Bioalternatives

In vitro and ex vivo efficacy studies of cosmetic products: oily skin, dry skin, hydration, skin ageing, pigmentation, hair growth, etc.

You are looking for validated or innovative efficacy assays to support your cosmetic claims? You wish to characterize your active ingredients, demonstrate the efficacy of your cosmetic formulations? Or would you like to test the safety of your products at an early stage? Our services will help you meet your objectives and support your projects.

With our strong expertise in numerous in vitro and ex vivo models and in skin physiology, our aim is to bring value to your cosmetic products:

• Screening of bioactive ingredients

• Mechanism of action and proof-of-concept

• in vitro safety assays (non-GLP)

• Repositioning or benchmarking studies

• in vitro or ex vivo evaluation of cosmetic formulations before clinical trials.

  
  

As your partner in innovation, we provide you with new concepts to substantiate your claims.

Let’s talk about it!

Skin ageing is a natural, multifactorial and still little-known process which results both from intrinsic factors (genomic, hormonal, etc.) and from external aggravating factors (UV radiation, pollution, food, free radicals, etc.). The latter are referred to as accelerated ageing or as photo-ageing in the particular case of UVs.
Intrinsic ageing and accelerated ageing have slightly different phenotypic characteristics. Intrinsic ageing is characterized by skin dryness, fine wrinkles and a loss of adipose tissue. Photo ageing is characterized by a significant loss of firmness and elasticity (loose skin), deeper wrinkles and pigmentation disorders.
In the past few years, significant progress has been made in the understanding of cell ageing mechanisms and we can now explore new dermo-cosmetics strategies which might prevent or decelerate the signs of ageing.

Bioalternatives has many in vitro models at your disposal:
intrinsic ageing model:
- “aged” human dermal fibroblasts (Hayflick model)
- “aged” dermal equivalent (Hayflick model)
accelerated ageing model:
- human dermal fibroblasts aged by oxidative stress (H2O2)
- human reconstructed skin in deficient medium
photo-ageing model:
- human dermal fibroblasts subjected to UVA, infrared or UV radiation
- photo-aged human full thickness reconstructed skin

Readout:
- cell renewal (cell proliferation, migration and differentiation)
- extracellular matrix synthesis and degradation (collagen, elastin, hyaluronic acid, MMPs, etc.)
- senescence marker expression
- free radical production

Example of Bioalternatives assays:
- Aged fibroblasts, reversion of aging, procollagen I and/or MMP-1 synthesis
Skin is the largest organ of the human body and the most exposed of external aggressions (chemical, UV, smoke, humidity etc.). All of these disturbances participate in the phenomenon of aging and the consequence is both phenotypic and structural modifications (reduction in the thickness of the skin, loss of cohesion, loss of elasticity, wrinkles, etc.).
Disturbances caused such as UV radiation cause epidermal stress contributing to the appearance of oxygen reactive species (ROS) capable of inactivating the phosphotase itself responsible for the activation of tyrosine kinase receptors. When activated, these receptors act on the map kinase pathway leading to the inhibition of procollagen synthesis as well as to the synthesis of matrix metalloproteases (MMP). MMPs are enzymes which act on the degradation of the components of the extracellular matrix (collagen, elastin, proteoglycans). This significant increase in MMP activity is the main factor influencing skin aging.
The purpose of this test is to measure by ELISA the level of expression of procollagen 1, collagen 1, an essential component of the extracellular matrix, and MMP-1 a collagenase protein responsible of the degradation of collagen within the extra cellular matrix, on aged Normal Human Dermal Fibroblasts.
- Aged fibroblasts, control of aging
Replicative aging/senescence is induced in fibroblasts by repeated subcultures (15-20 passages) of normal human dermal fibroblasts. This method modifies the metabolic activity and the gene expression profile of the cells generating a profile of aged fibroblasts characterized by:
- reduced cell proliferation,
- reduced synthesis of collagens and other ECM components,
- increased production of ECM degrading enzymes like MMPs,
- increased beta-galactosidase activity,
- expression of specific aging markers.
The purpose of this assay is to evaluate, via RT-qPCR  technology, the potential anti-aging effect of the compound on aged dermal fibroblasts (Hayflick model) versus normal human dermal fibroblasts (NHDF) at the transcriptional level (modulation of gene expression). More specifically, the effects of the compound on aged fibroblasts are analyzed using the RT-qPCR technology. Extracted mRNA is analyzed on a dedicated PCR array (expression of a set of « aging » genes, mQPA-NHDF AGING-16 or 64).
- Fibroblasts, prevention of aging, procollagen I and/or MMP-1 synthesis (H2O2 stimulation)
Skin ageing is characterized by dermis changes resulting from complex mechanisms. These mechanisms combine a decrease of the synthesis of extracellular matrix components, particularly collagen I, and an increase of protease expression and/or activity. Proteases such as MMPs (matrix metalloproteinases) are matrix-degrading enzymes.
There are multiple consequences to these changes: increase of stiffness of the collagen network and decrease of matrix volume, which have both an impact on the biomechanical properties of dermis.
The purpose of this assay is to evaluate, via an ELISA test, the capacity of compounds (molecules, active ingredients, extracts, etc.) to prevent the decrease of procollagen I production and the increase of MMP-1 (matrix metalloproteinase-1) production in a model of fibroblasts, prematurely aged by H2O2 treatment.
- And more other tests

Other additional information (graphics, illustrations) are also provided on our website
Contact us!

At Bioalternatives, we are committed to developing effective in vitro alternative options to animal experimentation methods, by offering a full range of solutions for the development of active ingredients and cosmetic formulations.
We offer customized technical solutions to guide your product research and support the claims of your cosmetic products. Our selection of in vitro testing solutions can be used to characterize your cosmetic products’ active ingredients, demonstrate the efficacy of your formulations, and test the safety of your cosmetic products at an early stage (for R&D purposes only).
With extensive experience in cosmetic product testing and state-of-the-art facilities, we are pleased to offer you dedicated project management support and consulting for your R&D process.

Bioalternatives provides services to support your research needs in biology and in in vitro pharmacology. For more than 20 years, we have been assisting scientists from the pharmaceutical and biotechnological industry on preclinical issues (exploratory research, in vitro modelling, discovery of therapeutic molecules).
Because each of our clients is unique and each study is based on different prerequisites and objectives, we focus on the customized technical solution that can best meet your requirements. We pay particular attention to the feasibility study and to the personalized management of your project, especially through regular and open communication between your study manager and our specialists.
in vitro pharmacological studies are our core business: by managing a plurality of projects, our scientists have acquired an expertise and know-how which can be applied to a wide range of products.

Pharmaceutical products and active ingredients:
- Natural extracts (plant, marine, bacterial)
- Synthetic molecules, peptides, proteins, glycoproteins, lipids
- Biotherapies and biosimilars (antibodies, growth factors)
- Formulations (creams, gels, lotions)
- Medical devices

in vitro pharmacological studies:
- 2nd or 3rd intention screening
- Activity profiling
- Proof-of-concept, study of mechanism of action
- Confirmatory assay
- Biosimilarity

Bioalternatives offers histology services.
As a researcher in the pharmaceutical industry or as a physician, you wish to carry out histological or immunohistochemical analysis as part of your research program or as part of your preclinical or clinical studies.

Do you have samples (tissues, human or animal biopsies), blocks or slides that are ready to be analyzed? Are you looking for a reactive high quality contract research organization that can provide you with expertise and flexibility?

Our laboratory and our special team dedicated to histology and immunohistochemistry methods would like to assist you with your studies:

Routine assays

• Preparation of samples to study:

  
      - cryo blocks or paraffin inclusion

  
      - generation of slices

• Methods:

      

• trichrome staining, specific staining

      

• immunolabeling (immunofluorescence or immunohistochemistry)

      

• Microscope observation and generation of images

      

• Image analysis: scoring, quantification
  

  
  Customized services

  - Development:

      

• staining

      

• immunolabeling (research and validation of antibodies)

      

• co-labeling

• Preparation of slices:

      

• Images, image mosaic, slide scanning, MET/MEB

      

• Quantification / image analysis (thickness, intensity, counts, etc.)

  
  

Our Histological staining services:

At Bioalternatives, we offer more than thirty standard (topographic) histological and special (descriptive) staining techniques.

These staining processes are carried out after a thorough preparation of samples and slides.

– With routine standard staining techniques, tissue morphology (structure, organization, dimension / nucleus, cytoplasm, collagen fibers) can be examined and tissue integrity or alteration can be evaluated.

– Special staining techniques allow us to go further in tissue observation and to visualize particular tissue components (lipids, melanin pigments, elastic fibers etc.).

Some tissue components can be highlighted in situ by using special staining techniques based on biochemical reactions between the dyes and the tissue components.

Example of Bioalternatives assays:

• RHE, epidermal morphology (Hematoxylin-Eosin staining):

  
  The purpose of this test is to observe the effect of compounds on RHE morphology
  After incubation, RHE were rinsed and fixed with a formaldehyde solution. Fixed tissues were dehydrated in multiple baths with increasing concentration of ethanol and then embedded in paraffin. Transversal sections were performed with a microtome.

  
  The sections were deparaffinized and stained with a solution of hematoxylin. Sections were rinsed and whitened using a diluted hydrogen chloride (HCl) solution, then stained using an eosin solution. After washes, the sections were rinsed, dehydrated and mounted in CV Ultra mounting medium

• Hair follicle, pigmentation:
  
  At the end of incubation, the selected hair follicles were rinsed and fixed with formaldehyde solution. Follicles were then dehydrated in multiple baths with increasing concentration of ethanol and then embedded in paraffin. The transversal sections were performed using a microtome and kept at room temperature until staining.
  The sections were deparaffinized, rinsed in acidified water and then incubated in silver nitrate solution. The sections were then washed and incubated in a silver nitrate, gelatin, and hydroquinone. After washing, the sections were incubated in a sodium thiosulfate solution. After washing, the sections were counter-stained with a Fast-red solution. Finally, the sections were washed again and mounted in CV Ultra medium.

• 3D sebocytes, lipid accumulation:
  
  Sebocyte differentiation is associated to an increase of lipid synthesis and accumulation. BIOalternatives has developed a human cell line (SEBO662) able to spontaneously differentiate into a sebaceous-like phenotype when cultured as a three-dimension (3D) epithelium.

• And more other tests

  
  

Other additional information (graphics, illustrations) are also provided on our website.

Contact us!

Bioalternatives offers immunolabeling services in order to detect the cell and tissue components in a tissue section. Proteins of interest (antigens) are detected by using polyclonal or monoclonal antibodies specifically directed against the target proteins.

Our laboratory can perform immunolabeling by using either the direct approach (antibody directly coupled to a marker) or the indirect approach (unlabeled primary antibody specifically recognized by a marker-labeled secondary antibody) for immunolabeling on different types of tissues: reconstructed tissues, human or animal explants and biopsies, paraffin embedded (FFPE) or frozen tissues.

The detection methods directly depend on the type of marker that is used. Our laboratory uses special in situ detection techniques: immunofluorescence and immunohistochemistry.

• Immunofluorescence is a technique which consists in detecting and localizing a protein of interest using a specific antibody (directly linked to a fluorophore or detected by a secondary fluorescent antibody). Immunolabeling of proteins of interest is generally associated to nuclear labeling by Hoechst staining or by propidium iodide staining. This technique, when associated to an image processing program (e.g. ImageJ), can also generate fluorescence intensity data that can be analyzed quantitatively. It is also possible to perform co-labeling with this technique.

• Immunohistochemistry by enzyme detection is the second special in situ detection technique, which consists in detecting and localizing a protein of interest within a tissue section, using a specific antibody which will be detected by an enzymatic reaction (such as peroxidase) that generates a red precipitate from an AEC-type chromogen (Aminoethyl carbazole).

  
  

Our laboratory has validated more than 120 markers and is at your disposal for any immunolabeling request
This is a non-exhaustive list of the validated biomarkers we offer:

Inflammation

• IL-1

• IL-8

• PGE2

Hydration and skin barrier
- Pro-collagen type I

- Collagen type I

- Collagen type III

- Collagen type VII

- Collagen IV

- Hyaluronic Acid

- Filaggrin

- E-cadhérine,Epithelial

Pigmentation

- Tyrosinase

Prolifération

- Ki67

Differentiation

- Cytokeratin-10

- Claudin 1

- Transglutaminase 1

Apoptosis
- Caspase 14

And more other markers

Other additional information (graphics, illustrations) are also provided on our website

Contact us!
 

Hydration and Skin Barrier

Skin hydration partly determines the aspect of the skin (dry, rough and oily skin, etc.). Through its barrier function, skin is our shield from external stress factors and prevents the evaporation of such essential elements as water, ions and amino acids.
Skin hydration is mostly linked to the skin barrier function. The role of a moisturizing skin care product is to act on skin surface to physically limit water loss (occlusive effect) or to act at the cellular level via different biological action models:
- reinforcement of the barrier function and of epidermal differentiation
- restoration of skin lipids
- stimulation of hygroscopic endogenous factors (water retention)
- improvement of cellular exchanges in solutes and in water
Hydration is a multifactorial process that cannot be understood through a single assay or model. It is therefore more conclusive to use a panel of complementary efficacy assays in order to study and demonstrate the hydrating properties of an active compound or cosmetic product.

Bioalternatives has many in vitro or ex vivo models at your disposal:
- normal human epidermal keratinocytes (NHEK)
- reconstructed human epidermis (RHE)
- sebocytes (SEBO662AR)
- full thickness skin (FTSK)
- skin explants (ex vivo)

On which we can evaluate case by case the hydrating effect of active compounds or formulations by measuring:
- the reinforcement of the barrier function
- the stimulation of the epidermal differentiation (e.g. filaggrin, involucrin, transglutaminase, cytokeratins)
- lipid synthesis (acid mantle):
    - sebaceous lipids
    - epidermal lipids (e.g. ceramides, cerebrosides and phospholipids)
- the expression or synthesis of epidermal extracellular matrix components:
    - glycosaminoglycans and hyaluronic acid
    - proteoglycans and ECM receptors
    - proteases (e.g. MMPs)
- the expression of markers of epidermal cohesion and intercellular cell junctions:
    - occluding junctions and attachment proteins (claudin, occludin, desmogleins, etc.)
    - dermoepidermal junction (e.g. integrin V, collagen IV, collagen VII, etc.)
    - gap junctions (e.g. connexins) and molecular channels (e.g. aquaporins)

Example of Bioalternatives assays:
- RHE, transepidermal permeation and barrier function
The epidermis provides a physical and permeability barrier which is continuously regenerated by the epidermal keratinocyte differentiation process. Multiple components can impact the barrier function quality including protein structure (claudin, desmoglein, filaggrin, etc.), lipids (ceramides) and proteases. When properly differentiated, the epidermis prevents water loss and provides a barrier to epidermal invasion of pollutants, toxins, allergens and bacteria.
The purpose of our assay is to evaluate, via radiodetection, the capacity of compounds (active ingredients or formulations) to reinforce the human reconstructed epidermis barrier function by analyzing caffeine diffusion.
- NHEK, epidermal barrier markers (mRNA)
The epidermis provides a physical and permeability barrier which is continuously regenerated by the epidermal keratinocyte differentiation process. Multiple components can impact the barrier function quality including protein structure (claudin, desmoglein, filaggrin, etc.), lipids (ceramides) and proteases. When properly differentiated, the epidermis prevents water loss and provides a barrier to epidermal invasion of pollutants, toxins, allergens and bacteria.
The purpose of our assay is to evaluate, via RT-qPCR technology, the capacity of the compounds to improve the epidermal barrier by analyzing gene expression level of 32 markers related to keratinocyte differentiation, tight junctions and adherens junctions, hyaluronic acid synthesis and other.
- NHEK, hyaluronic acid synthesis / release
Hyaluronic acid, a sulfate-free glycosaminoglycan, is one of the major components of the extracellular matrix. Hyaluronic acid fills the inter-cellular spaces in skin. It plays an important role in maintaining skin hydration and turgescence, due to its capacity to capture water molecules and ions.
The purpose of our in vitro assay is to evaluate, via an ELISA test, the stimulating effects of compounds (molecules, active ingredients, extracts, etc.) on hyaluronic acid production (synthesis and release) by normal human epidermal keratinocytes.

- And more other tests

Other additional information (graphics, illustrations) are also provided on our website
Contact us!

Atopic dermatitis, also known as atopic eczema, is a recurrent inflammatory skin disease that affects children in particular. It is due to an epidermal barrier dysfunction and to a hypersensitivity to environmental factors. Since the 2000s, the discovery of a preventive treatment for this severe dermatosis has been a special line of research in the pharmaceutical and dermocosmetics industry.

Using its long-standing expertise in skin inflammation and in in vitro models, Bioalternatives initiated a research program on atopic dermatitis several years ago. This program has made it possible for us to develop and validate predictive in vitro pharmacological assays in order to limit attrition in clinical trials.

The in vitro pharmacological assays listed below can be used for screening or evaluating potential modulators of atopic dermatitis (APIs, biosimilars, formulations, medical devices):
- Immune response (Th2/Th22 cytokine release, Ig production, etc.)
- Skin sensitization (basophil activation, histamine release, neuropeptide production, etc.)
- Activation of inflammatory response of keratinocytes or reconstructed epidermis
- Epidermal differentiation, skin integrity and lesions, barrier function and antimicrobial defenses
- Analysis of specific markers by immunoassays or Rt-qPCR-qPCR

Bioalternatives cells and tissues models:
- Reconstructed Epidermis, Bioalternatives (RHE)
- Normal Human Epidermal Keratinocytes (NHEK)
- Mast cells
- CD4+ T cells

Readout:
-RHE (basal or stimulated):
Filaggrin
-NHEK (stimulated):
TSLP release
Atopic dermatitis gene expression
-Mast cells:
Histamine release
-CD4+ T cells (stimulated):
IL-10 release
IL-4 release
or other Th2 cytokines
TNF- alpha release

Example of Bioalternatives assays:
- Gene expression (cytokine stimulation)
The purpose of our assays is to evaluate, via qPCR, the effect of compounds (molecules, active ingredients, extracts, etc.) on gene implicated in atopic dermatitis expression in normal human keratinocytes. A mix of pro-inflammatory cytokines is used to stimulate the cells in order to recreate the pathological tissue environment found in the chronic phase of atopic dermatitis.
- Filaggrin expression (cytokine stimulation)
The purpose of this test is to evaluate via immunohistfluorescence or immunohistochemistry the effect of compounds (molecules, active ingredients, extracts, etc.) on filaggrin expression in Reconstructed Human Epidermis (RHE)
A mix of pro-inflammatory cytokines is used to stimulate the RHE in order to recreate the pathological tissue environment found in the chronic phase of atopic dermatitis. The markers are then immunolabeled and the intensity of fluorescence is quantified.
- Mast cells degranulation, histamine release (compound 48/80 stimulation)
The purpose of this test is to evaluate via ELISA the effect of compounds (molecules, active ingredients, extracts, etc.) on histamine release in murine mast cells.
- And more other tests

Other additional information (graphics, illustrations) are also provided on our website
Contact us!

Our existing in vitro models can be used for regulatory safety and toxicity testing of your cosmetics and ingredients.

Such tests can include:
Cytotoxicity, including skin/eye irritation and corrosion
Phototoxicity (chemically induced skin irritation)
Mutagenicity (DNA damage)

Bioalternatives cells and tissues models:
2D models
suspended cells
3D models
Skin explant

Example of Bioalternatives assays:
- Cytotoxicity assay
This in vitro preliminary cytotoxicity assay evaluates the non-cytotoxic concentrations of a tested compound (molecule, active ingredient, extract) for a specific cell or tissue model (monolayer, RHE, skin explant) and associated culture conditions (culture medium, density, time and frequency of treatment, etc.). This assay is an essential precondition for carrying out biological assays (in vitro pharmacological assays).
Once the tested compound has been given a concentration range, this assay allows cell or tissue integrity to be measured by microscopic observation and cell viability quantification to be evaluated by using MTT reduction (vital staining) or WST-8 (water-soluble tetrazolium dye).
- Phototoxicity assay
Bioalternatives offer to measure the phototoxicity of our test compound on 3T3 murine fibroblasts in absence and in presence of UVA irradiation. Cell viability, after test compound incubation, is assessed by using the neutral red uptake method.
The phototoxic effect of the test compound will be assessed according to the Commission Directive 2000/33/EC Annex II “3T3/neutral red uptake” and the OECD guideline 432 from the 13 April 2004.
- Cutaneous irritation assay, OCDE 42 bis
Bioalternatives offer to determine the skin sensibilization effect of your compounds (molecules, active ingredients, extracts, etc.)
In this model, skin irritation is measured on reconstructed epidermis with a measurement of cell viability by photometry (MTT).
- And more other tests

Other additional information (graphics, illustrations) are also provided on our website
Contact us!

Are you looking for solutions to support your claims? in vitro assays are highly effective for establishing proof-of-concept of your compounds (e.g. extracts, active ingredients, formulations). Bioalternatives, a premier in vitro dermatological testing service laboratory, can assist you at every step of the pre-clinical development of your products: screening of bioactive compounds, proof-of-concept studies, repositioning of active ingredients.

Our knowledge of skin biology and its application to new target assessment, mechanistic understanding and claim substantiation are without question of the highest standards in the industry :

- Epidermal regeneration

- Skin barrier and hydration

- Skin pigmentation

- Skin aging

- Skin protection

- Skin vascularization

- Skin inflammation

- Sebaceous gland regulation

- Adipocyte metabolism - Hair regeneration

As a researcher in the pharmaceutical industry or as a physician, you wish to carry out transcriptome analysis as part of your preclinical or clinical studies.
Do you have biological samples (cells, tissues, human or animal biopsies), RNA or cDNA that are ready to be analyzed? Are you looking for a reactive high-quality contract research organization that can provide you with expertise and flexibility?
Our laboratory and our special team dedicated to molecular biology methods would like to assist you with your studies.

Routine assays:
- Preparation and quality control on RNA (total RNA and microRNA), cDNA or DNA
- Gene expression analysis by RT-qPCR (mRNA and microRNA) or PCRarrays (SYBR Green or Taqman)
- Whole genome or miRNome expression analysis (Affymetrix platform)
- Bioinformatic analysis (Biological processes and pathways analysis)

Customized services:
- Design and validation of PCR primers and probes
- Design and preparation of qPCRarrays (384-well format)
- in situ hybridization
- Genome editing, construction of modified cell lines
- Target gene silencing (SiRNA/ShRNA)
- Target gene overexpression

Our PCR arrays:
PCR arrays are dedicated to gene expression analysis by quantitative PCR. They are sets of primers, on the same support (384-well microplate), selected according to a theme or a given biological process. Gene expression analysis by PCR arrays is considered as the most effective and cost-efficient method for studying a panel of targeted genes.
This method can be used for applied research (sample or biological model characterization, identification of biomarkers, etc.) or for the discovery and selection of active ingredients. PCR arrays can also be used for the validation of results obtained after transcriptomic analysis.

- Standard PCR arrays:
Bioalternatives has a wide range of thematic ready-to-use PCR arrays (8 to128 genes) to perform gene expression studies:
Tissue inflammation
Oxidative stress
Ageing
Angiogenesis
Psoriasis
Atopic dermatitis
Innate immunity and skin defense mechanisms
Adipocyte differentiation and conversion
Extracellular matrix synthesis and degradation (fibroblasts)
Epidermal differentiation (keratinocytes)
Cell junction and communication
Pigmentation
UV
etc.

- Our qPCR services include:
Biological sample preparation and recovery
Total RNA, mRNA and microRNA extraction and quality control using electrophoresis (Agilent)
Reverse transcription and qPCR by SYBR Green or TaqMan probe
Raw data
Quality control and data normalization

At Bioalternatives, we can also design and validate custom PCR arrays dedicated to the design of tailored arrangements that include the selection of your genes of interest (8 to 128 genes).

Do not hesitate to contact us for any additional information.

The human skin serves as the first natural physical barrier against external assaults while concomitantly providing a home for more than 1000 billion beneficial microorganisms. These microorganisms form the commensal resident skin flora also known as the cutaneous microbiota. The microbiota lives a symbiotic relationship with its host and interacts constantly with the adaptive immune system, thus creating a complex ecosystem which is unique to each individual but highly variable depending on several factors such as (temperature, ph, humidity, body area, life period, diet, etc.). This fragile balanced ecosystem helps to maintain healthy skin and protects it against the adhesion and development of pathogens. Its deterioration or imbalance can cause skin disorders (dry skin, oily skin, etc.) and amplify pathologies (psoriasis, atopic dermatitis, acne, etc.)
Thanks to its expertise in cellular and tissue engineering, Bioalternatives has developed models that are suitable for analyzing the interactions between microbiota and skin and evaluate the efficacy of your products from in silico to clinical sample bioanalysis.

Microbiota-friendly studies:
- Bacterial growth and yeast growth: Photometry (OD), Colony-forming unit (CFU), qPCR (SYBR Green & TaqmanTM)
- Bacterial adhesion on RHE surface: Colony-forming unit (CFU), qPCR (SYBR Green & TaqmanTM), Radioactivity, Scanning electron microscopy)
- Bacterial & yeast quantification & identification:  Traditional microbiology on agar, Targeted qPCR, Non targeted metagenomic (MiSeq)

Anti-microbial studies:
- Biological target, active molecule: SELNERGY
- Bacterial viability, Bacterial growth /Yeast growth, Bacterial identification: Photometry (OD) - MIC / MBC / TKC, Colony-forming unit (CFU), qPCR (SYBR Green & TaqmanTM)
- Bacterial adhesion on RHE surface, epidermal protein analysis (antimicrobial peptides), inflammation assay (cytokine release): Colony-forming unit (CFU), qPCR (SYBR Green & TaqmanTM), Radioactivity, Scanning Electron Microscopy
- Wound healing delay after bacterial infection: Wound diameter, tissue morphology (HES)
- Bacterial quantification, bacterial identification: Traditional microbiology on agar, targeted qPCR, non targeted metagenomic (MiSeq).

Bioalternatives offers a wide range of microorganisms tested by our laboratory and available for in vitro testing:
GRAM (+)
Staphylococcus aureus

Staphylococcus epidermidis
Cutibacterium acnes
Corynebacterium xerosis
Streptococcus pyogenes
Mix of several strains (upon request)
Other strains upon request
GRAM (-)
Pseudomonas aeruginosa
Other strains upon request
VIRUSES
Herpes simplex virus type 1 (HSV-1)
Human Rhinovirus 16 (HRV-16)
YEAST
Malassezia

Example of Bioalternatives assays:
- Bacterial enumeration
The purpose of our test is to evaluate the effect of the compounds (molecules, active ingredients, extracts, etc.) on stimulating or inhibiting bacterial proliferation. This is done by bacterial enumeration using qPCR (DNA quantification) or by bacterial counting using colony forming unit (CFU). Products can be tested at different concentrations and on different time-point.
- RHE, adhesion of a bacterial mix (basal)
The purpose of our test is to evaluate via qPCR quantification the effect of compounds (molecules, active ingredients, extracts, etc.)  on the adhesion of a mix of 4 bacterial strains on reconstructed human epidermis.
(This test can also be realized with an uncial strains)
- NHDF, Cytokine/chemokine release
The purpose of our test is to evaluate via ELISA quantification the effect of compounds (molecules, active ingredients, extracts, etc.)  on the inflammatory response induced by bacterial strain in human fibroblasts.
(This test can also be realized in human sebocytes cell line)
- And more other tests

Other additional information (graphics, illustrations) are also provided on our website
Contact us!

Inflammation is a biological response of the immune system that can be triggered by a variety of factors, including pathogens, damaged cells and toxic compounds.

Our laboratory can support anti-Inflammatory project in the following research areas:

Cell and tissue damage:
Acute inflammation or stress: innate immunity & epithelial barrier response against damage
Defense mechanisms
Chronic inflammation, stress or disease: tissue response, damage, ageing, etc.

Regulation of immune and inflammatory responses:
Recruitment, proliferation, differentiation, polarization and function of immune cells
Cytokine, chemokine and growth factor release

Tissue Regeneration:
Tissue repair, wound healing
Regeneration, proliferation and migration
Differentiation process
Function and recovery

Bioalternatives cells and tissues models:

Our immune blood cells
Primary cells (human)
Whole blood
PBMC
PMN
CD4+ T lymphocytes
CD19+ B lymphocytes B
CD14+ monocytes, M1 and M2 macrophages
Dendritic and langerhans cells
Granulocytes
Basophils
Primary cells (murin)
Splenocytes
Thymocytes
Mast cells
Cell lines
U937
THP1
HL-60
Jurkat

Readout:
-Macrophage-like U-937 (basal or stimulated)
IL-1 beta release
IL-6 release
IL-8 release
TNF- alpha release
Annexin V / PI
PGE2 release
TXB2 release
-Thymocytes (basal or stimulated)
Active caspase 3
-THP-1 (basal or stimulated)
Caspase-1 activity
IL-1 beta release
-T cells, (basal or stimulated)
IFN- gamma release
CD25 expression
-PMN (basal or stimulated)
IL-8 release
IL-1 release beta
CD11b/CD62L expression
-PBMc (basal or stimulated)
IFN- gamma release
PI3K/AKT activation
Cytokine release
Inflammation genes
-Mast cells (basal or stimulated)
Histamine release 
-HL-60
mRNA expression
Arachidonic acid metabolite release
-CD4+ T cells
Cytokine release
-CD19+ B cells
IL-6 release
Ig production

Example of Bioalternatives assays:
- Blood, human basophil activation assay
Immediate-type hypersensitivity is characterized by allergic reactions following the contact with an allergen. This allergic reaction can be mediated or not by immunoglobulin E (IgE) class of antibodies. The cross-linking of two IgE receptors (FceRI) by an allergen induces intracellular signaling pathways, thus resulting in the basophil activation. The ultimate step of basophil activation is the degranulation resulting in histamine release. Such histamine release is responsible for the appearance of allergy reactions.
The degranulation step consists in the fusion of cytoplasmic granules with the basophil plasma membrane. CD63 is then exposed to the extracellular matrix and it is considered as a specific marker of activated basophils.
The purpose of our test is to evaluate via flow cytometry the effect of compounds (molecules, active ingredients, extracts, etc.) on CD63 expression in CCR3+ basophils.
- Mast cells degranulation, histamine release (compound 48/80 stimulation)
Mast cells and basophils represent the most relevant source of histamine in the immune system. Histamine is stored in cytoplasmic granules. Histamine release is regulated by several activating and inhibitory receptors. In conventional allergy, presence of allergen specific IgE activate mast cell degranulation and histamine release.
The purpose of our test is to evaluate via ELISA the effect of compounds (molecules, active ingredients, extracts, etc.) on histamine release in rat stimulated mast cells.
- Peripheral Blood Mononuclear Cells stimulated monocytes, cytokine release (LPS or anti-CD3/anti-CD28 stimulation)
The purpose of our test is to evaluate via flow cytometry (multiplex) the effect of compounds (molecules, active ingredients, extracts, etc.)  on cytokine in stimulated Peripheral Blood Mononuclear Cells (PBMC).
- And more other tests

Other additional information (graphics, illustrations) are also provided on our website
Contact us!

Acne is a chronic inflammatory pathology of the pilosebaceous follicle under hormonal dependence that includes:
-abnormal increase of sebaceous lipid production (sebum)
-formation of microcomedones due to an abnormal epidermal differentiation, a likely consequence of abnormal androgen (testosterone) metabolism
-accumulation of peroxidized lipids on the surface (blackheads) or inside the comedo (whitehead)
formation of infectious and non-infectious inflammatory lesions and a skin wound healing process

Bioalternatives provides you with a range of innovative in vitro pharmacological models and assays, more particularly dedicated to the evaluation of the pharmacological efficacy (screening, profiling, proof-of-concept) of your products (APIs, biosimilars, fomulations, medical devices):
-Sebaceous gland regulation and hyperseborrhea
-Androgen metabolism
-Abnormal epidermal differentiation
-Lipid peroxidation
-Inflammatory, lesional or microbial response
-Infection, immune system and skin defenses

Bioalternatives cells and tissues models:
-Human sebocytes: SEBO662 and SEBO662AR cell lines
-Normal human epidermal keratinocytes (NHEK)
-Normal human dermal fibroblasts (NHDF)
-Human follicle dermal papilla cells (HFDPC)
-Reconstructed human epidermis (RHE)

Readout:
-SEBO662 (basal)
testosterone metabolism
5- alpha reductase activity
-SEBO662AR (basal or stimulated)
Lipogenesis
-NHEK (Basal)
Antimicrobial peptide

Example of Bioalternatives assays:
- Keratinocytes antimicrobial peptide release
Elafin and beta-defensin 2 are potent antimicrobial peptides which are notably expressed in normal human keratinocytes. A high level of expression of these two peptides in keratinocytes is frequently associated with inflammatory skin lesions.
The purpose of our test is to evaluate via ELISA the effect of compounds (molecules, active ingredients, extracts, etc.)  on peptide release (beta-defansin, elafin) in human keratinocytes.
- Sebocytes, testosterone metabolism & 5-a reductase activity
Acne is a multiparametric skin disorder in which sebum hypersecretion induced by circulating testosterone is involved. Testosterone is converted, by 5α-reductase, into dihydrotestosterone (DHT), one of its biologically active metabolites. Moreover, an excess of sebum (hyperseborrhea) also favors the development of bacteria (i.e. Propionibacterium acnes) which in turn induces inflammation. The inhibition of 5α-reductase enzymatic activity and/or inflammation is known to reduce acne in human skin.
The purpose of our assays is to evaluate, via [14C]-testosterone incorporation, the effect of compounds (molecules, active ingredients, extracts, etc.) on 5-alpha reductase activity in human sebocytes 
- Sebocyte, lipogenesis
Acne is an inflammatory pathology linked to an abnormal production of sebum by the sebocytes. Sebum is produced because of a complex process of lipid synthesis, lipogenesis and then released by holocrine secretion. The production is under hormonal control such as testosterone known to stimulate the production of sebum. The stimulation by a complete testosterone results in an even stronger increase of the lipid synthesis and storage.
The purpose of our assays is to evaluate, via fluorescence, the effect of compounds (molecules, active ingredients, extracts, etc.) on lipid production in a model of human sebocytes cell line
- And more other tests

Other additional information (graphics, illustrations) are also provided on our website
Contact us!

Bioalternatives developed and validated in vitro cell and tissue models (e.g. normal or aged dermal fibroblasts, full thickness skin) and assays for investigating the anti-aging effect of cosmetics compounds, such as:
-cell migration
-proliferation or differentiation assays
-extracellular matrix synthesis or degradation assays (e.g. collagen or tropoelastin synthesis/ expression, MMP activities)
-gene expression assays
-reinforcement of dermis cohesion and firmness
-production of extracellular matrix components and improvement of cell-matrix interactions (collagen I, collagen III), elastin, fibronectin, proteoglycans, glycoaminoglycans  and hyaluronic acid proteases (MMPs) etc.).
-improvement of dermo-epidermal junction quality and cell adhesion (integrins, laminin V, collagen IV, collagen VII, desmogleins, etc.).

Dermal arterioles and venules are composed of endothelial cells, connective tissues (collagen, elastin) and of smooth muscle cells which allow vessel vasoconstriction and vasodilation. Dermal capillaries are composed of a single layer of endothelial cells and of pericytes surrounded by a basal membrane.
Angiogenesis involves the formation of capillaries from preexisting microvessels and therefore contributes to vascular remodeling and maturation. It plays a pivotal function in a variety of normal and pathological conditions such as embryonic development, the menstrual cycle, hair cycle, wound healing, arthritis, psoriasis, proliferative diabetic retinopathy, atherosclerosis, post ischemic vascularization of the myocardium and tumor growth and metastasis.
Microcirculation can be altered by external factors and also skin ageing, with a progressive decrease of new blood vessel formation.

Bioalternatives can evaluate the effect of active cosmetic compounds on skin microvascularization by measuring:
-Viability, proliferation and migration of endothelial cells
-Cell differentiation and pseudotube (angiogenesis, neovascularization) formation
-Protection of endothelial cells against stress (inflammation, infection, etc.) 
-Veinotonic activity of smooth muscle cells
-Angiogenic factor release (e.g.VEGF)

Bioalternatives cells and tissues models:
-Human umbilical vein endothelial cells (HUVEC)
-Human dermal microvascular endothelial cells (HMVEC-d)
-HMVEC-d and NHDF coculture (ENDOF)
-Human aortic smooth muscle cells (AoSMC)
-Other skin cells (NHEK, NHDF, etc.)

Readout
-NHDF:
VEGF release
-HUVEC:
ICAM-1 expression

Example of Bioalternatives assays:
- Differentiation/pseudotube formation (VEGF stimulation, anti-angiogenic agents)
Angiogenesis involves the formation of capillaries from preexisting microvessels and therefore contributes to vascular remodeling and maturation. It plays a pivotal function in a variety of normal and pathological conditions such as embryonic development, the menstrual cycle, hair cycle, wound healing, arthritis, psoriasis, proliferative diabetic retinopathy, atherosclerosis, post ischemic vascularization of the myocardium and tumor growth and metastasis. The initiation of the physiopathological angiogenic response, known as the ‘angiogenic switch’, depends on the dynamic balance between exogenous or endogenous stimuli (pro-angiogenic factors) and inhibitors (anti-angiogenic factors) acting in the immediate environment of endothelial cells.
The in vitro assay proposed below consists in a co-culture model of normal human endothelial cells with normal human dermal fibroblasts. After several days of incubation, the treatment of the co-culture with the pro-angiogenic factor VEGF results in the organization of endothelial cells in pseudotubes. Therefore, under VEGF-stimulated condition, this model is suitable to evaluate test compounds capable of inhibiting the formation of this pseudotube network. The pseudotube formation in this model is analyzed by image analysis further to the specific immunolabelling of endothelial cells only using an anti-VWF antibody.
Remark : Under basal conditions (in absence of VEGF), our co-culture model can also be used to screen compounds capable of promoting the development of the pseudotube.
- Veinotonic activity
Vascular smooth muscle cells play an essential role on the microvascular system and most vasoconstrictors/ veinotonic exert their effects by acting directly on the smooth muscle cells of the vascular wall. The contraction of smooth muscular cells, in most cases linked to an increase of cytoplasmic calcium, causes the vasoconstriction of nearby blood vessels.
The purpose of this assay is to evaluate, via time-lapse fluorescence microscopy, the capacity of the compounds to stimulate the contraction of primary smoth vascular muscle cell by measuring their capacity to induce the mobilization of intra-cellular calcium.
- VEGF release
The purpose of our assay is to evaluate, via ELISA the effect of compounds (molecules, active ingredients, extracts, etc.) on VEGF release in normal human fibroblast.

- And more other tests

Other additional information (graphics, illustrations) are also provided on our website
Contact us!

Cell and tissue engineering, development of new comple in vitro mammalian models:

• Tissue microdissection
• Primary cell isolation
• Cell line construction
• Co-culture model system
• Organotypic explant culture
• Three dimensional reconstruction
• Phenotypic and functional characterization

Cellular and molecular pharmacology assays for hit-to-lead selection of active pharmaceutical ingredients and claim substantiation of healthcare products:

• cell-based assays
• Screening
• Profiling
• Proof-of-concept
• Mechanism of action
• Benchmarking
• Safety
• Testing of formulations prior clinical trial

Services for the development, customization and optimization of innovative research methods and protocols :

• Literature review
• Feasability study
• Protocol design
• Method validation
• Multiplexing, miniaturization and optimization

Biological sample collection, processing, storage and information management:

• Study design
• Sample Collection
• Method develoment and validation (tissue microarrays, cDNArrays, PCRarrays, immunoassays, biochemical assays)
• Analysis service
• Biobanking (RNA, cDNA, cells, frozen tissues, embedded tissues)
• Data management

Cell migration is commonly assessed using the scratch assay which measures the movement of cells into an artificial wound made on a confluent cell monolayer to create a cell-free area. As an alternative to the scratch assay which has high variability, the Oris™ Cell Migration Assay allows the formation of homogeneously sized cell-free areas into which migration can occur without releasing factors from dead cells or damaging the underlying extracellular matrix.

- BrdU cell proliferation assay

- 3H thymine incorporation assay 

Cell-Based Gene Expression Assays using qPCR (SYBR Green or TaqMan) or microrrays (Affymetrix);