VISUAL SCIENCES RESEARCH CENTER

Tissue Culture and Hybridoma Core Facility

Ram H. Nagaraj, Ph.D, Director

Departments of Ophthalmology and Pharmacology

phone #368-2089

ram.nagaraj@case.edu

Dawn Smith, B.Sc., Module Manager

Department of Ophthalmology

phone #368-0790

dawn.smith@case.edu

Denice Major, B.A., Co-Manager, Hybridoma

Department of Ophthalmology

phone #368-6385

denice.major@case.edu

Located in The VSRC Consolidated Laboratory Space in the Institute of Pathology

The Tissue Culture (TCH) Module will provide visual science investigators with facilities and technical assistance for the maintenance and propagation of ocular and other cell types. The hybridoma division is involved in the development of monoclonal antibodies to proteins and other antigens of relevance to ocular disease.

Availability of primary and immortalized cell lines from normal and diseased humans is crucial for studies on the molecular basis of ocular pathologies. Services provided by the TCH core facility, such as, development, maintenance and provision of cultured cells is a cost-effective use of manpower and resources. The broad functions of Tissue Culture Module are 1) isolation of primary cells from ocular tissue; 2) maintenance and passage of established cell lines, including storage; 3) large scale amplification of cell lines; 4) transient transfection of cells; 4) generation of stably transfected cell lines; 5) training in growth and maintenance of cells; and 6) strategic advice in planning experiments and determining their feasibility.

Specialized cell types currently stored include human and bovine retinal capillary endothelial cells, human and bovine retinal capillary pericytes, a rat retinal Muller cell line, a human retinal pigmented epithelial cell line, human and rabbit corneal epithelial cells, a rat retinal ganglion cell line, human umbilical vein and aortic endothelial cells, and mouse and human lens epithelial cells and cell lines.

Equipment & Services Available

The Tissue Culture Manager will be available to assist in the following:

• -Preparation of specialized media.
• -Isolation, purification, and characterization of the ocular cells for primary culture.
• -Subculture and propagation of established cell lines.
• -Mycoplasma testing.
• -Cryopreservation of cells.

The Hybridoma Manager will be available to assist with the following:

• -Consultation on Immunization
• -Fusion of splenocytes with myeloma cells
• -Consultation and assistance on screening clones
• -Subcloning and cryopreservation of hybridomas
• -Determine isotype of cloned hybridoma cell lines
• -Large-scale production of antibodies from cell culture supernatants
• -Consultation on purification of antibodies
• -Assistance with characterization

Equipment available in the Tissue Culture Module:

• -Analytical balance.
• -Benchtop microcentrifuge.
• -Eppendorf tabletop centrifuge.
• -4 single chamber water jacket CO2 incubators.
• -Nikon TS 100 with Fluorescence and Phase  
• -Olympus CK2 inverted phase microscope.
• -Tabletop Steam sterilizer.
• -2 biological saftey cabinets classII, type A.
• -2 cryobiological storage vessels.

A limited supply of standard consumable tissue culture quality plasticware will be available for use. Culture media, serum, growth factors, and antibiotics will be supplied by the investigator from the individual grant sources. Limited funds are available for these purposes for investigators currently without grant support. Arrangements for the use of these funds must be made with the Tissue Culture and Hybridoma Module Director.