Routine maintenance of cell cultures
Continuous and primary cell cultures are routinely maintained for individual investigators. Cell cultures are monitored every eight weeks for mycoplasma and endotoxin contamination using commercial testing kits. Backup stocks of cell cultures are stored in one of six liquid nitrogen storage tanks housed in our common equipment facility (E7.238). Backup stocks are rederived every six months. The PI’s laboratory has a cryopreserved cell bank containing:
- 15 murine tumor cell lines
- 20 murine hybridomas
- 6 E6/E7 transformed corneal epithelial and endothelial cell lines
- 12 human uveal melanoma cell lines
- 30 different normal human and murine cell lines
Hybridoma propagation and monoclonal antibody purification
Monoclonal antibodies are prepared from:
- Existing hybridomas obtained from other investigators
- Hybridomas obtained from American Type Culture Collection (ATCC, Rockville, MD)
- Hybridomas produced de novo by in vivo immunization of rats or mice with purified proteins, or peptides provided by participating investigators in this core facility
Our hybridoma bank contains over 20 murine hybridomas. Moreover, we have extensive experience in the purification and characterization of numerous murine monoclonal antibodies for in vitro and in vivo use. We have also generated monoclonal antibodies de novo by in vivo immunization and subsequent in vitro fusion of heterologous fusion partners from Chinese hamsters and rats, and produced IgA-secreting hybridomas de novo. Depending on the quantity of monoclonal antibodies required, hybridomas are propagated in either static flat tissue culture flasks or in roller bottle cultures. Monoclonal antibodies are purified by affinity column chromatography using protein G charged columns. The affinity purified monoclonal antibodies are dialyzed against deionized water, concentrated on a vacuum concentrator, resuspended in sterile PBS, and the protein concentration determined by BCA. The purity of the monoclonal antibodies is confirmed by western blotting with commercial anti-mouse IgG or IgM, antibodies, depending on the isotype of the monoclonal antibody.
Primary cell cultures and immortalization of ocular cell lines
Members of our Department of Ophthalmology have extensive experience in developing primary cell cultures from human and murine ocular tissues. These include human retinal pigment epithelial cells, murine iris/ciliary body cells, human and murine corneal epithelial cells, human and murine keratocytes, and human and murine corneal endothelial cells. Ocular cell lines established from various mutant, transgenic, and knockout mice have enormous potential for a wide variety of experiments. The tissue culture/hybridoma module are a valuable resource for such initiatives.
Investigators in our department, including the PI, have extensive experience in developing immortalized ocular cell lines using cells transformed with either the E6/E7 papilloma virus oncogene, the gene encoding the SV40 large T antigen, or the gene encoding telomerase.
Mycoplasma and endotoxin screening
Some investigators may wish to maintain their more fastidious cell cultures within their own laboratory, but still utilize the module’s screening service for detecting mycoplasma and endotoxin contamination. In addition to regular screening of cell cultures maintained in the core facility, we offer ad hoc screening of cell cultures that are independently maintained by investigators. Commercial kits are used to detect endotoxin and mycoplasma contamination. Cells found to be contaminated with either of these agents are destroyed and replaced with frozen stocks. Valuable cell lines that are contaminated with mycoplasma and which do not have backup frozen stocks are treated with Plasmocin (InvivoGen) to purge mycoplasma. In our hands, this has been a very effective method for eliminating mycoplasma in cell cultures.