HOW TO CULTURE HUMAN ES AND iPS CELLS ON BIOLAMININ SUBSTRATES 

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

In the developing embryo, the alpha-1 and alpha-5 laminins are the first extracellular matrix proteins to be expressed. Laminin 111 is mostly expressed in extra-embryonic basement membranes and stimulates cells to commit to differentiation. Contrary, laminin 521 and laminin 511 are expressed around the stem cells in the inner cell mass and support survival and self-renewal of the pluripotent stem cells (Domogatskaya, 2012; Rodin, 2014a). The alpha-5 laminins are also produced endogenously by human pluripotent stem cells (hPSC) cultured in vitro and are critical autocrine and paracrine factors that regulates their survival and self-renewal (Laperle, 2015). 

Human recombinant laminin 521, Biolaminin 521 LN (LN521) is a chemically defined and animal component-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 (Rodin, 2014a). 

LN521 homogenizes and stabilizes pluripotent gene expression profiles between human ES cell lines

In an article by Albalushi et al., the authors tested the effect of the LN521 substrate on cultured hES cells. Five different human embryonic stem (hES) cell lines, originally derived on human foreskin fibroblasts (hFFs), were cultured on hFF, Matrigel or laminin-521. Culture on LN521 significantly reduced the variation of pluripotency marker expression compared to culture on hFFs. Moreover, hES cells cultured on LN521 were more homogenous, attached better and grew faster compared to hES cells cultured on Matrigel (Albalushi, 2017). The results show a positive effect of LN521 in homogenizing and stabilizing pluripotent gene expression profiles between hES cell lines and provide the first step towards generating more controllable, robust and clinically safe culture conditions.

Robust, long-term expansion of high quality human ES and iPS cells on LN521 

The natural cell-matrix interaction triggers authentic biorelevant signals and gives homogenous and genetically stable PSC cultures with a more uniformed gene expression. The cells migrate quickly on LN521, which correlates with the high survival of the cells. Cells amplify faster compared to other matrices, with about 10-fold expansion in 4 days (Rodin, 2014a). LN521 also support high post-thawed survival of cryopreserved pluripotent stem cells (Miyazaki, 2013). Due to faster growth rate and higher cell yield, the LN521 culture system is more cost-effective compared to other feeder-free matrices.

Human ES and iPS cells grow as a monolayer on LN521, providing you with a homogenous, high-quality cell culture (Rodin, 2014a). Compared to conventional colony passaging, hPSCs can be cultured on LN521 to high confluency without risk of spontaneous differentiation and there is no need to clear the culture from unwanted, differentiated cells (Rodin, 2014a; Rodin, 2014b).  

LN521 regulates signaling pathways that promotes hPSC survival and self-renewal 

LN521 has strong interaction to the α6β1 integrin, a key mediator of signalling pathways that regulates, survival, proliferation, migration and differentiation. LN521 binding to the α6β1 which induces the PI3K/Akt signaling pathway which in pluripotent stem cells promotes survival and efficient long-term self-renewal (Rodin, 2014a; Rodin, 2014b). Activation of integrin α6β1 by alpha-5 laminins have also show to regulate focal adhesion kinase (FAK) signaling in hPSCs and disruption of this pathway results in hPSC differentiation (Villa-Diaz, 2016). Laminin 511 binds the same integrin but the α6β1 integrin mediating effects of laminin 521 is much stronger than that of laminin 511 which results in more robust hPSC expansion on laminin-521 (Rodin, 2014a). 

A flexible culture system that is easy to control

Due to the authentic biorelevant signals provided by the culture matrice, LN521 is a flexible culture system. LN521 works with most commercial medias and hPSCs can routinely be cultured as single cells 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 medias (Rodin, 2014a). The biorelevant support provided by the LN521 matrix also have a protective effect against DNA damage caused by suboptimal culture conditions, such as high-density culture and pH changes (Jacobs, 2016). This allows for weekend-free feeding, drastically reducing work load, time and cost for growing your cells. 

LN521 supports clonal culture

With the support from the LN521 matrix, cells can be passaged in very low cell densities and can be cultured to high confluence without phenotypic alterations (Rodin, 2014a; Rodin, 2014b). LN521 also supports efficient clonal culture and is an excellent substrate for iPSC reprogramming (Uhlin, 2017; Tano, 2014; Lu, 2014). Clinically compliant human embryonic stem cell lines can even be derived from a single blastomere and there is no longer any need to destroy the embryo (Hovatta, 2014; Damdimopoulou, 2015). Importantly, culture on LN521 primes the hPSCs and gives more efficient differentiation and enhances cell maturation, polarization and organization (Cameron, 2015; Penton, 2016).

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. The authors also provide data around critical steps and reagents included for easier handling of more difficult lines and a useful trouble shooting guide (Rodin, 2014b).