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>Growth Factors for Induced Pluripotent Stem Cell Culturing
Cancers 2022, 14, 2266
The classical approach to generate iPSC-derived T cells starts from:
1. Co-culturing of iPSCs derived from healthy donors with murine bone marrow stromal cell line (C3H10T1/2 or OP9) to generate CD34+ hematopoietic progenitor cells.
2. Hematoproietic progenitors are enriched and co-cultured with OP9 cell line overexpressing DLL-1/DLL-4, with additional cytokines to drive T cell differentiation.
To generate iPSC-derived NK cells:
1. The iPSCs derived from healthy donors are co-cultured with the murine bone marrow stromal cell line M210-B4 for ~20 days to allow for the generation of CD34 + hematopoietic progenitors.
2. CD34 + hematopoietic progenitors are then enriched and co-cultured with a monolayer of murine AFT024 (a fetal liver-derived stromal cell line) or EL08-1D2 stroma cells with defined cytokines for 4 to 5 weeks, eventually generating mature NK cells.
Formation of iPSC-derived macrophages Mφs requires a more complex methodology:
1. Embryoid body(EBs) formation is the most commonly used method for differentiating iPSC-derived Mφs, utilizing specific modifications such as rotary suspension, microwell approaches, microfluidic hanging drop chips, and others.
2. After formation of iPSC-derived EBs, myeloid Mφs are further induced by supplementation of cytokines, including GM-CSF, M-CSF, IL-3, and IL-4.
Transitioning from Premium to GMP
Cytokines are critical raw materials used in cell culturing for cell and gene therapy (CGT) drugs. Usually, in the preclinical stage, both safety and quality for raw materials are not prioritized. In this case, research-use only products can be used. However, when progressing into later drug development stages such as CMC or clinical phases, it is necessary to replace the raw materials with GMP-grade materials to be compliant with regulatory guidelines. In this transitional period, there is a significant amount of energy dedicated to re-evaluating and validating new raw materials.
To ease this transition from the preclinical development into the clinical stage, we offer several grades of cytokines that have been evaluated to be near-identical in bioactivity, along with the documentation required from GMP products. We recommend using our premium-grade raw materials in the early development stage to seamlessly transition to our GMP-grade raw materials when entering CMC or clinical phases and minimize the number of re-evaluation and validation studies performed.
GMP Human IL-15 Protein on SDS-PAGE under reducing (R) condition. The gel was stained overnight with Coomassie Blue. The purity of the protein is greater than 95%.
Human IL-2, premium grade on SDS-PAGE under reducing (R) condition. The gel was stained overnight with Coomassie Blue. The purity of the protein is greater than 95%.
Human DLL4 Protein, Fc Tag, premium grade on SDS-PAGE under reducing (R) condition. The gel was stained overnight with Coomassie Blue. The purity of the protein is greater than 95%.
Human VEGF165, premium grade (Cat. No. VE5-H4210) stimulates proliferation of human umbilical vein endothelial cells (HUVEC). The ED50 for this effect is 4.216-9.281 ng/mL.
Human IL-2, premium grade (Cat. No. IL2-H5215) stimulates proliferation of CTLL-2 cells. The EC50 for this effect is 1.996 ng/mL, corresponding to a specific activity of 5 ⅹ10^6 IU/mg, which is calibrated against Interleukin-2 (Human, rDNA derived) (2nd International Standard) (NIBSC code: 86/500).
Immobilized Human DLL4 Protein, Fc Tag, premium grade (Cat. No. DL4-H5259) at 1 μg/mL (100 μL/well) can bind Monoclonal Anti-Human DLL4 Antibody, Human IgG1 with a linear range of 1-16 ng/mL.
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