Occurrence and Control of Sporadic Proliferation in Growth Arrested Swiss 3T3 Feeder Cells

March Occurrence and Control of Sporadic Proliferation in Growth Arrested Swiss 3T3 Feeder Cells Rishi Man Chugh 0 1 2 Madhusudan Chaturvedi 0 1 2 Lakshmana Kumar Yerneni 0 1 2 0 Cell Biology Laboratory, National Institute of Pathology (ICMR) , New Delhi , India 1 Funding: The study was supported by extra mural Research grant 2009-02800 to LKY by the Indian Council of Medical Research, (http://www.icmr.nic.in) New Delhi, India. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript 2 Academic Editor: Mauro Picardo, San Gallicano Dermatologic Institute , ITALY Growth arrested Swiss mouse embryonic 3T3 cells are used as feeders to support the growth of epidermal keratinocytes and several other target cells. The 3T3 cells have been extensively subcultured owing to their popularity and wide distribution in the world and, as a consequence selective inclusion of variants is a strong possibility in them. Inadvertently selected variants expressing innate resistance to mitomycin C may continue to proliferate even after treatment with such growth arresting agents. The failure of growth arrest can lead to a serious risk of proliferative feeder contamination in target cell cultures. In this study, we passaged Swiss 3T3 cells (CCL-92, ATCC) by different seeding densities and incubation periods. We tested the resultant cultures for differences in anchorage-independent growth, resumption of proliferation after mitomycin C treatment and occurrence of proliferative feeder contaminants in an epidermal keratinocyte co-culture system. The study revealed subculture dependent differential responses. The cultures of a particular subculture procedure displayed unique cell size distribution and disintegrated completely in 6 weeks following mitomycin C treatment, but their repeated subculture resulted in feeder regrowth as late as 11 weeks after the growth arrest. In contrast, mitomycin C failed to inhibit cell proliferation in cultures of the other subculture schemes and also in a clone that was established from a transformation focus of super-confluent culture. The resultant proliferative feeder cells contaminated the keratinocyte cultures. The anchorage-independent growth appeared in late passages as compared with the expression of mitomycin C resistance in earlier passages. The feeder regrowth was prevented by identifying a safe subculture protocol that discouraged the inclusion of resistant variants. We advocate routine anchorage-independent growth assay and absolute confirmation of feeder disintegration to qualify feeder batches and caution on the use of fetal bovine serum. - Competing Interests: The authors have declared that no competing interests exist. Large quantities of cultured epithelial autografts (CEA) for clinical use in the treatment of extensively burned patients are speedily grown from the adult epidermal keratinocytes over the growth arrested Swiss mouse embryonic 3T3 dermal fibroblasts [1]. These cells are superior in supporting the growth of other target cells as well [2, 3]. The original inactivation method involved -irradiation, although a more convenient option has been the treatment with mitomycin C (MC) [3]. The growth arrested 3T3 fibroblasts reportedly survived in CEA and elicited immunogenicity in recipient resulting in complete graft breakdown [4]. Reasonably the viable feeders can result either from the mitotically inactive yet surviving feeders or the proliferating ones. Although, there is evidence of proliferation in other growth arrested mouse embryonic feeders, but there are no specific studies to link the persistence of the viable 3T3 feeders with the failure of growth arrest [5]. The 3T3 cells have the potential to undergo spontaneous transformation depending on subculture, confluence state, and type and concentration of serum [6, 7]. Repeated and inconsistent passaging of cell cultures leads to the accumulation of specific transformed variants and display of altered characteristics [8]. Selective accumulation of such variants, particularly in late passage cultures of 3T3 is a strong possibility as they have been extensively subcultured due to their popularity and wide distribution through several channels in the world [8]. But indications of transformation such as loss of contact inhibition and presentation of phenotypic differences may not readily be apparent, when the transformed variants are less frequent. However, few variants with innate resistance to growth arrest may continue to even after exposure to MC. Such proliferative feeders then become visible and contaminate the target cell cultures. We hypothesize that the presence of such variants in 3T3 cell cultures is a potential cause for failure of growth arrest. We, therefore, propose to investigate if the proliferative feeder contamination of target cells is dependent on the adopted subculture protocol for 3T3 cells and identify preventive strategies. The identified solutions can help in eliminating apprehensions on feeder dependant culture system [9], which is the most efficient and economical method to culture stem cells compared with feeder-free systems [10]. We observed that it was essential to validate each lot of the growth arrested 3T3 cells through confirmation of the complete disintegration of feeders before qualifying them as safe feeders. Materials & Methods 3T3 fibroblast culture The frozen SWISS 3T3 cells (CCL-92, ATCC) supplied at 115th passage (designated as zero passage) were quickly thawed and grown in 3T3-CBS medium consisting of DMEM with 1.5 grams of sodium bicarbonate per liter and 10% calf bovine serum (CBS) in a humidified 5% CO2 atmosphere at 37C. The cells were serially subcultured until 6 passages to establish cryopreserved master and working banks (S1 Fig.). The frozen cells were incubated for 4 days to compensate for the initial slow growth while the subsequent cultures were passaged at uniform intervals of 3 days. The cultures were negative for Mycoplasma [11]. The working bank cultures were subjected to specific subculture schemes which were determined after several rounds of preliminary experiments. Initially, 7th passage (P7) cultures were setup in T75 flasks from working bank by seeding 3000 cells per cm2 and were subcultured by three schemes (S2 Fig.). Two of the schemes denoted as 3K3D and 3K4D, represented incubation of 3000 plated cells per cm2 for 3 and 4 days, respectively. The third scheme of 4K3D involved 3 days incubation of 4000 cells plated per cm2. The resultant cells were tested for anchorage-independent growth and stability of growth arrest by MC treatment. Further, the 3K3D cells that exhibited irreversible growth arrest and no anchorage-independent growth were repeatedly subcultured by 3K3D procedure to determine the number of passages that these characters persisted. Simultaneously, the cells were also passaged serially by the 4K3D scheme for comparison to match with the (...truncated)