Recombinant human fgf10

Manufactured by Thermo Fisher Scientific

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Recombinant human FGF10 is a growth factor protein produced in a laboratory setting. It plays a role in the regulation of cell growth, differentiation, and angiogenesis.

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15 protocols using recombinant human fgf10

Expansion of Human Liver Organoids

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After 1 week of seeding the organoids, isolation media were changed to human liver expansion media (EM; Ad+++ supplemented with 1X B27 supplement without retinoic acid (Gibco), 1X N2 supplement (Gibco), 1.25 mM N-acetyl-L-cysteine (Sigma), 20% (vol/vol) Rspo-1 conditioned medium, 1.25% (vol/vol) Wnt3a conditioned medium [Barker et al., 2010 (link)], 10 mM nicotinamide (Sigma), 10 nM recombinant human (Leu15)-gastrin I (Sigma), 50 ng/ml recombinant human EGF (Peprotech), 100 ng/ml recombinant human FGF10 (Peprotech), 25 ng/ml recombinant human HGF (Peprotech), 10 μM forskolin (Sigma), and 5 μM A8301 (Tocris)) (Huch et al., 2015 (link)).
EM was changed twice a week, and cultures were split every 7–10 days according to organoid density. For passaging (1:4-1:8, depending on growth rate of the culture), organoids were resuspended in 10 ml Ad+++, incubated in ice for 10 min, and collected by centrifugation (5 min at 200 xg). Subsequently, organoids were incubated for 1–2 min in TrypLE Express at RT and mechanically disrupted by pipetting. After a further wash in Ad+++, cells were resuspended in BME solution and seeded in 24- or 48-well suspension plates. After BME solution had solidified, wells were filled with 500 µl (24 wells) or 250 µl (48 wells) of human liver organoid expansion medium.

De Crignis E., Hossain T., Romal S., Carofiglio F., Moulos P., Khalid M.M., Rao S., Bazrafshan A., Verstegen M.M., Pourfarzad F., Koutsothanassis C., Gehart H., Kan T.W., Palstra R.J., Boucher C., IJzermans J.N., Huch M., Boj S.F., Vries R., Clevers H., van der Laan L.J., Hatzis P, & Mahmoudi T. (2021). Application of human liver organoids as a patient-derived primary model for HBV infection and related hepatocellular carcinoma. eLife, 10, e60747.

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Organoid Generation from Primary Hepatocytes

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Three lots of PHHs (lots DOO; Celsis, HC10-10; XENOTECH, HC4-24; XENOTECH) were used to generate organoids (Table S1). PHHs were washed with cold Advanced DMEM/F12 (Thermo Fisher Scientific) and spun at 400 g for 5 min. The cell pellet was mixed with Matrigel (growth factor reduced, Corning) and 1 × 10⁴ cells were seeded per well in a 24-well plate. After the Matrigel had solidified, 500 μl of organoid expansion medium was added to each well. The organoid expansion medium was prepared as described in a previous report [13 (link)]. Briefly, Advanced DMEM/F12 was supplemented with 1% Antibiotic Antimycotic Solution and 1 × GlutaMAX (GIBCO), 10 mM HEPES (Nacalai Tesque), 2% B27 supplement (GIBCO), 1.25 mM N-Acetylcysteine (Sigma), 10 mM Nicotinamide (Sigma), 10 nM recombinant gastrin (Merk), 50 ng/ml EGF (R&D), 10% R-Spondin1 conditioned medium (homemade), 100 ng/ml recombinant human FGF10 (peprotech), 25 ng/ml recombinant human HGF (R&D), 5 μM A83-01 (Wako), and 10 μM Forskolin (Wako). During cultivation, the medium was refreshed every 3 days. For the establishment of the organoids, the medium was supplemented with 25 ng/ml Recombinant Noggin (R&D), 7.5 ng/mL Recombinant Wnt3a (R&D) and 10 μM Y27632 (Wako) for the first 3–4 days. Passage was performed in a 1:3 split ratio once every 10–14 days. Organoids passaged 5–10 times were used in all experiments.

Tong Y., Ueyama-Toba Y, & Mizuguchi H. (2023). Biliary epithelial cell differentiation of bipotent human liver-derived organoids by 2D and 3D culture. Biochemistry and Biophysics Reports, 33, 101432.

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Establishing Organoid Culture Conditions

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Advanced Dulbecco’s modified Eagle’s medium/F12, Antibiotic-Antimycotic (Anti-Anti), and B-27 supplement were from Thermo Fisher Scientific (Waltham, MA); recombinant human R-spondin-1, recombinant human EGF, recombinant human HGF, recombinant human FGF10, and recombinant human noggin were from PeproTech (Cranbury, NJ):, gastrin, N-acetylcysteine, Y27632, nicotinamide and A83-01 were from Millipore-Sigma (Burlington, MA); human recombinant Wnt3A, Forskolin and human recombinant IL-17A were from R&D Systems (Minneapolis, MN). Matrigel (354230) and cell recovery solution were from Corning (Kennebunk, ME, USA).

Garcia Moreno A.S., Guicciardi M.E., Wixom A.Q., Jessen E., Yang J., Ilyas S.I., Bianchi J.K., Pinto e Vairo F., Lazaridis K.N, & Gores G.J. (2023). IL-17 Signaling in Primary Sclerosing Cholangitis Patient-Derived Organoids. Research Square.

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Ngn3-GFP Mouse Pancreatic Organoid Protocol

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Ngn3-GFP mouse pancreatic organoids were prepared as previously described [19 (link),43 (link)] with minor modifications. Briefly, the pancreas was dissected and minced into pieces, followed by incubation with digestion solution (wash medium with 0.125 mg/mL collagenase IV, 0.125 mg/mL dispaseⅡ, and 0.1 mg/mL DNase I) for 1 h at 37 °C. Isolated ducts were embedded in Matrigel covered with the culture medium (5% R-spondin2-conditioned medium, 5% Noggin-conditioned medium, 2% B27 supplement (without vitamin A), 100 ng/mL recombinant human FGF10 (Peprotech, Rocky Hill, NJ, USA), 50 ng/mL recombinant mouse epidermal growth factor (Peprotech), 10 mM nicotinamide (FUJIFILM WAKO, Osaka, Japan), 1 mM N-acetylcysteine (Sigma Aldrich, St. Louis, MO, USA), and 10 nM recombinant human [Leu15]-gastrin1 (Sigma Aldrich) in the basal medium). Every 2–3 days, the medium was replaced with fresh medium.

Kimura-Nakajima C., Sakaguchi K., Hatano Y., Matsumoto M., Okazaki Y., Tanaka K., Yamane T., Oishi Y., Kamimoto K, & Iwatsuki K. (2021). Ngn3-Positive Cells Arise from Pancreatic Duct Cells. International Journal of Molecular Sciences, 22(16), 8548.

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Establishment and Aging of Gastric Organoids

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Gastric organoids were established according to a previous report from Nanki and colleagues (14 (link)). Briefly, stomachs removed from mice were minced with surgical scissors and processed by Liberase TH (Roche Diagnostics). The processed cells were suspended in Matrigel (Corning), plated onto 48-well multiple-well plates, and cultured in WRC+ medium, which consisted of Advanced DMEM/F-12 medium (Invitrogen) supplemented with GlutaMAX-I (Invitrogen), HEPES (Invitrogen), Penicillin-Streptomycin (Invitrogen), B-27 and N-2 supplement (Invitrogen), N-Acetylcysteine (Sigma-Aldrich), Valproic acid (Sigma-Aldrich), Recombinant murine EGF and Noggin (Peprotech), Afamin/Wnt3a CM (MBL Life Science), Chir99021 (R&D Systems), Recombinant human FGF10 (Peprotech), Y-27632 and SB431542 (R&D Systems), R-Spondin1 conditioned medium (generated from Cultrex HA-R-Spondin1-Fc 293T cell line; R&D Systems). The organoids were cultured for 5 to 7 days and passaged using TrypLE Express (Invitrogen). In the indicated experiments, the aged gastric organoids were cultured with or without 2 μg/mL recombinant Dickkopf3 (Dkk3; Peprotech) in the medium that lacked Wnt3a, R-Spondin1, and Chir99021 (WRC− medium).

Takeuchi A., Asano N., Imatani A., Saito M., Jin X., Saito M., Kanno T., Hatta W., Uno K., Koike T, & Masamune A. (2022). Suppressed Cellular Senescence Mediated by T-box3 in Aged Gastric Epithelial Cells may Contribute to Aging-related Carcinogenesis. Cancer Research Communications, 2(8), 772-783.

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Establishing Long-term HGSC Organoid Cultures

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Long-term HGSC organoid cultures were established and characterized earlier as described (29 (link)). The samples were cultured in 7.5 mg/ml BME-2 matrix (Cultrex, BioTechne) in sample-specific media (29 (link))—for EOC883 and EOC172, Medium 1 [Advanced DMEM/F12 (#12634010, Gibco), supplemented with 100 μg/ml Primocin (#ant-pm-1, InvivoGen), 10 mM HEPES (#15630080, Gibco), 1 mM N-acetylcysteine (#A7250, Sigma), 1× GlutaMAX (#35050061), 1× B-27 Supplement, 0.5 μM SB202190 (#HY-10295, MedChemExpress), 0.5 μM A83-01 (#SML0788, Sigma), 10 ng/ml recombinant human FGF-10 (#100-26, PeproTech), 10 ng/ml recombinant human FGF-4 (#100-31, PeproTech), 100 nM β-estradiol (#E2758, Sigma) and 5 mM nicotinamide (#N0636, Sigma)]; for EOC989, EOC540 and EOC382, Medium 2 (Medium 1 supplemented with 5 ng/ml EGF, 5 μM heregulin-1β, 0.5 μg/ml hydrocortisone and 5 μM forskolin).

Benada J., Bulanova D., Azzoni V., Petrosius V., Ghazanfar S., Wennerberg K, & Sørensen C.S. (2023). Synthetic lethal interaction between WEE1 and PKMYT1 is a target for multiple low-dose treatment of high-grade serous ovarian carcinoma. NAR Cancer, 5(3), zcad029.

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Isolation and Culture of Mouse Pancreatic Tumor Cells

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Mouse pancreatic tumour cells were isolated from the tumour bulk of mice older than 8 weeks as previous described^{17 (link),19 (link)}. Briefly, mouse pancreatic tumours were minced and digested by enzymatic dissociation with 5 mg/ml Collagenase type XI (Gibco), 1 mg/ml Dispase (Gibco), 1% FBS (Gibco) in DMEM medium (Gibco) at 37 °C for a maximum of 16 hrs. Isolated material was incubated with TrypLE (Gibco) at 37 °C for 10 min, embedded into growth factor-reduced Matrigel (Corning), and cultured in mouse complete medium (AdDMEM/F12 (Gibco) supplemented with 1% penicillin/streptomycin (Gibco), 1% GlutaMAX (Gibco), 10 mM HEPES (Gibco), 1:50 B27 supplement (Gibco), 1.25 mM N-Acetylcysteine (Sigma), 10% (vol/vol) Rspo1-conditioned media, 10 mM Nicotinamide (Sigma), 10 nM recombinant human-gastrin I (Tocris), 50 ng/ml recombinant mouse EGF (Gibco), 100 ng/ml recombinant human FGF10 (Peprotech), 0.5 µM A83-01 (Tocris), and 100 ng/ml recombinant human Noggin (Peprotech)). Mouse complete medium was changed twice a week, and cultures were split upon the attainment of dense culture. Passage was performed in a 1:4–1:8 split ratio.

Filippini D., Agosto S.D., Delfino P., Simbolo M., Piro G., Rusev B., Veghini L., Cantù C., Lupo F., Ugel S., De Sanctis F., Bronte V., Milella M., Tortora G., Scarpa A., Carbone C, & Corbo V. (2019). Immunoevolution of mouse pancreatic organoid isografts from preinvasive to metastatic disease. Scientific Reports, 9, 12286.

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Intratracheal Instillation of Urban PM

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The PM (1649b) was obtained from the Standard Reference Material Program, and this material is certified by the National Institute of Standards and Technology (MD, USA). The PM used in the present study primarily comprised of common components of urban PM, including pesticides, dioxins, polycyclic aromatic hydrocarbons and polychlorinated biphenyl congeners. The PM was used for murine intratracheal instillation [63 (link)]. The recombinant human FGF10 was obtained from PeproTech (Shanghai, China). The anti-HMGB1, anti-TLR4 and anti-GAPDH were obtained from Cell Signaling Technology (MA, USA). The anti-pFGFR2 was obtained from Affinity Biosciences. Beyotime (Shanghai, China) was the source of all other western blot reagents. The ELISA kits for defined antigens (IL-6, IL-8, TNF-α and HMGB1) were obtained from Boyun Biotechnology (Shanghai, China). The real-time polymerase quantitative chain reaction (RT-qPCR) primers were obtained from Sangon Biotech (Shanghai, China). All other RT-qPCR reagents were obtained from Takara Bio (Shiga, Japan).

Liu L., Song C., Li J., Wang Q., Zhu M., Hu Y., Chen J., Chen C., Zhang J.S., Dong N, & Chen C. (2020). Fibroblast growth factor 10 alleviates particulate matter-induced lung injury by inhibiting the HMGB1-TLR4 pathway. Aging (Albany NY), 12(2), 1186-1200.

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Multicomponent Cell Culture Protocol

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Acetone, ethanol, and IPA were purchased from Millipore. The SU-8 and SU-8 developers were purchased from MicroChem. The amorphous fluoroplastic solution was purchased from Chemours Company. Pluronic F127 was purchased from Sigma-Aldrich (Oakville, ON, USA). Silicone oil (1 Cst) was purchased from Clearco, USA. Fetal bovine serum (FBS), PBS, collagenase II, Hank’s Balanced Salt Solution (HBSS), Earle’s balanced salt solution (EBSS), DMEM/F12, Glutamax, HEPES, and 1:50 B27 were purchased from Gibco. Cis-diammineplatinum (II) dichloride, EP, Y27632, dexamethasone, penicillin/streptomycin, N-acetyl-l-cysteine, nicotinamide, insulin, hydrocortisone, cholera toxin, and hyaluronidase were purchased from Sigma. Wzb117 was purchased from Selleckchem. Recombinant human EGF, recombinant human FGF10, and recombinant human HGF were purchased from Peprotech. RBC lysis buffer, EthD-1, erythrocyte lysate, and Cell Tracker™ Green CMFDA Dye were purchased from Invitrogen. StemMACS iPS-Brew XF medium was purchased from Miltenyl Biotec (USA). Dimethyl sulfoxide, Sor, Reg, Apa, Len, and DNase I were purchased from Solarbio. Forskolin and A8301 were purchased from Tocris.

Zhai J., Liu Y., Ji W., Huang X., Wang P., Li Y., Li H., Wong A.H., Zhou X., Chen P., Wang L., Yang N., Chen C., Chen H., Mak P.I., Deng C.X., Martins R., Yang M., Ho T.Y., Yi S., Yao H, & Jia Y. (2024). Drug screening on digital microfluidics for cancer precision medicine. Nature Communications, 15, 4363.

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Endometrial Organoid 3D Culture

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Endometrial tissues were minced into 1 cm³ and dissociated into single-cell suspensions by incubation with 50 ug/ml Liberase (Roche, Swiss) in HBSS (Gibco, USA) at 37 °C for 30 min. The mixture was passed through micro-pored strainers with different sizes (SPL, South Korea) to obtain epithelial-enriched fraction as previously described 27 (link). For 3D culture, cells were suspended in DMEM/F12, mixed with 6 mg/ml TeloCol - 6 (Advanced BioMatrix, USA) and added neutralization Solution (Advanced BioMatrix, USA) at ratio of 1:1 (v/v), finally seeded in 48-well culture plates. After solidification, the droplets were then maintained with organoids culture medium consisted of DMEM/F12 (Gibco, USA), 1 X N2 supplement (Gibco, USA), 1 X B27 supplement (Thermo, USA), 1 % P/S (Invitrogen, USA), 1.25 mM N-Acetyl-L-cysteine (Sigma, USA), 1 X L-glutamine (Gibco, USA), 10 µM Y27632 (Biogems, USA), 10 µM A83-01 (Peprotech, USA), 1 mM Nicotinamide (Biogems, USA), 50 ng/mL recombinant human EGF (Peprotech, USA), 100 ng/mL recombinant human Noggin (Peprotech, USA), 500 ng/mL recombinant human R-spondin1 (Peprotech, USA), 100 ng/mL recombinant human FGF-10 (Peprotech, USA), 50 ng/mL recombinant human HGF1 (Peprotech, USA), and grown at 37 °C under 5 % CO₂ as previously described 27 (link), 29 (link). The culture medium was changed every 2 days and passaged every 6-7 days.

Hwang S.Y., Lee D., Lee G., Ahn J., Lee Y.G., Koo H.S, & Kang Y.J. (2024). Endometrial organoids: a reservoir of functional mitochondria for uterine repair. Theranostics, 14(3), 954-972.

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