Cell Center Services Facility is the service component of the Cell Center, provides training and services in various cell culture and associated procedures including Mycoplasma and Endotoxin testing. The cell culture service includes cell culture at various scales, large scale growth of hybridoma and other cell lines followed by antibody purification by protein G column and the generation of lymphoblastoid cell lines by EBV induced transformation of lymphocytes. In addition, the facility prepares specialized cell culture media, drosophila media, and various molecular biological reagents.
Recently cell transfection and selection service has been introduced at the facility.
Role: Technical Supervisor, Cell Center Services
Role: Research Technician, Cell Center Services
Drosophila culture in vials and bottles using the method of Spradling.
Sterile custom formulations of commercial powder bases.
Screen supernatants by ELISA for subcloning of positive clones and other purposes.
The facility trains laboratory personnel in basic sterile and tissue culture techniques.
"The facility prepares media for Drosophila culture in vials and bottles using the method of Spradling. We also make a modified version where the molasses is replaced by dextrose."
"Epstein Barr Virus (EBV) transformation is a reliable method to immortalize mammalian cells. It is most often used to obtain cell lines from human lymphocytes that serve as a permanent source for DNA isolation and protein isolation. The technique has found widespread use in clinical trials as the principal method of generating a permanent source of patient DNA for genotyping. Patient genotyping is becoming more popular as the genetic causes of diseases are being unraveled at increasing speed.
EBV is the causative agent of human infectious mononucleosis , a childhood disease. More than 90% of adults are a carrier of EBV. Adults usually show no EBV symptoms.
EBV is a Herpesvirus and its genome consists of a 172kb linear double stranded DNA which has been completely sequenced. EBV molecular biology and pathogenesis are extensively studied and the roles of many crucial EBV and host cell genes in pathogenesis are known. EBV infects only certain mammalian epithelial cells and B lymphocytes. In vitro EBV immortalizes B-cells by activating a number of cell cycle regulating genes as well as B-cell specific genes including immunoglobulin genes. Usually B-cell infections are latent and only strong stimulation can cause the lytic cycle. EBV DNA is replicated as an episomal ring and 10-20 copies per cell are typical.
IMPORTANT: When working with EBV (or any other infectious agent) negative control blood samples may NOT be obtained from laboratory personnel in order to minimize the risk of infections with one's own EBV-transformed blood cells against which the body would not be able to mount an immune response."
"We test water or samples of media to determine the level of endotoxin using the LAL (Lumilius Amebosite Lysate) gel clot assay from Associates of Cape Cod, MA. At least 200 µl samples should be submitted in endotoxin free container. Generally radiation sterilized tissue culture plasticware is endotoxin free, but not autoclaved glassware. Three dilutions per sample are tested routinely, along with a set of blanks and positive controls."
"Growing cell lines at different scales from our cell bank upon request"
"The Cell Center Service Facility will assist with monoclonal antibody production, including antigen preparation, immunization, fusion, screening and cloning. Our high efficiency fusion protocol typically results in about 80% hybridoma containing wells per fusion. Immunization is outsourced, however injections are performed to our specifications. Isolation of spleen cells, fusion and all following cell culture work is done at the Cell Center Service Facility."
"The CCS will grow mammalian or insect cells for monoclonal antibody production, recombinant protein production or to harvest large numbers of cells. To this end there are several options available:
• Roller bottle culture: The CCSF can grow suspension or adherent cells in roller bottles and produce up to 10L of antibody/recombinant protein containing supernatant. Or we can provide the researcher with the cell pellet.
• CelLine Flask culture: Adherent or suspension cells can be grown in the CelLine flask. There are two different protocols that can be utilized. One is the 14/14 protocol, where cells are grown for two weeks in the flask and about 15ml of very concentrated supernatant will be harvested and given to the researcher after the two-week period. The other is the 7/21 protocol, where the researcher is provided with about 15ml of concentrated supernatant every week for as long as the researcher desires.
• Hollow-fiber bioreactor: This method of growing cells is best when multi-milligram amounts of antibody or recombinant protein need to be produced. The hollow-fiber cartridge is inoculated with cells and cells are grown on a continuous basis. 20 - 70ml of highly concentrated supernatant are harvested every day (except on weekends) and frozen immediately. Proteins produced in the hollow-fiber bioreactor are typically more homogeneous and generally of higher quality than proteins produced in other systems. A hollow-fiber cartridge can be kept going for as long as the researcher desires."
Storage of specimen vials in liquid Nitrogen.
"This test is extremely rapid, highly sensitive and is generic for all common mycoplasma strains. Mycoplasma have unique metabolic processes that derive energy from their host cells. The method specifically detects enzymes involved in mycoplasma metabolism that are absent in host cells. By using substrates for the mycoplasma specific enzymes, ADP is converted to ATP. The resultant ATP is conveniently detected using the bioluminescent reaction catalyzed by the Firefly Luciferase enzyme. Any increase in light output from this reaction, as measured by a luminometer, will indicate the presence of mycoplasma.
We run a mycoplasma test every Friday morning. Please note that samples should arrive at our facility by Thursday afternoon before 4:30 pm to be included in Friday’s testing. You will receive results that Friday afternoon."
Produce and purify antibodies from culture supernatants or ascites. Hybridoma cells can be grown either in roller bottles or in hollow fiber cartridges. The culture supernatant (1 liter or more) is precipitated by ammonium sulphate and IgG is purified by protein G chromatography through FPLC.
"The facility prepares and sterilizes custom formulations as long as a commercial powder base is available as a reasonable starting point."
"The CCS will transfect suspension 293 cells (or another cell line as requested). We will then scale up the cells for protein production and provide the researcher with supernatant and/or cell pellet. Upon request we are able to purify antibodies or tagged recombinant proteins.
For selection of stable cell lines we will select with G418 and pick up to 24 positive clones. Expression of the specific recombinant protein is verified by Western blot with an antibody that is to be provided by the researcher.
Please call us with inquiries about customized orders."