Growth properties of DH82 and RF/6A cell lines under standard laboratory conditions
DH82 Cell line has been utilized to grow Ehrlichia canis and RF/6A for drug evaluation in short-term cultures, as well as for replicating Anaplasma marginale. However, specific in vitro culture development conditions are unknown.Several experiments were designed to solve inquiries of such procedure. Both cell lines were acquired from ATCC and put into culture. Na Pyruvate, NaHCO3 and fetal calf serum were used to enrich MEM culture media and incubated at 37 °C on 5% CO2 - air humid mixture atmosphere. First assays used 24 well plates and were focused on determination of a minimum initial cell concentration and cell density. In the former, averages of 62,500 cell/well for DH82 & 8,836 for RF/6A; were found. Later, experiments to identify a minimum cell density started with 5, 10, 20 & 40 cells/mm2, harvesting with a EDTA-Trypsin solution when confluence be reached. Growth indexes of 3,319.32, 1,956.70, 870.73 & 422.14 times and of 62.38, 63.51, 25.31 & 12.16 times, were respectively found for DH82 & RF/6A cell lines. For kinetics studies, 35 mm Ø sterile Petri dishes were used. Cultures were set with 20 cell/mm2 seed density for DH82 and 10 cell/mm2 for RF/6A. Dishes were randomly separated into two groups, with and without culture media periodical replacement. Maximum growth was observed at 336 and 315 h with 42.9 and 36.9 h doubling time in culture, respectively for DH82 & RF/6A cell lines. Generated data provided a model to define an in vitro growth pattern for future studies.
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Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias
2016
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rev-remexcp-article41512017-07-17T18:33:53Z Growth properties of DH82 and RF/6A cell lines under standard laboratory conditions Propiedades de crecimiento de las líneas celulares DH82 y RF/6A bajo condiciones normales de laboratorio Machuca Figueroa, Samara Granjeno Colín, Gabriela Rodríguez Camarillo, Sergio Darío Agustín Vega, Carlos *, Murguía Cell culture; DH82; RF/6A; Doubling time. Cultivo celular; DH82, RF/6A; Intervalo de duplicación DH82 Cell line has been utilized to grow Ehrlichia canis and RF/6A for drug evaluation in short-term cultures, as well as for replicating Anaplasma marginale. However, specific in vitro culture development conditions are unknown.Several experiments were designed to solve inquiries of such procedure. Both cell lines were acquired from ATCC and put into culture. Na Pyruvate, NaHCO3 and fetal calf serum were used to enrich MEM culture media and incubated at 37 °C on 5% CO2 - air humid mixture atmosphere. First assays used 24 well plates and were focused on determination of a minimum initial cell concentration and cell density. In the former, averages of 62,500 cell/well for DH82 & 8,836 for RF/6A; were found. Later, experiments to identify a minimum cell density started with 5, 10, 20 & 40 cells/mm2, harvesting with a EDTA-Trypsin solution when confluence be reached. Growth indexes of 3,319.32, 1,956.70, 870.73 & 422.14 times and of 62.38, 63.51, 25.31 & 12.16 times, were respectively found for DH82 & RF/6A cell lines. For kinetics studies, 35 mm Ø sterile Petri dishes were used. Cultures were set with 20 cell/mm2 seed density for DH82 and 10 cell/mm2 for RF/6A. Dishes were randomly separated into two groups, with and without culture media periodical replacement. Maximum growth was observed at 336 and 315 h with 42.9 and 36.9 h doubling time in culture, respectively for DH82 & RF/6A cell lines. Generated data provided a model to define an in vitro growth pattern for future studies. La línea celular RF/6A ha sido utilizada en estudios de corto plazo evaluando fármacos o infecciones experimentales con Anaplasma marginale; en contraste, DH82 es utilizada para la multiplicación de Ehrlichia canis. No obstante, se desconocen condiciones específicas de su crecimiento, por lo que se diseñaron varios experimentos para resolver interrogantes de su propagación. Ambas líneas, se adquirieron de la American Type Culture Collection, mantenidas en Medio Mínimo Esencial suplementado con suero fetal bovino, piruvato de Na y NaHCO3 e incubadas en atmósfera de 5% de CO2 en aire, a 37 °C. Los primeros ensayos, en placas de 24 pozos, esclarecieron los valores de dosis mínima inicial, que fueron 62,500 y 8,836 células/pozo para DH82 y RF/6A; así como los de densidad de siembra; cultivos con concentraciones de 5, 10, 20 y 40 células por mm2, cosechados con solución Tripsina-EDTA al alcanzar >95% de confluencia. Los índices estimados fueron: 3,319.32, 1,956.70, 870.73 y 422.14 para DH82 y 62.38, 63.51, 25.31 y 12.16 veces con RF/6A. La cinética del crecimiento, en cajas de Petri de 35 mm Ø, incluyó la siembra de 20 células/mm2, cambio del medio cada 63 h y cosecha cada 21 h para DH82; para RF/6A; la siembra fue 10 células/ mm2, cambio de medio cada 45 h y cosecha cada 15 h. El máximo crecimiento se observó hasta las 336 y 315 h con tiempos de duplicación de 42.9 y 36.9 h respectivamente para DH82 y RF/6A. Los datos permitieron proponer un modelo patrón de cultivo, para estudios futuros. Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias 2016-02-17 info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion application/pdf https://cienciaspecuarias.inifap.gob.mx/index.php/Pecuarias/article/view/4151 10.22319/rmcp.v7i1.4151 Revista Mexicana de Ciencias Pecuarias; Vol. 7, Núm. 1 (2016): Enero-Marzo; 85-104 Revista Mexicana de Ciencias Pecuarias; Vol. 7, Núm. 1 (2016): Enero-Marzo; 85-104 2448-6698 2007-1124 spa https://cienciaspecuarias.inifap.gob.mx/index.php/Pecuarias/article/view/4151/3422 http://creativecommons.org/licenses/by-nc-sa/4.0 |
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Machuca Figueroa, Samara Granjeno Colín, Gabriela Rodríguez Camarillo, Sergio Darío Agustín Vega, Carlos *, Murguía |
| spellingShingle |
Machuca Figueroa, Samara Granjeno Colín, Gabriela Rodríguez Camarillo, Sergio Darío Agustín Vega, Carlos *, Murguía Growth properties of DH82 and RF/6A cell lines under standard laboratory conditions |
| author_facet |
Machuca Figueroa, Samara Granjeno Colín, Gabriela Rodríguez Camarillo, Sergio Darío Agustín Vega, Carlos *, Murguía |
| author_sort |
Machuca Figueroa, Samara |
| title |
Growth properties of DH82 and RF/6A cell lines under standard laboratory conditions |
| title_short |
Growth properties of DH82 and RF/6A cell lines under standard laboratory conditions |
| title_full |
Growth properties of DH82 and RF/6A cell lines under standard laboratory conditions |
| title_fullStr |
Growth properties of DH82 and RF/6A cell lines under standard laboratory conditions |
| title_full_unstemmed |
Growth properties of DH82 and RF/6A cell lines under standard laboratory conditions |
| title_sort |
growth properties of dh82 and rf/6a cell lines under standard laboratory conditions |
| description |
DH82 Cell line has been utilized to grow Ehrlichia canis and RF/6A for drug evaluation in short-term cultures, as well as for replicating Anaplasma marginale. However, specific in vitro culture development conditions are unknown.Several experiments were designed to solve inquiries of such procedure. Both cell lines were acquired from ATCC and put into culture. Na Pyruvate, NaHCO3 and fetal calf serum were used to enrich MEM culture media and incubated at 37 °C on 5% CO2 - air humid mixture atmosphere. First assays used 24 well plates and were focused on determination of a minimum initial cell concentration and cell density. In the former, averages of 62,500 cell/well for DH82 & 8,836 for RF/6A; were found. Later, experiments to identify a minimum cell density started with 5, 10, 20 & 40 cells/mm2, harvesting with a EDTA-Trypsin solution when confluence be reached. Growth indexes of 3,319.32, 1,956.70, 870.73 & 422.14 times and of 62.38, 63.51, 25.31 & 12.16 times, were respectively found for DH82 & RF/6A cell lines. For kinetics studies, 35 mm Ø sterile Petri dishes were used. Cultures were set with 20 cell/mm2 seed density for DH82 and 10 cell/mm2 for RF/6A. Dishes were randomly separated into two groups, with and without culture media periodical replacement. Maximum growth was observed at 336 and 315 h with 42.9 and 36.9 h doubling time in culture, respectively for DH82 & RF/6A cell lines. Generated data provided a model to define an in vitro growth pattern for future studies. |
| publisher |
Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias |
| publishDate |
2016 |
| url |
https://cienciaspecuarias.inifap.gob.mx/index.php/Pecuarias/article/view/4151 |
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