The cell line HTR8/SVneo (ATCC® CRL3271™) is a normal human extravillous trophoblast cell line that was created upon transfection of cells derived from human first-trimester placenta, with the gene that encodes simian virus 40 large T antigen. The HTR8/SVneo cell line was cultured in RPMI 1640 medium that was supplemented with 5% FBS, penicillin-streptomycin, and amphotericin B (Invitrogen, Cergy Pontoise, France). The cells were maintained in a humidified incubator at 37 °C with 5% CO2. In the figures, the HTR8/SVneo cell line is referred to as HTR.
Crl 3271
Manufactured by American Type Culture Collection
Sourced in United States
The CRL-3271 is a cell culture media product offered by American Type Culture Collection (ATCC). It is a basal medium designed to support the growth and maintenance of a variety of mammalian cell lines. The formulation and composition of CRL-3271 are specifically tailored for this purpose, providing the necessary nutrients and growth factors required for cell proliferation and viability.
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Lab products found in correlation
Htr 8 svneo, by American Type Culture Collection (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Amphotericin b, by Thermo Fisher Scientific (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Rpmi medium, by Thermo Fisher Scientific (1 mentions)
Cc 2935, by Lonza (1 mentions)
Cc 3156, by Lonza (1 mentions)
Bewo cells, by RIKEN Cell Bank (1 mentions)
Rpmi 1640 culture medium, by Nacalai Tesque (1 mentions)
Mycoalert, by Lonza (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Rpmi 1640 growth medium, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Trypsin edta, by Merck Group (1 mentions)
Pro ox 110 oxygen controlling regulator, by Biospherix (1 mentions)
Rpmi 1640 medium, by GE Healthcare (1 mentions)
Penicillin streptomycin p s, by Thermo Fisher Scientific (1 mentions)
Ampflstr identifiler plus pcr amplification kit, by Thermo Fisher Scientific (1 mentions)
3730xl genetic analyzer, by Thermo Fisher Scientific (1 mentions)
12 protocols using crl 3271
1
Culturing Human Trophoblast Cell Line
2
Endometrial Stromal Cell Lines and Trophoblast Immortalization
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In this study, two endometrial stroma cell lines were used: the human EnS cell line St-T1 known to be a reliable model for human endometrium and early decidua [27 (link), 28 (link)] (a generous gift from Professor Brosens, University of Warwick, Coventry, UK) [29 (link)]. This cell line was further used in our laboratory to generate EnS cell line with inducible, stable knock-down (kd) for Sdc-1 named KdS1 as published before [25 (link)]. Furthermore, HTR8/SVneo (ATCC® CRL3271™) immortalized first-trimester trophoblast cells were used [30 (link)].
3
Zika Virus Infection of Placental Cell Lines
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The ZIKV strain H/PF/2013 from French Polynesia, which belongs to the recent epidemic lineage, was used in this study. The virus was grown in C6/36 cells and titrated by plaque assay on Vero cells (titer: 1.5 × 107 PFU/mL). Three human placental cell lines were used for virus growth kinetics. HTR-8/SVneo (ATCC® CRL-3271™) and Swan7121 (link) are immortalized first trimester trophoblast cell lines and VSC is a primary villous stromal cell line. HTR-8/SVneo and Swan71 cells were obtained from the Department of Obstetrics, University of Jena. VSC cells were obtained from the Department of Developmental Pathology, University of Bonn. VSC cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco®, Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal calf serum (FCS) (Biochrom, Berlin, Deutschland) and 2 mM glutamine (Gibco®). Swan71 cells were cultivated in DMEM medium containing 10% FCS. HTR-8/SVneo cells were cultivated in RPMI medium (Gibco®) containing 10% FCS. All cells were incubated at 37 °C with 5% CO2.
4
Placental Microfluidic Culture System
5
Cell Culture of HTR-8 and BeWo Cells
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HTR-8/SVneo cells (subsequently referred to as HTR-8 cells) were obtained from ATCC® (CRL3271™) and cultured in RPMI-1640 culture medium (Nacalai Tesque, Japan). BeWo cells were obtained from Riken Cell Bank (Ibaraki, Japan) and cultured in Dulbecco’s Modified Eagle Medium/Ham F-12 culture medium (Nacalai Tesque, Japan). The medium was supplemented with 10% fetal bovine serum (FBS), penicillin (100 IU/mL), and streptomycin (100 mg/mL).
6
Culturing and Characterizing HTR-8/SVneo Cells
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HTR-8/SVneo cells (ATCC, CRL-3271™, Lot# 64275781, Manassas, VA, USA) were cultivated in RPMI-1640 medium (Gibco; 31870074, Carlsbad, CA, USA) containing 5% fetal bovine serum (FBS; PanBiotech; P40-38100, Aidenbach, Germany) and 1% glutamax (Gibco; 35050061, Carlsbad, CA, USA). The cells were sub-cultured every 3–5 days. CASY cell counter and analyzer (CASY; Innovatis Technologies Inc., Fairfax, VA, USA) was used to determine the cell number. The conditions for cell cultivation in the incubator were 37 °C and 5% CO2. Mycoplasma contamination was tested on a regular basis (MycoAlert; Lonza, Basel, Switzerland). HTR-8/SVneo cells from passages 86 to 96 were used.
Human placental endothelial cells (HPEC) and human trophoblast cells (hTC) used in the immunoblot (Figure 1 ) were isolated from healthy placentas according to previous studies [4 (link),52 (link)].
Human placental endothelial cells (HPEC) and human trophoblast cells (hTC) used in the immunoblot (
7
HSPA5 Gene Silencing in HTR8/SVneo Cells
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Gene silencing of HSPA5 was performed in the human placental trophoblast cell line HTR8/SVneo (CRL-3271™; ATCC, Manassas, Virginia). The procedure for gene silencing was described in detail previously.40 (link) Cells were grown in RPMI-1640 growth medium (Thermo Fisher Scientific, Waltham, Massachusetts). The growth medium was supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, Missouri) and 1× penicillin/streptomycin (Sigma-Aldrich). Cells were grown at 37 °C (5% CO2, humidified atmosphere), and 0.05% trypsin/0.02% EDTA was used in subculturing. HTR8/SVneo cells were reverse and forward transfected with siRNAs targeting HSPA5 (sense GAUAAUCAACCAACUGUUA, antisense UAACAGUUGGUUGAUUAUC) (Sigma-Aldrich). For the control, MISSION siRNA Universal Negative Control #1 (Sigma-Aldrich) was transfected in the same way as the siRNAs that targeted HSPA5. Lipofectamine 3000 (Invitrogen, Carlsbad, California) was used as a transfection reagent. Cells (100,000/well) were incubated with 10 nM siRNA concentration in the reverse transfection. Forward transfection was performed after 24 h of incubation. In the forward transfection, cells were transfected again with siRNA concentrations of 10 nM. Cells were incubated with siRNAs for 48 h and then harvested with 1× trypsin–EDTA (Sigma-Aldrich).
8
Hypoxic Culture of HTR8/Svneo Cells
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The human HTR8/Svneo first trimester trophoblast cell line was purchased from the American Type Culture Collection (CRL-3271; Lot #70016636, ATCC, Manassas, VA, USA) and cultured in RPMI-1640 medium (GE Healthcare, Piscataway, NJ, USA), supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1 mmol/L sodium pyruvate, and 1% penicillin/streptomycin (P/S) (all Gibco, ThermoFisher, Waltham, MA, USA). Cells were maintained at 37 °C in a humidified incubator with 5% CO2. For hypoxia experiments, cells were seeded in the appropriate cell culture plastic wells and maintained at 21%, 8%, 3%, or 1% O2 for 24 h to 72 h prior to experiments. Hypoxic chamber C-474 equipped with Pro-Ox 110 oxygen controlling regulator (BioSpherix, Parish, NY, USA) was used.
9
Microsatellite Genotyping of Cell Lines
10
Immortalized Trophoblast Cell Lines
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Swan 71 cells, immortalized by human telomerase, were constructed by Gil Mor lab at Yale University.[84 (link)
] HTR‐8/SVneo cells were commercially available (CRL‐3271, ATCC, USA). Swan 71 cells and HTR‐8/SVneo cells were, respectively, cultured in DMEM medium (Gibco, Invitrogen, Carlsbad, CA, USA) and RPMI 1640 medium (Gibco, Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS (fetal bovine serum) at 37 °C with 5% v/v CO2. Swan 71 cells were also synchronized in the G2 phase using double‐thymidine block and release protocols.[85
] Briefly, trophoblast cells were incubated in medium containing 2 mm thymidine (Sigma–Aldrich) for 18 h; then, in fresh medium for 9 h; and then, incubated in medium containing 2 mm thymidine for 18 h to synchronize cells in the S phase. Subsequently, the cells were incubated in fresh medium for 5 h to synchronize cells in the G2 phase, which were further verified by flow cytometry analysis.
] HTR‐8/SVneo cells were commercially available (CRL‐3271, ATCC, USA). Swan 71 cells and HTR‐8/SVneo cells were, respectively, cultured in DMEM medium (Gibco, Invitrogen, Carlsbad, CA, USA) and RPMI 1640 medium (Gibco, Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS (fetal bovine serum) at 37 °C with 5% v/v CO2. Swan 71 cells were also synchronized in the G2 phase using double‐thymidine block and release protocols.[85
] Briefly, trophoblast cells were incubated in medium containing 2 m
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