Why is using biologically relevant matrix so important for your research?

Basement membranes (BM) are sheet-like extracellular matrix structures that is the foundation for cells to grow on. The BM composition is highly cell-surface selective and laminins are key proteins. In addition to their central role in BM structural organization, laminin is essential for modulation of vital cellular responses, such as cell adhesion, differentiation, migration, phenotype stability, and resistance to apoptosis. Without the right combination of laminin isoforms cells and tissues becomes dysfunctional.

Biorelevance is about emphasizing nature. Our laminin cell culture matrices allow you to imitate the natural, cell-specific cell-matrix interaction in vitro, leading to enhanced cell maturation, cell organization and improved cell functionality. We offer an expansive portfolio of chemically defined and xeno-free laminin proteins for a variety of applications, including reliable expansion of pluripotent cell and differentiation and maintenance of specialized cell types, such as hepatocytes, skeletal muscle cells and different neural cells. The impact of our laminin matrices on cell culture quality has been scientifically validated in many high-impact journals. 

What are the major advantages of using human recombinant laminins vs. truncated or tissue-originated laminin products?

The BioLamina laminins are the only original full length, recombinant laminins on the market, with all the functional domains intact. Laminins isolated from tissue is an impure mix of several ECM proteins. Moreover, during isolation the proteins tend to be heavily degraded with the consequence of structural integrity and lost function. A fractionated or truncated laminin molecule or laminins isolated from tissue, lack many of the laminin domains which is needed by the cells for the proper extracellular network to form and to for stimulation of correct cellular signal transductions. Hence, only the intact, full length laminin can create a more authentic cell culture environment.

For reference about the difference in quality and function of commercially available recombinant versus isolated preparations of laminins see Wondimu et al., 2006.

What is the purity of Laminin products?

The purity of our laminin products is greater than 95%, as assessed by SDS-PAGE.

Which antibodies do you recommend for recognizing different laminin chains in tissue?

As a tool to identify and study the expression pattern of different laminin subunits present in a specific tissue we recommend to use the Laminin Marker Panel of PrecisA Monoclonals from Atlas Antibodies.

What are the molecular weights of the different laminin isoforms?

The molecular weights (MWs) of the different laminins isoforms are stated in the table below. Due to the complex post-translational modifications such as glycosylation of the laminin molecules, the MWs of each laminin is deduced by amino acid sequences used as reference.
  

 
How do I store the laminin protein properly?

The laminin stock solution is stable for 3 years when stored at -20°C to -80°C. Thawed laminin stock is stable for at least 3 months when stored at +2°C to +8°C under aseptic conditions. Avoid long exposure of the protein to ambient temperatures. Repeated freeze/thaw should be avoided. Laminin coated plates are recommended to be used as fresh as possible but, for your convenience, coated plates and diluted coating solution can be kept at 2-8°C for up to 4 weeks. Seal the plates (e.g. with Parafilm®) to prevent evaporation. The laminin matrix will be inactivated if let dry.  

521-To-Go™ pre-coated plates with can be stored at room temperature (+15°C to +25°C) with maintained functionality for at least 1 year.

How long can I store the thawed laminin laminin stock solution?

Thawed laminin stock solution (100 ug/mL) is stable for at least 3 months when stored at +2°C to +8°C under aseptic conditions. 
 

How long can I store laminin coated plates?

Laminin coated plates are recommended to be used as fresh as possible but, for your convenience, coated plates and diluted coating solution can be kept at 2-8°C for up to 4 weeks. Seal the cultureware (e.g. with Parafilm®) to prevent evaporation and contamination. The laminin matrix will be inactivated if let dry. Adjust the volume by adding DPBS (Ca++/Mg++).

Unused wells from coated plates stored at 37°C are not recommended to be re-used.

How stable is recombinant laminin at room temperature?

Laminins proteins should be handeled with care and unnecessary exposure of the protein to ambient temperatures should be avoided. If possible, keep the sample on ice during work.

How do I use laminin for cell culture?

Coat cultureware with laminin solution according to the instruction. Dilute the thawed laminin stock solution with 1xDPBS (Ca++/Mg++) and add the diluted laminin solution to tissue culture-treated cultureware. Seal the cultureware (e.g. with Parafilm®) to avoid evaporation. Incubate at +2°C to +8°C over night or, if a more rapid coating is required, incubate at +37°C for 2 hours. For culture of cells, suck away the excess laminin coating solution and add the cell suspension to the laminin coated well. No washing is required. If not used immediately, coated plates and diluted coating solution can be kept at +2°C to +8°C for up to 4 weeks. Do not allow the coated surface to dehydrate as that will inactivate the laminin coating.

What is the recommended laminin coating concentration?

For culture of hESC or iPSC lines, a coating concentration of 5 ug/mL is recommended as a starting concentration. The optimal coating concentration is cell type-dependent and should be optimized empirically for specific isoforms and cell lines. For many MSC lines and for many neural applications, a coating concentration as low as 1 ug/mL often can be used. For culture of some specialized cells, such as cardiomyocytes, a higher coating concentration (10 ug/mL) might be requied. Too low coating could result in slow growth or an uneven cell spread. 

I have issues with bad cell attachment when transfering my pluripotent stem cells from feeder cells or from feeder-free culture substrates to laminin. Hwat can I do?

If the transfer from other culture substrates are problematic, please follow the instruction for transfer to laminin. Some fay aspects are listed here:

  • We recommend to transfer the cells as single cells (or as small clumps) and always with the addition of apoptosis inhibitor, such as ROCKi for the first few (3-5) passages. Once the cells are adapted to the LN-521 matrix, the cells can be cultured as single cells without ROCKi. This may take up to 5 passages - give it some time.
  • If the cells are hard to adapt, try increasing the coating concentration to 10 ug/ml and seed at a higher cell density 50,000 – 100,1000 cells/cm2. Once the cells are adapted a lower coating and seeding concentration often can be used.
  • It is important that the cells transferred to the LN-521 matrix are of high quality. LN-521 also support specialized cell types so carefully select only undifferentiated cell areas for transfer. 
  • LN-521 work well in combination with most commercial media brands. However, it is to be expected that cell morphology will look different dependent on the medium used for culture. 

After seeding, I experience an uneven cell spread: What could be the reason for that?

An uneaven cell spread is often caused by an unsufficient laminin coating and could be caused by the following:

  • A too low coating concentration is beeing used. Increase the coating concentration untill you reach a high enough to support an even cell growth.
  • DPBS Ca--/Mg-- have been used to dilute the laminin stock solution. We recommend to use DPBS with Ca2+ and Mg2+ since divalent cations are important for the protein structure and function. 
  • Issues with the cultureware plastic. Most plastic work well for laminin coating but we know the laminin coating is not compatible with some NUNC-plates. SARSDET and Corning plate usually work well.
  • Bad coating coverage/the plate has dried out. Ensure that the entire surface is covered by the laminin coating solution when preparing fresh plates. Also, do not let the plate dry out as this will inactivate the laminin coating. Too long time in the incubator or long storage without sealing could cause too much evaporation so that part of the plate dries out (often center).

Does the coating concentration affect the properties of the cells?

Too low coating will result in slow growth and an uneven cell spread. Otherwize, no significant impact on cell properties has been observed.

Is there a difference in performance between the slow coating and the fast coating?

No significant difference on cell survival and proliferation rate has been observed.

Are BioLamina's laminin proteins optimized for any specific plastic brands?

The laminin coating is not optimized for certain plastic brands or well formats, however out of the brands tested there are a few with which the laminin coating seem to be functioning better (e.g. Falcon, Sarstedt, Corning) than others. Some Nunc culturewares are not compatible with laminin coating. 

Can the laminin be used to coat other surfaces, such as glass?

Yes, laminin can be used for coating glass with good cell attachment and maintained cell functions. Coat glassware as you normally coat your cultureware, however, 24-48 hour coating at +2°C to +8°C is recommended for a more reliable coating. Seal the coated glassware to avoid evaporation. Make sure all the surface is completely covered by the laminin coating solution as uncoated surface will not support cell growth. Please see Miyanari et al., 2013 for coating reference. 

Can I re-use the coating solution?

Due to contamination risks and uncontrollable coating concentration issues we do not recommend to re-use the coating solution but rather to evaluate the optimal coating concentration for you cells and application by titration. Often a lower concentration than the recommended starting concentration could be used. For culture of hESC or iPSC lines, a coating concentration of 5 ug/mL is recommended as a starting concentration. For many MSC lines and for many neural applications, a coating concentration as low as 1 ug/mL often can be used. Note that the optimal coating concentration is cell type-dependent and should be optimized empirically for specific isoforms and cell lines.

How long does the laminin coating last in culture? Do I have to add extra laminin to long-term cultures?

The laminin coating is functional for at least one month in culture. For long-term culture, if cell detachment is noticed, we recommend to add 1-5 ug/mL extra laminin to the medium.

Can I mix the laminins with other matrix proteins, such as collagen?

We don't have any data for this, but to ensure the best coating, one should do titration to optimize the mix ratio and coating time. 

Can I culture pluripotent stem cells as single cells on laminin-521 without use of apoptosis inhibitors? 

Yes! Laminin-521 successfully recreates the biologically relevant hPSC milieu in vitro and via integrin binding, laminin-521 induces the PI3K/Akt signaling pathway, promoting high survival and robust long-term self-renewal of human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC). Due to the biologically relavant support from the laminin matrix, pluripotent stem cells can be cultured as single cells without need for apoptosis inhibitors, such as ROCKi. When transfered from feeder-cells or feeder-free substraes to laminin, the cells migh need an adaptation period. It migh take up to 5 passages so give it some time. Once the cells are adapted to the LN-521 matrix, the cells can be cultured as single cells without ROCKi. 

Which enzymes do you recommend for passaging PSCs growing on laminin?

You can use a dissociation reagent of choice (e.g. TrypLE select, Trypsin-EDTA, EDTA, Accutase). The incubation time depends on the solution used to dissociate the cells and the specific cell line. Please not that cells often attach tighter on laminin compared to other matrices and scraping or pipetting without first loose up cells can affect cell integrity and viability which could result in less attachment next day. Less confluent cells need shorter treatment time whereas more confluent cells might need longer treatment time. The cells should detach easily without too much pipetting needed. Stem cells are sensitive and too long exposure to dissociation enzymes or too much mechanical force applied may result in lower cell viability. 

Does the laminin-521 only work with certain hPSC media?

The robust support of pluripotent stem cells by the laminin-521 culture substrate allow you to work with your medium and enzyme of choice. As a result, your protocols can easily be made totally defined and xeno-free. We have successfully tested many different commercial media, such as NutriStem™, mTeSR™1 & TeSR™2 and Essential 8™. However, it is to be expected that cell morphology will look different dependent on the medium used for culture.  

If you are using AF Nutristem XF for feeder containing culture, remember to switch to NutriStem XF/FF when using LN-521 as coating material.

Can I passage my stem cells as clumps using the stem cell matrix?

Yes, laminin-521 supports both single cell and clump passaging. However, we recommend single-cell passage or passage as small clumps since it is a much easier and more reliable method that allows standardized cultures. Each cell will have equal contact with the coating and the medium resulting in an homogenous environment. LN-521 is the natural niche protein for the cells and enables cell-cell contact since it promotes high cell migration. The survival rate after single cells seeding is high. When using LN-521 no treatment with apoptosis inhibitors, such as Rho-kinase (ROCK) inhibitor or blebbistatin, is needed to prevent anoikis. Hence, the conventional method where colony state is maintained to prevent apoptosis due to failure to adhere at re-seeding is unnecessary. Single cell passaging also decreases the risk of spontaneous differentiation.

Which range of seeding density do you recommend for human pluripotent stem cells?

Optimal seeding densities will vary from one cell line to another and should be determined empirically for your system. Laminin-521 has been shown to support cell survival of as low as 5,000 cells/cm2. However, we generally recommend seeding your cells at a concentration of 30,000-50,000 cell/cm2 or split your cells with a ratio of 1:10 to 1:30. 

How long does it take for the seeded cells to attch to the laminin matrix?

Most cells should have attached within 1 hour post-seeding and the cells should be evenly distributed over the entire plate. If there is a lot of cell death after seeding, the cells have most likely been treated too harsh during splitting. After cell attachment the cell start to migrate and should have formed small colonies 24h post-seeding. The cells will continue to expand as a homogenous monolayer and are ready to be passaged when cell culture is 60-99% confluent. Depending on the cell line, seeding density and on the medium used, cultures are usually passaged 3-6 days after seeding.

Can I make clonal cultures on laminin-521?

Clonal cultures can efficiently be done with CLONEstem-To-Go™, which is a product based on the LN-521 matrix in combination with other natural stem cell niche molecules. CLONEstem-To-Go™ allows efficient clonal derivation (about 20% success rate), clonal survival and long-term self-renewal of hESC under defined and xeno-free conditions. Furthermore, CLONEstem-To-Go™ even allows hESC derivation from a single blastomere without the need to destroy the embryo. The science behind this is further described in an article in Nature Communications by Rodin and colleagues (Rodin et al., 2014).

Why do I see that cells stick to the outer rim of the wells in 96-well plates?

This is probably caused by the high surface tension created in the smaller well formats. Try to slighly increase the coating volume. 

Do I need to pre-warm all solutions before usage and why?

The solutions should be pre-warmed since a large variation in temperature is stressful for the cells.

Can I use BioLamina’s human recombinant laminins for my animal ES and iPS cells?

Yes, most likely. Laminins, as well as many other basal membrane proteins, are highly conserved proteins. Customers working with mouse and monkey stem cells are successfully using our human recombinant laminins.
 

When culturing my mESC feeder-free on laminin-511 and laminin-521, do I have to add LIF to prevent the cells from differentiating?

No, laminin-511 and laminin-521 supports self-renewal of mouse ES cells in the absence of feeder cells, LIF, or other differentiation inhibitors, even at low cell density. Both laminin isoforms successfully support both naive and primed stem cells since it has been shown to activate the PI3/Akt pathway while the MAPK/ERK pathway is unaffected (Rodin et al., 2014).

How do I transfer my cells from feeder cells to the laminin-521 culture substrate?

We recommend to transfer the cells as single cells (or as small clumps) and always with the addition of ROCKi for the first few (3-5) passages. Once the cells are adapted to the LN-521 matrix, the cells can be cultured as single cells without ROCKi. This may take up to 5 passages. If the cells are hard to adapt, try increasing the coating concentration to 10 ug/ml and seed at a higher cell density 50,000 – 100,1000 cells/cm2. Once the cells are adapted a lower coating and seeding concentration often can be used.

It is important that the cells transferred to the LN-521 matrix are of high quality. LN-521 will generally also support differentiated cells so carefully select only undifferentiated cell areas for transfer. 

When changing from feeders, the cells might display a different morphology for the first few passages, likely due to the packed monolayer the cells form when confluent, rather than thick colonies as seen on feeders. It is important that the cells are of high quality when being transferred from feeders to laminin-521. Laminin-521 support maintenance of pluripotent cells but may also support some differentiated cells. Therefore, it is important that only undifferentiated cell colonies are being transferred.

Can cells, which have been growing feeder-free substrates, such as Matrigel, be directly transferred onto Laminin? Will cells look the same after transfer?

When moving you cells to laminin-521 from another feeder-free matrix (i.e. Matrigel or vitronectin), generally no specific adaptation is needed.We recommend to transfer the cells as single cells (or as small clumps) and always with the addition of ROCKi for the first few (3-5) passages. If the cells are hard to adapt, try increasing the coating concentration to 10 ug/ml and seed at a higher cell density 50,000 – 100,1000 cells/cm2. Once the cells are adapted a lower coating and seeding concentration often can be used.

It is important that the cells transferred to the LN-521 matrix are of high quality. Laminin-521 support maintenance of pluripotent cells but may also support some differentiated cells. Therefore, it is important that only undifferentiated cell colonies are being transferred.

Cell morphology should not change much, however, you should be prepared for a different growth pattern. Laminin-521 promotes high migration which is vital for cell survival. That will render a more even spread of the cells as compared to feeders and other feeder-free matrices. Also the cells will likely grow faster.

When thawing hPSCs grown on the laminin, what is the recovery like?

The post-thaw cell survival is high when supported by laminin-521, especially when freeze-thawed as single cells. This is nicely described in a recent publications by Miyazaki and colleagues, where they show that hPSC that were freeze-thawed a s colonies showed markedly decreased survival compared to cells freeze-thawed as single cells which retained the majority of their viability (Miyazaki et al., 2013).

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We have distributors in selected regions. Please see our distributor list. We ship directly to all other countries.