WO2016181114A1

WO2016181114A1 - Personalised media

Info

Publication number: WO2016181114A1
Application number: PCT/GB2016/051301
Authority: WO
Inventors: Roderick BENSON; Gareth Griffiths; Joanna DUTKOG-WOZDZ
Original assignee: Imagen Therapeutics Limited
Priority date: 2015-05-08
Filing date: 2016-05-06
Publication date: 2016-11-17
Also published as: GB2543374B; GB201507894D0; GB2543374A; GB201607940D0

Abstract

This present invention relates to a method of culturing cells, wherein the cell culture media comprises a body fluid preferably derived from the same species as the cells being cultured. The body fluid may be derived from the same subject as the cells being cultured, thereby providing a reflection of the in vivo cellular environment. The invention also relates to a media for use in culturing cells in vitro. The cells may be cancer cells.

Description

PERSONALISED MEDIA

This invention lies in the field of personalised medicine, and more specifically relates to a method for culturing cells, for example hyperproliferative (e.g. cancer) cells, in vitro to identify an optimal treatment. The present invention also relates to a media for use in culturing cancer cells in vitro.

BACKGROUND

Personalised medicine is useful in selecting appropriate and optimal therapies for an individual and has potential particularly in selected suitable cancer therapies. Previously drugs have been supplied in the belief that the same drug will be effective in everyone.

However, in recent times it has been appreciated that subjects respond differently to different therapies, and by tailoring therapy, treatment can be more effective and costs can be saved. In many cases, personalised medicine is based upon genomic analysis of the individual to predict responsiveness. Another approach is to test a biopsy from an individual for responsiveness to a variety of possible treatments. The degree of cell death in the biopsy indicates effectiveness of a particular treatment.

For example, for cancer therapy, cancer cells m a y b e obtained from a subject having a cancerous tumor, cultured in an appropriate medium and exposed to a candidate therapeutic agent. The effect of the therapeutic agent on the survival and proliferation of the cancer cells is determined in order to assess the effectiveness of the therapeutic agent on a subject. Using such a method, a treatment can be selected for the subject based at least in part on a prediction as to whether a particular therapeutic agent will be effective for the subject.

WO2013/069010 describes a method for optimizing cancer treatment by testing the anti- tumorigenic activity of a pharmaceutical compound on the growth of primary cancer cells derived from ascites fluid.

WO2014197543 also describes an assay for determining the effectiveness of a proposed cancer treatment, by measuring mRNA and protein expression in cancer cells on exposure to a candidate therapeutic compound, and comparing the expression to a control. The culture medium in which the cells are grown may include serum. EP2504698 describes a method of deriving tumour cells for a personalised medicine assay in relation to a solid epithelial tumour, where tumour cells are obtained from a body fluid such as ascites fluid, serum or lymph. The present invention aims to provide an improved personalised medicine assay. BRIEF SUMMARY OF THE DISCLOSURE

In a first aspect of the present invention, there is provided a method of culturing a mammalian cell, wherein the method comprises maintaining the cell in a culture medium comprising i) a pH-buffered isotonic salt solution; and ii) body fluid derived from a postpartum mammalian subject of the same species as the mammalian cell.

The first aspect also provides a method of culturing a sample of cancer cells of a subject, wherein the method comprises maintaining the cancer cells in a culture medium comprising i) a pH-buffered isotonic salt solution; and ii) autologous body fluid. Preferably, the body fluid is blood serum, ascites fluid or cerebrospinal fluid. More preferably, the body fluid is blood serum.

In a second aspect of the present invention, there is provided a cell culture medium for use in culturing a sample of cells in vitro, wherein the culture medium comprises a basal medium, B27, insulin, transferrin, hydrocortisone, BSA, EGF and FGF. Where the cells are derived from a subject, the culture medium may also comprise a body fluid. The body fluid may be autologous, such that it is derived from the same subject as the sample of cells, or it may be non-autologous but derived from a subject of the same species as the subject from which the sample of cells is derived. The body fluid may be ascites fluid, blood serum or cerebrospinal fluid. The basal medium may be any suitable medium, for example DMEM, preferably DMEM/F12. More preferably, the body fluid is blood serum.

In a third aspect of the present invention, there is provided a cell culture medium supplement for use in culturing a sample of cells of a subject in vitro, wherein the supplement comprises insulin, transferrin, hydrocortisone, BSA, EGF and FGF. The supplement may optionally also comprise B27. The supplement may also comprise a body fluid. The body fluid may be autologous, such that it is derived from the same subject as the sample of cells, or it may be non-autologous but derived from a subject of the same species as the subject from which the sample of cells is derived. The body fluid may be ascites fluid, blood serum or cerebrospinal fluid. In an embodiment, the body fluid is blood serum. A further aspect of the invention relates to methods of preparing a cell culture media culturing a sample of cells in vitro, wherein the method comprises combining a basal medium, B27, insulin, transferrin, hydrocortisone, BSA, EGF and FGF and optionally a body fluid. The body fluid may be autologous, such that it is derived from the same subject as the sample of cells, or it may be non-autologous but derived from a subject of the same species as the subject from which the sample of cells is derived. Preferably, the body fluid may be ascites fluid, blood serum or cerebrospinal fluid. More preferably, the body fluid is blood serum.

BRIEF DESCRIPTION OF THE DRAWINGS

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

Figure 1 is a photograph of breast cancer cells grown in DM EM with 20% patient ascites fluid;

Figure 2 is a photograph of colon cancer cells cultured in DMEM and 20% patient ascites. This sample is very unusual as colon cancer very rarely produces ascites fluid.

Figure 3 is a photograph of ovarian cancer cells cultured in B27 with additives, focused on overlying spheroids.

Figure 4 shows ovarian cancer cells cultured in media with B27, and insulin, transferrin, hydrocortisone, BSA, EGF and FGF on a haemocytometer after trypsinization. Cells have been stained with Trypan blue to detect dead necrotic cells. Most cells phase bright demonstrating intact healthy cell membranes.

Figure 5 shows an ovarian cancer sample growing in the medium of the present invention media but in 3D spheroid mode.

Figure 6 shows phase bright breast cancer cells derived from primary patient tissue sample in B27 modified media. Trypan blue negativity indicates the cell membranes are intact and the cells are viable.

Figure 7 shows a dosing design of a 384 microtitre plate (out of a total of two plates) which shows the layout for 28 therapeutic agents tested and the relevant plate controls.

Figure 8 shows the comparison of culturing MCF7 cells for 5 days in DMEM-F12 containing either 10 % FBS (row A) or 10 % human serum (row B) or 20% human serum (row C).

Figure 9 shows the comparison of culturing Hela cells for 5 days in DMEM-F12 containing either 10 % FBS (row A) or 10 % human serum (row B) or 20% human serum (row C). Figure 10 shows Human ovarian cancer cells grown in 10% human serum for 72 hours. Figure 11 shows cell death in MCF7 cells at 5 days as determined by automated fluorescence microscopy (High Content Analysis).

Figure 12 shows the cell death in Hela cells at 72 hours and 5 days (panel A) or at 48 hours (panel B) as determined by automated fluorescence microscopy (High Content Analysis see figure 5 for details).

Figure 13 shows the cell count for MCF7 (left panel) and Hela cells (right panel) after 72 hours (Hela) and 5 days (both cell lines) in culture.

Figure 14 shows the plate layout design used to prove the principle of growing cancer cell lines and patient cancer cells in patient-derived serum.

DETAILED DESCRIPTION

Personalised medicine has developed from recognition of the great biological diversity which exists between different cancers. The present inventors have recognized that cells can be maintained in vitro in cell culture medium comprising body fluid from a mammal. The present inventors recognize the value of replicating the in vivo environmental signature of the cells to improve personalization in screening for therapeutic compounds. The provision of body fluid in a culture medium allows the cells to grow in an environment which more closely resembles the in vivo environment of the cells.

Traditionally, most in vitro cell work has been conducted in pH-buffered isotonic saline solutions which contain 10% Fetal Bovine Serum (FBS). The present invention is partly based upon the realization that using FBS as a growth factor additive will most likely result in a set of growth signals which are different from those which the cancer cells are exposed to in vivo. This can result in the cells responding differently to a particular therapeutic agent in a drug screening assay than how they may respond in a subject, therefore leading either to assay false negatives or positives.

The terms cancer, tumor and hyperproliferative disorders are used herein interchangeably. By "cancer", "tumor" and "hyperproliferative disorders" are meant conditions characterized by abnormal proliferation of cells, particularly hyperproliferation. Also included are hyperproliferative disorders such as psoriasis and hyperplasia.

Thus, the present invention provides a method of culturing a mammalian cell, wherein the method comprises maintaining the cell in a culture medium comprising i) a pH-buffered isotonic salt solution; and ii) body fluid derived from a post-partum mammalian subject of the same species as the mammalian cell. In an embodiment, the present invention provides a method of culturing a mammalian cell, wherein the method comprises maintaining the cell in a culture medium comprising i) a pH- buffered isotonic salt solution; and ii) autologous body fluid derived post-partum from the same subject as the cell. In an embodiment, the cell is a cancer cell. In an embodiment, the cell is provided in a sample derived from a subject, for example tissue or fluid, such as ovarian tissue or ascites fluid. Other fluids and tissues are described herein. In an embodiment, the cell may be human or canine. In an embodiment, the culture medium produced from a combination of i) and ii) does not comprise fetal bovine serum.

In an embodiment, the present invention provides a method of culturing a sample of cancer cells of a subject, wherein the method comprises maintaining the cancer cells in a culture medium comprising i) a pH-buffered isotonic salt solution; and ii) autologous body fluid. Preferably, the body fluid is blood serum, ascites fluid or cerebrospinal fluid. More preferably, the body fluid is blood serum. In an embodiment, the culture medium does not comprise fetal bovine serum.

Thus, in an embodiment there is provided a method of culturing a sample of mammalian cancer cells derived from a subject, wherein the method comprises maintaining the cells in a culture medium comprising i) a pH-buffered isotonic salt solution; and ii) autologous blood serum derived post-partum from the mammalian subject.

Thus, in an embodiment there is provided a method of culturing a sample of mammalian ovarian cancer cells derived from ascites fluid of a subject, wherein the method comprises maintaining the cells in a culture medium comprising i) a pH-buffered isotonic salt solution; and ii) autologous blood serum derived post-partum from the mammalian subject.

By treatment of such a disorder is meant the application or administration of a therapeutic agent to a subject, for the purpose of curing, ameliorating or alleviating a condition, for example a hyperproliferative condition. This may include any suitable therapy. For a hyperproliferative condition, this may include i) inhibiting or slowing the proliferation or growth of hyperproliferative (e.g. cancer or tumour) cells or ii) killing hyperproliferative cells. The desired effect of a therapeutic agent is to inhibit growth of a population of hyperproliferative cells (i.e. a cancer or tumour); decrease the size of a population of hyperproliferative cells; eliminate a population of hyperproliferative cells; and/or inhibit or prevent migration of such cells to other parts of the subjects' body. Treatment is not necessarily curative, and may or may not comprise complete obliteration of such a cell population in a subject. An effective treatment is one which has one or more of the above mentioned effects on a population of hyperproliferative cells.

The terms "subject", "patient" and individual are used interchangeably herein, and may include any animal, e.g., mammals such as human, bovine, canine, ovine, feline, non- human primate, porcine, etc. The su bj ect m ay be can i ne . The su bject m ay be a h u m an . The su bj ect may be fem ale . Th e s ubj ect m ay be a female human having an ovarian cancer tumor. A subject may be an infant, a child or an adult human, or an infant, young or adult animal.

A subject may be one which has been previously diagnosed as having a hyperproliferative disorder. The subject may or may not have received treatment for the disorder.

By cell is meant one or more cells, and includes without limitation a singular cell, a population of cells, a cell line, and a body sample comprising a cell. A cell line is a culture developed in vitro from a single cell, and consists of cells having a uniform genetic make up. A population of cells may be uniform or non-uniform in terms of cell types and genotype of the cells. A cell derived from a subject, preferably provided as a sample for use in the invention, may be not have undergone any significant number (2, 3, 4, 5, or more) in vitro passages prior to use in a method of the invention. Reference to a cell derived from a subject does not include a cell line. A cell may be a normal cell or a hyperproliferative cell, which may also be referred to as a cancer cell. Hyperproliferative cells may be primary or secondary cancer cells. A "sample" obtained from a subject is any fluid or tissue comprising a cell. By derived or obtained means taken from or removed from a subject, using any suitable means. A sample may include a fluid or tissue comprising a hyperproliferative cell. To avoid confusion with the body fluid provided with the isotonic salt solution for cell culture, the body fluid from which cells are derived is referred to herein as the sample or body fluid sample. This may be the same or different to the body fluid used for the culture medium.

The sample may be obtained from any body tissue or fluid, and may contain hyperproliferative cells for culture. A sample may be obtained from stomach, colon, prostate, pancreas, heart, liver, colon, gastrointestinal, endocrine organ, muscle, bone, neuroendocrine lung, brain, blood, breast, ovary, fallopian tube, testicles, skin, or any other tissue or cells including immune cells, neural, endothelial, fibroblasts, or other epithelial and stromal tumors. Samples obtained from tissue may include solid tumours or cells derived from a bodily fluid including but not limited to abdominal ascites. A suitable sample from use in the present invention is ovarian cancer cells derived from ascites fluid that has been drained from the patient for the purposes of symptomatic relief. Samples obtained from ascites fluid may also contain cancer cells that have been derived from the stomach, colon, pancreas, liver, or lymphatic tumour cells.

A body fluid for use in the culture medium may be selected from serum, plasma, cerebrospinal or ascites fluid. In an embodiment, ascites fluid is used, preferably derived from the abdomen. The body fluid may be derived from a post-partum mammalian subject. Post-partum includes body fluid of infants, children and adults but excludes fetal body fluid (for example fetal bovine serum). The body fluid used in the cell culture medium as described herein may be the same as, or independent to, any body fluid in the sample (residual or otherwise). In an embodiment, the body fluid in which the cells are cultured is autologous, meaning that it is derived from the same subject as the cell being cultured. The autologous body fluid may be from the same part of the body as the cell being cultured, or from a different part. The autologous body fluid does not necessarily have to be from the same part of the body as the cell being cultured. Therefore the sample and autologous body fluid may be obtained from different parts of the same subject. In an embodiment, the cells are hyperproliferative cells from a body tissue or fluid where a hyperproliferative disorder is present or in close proximity, and the body fluid is serum. For example, the cell may be ovarian cells derived from ascites fluid and the body fluid used for cell culture may be blood serum, both obtained from the same subject.

However, it is also envisaged that non-autologous body fluid may be used in the cell culture medium. In such an embodiment, it is suitable that the non-autologous body fluid is from the same species as the cell being cultured. Therefore, for example, a human body fluid from a first subject may be used in media to culture a cell from a second human subject. Similarly, a canine body fluid from a first canine subject may be used in media to culture a cell from a second canine subject, and so on. Using body fluid from the same species in cell culture media enables the cells to be cultured in an environment with a closer growth factor signature to the original in vivo environment from which they were derived. Any suitable method may be used to obtain a sample or body fluid from a subject. Typical methods include performing a biopsy or needle aspiration. Patient blood may be obtained using standard phlebotomy techniques. The combination of a pH-buffered isotonic salt solution and a body fluid provides a cell culture media. The resulting cell culture media suitably comprises between 1.0% and 100% v/v of body fluid, more preferably 1.0% to 40%, more preferably 5-30%v/v, more preferably 5-20%. Preferably, where the body fluid is ascites fluid, it is provided at 10- 20%, preferably at a concentration of 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20% v/v with the isotonic media solution. Where the body fluid is serum it may suitably be provided at between 5-20%, more suitably 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25%.

A body fluid may be processed, for example centrifuged e.g. at 5000 rpm to remove any blood or cancer cells, prior to addition to the media.

A therapeutic agent which may be tested in a method of the invention may be any molecule, chemical entity, composition, drug, therapeutic agent, chemotherapeutic agent, or biological agent capable of treating a hyperproliferative condition as described above. Also included are potential therapeutic candidates, which may have the potential for treatment as described above. The term includes small molecule compounds, antisense reagents, siRNA reagents, antibodies, enzymes, peptides organic or inorganic molecules, cells, natural or synthetic compounds and the like. The term "therapeutic agent" may include a single agent or a combination of two or more agents. A therapeutic agent may be a known therapeutic agent or a candidate agent.

The isotonic media solution and body fluid in combination form a cell culture media, in which the cells obtained from the subject are maintained. The isotonic media solution may be a nutritive solution which supports the growth and proliferation of the cells in vitro, nd may be referred to herein as a cell culture medium or a basal medium. These terms include the plural, media. The isotonic media solution may be a buffered or balanced salt solution. It may be any media available to the person skilled in the art. Preferred media include basal media such as DMEM Hanks and RPMI, optionally supplemented with one or more nutrients. Preferably, the media does not comprise animal serum other than the autologous body fluid derived from the same subject as the cell sample. Thus, the media may not comprise FBS (fetal bovine serum). In the present invention, the culture media may comprise a basal media, a body fluid and insulin, transferrin, hydrocortisone, BSA, EGF and FGF. The basal media may be any suitable basal media, for example DMEM and/or RPMI. Preferably, the culture media is as described herein.

By "cell culture" or "culture" is meant the maintenance of cells in vitro. It i ncl u des the s u p port of individual cells, as well as cell populations, for example in the form of tissues or tissue samples. It also includes the culture of 3D cell spheroids derived either by using low adherence cell microtitre plates or microtitre plates that are shaped to encourage cancer cell aggregation. In a method of the invention, the sample and body fluid may be obtained separately, or in combination. Where the cell sample is derived from a body fluid e.g. ascites fluid, then a single sample may be obtained to provide the cells for culture and the autologous body fluid for addition to the culture media. Alternatively, the cells may be extracted from the body fluid and a different sample of body fluid may be obtained for the cell culture. Where the cells are obtained from a tissue, or from a solid tumour for example, then typically the body fluid will be obtained in a separate procedure to obtaining the cell sample.

Preferably, where a sample of cells are derived from a solid tumour, the body fluid is preferably patient blood serum, preferably derived from removing 100 ml of patient blood. Where the sample of cells are derived from ascites fluid, the body fluid is preferably ascites fluid from which the cancer is derived at a preferred media concentration of 20% v/v. Preferably, where the cancer is a glioblastoma, the autologous body fluid is cerebrospinal fluid. A method of the invention may comprise the step of obtaining a sample of cells from the subject, for example using a method as described herein. A method of the invention may also comprise the step of obtaining an autologous body fluid from the same subject as the sample of cells. These steps may be the same step or different steps i.e. temporally or physically separated.

Thus a method of invention may comprise:

i) adding body fluid to the sample;

ii) maintaining the sample in culture comprising the body fluid. The body fluid may be autologous. The body fluid may be combined with a cell culture medium prior to application to the sample. Thus, a method of the invention may comprise:

i) combining a body fluid with a cell culture medium or a component thereof;

ii) resuspending the sample in the combination prepared in i); and

iii) maintaining the sample in culture.

Alternatively, a method of the invention may comprise:

i) combining a body fluid comprising a sample from a subject with a cell culture media or a component thereof;

ii) maintaining the sample in culture.

The cell culture medium with which the body fluid is combined may be a basal medium, for example as described herein, and may optionally comprise one or more additives for example as described herein. In an embodiment, the cell culture medium may be a basal medium and one or more of B27, insulin, transferrin, hydrocortisone, BSA, EGF and FGF. Alternatively, the body fluid may be combined with any one or more of insulin, transferrin, hydrocortisone, BSA, EGF and FGF to produce a combination which may be added to a basal medium.

A method of the invention may further comprise the step of refreshing the medium at intervals during the culture process.

The sample may be maintained in culture for 1 , 3, 5, 12, 24, 48 or 72 hours or more. Preferably, the effect of a therapeutic agent on cell survival and growth is determined after at least 24, 48 or 72 hours of continuous therapeutic agent exposure, preferably at 96 hours after initial exposure. Initial exposure may comprise seeding into 384 well microtitre plates. In other examples, for example when cells are cultured in 3D configuration, the treatment period can be extended, for example up to a maximum of 14 days continuous treatment with the therapeutic agent, preferably at least 7, 8, 9, 10, 1 1 , 12 or 13 days continuous treatment.

Cell culture conditions will be known to a person skilled in the art, and preferably are those which assist cell survival. Typical conditions will be a humidified incubator, temperature 37°C and a carbon dioxide concentration of about 5%. A method of the invention may further comprise the step of isolating the cells from the sample, for example to avoid contamination with other (e.g. non-cancer) cells which may be present. This may be particularly relevant where a fluid sample is used as a source of the desired cells. Techniques for isolating the cells from a fluid sample are known to those skilled in the art and include for example centrifugation of sample through appropriate Percoll gradients and growing sample in defined selection media which may include the addition of appropriate supplements, e.g. B27. Typically, after 24 to 48 hours, the cells are removed from culture. Single cell suspensions comprising the desired cell concentrations may be selected for the assay. A preferred concentration may be in the range of 50-200 cells per μΙ, preferably 70-150 cells per μΙ; more preferably between 80 and 120 cells per μΙ, more preferably 90 to 110 cells per μΙ. A preferred aliquot size will depend upon the size of the microwells, but may be approximately 30 μΙ, therefore comprising approximately 3000 cells.

The method of the invention may further comprise the step of assaying the effect of a therapeutic compound on the growth and/or survival of the cell sample in culture. Thus, the present invention may comprise a method for assessing the efficacy and/or potency of a therapeutic compound as an anti-hyperproliferative therapeutic for a subject, wherein the method comprises:

a) culturing a sample of cells of a subject in vitro as described herein; and

b) contacting cell culture with a therapeutic agent;

c) assaying the effect of the therapeutic agent on the growth and/or survival of the cells; d) determining the potency and/or efficacy of the therapeutic agent being tested.

Preferably, the culture medium is as described herein, A therapeutic agent may be added to the cell culture, and the growth, survival and proliferation of the cells determined at one or more time intervals.

A therapeutic agent may be applied to a sample of cells at one or more different doses.

A method of the invention may comprise producing a dose-response curve for a therapeutic agent.

The relevance of the potency indicated by the assay of the present invention may be calibrated using historical sample testing data. This includes comparing a dose response curve derived from an assay of the present invention to a clinical response, such that the parameters of an in vitro non-responder and an in vitro responder can be determined from the clinical outcome data. Determining the effect of a therapeutic agent may be performed using tests for cell growth, survival and/or proliferation as described herein and may optionally comprise comparing the result to a predetermined threshold value which is indicative of therapeutic efficacy. The effect may alternatively or additionally be compared to the effect of a control compound on the growth and/or survival of the cells; and/or compared to the effect of one or more other therapeutic compounds. A method of the invention may further comprise the step of selecting the most effective compound for treatment.

Many suitable methods of assessing the effect of a therapeutic agent on the growth and/or survival of a cell population will be known and available to a person skilled in the art. Examples of such methods include proliferation assay (for example using a XTT cell proliferation kit (Biological industries); apoptosis assays (including for example staining dead cells followed by flow cytometry to determine number of dead cells); in vivo- in-ova drug response assay; and migration and invasion assay, cell number counts, and analysing cell cycle profiles. A preferred method comprises measuring an early activation marker of apoptosis (BAK) (Griffiths, G.J. et al. Cell damage-induced conformational changes of the pro-apoptotic protein Bak in vivo precede the onset of apoptosis. J. Cell Biol. 144, 903-914 (1999)) and the activation of caspase 3. Specifically, monoclonal antibodies to the N-terminus of Bak are used to detect revealing of a concealed epitope at the N terminus, by detecting immunofluorescence following binding of the monoclonal antibody to the revealed epitope. An increase in immunofluorescence indicated, shown using standard techniques such as flow cytometry, is a marker of the onset of cell death. Preferred monoclonal antibodies are monoclonal antibody designated as Ab-1 and raised against the peptide sequence amino-acids 1-52 (AM03; Calbiochem- Novabiochem Ltd.), or Ab-2 (AM04; Calbiochem-Novabiochem Ltd.) also made to the same peptide. Another preferred method is a live cell assay, in particular comprising using a nuclear dye which can only cross cell membranes that have lost integrity. Another suitable method is to measure the number of population doublings, as a measure of cell proliferation during continuous culture.

The method of assaying the effect of a therapeutic compound on the growth and/or survival of a cell in culture may be repeated for different therapeutic compounds, for example until an effective compound is identified. Alternatively, a plurality of therapeutic compounds may be tested simultaneously on different aliquots of the culture, for example in a multi-well plate or high throughput assay. Automated procedures may be used, for example a Hamilton Star liquid handling dosing robot may be used to place sample aliquots into a multi-well plate, such as a 384 microwell plafe. Some or all of the wells in a muitiwel! plate may be dosed with sample. Preferably, the remaining wells may comprise a fluid to protect against fluid evaporation, for example phosphate buffered saline.

Thus, the method may comprise the step of splitting a cell culture into multiple aliquots; contacting each simultaneously with a different therapeutic agent or combination of agents. The method may also comprise the step of comparing the effect of the different therapeutic agents to identify the most effective.

Thus, in a preferred embodiment of the present invention, there is provided a method of assaying the effect of a therapeutic compound on the growth and/or survival of a ceil in culture, the method comprising:

a) culturing a mammalian cell in vitro as described herein; and

b) splitting a cell culture into multiple aliquots;

b) contacting each simultaneously with a different therapeutic agent or combination of agents

c) assaying the effect of the therapeutic agent on the growth and/or survival of the cells; d) determining the effect of the therapeutic agent;

e) comparing the effect of the different therapeutic agents to identify the most effective. An effective therapeutic agent is one which the tumour cells are sensitive to, i.e. in response to which they exhibit a reduction in growth, survival or proliferation. A low effect of a sample to a therapeutic agent may be correlated with a predicted poor response of the subject to the therapeutic agent, and a high effect may be correlated with a good response.

In addition, a method of the invention may be used to simultaneously analyse s a m p l e s from multiple subjects (e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 50, 100, 1000, 10,000, or more subjects) for example in a high throughput format. The invention may further comprise producing a workbook comprising a dose response curve for each therapeutic agent tested in the assay. Preferably, each dose response curve is a 3, 4, or 5 point dose response curve. The invention may further comprise the step of ranking the therapeutic compounds in terms of potency and/or efficacy to induce cell death, and/or to suppress cell growth and/or proliferation.

The present invention may further comprise providing a shortlist of therapeutic agents to a patient.

A method of the invention may comprise comparing the growth, survival and/or proliferation of a cell culture exposed to a therapeutic agent with the growth, survival and/or proliferation of a control cell culture. A control cell may be a cell culture of a sample of a subject as described herein, which has not been exposed to a therapeutic agent. Alternatively, a control sample may comprise normal cells isolated from the same subject and preferably same tissue, or may be an established cell line, for example from a subject with a known response. Based on a comparison of the growth, survival and/or proliferation with a control sample, effectiveness of a therapeutic agent can be assessed.

A therapeutic window for treatment of a particular condition e.g. cancer may be determined by directly comparing the response of the patient-derived cells to a particular therapeutic agent to the response of normal blood cells derived from the same patient to the same agent.

A method of the invention may also comprise a step of detecting the therapeutic agent in a sample. For example, where a therapeutic agent is an antibody, fluorescence may be used to determine presence of the antibody. As with the other methods described herein, the method can further include treating the subject with one or more therapeutic agents identified using the method of the invention, optionally in combination with one or more other therapies for example chemotherapy, radiation therapy, surgery, etc. After the subject is treated with a therapeutic agent, at one or more (e.g., one, two, three, four, etc.) time points, the subject or a sample from the subject (e.g., a biopsy, culture) can be analyzed to determine the subject's response to the therapeutic agent, for example using a method as described herein or any other suitable method, for example measuring tumour size or assaying for tumour markers. The present invention also relates to a cell culture medium, comprising a basal medium, and B27, insulin, transferrin, hydrocortisone, BSA, EGF and FGF. The cell culture medium may also comprise one or more additional ingredients, for example estrogen. The term "ingredient" refers to any compound, whether of chemical or biological origin, that can be used in cell culture media to maintain or promote the growth, proliferation or survival of a cell. The terms "component," "nutrient" and ingredient" can be used interchangeably and are all meant to refer to such compounds. The ingredients of the cell culture medium may be aqueous-based, comprising a number of ingredients in water, liquid, and/or an aqueous solution. One or more ingredients may be dried or frozen, and reconstituted.

Typical ingredients include amino acids, salts, metals, sugars, lipids, nucleic acids, hormones, vitamins, fatty acids, a n d proteins etc. Thus, the media of the invention may additionally comprise one or more antioxidants; nucleotide synthesis and salvage pathway precursors; lipid synthesis precursors; agonists of intracellular cAMP level; hormones, growth factors, amino acid supplements, vitamins, trace minerals, inorganic salts, energy sources (e.g. for glycolysis), antibiotics and pH indicators. Particular ingredients may include amino acids, vitamins, inorganic salts, adenine, D-glucose, N-[2- hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES), hydrocortisone, insulin, lipoic acid, phenol red, phosphoethanolamine, putrescine, sodium pyruvate, triiodothyronine (T3), and thymidine. Each of these ingredients may be obtained commercially, for example from Sigma (Saint Louis, MO). Other ingredients that promote or maintain cell growth, survival and/or proliferation may be included, and will be known to persons skilled in the art. Some or all of the above ingredients may be provi ded i n a basal medium or media supplement.

A basal medium as used herein will be any basic nutrient media for cell culture, which can be supplemented as described herein. Basal media are known and available to persons skilled in the art, and include for example MEM Eagle, H (1959) Science 130: 432), Ham's F12, DMEM or RPMI 1640 (Moore et al (1967) J.A.M.A 199:519). Preferred basal media for use in the present invention is DMEM, preferably DMEM/F12. The basal media may be 90% up to 99.5% of the volume of the complete media.

To this basal medium, B27, insulin, transferrin, hydrocortisone, BSA, EGF and FGF may be added to formulate the complete culture media of the present invention. One or more of these supplements may be added to the basal medium, either directly or pre-mixed with one or more of the other supplements. One or more additional ingredients may be added, for example estrogen. Typically, the medium ingredients can be added in any order.

B27 is a cell culture supplement available from Life Technologies. Preferably, the supplement lacks vitamin A. Other similar supplements are available in the art, and may be used. Insulin may include insulin or an insulinomimietics for example somatomedins or insulin like GF such as IGF1 or IGF2 or vandate. Insulin may be provided in the final media at a concentration of 1-20, more preferably 5 to 15, more preferably 8 to 12, or 10 mg/ml. In 100ml of media (excluding the autologous body fluid) there will preferably be between 50- 150, more preferably 70-120, more preferably 90-110, more preferably about 100μΙ insulin.

Transferrin may be provided to provide protection against heavy metals. Suitable alternatives may be known to persons skilled in the art. Transferrin may be provided in the final media at a concentration of 1-10, more preferably 2 to 8, more preferably 3 to 7, or 5mg/ml. In 100ml of media (excluding the autologous body fluid) there will preferably be between 100-250, more preferably 130-220, more preferably 100-190, more preferably 140-160, more preferably about 150μΙ transferrin.

Hydrocortisone or similar glucocorticoids which act on metabolism of glucides may be provided. Alternatives include for example dexamethasone, prednisolone or triamcinolone. Hydrocortisone may be provided in the final media at a concentration of 1- 100, more preferably 20 to 80, more preferably 30 to 70, or 50mg/ml. In 100ml of media (excluding the autologous body fluid) there will preferably be between 50-150, more preferably 70-120, more preferably 90-110, more preferably about 100μΙ hydrocortisone.

BSA may be provided at a concentration of 5-10%, more preferably 7.5% solution. In 100ml of media (excluding the autologous body fluid) there will preferably be between 30- 100ul, more preferably 50-80, more preferably about 70μΙ BSA. EGF may be provided to allow proliferation and differentiation. It may be provided in the final media at a concentration of 1-100, more preferably 20 to 80, more preferably 30 to 70, or 50mg/ml. In 100ml of media (excluding the autologous body fluid), there may be provided 5-30, more preferably 10-25, more preferably about 20μΙ EGF.

FGF may be provided to allow proliferation and differentiation. It may be provided in the final media at a concentration of 1-100, more preferably 20 to 80, more preferably 30 to 70, or 50mg/ml. In 100ml of media (excluding the autologous body fluid), there may be provided 5-30, more preferably 10-25, more preferably about 20μΙ EGF2-20, more preferably 5-15, more preferably about 20μΙ FGF. A person skilled in the art will readily be able to determine if any listed component is necessary and/or optimum by eliminating a component or changing the concentration of a component, one at a time, and comparing the effect on cell growth, survival and proliferation to that with the original medium. One or more components may also be substituted by other chemicals of similar properties when necessary. Similarly, a skilled person will be able to determine the optimal level of any given component for a particular cell type, by, for example, testing a range of concentrations for each component. Suitable methods are known and available in the art, for example, Ham. Methods for Preparation of Media, Supplements and Substrata for Serum-Free Animal Culture, Alan R. Liss Inc. New York, pp. 3-21 , 1984) and Waymouth (Waymouth. C Methods for Preparation of Media, Supplements and Substrata for Serum-Free Animal Culture Alan R. Liss, Inc., New York, pp. 23-68, 1984. In this manner, the culture media of the present invention may be optimized for different cell types. Such optimized media are within the scope of the present invention Where any of the ingredients of the present medium can exist in different forms, (e.g., different naturally occurring or non-natu rally occurring forms), it is envisaged that they can be used as substitutes for one another. In addition, an ingredient of the present medium may be substituted by any form having similar activity. The pH of the final medium will preferably in in the region of 7,0-7.6, preferably about 7.1-7,5, and most preferably about 72-7.4 The osmolarity of the medium should also be adjusted to about 275-350 mOsm, preferably about 285-325 mOsm, and most preferably about 280- 310 mOsm. The ingredients or supplements can be provided in liquid or dry form, and in the latter case may be dissolved prior to addition to the basal media or may be added in dry form. The cell culture medium may be prepared as a 1-100x formulation, most preferably as a 1 -50x, which is then diluted appropriately into culture medium to provide a 1 x final formulation in the complete media of the present invention. A "1x formulation", meaning some or all the ingredients are found in at working concentrations. A cell culture medium used for the in vitro culture of cells is a 1x formulation by definition. When a number of ingredients are present, each ingredient in a 1x formulation has a concentration about equal to the concentration of those ingredients in a cell culture medium. A "10 x formulation" may contain a number of additional ingredients at a concentration about 10 times that found in the 1x formulation. It follows that a 25x formulation will contain ingredients at 25 fold that of a 1x formulation and so on. The osmolarity and pH of the media formulation may vary

Where a media ingredient is prepared as a separate concentrated solution an appropriate amount of each concentrate is combined with a diluent to produce a 1 x formulation. An suitable aqueous solution may be used. Typically, the diluent used is deionized water but other solutions including aqueous buffers, aqueous saline solution, or other aqueous solutions may be used according to the invention. The culture medium of the present invention may be sterilized to prevent unwanted contamination. Method for sterilization are known in the art, and include for example filtration through a low protein-binding membrane filter of about 0.1-1.0 pm pore size (available commercially, for example, from Millipore, Bedford, Mass.). Preferred methods avoid heating of the ingredients, which may cause degradation. Alternatively, single ingredients or combinations thereof may be sterilized and stored as sterile solutions, which can then be mixed, preferably under sterile conditions, to produce a medium of the present invention.

Examples

Example 1

Sample preparation: 2 bottles of light yellow solution were obtained as patient sample of ascites fluid. One bottle was divided between 9 flasks (20 ml of ascites +10 ml DMEM/F12 in each) and put into the incubator. The leftover solution from the bottle was put into the fridge. To the second bottle, dispase was added and left in the incubator overnight. Next day the second bottle was diluted in PBS 50:50 and spun down and the cells were put into 3 flasks - 2 with 20% ascites solution and 1 with B27 containing full supplements as described in example 2 below. The tissue type was ovarian, and the samples as described above were tested using the master ovarian 56 drug master plate. At a single 72 hour time point after the cells have been dosed and are scoring the number of dead cells in the control and treated population. The best performing drugs to induce cell death out of all those tested were the proteasome inhibitors bortezomib and carfilzomib. Best licensed therapy was paclitaxel in terms of producing a death response at relatively low doses. However, this drug did not demonstrate very good maximum efficacy

Example 2

Ovarian Ascites sample obtained on the 23/05/2014. Lots of cell clumps in various sizes were observed. After spinning and washing some cells were frozen and the rest as put into 4 flasks (2 with media comprising DMEM/F12, 1 % P/S and B27, EGF, Insulin, Hydrocortisone, and transferrin; and 2 with 10% FBS for comparative purposes). The media of the first two flasks consisted of:

C001880T1AFa Ovarian Ascites Masterplate comparison of two medias

Drug dosed for a 48 hour time period. The sample consisted of spheroids/ clumps.

Figures 1 to 6 show phase contrast microscope images of primary cancer cell cultures are shown indicating successful tissue culturing of patient cancer biopsies and ascites fluid. From these cultures cells are further passaged into a microtitre 384 well plate where they are dosed with 56 different chemotherapies using a Hamilton STAR liquid handling robot.

Figure 7: Dosing design of the first 384 microtitre plate (out of a total of two plates) which shows the layout for the first 28 compounds tested plus the relevant plate controls. The plate map on the left shows each compound being tested using 5 log doses (right plate map) in duplicate (duplicate samples in rows doses in columns). So, for example, the drugs tested in the first section of the first plate (rows B-0 and columns 2-6) are Paclitaxel, docetaxel, doxorubicin), gemcitabine, cisplatin, topotecan and etoposide (in the black and white reproduction of the plate map cisplatin, topotecan and etoposide can be distinguished by darkening tones of grey each drug occupying 10 wells of the 384 well plate). The standard 5 point dose response curve (right plate map) is 1 nM (column 2), 10 nM (column 3), 100 nM (column 4), 1000 nM (column 5) and 10000 nM (column 6). Note the stock solutions of some drugs are not 10 mM but 100 mM or 1 mM; meaning that the dose response curves for these drugs start at 10 nM and reach a top dose of 100 μΜ or start at 0.1 nM and reach a top dose of 1000 nM (seen grey changes moving down the rows of a particular column). The plate controls for these experiments are located in the centre of the plate in columns 12 (positive) and 13 (negative vehicle control) respectively. The two positive controls used are benzothenium chloride (100 μΜ) and stauroporine (100 nM) respectively.

Table 1 above: Demonstration that culturing ovarian cancer in 10% ascites solution before plating in B27 prior to dosing results in an assay that predicts clinical outcome. Each sample was tested against 56 different drugs or combinations. The table above lists the type of cancer the ascites has developed from, if the sample tested was received prior to treatment, during or after treatment. Response indicates if the patient's tumour shrank or showed a decrease in cancer biomarkers. The final column dictates whether the actual response was correctly predicted by the assay. All rows except C002307 indicate which samples were correctly predicted; C002307 indicates a false prediction. In sample C002287 the assay showed correctly there would be no response. A stable cell line derived from this sample after several months growing in FBS showed incorrectly it would respond demonstrating the importance of assaying cancer cells as soon as possible after extracting from the patient in order to minimize patient phenotypic drift.

Example 3 It is recognised by those knowledgeable in the art that cells derived from a cancer patient, when grown in laboratory conditions, do not always behave exactly the same way as when they were in the patient. While several hypotheses have been proposed to explain this discrepancy, the two most obvious causes of in vitro-type artefacts are : 1) cellular phenotypic drift as cells are kept for long periods of time in culture and 2) the in vitro environment provided for the cancer cells poorly matches the in vivo environment, especially in relation to the cancer cells' communication with normal stromal tissue and their exposure to artificial growth media and serum, in particular, foetal bovine serum (FBS) which is normally added to cells when cultured in vitro.

In order to address the second challenge of recapitulating an in vivo environment, which is more consistent with the environment of the cancer when it is in the patient, the present example explores the replacement of FBS with the media of the present invention and employing the step of culturing the patient's cancer cells in their own blood serum. The advantage of this second technique is that the cancer cells are exposed to a growth factor profile which was derived from the patient entirely rather than a growth factor profile that has been derived from an unborn calf.

The following experiments were performed:

1) culturing 2 standard mammalian tissue cell lines (Hela and MCF7) in standard 10% FBS and comparing their growth and cell death signatures directly with growing the same cells in 10% and 20% patient-derived serum; 2) culturing patient-derived tissue in the same patient serum. In all cases it was possible to successfully culture patient cells in patient serum.

Method for culturing Cell Lines and Patient Cells in Patient-Derived Serum: Human blood was received from the Manchester BRC biobank (Manchester Royal Infirmary, Manchester, M13 9WL) in red capped BD (clot activator) tubes (Becton Dickinson New Jersey). The sample was then transferred to 15 or 50 ml falcon centrifuge tubes and centrifuged for at 1000 g for 10 minutes at 10°C. The serum was decanted into a fresh tube and the pellet discarded. In order to disable complement, the serum was heat inactivated by placing the tube in a heated water bath at 56°C for 1 hour. The serum was then spun down a second time at 1000g for 5 minutes and the resultant supernatant was added to DMEM-F12 media (Life Technologies Paisley UK) to make a final working media of either 10 or 20% (serum to media).

The plate design is shown in Figure 14. Cells were analysed after 72 hours (light grey) or 5 days (dark grey, left map) and cultured in 10% FBS (light grey) or patient serum (dark grey) or 20% patient serum (light grey striping, right map) and multiple well replicates were analysed. In a separate experiment these data were compared directly with cells grown in serum free conditions (plate map not shown).

Hela, MCF7 or patient-derived cells were then prepared in standard DMEM-F12 media containing 10% FBS and cells were plated into a 96 well plate (100 μΙ per well). Only the inner 60 wells of the 96 well plate was used to avoid plate edge effects. Once the cells had stuck down, the media in the human serum experimental wells was replaced with DMEM- F12 containing the appropriate concentration of human serum (10 or 20% as described above) or DMEM containing no serum which acted as a positive control. After 72 hours and 5 days, the cells were assessed using a standard cell death assay to determine cell viability and proliferation. Using this methodology, it was possible to ascertain how well patient serum provided a growing medium for cell lines and patient cancer cells compared to the normal standard tissue culture conditions used by those knowledgeable in the art. Figure 8 shows the results of culturing MCF7 cells for 5 days in DMEM-F12 containing either 10 % FBS (row A) or 10 % human serum (row B) or 20% human serum (row C). The cells were dual stained with Hoescht 33342 which enters all cells and stains the nuclei and Draq 7 which is a far red dye that will only stain cell nuclei when the cells have lost membrane integrity which is indicative of cell death (secondary necrosis). Single channel images of Hoechst staining are shown in column A, Draq 7 staining (column B) and the pseudo colour composite image is shown in column C. It is clear from these images that the long term culture of MCF7 cells in human serum is optimal at 10% with 20% resulting in a higher death rate at 5 days.

Figure 9 shows the comparison of culturing Hela cells for 5 days in DMEM-F12 containing either 10 % FBS (row A) or 10 % human serum (row B) or 20% human serum (row C). The cells were dual stained with Hoescht 33342 and Draq 7 (see previous figure for details). Single channel images of Hoechst staining are shown in column A, Draq 7 staining (column B) and the pseudo colour composite image is shown in column C. It is clear from these images that the long term culture of Hela cells is no worse in human serum than it is in FBS with both conditions showing higher rates of cell death at 5 days than MCF7.

Figure 10 shows Human ovarian cancer cells grown in 10% human serum for 72 hours. As in figures 8 and 9 the cells were dual stained with the fluorescent probes Hoechet 33342 which stains all cell nuclei (left image) or Draq 7 which only stains the nuclei of dead cells (centre image). The pseudo colour composite of the left and centre images is shown in the right image. It is clear from these images that it is possible to culture patient cells in patient serum for the required duration necessary to perform an in vitro chemotherapy drug screen.

Figure 11 shows cell death in MCF7 cells at 5 days as determined by automated fluorescence microscopy (High Content Analysis). Briefly the 96 well plates were scanned on an Arrayscan VTI using the compartmental analysis algorithm. The cells were identified by the algorithm using the blue Hoechst stain and overall cell number determined. The number of cells positive for the non-membrane permeable dye Draq 7 was also determined and the percentage of Draq 7 positive to overall cell count was calculated. This percentage is a direct measure of cell death (axis ordinate). The three conditions compared at 5 days were 10% FBS (blue bar) 10% human serum (red bar) and 20% human serum (green bar). Each bar represents the mean±SEM of plate replicates. Figure 12 shows the cell death in Hela cells at 72 hours and 5 days (panel A) or at 48 hours (panel B) as determined by automated fluorescence microscopy (High Content Analysis see figure 5 for details). In panel B the effect of serum starvation (brown bar) is being directly compared to culturing cells in FBS (blue bar). Each bar represents the mean±SEM of plate replicates.

Figure 13 shows the cell count for MCF7 (left panel) and Hela cells (right panel) after 72 hours (Hela) and 5 days (both cell lines) in culture. The cell counts are shown on the ordinate while the time points are shown on the abscissa. Cells were either cultured in 10% FBS (blue bars), 10% human serum (red bars) or 20 % human serum (green bars). Data represents the mean±SEM of plate replicates.

The results show that both cell lines and patient cells can be cultured in human patient serum for up to 5 days. The viability of cells after 5 days was more intrinsic to the cell line rather than the culture medium, with MCF7 showing very low death rates in either patient serum or FBS (Figure 8 and Figure 1 1). While Hela cells showed more death than MCF7 at 5 days (Figure 9 and Figure 12), the death rate in 10% and 20% human serum was still under 20% (16 ± 1 % Mean ± SEM) for 10% and (12 ± 0.4%) for 20% human serum respectively, although both conditions were significantly higher than when cells were grown in 10% FBS (8.5 ± 0.2% Mean ± SEM; p<0.01 One-way ANOVA). These data are significantly under the death rate of Hela cells at 48 hours when serum is removed from the media (43.6 ± 9.2% Mean ± SEM). The patient cancer sample showed good viability at 72 hours (Figure ).

The cell proliferation data is shown in Figure 13. It is clear from these data that for both Hela and MCF7 cells there is a substantial number of viable cells in all culture conditions on the plates demonstrating that it is possible to culture human cells and cell lines in patient-derived blood serum rather than using FBS.

The data demonstrates conclusively that it is possible to culture patient cells in blood serum which is also derived from a human patient (autologous blood serum), preferably the same patient from which the cancer cells were derived. While the cancer cells do not proliferate as quickly in human serum, this result may in fact not be detrimental because it suggests that the traditional wisdom of growing cells in FBS could create an unnaturally rich growth factor environment where cells are inadvertently altered away from their native biology. This could result in an intrinsic growth rate which is less representative of the natural in vivo situation. It is important to consider that FBS has been used for many years to speed up the production of cells in tissue culture. Yet the rate of cell division in lab tissue culture is extremely rapid and equivalent rates in a patient would quickly lead to large tumour growth and death. The fact that in many patients, tumour growth proceeds over months or even sometimes years, rather than days or weeks, suggests that the lower growth rates observed in our experiments may be closer to in vivo biology than the FBS tissue culture models. 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.

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.

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.

The paragraphs following are not claims but represent preferred aspects and embodiments of the invention:

1. A method of culturing a sample of cancer cells of a subject, wherein the method comprises maintaining the cancer cells in a culture medium comprising i) a pH- buffered isotonic balanced salt solution; and ii) autologous body fluid.

2. A method according to paragraph 1 wherein the body fluid is ascites fluid, patient blood serum or cerebrospinal fluid.

3. A method according to paragraph 1 or 2, comprising:

i) adding to a cell sample obtained from a subject, an autologous body fluid; ii) maintaining the sample comprising in a culture medium comprising i) a pH- buffered isotonic balanced salt solution; and ii) the autologous body fluid.

4. A method according to paragraph 1 or 2 comprising:

i) combining an autologous body fluid with a cell culture medium or a component thereof;

ii) resuspending the sample in the combination prepared in i); and

iii) maintaining the sample in culture.

5. A method according to any one of paragraphs 1 to 4 further comprising i) exposing the sample to a candidate therapeutic compound and ii) assaying the effect of a therapeutic compound on the growth and/or survival of the hyperproliferative cell sample in culture.

6. A method according to paragraph 5, wherein step i) and ii) of paragraph 5 are repeated for different candidate therapeutic compounds.

7. A method according to any one of paragraphs 1 to 4 comprising i) splitting a cell culture into multiple aliquots; ii) exposing each aliquot to a candidate therapeutic compound and iii) assaying the effect of a therapeutic compound on the growth and/or survival of the cell sample in culture.

8. A method according to paragraph 7, comprising the step of selecting the most effective compound for treatment.

9. A method according to any one of paragraphs 5 to 8 wherein the effect is determined using a method selected from the group consisting of: a proliferation assay; an apoptosis assay; in vitro- drug response assay; migration and invasion assay; cell number counts (live or dead); and analysing cell cycle profiles.

10. A method according to paragraph 9 wherein the assay is an apoptosis assay and comprises measuring an early activation marker of apoptosis (BAK) and the activation of caspase 3.

1 1. A method according to paragraph 9 wherein the assay is a live cell assay. 12. A method according to paragraph 1 1 wherein the live cell assay comprises using a nuclear dye which can only cross cell membranes that have lost integrity.

13. A method according to any one of paragraphs 6 to 12 further comprising comparing the effect of the different therapeutic agents to identify the most effective.

14. A method according to any one of paragraphs 1 to 13 comprising simultaneously analysing s a m p l e s from 2 , 3 , 4 , 5 , 1 0 , 1 5 , 2 0 , 2 5 , 3 0 , 3 5 , 4 0 , 5 0 , 1 0 0 , 1 0 00 , 1 0 , 0 0 0 or more subjects.

15. A method according to any one of the previous paragraphs further comprising treating the subject with one or more identified therapeutic agents.

16. A method according to any one of the previous paragraphs wherein a therapeutic agent is any molecule, chemical entity, composition, drug, therapeutic agent, chemotherapeutic agent, or biological agent.

17. A method according to paragraph 16 wherein a therapeutic agent is a small molecule compound, antisense reagent, siRNA reagent, antibody, enzyme, peptide, organic or inorganic molecule, cell, natural or synthetic compound, and the like.

18. A method according to any one of the previous paragraphs wherein a therapeutic agent includes a single agent or a combination of two or more agents.

19. A method according to any one of the previous paragraphs wherein the subject is a mammal.

20. A method according to paragraph 19 wherein the subject is a human.

21. A method according to paragraph 20 wherein the s ubj ect i s a female human having an ovarian cancer tumor.

22. A method according to any one of paragraphs 1 to 20 wherein the subject has been previously diagnosed as having a hyperproliferative disorder.

23. A method according to any one of the previous paragraphs wherein a sample is a cell or tissue or a body fluid comprising a population of hyperproliferative cells.

24. A method according to paragraph 23 wherein the sample is obtained from stomach, colon, prostate, pancreas, heart, liver, colon, gastrointestinal, endocrine organ, muscle, bone, neuroendocrine lung, brain, blood, breast, ovary, fallopian tube, testicles, skin, or any other tissue or cells including immune cells, neural, endothelial, fibroblasts, or other epithelial and stromal tumors.

25. A method according to paragraph 24 wherein the sample is ascites fluid.

26. A method according to paragraph 25 wherein the sample comprises cancer tissue derived from any part of a patient as defined in paragraph 24. 27. A method according to any one of the preceding paragraphs wherein the sample is cultured a media which comprises between 1.0% and 100% v/v of autologous body fluid, more preferably 1.0% to 40%, more preferably 5-30%v/v, more preferably 5-20%.

28. A method according to any one of the previous paragraphs wherein media comprises 10-20% autologous ascites fluid or 5-10% autologous blood serum or 10-20% cerebrospinal fluid.

29. A method according to any one of the previous paragraphs comprising removing cells from the autologous body fluid.

30. A cell culture medium for use in culturing a sample of cells of a subject in vitro, wherein the culture medium comprises a basal medium and B27, insulin, transferrin, hydrocortisone, BSA, EGF and FGF.

31. A culture medium of paragraph 30 further comprising an autologous body fluid.

32. A culture medium according to paragraph 31 wherein the body fluid is ascites fluid or blood serum or cerebrospinal fluid.

33. A culture medium according to any one of paragraphs 30 to 32 wherein the basal media is DMEM/F12.

34. A culture medium according to any one of paragraphs 30 to 33 wherein the media does not comprise FBS (fetal bovine serum).

35. A cell culture medium supplement for use in culturing a sample of cells of a subject in vitro, wherein the supplement comprises insulin, transferrin, hydrocortisone, BSA, EGF and FGF.

36. A cell culture medium supplement according to paragraph 35 further comprising estrogen.

37. A cell culture medium supplement according to paragraph 35 wherein the supplement further comprises an autologous body fluid.

38. A cell culture medium supplement according to paragraph 36 wherein the body fluid is ascites fluid or serum.

39. A cell culture medium or supplement according to any one of paragraphs 30 to 38 further comprising one or more ingredients selected from: amino acids, salts, metals, sugars, lipids, nucleic acids, hormones, vitamins, fatty acids, a n d proteins.

40. A cell culture medium or supplement according to any one of paragraphs 30 to 39 further comprising one or more ingredients selected from: one or more antioxidants; nucleotide synthesis and salvage pathway precursors; lipid synthesis precursors; agonists of intracellular cAMP level; hormones, growth factors, amino acid supplements, vitamins, trace minerals, inorganic salts, energy sources (e.g. for glycolysis), antibiotics and pH indicators.

A cell culture medium or supplement according to any one of paragraphs 30 to 40 wherein in 100ml of media or supplement, excluding autologous body fluid, there is provided:

i) 95% up to 99.5% basal media;

ii) between 50-150, more preferably 70-120, more preferably 90-1 10, more preferably about 100μΙ insulin;

iii) between 100-250, more preferably 130-220, more preferably 100-190, more preferably 140-160, more preferably about 150μΙ transferrin;

iv) between 50-150, more preferably 70-120, more preferably 90-1 10, more preferably about 00μΙ hydrocortisone;

v) between 30-100ul, more preferably 50-80, more preferably about 70μΙ BSA;

vi) 5-30, more preferably 10-25, more preferably about 20μΙ EGF; and vii) 5-30, more preferably 10-25, more preferably about 20μΙ EGF2-20, more preferably 5-15, more preferably about 20μΙ FGF.

A cell culture medium or supplement according to any one of paragraphs 30 to 41 wherein in the final media: insulin is provided at a concentration of 1-20 mg/ml; transferrin is provided at a concentration of 1-10mg/ml; hydrocortisone is provided at a concentration 1-100mg/ml; BSA is provided at 5-10% solution; EGF is provided at a concentration of 1-100mg/ml; and FGF is provided at a concentration of 1-100mg/ml.

A cell culture medium or supplement according to any one of paragraphs 30 to 42 wherein in the final media insulin is provided at a concentration of 10 mg/ml; transferrin is provided at a concentration of 5mg/ml; hydrocortisone is provided at a concentration 50mg/ml; BSA is provided at 7.5% solution; EGF is provided at a concentration of 50mg/ml; and FGF is provided at a concentration of 50mg/ml. A method of preparing a cell culture media for culturing a sample of cells of a subject in vitro, wherein the method comprises combining a basal cell culture media, B27, insulin, transferrin, hydrocortisone, BSA, EGF and FGF and optionally an autologous body fluid.

A method according to paragraph 44 wherein the body fluid is ascites fluid or serum.

Claims

1. A method of culturing a mammalian cell, wherein the method comprises maintaining the cell in a culture medium comprising i) a pH-buffered isotonic salt solution; and ii) body fluid derived from a post-partum mammalian subject of the same species as the mammalian cell.

2. A method according to claim 1 wherein the cell is of a mammalian cell line.

3. A method according to claim 1 wherein the cell is derived from a subject.

4. A method according to claim 3 wherein the cell is derived from a subject having a hyperproliferative disorder.

5. A method according to any one of claims 1 , 3 or 4 wherein the cell is a cancer cell.

6. A method according to claim 1 or claims 3 to 5 wherein the cell is provided in a sample.

7. A method according to any one of claims 1 to 6 wherein the body fluid is i) an autologous body fluid, derived from the same subject as the cell; or ii) a non- autologous body fluid, derived from a different subject to the cell.

8. A method according to any one of the preceding claims wherein the body fluid is blood serum, ascites fluid or cerebrospinal fluid.

9. A method of culturing a sample of cancer cells of a subject, wherein the method comprises maintaining the cancer cells in a culture medium comprising i) a pH- buffered isotonic salt solution; and ii) autologous blood serum.

10. A method according to any one of the preceding claims, comprising: iii) adding the body fluid to the cell;

iv) maintaining the combination of i) in a culture medium comprising a pH- buffered isotonic salt solution and the body fluid.

11. A method according to any one of claims 1 to 9 comprising: iv) combining the body fluid with a cell culture medium or a component thereof;

v) resuspending the cell in the combination prepared in i); and

vi) maintaining the cell in culture.

12. A method according to any one of the previous claims further comprising i) exposing the cell to a candidate therapeutic compound and ii) assaying the effect of a therapeutic compound on the growth and/or survival of the cell in culture.

13. A method according to claim 12, wherein step i) and ii) of claim 5 are repeated for different candidate therapeutic compounds.

14. A method according to any one of the preceding claims comprising i) splitting a cell culture medium into multiple aliquots; ii) exposing each aliquot to a candidate therapeutic compound and iii) assaying the effect of a therapeutic compound on the growth and/or survival of the cell in culture.

15. A method according to claim 14, comprising the step of selecting the most effective compound for treatment.

16. A method according to any one of claims 12 to 15 wherein the effect is determined using a method is selected from the group consisting of: a proliferation assay; an apoptosis assay; in vitro- drug response assay; migration and invasion assay; a live cell assay; cell number counts (live or dead); and analysing cell cycle profiles.

17. A method according to claim 15 wherein the selected method is an apoptosis assay comprising measuring an early activation marker of apoptosis and the activation of caspase 3, preferably comprising contacting the cell with an antibody specific for .

18. A method according to claim 17 wherein the early activation marker is BAK.

19. A method according to claim 16 wherein the assay is a live cell assay.

20. A method according to claim 19 wherein the live cell assay comprises contacting the cell with a nuclear dye which can only cross cell membranes that have lost integrity.

21. A method according to 14 wherein the therapeutic compound is an antibody.

22. A method according to claim 21 wherein the distribution of a therapeutic antibody is determined by immunocytochemistry and High Content Analysis, and wherein the method further comprises a cell death assay.

23. A method according to 21 wherein the antibody is specific for cellular antigen, and the method further comprises a cell death assay.

24. A method according to 22 where the cellular antigen is extracellular and the cell death assay is a live cell assay that measure the loss in membrane integrity.

25. A method according to claim 23 and 24 wherein cell death assay is a live cell assay which measures the loss in membrane integrity.

26. A method according to any one of claims 12 to 22 further comprising comparing the effect of the different therapeutic agents to identify the most effective.

27. A method according to any one of claims 1 to 25 comprising simultaneously analysing the growth and/or survival of 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 50, 100, 1000, 10,000 mammalian cells, wherein each cell is derived from a different subjects.

28. A method according to any one of claims 3 to 26 further comprising treating the subject from which the cell is derived with one or more identified therapeutic agents.

29. A method according to any one of the previous claims wherein a therapeutic agent is any molecule, chemical entity, composition, drug, therapeutic agent, chemotherapeutic agent, or biological agent.

30. A method according to claim 22 wherein a therapeutic agent is a small molecule compound, antisense reagent, siRNA reagent, antibody, enzyme, peptide, organic or inorganic molecule, cell, natural or synthetic compound, and the like.

31. A method according to any one of the previous claims wherein a therapeutic agent includes a single agent or a combination of two or more agents.

32. A method according to claim 1 or 3 to 30 wherein the cell is derived from a non-human subject, preferably a canine species.

33. A method according to any one of claims 1 or 3 to 30 wherein the subject is a human.

34. A method according to claim 32 wherein the su bject is a female human having an ovarian cancer tumor.

35. A method according to any one of claims 3 to 33 wherein the subject has been previously diagnosed as having a hyperproliferative disorder.

36. A method according to any one of the previous claims wherein the cell is provided in a sample, wherein the sample is a cell or tissue or a body fluid comprising a population of hyperproliferative cells.

37. A method according to claim 35 wherein the sample is obtained from stomach, colon, prostate, pancreas, heart, liver, colon, gastrointestinal, endocrine organ, muscle, bone, neuroendocrine lung, brain, blood, breast, ovary, fallopian tube, testicles, skin, or any other tissue or cells including immune cells, neural, endothelial, fibroblasts, or other epithelial and stromal tumors.

38. A method according to claim 36 wherein the sample is ascites fluid.

39. A method according to claim 37 wherein the sample comprises cancer tissue derived from any part of a subject as defined in claim 30.

40. A method according to any one of the preceding claims wherein the cell is cultured a media which comprises between 1.0% and 100% v/v of body fluid, more preferably 1.0% to 40%, preferably 5-30%v/v, or preferably 5-20%.

41. A method according to claim 39 wherein the cell is cultured a media which comprises between 10% v/v or 20% v/v of body fluid, preferably wherein the body fluid is blood serum.

42. A method according to any one of the previous claims wherein media comprises 10- 20% autologous ascites fluid or 5-20% autologous blood serum or 10-20% cerebrospinal fluid.

43. A method according to any one of the previous claims comprising removing cells from the autologous body fluid.

44. A cell culture medium for use in culturing a sample of cells of a subject in vitro, wherein the culture medium comprises a basal medium and B27, insulin, transferrin, hydrocortisone, BSA, EGF and FGF.

45. A culture medium of claim 43 further comprising a body fluid, preferably wherein the body fluid is autologous or non-autologous; further preferably wherein an autologous body fluid derived from the same subject as the sample of cells to be cultured.

46. A culture medium according to claim 44 wherein the body fluid is ascites fluid or blood serum or cerebrospinal fluid.

47. A culture medium according to any one of claims 43 to 45 wherein the basal media is DMEM/F12.

48. A culture medium according to any one of claims 43 to 46 wherein the media does not comprise FBS (fetal bovine serum).

49. A cell culture medium supplement for use in culturing a sample of cells of a subject in vitro, wherein the supplement comprises insulin, transferrin, hydrocortisone, BSA, EGF and FGF.

50. A cell culture medium supplement according to claim 48 further comprising estrogen.

51. A cell culture medium supplement according to claim 48 or 49 wherein the supplement further comprises a body fluid, preferably wherein the body fluid is autologous or non-autologous; further preferably wherein an autologous body fluid derived from the same subject as the sample of cells to be cultured.

52. A cell culture medium supplement according to claim 48 wherein the body fluid is ascites fluid or serum.

53. A cell culture medium or supplement according to any one of claims 43 to 51 further comprising one or more ingredients selected from: amino acids, salts, metals, sugars, lipids, nucleic acids, hormones, vitamins, fatty acids, a n d proteins.

54. A cell culture medium or supplement according to any one of claims 43 to 52 further comprising one or more ingredients selected from: one or more antioxidants; nucleotide synthesis and salvage pathway precursors; lipid synthesis precursors; agonists of intracellular cAMP level; hormones, growth factors, amino acid supplements, vitamins, trace minerals, inorganic salts, energy sources (e.g. for glycolysis), antibiotics and pH indicators.

. A cell culture medium or supplement according to any one of claims 43 to 53 wherein in 100ml of media or supplement, excluding autologous body fluid, there is provided: viii) 95% up to 99.5% basal media;

ix) between 50-150, more preferably 70-120, more preferably 90-110, more preferably about 100μΙ insulin;

x) between 100-250, more preferably 130-220, more preferably 100-190, more preferably 140-160, more preferably about 150μΙ transferrin;

xi) between 50-150, more preferably 70-120, more preferably 90-110, more preferably about 100μΙ hydrocortisone;

xii) between 30-1 OOul, more preferably 50-80, more preferably about 70μΙ BSA;

xiii) 5-30, more preferably 10-25, more preferably about 20μΙ EGF; and xiv) 5-30, more preferably 10-25, more preferably about 20μΙ EGF2-20, more preferably 5-15, more preferably about 20μΙ FGF.

56. A cell culture medium or supplement according to any one of claims 43 to 54 wherein in the final media: insulin is provided at a concentration of 1-20 mg/ml; transferrin is provided at a concentration of 1-10mg/ml; hydrocortisone is provided at a concentration 1-100mg/ml; BSA is provided at 5-10% solution; EGF is provided at a concentration of 1-100mg/ml; and FGF is provided at a concentration of 1-100mg/ml.

57. A cell culture medium or supplement according to any one of claims 43 to 55 wherein in the final media insulin is provided at a concentration of 10 mg/ml; transferrin is provided at a concentration of 5mg/ml; hydrocortisone is provided at a concentration 50mg/ml; BSA is provided at 7.5% solution; EGF is provided at a concentration of 50mg/ml; and FGF is provided at a concentration of 50mg/ml.

58. A method of preparing a cell culture media for culturing a sample of cells of a subject in vitro, wherein the method comprises combining a basal cell culture media, B27, insulin, transferrin, hydrocortisone, BSA, EGF and FGF and optionally a body fluid, preferably wherein the body fluid is autologous or non-autologous; further preferably wherein an autologous body fluid derived from the same subject as the sample of cells to be cultured.

59. A method according to claim 57 wherein the body fluid is ascites fluid or serum.