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Recombinant human albumin

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Recombinant human albumin is a laboratory-produced protein that is structurally and functionally equivalent to the natural albumin found in human blood. It is used in various in vitro applications, such as cell culture and protein purification, to provide a stable and physiologically relevant environment for biological processes.

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Lab products found in correlation

L ascorbic acid 2 phosphate, by Merck Group (13 mentions) Chir99021, by Merck Group (7 mentions) Rpmi 1640 medium, by Thermo Fisher Scientific (5 mentions) Rpmi 1640, by Thermo Fisher Scientific (5 mentions) Chir99021, by Bio-Techne (4 mentions) Wnt c59, by Bio-Techne (4 mentions) Lactate, by Merck Group (3 mentions) L ascorbic acid, by Thermo Fisher Scientific (3 mentions) L ascorbic acid, by Merck Group (3 mentions) Iwp 2, by Merck Group (3 mentions) L lactic acid, by Merck Group (2 mentions) Rpmi 1640 no glucose, by Thermo Fisher Scientific (2 mentions) Rpmi 1640, by Merck Group (2 mentions) Bone morphogenetic protein 4 (bmp4), by Thermo Fisher Scientific (2 mentions) Vascular endothelial growth factor (vegf), by Novoprotein (2 mentions) Basic fibroblast growth factor (bfgf), by Novoprotein (2 mentions) Data analysis software v8.2, by Molecular Devices (2 mentions) B27 supplement, by Thermo Fisher Scientific (2 mentions) Rpmi 1640 medium glutamax supplement hepes, by Thermo Fisher Scientific (2 mentions) Matrigel, by BD (2 mentions)

28 protocols using recombinant human albumin

1

Directed Cardiac Differentiation of hiPSCs

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hiPSCs (>p20) were split at 1:12 to 1:15 ratios using EDTA as above and grown for 3–4 days at which time they reached ~75% confluence. Media was changed to CDM36 (link), consisting of RPMI 1640 (10–040–CM, Corning), 500 µg ml−1Oryza sativa–derived recombinant human albumin (Oryzogen Sciencell), and 213 µg ml−1L–ascorbic acid 2–phosphate (Sigma–Aldrich). Media was changed every other day (48 h). For days 0–2, media was supplemented with 6 µM CHIR99021 (MedChem Express)19 , 20 . On day 2, media was changed to CDM3 supplemented with 2 µM Wnt–C59 (Biorbyt). Media was changed on day 4 and every other day for CDM3. Contracting cells were noted from day 7. At day 10, media was changed to CDM3L made with using RPMI 1640 no glucose (11879–020, Life Technologies), 500 µg ml−1 recombinant human albumin, and 213 µg ml−1L–ascorbic acid 2–phosphate supplemented with 4 mM L–lactic acid (Sigma–Aldrich). At day 15, media was changed to CDM3M consisting of RPMI 1640 no glucose, 500 µg ml−1 recombinant human albumin, 213 µg ml−1L–ascorbic acid 2–phosphate supplemented with 10 mM D–galactose (Sigma–Aldrich)21 (link), 4 mM L–lactic acid, 1 mM sodium pyruvate (Life Technologies), 20 µg ml−1 insulin (Life technologies), 1 × chemically defined lipid concentrate (Life Technologies), and 200 ng ml−1 tri–iodo–L–thyronine (Sigma–Aldrich)22 (link).
Burridge P.W., Li Y.F., Matsa E., Wu H., Ong S., Sharma A., Holmström A., Chang A.C., Coronado M.J., Ebert A.D., Knowles J.W., Telli M.L., Witteles R.M., Blau H.M., Bernstein D., Altman R.B, & Wu J.C. (2016). Human Induced Pluripotent Stem Cell–Derived Cardiomyocytes Recapitulate the Predilection of Breast Cancer Patients to Doxorubicin–Induced Cardiotoxicity. Nature medicine, 22(5), 547-556.
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2

Efficient Generation of iPSC Clones

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Cells were individualized with Accutase 4–5 days after electroporation and resuspended in DMEM/F12 media immediately before experiments. For analysis, individualized cells were analyzed on an LSR Fortessa (BD) using 488nm laser for EGFP and 561nm laser for tdTomato. Data analysis was performed using FlowJo® v10.4.1. For FACS sorting, individualized cells were put on a SH800 cell sorter (Sony) fitted with a 130 μm chip and GFP negative cells were sorted directly into a well of a 24-well plate coated with matrigel and filled with 1.5 ml E8 media supplemented with 10 μM Y-27632 and 25 μg/mL recombinant human albumin (Sigma). Sorted cells were then cultured in tissue culture incubator with 5% CO2 at 37°C. Media was changed to regular E8 one day after sorting and daily media change with E8 was carried out thereafter. For isolating single cell clones, GFP-negative and tdTomato-positive cells were sorted directly into 96-well plate (one cell per well) coated with matrigel and filled with 150 μL E8 media supplemented with 10 μM Y-27632 and 25 μg/mL recombinant human albumin (Sigma). Media was changed to regular E8 two days after sorting and media change with E8 was carried out every two days thereafter.
Dolan A.E., Hou Z., Xiao Y., Gramelspacher M.J., Heo J., Howden S.E., Freddolino P.L., Ke A, & Zhang Y. (2019). Introducing a spectrum of long-range genomic deletions in human embryonic stem cells using Type I CRISPR-Cas. Molecular cell, 74(5), 936-950.e5.
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3

Human iPSC Differentiation Protocol

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The differentiation protocol started when human iPS cells reached 80–90% of confluency (day 0). Cells were fed with RPMI-1640-Medium-GlutaMAX Supplement-HEPES (Gibco, 72400-021) supplemented with 0.5 mg ml−1 human recombinant albumin (Sigma-Aldrich, A9731), 0.2 mg ml−1l-ascorbic acid 2-phosphate (Sigma-Aldrich, A8960) and 4 μM CHIR99021 (Sigma-Aldrich, 361559). After 48 h (day 2), medium was replaced by RPMI-1640-Medium-GlutaMAX Supplement-HEPES supplemented with 0.5 mg ml−1 human recombinant albumin (Sigma-Aldrich, A9731), 0.2 mg ml−1l-ascorbic acid 2-phosphate and 5 μM IWP2 (Sigma-Aldrich, 681671). On day 4 and day 6, cells were refreshed with RPMI-1640-Medium-GlutaMAX Supplement-HEPES supplemented with 0.5 mg ml−1 human recombinant albumin and 0.2 mg ml−1l-ascorbic acid 2-phosphate. From day 8 onwards, the medium of the cells was refreshed every 3–4 days with RPMI-1640-Medium-GlutaMAX Supplement-HEPES supplemented with B-27 Supplement (50×)-serum free (Gibco, 17504001).
Kyriakopoulou E., Versteeg D., de Ruiter H., Perini I., Seibertz F., Döring Y., Zentilin L., Tsui H., van Kampen S.J., Tiburcy M., Meyer T., Voigt N., Tintelen V.J., Zimmermann W.H., Giacca M, & van Rooij E. (2023). Therapeutic efficacy of AAV-mediated restoration of PKP2 in arrhythmogenic cardiomyopathy. Nature Cardiovascular Research, 2(12), 1262-1276.
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4

Purification of Cardiac Myocytes

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Differentiating CM cultures were purified via metabolic selection between d10 and d12 based on previously described methods4 (link). Briefly, cultures were rinsed with PBS and incubated with “no glucose” medium for 48 h (glucose-free RPMI (ThermoFisher Scientific 11879020) supplemented with 4 mM lactate (Sigma L4263), 0.5 mg/mL recombinant human albumin (Sigma A6612), and 213 μg/mL l-ascorbic acid 2-phosphate (Sigma A8960))3 (link). Occasionally, cultures required 1–2 days of additional selection to more efficiently eliminate non-CMs. At the end of the selection period, cultures were dissociated into single cells using 0.25% trypsin/EDTA followed by quenching with stop buffer (DMEM, 20% FBS, 20 μg/mL DNAse I (Millipore 260913)) and replated onto fresh Matrigel-coated dishes to remove dead cells and debris.
Shadrin I.Y., Allen B.W., Qian Y., Jackman C.P., Carlson A.L., Juhas M.E, & Bursac N. (2017). Cardiopatch platform enables maturation and scale-up of human pluripotent stem cell-derived engineered heart tissues. Nature Communications, 8, 1825.
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5

Western Blot Analysis of Retinol-Binding Protein

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Apo-rRBP, serum-derived RBP (Athens Research and Technology, Cat # 16–16-180216) and recombinant human albumin (Sigma, Cat # A9731) were diluted into LDS 4X Sample buffer (NuPAGE), boiled and loaded into 4–12% Bis-Tris SDS-PAGE gel (NuPage) at a total protein concentration of 75 ng per well. SDS-PAGE was run in MES Running Buffer (50 mM MES, 50 mM Tris Base, 0.1% w/v SDS, 1 mM EDTA, pH 7.3) at 200 V for 35 minutes. Samples were transferred to 0.45 micron polyvinylidene difluoride membranes at 30 V for one hour before being collected and washed 3X at 5 minutes each in TBST (20 mM Tris, 135 mM NaCl, 0.1 % v/v Tween-20, pH 7.6). Membranes were blocked for 1 hour at room temperature in blocking buffer (5% w/v non-fat dry milk (LabScientific) dissolved in TBST) before incubation at 4 °C overnight with either mouse monoclonal anti-human RBP4 (Abnova, 1:500 dilution, Cat # MAB3211) in blocking buffer or blocking buffer alone (for no-primary controls). After overnight staining, all membranes were washed 3X at 5 minutes each in TBST before secondary antibody incubation at room temperature for 1 hour in the dark with goat anti-mouse IgG IRDye 800CW (Li-Cor, 1:5000 dilution, Cat # 926–32210) antibody in blocking buffer. Probed membranes were washed 3X at 5 minutes each in TBST and subsequently imaged using a Li-Cor Odyssey Imager.
Est C.B, & Murphy R.M. (2019). Retinol Binding Protein IV Purified from Escherichia coli Using Intein-Mediated Cleavage as a Suitable Replacement for Serum Sources. Protein expression and purification, 167, 105542.
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6

Cardiomyocyte Differentiation from iPSCs

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The iPSCs (>p20) were passaged using EDTA and grown until the cells reached ∼75% confluency. The medium was changed to CDM3, which consists of RPMI 1640 (Life Technologies), Oryza sativa–derived recombinant human albumin (500 μg ml−1; Sigma-Aldrich), and l-ascorbic acid 2-phosphate (213 μg ml−1; Sigma-Aldrich). Medium was changed every other day (48 hours). At days 0 to 2, medium was supplemented with 6 μM glycogen synthase kinase 3-β inhibitor CHIR99021 (APExBio). On day 2, the medium was changed to CDM3 supplemented with 2 μM Wnt inhibitor Wnt-C59 (Calbiochem Research Biochemicals). The medium was changed on day 4 and every other day for CDM3 cultures. The contracting cells were observed from day 7. At day 10, the medium was changed to CDM3L, which consists of RPMI 1640 no glucose (Life Technologies), recombinant human albumin (500 μg ml−1), and l-ascorbic acid 2-phosphate (213 μg ml−1) supplemented with 4 mM l-lactic acid (Sigma-Aldrich). CM formation was further confirmed by staining for NKX2.5 (Cell Signaling Technology no. 8792S) and cardiac troponin (catalog no. AB8295, Abcam).
Sequiera G.L., Srivastava A., Sareen N., Yan W., Alagarsamy K.N., Verma E., Aghanoori M.R., Aliani M., Kumar A., Fernyhough P., Rockman-Greenberg C, & Dhingra S. (2022). Development of iPSC-based clinical trial selection platform for patients with ultrarare diseases. Science Advances, 8(14), eabl4370.
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7

SARS-CoV-2 Nanobody Stability Assessment

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NIH-CoVnb-112 expressed in Pichia pastoris was diluted from a concentrated stock solution into apheresis derived pooled human plasma (#IPLA-N, Innovative Research) to a final concentration of 5 µM and incubated at 37 °C for either 24 hr or 48 hr with gentle rotation. An identical sample set was prepared at 5 µM in a solution containing 35 mg/mL recombinant human albumin (#A9731, Sigma-Aldrich) and incubated at 37 °C for either 24 hr or 48 hr with gentle rotation. A no-incubation control for each the plasma and recombinant human albumin conditions was prepared at 5 µM. The samples were prepared in a manner providing all conditions complete at the same time. The samples were diluted 1:10 with 1xPBS to yield a final nanobody concentration of 500 nM and Bio-layer Interferometry was performed using immobilized biotinylated SARS-CoV-2 S protein RBD to determine retention of binding potential.
Esparza T.J., Martin N.P., Anderson G.P., Goldman E.R, & Brody D.L. (2020). High affinity nanobodies block SARS-CoV-2 spike receptor binding domain interaction with human angiotensin converting enzyme. Scientific Reports, 10, 22370.
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8

Cardiomyocyte Differentiation from hESCs

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For differentiation into CMs, hESCs were seeded into 1% Geltrex (Thermo Fisher Scientific, Waltham, MA, USA)-coated tissue culture plates and grown to 70% cell confluency. For mesodermal induction, 6 μM CHIR99021 (Tocris, Bristol, UK) was treated for 2 days in CM differentiation medium consisting of RPMI-1640, 212 μg/ml l-ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA), and 500 μg/ml recombinant human albumin (Sigma-Aldrich). After mesodermal induction, 2 μM Wnt-C59 was administered for another 2 days. The differentiated CMs were maintained in RPMI-1640 supplemented with 212 μg/ml l-ascorbic acid and 1x B27 supplement (Thermo Fisher Scientific). The antibodies used in this study are listed in Supplementary Table S1. Human Cardiomyocyte Total RNA (Cat No. 36044-15RNA) was purchased from CELPROGEN.
Lee M.O., Lee S.G., Jung C.R., Son Y.S., Ryu J.W., Jung K.B., Ahn J.H., Oh J.H., Lee H.A., Lim J.H., Kim J., Jang I., Choi J., Jung J., Park K., Lee B., Kim D.S., Son M.Y, & Cho H.S. (2021). Development of a quantitative prediction algorithm for target organ-specific similarity of human pluripotent stem cell-derived organoids and cells. Nature Communications, 12, 4492.
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9

Cardiac Differentiation of Human iPSCs

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Using a previously established protocol, cardiac differentiation of human iPS cells was initiated in 90% confluent cell monolayers by replacing the mTeSR™1 medium with CDM3 (chemically defined medium) with 3 components: RPMI Medium 1640 (1×, Gibco), 500 μg mL−1 of recombinant human albumin (Sigma-Aldrich) and 213 μg mL−1 of l-ascorbic acid 2-phosphate (Sigma-Aldrich), supplemented with 1% penicillin/streptomycin.61 (link) Medium was changed every 48 hours. For the first 48 hours, the medium was supplemented with 3 mM of glycogen synthase kinase 3 inhibitor CHIR99021 (Tocris). On day 2, the culture was switched to CDM3 medium supplemented with 2 mM of the Wnt inhibitor Wnt-C59 (Tocris). After day 4 of differentiation, the medium was changed to CDM3 with no supplements. Contracting cells were noted around day 10, when medium was changed to RPMI 1640 supplemented with B-27™ (50×; Gibco), and were used in experiments without selection for cardiomyocytes.
Chramiec A., Teles D., Yeager K., Marturano-Kruik A., Pak J., Chen T., Hao L., Wang M., Lock R., Tavakol D.N., Lee M.B., Kim J., Ronaldson-Bouchard K, & Vunjak-Novakovic G. (2020). Integrated human organ-on-a-chip model for predictive studies of anti-tumor drug efficacy and cardiac safety. Lab on a chip, 20(23), 4357-4372.
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10

Chemically Defined Differentiation of hESCs into Endothelial Cells

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When hESCs reach 90% density, the cell culture medium was changed to the chemically defined differentiation medium CDM3, which consists of RPMI 1640 medium (Thermo Fisher, USA), 500 μg/mL recombinant human albumin (Sigma-Aldrich, USA), and 213 μg/mL L-ascorbic acid 2-phosphate (Sigma-Aldrich, USA). According previous description, the cells were successively treated with 6 μM CHIR99021 (Sigma-Aldrich, USA) for 2 days (day 0–1), 50 ng/mL bFGF (Novoprotein, USA) for 1 day (day 2), as well as a combination of 50 ng/mL VEGF (Novoprotein, USA) and 25 ng/mL BMP4 (Peprotech, USA) for 3 days (day 3–5).43 (link) All the hESC-differentiated cells were collected on day 6 for subsequent MACS and flow cytometry analysis.
Ding F., Wu H., Han X., Jiang X., Xiao Y., Tu Y., Yu M., Lei W, & Hu S. (2023). The miR-148/152 family contributes to angiogenesis of human pluripotent stem cell- derived endothelial cells by inhibiting MEOX2. Molecular Therapy. Nucleic Acids, 32, 582-593.
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