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LAMININ-521LAMININ-221 and LAMININ-211 


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PROTOCOL

Long-term expansion and differentiation of myogenic progenitors on laminin


CUSTOMER TESTIMONIALS

"Laminin-521 shifts away from the paradigm that muscle cells lose their ability to differentiate after culturing. Muscle differentiation on laminin-521 is nothing short of spectacular, yielding increased numbers of nuclei per myotube and improved myotube organization. Surprisingly, muscle cells expanded on laminin-521 maintain their ability to differentiate into myotubes after long-term passaging, better-resembling differentiation of freshly isolated cells."

                                           Dr. Christopher Penton, Icagen, Inc., USA

         
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ARTICLES

HOW TO CULTURE MUSCLE CELLS ON BIOLAMININ SUBSTRATES

 Get faster proliferation and better differentiation of skeletal muscle cell culture on human recombinant laminin-521Laminin 211, 221 and 521 form an important part of the adult skeletal muscle microenvironment

Laminin proteins are key components of the basal lamina surrounding muscle stem cells and adult cells (Sanes, 2003 and Yin, 2013). Laminins are not only structural proteins of the basal lamina but are also signaling molecules that are important for the adhesion, localization, and function of muscle cells in their niche. Laminin-211, -221, -511 and -521 are the main laminin isoforms expressed in the basal lamina of striated muscles. Laminin 421 and laminin 111 are also expressed in small amounts, preferably at junctional regions (Rogers & Nishimune, 2016). Laminin 211 and laminin 221 are the most abundant laminin isoform in the adult skeletal muscle basement membrane with integrin α7β1 as the main receptor (Holmberg & Durbeej, 2013) and an expression deficiency in the alpha-2 laminins results in muscular dystrophy, accompanied by a dilated cardiomyopathy (Oliviéro, 2000). Laminin 211 is located within the basement membrane that surrounds the sarcolemma, whereas laminin 221 predominates in the neuromuscular and myotendinous junctions.

Laminin is crucial for normal muscle function, evident from naturally occurring mutations in laminin genes. Mutations in the gene encoding laminin a2 chain are the most common cause of congenital muscular dystrophy type 1A (Gawlik & Durbeej, 2011). In a publication by Fontes-Oliveira et al. (2017), the authors show that the absence of laminin α2 chain leads to downregulated PGC1α expression, which impairs mitochondrial biogenesis, causing a reduction of mitochondrial content that finally leads to a bioenergetic inefficiency in myoblasts and myotubes from MDC1A patients. By culturing MDC1A myotubes on recombinant Biolaminin 211, the mitochondrial function is restored. Laminin expression in human muscle tissue Basal respiration, maximum respiration, and ATP production, as well as basal mitochondrial respiration and maximal mitochondrial respiration capacity, are normalized to control levels in the presence of laminin 211.

Due to their vital role for muscle tissue structure, maintenance, and function, laminin serve as an important tool in protocols aiming to improve cell therapy for muscular deceases. Moreover, the intramuscular injection of laminin has shown to improve muscle regeneration and the efficiency of myoblast transplantation (Riederer, 2015). 

Laminin β-2 immunohistochemical staining of human skeletal muscle shows strong membranous immunoreactivity in myocytes. Anti-LAMB2 antibody
AMAb91096 (Atlas Antibodies).


Biolaminin 521 and 221 are superior substrates for both short-term and long-term myogenic cell expansion and differentiation

During myogenesis, laminin 521 and laminin 511 are the main isoforms surrounding myogenic progenitors (Gawlik & Durbeej, 2011). Indeed, recent data presented by researchers at Icagen Inc. demonstrate that the Biolaminin 521 cell culture matrix maintains the differentiation potential of mouse and human satellite cell-derived myoblasts, even during long-term culture expansion (Penton, 2016). Biolaminin 521 supports increased proliferation during expansion and superior differentiation with myotube hypertrophy, larger myotubes and higher amounts of nuclei per myotube. Moreover, Penton et al. show that Biolaminin 521 supports more consistent and reliable differentiation over long-term culture and is the only substrate facilitating high-level fusion following long-term culture. Biolaminin 521 supports increased differentiation potential without altering the traditional Pax7/MyoD paradigm and the results are translational across several mouse backgrounds, human cells, and disease states. 

Researchers at Stanford that developed artificial muscle fibers (AMFs) that mimic the native myofiber of the MuSC niche show that Biolaminin 211 maintains muscle stem cells (MuSCs) in a potent, quiescent state in vitro. AMFs coated with recombinant Biolaminin 211 showed prolonged quiescence in vitro and enhanced potency in vivo (Quarta, 2016). 

 

An improved method for culturing myotubes on laminins for the robust clustering of postsynaptic machinery

Motor neurons from specialized synapses with skeletal muscle fibers called neuromuscular junctions (NMJs). The function of this type of synapse is to transmit signals from the central nervous system to muscles and thus stimulate their contraction. Muscle cells form a thick ECM around the fiber that contains various laminins, collagens, fibronectin, and other glycoproteins. The basal lamina (BL) at the synaptic cleft has a specific molecular composition that contains laminin α4, α5, and β2 isoforms that are mostly absent in extrasynaptic regions of muscle fibers. These ECM components are crucial for the proper development of NMJs and it has been shown that the laminin-dystroglycan interaction is crucial for regulating NMJ developmental remodeling. 

Little is known about the mechanisms of postsynaptic machinery remodeling in vivo. Mouse laminin-111 has been routinely used to induce AChR clustering and is the only in vitro system where the AChR clusters undergo developmental remodeling that resembles mature NMJs, providing the model to study the underlying mechanisms. In a publication in Scientific Reports (Pęziński, 2020) the authors present an improved protocol for culturing C2C12 muscle cells that reproducibly promote the formation of complex AChR clusters. The authors tested several laminin isoforms and found that laminin-121, laminin-211, laminin-221, laminin-511, and laminin-521 induced significantly more AChR clusters in C2C12 myotubes than the commonly used laminin-111. Moreover, they found that clusters of postsynaptic machinery that were formed in C2C12 myotubes cultured on laminin-121 and laminin-221 were the most developed. Laminin-421 and laminin-511 were the isoforms that promoted the formation of the most podosome-containing AChR clusters in human primary myotubes. Myotubes that were derived from human primary myoblasts obtained from human biopsies also formed AChR clusters in vitro that underwent the remodeling process, thus demonstrating the potential utility of this methodology for further studies that seek to improve diagnoses of neuromuscular disorders and elucidate their underlying mechanisms. Thus, this novel method may facilitate the identification of novel synaptic regulators and the high reproducibility of culturing and robust formation of AChR clusters are important prerequisites for establishing high-throughput screening. The protocol is also useful for obtaining and freezing a large number of cell stocks and utilizing cells for experimentation with a constant and low passage number, which significantly increases experimental reproducibility. The method can be implemented in different formats, such as permanox slides, glass surfaces as well as multi-well culturing dishes. Collectively, these results demonstrate an advanced method for culturing myotubes that provide an important basis for high-throughput genetic screening and potential drug development for screening for potential therapeutic targets for neuromuscular disorders.

BIOLAMININ KEY ADVANTAGES

  • Laminin 211 is the most abundant laminin isoform in the basement membrane of adult muscle

  • Laminin 211 has been shown to contribute to maintaining muscle stem cells (MuSCs) in a potent, quiescent state in vitro and enhanced potency in vivo

  • Culture of MDC1A myotubes on recombinant Biolaminin 211 restores the mitochondrial function
  • Laminin 521 is one of the main isoforms surrounding myogenic progenitors 

  • Culture on Biolaminin 521 gives larger myotubes and higher amounts of nuclei per myotube

  • Increased differentiation potential on Biolaminin 521 without altering the traditional Pax7/MyoD paradigm

  • More consistent and reliable differentiation on Biolaminin 521, even after long-term culture

  • Biolaminin-121, -211, -221,-511, and -521 induced significantly more AChR clusters in C2C12 myotubes than the commonly used laminin-111. Laminin-221 was the most potent for inducing AChR clusters, whereas laminin-121, laminin-211, and laminin-221 afforded the highest percentages of topologically complex assemblies.

  • Human primary myotubes that were derived from myoblasts obtained from patient biopsies cultured on Biolaminin-121, -211, -221,-511, and -521 were also able to form AChR clusters with a complex topology that contained synaptic podosomes. 

  • Translatability across several mouse backgrounds, human cells, and disease states

  • Defined and animal origin-free matrix, suitable for cell therapy development and clinical trials

  • x
  • Expansion of human PSC

  • Mesenchymal stem cells

  • Clonal cell culture applications

  • Eye cells

  • Cardiac cells

  • Neural cells

  • Skeletal muscle cells

  • Kidney cells

  • Hepatic cells

  • Cancer cells

  • Lung cells

  • Animal stem cells

  • Endothelial cells

  • Pancreatic cells

  • Intestinal cells

  • Normal and cancerous mammary cells

  • Epithelial cells

Biolaminin 521 CTG

Biolaminin 521 CTG cell therapy grade cell culture matrix makes pluripotent stem cell culture easy. A defined, animal origin-free and biologically relevant cell culture system for better cell models.

  • x
  • Expansion of human PSC

  • Clonal cell culture applications

  • Eye cells

  • Cardiac cells

  • Neural cells

  • Skeletal muscle cells

  • Kidney cells

  • Hepatic cells

  • Cancer cells

  • Lung cells

  • Animal stem cells

  • Mesenchymal stem cells

  • Endothelial cells

  • Pancreatic cells

  • Intestinal cells

  • Normal and cancerous mammary cells

  • Epithelial cells

Biolaminin 521 MX

Biolaminin 521 MX research-grade cell culture matrix makes pluripotent stem cell culture easy. A defined, animal origin-free and biologically relevant cell culture system for better cell models.

  • x
  • Expansion of human PSC

  • Mesenchymal stem cells

  • Clonal cell culture applications

  • Eye cells

  • Cardiac cells

  • Neural cells

  • Skeletal muscle cells

  • Kidney cells

  • Hepatic cells

  • Cancer cells

  • Lung cells

  • Animal stem cells

  • Endothelial cells

  • Pancreatic cells

  • Intestinal cells

  • Normal and cancerous mammary cells

  • Epithelial cells

Biolaminin 521 LN

Biolaminin 521 LN research-grade cell culture matrix makes pluripotent stem cell culture easy. A defined, animal origin-free and biologically relevant cell culture system for better cell models.