The HEKs were derived from neonatal foreskin and grown in serum-free EpiLife™ cell culture medium (Life Technologies) containing 0.06 mmol/L calcium chloride, 1% human keratinocyte growth supplement S7 (Life Technologies), and 1% gentamicin/amphotericin as described previously.9 To investigate the effects of the B. longum, GOS, and a synbiotic mixture (B. longum and GOS) on the mRNA expression of filaggrin (FLG) and loricrin (LOR), HEKs were differentiated in the presence of 1.3 mmol/L CaCl2 for 3 days. Cells were then incubated with B. longum (0.01, 0.05, or 0.1 CFU/cell), GOS (0.01%), a synbiotic mixture, a combination of IL-4 (5 ng/mL, R&D Systems, Minneapolis, MN, USA) and IL-13 (5 ng/mL, R&D Systems), or interferon (IFN)-γ (10 ng/mL, R&D Systems) for additional 24 hours. To investigate the effects of BLEVs on the expression of FLG and LOR, HEKs were differentiated in the presence of 1.3 mmol/L CaCl2 for 3 days. HEKs were then incubated with BLEVs (0, 25, 50, or 100 ng/mL) for additional 24 hours. All reagents including B. longum, GOS, cytokines, and BLEVs were diluted using the serum-free EpiLife™ cell culture medium (Life Technologies) for in vitro experiments.
Epilife cell culture medium
Manufactured by Thermo Fisher Scientific
Sourced in United States, Spain
EpiLife™ cell culture medium is a serum-free, defined medium designed for the culture of human epidermal keratinocytes. It provides a physiologically relevant environment for the growth and differentiation of keratinocytes.
Automatically generated - may contain errors
Lab products found in correlation
Epilife defined growth supplement, by Thermo Fisher Scientific (2 mentions)
Keratinocyte sfm medium, by Thermo Fisher Scientific (2 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (2 mentions)
Bicinchoninic protein assay kit, by Thermo Fisher Scientific (1 mentions)
Interferon ifn γ, by R&D Systems (1 mentions)
Human keratinocyte growth supplement s7, by Thermo Fisher Scientific (1 mentions)
Interleukin 4 (il 4), by R&D Systems (1 mentions)
Il 13, by R&D Systems (1 mentions)
Hconepics, by Innoprot (1 mentions)
Im ocular epithelial cell medium kit, by Innoprot (1 mentions)
5 protocols using epilife cell culture medium
1
Skin Cell Response to Synbiotic Mixture
2
Cultivation and Characterization of Human Keratinocytes
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Human primary keratinocytes were purchased from Thermo Fisher Scientific. Cells were grown in a 37°C incubator in serum-free Epi-Life cell culture medium (Gibco) supplemented with Epi-Life Defined Growth Supplement containing 0.05 mM Ca2+. NTERT 2G keratinocytes were a gift from the laboratory of Johann Gudjonsson (University of Michigan, Ann Arbor, Michigan, USA) and cultured in Keratinocyte SFM medium supplemented with EGF and BPE (Gibco) prior to use for luciferase and infection studies. Complete keratinocyte-associated methods, including circadian synchronization, siRNA experiments, and infections, can be found in Supplemental Methods .
3
Cultivation and Characterization of Human Keratinocytes
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Human primary keratinocytes were purchased from Thermo Fisher Scientific. Cells were grown in a 37°C incubator in serum-free Epi-Life cell culture medium (Gibco) supplemented with Epi-Life Defined Growth Supplement containing 0.05 mM Ca2+. NTERT 2G keratinocytes were a gift from the laboratory of Johann Gudjonsson (University of Michigan, Ann Arbor, Michigan, USA) and cultured in Keratinocyte SFM medium supplemented with EGF and BPE (Gibco) prior to use for luciferase and infection studies. Complete keratinocyte-associated methods, including circadian synchronization, siRNA experiments, and infections, can be found in Supplemental Methods .
4
Bifidobacterium longum Interacts with Keratinocytes
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B. longum was grown in tryptic soy broth, and 5 × 106 colony-forming units (CFU) /flask (150 cm2) of B. longum were incubated with differentiated human primary epidermal keratinocytes (HEKs) in serum-free Epilife™ cell culture medium (Life Technologies, Grand Island, NY, USA) for 48 hours. Then, culture medium was centrifuged at 8,000 rpm for 20 minutes. The supernatant was filtered using a 0.22-μm bottle-top filter to remove cellular debris and stored at −80°C until use. Non-conditioned control medium was generated in the same manner as above, except that no cells were cultured on the plates. Protein concentrations of BLEVs were measured using a Bicinchoninic Protein Assay Kit (ThermoFisher Scientific, Rockford, IL, USA).
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B. longum was grown in tryptic soy broth, and 5 × 106 colony-forming units (CFU) /flask (150 cm2) of B. longum were incubated with differentiated human primary epidermal keratinocytes (HEKs) in serum-free Epilife™ cell culture medium (Life Technologies, Grand Island, NY, USA) for 48 hours. Then, culture medium was centrifuged at 8,000 rpm for 20 minutes. The supernatant was filtered using a 0.22-μm bottle-top filter to remove cellular debris and stored at −80°C until use. Non-conditioned control medium was generated in the same manner as above, except that no cells were cultured on the plates. Protein concentrations of BLEVs were measured using a Bicinchoninic Protein Assay Kit (ThermoFisher Scientific, Rockford, IL, USA).
5
In Vitro Tolerance of Human Cell Lines
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Two different human cell lines were employed to assess the in vitro tolerance of the developed preparations. Immortalized human corneal epithelial cells (HCECs; Evercyte GmbH, Vienna, Austria) were maintained at 37 °C under 5% CO2 in a humid atmosphere (95%). The medium was changed every 48–72 h. HCECs were kept in an EpiLife® cell-culture medium (Life Technologies, Madrid, Spain) with EDGS® 1X (Life Technologies, Madrid, Spain) and penicillin–streptomycin 1% (Life Technologies, Madrid, Spain) as supplementation. Likewise, immortalized human conjunctival epithelial cells (HConEpiCs; Innoprot, Bizkaia, Spain) were maintained under the same temperature, humidity, and CO2 conditions, while the medium was changed every 48 h. HConEpiCs were cultured using the IM-Ocular Epithelial Cell Medium Kit (Innoprot, Bizkaia, Spain). To ensure correct cell attachment and maintenance, the flasks were coated with collagen I (1 mg/mL) (Innoprot, Bizkaia, Spain).
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