The disclosure relates to a screening assay comprising at least two different cell types and its use in the testing of xenobiotic agents on lung cell function.

The lungs are the major organ of respiration and are unique in their exposure to adventitious infective agents and substances that affect lung function there being a direct connection between the lungs and the external environment via the mouth and trachea. The human lungs are paired organs; the left lung is divided into two lobes and the right into three lobes. The trachea divides into a pair of bronchi which terminate in bronchioles which end in the alveoli which are well served by blood vessels that conduct inhaled oxygen to the vascular system and essential organs and remove the waste products of cellular respiration for exhalation and removal from the body. Lung tissue is fragile and susceptible to damage by toxic substances and infective agents.

There are a number of disease conditions that affect the lungs. For example, chronic respiratory diseases are a major debilitating group of diseases that are often fatal if not treated. Inflammatory diseases such as chronic respiratory disease, pneumonia, fibrosing alveolitis, chronic bronchitis, bronchiectasis, emphysema, asthma, silicosis and other pneumoconiosis and tuberculosis are major causes of death in the population. Chronic Obstructive Pulmonary Disease [COPD] is a leading cause of death worldwide and can be caused by noxious particles or gas commonly found in tobacco smoke. The pathogenesis of COPD is characterized by airflow obstruction due primarily to inflammation and comprises both chronic bronchitis and emphysema both of which lead to lung tissue destruction. In addition COPD patients suffer not just from lung related damage but also to cardiovascular disease. Lung cancer is a generic term to describe cancers of lung tissue. The majority of lung cancers are carcinomas which are derived from epithelial cells. Of this type the cancer is either a small cell lung carcinoma or a non-small cell lung cancer. Other classes of lung cancer include carcinoid, sarcoma and metastatic cancers of different tissue origin. Lung cancers are amongst the most common cancers there being a strong correlation between the incidence of lung cancer and smoking tobacco. The prognosis of lung cancer patients is not good even with treatment.

There is therefore a desire and need to identify therapeutic agents that ameliorate disease symptoms associated with the lungs and related organs.

There is also a desire to move away from animal testing of medicinal agents. The total number of animals used for experimental purposes in the European Union in 2008 was above 12 million (EU, 2010). Rodents and rabbits represented more than 80% of the total number of animals used in tests for the production and quality control of products for human medicine and dentistry and for veterinary medicine. The largest percentage (45%) of use of animals in toxicological and other safety evaluations was due to acute/sub-acute toxicity testing. There are scientific reasons for moving away from animal studies in the field of inhalation toxicology which include their extreme cost and time, which lead to a very low sample throughput, and the difficulties in extrapolating across species. There are also significant problems in extrapolating high-dose animal studies to low-dose chronic exposures (inhalation and instillation), since the capacity of the airway epithelium to deactivate compounds can be saturated at the high concentrations and short times often tested on laboratory animals (Gerde, 2005).

This disclosure relates to an in vitro co-culturing method to test the efficacy and/or toxicity of xenobiotic agents on lung cell metabolism. The co-culturing of cells is known in the art. For example, WO2010/1 18352 discloses a three dimensional construct for the co-culturing of fibroblasts with cardiac muscle cells or cardiac stem cells and the use of the construct in the treatment of cardiac disorders. WO2010/042669 discloses co-cultures of differentiated cells from induced pluripotent stem cells and the use of the co-cultures to identify therapeutic agents. WO2009/024748 discloses the co-culture of induced mesenchymal stem cells with neural stem cells and differentiation of the neural stem cells into neurones. WO2002/48318 discloses the co-culture of hepatocytes and liver stellate cells and the use of the co-culture in toxicological testing of agents. It is apparent that co-culturing of different cells is known. We disclose a co-culture of lung derived cells, for example lung epithelial cells with hepatocytes, to provide a cell based model for testing agents with biological activity. Our model comprises a fully differentiated, / ^' n-wVo-like model of the human bronchial epithelium. The cell based model is well-suited as an in vitro replacement alternative for in vivo high-throughput (HTP) inhalation toxicology testing.

Statements of Invention

According to an aspect of the invention there is provided an in vitro cell-based screening method for the testing of xenobiotic agents that have a cellular modulating activity comprising the steps:

i) providing a cell culture vessel comprising

a cell culture including at least two cell types wherein the cell-types are mammalian lung epithelial cells and mammalian hepatocytes and further wherein said mammalian lung epithelial cells are cultured at an Air-Liquid Interface to provide a muco-ciliary lung epithelium having an apical surface into which mucin is secreted; and a cell culture medium adapted for the culture of said cell types;

ii) contacting the cell culture with at least one test agent;

iii) analyzing the biological effect of said agent[s] on at least one biomarker and/or the metabolite profile of said culture; and iv) comparing said biomarker expression and/or metabolite profile to a control cell culture which has not be contacted with said agent[s].

Reference herein to the term Air-Liquid Interface (ALI) refers to the culture of cells such that their basal membrane is in contact with, or submerged in, liquid and their apical membrane is in contact with the air. As will be appreciated by those skilled in the art, such culture arrangement mimics that of the lung epithelia of the airway, taking into account the basic structural and functional organisation of the respiratory epithelium. Advantageously, the cells consequently demonstrate apical-basal polarity in their differentiation resulting in the development of directional polarised metabolic processes as seen in vivo, such as but not limited to, cilia and mucin secretion.

The term "xenobiotic agent" is given a broad definition and includes not only compounds but also gaseous agents and particulate agents that are potentially damaging to lung tissue. These can be found in tobacco smoke and other recreational drugs [e.g. cannabis] and in air pollution. Typically xenobiotic agent encompasses pharmaceutically active agents used in human and veterinary medicine. Included are environmental toxins [e.g. herbicides, insecticides, industrial chemicals] that may be inhaled by subjects and have biological activity toward lung tissue. "Biomarkers" that are typically measured are for example cytochrome P450s which are known to be associated with xenobiotic metabolism. Cytochrome P450s are a very large family of enzymes responsible for the oxidation, peroxidation and reduction of a vast number of xenobiotics and intermediate metabolites. P450s are known to be involved in the metabolism and detoxification of drugs as well as the biosynthesis of primary and secondary metabolites. "Metabolite profiling" is undertaken using known methods; for example, high performance liquid chromatography and mass spectroscopy. In addition light, electron and fluorescent microscopy will be undertaken to analyse cell ultra structure after exposure to test xenobiotic agents.

The co-culture of lung and liver cells enables one to measure the biotransformation of xenobiotic agents that may affect lung or liver function. In a preferred method of the invention said lung epithelial cells and hepatocytes are primate, preferably human.

In a preferred embodiment of the invention said lung epithelial cells are primary lung epithelial cells.

In a preferred embodiment of the invention said hepatocytes are primary hepatocytes. In a preferred method of the invention said lung epithelial cells and/or said hepatocytes are autologous.

In an alternative preferred method of the invention said lung epithelial cells and/or said hepatocytes are allogenic.

In a preferred method of the invention said lung epithelial cells are bronchiole epithelial cells. In an alternative preferred embodiment of the invention said lung epithelial cells are isolated from diseased tissue.

Diseased lung tissue may be obtained from subjects suffering from for example, chronic respiratory disease, COPD, pneumonia, fibrosing alveolitis, chronic bronchitis, bronchiectasis, emphysema, asthma, silicosis and other pneumoconiosis and tuberculosis.

In yet a further preferred embodiment of the invention, said test agent can contact the cell culture by adding it to said cell culture medium. Alternatively, and more ideally, said test agent can contact the cell culture by adding it to the apical surface of said mammalian lung epithelium cells. Advantageously, this permits delivery of vapour-like test agents, such as but not limited to, gases and dry airborne powders, in addition to soluble test-agents. As will be appreciated by those skilled in the art, this is much more representative of events that occur in-vivo wherein the bronchial epithelium of the lung is one of the first lines of defence to inhaled particles. Consequently, this therefore provides a model ideally suited to study the toxicology of inhaled substances and their subsequent biotransformation. In a preferred method of the invention said cell culture vessel further comprises fibroblasts; preferably lung fibroblasts. As will be appreciated by those skilled in the art, the co-culture of cells with fibroblasts improves longevity of hepatocytes and lung cells. Moreover, the presence of fibroblasts also permits investigation into, but not limited to, fibrotic lung diseases or the like. In a preferred method of the invention said fibroblasts are human and autologous or allogenic.

In a preferred method of the invention said cell culture includes a cell support substrate.

In a preferred method of the invention said cell support substrate comprises collagen.

In a preferred method of the invention said cell culture support is Matrigel ^tm .

According to a further aspect of the invention there is provide a cell culture vessel comprising:

i) a co-culture of cells wherein said co-culture comprises at least two cell types wherein the cell-types are mammalian lung epithelial cells and mammalian hepatocytes and further wherein said mammalian lung epithelial cells are cultured at an Air-Liquid Interface to provide a muco-ciliary lung epithelium having an apical surface into which mucin is secreted; and

ii) ii) a cell culture medium adapted for the culture of said cell types. In a preferred embodiment of the invention said lung epithelial cells and hepatocytes are primate, preferably human.

In a preferred embodiment of the invention said lung epithelial cells and/or said hepatocytes are autologous.

In an alternative preferred embodiment of the invention said lung epithelial cells and/or said hepatocytes are allogenic. In a preferred embodiment of the invention said cell culture vessel further comprises fibrobalsts; preferably lung fibroblasts.

In a preferred embodiment of the invention said fibroblasts are human and autologous or allogenic.

According to a further aspect of the invention there is provided a cell culture vessel according to the invention for use in the testing of biologically active xenobiotic agents.

According to a further aspect of the invention there is provided a cell array wherein said array comprises a plurality of cell culture vessels comprising a co- culture of at least two cell types wherein said cell types are mammalian lung epithelial cells and mammalian hepatocytes and further wherein said mammalian lung epithelial cells are cultured at an Air-Liquid Interface to provide a mucociliary lung epithelium having an apical surface into which mucin is secreted; and a cell culture medium adapted for the culture of said cell types.

The screening of large numbers of agents requires preparing arrays of cells for the handling of cells and the administration of agents. Assay devices, for example, include standard multiwell microtitre plates with formats such as 6, 12, 48, 96 and 384 wells which are typically used for compatibility with automated loading and robotic handling systems. Typically, high throughput screens use homogeneous mixtures of agents with an indicator compound which is either converted or modified resulting in the production of a signal. The signal is measured by suitable means (for example detection of fluorescence emission, optical density, or radioactivity) followed by integration of the signals from each well containing the cells, agent and indicator compound.

According to a further aspect of the invention there is provided a method for the high through put screening of biologically active xenobiotic agents comprising the steps:

i) providing an array according to the invention;

ϋ) contacting the array with a plurality of agents to be tested;

ill) collating activity data obtained following (ii) above; iv) converting the collated data into a data analysable form; and optionally

v) providing an output for the analysed data. A number of methods are known which image and extract information concerning the spatial and temporal changes occurring in cells or nuclei expressing, for example fluorescent molecules and other markers of gene expression (see Taylor et al Am. Scientist 80: 322-335, 1992). Moreover, US5, 989,835 and US09/031 ,271 , both of which are incorporated by reference, disclose optical systems for determining the distribution or activity of fluorescent reporter molecules in cells for screening large numbers of agents for biological activity. The systems disclosed in the above patents also describe a computerised method for processing, storing and displaying the data generated.

According to a further aspect of the invention there is provided a method for the formation of a three dimensional cell model comprising:

i) providing a cell culture vessel comprising confluent or near confluent human fibroblasts and a collagen based cell support substrate in cell culture medium;

ii) contacting the human fibroblasti/collagen based cell support substrate with a co-culture comprising human lung epithelial cells, preferably bronchiole epithelial cells, and human hepatocytes wherein said lung epithelial cells are cultured at an Air-Liquid Interface to provide a muco-ciliary lung epithelium having an apical surface into which mucin is secreted;

iii) culturing the cell culture in ii) to form a three dimensional cell model; and optionally

iv) monitoring the differentiation state of cells comprising the three dimensional cell model.

In a preferred method of the invention the three dimensional cell model is contacted with at least one xenobiotic agent. In a preferred method of the invention the human fibroblasts, human lung epithelial cells and/or human hepatocytes are autologous.

In an alternative preferred method of the invention the human fibroblasts, human lung epithelial cells and/or human hepatocytes are allogenic.

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does 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.

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.

Moreover, unless stated otherwise, any feature disclosed herein may be replaced by an alternative feature serving the same or a similar purpose. No admission is made that any reference referred to herein constitutes prior art. Further, no admission is made that any of the prior art constitutes part of the common general knowledge in the art.

An embodiment of the invention will now be described by example only and with reference to the following figures: Figure 1 illustrates the production of acetaminophen, 24 hours post-treatment with its substrate probe Phenacetin; as determined by HPLC. HPLC analysis was performed on culture media from hHeps alone , NHBE cultures alone and NHBE- Hepatocyte co-culture, i.e. "Metabo-Lung" . Error bars express standard deviations. Acetaminophen was generated in hHep mono-cultures following exposure to 100μΜ of Phenacetin. NHBE cultures did not BT Phenacetin (100μΜ) but co-cultured with hHeps yielded the marker metabolite;

Figure 2 illustrates the production of hydroxydiclofenac following BT of Diclofenac, 24 hours post-treatment, as determined by HPLC. HPLC analysis was performed on culture media from Hepatocytes alone , NHBE cultures alone and NHBE-Hepatocyte co-culture . Error bars express standard deviations. NHBE cultures exhibited inherent metabolic activity when challenged with 50μΜ of Diclofenac, generating the target metabolite, but when co-cultured with Hepatocytes, produced more Hydroxydiclofenac;

Figure 3 illustrates a graph comparing hepatocyte culture viability (ATP assay; luminescence) when cultured for 24 hours in either their own media (Hep media; In Vitrogen) or NHBE cell media (ALI; Prytherch, 2010). Four different hepatocyte concentrations were tested to determine the optimum seeding density. Pilot work determined that a seeding density of 0.5 million cells per well in the ALI media supported the growth of both Hepatocytes and NHBEs;

Figure 4 illustrates microscopical images of NHBE model. (Top): LM image stained with toluidine blue showing pseudo-stratified epithelium of muco-ciliary phenotype. Cell types: Ciliated cell (CC), goblet cell (GC), Clara Cell (CIC), basal cell (BC) and intermediate cell (IC). (Bottom, L-R): TEM revealing cell-cell junction and complexes (tight (TJ) and adherens junction (AJ) and desmosome (D)). SEM of surface denoting cilia and mucin globules. Confocal image of apex stained for two TJ proteins, ZO-1 and occludin ;

Figure 5 illustrates a schematic diagram of tissue-engineering Stages 1 -3 to optimise and validate the Metabo-Lung model into a viable in vitro alternative to animal use for inhalation toxicology testing; and Table 1 is a summary of the cell culture medium and sources of cell medium components.

Materials and Methods

INITIAL SEEDING AND CULTURE TO CONFLUENCE OF NHBE CELLS

Normal human bronchial epithelial (NHBE) cells were obtained from nonsmoking, post-mortem individuals by enzymatic digestion (sourced from Lonza, Basel, Switzerland; but can be obtained from other companies or as direct tissue donations).

NHBE cells were isolated in BEGM ^® (Bronchial Epithelial Growth Medium: 10ml BEGM: 10ml BEBM, 40μΙ Bovine Pituitary Extract and 10μΙ of insulin, hydrocortisone, transferrin, triodothyronine, epinephrine, epidermal growth factor, retinoic acid and Gentamicin/Amphoteracin) and received as a cryovial (>500,000 cells). BEGM ^® was prepared with Bronchial Epithelium Basal Medium (BEBM) and BEGM ^® SingleQuots ^® . The cryovial of NHBE was sprayed with 70% ethanol, placed in the cell culture hood, dried, partially open the lid to relieve the pressure and re-sealed. The cryovial of cells was thawed (2 minutes, partial submersion in a 37°C water bath) and suspended in 30ml BEGM ^® and then seeded into 2x T75 cell culture flasks (-3000 cells/cm ² ). NHBE cells were incubated at 37°C and 5% CO ₂ . During the first overnight incubation (-16 - 24 hours) the NHBE cells attached to the base of the flask. 24 hours later the BEGM ^® (15ml/T75) was aspirated and replaced with 15ml fresh BEGM ^® medium per T75 following this the BEGM ^® medium was replaced (20ml/T75) every third day until the cells reach 65 - 75% confluency (- 2 - 3 days).

SECOND PASSAGE

Confluent cultures were rinsed twice with 5ml HBSS (37°C). Trypsinisation of cells was achieved by addition of 3ml Trypsin-EDTA solution for 3 - 5 minutes (RT), until 90% of the cells were detached. FCS (10%) in RPMI (30ml) was added to neutralise the trypsinisation and the cell suspension was removed into a 50ml universal centrifuge tube. Cell suspensions were centrifuged (72 x g for 4 minutes) and the supernatant removed. Cell pellets were re-suspended in 2ml BEGM ^® (37°C) and added to the remaining 178ml BEGM ^® (37°C). BEGM ^® -cell suspension (30ml) was placed into each of six T175 cell culture flasks. During an overnight incubation (-18 hours) the NHBE cells attached to the base of the T175 flasks. The BEGM ^® (30ml) was replaced with 30ml fresh BEGM ^® medium. BEGM ^® (30ml) was replaced every third day until the cells reached 70% confluency (- 2 - 3 days).

SEEDING INTO CELL CULTURE INSERTS

Confluent cell cultures were rinsed twice with 5ml HBSS (37°C). Trypsinisation of cells from the collagen layer was achieved by addition of 4ml Trypsin-EDTA solution for 3 - 5 minutes (RT), until 90% of the cells were detached. FCS (10%) in RPMI (30ml) was added to neutralise the trypsinisation and the cell suspension was removed into a 50ml universal centrifuge tube. Cell suspensions were centrifuged (72 x g for 4 minutes) and the supernatant removed. Cell pellets were re-suspended and combined in a 4ml BEGM ^® (37°C). The cell concentration of the BEGM ^® -cell suspension was determined by using a 5x5 grid on a haemocytometer. The required seeding density for imputing into inserts was 0.5 million cells/ml. BEGM ^® -cell suspension was added to the required volume of BEGM ^® for seeding and 150μΙ BEGM ^® -cell suspension was placed into each cell culture insert, with 500μΙ of BEGM ^® placed in each well beneath the insert (24- well cell culture plate). During the overnight incubation (-18 hours) the NHBE cells attached to the insert membrane. NHBE cells were seeded into Millipore ^® inserts, as well as into 6x Transwell ^® inserts, as Transwell ^® inserts allow visualisation. CONFLUENT INSERT CULTURES

Following seeding into inserts, apical media was removed to create an ALI culture (Day 1 ). The basal media (BEGM ^® ) was replaced with 300μΙ of ALI media (20ml ALI media: 10ml BEBM, 10 ml DMEM, 80μΙ Bovine Pituitary Extract and 20μΙ of insulin, hydrocortisone, transferrin, triodothyronine, epinephrine, Gentamicin/Amphoteracin, epidermal growth factor and retinoic acid [Sigma]). Basal ALI media was changed daily, adding 300μΙ of fresh ALI media into each well for the first week, then 6 days a week thereafter. Once the cells began to show mucin secretion (a layer of clear viscous liquid on the apical surface of the cultures; -Day 15), it was carefully removed by aspiration every second day to prevent mucin accumulation (as there was no muco-ciliary escalator to remove the mucin). INITIAL CULTURE OF HEPATOCYTES

Hepatocytes (Invitrogen, HMCPMS; 5 - 8 million cells per vial) were received as a cryovial, this was sprayed with 70% ethanol, placed in the cell culture hood, wiped dry, the lid was partially opened to relieve the pressure and then re-sealed. The cryovial of cells were thawed (2 minutes, submersion in a 37°C water bath) and then suspended in 50ml Cryo-preserved Hepatocyte Recovery Media (CHRM; 37°C) then centrifuged (100 x g for 10 minutes). The supernatant was carefully aspirated and cell pellet was re-suspended in 3ml Cryo-preserved Hepatocyte Plating Media (CHPM; on ice). Cell viability was determined with Trypan Blue and 500μΙ of 0.6 million cells/ml were seeded into collagen-coated 24-well plates and incubated for at least 6 hours. CHPM will then be removed and replaced with 500μΙ Hepatocyte media (Williams E media and cell maintenance supplements; 37°) and cultured for at least 6 hours before experiments can begin.

CO-CULTURE OF NHBE MODEL AND HEPATOCYTES

NHBE cells can be used in testing between Days 24 - 33 in ALI culture. Prior to adding test compound the Hepatocyte media is removed from the well and replaced with 300μΙ of ALI media. To co-culture simply remove the NHBE cell culture insert from the 24-well plate and place in the collagen-coated 24-well plate containing the Hepatocytes with ALI media. In the co-culture model test compounds (50μΙ) are added to the apical surface of the NHBE cells. The controls will include: 1 ) addition of the compound to the apical surface of NHBE cells cultured alone; 2) addition of the compound to the ALI media where Hepatocytes are cultured alone; 3) as well as exposure to carrier solutions in both co- and mono-cultures.

TABLE 1

N H BE & HEPATOCYTE CO-CULTURE METHOD

MATERIALS & SOLUTIONS SUPPLIER

NHBE Cells, Cryopreserved in Bronchial Epithelial

Growth Medium ^® (BEGM ^® ),

Lonza Group Ltd., with Retinoic Acid

Switzerland

Bronchial Epithelial Basal Medium (BEBM)

(Formerly Cambrex BEGM ^® SingleQuots ^®

Bioscience, Workingham,

Dulbecco's Modified Eagle's Medium (DMEM), with

Berkshire, UK)

4.5g/L Glucose, L-Glutamine, without Pyruvate

Penicillin-Steptomycin-Amphoteracin B

T75 and T175 Cell Culture Flasks Fisher Scientific, UK

Hank's Balanced Salt Solution (HBSS)

Trypsin-EDTA

RPMI-1640

Human Hepatocytes: HMCPMS, 5 - 8 million cells

Cryo-preserved Hepatocyte Recovery Media (CHRM) Invitrogen Ltd., Paisley, Cryo-preserved Hepatocyte Plating Media (CHPM) Scotland

Trypan Blue 0.4%

Collagen-coated 24-well plate

Williams E Media

Cell Maintenance Supplement Pack

JRH Bioscience, Kansas,

Foetal Calf Serum (FCS)

USA

Millicell Hanging Cell Culture Insert, Polyethylene

Millipore Ltd., Watford, UK Terephthalate; 6.5mm diameter, 0.4μηη pore size

24-Well Cell Culture Plate Grenier Bio-one Ltd., UK

Retinoic Acid Sigma, Dorset, UK