A novel method for large-scale culture of Plasmodium falciparum asexual blood stage and gametocytes in a Wave Bioreactor cell culture system
Notice bibliographique
Résumé
The clinical manifestations of Plasmodium falciparum malaria occur when parasites are multiplying within erythrocytes. During this stage, some parasites develop into sexual forms (male and female gametocytes) that are transmitted to the mosquito during their feeding on blood. The development of new drugs or vaccines targeting both asexual and sexual blood stage parasites is important to strengthen existing tools for fighting against malaria, and this is an essential component of the overall strategy of malaria eradication. However, there are still many unsolved questions concerning the cellular development and cell biology of P.falciparum asexual parasites, as well as for the onset and the development of the gametocyte stage. This is partly because of the inability to obtain good workable quantities of parasite material. Routine bulking-up of cultures in most laboratories involves establishing multiple culture flasks, which is very time consuming and labour intensive. To obtain large quantities of parasite material with high consistency, we recently reported the use of the Wave Bioreactor system for the culture of asexual blood stage of P. falciparum in suspension [1]. Controlled wave-induced motion within the bioreactors provides low-shear, favourable hydrodynamic conditions and very efficient gas transfer that is ideal for parasite cultivation. We used the Wave Bioreactor™ 20/50 EHT which is composed of a single-use plastic Cellbag, the cultivation chamber, that sits on a heated and rocking platform. We established important parameters for maintaining P. falciparum cultures, such as rocking motion, temperature and gas flow. Moreover, by monitoring parasite growth along with glucose consumption, pH dynamic and lactic acid production we characterized the development of the parasites within the bioreactor. We showed that malaria parasites grow far better in these rocking cultures than in static flask cultures in terms of preserving parasite cell synchronicity and reducing the number of multiple infected erythrocytes. Finally, we established a simple and straightforward protocol for bulk cultures of up to 1L of culture of P. falciparum within five days. To take advantage of the benefits of P. falciparum culture in suspension, we have now developed a simple protocol for gametocyte production and isolation from 1L culture in the wave bioreactor. This protocol includes 2 phases; the growth of the asexual parasites to stress conditions to induce gametocytes, followed by the maturation of gametocytes. In our experiments, approximately 8-10% of the asexual parasites commit to gametocytes. We reproducibly obtained mature gametocytes within 10 to 13 days (males in 10-12 days and females in 11 to 13 days). Batches of matures gametocytes can be collected from the same wave bag culture over several days. Finally, exflagellation tests confirm that gametocytes produced in the wave bioreactor are viable and, therefore, likely infectious for mosquitoes. The use of the Wave Bioreactor is a breakthrough method that will allow large scale production of Plasmodium falciparum asexual blood stage and gametocytes for antigen or organelle isolation, high throughput screening of compound libraries, for infection of mosquitoes and for whole cell blood-stage malaria vaccine development under GMP compliant procedures.
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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 | 0,002 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,001 | 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 | 0,001 |
| 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 ».