Three-dimensional culture of chicken primordial germ cells in chemically defined media containing the functional polymer FP003

Scalable production of avian suspension cell exhibits a valuable potential on therapeutic application by producing recombinant protein and as the substrate for virus growth. This study sought to establish a system with chemically defined components for three-dimensional (3D) culture of chicken primordial germ cells (cPGCs), a pluripotent avian cell type. cPGCs were cultured in medium supplemented with the functional polymer FP003. Viscoelasticity was low in this medium, and cPGCs did not sediment,and consequently their expansion was improved. The total number of cPGCs increased by 17-fold after 1week of culture in 3D-FAot medium, an aseric chemically defined medium containing FP003, indicating that this medium enhances the expansion of cPGCs. Moreover, cPGC cell lines stably expressed the germline-specific reporter VASA:tdTOMATO, as well as other markers of cPGCs, for more than 1 month upon culture in 3D-FAot medium, indicating that the characteristics of these cells are maintained. cPGCs harboring both PGK:EGFP and VASA:tdTOMATO robustly expressed both fluorescent proteins upon culture in 3D-FAot, suggesting that this approach is perspective for recombinant protein production. In summary,this novel 3D culture system can be used to efficiently expand cPGCs in suspension without mechanical stirring or loss of cellular properties. This system provides a platform for large-scale culture ofcPGCs in industry.


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118 Materials and methods 119 Incubation of chicken eggs 120 To isolate cPGCs, specific pathogen-free chicken (Gallus gallus) eggs were 126 127 128 Preparation of culture media 129 The three types of culture media used in this study were prepared as described by . One-third of the medium was replaced by fresh medium every 2 days.
160 Cells were sub-cultured into a larger dish in fresh medium when they became confluent.
161 cPGCs are suspension cells, and therefore did not require trypsinization during passage. 256 Media containing and lacking FP003 are referred to as 3D and 2D media, respectively.
257 Polystyrene beads were used to mimic cells (Fig 1A). After shaking, beads sedimented 258 in 2D medium, but remained suspended in 3D media containing 0.010 %, 0.012 %, and 259 0.016 % FP003. Moreover, viscoelasticity was 0.9 mPa/s in 2D medium and 5.6 mPa/s 260 in 3D medium containing 0.016 % FP003 (Fig 1B). These results suggest that addition  (Fig 2A). After 96 hr, growth tended to decrease in all groups 281 as cells became confluent (Fig 2A). The fold increase in the total cell number after 48 282 hr was significantly lower for cPGCs cultured in 2D medium than for cPGCs cultured 283 in 3D medium containing each of the three concentrations of FP003 (Fig 2B). After 96 284 hr, the fold increase in the total cell number for cPGCs cultured in 3D medium 285 containing 0.016 % FP003 was twice that for cPGCs cultured in 2D medium (Fig 2B).
286 The fold increase in the total cell number was highest for cPGCs cultured in 3D medium 287 containing 0.012 % FP003 after 48 hr, but for cPGCs cultured in 3D medium containing

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To determine the optimal cell seeding density, we seeded cPGCs at five densities 308 in FAcs medium containing 0.016 % FP003. cPGCs seeded at densities of 5 × 10 4 , 1 × 309 10 5 , and 2 × 10 5 cells/mL expanded (Fig 2C). A seeding density of 1 × 10 5 cells/mL 310 was optimal for the proliferation of cPGCs. Using this seeding density, the total cell 311 number was 6-fold higher at 72 hr than at 24 hr ( Fig 2C). cPGCs cultured in polymer-312 containing 3D medium were difficult to isolate from suspension by only centrifugation 313 when compared to those in 2D medium (Fig 2D). To harvest cPGCs in 3D medium, 314 samples were centrifuged at 2000 × g following addition of up to 20 vol % citrate/PBS 315 in order to dissociate polymer-ion structures. Cells were as efficiently harvested by this 316 method as by centrifugation for 5 min at 500 × g in 2D medium (Fig 2E and 2F).

318 Comparison of the growth of cPGCs between serum-containing
319 and chemically defined media 320 We plated a low number (1 × 10 4 ) of cPGCs in serum-containing (FAcs) or 321 chemically defined (FAot or FAits) medium and cultured the cells for 1 week. The 322 proliferation of cPGCs cultured in these three types of media differed at various time