Laminin Cell Culture Matrices - These extracellular matrix proteins are the key to efficiently harvesting and reliably culturing stem cells and specialized cells (2023)

Since the generation of the first lineage of human embryonic stem cells, researchers in the fields of regenerative medicine, basic biomedical research and drug discovery have strived for more reliable and reproducible methods of culturing and/or differentiating human pluripotent stem cells (hPSCs). cells. Years of development have resulted in better defined culture methods and although significant media improvements have been made, undefined culture media are still used and few are natural and cell type specific.

Reliable derivation and expansion of high quality hPSCs under feeder-free, defined and xeno-free conditions, rapid scale-up, efficient and reproducible differentiation and maintenance - all these areas are important for a stem cell researcher, but many struggle to achieve it. In addition, for therapeutic applications, all these steps must meet clinical requirements. Mimicking the natural cell environment is the key to success and the growing media play a crucial role. Here I will discuss the use of BioLamina's biologically relevant human recombinant laminin cell culture matrices and how they can be used to mimic the cell-specific physiological microenvironment on a cell culture dish. We call it biorelevance. Let me show you how laminins can pave the way to successful cell therapy.

Laminin Biology

Most organized cells in the body grow on specialized extracellular matrices (ECMs) called basement membranes, and different cells and tissues require their specific ECM composition for survival and proper function. Laminins (>16 different isoforms) are the major cell-type specific proteins of the ECM and are both spatially and temporally regulated. Cells bind to laminins via cell surface receptors such as integrins, which regulate vital cellular responses such as cell attachment, survival, proliferation and differentiation. Laminins are complex proteins and BioLamina is the only company able to supply various full-length recombinant laminin isoforms with biological activities intact. Our chemically defined and xeno-free laminin cell culture substrates correctly mimic natural cell-matrix interactionin-vitro, and can be used for many different applications, such as B. the expansion of pluripotent cells and the differentiation and maintenance of specialized cell types.

Laminin-521 Stem Cell Matrix provides efficient derivation and robust and reliable expansion of pluripotent stem cells

The expression of laminin chains can be observed as early as four-cell stage embryos, and in the blastocyst, laminins containing an α5 chain, such as laminin-521 and laminin-511, are abundant around the pluripotent human embryonic stem (ES) cell inner cell mass .

The laminins containing the α5 chain also surround many adult stem cells, such as B. cells in crypts of intestinal villi, muscle precursors and hair follicles. The prominent localization of laminins in the stem cell niches suggests that they are important for stem cell growthlive.In 2014, Rodin et al. first described an efficient xeno-free and chemically defined protocol for monolayer cultivation of hPSC on laminin-521 (Rodin et al., Nature Protocols., 2014). Via the specific binding of cellular receptors to hPSCs, laminin-521 triggers authentic cell signaling pathways that enable high survival and rapid expansion of hPSC single cell cultures without the addition of ROCK inhibitors or other inhibitors from Anoikis. hPSCs in single cell suspension plated on laminin-521 rapidly attach, migrate and form small monolayer colonies. The cells rapidly divide and expand until the entire dish is covered by a confluent monolayer. hPSC cultured in thick multilayer colonies often require manual, random removal of undifferentiated parts. In contrast, hPSCs were cultured

on laminin-521 grow as a homogeneous monolayer with minimal risk of spontaneous differentiation or genetic abnormalities. Additionally, the low batch-to-batch variation in laminin substrates leads to more consistent and reproducible experiments. The biorelevant culture environment produces hPSC cultures with more uniform gene expression and yields a priming effect that improves cell maturation, polarization, and functional organization in many cellular applications. The laminin-521 matrix also enables efficient clonal derivation, clonal survival, and long-term self-renewal of human ES cells. Human ES cell lines can even be derived from a single blastomere under chemically defined and xeno-free conditions on LN-521 (Rodin et al., Nature Communications, 2014) without destroying the embryo and thereby circumventing the ethical issues associated with humans ES cells. Laminin-521 enables flexible and user-friendly culture protocols, where cells can be seeded at low densities (>5,000 cells/cm²).²) and cultured to high (up to 99%) confluency. In addition, the Laminin 521 growing medium is compatible with any medium, any dissociation agent (e.g. enzymes and EDTA) and supports weekend-free feeding.

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Biology holds the key to the proper use of hES and iPS cells and paves the way for successful cell therapies

The field of stem cell therapy is still in its infancy and there are currently no GMP-compliant substrates available that provide reliable differentiation performance. Cell therapy companies still rely on undefined and non-human matrices. However, increasing emphasis is now being placed on cell culture products that better relate to future clinical translation. Due to the validated functionality and superior biological properties, human recombinant laminins in combination with optimized differentiation protocols offer exciting prospects for regenerative medicine. Laminin substrates have significantly advanced the field of stem cell research and have become a substrate of choice for both basic and therapeutic-focused research and companies. This has been highlighted in a number of high impact scientific articles over the past two years and some examples are detailed below:

High yield of clinically compliant dopaminergic neurons

Current treatment regimens for Parkinson's disease are primarily based on drugs that mimic dopaminergic signaling in the brain. These dopamine agonists and precursors work well initially but are limited by the development of loss of function along with increased side effects during treatment. The transplantation of dopaminergic neurons is a potential major advance in the treatment of this disease. The group of dr. Malin Parmar from Lund University, Sweden, recently published a groundbreaking article in Cell Stem Cell, in which she presented a fully defined and GMP-compliant method for dopaminergic cell differentiation of human ES cell lines on Biolamina Laminin-111 (Kirkeby et al., Cell Stem Cell, 2016). Their protocol provides a dramatic (>40-fold) increase in cell yield of high-quality, clinically relevant human dopaminergic neurons and significantly reduces the cost of preparing the cells for transplantation. Their method is so effective that the cell material from a 6-well plate is sufficient to produce material for the treatment of 500 patients. With the help of large grants, this research group, in collaboration with BioLamina and other partners, aims to make the transition to a cell therapy for Parkinson's disease within a few years.

Efficient hepatocyte maturation and cell organization

Stem cell-derived hepatocytes are a promising tool to create more predictive in vitro toxicity models for preclinical safety assessments and could become a more accessible, commercial alternative to organ donation. In November 2015, the group of Dr. David Hay from the University of Edinburgh, Scotland, a paper in which they show a defined process for differentiating human ES cells into hepatocytes using a combination of laminin-521 and laminin-111 as culture substrates (Cameron et al., Stem Cell Reports, 2015). Their results demonstrate efficient hepatocyte specification, polarized organization, enhanced maturation, and significant improvement and stabilization in cell functions. Their results represent a significant advance and bring cell-based hepatocyte therapies closer to biomedical applications.

JoVE Articlefor the defined and scalable generation of hepatocyte-like cells from human pluripotent stem cells.

Maintaining the differentiation potential of satellite cell-derived myoblasts during long-term culture

Large-scale expansion of myogenic progenitors is needed to support the development of high-throughput cellular assaysin vitro,and to advance genetic engineering approaches needed to develop cell therapies for rare muscle diseases. A recent publication by researchers at Icagen, Inc., USA shows that laminin-521 is a superior substrate for myogenic cell culture applications (Penton et al., Skeletal Muscle, 2016). Laminin-521 dramatically improves cell proliferation and preserves the differentiation potential of human satellite cell-derived myoblasts. Laminin-521 appears to increase differentiation potential without altering the traditional Pax7/MyoD paradigm and supports the formation of larger myotubes and higher amounts of nuclei per myotube. Importantly, laminin-521 provides more consistent and reliable differentiation even over long-term culture expansion.

Clinically compliant hESC-derived retinal pigmented epithelial cells (RPE).

Age-related macular degeneration is the leading cause of severe vision loss in the western world, with the economic burden on healthcare systems exceeding that of stroke. The RPE monolayer of cells is essential for normal neuroretinal and choroidal functions. In early 2016, the groups of Dr. Fredrik Lanner, Outi Hovatta and Anders Kvanta from Karolinska Institutet, Sweden published a xenofree and defined protocol for the production of human ES cell-derived RPE cells on laminin-521 (Plaza Reyes et al., Stem Cell Reports, 2015) . Suspension transplants of the laminin-521 RPE cells into a bigeye rabbit model show efficient cell integration as a polarized subretinal monolayer that was able to rescue overlying photoreceptors from induced damage. Work is underway to create new GMP-grade xenofree human ES cell lines for the construction of an HLA-matched hESC/RPE library using BioLamina laminins for transfer to a clinical setting.

Read more about our differentLaminin Cell Applications

In summary, by providing cells with their biorelevant matrix proteins, the natural cellular interactions required for cell adhesion, proliferation, organization and survival are stimulated. The derivation and expansion of hPSCs and subsequent cell specialization on specific laminin substrates could help bridge the gap between academic stem cell research and the development of human cell therapies. To further facilitate the transition of these preclinical research protocols into clinical settings, BioLamina is currently developing a clinical grade product of Laminin-521 (MX521). A clinical-grade laminin-111 (MX111) is also under development to address the need for dopaminergic neuron and hepatic cell therapy protocols. We believe that recombinant laminin culture media are one of the essential components for unlocking the great potential of stem cells and that the research examples highlighted here will be followed by many more pioneering works for other applications. Please contactScientific Supportor visit ourscience roomfor further data and publications based on human recombinant laminins.

Cover picture: Human embryonic stem cells on laminin-521. Courtesy of Dr. Liselotte Antonsson, Karolinska Institute, Sweden