C - Chemistry – Metallurgy – 12 – N
Patent
C - Chemistry, Metallurgy
12
N
C12N 5/08 (2006.01) C12N 5/06 (2006.01)
Patent
CA 2114311
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. -2-
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