irregular large nucleus with a prominant nucleolus. The WB cell line exhibited immunoglobulin (Ig) G with Kappa (K) light-chain, the CD19 and CD20 pan-B antigens, and activation markers DR, DQ, Leu 8, CD23, CD25, CD38, and CD71. Expression of both Leu 8 and CD38 antigens indicated that the cell line was highly activated with inter-cell and intra-cellular signals operational. The cell line did not express CD10, t~rm;n~l deoxynucleotidyl transferase, and the common myelomonocytic and T-cell lineage antigens. After stabilization in serum-free cultures for 6 months, the cell line was subjected to progressive adaptation into a protein-free RPMI
1640 medium. Surprisingly, the WB cells maintained normal growth and replication amongst apoptotic ones in this stringent environment for more than 6 months, a feat yet achieved by other mammalian cell lines. These protein-independent cells developed ringed nuclei (Figure 2), binucleism (Figure 3), with numerous dense elongated mitochondria and inclusion bodies (Figure 4). Also, their phenotype showed abrogation of DR, DQ, Leu 8, CD23, CD25, CD38. and CD71.
~, D
211~311 Phenotypic characterization of the previously established WEE-l B-cell line cloned by manual selection of B-cell clusters from PBMC cultures grown in teflon (trade-mark) jars demonstrated the acquisition of the transferrin receptor (CD71) with long-term in vitro culture. Since endocytosis of transferrin was functional and adaptation of the WEE-1 cells to serum-free culture attainable, I exploited this capacity of the pre-activated B-cells to effect an efficient iron transporter for their selective expansion from other PBMC.
Separated PBMC were cultured at 106 per ml in 15 ml serum-free medium in duplicate tissue culture (TC) flasks incubated upright to maintain microaerophilic and microadhesive conditions. Initially, the cultures developed T-cells, and mature monocytes which adhered to the bottom of the TC flasks; after 8 weeks many buoyant B-cell clusters became apparent.
Within a week, enough clusters predominate to allow a 1:2 split twice weekly in fresh serum-free medium. After several subcultures, the monoclonal WB cell line exhibited a doubling time of approximately 30 hours, and its viability was consistently more than 95%.
Since transferrin-independent uptake of iron-organic anion chelates by a membrane-based transport system could be functional in cultured cell lines, the WB cell line was gradually weaned into a protein-free RPMI 1640 basal medium by doubling subcultures. At the onset there was enhanced proliferation, followed by cultures harbouring a few cells that underwent programmed cell death (apoptosis). This ph~nnm~nnn declined with continued subcultures, and after three months, apoptosis was min;m~l. By phase-contrast microscopy, these cells appeared more transparent than their parental counterparts; a consequence of protein deprivation in spite of sustained equivalent rates of growth and replication.
The conditioned medium (CM) obtained from either the serum-free or the protein-free WB cell culture contained two novel bioactive proteins one of which resembled the 21 kilodaltons (Kd) WEE-l blood tumour necrosis factor (BTNF or TNF-y) with similar cytotoxic function, and the other a 53 Kd growth factor. This 53 Kd B-cell specific activator protein (BSAP) enabled a number of established haematopoietic cell lines to proliferate in a protein-free medium beyond two months. The WB cell line could provide a unique resource for simple purification of such invaluable cell products, and production of human monoclonal antibodies for therapeutic use.
D
Claims (3)
1. (i) The Epstein-Barr virus (EBV) negative monoclonal B-cell line, designated WB, is derived from peripheral blood mononuclear cells (PBMC), (ii) that the WB cells can sustain autonomous growth and replication in semi-suspension culture in a protein-free RPMI 1640 basal medium, (iii) that the cell line secretes a 21 Kd blood tumour necrosis factor (BTNF or TNF-y), and a 53 Kd B-cell specific activator protein (BSAP), and (iv) that the cell line when maintained in protein-free RPMI 1640 medium, exhibits ringed nuclei and binucleism.
2. (i) A cell line as defined in claim 1 which expresses immunoglobulin G with Kappa lightchain, pan-B antigens (CD19 and CD20), Leu 8 cell adhesion molecule, CD38 transmembrane signal molecule, and activation markers DR, DQ, CD23, CD25, CD71, (ii) that the cell line when maintained in protein-free RPMI 1640 medium showed abrogation of DR, DQ, Leu8, CD23, CD25, CD38, and CD71.
3. A cell line as defined in claims 1 or 2 which exhibits pleiomorphic 10u to 20u fusiform cells with irregular large nuclei, and when maintained in protein-free RPMI 1640 medium exhibits numerous cytoplasmic inclusion bodies, and dense elongated mitochondria.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA002114311A CA2114311C (en) | 1991-11-25 | 1991-11-25 | Monoclonal wb b-cell line |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA002114311A CA2114311C (en) | 1991-11-25 | 1991-11-25 | Monoclonal wb b-cell line |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2114311A1 CA2114311A1 (en) | 1993-05-26 |
| CA2114311C true CA2114311C (en) | 1996-02-27 |
Family
ID=4152799
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002114311A Expired - Fee Related CA2114311C (en) | 1991-11-25 | 1991-11-25 | Monoclonal wb b-cell line |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2114311C (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6824767B2 (en) | 1994-11-07 | 2004-11-30 | Human Genome Sciences, Inc. | Tumor necrosis factor-gamma |
| US6599719B2 (en) | 1994-11-07 | 2003-07-29 | Human Genome Sciences, Inc. | Nucleic acid molecules encoding tumor necrosis factor-gamma-alpha |
| ATE364614T1 (en) * | 1994-11-07 | 2007-07-15 | Human Genome Sciences Inc | TUMOR NECROSIS FACTOR GAMMA |
-
1991
- 1991-11-25 CA CA002114311A patent/CA2114311C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CA2114311A1 (en) | 1993-05-26 |
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