The CEINGE Cell Culture Facility activity is focused on the acquisition, manipulation, characterization and cryopreservation of human and animal cellular models and/or of cell suspensions derived from peripheral blood, bone marrow and fresh tissue biopsies. The CEINGE Cell Culture Facility work is based on application of good laboratory practice rules in Cell Culture, such as manipulation of only one cell line at a time, proliferation rate measurement by Population Doublings formula application, and monitoring of morphological appearance of each cell line.
The Facility is able to offer services such as human and animal cell lines supply, cryopreservation of stabilized or fresh tissue-derived cells, detection of mycoplasma in cell lines and/or reagents, mycoplasma eradication in cell lines, and creation of stable lymphoblastoid cellular models starting from human peripheral blood samples.
1. Human and Animal Cell Lines supply
CEINGE cell line collection can be supplied in three forms: cryopreserved, in active growth or as a cellular pellet. Cryopreserved cell lines are provides as frozen ampoules derived from liquid nitrogen (-196 ° C). The delivery of actively growing cell lines as well as cell pellet is performed after revitalization, cellular count, viability evaluation, and maintenance in optimal culture conditions of the cell line itself. All cell lines are controlled for: 1) microbiological status by mycoplasma detection assays; 2) morphology, by monitoring in microscopy; 3) proliferative capacity, through accurate and optimal manipulation in vitro as suggested by good laboratory practice in Cell Culture.
2. Cell lines amplification, count and viability evaluation, and cryopreservation
Human and animal cell lines can be delivered to the Cell Culture Facility, where they are appropriately amplified and cell counts and viability are defined. As regards cryopreservation procedure, the samples are resuspended in appropriate freezing solutions, transferred to freezers at -80 ° C within specific supports that allow the gradual descent of the temperature (about 1° C/ minute) and finally stored in tanks of liquid nitrogen ( -196 ° C).
3. Isolation of mononuclear cells from peripheral blood and/or bone marrow samples, with cell count and viability evaluation
The mononuclear cells contained within each blood sample can be separated from the rest of the cell populations by the use of Ficoll-Paque gradient. The mononuclear cell component is then harvested and its viability and count are determined. The so purified cells can be cryopreserved on request.
4. Cryopreservation of cells isolated from peripheral blood and/or bone marrow and/or solid tissue, with cell count and viability evaluation
Cell Culture Facility offers the service of cryopreservation and cryoconservation of cell suspensions derived from peripheral blood, bone marrow or tissue biopsies, with evaluation of viability and cell count.
5. Human continuous lymphoblastoid cell lines stabilization by EBV infection
Peripheral blood mononuclear cells are isolated by Ficoll-Paque gradient and cultured with Epstein-Barr Virus (EBV). Once infected by the Epstein-Barr virus, B-lymphocytes acquire characteristic morphological signs visible under the microscope. EBV-infected B-lymphocytes are then amplified until a stable lymphoblastoid cell line is created, which is then cryopreserved on request.
6. Mycoplasma detection in cell lines and/or in reagents
CEINGE Cell Culture Facility is able to detect mycoplasma contamination in cell lines by means of two types of assays: a bioluminescent assay and an agar culture assay.
The Bioluminescent Assay is a rapid detection method of mycoplasma contamination and is based on evaluation of ATP consumption by cell lines. The rationale is that a mycoplasma-positive cell line will consume more ATP than a mycoplasma-free cell line.
The Agar Culture Assay is able to reveal the contamination derived by different species of mycoplasma in cell lines and it is performed setting up a double culture arm: one in anaerobiosis and the other one in aerobiosis. Anaerobiosis arm is performed by culturing supernatants of potentially contaminated cell lines on solid agar. At the same time, aerobiosis arm is prepared by culturing supernatants of cell lines in liquid broth. After 14 days, the liquid broth is transferred on solid agar for another 14 days. After 28 days of culture, this procedure of mycoplasma detection is able to reveal the presence of even one colony of mycoplasma.
7. Mycoplasma eradication in cell lines
Each mycoplasma-positive cell line is subjected to a standardized protocol based on the use of 5 different antibiotics. After pharmacologic treatments, each sub-culture is maintained for 30 days in antibiotic-free culture condition and finally mycoplasma detection tests (both bioluminescent and culture assay on agar) are performed to verify antibiotics efficacy.