Skin model

Abstract

A three dimensional (3-D) model comprising a scaffold and autologous skin cells, the invention also provides methods of predicting immunogenicity and hypersensitivity or allergic or adverse immune reactions to potential therapeutic compounds, biologies, cosmetics and chemical sensitizers using the 3-D model of skin cells. The methods provide an in vitro assay employing autologous blood derived cells in the 3-D skin equivalent model and is of particular utility in the identification and prediction of skin sensitizers and in particular agents that may cause allergic contact dermatitis. The assay of the present invention provides inter alia methods of screening library compounds for sensitizing activity, identifying optimal therapeutics, especially but not exclusively, monoclonal antibodies and kits therefor.

Claims

1. An in vitro method for identifying chemical compounds that are sensitizers or for discriminating between chemical compounds that are sensitizers and non-sensitizers, the method comprising: (i) preparing a donor blood sample so as to isolate a population of T cells and monocyte-derived dendritic cells therefrom; (ii) incubating the monocyte-derived dendritic cells with a test compound; (iii) incubating the compound treated monocyte-derived dendritic cells with a population of T cells isolated in step (i); (iv) establishing a 3-D skin equivalent model that comprises a monoculture of fibroblasts grown in monoculture on matrix for at least 21 days and subsequently seeded with keratinocytes, wherein both fibroblasts and keratinocytes are isolated from an autologous biopsy sample obtained from the same donor; (v) incubating the mixed T cell and compound treated monocyte-derived dendritic cells with the 3-D skin equivalent model of step (iv) that comprises keratinocytes and fibroblasts isolated from an autologous biopsy sample obtained from the same donor; and (vi) assessing hypersensitivity and allergic reactions by graded histological changes in the 3-D skin equivalent model as compared to a control, wherein an increase of a graded histological reaction in the 3-D skin equivalent model identifies the test compound as a sensitizer, wherein the 3-D skin equivalent model comprises a scaffold, wherein the scaffold comprises a polystyrene support, and wherein the chemical compound is selected from a chemical, therapeutic, pharmaceutical, biologic, antibody or cosmetic agent, substance, preparation or composition.

2. The method of claim 1, wherein the T cells and monocyte-derived dendritic cells (DCs) are isolated from peripheral blood mononuclear cells (PBMC).

3. The method of claim 1, wherein the first incubating step of step (ii) is for between 2 to 24 hours.

4. The method of claim 1, wherein the second incubating of step (iii), comprising incubating DCs with a population of T cells isolated in step (i) is for between 3-7 days using the same culture conditions as for step (ii) except that 10% heat inactivated autologous serum, human AB serum, or an equivalent thereof is used in step (iii) and foetal calf serum is used in step ii).

5. The method of claim 1, wherein the step of assessing hypersensitivity and allergic reactions in the 3-D skin equivalent model by graded histological changes comprises assessment of vacuolisation of epidermal cells, damage to basal keratinocytes and connection between the epidermis and dermis.

6. The method of claim 1, wherein the control value may be derived from the group comprising: (i) a further or second set of monocyte-derived dendritic cells that have been incubated in step (ii) with a compound that is a known non-sensitizer; (ii) a further or second set of monocyte-derived dendritic cells that have been incubated in step (ii) with no additional chemical compounds; (iii) a 3-D skin equivalent model that has been incubated with autologous lymphocytes; or (iv) a skin 3-D skin equivalent model that has been incubated with compound alone at the same concentrations as that used in step (ii).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

(2) FIG. 1 shows histopathological changes for different grades of skin graft versus host reaction (GVHR); FIG. 1A shows Grade I skin GVHR showing very mild vacuolisation of epidermal cells (Negative reaction); FIG. 1B shows Grade II skin GVHR showing diffuse vacuolisation of epidermal cells (Positive reaction); FIG. 1C shows Grade III skin GVHR showing cleft formation between the epidermis and dermis caused by confluent vacuolar damage to basal keratinocytes (Positive reaction) and; FIG. 1D shows Grade IV skin GVHR showing the complete separation of the epidermis and dermis (Positive reaction).

(3) FIG. 2 shows 3D skin equivalent sections that have been formalin fixed. Sections were paraffin embedded, sectioned and stained with haematoxylin and eosin.

DETAILED DESCRIPTION

(4) Reference herein to scaffold means any 3-D structure either native/natural or tissue engineered that is capable of supporting cell migration, cell infiltration and/or cell proliferation.

(5) Reference herein to autologous means that the blood derived products and skin explants are derived or collected from the same individual.

(6) Reference herein to a sensitizer includes any chemical compound or chemical agent or antibody that causes a substantial proportion of exposed people or animals to develop an allergic reaction in normal tissue after single or repeated exposure to the said compound, antibody or chemical agent.

(7) Reference herein to an allergen and allergenic includes any foreign substance such as an environmental substance or chemical that is capable of inducing allergy or a specific hypersensitive reaction in the body. Common allergens include plant pollens, spores of mold, animal dander, house dust, foods, feathers, dyes, soaps, detergents, cosmetics, plastics, and drugs. Allergens can enter the body by, for example, being inhaled, swallowed, touched, or injected.

(8) Reference herein to a chemical compound is intended to include a chemical, therapeutic, pharmaceutical, biologic, antibody or cosmetic agent, substance, preparation or composition.

(9) Reference herein to a biologic is intended to include a preparation, such as a drug, a vaccine, serum or an antitoxin, that is synthesized from living organisms or their products and used as a diagnostic, preventive, or therapeutic agent.

(10) Reference herein to biological therapeutic products includes, but is not limited to, antibodies, e.g. monoclonal antibodies, antibody conjugates, Fc fusions; or proteins or protein based therapeutics.

(11) We have previously shown that following incubation of chemicals with blood derived dendritic cells or mononuclear cells with monoclonal antibodies then interaction with T cells (in the first instance for chemical sensitisation) and then with skin in the second phase of the reaction it is possible to observe a histopathological damage read out. The prejudice in the art was that it was only possible to show skin sensitisation or allergy to chemicals or other allergies using skin dendritic cells. The prejudice arises from the fact that far fewer dendritic cells can be obtained from skin biopsies than can be prepared from monocytes in whole blood. In the present invention, with improved technology to enable 3-D skin equivalent models to be developed, we provide for the first time an autologous 3-D skin equivalent model that surprising comprises less cells, with increased longevity that can be used as a robust and accurate system for detecting allergic and sensitizing reactions.

(12) The present invention conveniently provides a 3-D skin equivalent model of autologous mammalian skin cells that can be used in an in vitro assay and methods which allows for the study of primary and secondary immune responses in the presence of potential sensitizing compounds thereby advantageously reducing the need for extensive animal testing.

(13) The present invention conveniently provides a 3-D skin equivalent model of autologous mammalian skin cells from a specified individual that can be used in an in vitro assay to assess that individual's response to a particular chemical compound or therapeutic agent and also to monitor the efficacy of any treatment regime.

(14) The present invention is of particular utility to an individual who is about to receive antibody or biologic therapy, in particular but not exclusively an individual suffering from chronic rheumatoid arthritis who is to receive antibody therapy. The present invention will allow a clinician to assess which antibody therapy will be tolerated and which antibody therapy may cause an allergic reaction. It is envisaged that the problem associated with unpredictable allergic reactions to antibody therapy will be overcome by the use of the 3-D skin equivalent model of autologous skin cells of the present invention.

(15) The ability to grown whole skin models from constituent cells rather than individual punch biopsy sample has many advantages. For example, using a single sample of skin tissue to create an autologous 3-D skin equivalent model with extended longevity it will be possible to use the 3-D model in multiple tests over a period of months rather than a single sample for use in only one assay. The 3-D skin equivalent model is viable over many months rather than weeks in the standard skin biopsy model. It is envisaged that a further advantage is that culturing under flow conditions will achieve a faster cell culture with increased proliferation rates and improved cell viability which will not only reduce the time taken and cost of testing but will also permit repeated dose testing for allergenicity. Using the longer viable 3-D skin equivalent model of the present invention will enable the efficacy of therapy to be assessed over time without the requirement for a further skin biopsy sample being taken from an individual, the only further requirement being a peripheral blood sample. This is of benefit to the individual undergoing the test as it will minimize any pain due to taking of a biopsy sample.

(16) The products and methods of the present invention are unique and gives insight into the use of a blood based assay on a 3-D autologous skin equivalent model for predicting response to chemical sensitizers and to investigate their potential allergic/inflammatory signals. The present invention provides a blood based assay and 3-D skin equivalent model that improves on the current techniques and provides a novel means of testing novel drugs for hypersensitivity and allergic reactions.

(17) The product and assay of the present invention provides the advantage over a heterologous three dimensional skin equivalent model as it uses blood with autologous immune cells and autologous skin cells enabling immune responses to be studied and cellular and molecular targets identified thus aiding in drug discovery, improving drug design and optimisation for drug dosage prior to a clinical trial.

(18) Viability Assay

(19) A dye exclusion method can be used to investigate cell viability. It is based on the principle that live cells possess intact cell membranes that exclude certain dyes, such as trypan blue, eosin, or propidium, whereas dead cells do not. Cells are treated with different concentrations of the test substance for a period of 24 hours. Cells are harvested and an aliquot of the cell suspension is mixed with trypan blue (1:1) and then visually examined to determine whether cells take up or exclude dye. The trypan blue chromopore is negatively charged and cannot react with a cell unless the cell membrane is damaged, therefore a viable cell has a clear cytoplasm whereas a non-viable cell has a blue cytoplasm. A total of 100 cells are counted. The unstained (viable) and stained (non-viable) cells are counted separately using a haemocytometer and viability recorded. By culturing cells in the presence of the test reagents any adverse effect of the reagents on cell viability can be observed. Cell viability of 70% or more is regarded as adequate for the methods of the present invention. Preferably, the cell viability is 75% or more or 80% or more.

(20) Preparation of Peripheral Blood Mononuclear Cells (PBMC)

(21) Peripheral Blood Mononuclear cells (PBMC) from blood obtained from healthy volunteers was prepared by density-gradient centrifugation using Lymphoprep solution (Axis-Shields) and diluted 1:1 in Earle's balanced salt solution (EBBS) (Sigma). Mononuclear cells were collected from the density medium:plasma interface and washed in cold PBS and counted using an Improved Neubauer cell counting chamber (Weber Scientific International Ltd., UK). Cell viability was assessed by trypan blue (Gibco).

(22) Separation of CD14+ Monocytes Using the MACS Technology

(23) The MACS (Magnetic-activated cell sorting) technology (Miltenyi Biotec) uses columns filled with magnetic particles to separate magnetically labelled cells. For the separation process these columns are placed in a strong magnetic field (QuadroMACS separator). Required amount of PBMC were transferred to a fresh 50 ml falcon tube, topped up with PBS and strained through a 100 m nylon filter to remove any clumps. A maximum of 10010.sup.6 mononuclear cells were washed and re-suspended in cold MACS buffer (PBS containing 0.5% FCS and 1 mM ethylene diamine tetracetic acid (EDTA) resusupended in 80 l buffer/1010.sup.6 cells. The cells were incubated at 2-8 C. for 20 minutes with 10 l/1010.sup.6 cells CD14 antibody coupled with magnetic microbeads. The cell suspension was added to the column allowing the negative cells to pass through for collection (as the T cell fraction and the positive cells (CD14+) were then collected and assessed for purity by flow cytometry analysis.

(24) Generation of Monocyte-Derived Dendritic Cells (moDC)

(25) CD14 positive monocytes purified by MACS separation were cultured in a 24 well plate at a density of 0.510.sup.6/ml in culture medium with 50 ng/ml GM-CSF and 50 ng/ml IL-4. After 3 days 400 l of the medium were carefully removed and 500 l fresh medium containing 50 ng/ml GM-CSF and 50 ng/ml IL-4 (Immunotools) were added and left for a further 3 days. After 6 days immature antigen presenting cells dendritic cells (DC) were either collected or allowed to mature by adding lipopolysaccharides (LPS) (0.1 g/ml, Sigma), IL-1 (10 ng/ml, Immunotools) and TNF (10 ng/ml, Immunotools) for a further 24 hours.

(26) Generation of Mature Fast DC

(27) CD14 positive selection cells were put into culture (0.3-0.510.sup.6 cells per well in 24 well plate) with RP-10 medium supplemented with IL-4 (50 ng/ml) and GM-CSF (50 ng/ml). After 24 hours maturation cytokines TNF- (10 ng/ml), IL-1 (10 ng/ml), IL-6 (10 ng/ml), 1 uM PGE2, Resiquimod (2.5 g/ml), CD40L (1 g/ml) and LPS (0.1 g/ml) were added to each well for a further 24 hours.

(28) T Cell Proliferation Assays

(29) Mature Fast DC treated and untreated with compounds as well as cells from both allogeneic and autologous sources in triplicate at a ratio of 1:10 (DC:T cells) in (200 l total volume) in 96-well round-bottomed plates for 5 days at 37 C. in a humidified 5% CO.sub.2 in air incubator. After 5 days, 40 l of supernatant was removed from the top of each triplicate well and stored at 20 C. for further cytokine analysis. [.sup.3H]-Thymidine (used at a concentration of 3.7 MBq/ml) was then added to each well using appropriate radiation protection methods and allowed to incubate for 16-18 hours at 37 C. in a humidified 5% CO.sub.2 in air incubator. Cells were harvested and subsequently counted using the 1450 MicroBeta TriLux Microplate Scintillation and Luminescence Counter (PerkinElmer). Data was interpreted using Graphpad Prism software.

(30) Skin Biopsy

(31) Sections from a 4 mm wide 2 mm deep skin punch biopsy were obtained from an individual. Ideally the skin punch biopsy is obtained from an area of medium skin thickness and low innervations so as to cause as little discomfort as possible to the individual. Autologous keratinocytes and fibroblasts are separated from the biopsy sample

(32) Generation of Keratinocytes and Fibroblasts

(33) Autologous keratinocytes and fibroblasts were generated from a 4 mm punch skin biopsy, the skin biopsies were incubated with dispase (final concentration 1 mg/ml) at 4 C. overnight. Following the incubation dispase was removed by washes then the epidermis was peeled from the dermis using sterile forceps. The epidermis was used for generating keratinocytes and dermis used for generating fibroblasts. The epidermis was incubated at 37 C. with Trypsin/EDTA and dermis incubated with collagenase (100 U/ml) to release the keratinocytes and fibroblasts from the tissue matrix into the supernatant. The cells were collected from supernatants following centrifugation. Collected keratinocytes and fibroblasts were cultured with appropriate medium in a 48 or 24 well culture plate as passage 0. The medium used for growing keratinocytes is EpliLIfe (Life technologies) and for fibroblasts is Dulbecco's Modified Eagles Medium (DMEM) (Sigma) containing 100 IU/ml penicillin, 100 g/ml streptomycin (Gibco UK) and 2 mM L-glutamine (Gibco UK) supplemented with 10% v/v heat inactivated foetal calf serum (FCS, Sera Lab) respectively. Both keratinocytes and fibroblasts would adhere to the plastic surface and have a monolayer growth. The cells were fed twice a week till they reached approx 80% confluence. Then the cells were removed from the culture wells using Trypsin/EDTA. The cells were then collected, washed and reseeded into a larger tissue culture well or a flask as passage 1 to expand further on for example 12 or 24 well scaffold inserts. The cells have been expanded up to passage 3 to obtain 0.5-210.sup.6 cells. The successful rate of generating keratinocytes from punch skin biopsies is 60%.

(34) The skin explant assay consisted of co-incubating the treated and untreated DC cells with T cells from the same donor for 7 days. After this time the T cells are added in 96 well plates or 12 or 24 well inserts to sections of the 3D human skin equivalent model. The skin equivalent model is co-incubated for three days and then routinely stained for histopathology. 3-D skin equivalent models incubated with medium alone or autologous cells alone are used as controls. The 3-D skin equivalent model is then routinely sectioned and stained for graded histopathological damage using a criteria which is very similar to that used and observed in the clinical setting with distinct pathological damage.

(35) In the present invention DC response to chemical sensitizers versus known non-sensitizers can be assessed by their effect on sensitized cells by assessment in vitro of graded skin damage.

(36) 3-D Skin Equivalent Model Culture

(37) Human dermal fibroblasts isolated from dermis of the skin are seeded onto a scaffold and cultured for 3 weeks to allow production of the extracellular matrix (ECM). Keratinocytes from the same piece of skin are isolated (0.5-510.sup.6) and cryopreserved and at 3 weeks thawed and seeded onto the scaffold, raised to the air-liquid interface, and assessed for growth over 2-5 months. To aid differentiation and growth and use of cell flow conditions at a rate of 100-500 ul/min to increase longevity is also employed.

(38) Processing Skin

(39) Preparation of 3T3 Cells

(40) 3T3 cells (3T3-J2 strain (ATCC# CCL-92)) were grown as feeder cells for keratinocytes. 3T3 cells were grown in 3T3 medium (DMEM supplemented with 10% new-born calf serum, 1% Penicillin-Streptomycin-Fungizone). 3T3 cells were maintained in sub-confluent culture to prevent spontaneous transformation. Medium was replaced every 3-4 days. A flask of 70% confluent 3T3 cells treated with Mitomycin-C (0.4 g/ml) for 2 hours at 37 C. was prepared before processing epidermis.

(41) Processing Skin

(42) Skin was washed and cleaned with PBS and fat removed leaving a thin layer of epidermis and dermis. Skin was then incubated overnight with Dispase (1 mg/ml) at 4 C. The next day skin was removed from the well and epidermis peeled backusing forceps.

(43) Processing Epidermis

(44) A flask of 70% confluent 3T3 cells was prepared in advance of processing the epidermis. The peeled epidermis was incubated for 5 minutes at 37 C. with Trypsin/EDTA (T/E). After 5 minutes a serological pipette was used to disrupt the tissue and release cells into the supernatant. Trypsin/EDTA was neutralised by adding 200 ul FCS and 10 mls PBS. Epidermis was removed with a tip and discarded. Supernatant was centrifuged at 500 g for 5 minutes. Supernatant was discarded and the cell pellet resuspended in 20 ml pre-warmed F-Media. 3T3 medium was removed from 3T3 cells treated with mitomycin-C, rinsed X2 with PBS and the resuspended pellet in F-Media was added to the flask. Cells were grown until keratinocytes became visible. Keratinocytes were then removed from the 3T3 cells by trypsination and further cultured in EpiLife medium until the required numbers of cells (210.sup.6) were acquired. Cells should be between passages 0-3.

(45) Processing Dermis

(46) The remaining dermis (after the epidermis peel) was cut into square pieces using a scalpel and placed in to 3 mls RF10 and collagenase enzyme (100 U/ml) and incubated overnight at 4 C. The next day tissue was disrupted using a serological pipette. The supernatant was passed through a cell strainer (100 micron) and centrifuged at 500 g for 5 minutes. The cell pellet was re-suspended in 1 ml DMEM (DMEM+Glut+P/S+20% FCS). Cells were cultured until the required numbers of fibroblasts (0.510.sup.6) were acquired. Cells should be between passages 0-7.

(47) 3D Skin Equivalent Model

(48) Alvetex Scaffold Preparation

(49) Alvetex was soaked in 70% ethanol for 5 minutes and washed twice in 8 mls DMEM for 2 minutes each. Alvetex was then placed into a 6 well plate.

(50) Adding Fibroblasts to Alvetex Scaffold

(51) Fibroblasts were grown to maximum of passage 7 in DMEM to 70% confluence. Fibroblasts were trypsinised with T/E (2 minutes at 37 C.). Cells were centrifuged as before and 0.510*6 cells were removed and placed in 100 ul volume DMEM. Fibroblasts were added to the centre of the scaffold. The plate was incubated for 3 hours to allow the cells to attach to the scaffold. The well was then flooded with 8 mls DMEM. Medium was replaced every 2 days for at least 21 days. After 21 days, fibroblast monolayer (dermis) was formed and ready for the addition of keratinocytes.

(52) Adding Keratinocytes to Alvetex Scaffold

(53) Keratinocytes were grown to maximum of passage 3 in Epilife to 70% confluence. Keratinocytes were then trypsinised with T/E (2 mins at 37 C.). Cells were centrifuged as before and 210*6 cells were removed and placed in 100 ul volume F-Media. Media was removed from the well containing the Alvetex scaffold and keratinocytes were added to the centre of the scaffold. The well was flooded with 4 mls F-Media. The plate was incubated for 3 hours to allow the cells to attach to the scaffold. The well was then flooded with another 5 mls F-Media. The plate was incubated for 3 days. After 3 days medium was removed and replaced with 4 mls F-Media to allow cells exposure to air-surface interface. Medium was replaced every 2 days for at least 14-18 days. After 18 days, 3D skin equivalent was cut out of the plastic holder and formalin fixed. Sections were paraffin embedded, sectioned and stained with haematoxylin and eosin as shown in FIG. 2.

(54) Throughout the description and claims of this specification, the words comprise and contain and variations of them mean including but not limited to, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

(55) Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

(56) The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.