Clonal stem cell culture

Biolaminin 521 successfully replicates the genuine human stem cell niche in vitro

In the developing embryo, the alpha-5 laminins (laminin isoforms 511 and 521) are the first extracellular matrix proteins to be expressed around the stem cells in the inner cell mass and support survival and self-renewal of the pluripotent stem cells. The alpha-5 laminins are also produced endogenously by pluripotent stem cells cultured in vitro and are critical autocrine and paracrine factors that regulate their survival and self-renewal. 

Human recombinant Biolaminin 521 LN (LN521) is a chemically defined and animal origin-free system for robust, long-term expansion of human pluripotent stem cells. It successfully replicates the genuine human stem cell niche in vitro and thereby provides a highly reliable and reproducible culture system to create sufficient populations of undifferentiated cells that, in turn, can be terminally differentiated. 

Robust, long-term expansion of human ES and iPS cells on Biolaminin 521

A detailed step-by-step protocol for secure transfer from feeders, true clone generation and expansion of hPSCs and freezing and thawing as single cells on LN521 has been published by Rodin et al. There are also critical steps and reagents included for easier handling of more difficult lines and useful troubleshooting guide.

Once the cell line is established, LN521 supports the robust, long-term, single-cell expansion of human pluripotent stem cells. Pluripotent stem cells grow as a monolayer on LN521 providing you with a homogenous, pluripotent cell culture without the need to dispose of unwanted differentiated cells. Cells amplify faster compared to other matrices, with about 10-fold expansion in 4 days. Due to the authentic biorelevant signals provided by the culture matrice, LN521 is a flexible culture system. It works with most commercial media, the cells can be passaged in very low cell densities and can be cultured to high confluence without phenotypic alterations or genetic alterations. Moreover, hPSCs can routinely be cultured as single cells on LN521 without need to add apoptosis inhibitors, such as ROCKi. Since laminin efficiently buffers endogenously produced growth factors LN521 also performs well together with growth-factor reduced media. The biorelevant support provided by the LN521 matrix also has a protective effect against DNA damage caused by suboptimal culture conditions, such as high-density culture and pH changes. This allows for weekend-free feeding, drastically reducing workload, time and cost for growing your cells.

Efficient generation of clinically compliant human ES cell lines on Biolaminin 521

In a publication by Rodin et al., the authors showed that LN521 supports efficient generation and large-scale expansion of clinically compliant human ES cell (hESC) lines. The biorelevant LN521 clonal stem cell culture protocol vastly improved the survival of completely dissociated human ES and iPS cells, even at clonal density. The cells quickly adhered to the LN521 matrix and started to divide with high survival. LN521 matrix in combination with E-cadherin even allowed clinically compliant hESC line derivation from single blastomere cells without a need to destroy the embryo.

High-efficiency iPSC reprogramming and expansion on Biolaminin 521

LN521 is an optimal defined and feeder-free matrix for the clinically compliant reprogramming and expansion of human iPS cells. Reprogramming on LN521 has successfully been performed by mRNA and Sendai-virus methods. Lu et al. show that LN521 achieves high-efficiency reprogramming and rapid, long-term self-renewal of iPSC with normal karyotype and pluripotency. Moreover, LN521 supported direct hiPSC lineage-specific differentiation. 

Tano et al. (2014) showed a novel approach where they use LN521 for direct and sensitive detection of trace amounts of residual undifferentiated hPSCs for cell therapy products. The highly efficient culture system detected colony-forming hPSCs spiked into primary human MSCs or neurons at a ratio as low as 0.001%–0.01%.

Matsumura and co-workers (2014) showed that LN521 increase the survival of single-cell hiPSC clones. The authors described increased survival and propagation of single hiPSC clones on LN521 in both culture and on a new microfluidic device by conditioning the medium.

Protocols:

Defined and animal origin–free, clonal culture, and expansion of hESC and iPSC on Biolaminin 521

Simplified and efficient cryopreservation of high-quality hPSCs