343. Non-Viral Transfection of Primary Human Fibroblasts in Suspension Microcarrier Culture
Notice bibliographique
Résumé
Suspension bioreactors have been employed for the large-scale production of recombinant biomacromolecules and expansion of primary tissue-derived cells for clinical applications. However, the bioprocess requires establishing a stably-expressing cell line in static culture first, which is a lengthy process that can add significant upfront cost and limit economies of scale. A transient-expression system is a simpler cost-effective platform for rapid production in a small-medium format, which could make it feasible for generating patient-specific cell-based products. However, current methods for the efficient transfection of primary cells involve either physical methods of delivery that are not adaptable for suspension culture, or viral vectors that are potentially mutagenic, which can present an even greater problem in suspension culture that tend to select for high proliferative tumorigenic clones. Thus, a method to efficiently transfect clinically-relevant primary cells directly in suspension culture by non-viral means is needed to streamline the derivation, expansion and production of cell-based product in one integrated bioprocess. In this study, we explored the feasibility of transfecting primary tissue-derived fibroblasts directly in microcarrier suspension culture using non-viral cationic reagents. We first developed an optimized non-viral transfection system by adopting the gWiz High Expression plasmid, which is capable of 5x higher expression than CAG-based episomal plasmids. We further evaluated several commercial cationic reagents for transfection of primary human fibroblast in static culture and found that XtremeGENE HP and TransIT-3D were among the most efficient transfection reagents (up to 60%). Next, in order to transfect anchorage-dependent cells in suspension, we evaluated a number of microcarriers for their suitability in culturing fibroblast in suspension. We focused on examining cell attachment efficiencies and growth rates on the microcarriers since these variables have the most significant impact on the overall transfection efficiencies. While all of the microcarriers surveyed in this study (i.e. polyGEMs with FACTIII, Collagen, Pronectin F, Glass, Plastic, or Plastic+, and CultiSphere S, Cytodex 3, Hillex II) were capable of supporting cell attachment, Cultisphere S and Hillex II had the most conducive surface for attachment (40% and 25% higher compared to tissue culture dishes). These differences in attachment efficiencies translated into different lag phase and exponential phase among the carriers when we subsequently assayed for growth rate on the carriers over a 12-day period using a MTT-based assay. Due to the differences in coating, charged surfaces, and growth rate on these microcarriers, in order to properly assess for their compatibility with cationic transfection reagent in facilitating transfection, we transfected cells at multiple time points (day 2, 3, 5 and 7 days post cell seeding) and found that transfection efficiencies correlated with the time frame in which cells were growing the fastest (Days 2-3); highest efficiencies were seen in cells cultured on Glass, Cytodex 3, CultiSphere S and Hillex II (~12-16%). In summary, we demonstrated here a first step towards the efficient transfection of primary tissue-derived human fibroblasts directly in a suspension microcarrier culture using cationic reagent; additional optimization is expected to bring the efficiencies comparable to those in static culture.
Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.
Comment cette classification a été obtenuedéplier
Prédiction distillée sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Apprise à partir de 10 348 étiquettes directes de Codex et de 10 348 étiquettes directes de Gemma. Le mode candidate est l'union des têtes enseignantes seuillées; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont ni des étiquettes humaines ni des étiquettes directes de modèles de pointe.
Scores Codex et Gemma par catégorie
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,000 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,000 | 0,000 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,000 | 0,000 |
| Science ouverte | 0,000 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,000 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,000 | 0,000 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule tête enseignante, pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».