Cell Lines, Primary Cultures
Cell lines maintain growth and specialized properties during prolonged or indefinite culture in the laboratory. Primary cell isolates are derived fresh from tissues and will grow and maintain specialized properties for a limited time, about 10 passages. We have experience with explants of liver, breast, ovary, lung, skin, spleen, lymph node and brain. Both cell lines and primary cultures can be stored frozen in liquid nitrogen and then put back into culture. These methods facilitate biological studies that are convenient, reproducible, and cost effective. Cell lines allow studies that may be difficult with whole organs or in vivo, such as mechanism of action, radioactive experiments, or system manipulation. Cell lines with desired properties are obtained from repositories such as the American Type Culture Collection, or derived by MuriGenics through selection techniques and/or DNA transfection.
Cell Culture, Medium Optimization, Serum-Free Adaptation
Each cell line must be matched to a particular growth medium. Small scale growth and maintenance in culture (1 mL to 100 mL) is carried out in tissue culture grade plastics, while scale-up utilizes roller bottles, porous bead supports or a hollow fiber bioreactor, or stir cells. Nonadherent cells are also economically grown up to 40 L in stir cell suspension culture. Some adherent cell types can be adapted to nonadherent growth resulting in a more simple production method. Adaptation to serum-free conditions allows convenient purification of a secreted protein product.
Cloning is used to obtain a stable culture of homogeneous cells. Over periods of many months in tissue culture, cells can change properties due to somatic gene mutation, and overgrowth of mutated cells. It may be desired to select a rare cell type or the few stably transfected cells in a transfection pool. Cloning is achieved by diluting a culture so that Â½ the wells in a 96 well plate contain one cell and Â½ contain no cells. At this low cell concentration, conditioned medium (medium from the same cell type harvested at high concentrations) is added to enhance growth. When the clone reaches suitable numbers, aliquots are frozen in order to retrieve cells with the same properties at a future date.
Genes can be introduced into cells by suitable molecular biology methods such as electroporation, cationic lipid reagents, or calcium phosphate. A gene can be transfected into cells simply with its regulatory elements or after making a construct to achieve high overexpression levels. Cells can also be transfected with conditionally expressed genes of interest. Transfections in mammalian cells can be transient or permanent. Transient expression lasts only a few days. Permanent expression requires cotransfection with a dominant selectable marker and several rounds of selection for the cell populations that stably integrate the transfected DNA into a cellular gene. This takes approximately three to four weeks.
- H & E
- Toluidine Blue
- Sirius Red
- Massonâ€™s Trichrome
We are able to customize an immunohistochemical assay for your particular research and development needs and/or to complement any of our disease model studies. Our services include screening antibodies in small animals, as well as optimizing our assays to ensure highly specific and robust expression.
Some of the validated assays that we perform frequently for our clients: