To maintain the pluripotency of the iPSC, special culture conditions are required. iPSC requires strict culture media and passaging methods to maintain a healthy and undifferentiated state. Two commonly used iPSC culture systems are feeder-dependent culture system and feeder-free culture system. If animal-derived feeder cells are used, clinical applications may not be achieved. Animal feeder cells have safety problems, difficulties in quality control, and high cost, which make it limited for large-scale culture of iPSC. To overcome these limitations, the feeder-free culture system is an optimal option for the researchers due to its xeno-free, low-cost, easy-handle, and batch-to-batch consistency.
The feeder-dependent culture system uses a layer of feeder cells that provide the iPSC with the growth factors and extracellular matrix proteins needed to maintain its health and expansion. There are currently two main sources of feeder cells, human and murine. When only researching on in vitro culture, it is not an important question whether the feeder cells are allogenic or xenogeneic for iPSC. For example, most researchers adopt the mouse embryonic fibroblasts (MEF) to maintain both murine iPSC and human iPSC in vitro. However, when used for clinical use, safety must be considered. At this time, it must be ensured that the feeder cells and iPSC cells are autologous, that is, human feeder cells should be used. For example, human dermal fibroblasts (HDF) are used as autologous feeder cells for human iPSC derived from HDF. Commonly used feeder cells are as follows:
1. Murine feeder cells: MEF, murine amniocytes.
2. Human feeder cells: HDF, human amniocytes.
To provide specific cytokines and factors for iPSC, feeder cells should be arrested and held in non-multiplying state before use. One method is to utilize chemicals (such as mitomycin-C [MC] and glutaraldehyde) or physical methods (such as X-ray irradiation, electric pulses, and γ-irradiation) to arrest the growth of feeder cells (Fig.1a). In some cases, live feeder cells without any treatment are applied (Fig.1b). For example, human fibroblasts are used as feeder cells for iPSC derived from human fibroblasts.
Fig.2 Preparation of feeder cells. (Llames, 2015)
Although feeder cells can provide the necessary material basis for the maintenance and growth of iPSCs, the clinical application of which is limited. iPSCs grown on feeder cells are at risk of contamination by pathogens and immunogenicity (especially when the feeder cells originate from murine). Besides, the high cost and the complex operation limits the use of feeder cells in large-scale iPSC culture. Under these circumstances, the feeder-free culture system is an alternative for researchers. The feeder-free culture system uses an alternative medium, which provides all the growth factors required for iPSC, such as SC-1 (Pluripotin), dual kinase (ERK1, MAPK3), and GTPase inhibitor. Commonly used feeder-free culture systems are listed below:
1. Biological materials
The biomaterial matrix modified by bioactive molecules is used to maintain the growth of stem cells through the mutual lease with cells, providing an artificial microenvironment for the growth of stem cells in vitro. The biological materials currently used are:
A. Matrigel
It is composed of growth factors and extracellular matrix (ECM) components secreted by Engelbreth-HolmSwarm mouse sarcoma cells.
B. Protein-modified substrates
Protein-modified substrates mainly include ECM proteins (such as laminin, recombinant laminin-511, fibronectin, etc.) and cadherins (E-cadherin and N-cadherin). Most proteins used for engineering substrates origin from animal sources or isolated from animal cell cultures.
2. Synthetic materials
A. Peptide-modified substrates
Synthetic peptides derived from the active domain of ECM proteins can be used to maintain iPSC self-renewal and pluripotency. Since the glycosaminoglycans on the iPSC surface are important for cell adhesion, peptide sequences with binding affinity to anionic polysaccharides can be used as substrates, such as heparin. Based on this finding, the heparin-binding peptide, GKKQRFRHRNRKG has been demonstrated as the most effective substrate for iPSC adhesion.
B. Synthetic polymer-grafted substrates
Compared with proteins and peptides, chemical polymers have the following advantages: repeated synthesis, controllable batch-to-batch variances, lower storage requirements, and simple operation. The chemical composition is coated in a petri dish with high stability and long-term storage. Therefore, synthetic polymer substrates are more suitable for large-scale culture of iPSC. For example, a synthetic polymer, poly(methyl vinyl ether-alt-maleic anhydride) (PMVE-altMA) and poly(acrylamide-co-propargyl acrylamide)-coated polystyrene flasks with coupled cRGDfK coating (with modifying two-polymer brush coating [poly(acrylamide-co-acrylic acid) and poly(acrylamide-co-propargyl acrylamide)] have been used for the long-term attachment and self-renewal of iPSC.
In all, the feeder-free culture system may be simpler to use, because cells can grow on simple matrix-coated plates, so you don't need to worry about the feeder cell removal steps before downstream experiments.
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Reference
For Research Use Only. Not For Clinical Use.
