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Laminin Culture Methods

Human stem cell based corneal tissue mimicking structures using laser-assisted 3D bioprinting and functional bioinks

Sorkioa A., Kochb L., Koivusaloa L., Deiwickb A., Miettinena S., Chichkovb B., Skottman H.
Biomaterials, 2018

In this study, the authors produced 3D cornea-mimicking tissues using human stem cells and laser-assisted bioprinting (LaBP). Human embryonic stem cell derived limbal epithelial stem cells (hESC-LESC) were used as a cell source for printing epithelium-mimicking structures, whereas human adipose tissue derived stem cells (hASCs) were used for constructing layered stroma-mimicking structures. The autors used two previously established LaBP setups based on laser induced forward transfer, with different laser wavelengths and appropriate absorption layers. Recombinant human laminin and human sourced collagen I served as the bases for the functional bioinks. For hESC-LESCs, bioink containing human recombinant laminin-521 was chosen, as laminin is a major component in LESC basement membrane in the native cornea. Three different types of corneal structures was printed: stratified corneal epithelium using hESC-LESCs, lamellar corneal stroma using alternating acellular layers of bioink and layers with hASCs, and finally structures with both a stromal and epithelial part. The printed constructs were evaluated for their microstructure, cell viability and proliferation, and key protein expression. The 3D printed stromal constructs were also implanted into porcine corneal organ cultures. Both cell types maintained good viability after printing. Laser-printed hESC-LESCs showed epithelial cell morphology, expression of Ki67 proliferation marker and co-expression of corneal progenitor markers p63α and p40. Importantly, the printed hESC-LESCs formed a stratified epithelium with apical expression of CK3 and basal expression of the progenitor markers. The structure of the 3D bioprinted stroma demonstrated that the hASCs had organized horizontally as in the native corneal stroma and showed positive labeling for collagen I. After 7 days in porcine organ cultures, the 3D bioprinted stromal structures attached to the host tissue with signs of hASCs migration from the printed structure. This is the first study to demonstrate the feasibility of 3D LaBP for corneal applications using human stem cells and successful fabrication of layered 3D bioprinted tissues mimicking the structure of the native corneal tissue.

 

Gelatine methacrylamide-based hydrogels – an alternative 3D cancer cell culture system

Kaemmerer E., Melchels F.P.W., Holzapfel B.M, Meckel T., Hutmacher D.W., Loessner D.
Acta Biomaterialia, 2014

The authors present a 3D biomaterial platform for the analysis of ovarian cancer spheroid growth that is an efficient semi-synthetic alternative, combining native ECM components and tuneable matrix properties, resulting in higher reproducibility, less complexity and better comparability between different groups than traditional cell monolayer approaches. In this study, gelatine methacrylamide-based hydrogels (GelMA) with added laminin 411 were established as in vitro and in vivo spheroid-based 3D cancer models. 

 

Development of a biomaterial associated with mesenchymal stem cells and keratinocytes for use as a skin substitute

Steffens D., Mathor M.B., Santi B.T.S., Luco D.P., Pranke P.
Regen. Med., 2015

Here they developed s scaffolds of poly-DL-lactic acid with and without the linkage of laminin 332, bringing together MSCs and keratinocytes aimed for treatment as a new skin substitute. Three groups of scaffolds were studied: 1) poly-DL-lactic acid (PDLLA), 2) hydrolyzed PDLLA (PDLLA/NaOH) and 3) PDLLA/Lam which is a PDLLA/NaOH scaffold linked to laminin protein. The scaffolds developed in this work have the characteristics for use as a new biomaterial that is completely adequate for application in tissue engineering for skin regeneration. The linkage of laminin was achieved and demonstrated. The binding of the laminin in the matrices made the biomaterial become more hydrophilic. The use of biocompatible and biodegradable polymers associated with the properties of laminin lead to an improvement in the adherence and viability of the cells, showing laminin 332 is beneficial for growth of MSCs and keratinocytes.

 

Human embryonic stem cell dispersion in electrospun PCL fiber scaffolds by coating with laminin-521 and E-cadherin-Fc

Leino M., Astrand C., Hughes-Brittain N., Robb B., McKean R., Chotteau V.
J Biomed Mater Res Part B 2017

Here, the authors describe a nonaggregate culture system of human embryonic stem cells inside electrospun polycaprolactone (PCL) fiber scaffolds combined with the defined extracellular proteins laminin-521, naturally occurring in the stem cell niche. PCL fiber scaffolds coated with recombinant human laminin-521 readily supported initial stem cell attachment and growth from a single-cell suspension. The combination of recombinant E-cadherin-Fc and laminin-521 sigthly improved cell dispersion rendering a uniform cell population. Finally, we showed that the cells cultured in Ecadherin-Fc- and laminin-521-coated PCL scaffolds could differentiate into all three germ layers. Importantly, we provided a chemically defined 3-D system in which pluripotent stem cells grown and differentiated avoiding the formation of cell aggregates.

 

Enhanced reseeding of decellularized rodent lungs with mouse embryonic stem cells

Lecht S., Stabler C.T., Rylander A.L., Chiaverelli R., Schulman E.S., Marcinkiewicz C., Lelkes P.I.
Biomaterials, 2014

Pre-treatment of decellularized lung with defined ECM proteins, to evaluate the efficacy of reseeding of mESC. All current decellularization methodologies result in significant alterations of the contents, and ratios of ECM proteins, though the exact degree of the loss of ECM glycoproteins, such as laminins. Following ECMs were tested: bovine collagen type IV; human collagen type IV; human pro-collagen type I; human collagen type I; rat collagen type I; human plasma-purified fibronectin; human thrombospondin-1; VCAM; vitronectin; bovine elastin; Matrigel-purified laminin 111; and human recombinant laminins 111, 211, 332, 411, 421, 511, 521. mESCs lack major integrin receptors for collagens but express high levels of functional receptors for LM and FN. Laminin next to FN appears to be the major pro-adhesive ECM protein for mESCs. The mESCs were found to adhere differentially in an isoform-dependent manner with the following order of potencies 511 = 521 >332 >421 >211 >111 >411. These findings further support the notion that a3b1 and a6b1 integrin receptors expressed on the mES cell surface are involved in the specific binding to LM in general and to LM 511 and 521. LM and FN interact more efficiently with collagen type I than with collagen IV or elastin 90% of the available Coll I in the decellularized lung matrix forms a complex with LM. in vitro that cell-derived FN and LM interact with Coll I with much higher efficiently than commercial FN or LM This finding may be explained by a possible partial loss of activity as a result of the purification procedures in contrast to the unprocessed CM.

 

Collaboration of 3D Context and Extracellular Matrix in the Development of Glomma Stemness in a 3D Model

Ma N.K.L., Kai Lim J., Fatt Leong M., Sandanaraj E., Ti Ang B., Tang C., Wan A.C.A.
Biomaterials, 2015

This work illustrates that different laminin isoforms have specific effects in promoting the stemness of glioma cells, in collaboration with a 3D context. U251 glioblastoma cells were cultured on electrospun polystyrene (ESPS) scaffolds coated with 7 different laminin isoforms (LAMscreen kit) to provide a 3D model for stem cell-related genes and proteins expression studies. The results indicate the influence of 3D (versus 2D) context on stemness markers and integrin expression, specifically, the upregulation of the laminin-binding integrins a6b4. By a colony forming assay, we showed enhanced clonogenicity of cells grown on ESPS scaffolds in collaboration with laminins 411, 421, 511 and 521. The present results demonstrate how 3D versus 2D context profoundly affects ECM signaling, leading to stemness.

 

Improved Human Pluripotent Stem Cell Attachment and Spreading on Xeno-Free Laminin-521-Coated Microcarriers Results in Efficient Growth in Agitated Cultures

Lam A.T., Li J., Chen A.K., Birch W.R., Reuveny S., Oh S.K.
BioResearch Open Access, 2015

Plastic and plastic plus microcarriers (MCs) coated with LN521 without additional positive charge for a defined, xeno-free, GMP-compatible, and scalable bioprocessing platform. Efficient cell attachment of both ES and iPS cell (87%) and spreading (85%), which leads to generation of cells/MC aggregates (400 um in size) and high cell yields (2.4–3.5 · 106 cells/mL) within 7 days in agitated plate and scalable spinner cultures. Long-term pluripotent cell expansion and maintenance of normal karyotype was demonstrated after 10 cell passages. Moreover, tri-lineage differentiation as well as directed differentiation into cardiomyocytes was achieved when placed back on 2D Ln521 (enzymatic dissociation). The authors show that LN521 enables efficient cell attachment and spreading of hPSCs that results in high cell yields (~3.5×10^6 cells/mL) within 7 days in agitated plate and scalable spinner cultures. This offers a defined, xeno-free, GMP-compatible, and scalable bioprocessing platform for the production of hPSC with the quantity and quality compliant for clinical applications. Use of LN521 on microcarriers also enabled a 34% savings in matrix and media costs over monolayer cultures to produce 10^8 cells.

     

Agitation conditions for the culture and detachment of hMSCs from microcarriers in multiple bioreactor platforms

Nienow A.W., Hewitt C.J., Heathman T.R.J, Glyn V.A.M, Fonte G. alo N., Hanga M.P, Coopman K., Rafiq Q.A.
Biochemical Engineering Journal. 2015

The authors used different bioreactors up to 2.5 L in scale, and successfully cultured hMSCs using the minimum agitator speed required for complete microcarrier suspension. In addition, we also reported a scaleable protocol for the detachment from microcarriers in spinner flasks where they use of a short period of intense agitation in the presence of enzymes such that the cells are detached without being damaged. Here, the same approach has been effective for 15 mL ambrTMbioreactors, 100 mL spinner flasks and 250 mL Dasgip bioreactors.  Two types of microcarrier was used, with (laminin-521) and without surface coatings, four different enzymes and three different growth media (with and without serum), a total of 22 different combinations. In all cases after detachment, the cells were shown to retain their desired quality attributes and were able to proliferate. Qasim Rafiq presented this data at BioLamina symposium 2015. It was found that human recombinant laminin LN-521 and recombinant fibronectin were the optimal ECM proteins for microcarrier coating. A serum-free expansion, harvest and xeno and DMSO-free cryopreservation process, using LN-521 coated microcarriers and FREEZEstemTM cryopreservation medium, was then developed which demonstrated > 5 fold increase in hMSC yield.

 

Live visualization of chromatin dynamics with fluorescent TALEs

Miyanari Y., Ziegler-Birling C., Torres-Padilla M.E.
Nature structural & molecular biology, 2013

The authors of these two Nature publications show that laminin can be coated directly on glass, which many other substrates and proteins can not. This enables growth of pluripotent stem cells as monolayeres even on glass, which is especially suitable for live imaging.

 

Control of ground-state pluripotency by allelic regulation of Nanog

Miyanari Y., Torres-Padilla M.E.
Nature Letter, 2012

Laminin-511 used to coat glass-bottomed dishes.

 

Dynamic Heterogeneity and DNA Methylation in Embryonic Stem Cells

Singer Z.S., Yong J., Tischler J., Hackett J.A., Altinok A., Surani M.A., Cai L., and Elowitz M.B.
Cell Press, 2014

Analyzis of gene expression in individual ESCs using single-molecule RNA-FISH and quantitative time-lapse movies. Fluorescence Time-Lapse Microscopy and Data analysis on reporter cells mixed with unlabeled parental cells at 1:9 ratio and plated at a total density of 20,000 cells/cm2 on glass-bottom plates (MatTek) coated with human laminin-511 >12 hr before imaging.

 

Early embryonic-like cells are induced by downregulating replication-dependent chromatin assembly

Ishiuchi T., Enriquez-Gasca R., Mizutani E., Bošković A., Ziegler-Birling C., Rodriguez-Terrones D., Wakayama T., Vaquerizas J.M., Torres-Padilla M-E.
Nature structural & molecular biology, 2015

mES cells cultured on coverslips coated with laminin-511for a RNA-FISH assay.

 

A Balance between Secreted Inhibitors and Edge Sensing Controls Gastruloid Self-Organization

Etoc F., Metzger J., Ruzo A., Kirst C., Yoney A., Ozair Z.M, Brivanlou A.H., Siggia E.D
Developmental cell, 2016

Used colonies of hESC grown on micropatterned substrate and differentiated with BMP4 to model patterning events in the human embryo. Coated micropatterned glass coverslips with LN521.

A High Proliferation Rate is Critical for Reproducible and Standardized Embryoid Body Formation from Laminin-521-Based Human Pluripotent Stem Cell Cultures

Dziedzicka D., Markouli C., Barbé L., Spits C., Sermon K., Geens M.
Stem Cell Rev and Rep., 2016

In this study the authors developed an efficient and standardized embryoid body (EB) formation protocol for human pluripotent stem cells (hPSC) cultured in a laminin-521-based xeno-free system. The cell proliferation rate of the cells growing on laminin-521 strongly affected the efficiency of aggregate formation, and recently passaged cells, as well as the addition of ROCK inhibitor, were essential for reproducible EB formation from hPSC single-cell suspensions. EBs could be obtained in a variety of differentiation media, in 96-well round-bottom plates and in hanging drops. The authors also showed that the medium used for differentiation influenced the differentiation outcome to a much greater extent than the number of cells used for the initial EB formation.

Optimization of slow cooling cryopreservation for human pluripotent stem cells

Miyazaki T., Nakatsuji N., Suemori H.
Genesis, 2013 

This is one of the first customer publications that demonstrates laminin-521 as an optimal xeno- and feeder-free matrix for pluripotent stem cells. The authors show cells should be cryopreserved as single cells for highest survival which is specifically supported by laminin-521 that promotes adhesion and self-renewal of fully dissociated single cells in the absence of ROCK inhibitor. They demonstrate 80-90% survival of hPSCs post-thawing and 60% survival following subculture on laminin-521, allowing for efficient and easy handling of cells and bulk storage of high-quality hPSCs.

A Balance between Secreted Inhibitors and Edge Sensing Controls Gastruloid Self-Organization

Etoc F., Metzger J., Ruzo A., Kirst C., Yoney A., Ozair Z.M, Brivanlou A.H., Siggia E.D
Developmental cell, 2016

Here, the authors use colonies of hESC grown on micropatterned substrate and differentiated with BMP4 to model patterning events in the human embryo. Coated micropatterned glass coverslips with LN521.

N-Terminomics identifies HtrA1 cleavage of thrombospondin-1 with generation of a proangiogenic fragment in the polarized retinal pigment epithelial cell model of age-related macular degeneration

Chen C-Y., Melo E., Jakob P., Friedlein A., Elsässer B., Goettig P., Kueppers V., Delobel F., Stucki C., Dunkley T., Fauser S., Schilling O., Iacone R.
Matrix Biology, 2018

Here, the authors investigate the impact of elevated HtrA1 levels on the retina pigment epithelial (RPE) secretome using a polarized culture system. The authors suggest a mechanism by which increased levels of HtrA1 may contribute to AMD pathogenesis. Human primary Retinal Pigmented Epithelium (RPE) cells (Sciencell, 6540) was seeded in transwells coated with Laminin 521. A model that recapitulates the structural, molecular and apical/basolateral signatures of adult RPE cells was achieved. Upregulation of HtrA1 alters the abundance of key proteins involved in angiogenesis and extracellular matrix remodeling. Thrombospondin-1, an angiogenesis modulator, was identified as a substrate for HtrA1 using terminal amine isotope labeling of substrates in conjunction with HtrA1 specificity profiling. HtrA1 cleavage of thrombospondin-1 was further corroborated by in vitro cleavage assays and targeted proteomics together with small molecule inhibition of HtrA1. While thrombospondin-1 is anti-angiogenic, the proteolytically released N-terminal fragment promotes the formation of tube-like structure by endothelial cells.

 

CCR2+CCR5+ T Cells Producing Matrix Metalloproteinase-9 and Osteopontin in the Pathogenesis of Multiple Sclerosis

Sato W., Tomita A., Ichikawa D., Lin Y., Kishida H., Miyake S., Ogawa M., Okamoto T., Murata M., Kuroiwa Y., Aranami T., Yamamura T.
Journal of Immunology, 2012

To recapitulate the glia limitans layered with parenchymal basal lamina experimentally, we coated the upper sides of Transwell membrane inserts. The upper sides of Transwell membrane inserts (8 mm; Corning) were coated with 10 mg/ml laminin-1 (Sigma) or 20 mg/ml laminin-121. After aspirating the laminin solutions, the membrane inserts were turned upside down, and normal human astrocytes (NHA) were seeded on the lower sides of the membrane inserts. T cells were stimulated, harvested, suspended in the fresh medium, and seeded onto the upper chambers. After 8 h, cell suspension was collected from the lower chambers after careful pipetting, and absolute numbers of migrated cells were calculated. The T-cell migration across the NHA layered with laminin-111 or -121 was less efficient compared with the migration across the untreated membrane or the membrane treated with laminin alone, thus, this model would exhibit barrier functions against the penetration of activated T cells.

 

A Balance between Secreted Inhibitors and Edge Sensing Controls Gastruloid Self-Organization

Etoc F., Metzger J., Ruzo A., Kirst C., Yoney A., Ozair Z.M, Brivanlou A.H., Siggia E.D
Developmental cell, 2016

Used colonies of hESC grown on micropatterned substrate and differentiated with BMP4 to model patterning events in the human embryo. Coated Costar Transwells with LN521.

 

TRPC3-GEF-H1 axis mediates pressure overload-induced cardiac fibrosis

Numaga-Tomita T., Kitajima N., Kuroda T., Nishimura A., Miyano K., Yasuda S., Kuwahara K., Sato Y., Ide T., Birnbaumer L., Sumimoto H., Mori Y., Nishida M.
Scientific Reports, 2016

iPSC-derived cardiomyocytes seeded as single cells onto laminin-211 coated stretch chamber dishes (Menicon).