Alvéole - Take care of your cells

Primo

The first multi-protein
photopatterning solution

Control several parameters in the cell microenvironment and study their impacts on cell development using an innovative multi-protein photopatterning solution.

Technology

For many years, studying the influence of the microenvironment on intracellular and intercellular mechanisms has been essential for research in cell and medical biology. Among the methods for controlling this microenvironment is the rapidly developing process of “micropatterning”, which involves creating protein patterns on which living cells are cultivated. However, current micropatterning techniques are tedious, complex and non-quantitative.

Based on this finding, the scientists at Alvéole developed an innovative multi-protein photopatterning technique to make experimental manipulations easier for researchers.

The PRIMO technique is based on LIMAP* technology (Light Induced Molecular Adsorption of Proteins) and combines a UV illumination system controlled by a dedicated software (named “Leonardo”) and a specific photoactivatable reagent (PLPP). Working together, these two key system components make it possible to generate, in only a few seconds, any multi-protein pattern on standard cell culture substrates.

*“Multiprotein Printing by Light-Induced Molecular Adsorption” Strale P.O. et al, Adv Mater. 2015

Applications

Primo was developed to enable you to design and conduct all the micropatterning experiments you can imagine, in 2D and also 3D. Simply select from amongst your computer’s files the pattern you want to use (no size or shape limitations). Primo then projects it on the cell culture substrate and allows you to generate the pattern with the protein of your choice.

We are gradually discovering the extent of the fields of application of this new technology and we are pleased to show you a few examples of some early experiments conducted by our users.

Custom micropatterning

Mouse teratocarcinoma cells plated on fibronectin micropatterns.

Single cell micropatterning

Fibroblasts plated on fibronectin micropatterns (after 2 weeks).

Studying the cytoskeleton

Embryonic fibroblasts from vimentin knockout mice on fibronectin + fibrinogen-A647 (blue) patterns of different shapes, actin labelled with phalloidin-A555 (red) and focal adhesions revealed via Anti-Paxillin Antibodies + secondary Antibodies coupled to A488 (green).(1)

Dynamic control of collective cell migration

HeLa cells were plated on a ring pattern of fibronectin. After addition of PLPP and UV-illumination of the center of the ring, the anti-adhesive molecules are removed and the cells start to migrate inwards.

Cell co-culture

S180 cells and MEF cells successively seeded on a Yin & Yang pattern (Yin : fibronectin, Yang : streptavidin incubated with biotinylated fibronectin).(2)

Patterning and neurobiology

Astrocyte-based assays, 1st without patterning
Astrocytes plated on a uniform fibronectin coating show heterogeneity in shapes.(3)
The microtubules were tagged with GFP, but their growth direction was heterogeneous and couldn’t be measured.(3)
Astrocyte-based assays, with patterning
Astrocytes were plated on fibronectin lines.(3)
The microtubules were tagged with GFP and then shown the same orientation, which allowed to measure microtubule growth rates.(3)
Axon guidance
Chicken brain explant positioned in the center of a wheel pattern of laminin labelled with alexa488 (green).(4)
Axon guidance on the spokes and around the circumference of the laminin wheel pattern (24 hours time-lapse video).(4)

Micro-organ-on-a-chip

3 hepatocytes HepG2 adhering on patterns of fibronectin on the sides and the bottom of a micro-well.(5)

Microfabrication for microfluidics

3D SU8 photo-resist mold for manufacturing microfluidic chips, achieved with PRIMO (virtual mask projection and UV light exposure).
Zoom on the SU8 mold.
  1. Courtesy of A.J. Jimenez and B. Vianay, Physics of cytoskeleton & Morphogenesis lab.
  2. Courtesy of V. Studer and P.-O. Strale. ”Multiprotein Printing by Light-Induced Molecular Adsorption“ Strale P.-O. et al., Adv Mater. 2015
  3. Courtesy of C. Delépine. ”Altered microtubule dynamics and vesicular transport in mouse and human MeCP2-deficient astrocytes“, Delépine C. et al., Hum. Mol. Genet. 2015.
  4. Courtesy of H. Ducuing, R. Moore, Y. Lecomte, P.-O. Strale and V. Studer.
  5. Courtesy of C. Stoecklin and V. Viasnoff

The Future

PRIMO allows precise, easy and rapid adjustment of protein micropatterns, either to study the effect of a drug or to imitate physiological conditions. It therefore opens up new possibilities for multiple areas of application, such as stem cell research, and cell-based assays for drug development and predictive toxicology*, to meet major public health challenges.

Cancer
Immunology
Neurobiology
Regenerative Medicine

*Cell-based assays performed using the micropatterning technique constitute an excellent alternative to animal testing.

Specifications

Three specific components

PRIMO: a UV illumination module. Mounted on a microscope, its optical imaging system projects your chosen pattern onto standard cell culture substrates.

PLPP: an exclusive specific photoactivatable reagent. When coupled with UV action, it enables the pattern to be created on the illuminated area of the substrate. Thus you can precisely apply the protein of your choice.

LEONARDO: the software that provides you with optimal control of the PRIMO module, and facilitates your experimental manipulations.

PRIMO® is a registered trademark of Alvéole SAS. PLPP™ is a trademark of Alvéole SAS.

The integrated photopatterning software

Unrivalled performance

Projected image
Image obtained by photopatterning 3 fluorescent dyes (PEG-FITC, PEG-TRITC, PEG-Atto 647) successively deposited*
Sequential photopatterning of Fibrinogen-A488 in green and Protein A-A647 in red onto PDMS micropillars microfabricated with PRIMO.

User Testimonials

Virgile Viasnoff

Associate Professor at MechanoBiology Institute - National University of Singapore, and Director of Research at CNRS

« My research work consists in understanding how microenvironmental cues influence, guide and shape cell-cell contacts. We are currently particularly interested in determining the role of the biophysical environment in the establishment of apico-basal polarity in mammary gland cells and in liver cells. The use of PRIMO in this context proved absolutely essential since it allowed us to create artificial microniches in 3D where we could control up to 150 combinations of environmental cues.

By printing our protein of choice and microfabricating intricate structures under the microscope, we are gradually uncovering how the geometrical and biophysical parameters drive the elongation of lumen into tubes in the context of hepatic development and diseases.

PRIMO is unique in its capabilities, and for us it is the perfect tool. Submicron-resolution, flexibility and versatility in terms of the proteins you can deal with. It is a very reliable tool to print proteins on surface. »

Combining topographical cues and biochemical cues at cell aggregate level by addition of a micro-pillars base layer. Patient-derived head and neck cancer cell aggregates (HN cells) were grown into 60 µm diameter and 30 µm height micro-niches with Laminin (green) homogeneously coated on the pit walls, and fibronectin (red) coated on the NOA 73 micro-pillars (2 m height and diameter).

Olivier Théodoly

Director of Laboratory “Adhesion & Inflammation” - INSERM

« The team is studying the recruitment mechanisms of white blood during an immune response. The complex orchestration of white blood cells traffic between blood/lymph systems, lymph nodes and inflammation zones relies on sophisticated but still poorly understood signaling routes that guide cells toward precise targets. Our aim is to develop in vitro experimentation to decipher guiding mechanisms involved in vivo.

PRIMO technology is particularly adapted to design in vitro microdevices patterned with controlled patches of the signaling proteins relevant for white blood cell migration. »

Primary human T lymphocytes on specific adhesion strips.

Benoit Vianay

CEA Engineer at the Physics of cytoskeleton & Morphogenesis lab (Cytomorpholab)

« We are working on the generation of 3D cellular microenvironments to reproduce hematopoietic niches. Primo will be used to generate 3D photopolymerised microenvironments and to pattern them to localize different cell populations involved in the hematopoiesis.

We initially chose to use PRIMO because it allows sequential multi patterning and makes gradient patterning possible. It works with a broad range of scale and its design creation is limitless. The technology is reproducible and robust after good practical and highly performant to prototype designs or protein combinations. »

RPE1 cells on an Eiffel Tower micropattern generated with PRIMO using Fibronectin and Fibrinogen-Alexa647.

Shrikrishnan Sankaran

Research Scientist at INM-Leibniz Insitute for New Material

SKBR3 cells spread over Fibronectin-Rhodamine patterns.

« We are interested in imaging subcellular localization of certain cell surface receptors and check whether they colocalize with focal adhesion complexes. For this purpose we are interested in making different types of patterns of Fibronectin with subcellular dimensions. We decided to use PRIMO because our lab uses photoresponsive ligands to make dynamic biomaterials, so in general this was a useful tool for most of our members. In particular, it suited my requirement of making patterns with different dimensions and features and being able to immobilize any protein of interest onto it. »

Mélanie Chabaud

Postdoctoral Fellow at UNSW Sydney

« My research project aims at unravelling how a T cell switches from a fast migratory state to a stationary state upon activation. To do so, I perform live cell imaging of T cells migrating inside micro-fabricated channels coated with activating molecules. However, with this approach, I do not control when and where a T cell encounters the activating molecules. The use of PRIMO allows me to print a “migratory zone” and an “activation zone” along micro-channels to visualize this transition. And I also chose to use PRIMO for its capacity to print proteins on 3D surfaces and because it enables me to fine-tune the printing area on a per experiment basis. »

Single lymphocyte (cell nucleus staining with Hoechst) migrating in a microchannel patterned with a migratory zone (fibronectin) and an activating zone (anti-CD3 Abs).

Oleh Halaidych

PhD Student at Leiden University Medical Center, Anatomy and Embryology Lab.

The team wanted to have a flexible system which they can use to pattern multiple proteins, gradients on different substrates. Here is Oleh’s opinion on our technology after a few weeks use: « If I should use one word to characterize PRIMO, I would say mind blowing, impressive or unbelievable. »