HOW TO CULTURE PANCREATIC CELLS ON BIOLAMININ SUBSTRATES

The basement membrane formed next to the beta-cells contains alpha-4 and alpha-5 laminin isoforms

Endocrine pancreatic β-cells do not form their own basement membrane (BM). Instead, the β-cells require endothelial signals for their differentiation and for normal function (Lammert, 2003; Yoshitomi & Zaret, 2004). During late embryonic development, β-cells express high levels of VEGF-A that attract endothelial cells that, in turn, form the vasculature of the islets (Lammert, 2003; Nikolova, 2006). β cells in VEGF-A deficient mice have shown to have reduced insulin gene expression compared to fully vascularized control islets (Nikolova, 2006).

The vascular basement membrane formed next to the β-cells contains alpha-4 and alpha-5 laminin isoforms (Sigmundsson, 2018; Sixt, 2001). Immunostaining of fetal human pancreas show that laminin 411 is expressed (Petajaniemi, 2002), however, adult human islets are surrounded by a double BM containing both laminin 511 and laminin 411 but where the BM facing the human endocrine islet is suggested to only contain laminin isoforms 511 and 521 (Virtanen, 2008). Integrin expression analysis in isolated mouse islets highlights integrin β1, αV, and α6 as the most abundant transcripts (Sigmundsson, 2018) where all three bind α5-laminins. A study by Kantengwa et al. also demonstrates an important role of β1 integrins in islet cell attachment and spreading in vitro (Kantengwa, 1997).

Pancreatic islets cultured on Biolaminin 521 expand and remain functional

Sigmundsson et al. have developed a novel method to grow and maintain normoxic and functional islets which may significantly enhance the efficacy of islet transplantation treatment for diabetes (Sigmundsson, 2018). A key component of this method is the coating with biologically relevant laminins. Islets cultured in vitro on Biolaminin 521 adhere and spread to form layers of 1-3 cells in thickness while maintaining cell-to-cell contacts. The cells remained normoxic and functional for at least 7 days in culture. In contrast, spherical islets kept in suspension developed hypoxia and central necrosis within 16 hours. Mouse islets plated on Biolaminin 521 could be cultured in a serum-free mTeSR1 medium for an extended period of time. The flattened islets start robust cell proliferation after a lag period of approximately two weeks in a serum-free culture on Biolaminin 521. Approximately 20% of islet cells showed a co-expression of insulin and glucagon. Transplantation mouse islets cultured on α5-laminin-coated polydimethylsiloxane membranes for 3–7 days normalized blood glucose already within 3 days in mice with streptozotocin-induced diabetes.

Laminin proteins stimulate beta-cell proliferation and insulin production in vitro

By using pancreas-specific laminins in cell culture, the biological environment of the pancreas can be mimicked, making pancreatic islet cells thrive. In a study by Dr. Doug Melton et al. they investigated which endothelial signals that promote β-cell proliferation and function. They show that laminin isoforms 411 and 511 stimulate β1 integrin signaling–dependent insulin production in vitro (Nikolova, 2006). The authors suggest that treating islets with these biologically relevant laminin isoforms prior to transplantation could help maintain insulin production until new capillaries are formed in transplanted islets (Nikolova, 2006).

Several studies have also revealed an essential role of cell-to-matrix interactions mediated by laminin 332 in the function of isolated pancreatic islet cells. Integrins a3β1 and a6β1 have been identified in rat pancreatic B cells (Bosco, 2000; Parnaud, 2006) and β cells cultured on a laminin 332-rich matrix spread out and secreted twice more insulin in response to glucose compared to cells cultured on plastic, poly-L-lysine (Parnaud, 2006). Furthermore, laminin 332 promote the proliferation of rat β-cells and protected the cells from apoptosis induced by pro-inflammatory cytokines (Hammar, 2004). In a follow-up publication by Armanet et al, the authors show that laminin 332 plays a physiologically relevant role in human islets since laminin 332 is expressed and secreted by human islets and since conditions inducing islet stresses, such as isolation procedure and cytokine exposure, enhance laminin-332 production (Armanet, 2009).

Primary pancreatic islets are cultured on laminin express insulin.
Courtesy of Dr. Anna Domogatsakya, Karolinska Institutet. 

Biolaminin 411 is a potent differentiation inducer of insulin-producing cells

In a publication by Qu et al. they show that laminin 411 acts as a potent differentiation inducer of UC-MSC into insulin-producing cells (Qu et al., 2014). In the article, they show that Biolaminin 411 induces the expression of pancreatic precursor markers and markedly up-regulate insulin expression, both at the mRNA and protein level. More importantly, the administration of Biolaminin 411-induced insulin-producing cells rapidly and significantly down-regulated fasting blood glucose levels, significantly reduced the HbA1c concentration and markedly improved the symptoms and survival of type-1 diabetic rats (Qu et al., 2014).