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Essential 8 media

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Essential 8 media is a serum-free, xeno-free medium formulated to support the culture and expansion of human pluripotent stem cells. It provides the essential components required for the maintenance of undifferentiated human embryonic stem cells and induced pluripotent stem cells.

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Lab products found in correlation

Glutamax, by Thermo Fisher Scientific (14 mentions) Geltrex, by Thermo Fisher Scientific (9 mentions) Growth factor reduced matrigel, by Corning (6 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions) Vitronectin, by Thermo Fisher Scientific (4 mentions) Non essential amino acids (neaa), by Thermo Fisher Scientific (4 mentions) Essential 6 media, by Thermo Fisher Scientific (3 mentions) Matrigel, by Corning (3 mentions) Accutase, by Merck Group (3 mentions) Accutase, by Thermo Fisher Scientific (2 mentions) Rock inhibitor y 27632, by Merck Group (2 mentions) Matrigel, by BD (2 mentions) Relesr reagent, by STEMCELL (2 mentions) Matrigel hesc qualified matrix, by Corning (2 mentions) Relesr, by STEMCELL (2 mentions) Induction media, by Thermo Fisher Scientific (2 mentions) N2 supplement, by Thermo Fisher Scientific (2 mentions) A13777, by Thermo Fisher Scientific (2 mentions) Puromycin, by Takara Bio (2 mentions) Lipofectamine stem, by Thermo Fisher Scientific (2 mentions)

Spelling variants of this product

Essential 8 (61 citations) Essential 8 Medium media (6 citations) Essential 8 Flex media (5 citations) Essential 8 culture media (2 citations) Essential 8 (E8) media change (2 citations) Essential 8 Flex Media Kit (2 citations)

53 protocols using essential 8 media

1

Fibroblast Reprogramming to hiPSCs

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A skin punch biopsy was performed and dermal fibroblasts were acquired from this biopsy under informed consent as outlined in and approved by Stanford’s Institutional Review Board (IRB) protocols. Fibroblasts were grown in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% pen/strep on gelatin-coated flasks. Fibroblasts were passaged using trypsin and 1 × 106 fibroblasts were electroporated using 10 µg of reprogramming episomal vectors35 (link) using the Neon® Transfection System. Fibroblasts were plated onto Matrigel™-coated 10 cm culture wells in DMEM supplemented with 10% FBS, 1% pen/strep, and hydrocortisone. Fibroblasts were then grown using Essential 7 (E7) media [Essential 6 (Invitrogen) with FGF2 (50 µg/L)] and sodium butyrate (0.2 mM NaB) (B5887 diluted in DMSO, Sigma) for thirteen days and then switched to Essential 8 (E8) media (Invitrogen) until hiPSC colonies were formed. hiPSC colonies were picked and expanded until passage 10 before being used in experiments outlined below. hiPSCs were tested to be mycoplasma negative using the Mycoalert Mycoplasma testing kits (LT07-318, Lonza).
Churko J.M., Garg P., Treutlein B., Venkatasubramanian M., Wu H., Lee J., Wessells Q.N., Chen S.Y., Chen W.Y., Chetal K., Mantalas G., Neff N., Jabart E., Sharma A., Nolan G.P., Salomonis N, & Wu J.C. (2018). Defining human cardiac transcription factor hierarchies using integrated single-cell heterogeneity analysis. Nature Communications, 9, 4906.
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2

Episomal Reprogramming of Dermal Fibroblasts

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Dermal fibroblasts were grown in fibroblast media (DMEM supplemented with 10% FBS and 1% pen/strep) on gelatin-coated T225 flasks. Fibroblasts were dissociated using trypsin and 3 × 106 fibroblasts were reseeded the day before electroporation. On the day of electroporation, one million fibroblasts were electroporated with a total of 10 μg of episomal vectors25 (link) (Supplementary Fig. 9A) using the Neon Transfection System (Invitrogen). Fibroblasts were plated onto a Matrigel-coated (356231, BD Biosciences) 10 cm dish in fibroblast media supplemented with hydrocortisone (Sigma H0396 diluted in DMEM/F12) for three days. Fibroblasts were then switched to essential 7 (E7) media (essential 6 (Invitrogen) with FGF2 (50 μg l-)) supplemented with sodium butyrate (0.2 mM NaB) (B5887 diluted in DMSO, Sigma) for 13 days. Fibroblasts were then switched to essential 8 (E8) media (Invitrogen) until hiPSC colonies were picked (day 30 after electroporation). Picked colonies were passaged at a 1:10 ratio using Accutase (Invitrogen) and reseeded in E8 containing a 10 μM ROCK inhibitor for the first day (Sigma, Y27632). Each day, hiPSCs were fed with fresh E8 media. All further culturing and passaging of hiPSCs was performed by this method.
Churko J.M., Lee J., Ameen M., Gu M., Venkatasubramanian M., Diecke S., Sallam K., Im H., Wang G., Gold J.D., Salomonis N., Snyder M.P, & Wu J.C. (2017). Transcriptomic and epigenomic differences in human induced pluripotent stem cells generated from six reprogramming methods. Nature biomedical engineering, 1(10), 826-837.
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3

Differentiation of hiPSCs into MSCs

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Differentiation and maintenance of the hiPSC-MSCs have been previously described (Winston et al., 2019 (link)). Simply, the hiPSCs were seeded on Geltrex-coated 6-well plates and maintained in Essential 8 (E8) media (Life Technologies, Ca# A1517001). Next, hiPSCs were differentiated into MSCs using a specified differentiation media consisting of 10 ng/ml bFGF (R&D Systems, Ca# 233-FB), 4 μM SB431542 (Stemgent, Ca# 04-0010-10), and 4 μM WNT agonist CHIR99021 (CHIR) (Stemgent, 04-2004) in Essential 6 (E6) media (Life Technologies, A1516401). The differentiated hiPSC-MSCs were maintained in the CTS StemPro MSC SFM media (Life Technologies, A1033201) and passaged every 6 days.
Winston T.S., Chen C., Suddhapas K., Tarris B.A., Elattar S., Sun S., Zhang T, & Ma Z. (2022). Controlling Mesenchyme Tissue Remodeling via Spatial Arrangement of Mechanical Constraints. Frontiers in Bioengineering and Biotechnology, 10, 833595.
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4

Differentiating iPSCs into Definitive Endoderm

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The joxm_1 IPSC line was cultured in Essential 8 (E8) media (LifeTech) according to the manufacturer’s instructions. For dissociation and plating, cells were washed × 1 with DPBS and dissociated using StemPro Accutase (Life Technologies, A1110501) at 37 °C for 3–5 min. Colonies were fully dissociated through gentle pipetting. Cells were washed × 1 with MEF medium [23 (link)] and pelleted gently by centrifuging at 285×g for 5 min. Cells were re-suspended in E8 media, passed through a 40-μm cell strainer, and plated at a density of 60,000 cells per well of a gelatin/MEF-coated 12-well plate in the presence of 10 μM Rock inhibitor—Y27632 [10 mM] (Sigma, Cat # Y0503—5 mg). Media was replaced with fresh E8 free of Rock inhibitor every 24 h post-plating. Differentiation into definitive endoderm commenced 72 h post-plating as previously described [23 (link)].
Linker S.M., Urban L., Clark S.J., Chhatriwala M., Amatya S., McCarthy D.J., Ebersberger I., Vallier L., Reik W., Stegle O, & Bonder M.J. (2019). Combined single-cell profiling of expression and DNA methylation reveals splicing regulation and heterogeneity. Genome Biology, 20, 30.
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5

Induced Pluripotent Stem Cell Maintenance

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(embryonic and induced) were maintained on Vitronectin coated dishes in Essential 8 (E8) media (Thermo) as previously described52 (link). Induced pluripotent stem cells were purchased from Coriell, Patient normal lines 41865, 41866 and patient ALS (A4V) lines, 35659, 35673 and 35677. Cells were used for differentiation between passage 30–50 and passaged two times every week. Cells were subjected to mycoplasma testing every 2–3 months.
Tchieu J., Calder E.L., Guttikonda S.R., Gutzwiller E.M., Aromolaran K.A., Steinbeck J.A., Goldstein P.A, & Studer L. (2019). NFIA is a gliogenic switch enabling rapid derivation of functional human astrocytes from pluripotent stem cells. Nature biotechnology, 37(3), 267-275.
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6

Induced Pluripotent Stem Cell Maintenance

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(embryonic and induced) were maintained on Vitronectin coated dishes in Essential 8 (E8) media (Thermo) as previously described52 (link). Induced pluripotent stem cells were purchased from Coriell, Patient normal lines 41865, 41866 and patient ALS (A4V) lines, 35659, 35673 and 35677. Cells were used for differentiation between passage 30–50 and passaged two times every week. Cells were subjected to mycoplasma testing every 2–3 months.
Tchieu J., Calder E.L., Guttikonda S.R., Gutzwiller E.M., Aromolaran K.A., Steinbeck J.A., Goldstein P.A, & Studer L. (2019). NFIA is a gliogenic switch enabling rapid derivation of functional human astrocytes from pluripotent stem cells. Nature biotechnology, 37(3), 267-275.
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7

Fibroblast Reprogramming into iPSCs

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Fibroblasts were reprogrammed into iPSC by viral transduction of cMyc, Oct4, Klf4 and Sox2 as described previously (17 (link)). Briefly, fibroblasts were transduced with viral particles and maintained on a mouse embryonic fibroblast feeder layer in hESC medium [KO-DMEM, 20% knockout serum replacement, 2 mm l-glutamine, 1 × nonessential amino acids, 50 μM 2-mercaptoethanol, 50 U ml−1 penicillin, 50 μg ml−1 streptomycin (all from Invitrogen), and 20 ng ml−1 FGF2 (Peprotech)] until colonies with iPSC morphology were observed. Colonies with iPSC morphology were mechanically picked and clonally expanded for further characterization. Karyotyping of iPSC was performed by Cell Guidance Systems, UK. Pluripotent stem cells (PSCs) were subsequently cultured under feeder-free conditions on Geltrex in Essential 8 media (Invitrogen). Cultures were fed daily and passaged every 5–7 days. The hESC line Shef 6 was obtained from the UK Stem Cell Bank, and control iPSCs, also generated using retroviral transduction, were obtained from the laboratory of Dr Tilo Kunath.
Sposito T., Preza E., Mahoney C.J., Setó-Salvia N., Ryan N.S., Morris H.R., Arber C., Devine M.J., Houlden H., Warner T.T., Bushell T.J., Zagnoni M., Kunath T., Livesey F.J., Fox N.C., Rossor M.N., Hardy J, & Wray S. (2015). Developmental regulation of tau splicing is disrupted in stem cell-derived neurons from frontotemporal dementia patients with the 10 + 16 splice-site mutation in MAPT. Human Molecular Genetics, 24(18), 5260-5269.
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8

Generation of Trisomy 21 iPSCs from Urine

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Dissociated single urine‐derived T21 cells (6 × 105 cells) were electroporated with 4.2 µl of Episomal iPSC Reprogramming vectors (Invitrogen, Waltham, MA, http://www.thermofisher.com/) using an Amax 4D‐Nucleofector device with P1 solution and the program EA‐104. The electroporated cells were transferred to Vitronectin (VTN‐N, Invitrogen) coated 100mm dish in N2B27 media with 3 µM CHIR99021 (Stemgent, Cambridge, MA, www.stemgent.com), 0.5 µM PD0325901 (Stemgent), 0.5 µM A‐83‐01 (Stemgent), 10 ng/ml hLIF (Invitrogen), 10 µM Y‐27632 (Stemgent), 100 ng/ml basic fibroblast growth factor (bFGF, Invitrogen). Half of the media were changed every day until 14 days after transfection and on day 15, media were switched into Essential 8 media (Invitrogen) until embryonic stem cell (ESC)‐like colonies were generated. From day 25 to 30 of transfection, ESC‐like colonies were picked and transferred to VTN‐N coated 12‐well plate for expansion and several passages were performed to establish T21‐iPSC lines. Each cell line was named CWRU1XXXi‐YYT; CWRU (Case Western Reserve University) is the institution name, 1XXX is the deidentified cell line number from each donor (starting at #1001, to prevent any potential confusion with other iPSC lines produced in our institution), YY is the clone number, and T represents trisomy, respectively, according to a proposed nomenclature convention 21.
M. Lee Y., Zampieri B.L., Scott‐McKean J.J., Johnson M.W, & Costa A.C. (2017). Generation of Integration‐Free Induced Pluripotent Stem Cells from Urine‐Derived Cells Isolated from Individuals with Down Syndrome. Stem Cells Translational Medicine, 6(6), 1465-1476.
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9

Differentiation of Human Embryonic Stem Cells to Induced Keratinocytes

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Human ESCs (H9, female, Stanford Stem Cell Bank), mutant, and transgenic cell lines were seeded in culture plates using Matrigel hESC-Qualified Matrix (BD Corning) and were maintained in Essential 8 media (Life Technologies). Colonies were passaged as clumps every 2 days using 0.5 mM EDTA. Mouse 3T3 J2 fibroblasts (male, ATCC) were maintained in DMEM with 10% FBS. Induced keratinocytes (iKCs) and Normal Human Keratinocytes (NHKs) were maintained in Defined Keratinocyte Medium (Life Technologies). RA/BMP4 differentiation was performed as previously described5 (link). Briefly, hESCs were switched to Essential 6 media supplemented with 5ng/ml BMP4 (R&D Systems) and 1μM RA (Sigma) for seven days. To further differentiate the cells to iKCs, the medium was changed to Defined Keratinocyte Serum Free Medium (DKSFM) with growth supplements containing EGF and FGF (Life Technologies) for 50 days total. To induce terminal keratinocyte differentiation, confluent keratinocytes were cultured in 2μM CaCl2 for 72 hours. All cells are regularly tested for Mycoplasma and were authenticated with sequencing.
Collier A., Liu A., Torkelson J., Pattison J., Gaddam S., Zhen H., Patel T., McCarthy K., Ghanim H, & Oro A. (2022). Gibbin mesodermal regulation patterns epithelial development. Nature, 606(7912), 188-196.
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10

Pluripotent Stem Cell Generation from Diverse Sources

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In this study we used the following hESCs: H1 (purchased from WiCell)39 (link); 207, 360, and 429 obtained from the Karolinska Institute47 . BJ (CRL-2522), CRL 2097 and Detroit 551 (CCL-110) fibroblasts were obtained from the American Type Culture Collection. For retroviral based hiPSC generation, hOCT4, hSOX2, hKLF4 and hcMYC, retrovirus viral particles were purchased from Vectalys and transduced at an MOI of 5 as previously described by Vallier and colleagues48 (link). For non-integrative Sendai virus mediated reprogramming, we used the CytoTune™-iPS 2.0 Sendai Reprogramming Kit (Life Technologies) containing polycistronic Klf4–Oct3/4–Sox2, cMyc, and Klf4, according to the manufactures instructions for feeder free derivation. For both retro- and Sendai virus reprogramming, hiPSC colonies were manually picked into Matrigel (Sigma) coated dishes, cultivated and expanded clonally in Essential 8 Media (Life Technologies). The hiPSC cell lines were verified for the expression of pluripotency related genes by immunofluorescence, FACS, karyotyping and RT-qPCR11 (link).
Siller R., Naumovska E., Mathapati S., Lycke M., Greenhough S, & Sullivan G.J. (2016). Development of a rapid screen for the endodermal differentiation potential of human pluripotent stem cell lines. Scientific Reports, 6, 37178.
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