Tissue Culture Shared ResourceServices & Fees
- Generation of Primary Tumor Cell Lines Using Conditionally Reprogrammed Cell (CRC) Technology.
CRC allows unlimited propagation of adult mammalian epithelial cells, and offers exciting opportunities for future cancer research with a significant and rapid impact on the understanding of epithelial biology. This technology allows rapid and conditional reprogramming of normal and tumor epithelial cells to a highly proliferative state during which they maintain their original karyotypes. Rho kinase inhibitor (ROCK inhibitor) (Y-27632), in combination with fibroblast feeder cells, induces normal and tumor epithelial cells from many tissues to proliferate indefinitely in vitro, without transduction of exogenous viral or cellular genes. This reprogramming of cell growth and differentiation is conditional, thereby allowing facile in vitro manipulation of cell behavior. Removal of these conditions restores the capacity for cell differentiation within.
The TCSR provides an EBV immortalization service. Blood from study subjects is used to generate lymphoblastoid cell lines. Details of how blood is to be drawn, shipped and stored are provided here.
Contamination of cell cultures with mycoplasma can be a significant problem since there are often no overt signs of the presence of the organism and infection is an important potential source of artifacts. For this reason the TCSR offers a mycoplasma testing service. It is the policy of the TCSR that all cell lines must be tested for mycoplasma before they can be cultured in shared tissue culture rooms. The TCSR provides a mycoplasma testing service. Samples for mycoplasma testing can be dropped off at the main TCSR lab in W314 in the Research building. A detailed description of how to prepare and store your samples can be found here.
Genetic Fingerprinting Service
This service is offered as a major quality control tool to authenticate and confirm the identity of cell lines used at Lombardi. TCSR uses the commercially available Cell ID™ System (Promega). This system uses short tndem repeat (STR) analysis of specific loci in the human genome through co-amplification and three-color detection of 10 loci (nine STR loci and Amelogenin for gender identification). There are many advantages to using this system, including its powerful level of discrimination and reproducibility. The loci collectively provide a genetic profile with a random match probability of 1 in 2.92 x 109. Inclusion of the Y-specific Amelogenin among the loci allows gender determination of the cell lines, which is an additional advantage for quality control.
Cell Culture Service and Cell Banking
A culture service is provided to assist investigators. In addition, a cell banking service is provided to assist investigators who receive frequent requests for cell lines they have generated.
Liquid nitrogen storage space
The TCSR maintains several liquid nitrogen freezers in which investigators can store stocks of their own cells/reagents. Inventory records are kept by each investigator. Investigators must contact the Manager of the TCSR to request liquid nitrogen storage space.
Tissue Culture reagents
The TCSR purchases, tests, and prepares several tissue culture reagents that are made available to investigators. These reagents can be obtained on a walk-in basis at W314 or through the completion of the appropriate request form.
- Fetal calf serum
- Charcoal stripped calf serum
- Horse serum
- Coulter Counters (models Z1 and Z2) are available 24/7 in shared rooms W404 and E517. (Use for counting total cells). For instructions, click here.
- Coulter Multi-sizer (model M3) available in W314A during business hours. Key can be signed out. (Use for counting cells and cell size analysis).
- Luminometer (Geneprobe, model Leader 50) is available in W314 during business hours. Key can be signed out. (Use for test tubes, but not for 96 well plates).
- xCELLigence System available in W423. Key can be signed out.
Shared culture space
The TCSR equips, maintains and monitors shared tissue culture laboratories for use by the LCCC researchers. Each shared lab is equipped with class II laminar flow hoods and several CO2 incubators. All the necessary ancillary equipment, such as inverted microscopes, Coulter counters and centrifuges are available in these laboratories. A quarantine room (W314A) where investigators can grow cells that they have obtained from collaborators, from publicly or privately available collections, or from the operating theater, until the cells are verified to be free from mycoplasma. This laboratory contains a class II hood, a double chamber CO2 incubator, a Coulter counter, and a microscope. Please contact the Manager of the TCSR to learn how to make use of this service.
Cancer cell line repository
The TCSR manages the Lombardi's cell line repository, which includes an extensive collection of normal and tumor cell lines (animal and human) stored in a series of liquid nitrogen freezers. These are made available to investigators either as frozen vials of cells or as growing cultures. A searchable database is available to the Lombardi Comprehensive Cancer Center investigators and can be found on the H drive.
Processing, Banking and Disbursement of Blood and Other Biofluid Specimens
This service is available to enable biobanking of blood and other body fluid specimens that are obtained from subjects enrolled in various studies at Lombardi. Blood samples are processed and aliquots of red blood cells, buffy coat, serum and plasma are stored at -80°C for future usage. The TCSR follows the guidelines of the NCI Best Practices for Biospecimen Resources (http://biospecimens.cancer.gov/practices/). A barcoding system is used to label aliquots and track specimens.
Upon request, the TCSR offers a formal training in the basic principles of tissue culture with hands on experience to new laboratory personnel and trainees joining the Cancer Center. In addition, advice and assistance is provided on a walk in basis. The Resource Director gives a yearly introductory lecture about the facility during the "Resource for Cancer Research" course, which is attended by new fellows, students and staff.
The following equipment is maintained by the core and is available for use by the investigators:
A number of other services are offered. These include DNA extraction from cell lines, and the preparation of cell pellets and lysates for various purposes. In addition, the resource will provide custom tissue culture related services to investigators.