5D Health Protection Group limited

multi award winning microbiology laboratory

Cell Biology and Tissue Engineering

Cell Biology, Culturing and Tissue Engineering

Our interdisciplinary team of scientists are very experienced in the analysis of cells, tissues, model development and the effects that different products and materials may have on different human cells. Due to our extensive knowledge in cell culturing and tissue engineering we can apply these skills to help in supporting our customers' products and also help to speed up the time taken to evaluate a product before and after development.

5D offer many different tests that can be applied to medical devices, actives and antimicrobials. In particular we are able to offer biocompatibility testing. We are able to offer both standard and modified tests in line with ISO 10993. ISO 10993 sets down a list of guidelines for the assessment of medical devices prior to a medical device coming to market. 

World Class Scientists in Cell Biology and Tissue Engineering

In vitro Cytotoxicity 

Cytotoxicity testing is one of the in vitro screening tests to investigate the effects of medical devices on cellular growth, cellular reproduction and the effects on cellular morphology. It is a relatively quick, simple highly sensitive array of tests without using animals. We offer a number of different in vitro cytotoxicity tests that are used to assess the cytotoxic effects of materials, actives, antimicrobials, antibiofilm agents, technologies and products on living cells. In reference to international requirements cytotoxicity investigates if materials and products have an inhibitory effect on mammalian or mouse cell lines. Furthermore cytotoxicity constitutes the first step in ensuring biocompatibility of a medical device for animal or human use.

Cytotoxicity tests can be used to support innovation and R&D pipelines as it will enable comparisons to be made of materials and prototypes to assist with product improvements and for investigating if significant changes in a manufacturing process can affect the safety of a product. 


As well as cytotoxicity other tests are required to investigate the biocompatibility of materials for animal and human use. Biocompatibility is an indication of how compatible a medical device may be in biological systems eg humans and animals. It is important that a device does not cause detrimental physiological effects. Local and systemic toxicity can have detrimental effects on many biological systems such as the immune system or nervous system. Some examples of standard biocompatibility tests that need to be undertaken for medical devices include sensitisation tests, irritation tests, acute systemic toxicity tests, and other tests that could include heamolysis, are appropriate to ensure devices are safe and effective. 

ISO 10993-5 is a test method that is used to assess the in vitro cytotoxicity of medical devices. The method involves evaluating the biological response of L929 mouse fibroblast cells following direct contact or through diffusion of impregnated chemicals from a medical device. 

Biological evaluation of medical devices – Part 5:Tests for in vitro cytotoxicity

Direct Contact Method

The direct contact method is used for products such as medical devices that may elute out actives. These could include medical devices such as wound dressings and contact lenses. Leachable chemicals from the test article will diffuse and the effects of these leachable will be investigate on cell monolayers of L-929 mouse fibroblasts. These cells are grown to about 80% confluent levels and test articles are then added directly onto these cells. Following incubation of 24 hours the cells are studied under microscopy for degeneration and cell lysis. The cells are then graded (0 to 4) to indicate low reactivity or severe reactivity to the test articles being evaluated. 

Extraction/MEM Elution Method

The extraction method/MEM Elution test involves taking an extract or leachable from the test article and adding this to cell lines. The extract or leachable is added directly to a monolayer of cells, usually L929 mouse fibroblasts, and incubated. This may be done for up to 48 hours. After incubation the cells are examined under a microscope to look at the cellular death and degeneration. Scoring is based on a grading system of 0 to 4 which correlates to the cytotoxic effects of the leachable agents.

Indirect Contact Method (Agrarose Overlay)

The indirect contact or agar overlay method is used for gels and often semi-solid ointments. In this method a layer of agar is placed over a monolayer of cells and left to solidify and the test article is then placed on top of the agar. Following incubation, a zone of lysed cells under and around the test article is examined microscopically after 24 hours incubation. 

Cytotoxicity-USP 87

USP 87 determines the biological reactivity of mammalian cell cultures following direct and indirect contact to a material or leachable extracts. 

In the agar diffusion test, materials are graded from 0 to 4 for reactivity. A grade of 0 implies that the material has no reactivity. A grade of 4 indicates a severe reactivity where a zone will extend greater than 1 cm beyond the test article. In the elution test a grade of 0 indicates no reactivity. A grade of 4 indicates a sever reactivity where there is nearly complete destruction of the cellular monolayer. 

Cell Insert Cytotoxicity Model

This is an in vitro cytotoxicity assay that is used to evaluate the effects of dilution factors on actives and antimicrobials that maybe used in medical devices used in wound care.

This assay takes into account the dilution effect on active agents.

MTT Assay

The MTT assay is a colorimetric assay cytotoxic assay that is quantitative by assessing the viability of cells. It uses the dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). This dye is yellow and water soluble. It is cleaved by the enzyme succinate dehydrogenase (mitochondria) to form formazan which is violet in colour.

When cells are living, this reaction occurs. If cells are dead however, the mitochondrial succinate dehydrogenase will not cleave MTT. This test is used when an extract is taken from a test article. This extract is added onto L-929 mouse fibroblasts at different concentrations i.e. undiluted, 1:2, 1:4 and 1:10. Following an incubation period of 24 hours MTT is added to the fibroblasts and the levels of formazan are measured.

The number of viable cells can be determined between undiluted and diluted extracts and controls. 

Cell Proliferation, Viability, Metabolic and Membrane Integrity Assays

For measuring the viability of cells we use assays that measure membrane integrity, metabolic activity and other metabolic markers. These may include MTT, Alamar Blue and water soluble tetrazolium salts. We also are able to measure ATP levels. For membrane integrity, assays we employ for customers include the use of lactate dehydrogenase (LDH), Trypan Blue, Calcein-AM, Propidium iodide/7-AAD and cell-impermeable DNA binding dyes. 

Cell Resealing Assay

This test involves disrupting the cell plasma membrane of different cell types and determining technologies that may help to prevent cellular necrosis. For this assay we use different techniques to investigate cellular integrity including a Live-Dead assay, a F-actin staining assay and a lactate dehydrogenase assay (LDH). 

Gel Contraction Assay

Wound healing involves a contraction phase which occurs after connective tissue deposition. This contraction process is mediated by specialised fibroblasts called myofibroblasts. It is a model that is used to study tissue contraction and investigate effects that materials and products may have on the rate and extent of the extracellular matrix contraction.

In Vitro Scratch Test Model

The in vitro scratch assay is considered a low cost and well established method to measure cell migration in an in vitro environment. The method involves making a “scratch” in a monolayer of cells.

Over time, images of the cells are captured as they migrate to close the scratch. This assay is used to investigate the effects of materials and products on cell-cell integration and effects on cellular motility and migration.

In Vitro Skin Irritation Assays (Reconstructed Human Epidermis)

Skin irritation is defined as the reversible damage of the skin following exposure to a test substance for up to 4 hours. Skin irritation in vitro tests provide a measurement of cell viability in reconstructed human epidermis (RhE) tissue following exposure to a treatment. Within cosmetic products there maybe ingredients that cause skin irritation. The in vitro test can be undertaken in EpiDerm™, epiCS®, LabCyte EPI-MODEL, EpiSkin™ and SkinEthic™ RHE tissue models which are composed of human epithelial cells. The end point of these tests is determined using a cell viability (MTT reduction assay) assay with and without histology. 

Following completion of the test, the material is classified as severe, moderate, mild or minimal / non-irritant. There are a number of other assays 5D use that can be used to evaluate the skin irritation of test articles. These include Time-to-Toxicity ET50, Cytokine Expression Assays and In vitro skin irritation/corrosion assays. 

In Vitro Skin Corrosion Assay (Reconstructed Human Epidermis)

The skin corrosion test is used to investigate the effects of substances that cause irreversible damage to skin. This in vitro test can be undertaken in RHE models including EPISKINTM (commercial skin product), or EpiDermTM. The end point of these test is determined using a cell viability (MTT reduction assay) test with and without histology. In addition to this, the CorrositexTM test can be employed to investigate skin corrosion which is a useful test for acids and bases. 

In Vitro Gingival Irritation Test

This in vitro test is undertaken in human gingival cells and is used to evaluate the potential of oral care products to irritate the gingival mucosa. As with the skin irritation test, the effects of products on cellular viability is measured by the enzymatic conversion of the dye MTT into a blue formazan salt.

Customer Testimonials

  • "We have found 5D Health Protection very informative and responsive to the work proposals, questions and final data. 5D Health were very helpful and clear in their support and advice throughout the study"
  • "5D provided an excellent service regarding product evaluation, consultancy and education"
  • "If anybody is looking to begin working with biofilms, they need to look at this course. It is run by top notch people, and they make it fun"
  • "Excellent communication of timelines"
  • “Always happy to discuss test methods and results with us”
  • “All testing was conducted as agreed, clear and professional report supplied”
  • "Very quick response to the initial request. Very professional and helpful advice to the requested studies. Very Supportive staff"
  • "If not for this course, we would likely never be able to get our lab up and running in a timely manner. This was a HUGE time saver in the long run"

5D Accomplishments, Partnerships and Collaborations

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  • Cardiff University


5D Health Protection Group Ltd

Accelerator Building
1 Daulby Street
L7 8XZ
United Kingdom


+44 (0) 151 702 9492

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