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

Abstract

Scalable production of avian cell lines exhibits a valuable potential on therapeutic application by producing recombinant proteins and as the substrate for virus growth due to the special glycosylation occurs in avian species. Chicken primordial germ cells (cPGCs), a germinal pluripotent avian cell type, present the ability of self-renewal, an anchorage-independent cell growth and the ability to be genetically modified. This cell type could be an interesting bioreactor system for industrial purposes. This study sought to establish an expandable culture system with defined components for three-dimensional (3D) culture of cPGCs. cPGCs were cultured in medium supplemented with the functional polymer FP003. Viscoelasticity was low in this medium but cPGCs did not sediment in culture and efficiencies of space and nutrient utilization were thus enhanced and consequently their expansion was improved. The total number of cPGCs increased by 17-fold after 1 week of culture in 3D-FAot medium, an aseric defined medium containing FP003 polymer, FGF2 and Activin A as growth factors and Ovotransferrin as protein. 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. In summary, this novel 3D culture system can be used to efficiently expand cPGCs in suspension without mechanical stirring, which is available for long-term culture and no loss of cellular properties was found. This system provides a platform for large-scale culture of cPGCs.

In vitro culture and characterization of duck primordial germ cells

“…All PGCs were maintained at 37°C in 5% CO 2 . FAcs (fibroblast growth factor 2 ( FGF2 ), Activin A and chicken serum) and FAot (FGF2, Activin A and ovotransferrin) media were prepared as described by Chen et al ( 2018 ). Briefly, FAcs medium was diluted Dulbecco's modified eagle medium ( DMEM ) (1:3 ratio of sterile deionized water and calcium-free high-glucose DMEM) containing 1× B-27 supplement, 2 mM GlutaMAX, 1× non-essential amino acids, 0.1 mM 2-mercaptoethanol, 1 mM sodium pyruvate, 0.2% chicken serum (all from Gibco®, Grand Island, NY, USA), 1× nucleosides (EMD Millipore, Billerica, MA, USA), 2 mg/mL ovalbumin (Sigma-Aldrich, St. Louis, MO, Germany), 0.1 mg/mL sodium heparin (Sigma-Aldrich), 25 ng/mL human Activin A, and 4 ng/mL human FGF2 (both from R&D Biosystems, Minneapolis, MN, USA).…”

Fate Decisions of Chicken Primordial Germ Cells (PGCs): Development, Integrity, Sex Determination, and Self-Renewal Mechanisms

“…In addition, several small molecules, such as blebbistatin and CHIR99021, induce the activation of the self-renewal of cultured PGCs by reducing apoptosis [ 159 , 160 ]. Furthermore, Chen et al established a three-dimensional culture system for chicken PGCs, enabling the efficient culture of chicken PGCs on a large scale [ 161 ].…”

Three-Dimensional Avian Hematopoietic Stem Cell Cultures as a Model for Studying Disease Pathogenesis

“…Very little attention has been given to the use of 3D cell cultures in avian models. It has been studied predominantly in embryonal cells, such as primordial germ cells ( Chen et al, 2018 ), intestinal epithelial cells ( Pierzchalska et al, 2019 ), or chicken bone marrow cells as support for osteogenesis of human stromal cells ( Yang et al, 2020 ). Therefore, our current knowledge about 3D stem cell cultures comes from studies in mammals.…”