Practice question

Mammalian antibody-secreting plasma cells are terminally differentiated, possessing an extensively expanded endoplasmic reticulum and unfolded protein response activated to sustain immunoglobulin production at up to 10,000 molecules per second, but these cells quickly trigger p53 dependent apoptosis and survive only days in vitro. To overcome limited lifespan, Georges Kohler and Cesar Milstein in 1975 created hybridoma technology that couples specificity donor with immortal partner. Splenic B lymphocytes isolated from hyperimmunized mouse provide functional heavy and light chain loci after somatic hypermutation and class switch recombination for high affinity binding. Murine myeloma lines like SP2/0-Ag14, NS0, and X63-Ag8.653 lack antibody secretion due to loss-of-function immunoglobulin genes but express constitutive telomerase, dysregulated N-Myc, loss of p16INK4a, autocrine IGF-1 signaling, and robust glycolytic metabolism supporting unlimited division in conventional media. Fusion using polyethylene glycol generates heterokaryons whose nuclei fuse, chromosome segregation yields stable hybrids secreting single idiotype. Cloning by limiting dilution at 0.3 cells per well ensures monoclonality, enabling establishment of master cell banks, GMP production, and consistent bioreactor harvests without repeated animal immunization.