Amniomax c 100 basal medium

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AmnioMax C-100 Basal Medium is a cell culture medium designed for the growth and maintenance of human amniotic fluid-derived cells. It provides the necessary nutrients and growth factors to support the in vitro culture of these cell types.

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26 protocols using amniomax c 100 basal medium

Bovid Tribe Comparative Cell Culture

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Our investigation included representatives of three bovid tribes (Hassanin and Douzery 1999 (link)): cattle (B.taurus, BTA, tribe Bovini), the spiral horned antelope species (tribe Tragelaphini), Tragelaphus angasii (TAN, Nyala), Tragelaphus imberbis (TIM, Lesser kudu), Tragelaphus scriptus (TSC, Bushbuck), Tragelaphus spekii (TSP, Sitatunga), Tragelaphus strepsiceros (TST, Greater kudu), Taurotragus derbianus (TDE, Derby Eland), and Taurotragus oryx (TOR, Common Eland). The third tribe, the Caprini, was represented by the sheep Ovis aries (OAR) and goat Capra hircus (CHI). Cell lines were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 13% AmnioMax C-100 Basal Medium, 2% AmnioMax C-100 supplement, 15% Fetal Bovine Serum (FBS), 100 U/ml and 100 μg/ml of penicillin/streptomycin antibiotic mixture, and 200-mM l-glutamine (all from Gibco, Thermo Fisher Scientific). Chromosome harvesting and metaphase preparations followed routine procedures. Genomic DNA isolation was performed using Quick-Gene DNA Tissue Kit S (Fujifilm Life Science) according to the manufacturer’s instructions.

Escudeiro A., Adega F., Robinson T.J., Heslop-Harrison J.S, & Chaves R. (2019). Conservation, Divergence, and Functions of Centromeric Satellite DNA Families in the Bovidae. Genome Biology and Evolution, 11(4), 1152-1165.

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Fibroblast Culture in Vertical Flasks

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AmnioMAX-C100 Basal Medium supplemented with 7.5% AmnioMAX-C100 Supplement, 7.5% HyCloneTM fetal bovine serum, 1% penicillin–streptomycin and 1% L-Glutamine 200 mM (ThermoFisher Scientific, Waltham, MA, USA) was used for all cell culturing. Incubation was performed at 37 °C with 5% CO₂ and around 90% relative humidity. Cultures were passaged twice before approximately 200,000 cells were seeded in T25 flasks and cultured for 3 days. Thereafter, growth medium was changed and cultures were incubated for another 24 hours. The culture flasks had to be irradiated in a vertical position hence the flasks were filled with medium immediately prior to delivery of the first of three fractions. The flasks were kept vertical and filled with medium until the cells were harvested 2 days later. Surface adherence, growth rate and lethality of the fibroblasts were previously verified to be identical in the vertical position compared with the same fibroblasts kept under standard culturing conditions.

Nielsen S., Bassler N., Grzanka L., Swakon J., Olko P., Andreassen C.N., Alsner J, & Sørensen B.S. (2018). Optimal reference genes for normalization of qPCR gene expression data from proton and photon irradiated dermal fibroblasts. Scientific Reports, 8, 12688.

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Cell Culture Conditions for Diverse Species

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CHM13 cells were cultured in complete AmnioMax C-100 Basal Medium (Thermo Fisher Scientific, 17001082) supplemented with 15% AmnioMax C-100 Supplement (Thermo Fisher Scientific, 12556015) and 1% penicillin-streptomycin (Thermo Fisher Scientific, 15140122). HG00733 cells were cultured in RPMI 1640 with l-glutamine (Thermo Fisher Scientific, 11875093) supplemented with 15% FBS (Thermo Fisher Scientific, 16000-044) and 1% penicillin-streptomycin (Thermo Fisher Scientific, 15140122). Chimpanzee (P. troglodytes; S006007) and macaque (M. mulatta; AG07107) cells were cultured in MEMα containing ribonucleosides, deoxyribonucleosides and l-glutamine (Thermo Fisher Scientific, 12571063) supplemented with 12% FBS (Thermo Fisher Scientific, 16000-044) and 1% penicillin-streptomycin (Thermo Fisher Scientific, 15140122). Orangutan (P. abelii; PR01109) cells were cultured in MEMα containing ribonucleosides, deoxyribonucleosides and l-glutamine (Thermo Fisher Scientific, 12571063) supplemented with 15% FBS (Thermo Fisher Scientific, 16000-044) and 1% penicillin-streptomycin (Thermo Fisher Scientific, 15140122). All cells were cultured in a humidity-controlled environment at 37 °C with 5% CO₂.

Logsdon G.A., Vollger M.R., Hsieh P., Mao Y., Liskovykh M.A., Koren S., Nurk S., Mercuri L., Dishuck P.C., Rhie A., de Lima L.G., Dvorkina T., Porubsky D., Harvey W.T., Mikheenko A., Bzikadze A.V., Kremitzki M., Graves-Lindsay T.A., Jain C., Hoekzema K., Murali S.C., Munson K.M., Baker C., Sorensen M., Lewis A.M., Surti U., Gerton J.L., Larionov V., Ventura M., Miga K.H., Phillippy A.M, & Eichler E.E. (2021). The structure, function and evolution of a complete human chromosome 8. Nature, 593(7857), 101-107.

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Immortalized Hydatidiform Mole Cell Line

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Cells from the complete hydatidiform mole CHM13 were originally cultured from one case of a hydatidiform mole at Magee-Womens Hospital (Pittsburgh, PA) as part of a research study that occurred in the early 2000’s (IRB MWH-20–054). At that time, the CHM13 cells were cultured, karyotyped using Q banding, and subsequently immortalized using human telomerase reverse transcriptase (hTERT). For this study, cryopreserved CHM13 cells were thawed and cultured in complete AmnioMax C-100 Basal Medium (ThermoFisher Scientific, Carlsbad, CA) supplemented with 1% Penicillin-Streptomycin (ThermoFisher Scientific, Carlsbad, CA) and grown in a humidity-controlled environment at 37°C, with 95% O2 and 5% CO2. Fresh medium was exchanged every 3 days and all cells used for this study did not exceed passage 10. Cells have been authenticated and tested negative for mycoplasma contamination.

Miga K.H., Koren S., Rhie A., Vollger M.R., Gershman A., Bzikadze A., Brooks S., Howe E., Porubsky D., Logsdon G.A., Schneider V.A., Potapova T., Wood J., Chow W., Armstrong J., Fredrickson J., Pak E., Tigyi K., Kremitzki M., Markovic C., Maduro V., Dutra A., Bouffard G.G., Chang A.M., Hansen N.F., Wilfert A.B., Thibaud-Nissen F., Schmitt A.D., Belton J.M., Selvaraj S., Dennis M.Y., Soto D.C., Sahasrabudhe R., Kaya G., Quick J., Loman N.J., Holmes N., Loose M., Surti U., Risques R.A., Graves Lindsay T.A., Fulton R., Hall I., Paten B., Howe K., Timp W., Young A., Mullikin J.C., Pevzner P.A., Gerton J.L., Sullivan B.A., Eichler E.E, & Phillippy A.M. (2020). Telomere-to-telomere assembly of a complete human X chromosome. Nature, 585(7823), 79-84.

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Immortalization of Hydatidiform Mole Cells

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Cells from a complete human hydatidiform mole, CHM13 (46X,X), were immortalized with human telomerase reverse transcriptase (hTERT) and cultured in complete AmnioMAX C-100 Basal Medium (ThermoFisher Scientific, Carlsbad, CA) supplemented with 15% AmnioMAX supplement (ThermoFisher Scientific, Carlsbad, CA) and 1% penicillin and streptomycin. Cells were maintained at 37°C in a humidified incubator with 5% CO₂.

VOLLGER M.R., LOGSDON G.A., AUDANO P.A., SULOVARI A., PORUBSKY D., PELUSO P., WENGER A.M., CONCEPCION G.T., KRONENBERG Z.N., MUNSON K.M., BAKER C., SANDERS A.D., SPIERINGS D.C., LANSDORP P.M., SURTI U., HUNKAPILLER M.W, & EICHLER E.E. (2019). Improved assembly and variant detection of a haploid human genome using single-molecule, high-fidelity long reads. Annals of human genetics, 84(2), 125-140.

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Cell Culture Conditions for Human and Primate Cells

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CHM1 cells were cultured in complete AmnioMax C-100 Basal Medium (Thermo Fisher Scientific, 17001082) supplemented with 15% AmnioMax C-100 Supplement (Thermo Fisher Scientific, 12556015) and 1% penicillin–streptomycin (Thermo Fisher Scientific, 15140122). HG00733 (Homo sapiens) cells were cultured in RPMI-1650 medium (Sigma-Aldrich, R8758) supplemented with 15% fetal bovine serum (FBS; Thermo Fisher Scientific, 16000-044) and 1% penicillin–streptomycin (Thermo Fisher Scientific, 15140122). Chimpanzee (P. troglodytes; Clint; S006007) and macaque (Macaque mulatta; AG07107) cells were cultured in MEM α containing ribonucleosides, deoxyribonucleosides and l-glutamine (Thermo Fisher Scientific, 12571063) supplemented with 12% FBS (Thermo Fisher Scientific, 16000-044) and 1% penicillin–streptomycin (Thermo Fisher Scientific, 15140122). Orangutan (P. abelii; Susie; PR01109) cells were cultured in MEM α containing ribonucleosides, deoxyribonucleosides and l-glutamine (Thermo Fisher Scientific, 12571063) supplemented with 15% FBS (Thermo Fisher Scientific, 16000-044) and 1% penicillin–streptomycin (Thermo Fisher Scientific, 15140122). All cells were cultured in a humidity-controlled environment at 37 °C under 95% O₂.

Logsdon G.A., Rozanski A.N., Ryabov F., Potapova T., Shepelev V.A., Catacchio C.R., Porubsky D., Mao Y., Yoo D., Rautiainen M., Koren S., Nurk S., Lucas J.K., Hoekzema K., Munson K.M., Gerton J.L., Phillippy A.M., Ventura M., Alexandrov I.A, & Eichler E.E. (2024). The variation and evolution of complete human centromeres. Nature, 629(8010), 136-145.

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Culturing Primary Fibroblasts from Skin Biopsy

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A primary fibroblast cell line was obtained from patient II‐3's skin biopsy. The growing cell line was cultured in AmnioMAX C‐100 Basal Medium (Thermo Fisher Scientific), which was supplemented with AmnioMAX C‐100 Supplement (Thermo Fisher Scientific) and Amphotericin B (Gibco) according to the standard laboratory procedures for human cell cultures. The fibroblasts were cultured to the confluence of about 80%; adherent cells were then harvested. The final cell pellets were subsequently used for RNA isolation.

Siavrienė E., Mikštienė V., Radzevičius D., Maldžienė Ž., Rančelis T., Petraitytė G., Tamulytė G., Kavaliauskienė I., Šarkinas L., Utkus A., Kučinskas V, & Preikšaitienė E. (2019). Novel GLI3 variant causes Greig cephalopolysyndactyly syndrome in three generations of a Lithuanian family. Molecular Genetics & Genomic Medicine, 7(9), e878.

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Placenta-Derived MSCs in Liver Fibrosis

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Placenta-derived MSCs (passage 3) were provided by Wake Forest Regenerative Medicine Clinical Center (RMCC, NC, USA). Pd-MSCs were cultured in α-modified Eagle’s medium (α-MEM, HyClone, MA, USA), supplemented with 15% FBS (HyClone, MA, USA), 17% AmnioMax C-100 Basal Medium (Thermo Fisher Scientific, NY, USA), 2% AmnioMax C-100 supplement (Thermo Fisher Scientific), 1% GlutaMax Supplement (Thermo Fisher Scientific), and 1% Gentamicin (Thermo Fisher Scientific). Human hepatic stellate cells were cultured in Dulbecco’s low glucose modified Eagle’s medium with 2% FBS and 1% Penicillin Streptomycin solution (HyClone, MA, USA). The processes of Pd-MSCs obtainment in RMCC complied with the Declaration of Helsinki. The mimics and inhibitors of miR-378c were purchased from RiboBio (Guangzhou, China) and transfected into cells with RNAiMAX transfection reagent according to the Invitrogen (CA, USA).

Zheng W., Bian S., Qiu S., Bishop C.E., Wan M., Xu N., Sun X., Sequeira R.C., Atala A., Gu Z, & Zhao W. (2023). Placenta mesenchymal stem cell-derived extracellular vesicles alleviate liver fibrosis by inactivating hepatic stellate cells through a miR-378c/SKP2 axis. Inflammation and Regeneration, 43, 47.

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Establishing Primary Fibroblast Cell Line

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A primary fibroblast cell line was obtained from a skin biopsy from the proband and the control individual (n = 1). The cell line was cultured in AmnioMAX C-100 Basal Medium (Thermo Fisher Scientific, Waltham, MA, USA) which was supplemented with AmnioMAX C-100 Supplement (Thermo Fisher Scientific, Waltham, MA, USA) and Amphotericin B (Gibco, Waltham, MA, USA) according to the laboratory procedures for human fibroblast cultures [14 (link)]. The final cell pellets were subsequently used for gene-expression and gene-editing assays.

Siavrienė E., Petraitytė G., Mikštienė V., Maldžienė Ž., Sasnauskienė A., Žitkutė V., Ambrozaitytė L., Rančelis T., Utkus A., Kučinskas V, & Preikšaitienė E. (2023). Molecular and Functional Characterisation of a Novel Intragenic 12q24.21 Deletion Resulting in MED13L Haploinsufficiency Syndrome. Medicina, 59(7), 1225.

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Culturing Human and Primate Cell Lines

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CHM13 cell cultured in complete AmnioMax C-100 Basal Medium (Thermo Fisher Scientific, 17001082) supplemented with 15% AmnioMax C-100 Supplement (Thermo Fisher Scientific, 12556015) and 1% penicillin-streptomycin (Thermo Fisher Scientific, 15140122). HG00733 cells were cultured in RPMI 1640 with L-glutamine (Thermo Fisher Scientific, 11875093) supplemented with 15% FBS (Thermo Fisher Scientific, 16000-044) and 1% penicillin-streptomycin (Thermo Fisher Scientific, 15140122). Chimpanzee (Pan troglodytes; Clint; S006007) and macaque (Macaca mulatta; AG07107) cells were cultured in MEM α containing ribonucleosides, deoxyribonucleosides, and L-glutamine (Thermo Fisher Scientific, 12571063) supplemented with 12% FBS (Thermo Fisher Scientific, 16000-044) and 1% penicillin-streptomycin (Thermo Fisher Scientific, 15140122). Orangutan (Pongo abelii; Susie; PR01109) cells were cultured in MEM α containing ribonucleosides, deoxyribonucleosides, and L-glutamine (Thermo Fisher Scientific, 12571063) supplemented with 15% FBS (Thermo Fisher Scientific, 16000-044) and 1% penicillin-streptomycin (Thermo Fisher Scientific, 15140122). All cells were cultured in a humidity-controlled environment at 37°C with 5% CO₂.

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